Synthesis and evaluation of ester derivatives of 10-hydroxycanthin-6-one as potential antimicrobial agents

Article abstract of DOI:10.3390/molecules21030390

As part of our continuing research on canthin-6-one antimicrobial agents, a new series of ester derivatives of 10-hydroxycanthin-6-one were synthesized using a simple and effective synthetic route. The structure of each compound was characterized by NMR, ESI-MS, FT-IR, UV, and elemental analysis. The antimicrobial activity of these compounds against three phytopathogenic fungi (Alternaria solani, Fusarium graminearum, and Fusarium solani) and four bacteria (Bacillus cereus, Bacillus subtilis, Ralstonia solanacearum, and Pseudomonas syringae) were evaluated using the mycelium linear growth rate method and micro-broth dilution method, respectively. The structure-activity relationship is discussed. Of the tested compounds, 4 and 7s displayed significant antifungal activity against F. graminearum, with inhibition rates of 100% at a concentration of 50 μg/mL. Compounds 5, 7s, and 7t showed the best inhibitory activity against all the tested bacteria, with minimum inhibitory concentrations (MICs) between 3.91 and 31.25 μg/mL. Thus, 7s emerged as a promising lead compound for the development of novel canthine-6-one antimicrobial agents.

Full text of DOI:10.3390/molecules21030390

molecules
Article
Synthesis and Evaluation of Ester Derivatives of
10-Hydroxycanthin-6-one as Potential
Antimicrobial Agents
Fei Zhao, Jiang-Kun Dai, Dan Liu, Shi-Jun Wang and Jun-Ru Wang *
College of Science, Northwest A & F University, Yangling 712100, China; zhaofeicaiwei@163.com (F.Z.);
daijkun@hotmail.com (J.-K.D.); ld127222@126.com (D.L.); 444795415@163.com (S.-J.W.)
* Correspondence: wangjunru@nwsuaf.edu.cn; Tel.: +86-29-8709-2829
Academic Editors: Shufeng Zhou and Wei-Zhu Zhong
Received: 22 February 2016 ; Accepted: 16 March 2016 ; Published: 21 March 2016
Abstract: As part of our continuing research on canthin-6-one antimicrobial agents, a new series
of ester derivatives of 10-hydroxycanthin-6-one were synthesized using a simple and effective
synthetic route. The structure of each compound was characterized by NMR, ESI-MS, FT-IR, UV, and
elemental analysis. The antimicrobial activity of these compounds against three phytopathogenic
fungi (Alternaria solani, Fusarium graminearum, and Fusarium solani) and four bacteria (Bacillus cereus,
Bacillus subtilis, Ralstonia solanacearum, and Pseudomonas syringae) were evaluated using the mycelium
linear growth rate method and micro-broth dilution method, respectively. The structure-activity
relationship is discussed. Of the tested compounds,
against F. graminearum, with inhibition rates of 100% at a concentration of 50
7s, and 7t showed the best inhibitory activity against all the tested bacteria, with minimum
inhibitory concentrations (MICs) between 3.91 and 31.25 g/mL. Thus, 7s emerged as a promising
4
and 7s displayed significant antifungal activity
µg/mL. Compounds
5,
µ
lead compound for the development of novel canthine-6-one antimicrobial agents.
Keywords: 10-hydroxycanthin-6-one; ester derivatives; antibacterial activity; antifungal activity
1. Introduction
The canthine-6-one alkaloids represent a fairly large subclass of
β-carboline. The first
member (Figure 1) in the series was isolated in 1952 from Pentaceras australis [
1
]. Since then,
more than 60 compounds with the characteristic tetracyclic core have been isolated from many
other species, principally from the Rutaceae and Simaroubaceae families, and recently from
fungi [
antiviral [3–5], anticancer [6–8], antiparasitic [
and antifungal [17 18] activity. Notably, canthine-6-one alkaloids have received considerable attention
2]. These compounds exhibited a broad spectrum of biological properties, including
9–
11], anti-inflammatory [12 13], antibacterial [14 16],
,
–
,
owing to their high antimicrobial potencies. Soriano-Agaton et al. [17] reported the antifungal
activity of four naturally occurring canthin-6-ones and their derivatives against five pathogenic
fungi. However, the structure-activity relationship (SAR) for the antifungal activity remained unclear.
Lagoutte et al. [18] investigated the mechanism of action of the antifungal canthin-6-one series in
Saccharomyces cerevisiae. They showed an affinity of canthine-6-ones for lipid droplets and an effect
on lipid metabolism suggesting a stimulation of alkyl chain desaturase enzyme systems. In 2007,
O’Donnell et al. [16] isolated two canthin-6-ones which displayed minimum inhibitory concentrations
(MICs) in the range 8–32
against multidrug-resistant and methicillin-resistant strains of Staphylococcus aureus. In a program
screening of antimicrobials from natural products and their derivatives, our group [14 19] synthesized
a series of hydrophilic derivatives of canthin-6-one having a peak MIC of 0.98 g/mL against S. aureus.
µg/mL against a panel of fast-growing Mycobacterium species and 8–64 µg/mL
,
µ
Molecules 2016, 21, 390; doi:10.3390/molecules21030390
www.mdpi.com/journal/molecules

Molecules 2016, 21, 390
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1
11
2
10
C
A
N 3
9
B
8
N
6
D
4
5
O
Figure 1. Structure of canthin-6-one.
Plant diseases caused by microorganisms have been recognized as a worldwide threat to crop
production [20], and they are extremely difficult to manage in agricultural production. Moreover,
the repeated use of conventional antimicrobials over many years has resulted in serious environmental
and resistance problems. Libraries of natural products are expected to provide a rich resource for the
discovery of new drugs [21]. In the past decades, natural product-based or -derived plant protectants
have attracted lots of attention due to their low toxicity and good biocompatibility [22].
To exploit the potential antimicrobial activities of compounds having a canthin-6-one skeleton,
a novel series of ester derivatives of 10-hydroxycanthin-6-one were synthesized and evaluated
for their bioactivity against three phytopathogenic fungi and four bacterial strains. Furthermore,
the structure-activity relationship was discussed.
2. Results and Discussion
2.1. Chemistry
The synthetic route used for the target compounds is outlined in Scheme 1. The natural
product 10-methoxycanthin-6-one (
5) was synthesized using a modification of previously reported
methods [23], with 5-methoxytryptamine (1) as the starting material. Another key parent compound,
10-hydroxycanthin-6-one (
an argon atmosphere, with anhydrous dichloromethane as solvent. At 0 C, a new series of ester
6
), was obtained by treating compound
5
with˝boron tribromide under
derivatives of 10-hydroxycanthin-6-one (7a–7x) was obtained in 48%–86% yield by the reaction of
compound 6 with the corresponding acyl chloride in the presence of triethylamine.
1
1
7a: R = C6H5
7g: R = 3 -ClC6H4
7m: R = 4 -FC6H4
7s: R = CH3
1
1
1
7b: R = 2 -pyridyl
7h: R = 4 -ClC6H4
7n: R = 2 -CF3C6H4
7t: R = CH3CH2
7u: R = CH3(CH2)2
7v: R = CH3(CH2)3
7w: R = CH3(CH2)4
7x: R = CH3(CH2)10
1
1
1
1
70: R = 3 -CF3C6H4
7c: R = 2 -CH3C6H4
7i: R = 2 ,4 -Cl2C6H3
1
1
1
7p: R = 4 -CF3C6H4
7d: R = 3 -CH3C6H4
7j: R = 4 -CH3OC6H4
1
1
1
7q: R = 1 -naphthyl
7e: R = 4 -CH3C6H4
7k: R = 2 -FC6H4
1
1
1
7r: R = 2 -naphthyl
7f: R = 2 -ClC6H4
7l: R = 3 -FC6H4
Scheme 1. Synthetic route and chemical structures of compounds
(a) i: benzaldehyde, methanol, r.t.; ii: NaBH4, r.t., 98%; (b)
dioxane = 3:2, DST, reflux, 83%; (c) HCOONH4, 5% Pd/C, methanol: toluene = 1:1, reflux, 75%;
4–
6
and 7a–7x. Reagents and conditions:
α
-ketoglutaric acid, p-TSA, dry toluene:
(d) 5% Pd/C, xylene, reflux, 90%; (e) BBr3, dry DCM,
dry DCM, 0 ˝C, 48%–86%.
´
78 ˝C to r.t., 63%; (f) acyl chloride, TEA,

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The structures of all compounds were elucidated by spectroscopic analysis, including ESI-MS,
1H-NMR spectra, 13C-NMR spectra, FT-IR, UV, and elemental analysis. All the spectral and analytical
data obtained were in agreement with the assigned structures.
2.2. Biological Evaluation
2.2.1. Antifungal Activity
The in vitro antifungal properties of synthesized compounds 4–6 and 7a–7x were evaluated against
three phytopathogenic fungi (Alternaria solani, Fusarium graminearum, and Fusarium solani) using the
mycelium linear growth rate method [24]. Thiabendazole (TBZ), a commercially available agricultural
fungicide, was used as a positive control. The results are summarized in Table 1.
Table 1. Average inhibition rates of compounds 4–6 and 7a–7x against three phytopathogenic fungi.
Compounds
Average Inhibition Rate ˘ SD (%)
No.
R
A. solani
F. graminearum
F. solani
4
5
6
-
-
-
61.2 ˘ 0.4 g
71.8 ˘ 0.5 b
2.0 ˘ 0.5 q
53.1 ˘ 0.7 i
66.6 ˘ 0.0 de
2.7 ˘ 0.7 q
45.8 ˘ 0.9 k
28.7 ˘ 0.8 l
2.1 ˘ 0.8 q
21.4 ˘ 0.8 n
5.7 ˘ 0.9 p
50.1 ˘ 0.8 j
50.6 ˘ 0.3 j
68.4 ˘ 0.7 c
54.0 ˘ 0.9 i
58.9 ˘ 1.3 h
64.4 ˘ 0.9 f
25.7 ˘ 0.7 m
8.3 ˘ 0.3 o
65.1 ˘ 1.1 ef
25.8 ˘ 0.2 m
68.4 ˘ 0.6 c
59.1 ˘ 0.9 h
66.9 ˘ 0.9 cd
49.9 ˘ 0.8 j
66.5 ˘ 0.9 de
72.2 ˘ 0.2 b
89.1 ˘ 0.6 a
100.0 ˘ 0.0 a
77.7 ˘ 0.4 d
74.5 ˘ 0.5 e
48.8 ˘ 0.4 j
47.0 ˘ 0.7 k
35.6 ˘ 0.4 n
43.6 ˘ 0.6 l
23.6 ˘ 0.2 q
31.8 ˘ 0.4 p
3.4 ˘ 0.3 s
53.4 ˘ 0.7 n
92.9 ˘ 0.1 a
57.9 ˘ 0.6 k
41.7 ˘ 0.7 q
79.8 ˘ 0.5 d
65.3 ˘ 1.5 i
64.1 ˘ 0.4 j
77.0 ˘ 0.6 e
68.1 ˘ 0.4 h
86.1 ˘ 0.6 c
72.2 ˘ 0.5 g
22.8 ˘ 0.6 u
41.9 ˘ 0.3 q
40.5 ˘ 0.4 r
49.4 ˘ 0.2 p
52.1 ˘ 0.9 o
13.5 ˘ 1.0 w
54.6 ˘ 0.3 m
22.9 ˘ 0.4 u
25.2 ˘ 0.7 t
20.3 ˘ 0.1 v
86.0 ˘ 0.2 c
80.1 ˘ 0.3 d
69.2 ˘ 0.9 h
74.2 ˘ 0.2 f
56.6 ˘ 0.3 l
29.8 ˘ 0.4 s
89.3 ˘ 0.4 b
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
C6H5
21-pyridyl
21-CH3C6H4
31-CH3C6H4
41-CH3C6H4
21-ClC6H4
31-ClC6H4
41-ClC6H4
21,41-Cl2C6H3
41-CH3OC6H4
21-FC6H4
3.1 ˘ 0.6 s
7.1 ˘ 0.5 r
6.3 ˘ 0.4 r
37.6 ˘ 0.5 m
33.0 ˘ 0.7 o
51.1 ˘ 0.6 h
80.5 ˘ 0.4 c
32.6 ˘ 0.8 po
80.1 ˘ 1.0 c
49.9 ˘ 0.6 i
50.2 ˘ 0.6 hi
100.0 ˘ 0.0 a
70.0 ˘ 0.3 f
44.4 ˘ 0.7 l
85.4 ˘ 0.4 b
55.1 ˘ 0.2 g
85.6 ˘ 0.4 b
100.0 ˘ 0.0 a
31-FC6H4
41-FC6H4
21-CF3C6H4
31-CF3C6H4
41-CF3C6H4
11-naphthyl
21-naphthyl
CH3
7s
7t
7u
7v
7w
7x
TBZ
CH3CH2
CH3(CH2)2
CH3(CH2)3
CH3(CH2)4
CH3(CH2)10
-
All values are expressed as means
of 50 g/mL for 72 h. The differences between data with the different lower case letters within a column are
˘
SD (n = 3). Tested fungi were treated with each compound at a concentration
µ
significant for the same tested fungus (p < 0.05).
As shown in Table 1, all the compounds displayed activity to some degree against each of
the tested fungi. In general, the compounds showed the highest activity against F. graminearum,
followed by F. solani. At a concentration of 50
activity, with inhibition rates of 49.9%–100%. Compounds
F. graminearum, with inhibition rates of 100%, equal to that of TBZ. Two compounds (
inhibition rates exceeding 70% against A. solani. Six compounds ( 7n 7p 7s 7v, and 7x) displayed
7g 7s, and 7t) had
7s, and 7t showed
µ
g/mL, 58% of the test items (47/81) revealed good
and 7s had the highest activity against
and 7x) had
4
5
4
,
,
,
,
more than 80% inhibition activity against F. graminearum, and four compounds (
over 80% inhibition activity against F. solani. Of these compounds, compounds
5
5
,
,
,

Molecules 2016, 21, 390
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broad-spectrum fungicidal activity against all the phytopathogenic fungi tested. Compound 7g
showed special activity against F. solani, displaying 86.1% inhibition activity, but had a low level of
activity against F. graminearum and A. solani. Compound 7p exhibited much higher activity against
F. graminearum than against F. solani and A. solani.
2.2.2. Antibacterial Activity
The antibacterial activity of the compounds was investigated using a previously reported
method [14,25]. First, compounds 4–6 and 7a–7x (10 µg) were screened for their antibacterial activity
against two Gram-positive (Bacillus cereus and Bacillus subtilis) and two Gram-negative bacterial strains
(Ralstonia solanacearum and Pseudomonas syringae) using the disk diffusion method. Penicillin sodium
was used as a positive control, and a dichloromethane poured disk as a negative control. The diameter
of the inhibition zone was assayed to make a preliminary evaluation of the antibacterial potential of
the tested compounds. The results are summarized in Table 2.
Table 2. Inhibition zones (mm) of some synthesized compounds (10 µg).
Gram-Positive Bacteria
Gram-Negative Bacteria
R. solanacearum P. syringae
Compounds
B. cereus
B. subtilis
5
6
7s
7t
7u
7v
7w
11.2 ˘ 0.5
10.9 ˘ 0.8
9.8 ˘ 0.4
16.9 ˘ 0.7
17.4 ˘ 0.1
13.9 ˘ 0.4
10.3 ˘ 1.1
8.5 ˘ 0.3
22.5 ˘ 0.2
12.6 ˘ 0.4
10.9 ˘ 1.0
8.5 ˘ 0.1
- *
-
12.2 ˘ 0.1
16.6 ˘ 0.2
13.5 ˘ 0.3
12.7 ˘ 0.2
9.4 ˘ 0.1
-
12.3 ˘ 0.2
18.4 ˘ 0.2
21.0 ˘ 0.4
13.0 ˘ 0.8
15.4 ˘ 0.2
12.5 ˘ 0.2
20.4 ˘ 0.8
13.0 ˘ 0.2
-
-
-
Penicillin Sodium
16.2 ˘ 0.2
All values are expressed as means ˘ SD (n = 3). * Inhibition zone < 8 mm.
The results revealed that compounds
inhibition zones < 8 mm), while compounds
both Gram-positive and Gram-negative bacteria. Compounds 7s and 7t showed higher antibacterial
activities than the parent compounds and . However, compounds 7u 7w exhibited equivalent or
weaker antibacterial activity than compound 5.
The MICs of compounds , and 7s 7w were determined in further studies using the micro-broth
4,
7a
–
7r, and 7x showed no antibacterial activity (growth
5,
6
, and 7s 7w exhibited moderate activity against
–
5
6
–
5
,
6
–
dilution method. The results are shown in Table 3.
Table 3. The in vitro antibacterial activity as MIC (µg/mL) for compounds 5, 6, and 7s–7w.
Gram-Positive Bacteria
Gram-Negative Bacteria
R. solanacearum P. syringae
15.62 7.81
Compounds
B. cereus
B. subtilis
5
6
7s
7t
7u
7v
7w
3.91
15.62
15.62
15.62
31.25
>125
>125
3.91
7.81
>125
15.62
7.81
31.25
125
>125
31.25
15.62
ND *
ND
>125
7.81
7.81
15.62
31.25
>125
0.98
62.5
7.81
ND
7.81
Penicillin Sodium
* Not determined.
Significant activity was observed for compounds
5
,
7s, and 7t against all the tested bacterial strains,
was active against the tested Gram-positive
with MICs between 3.91 and 31.25 g/mL. Compound
µ
5
bacteria, with MICs equivalent to that of penicillin sodium. However, none of the investigated
derivatives were found to be superior to the reference drug.

Molecules 2016, 21, 390
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2.3. Structure-Activity Relationship
Based on the results of the present study, the structure–activity relationship (SAR) for the
antifungal activity may be summarized as shown in Figure 2. Comparison of the antifungal activities of
compound 4 and 10-methoxycanthin-6-one (5) suggests that the conjugated unsaturated bonds of rings
C and D (see Figure 1) are non-essential. The aliphatic ester derivatives of 10-hydroxycanthin-6-one
had much higher antifungal activity than did the aromatic ester derivatives.
Figure 2. Structure-activity relationship for the antifungal activity.
The weak antibacterial activity of compound 4 suggests that the conjugated unsaturated bonds
of rings C and D play an important role in antibacterial activity. The introduction of aromatic esters
decreases the antibacterial activity. Other compounds with a shorter or longer aliphatic chain did not
show any distinct effect, except for compound 7x (Figure 3).
Figure 3. Structure-activity relationship for the antibacterial activity.
3. Materials and Methods
3.1. General Details
All the reagents and solvents were obtained locally and were of analytical grade or were purified
according to standard methods before use. The tested fungi and bacteria (B. cereus, B. subtilis,
R. solanacearum, P. syringae, A. solani, F. graminearum, and F. solani) were provided by the Institute of
Pesticide Science, Northwest A&F University, China. Melting points were determined using a digital
1
melting-point apparatus and were uncorrected. H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra
were recorded using a Bruker Avance III 500 MHz instrument (Bruker, Madison, WI, USA), with
TMS as the internal standard and CDCl3 or dimethylsulfoxide (DMSO-d6) as the solvent. Chemical
shifts (δ values) and coupling constants (J values) are given in this paper in ppm and Hz, respectively.
ESI-MS was carried out using a Thermo Fisher LCQ Fleet instrument (Shimadzu, Beijing, China).
Elemental analysis of the synthesized compounds was undertaken using a CE-440 elemental analyzer
(EAI, North Chelmsford, MA, USA). Ultraviolet spectra (UV) were obtained using a Thermo Scientific
Evolution 300 UV-vis spectrophotometer (Shimadzu, Beijing, China). Infrared spectra (IR) were
recorded on a Bruker TENSOR 27 spectrometer (Bruker, Madison, WI, USA) with KBr disks.

Molecules 2016, 21, 390
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3.2. Synthesis of Target Compounds
3.2.1. Synthesis of Compound 2
A previously described method [26] was used to synthesize compound
2. A solution of
5-methoxytryptamine (1) (4.76 g, 25 mmol) and benzaldehyde (3.06 mL, 30 mmol) in methanol was
vigorously stirred at room temperature for 36 h. Solid NaBH4 (2.84 g, 75 mmol) was added to the
resulting solution, and the mixture stirred at room temperature for 1 h. The reaction was quenched
by adding saturated aqueous NaHCO3 (100 mL), and the mixture concentrated under vacuum until
approximately 50 mL remained. The residue was filtered, and the filter cake was washed with CH2Cl2.
The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (3
ˆ
50 mL). The
combined organic phases were washed with brine (3 50 mL) and dried over anhydrous Na2SO4. The
ˆ
organic solvent was removed under vacuum to give the title compound as a brown solid (6.87 g, yield
1
98%, m.p. 71.2–72.3 ˝C), which was used without further purification. H-NMR (500 MHz, CDCl3)
δ
: 8.06 (s, 1H), 7.37–7.28 (m, 2H), 7.26–7.18 (m, 4H), 7.02 (d, J = 2.5 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H),
6.84 (dd, J = 8.8, 2.4 Hz, 1H), 3.87–3.76 (m, 5H), 3.01–2.93 (m, 4H), 1.87(s, 1H). 13C-NMR (125 MHz,
CDCl3) δ: 153.9, 140.1, 131.6, 128.4, 128.2, 127.9, 127.0, 122.8, 113.5, 112.3, 111.9, 100.8, 56.0, 53.9, 49.3,
25.8. ESI-MS m/z: [M + H]+(found) = 281.16, [M + H]+(calcd) = 281.37. Elemental analysis calculated for
1
C18H20N2O (%): C, 77.11; H, 7.19; N, 9.99 (found: C, 77.02; H, 7.23; N, 10.05). H-NMR, 13C-NMR, and
FT-IR spectra for some target compounds were showed in the Supplementary Materials.
3.2.2. Synthesis of Compound 3
A mixture of compound
2 (5.61 g, 20 mmol), α-ketoglutaric acid (3.51 g, 24 mmol),
p-toluenesulfonic acid (0.69 g, 4 mmol), and a solution of toluene/dioxane (60 mL 3:2 v/v) was
heated at 180 ˝C for 48 h, using a Dean-Stark trap for water removal. Upon completion, the reaction
mixture was cooled to room temperature and the organic solvent removed. The residue was dissolved
in a mixture of CH2Cl2 (60 mL) and saturated aqueous NaHCO3 (80 mL). The aqueous phase was
separated and the product extracted with CH2Cl2 (3
washed with brine (3 50 mL) and dried over anhydrous Na2SO4. The obtained crude product
was chromatographed on silica gel eluting with dichloromethane to afford compound as a white
powder (5.75 g, yield 83%, m.p. 192.5–193.7 ˝C). 1H-NMR (500 MHz, CDCl3)
: 8.26 (d, J = 8.8 Hz,
ˆ
50 mL). The combined organic phases were
ˆ
3
δ
1H), 7.43–7.27 (m, 5H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 4.23 (d, J = 13.5 Hz,
1H), 3.85 (s, 3H), 3.56–3.52 (m, 1H), 3.39 (d, J = 13.5 Hz, 1H), 3.25–3.15 (m, 1H), 2.91–2.68 (m, 3H),
2.61–2.47 (m, 3H), 1.96–1.85 (m, 1H). 13C-NMR (125 MHz, CDCl3)
δ: 167.7, 156.9, 138.5, 135.4, 130.5,
129.6, 128.9, 128.5, 127.3, 116.9, 113.4, 112.0, 101.8, 57.7, 57.3, 55.7, 50.1, 32.8, 27.6, 21.4. ESI-MS m/z:
[M + H]+(found) = 347.23, [M + H]+(calcd) = 347.43. Elemental analysis calculated for C22H22N2O2 (%):
C, 76.28; H, 6.40; N, 8.09 (found: C, 76.12; H, 6.51; N, 8.03).
3.2.3. Synthesis of Compound 4
To a flask was added compound
3 (5.75 g, 16.6 mmol), HCOONH4 (10.52 g, 167 mmol), Pd/C
˝
(5%, 1.5 g), methanol (25 mL), and toluene (25 mL). The mixture was stirred vigorously at 80 C.
After 4 h, the reaction was quenched by addition of saturated aqueous NaHCO3 until pH 8.5
was reached. The mixture was filtered, and the filter cake was washed with copious amounts of
dichloromethane and methanol until TLC indicated complete removal of the product. The combined
filtrates were concentrated under vacuum until approximately 50 mL remained, and then extracted
with CH2Cl2 (3
dried over anhydrous Na2SO4. The sample was concentrated and purified using silica gel column
chromatography, eluting with ethyl acetate-methanol (v/v = 2:1) to give product as a green solid
(3.19 g, yield 75%, m.p. > 250 ˝C). 1H-NMR (500 MHz, CDCl3)
8.24 (d, J = 8.9, 1H), 6.92–6.85 (m,
2H), 4.02–3.95 (m, 1H), 3.86 (s, 3H), 3.56–3.49 (m, 1H), 3.18–3.10 (m, 1H), 2.86–2.67 (m, 3H), 2.66–2.58
(m, 1H), 2.33–2.24 (m, 1H), 1.85–1.74 (m, 2H). 13C-NMR (125 MHz, CDCl3)
: 167.8, 156.9, 136.9, 131.0,
ˆ
80 mL). The combined organic phases were washed with brine (3
ˆ
80 mL) and
4
δ
δ

Molecules 2016, 21, 390
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129.2, 116.8, 112.8, 112.0, 101.9, 55.8, 50.7, 43.9, 33.1, 29.2, 21.7. ESI-MS m/z: [M + H]+(found) = 257.09,
[M + H]+
= 257.31. Elemental analysis calculated for C15H16N2O2 (%): C, 70.29; H, 6.29; N, 10.93
(calcd)
(found: C, 70.33; H, 6.26; N, 10.89).
3.2.4. Synthesis of Compound 5
To a stirred solution of compound
4 (2.56 g, 10 mmol) in xylene was added Pd/C (5%, 1.8 g),
and the mixture was heated at reflux for 4 days. The mixture was then cooled and filtered, and
the filtered solid washed carefully with dichloromethane and methanol. The combined filtrates
were concentrated under vacuum and purified using column chromatography on silica gel using
dichloromethane–acetone (v/v = 5:1) as eluent to give compound
5
as a bright yellow solid (2.25 g,
(KBr) cm´1: 1676 (C=O), 1450 (C=N). UV
(lg ) (DCM)
: 8.84 (d, J = 5.0 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H),
8.30 (d, J = 5.0 Hz, 1H), 8.12 (d, J = 9.8 Hz, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.34 (dd, J = 8.9, 2.6 Hz, 1H),
6.99 (d, J = 9.8 Hz, 1H), 3.92 (s, 3H). 13C-NMR (125 MHz, CDCl3)
: 159.3, 157.9, 145.7, 139.3, 136.4,
133.8, 132.5, 130.3, 129.0, 125.7, 118.0, 118.0, 116.4, 106.5, 56.0. ESI-MS m/z: [M + H]+(found) = 251.25,
[M + H]+
= 251.26. Elemental analysis calculated for C15H10N2O2 (%): C, 71.99; H, 4.03; N, 11.19
˝
yield 90%, m.p. 202.9–204.1 C). IR,
υ
λ
max
ε
max
380 (3.91) nm. 1H-NMR (500 MHz, DMSO-d6)
δ
δ
(calcd)
(found: C, 72.03; H, 4.12; N, 11.07).
3.2.5. Synthesis of Compound 6
To a solution of 10-methoxycanthin-6-one (2.25 g, 9 mmol) in anhydrous dichloromethane (100 mL)
was added dropwise boron tribromide (8.50 mL, 90 mmol) under argon at
stirred overnight at room temperature. The reaction was cautiously quenched with iced water (80 mL).
The solids were filtered and the filter cake washed with dichloromethane and then purified using
´
78 ˝C, and the mixture
silica gel column chromatography to give 10-hydroxycanthin-6-one (
6
) as a yellow powder (1.34 g,
yield 63%, m.p. > 250 ˝C). IR,
υ
(KBr) cm´1: 3424 (OH), 1691 (C=O), 1436 (C=N). UV
λ
max
(lg
ε)
max
1
(DCM) 375 (3.89) nm. H-NMR (500 MHz, DMSO-d6)
δ: 10.01 (s, 1H), 8.79 (d, J = 5.0 Hz, 1H), 8.30
(d, J = 8.8 Hz, 1H), 8.23 (d, J = 5.0 Hz, 1H), 8.08 (d, J = 9.8 Hz, 1H), 7.67 (d, J = 2.5 Hz, 1H), 7.19 (dd,
J = 8.8, 2.5 Hz, 1H), 6.96 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, DMSO-d6)
δ 158.9, 156.2, 146.1, 139.7,
136.2, 132.4, 132.2, 130.0, 129.0, 126.0, 119.0, 117.7, 117.4, 109.5.ESI-MS m/z: [M + H]+(found) = 237.21,
[M + H]+(calcd) = 237.23. Elemental analysis calculated for C14H8N2O2 (%): C, 71.18; H, 3.41; N, 11.86
(found: C, 71.20; H, 3.45; N, 11.77).
3.2.6. General Procedure for the Synthesis of Compounds 7a–7x
According to the previously reported method [27], compounds 7a
–7x were prepared through the
direct condensation of 10-hydroxycanthin-6-one ( ) with the corresponding acyl chloride. The general
6
procedure was as follows. A solution of benzoyl chloride (0.05 mL, 0.45 mmol) in CH2Cl2 (5 mL) was
added slowly to an ice-cooled mixture of 10-hydroxycanthin-6-one (0.71 g, 0.3 mmol) and triethylamine
(0.21 mL, 1.5 mmol) in anhydrous CH2Cl2 (30 mL) over 15 min with stirring. The reaction mixture was
stirred overnight at room temperature to provide a clear solution, and then quenched with saturated
aqueous NaHCO3 (50 mL). The organic phase was separated, washed with brine (3
finally dried over anhydrous Na2SO4. The solvent was removed and the residue purified by column
ˆ
50 mL), and
chromatography on silica gel, eluting with dichloromethane–acetone (v/v = 20:1) to afford 7a as a pale
˝
yellow powder (0.82 g, yield 80%, m.p. 220.4–221.2 C). IR,
υ
(KBr) cm´1: 1685 (C=O), 1669 (ester),
max
1
1420 (C=N). UV
λ
max
(lg ε) (DCM) 364 (4.25) nm, 383 (4.24) nm. H-NMR (500 MHz, CDCl3) δ 8.87 (d,
J = 5.0 Hz, 1H), 8.75 (d, J = 8.7 Hz, 1H), 8.33–8.25 (m, 2H), 8.09 (d, J = 9.8 Hz, 1H), 8.05 (d, J = 2.3 Hz,
1H), 7.97 (d, J = 5.0 Hz, 1H), 7.76–7.69 (m, 1H), 7.63–7.56 (m, 3H), 7.04 (d, J = 9.8 Hz, 1H). 13C-NMR
(125 MHz, CDCl3) δ165.3, 159.3, 148.7, 145.9, 139.6, 137.0, 136.4, 134.0, 130.3, 130.0, 129.8, 129.1, 129.0,
128.7, 125.5, 124.5, 118.0, 116.7, 116.2. ESI-MS m/z: [M + H]+(found) = 341.35, [M + H]+(calcd) = 341.34;
Elemental analysis calculated for C21H12N2O3 (%): C, 74.11; H, 3.55; N, 8.23 (found: C, 74.09; H, 3.45;
N, 8.26).

Molecules 2016, 21, 390
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6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl nicotinate (7b): Yield: 64%; yellow solid; m.p.
˝
235.8–236.8 C; IR, υ
(KBr) cm´1: 1739 (C=O), 1683 (ester), 1588 (C=N). UV λ
(lg ε) (DCM) 364
max
max
1
(4.04) nm, 382 (4.03) nm. H-NMR (500 MHz, CDCl3)
δ 9.45 (d, J = 2.2 Hz, 1H), 8.90 (dd, J = 4.9, 1.7 Hz,
1H), 8.84 (d, J = 5.0 Hz, 1H), 8.73 (d, J = 8.8 Hz, 1H), 8.50 (dt, J = 7.9, 2.0 Hz, 1H), 8.06–8.00 (m, 2H), 7.93
(d, J = 5.0 Hz, 1H), 7.56 (dd, J = 8.8, 2.3 Hz, 1H), 7.51 (ddd, J = 8.0, 4.9, 0.9 Hz, 1H), 6.99 (d, J = 9.8 Hz,
1H). 13C-NMR (125 MHz, CDCl3)
δ
164.0, 159.3, 154.3, 151.5, 148.2, 146.0, 139.8, 137.7, 137.2, 136. 6,
132.6, 129.6, 129.0, 125.6, 125.3, 124.3, 123.6, 118.1, 116.6, 116.1. ESI-MS m/z: [M + H]+(found) = 342.34,
[M + H]+
= 342.33; Elemental analysis calculated for C20H11N3O3 (%): C, 70.38; H, 3.25; N, 12.31
(calcd)
(found: C, 70.25; H, 3.13; N, 12.28).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 2-methylbenzoate (7c): Yield: 85%; yellow powder; m.p.
˝
214.7–215.8 C; IR, υ
(KBr) cm´1: 1715 (C=O), 1667 (ester), 1423 (C=N). UV λ
(lg ε) (DCM) 364
max
max
1
(4.26) nm, 382 (4.24) nm. H-NMR (500 MHz, CDCl3)
δ 8.84 (d, J = 4.9 Hz, 1H), 8.72 (d, J = 8.8 Hz, 1H),
8.23 (d, J = 7.9 Hz, 1H), 8.04 (d, J = 9.8 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.94 (d, J = 5.0 Hz, 1H), 7.57–7.49
(m, 2H), 7.40–7.33 (m, 2H), 7.00 (d, J = 9.8 Hz, 1H), 2.72 (s, 3H). 13C-NMR (125 MHz, CDCl3)
δ
165.8,
159.4, 148.7, 146.0, 141.6, 139.7, 137.0, 136.5, 133.1, 132.6, 132.1, 131.3, 129.8, 129.0, 128.1, 126.1, 125.6,
124.6, 118.0, 116.6, 116.3, 22.0. ESI-MS m/z: [M + H]+(found) = 355.33, [M + H]+(calcd) = 355.37; Elemental
analysis calculated for C22H14N2O3 (%): C, 74.57; H, 3.98; N, 7.91 (found: C, 74.62; H, 4.03; N, 7.88).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 3-methylbenzoate (7d): Yield: 80%; pale yellow powder;
˝
m.p. 183.2–183.9 C; IR,
υ
(KBr) cm´1: 1738 (C=O), 1636 (ester), 1420 (C=N). UV
λ
max
(lg ε)
max
(DCM) 364 (4.26) nm, 382 (4.24) nm. 1H-NMR (500 MHz, CDCl3)
δ
8.81 (dd, J = 6.6, 4.8 Hz, 1H),
8.69 (t, J = 8.9 Hz, 1H), 8.07–7.97 (m, 4H), 7.90 (dd, J = 8.9, 4.9 Hz, 1H), 7.54 (dd, J = 8.8, 2.3 Hz,
1H), 7.48 (d, J = 7.7 Hz, 1H), 7.42 (td, J = 7.5, 2.9 Hz, 1H), 6.98 (dd, J = 9.8, 5.9 Hz, 1H), 2.46
(d, J = 2.3 Hz, 3H). 13C NMR (125 MHz, CDCl3)
δ
165.4, 159.3, 148.7, 145.9, 139.6, 138.6, 137.0,
136.5, 134.7, 132.5, 130.8, 129.7, 129.1, 129.0, 128.6, 127.4, 125.5, 124.5, 118.0, 116.6, 116.2, 21.3. ESI-MS
m/z: [M + H]+ = 355.35, [M + H]+
= 355.37; Elemental analysis calculated for C22H14N2O3
(found)
(calcd)
(%): C, 74.57; H, 3.98; N, 7.91 (found: C, 74.62; H, 3.96; N, 7.97).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 4-methylbenzoate (7e): Yield: 83%; pale yellow powder;
˝
m.p. 227.9–228.5 C; IR,
υ
(KBr) cm´1: 1732 (C=O), 1636 (ester), 1421 (C=N). UV
λ
max
(lg
ε
) (DCM)
max
1
364 (4.22) nm, 382 (4.20) nm. H-NMR (500 MHz, CDCl3)
δ 8.83 (d, J = 5.0 Hz, 1H), 8.71 (d, J = 8.8 Hz,
1H), 8.14 (d, J = 8.1 Hz, 2H), 8.03 (d, J = 9.8 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.93 (d, J = 5.0 Hz, 1H), 7.55
(dd, J = 8.8, 2.3 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 9.8 Hz, 1H), 2.48 (s, 3H). 13C-NMR (125 MHz,
CDCl3)
δ 165.3, 159.4, 148.8, 146.0, 144.9, 139.7, 137.0, 136.5, 132.6, 130.3, 129.8, 129.5, 129.0, 126.4, 125.5,
124.6, 118.0, 116.6, 116.3, 21.8. ESI-MS m/z: [M + H]+(found) = 355.35, [M + H]+(calcd) = 355.37; Elemental
analysis calculated for C22H14N2O3 (%): C, 74.57; H, 3.98; N, 7.91 (found: C, 74.43; H, 3.99; N, 7.86).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 2-chlorobenzoate (7f): Yield: 72%; pale yellow powder;
˝
m.p. 208.9–210.3 C; IR,
υ
(KBr) cm´1: 1744 (C=O), 1676 (ester), 1416 (C=N). UV
λ
(lg
ε
) (DCM)
8.84 (d, J = 5.0 Hz, 1H), 8.72 (d, J = 8.8 Hz,
1H), 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 8.06–8.02 (m, 2H), 7.94 (d, J = 5.0 Hz, 1H), 7.59–7.52 (m, 3H), 7.46–7.41
(m, 1H), 6.99 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, CDCl3)
164.1, 159.4, 148.3, 146.0, 139.7, 137.1,
max
max
1
364 (4.31) nm, 382 (4.32) nm. H-NMR (500 MHz, CDCl3)
δ
δ
136.5, 134.6, 133.6, 132.6, 132.0, 131.5, 129.7, 129.0, 128.9, 126.9, 125.6, 124.4, 118.1, 116.7, 116.1. ESI-MS
m/z: [M + H]+(found) = 375.33, [M + H]+(calcd) = 375.78; Elemental analysis calculated for C21H11ClN2O3
(%): C, 67.30; H, 2.96; N, 7.47 (found: C, 67.22; H, 2.98; N, 7.53).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 3-chlorobenzoate (7g): Yield: 75%; pale yellow powder;
˝
m.p. 240.2–241.1 C; IR,
υ
(KBr) cm´1: 1742 (C=O), 1677 (ester), 1422 (C=N). UV
λ
max
(lg
ε
) (DCM)
max
1
364 (4.33) nm, 382 (4.34) nm. H-NMR (500 MHz, CDCl3)
δ 8.85 (d, J = 5.0 Hz, 1H), 8.75 (d, J = 8.8 Hz,
1H), 8.24 (t, J = 1.8 Hz, 1H), 8.14 (dt, J = 7.8, 1.2 Hz, 1H), 8.05 (d, J = 9.8 Hz, 1H), 8.02 (d, J = 2.2 Hz,
1H), 7.95 (d, J = 5.0 Hz, 1H), 7.69–7.63 (m, 1H), 7.56 (dd, J = 8.8, 2.3 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H),
7.01 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, CDCl3)
δ 164.1, 159.4, 148.4, 146.0, 139.8, 137.2, 136.6,

Molecules 2016, 21, 390
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135.0, 134.0, 132.6, 131.0, 130.3, 130.1, 129.7, 129.0, 128.4, 125.6, 124.4, 118.1, 116.6, 116.1. ESI-MS m/z:
[M + H]+ = 375.33, [M + H]+
= 375.78; Elemental analysis calculated for C21H11ClN2O3 (%):
(found)
(calcd)
C, 67.30; H, 2.96; N, 7.47 (found: C, 67.33; H, 3.04; N, 7.32).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 4-chlorobenzoate (7h): Yield: 69%; pale yellow powder;
˝
m.p. 239.8–240.7 C; IR,
υ
(KBr) cm´1: 1734 (C=O), 1684 (ester), 1420 (C=N). UV
λ
(lg
ε
) (DCM)
8.82 (d, J = 5.0 Hz, 1H), 8.71 (d, J = 8.8 Hz,
1H), 8.18 (d, J = 8.6 Hz, 2H), 8.03 (d, J = 9.8 Hz, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.91 (d, J = 5.0 Hz, 1H),
7.57–7.49 (m, 3H), 6.99 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, CDCl3)
164.4, 159.3, 148.4, 146.0, 140.6,
139.7, 137.1, 136.5, 132.6, 131.6, 129.7, 129.1, 129.0, 127.6, 125.6, 124.4, 118.0, 116.6, 116.1. ESI-MS m/z:
[M + H]+ = 375.35, [M + H]+
= 375.78; Elemental analysis calculated for C21H11ClN2O3 (%):
max
max
1
364 (4.53) nm, 382(4.52) nm. H-NMR (500 MHz, CDCl3)
δ
δ
(found)
(calcd)
C, 67.30; H, 2.96; N, 7.47 (found: C, 67.36; H, 2.95; N, 7.53).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 2, 4-dichlorobenzoate (7i): Yield: 78%; pale yellow
powder; m.p. 246.2–247.1 ˝C; IR,
υ
(KBr) cm´1: 1738 (C=O), 1676 (ester), 1423 (C=N). UV
λ
max
max
1
(lg
ε
) (DCM) 364 (4.23) nm, 383 (4.25) nm. H-NMR (500 MHz, CDCl3)
δ
8.84 (d, J = 5.0 Hz, 1H), 8.73 (d,
J = 8.8 Hz, 1H), 8.09 (d, J = 8.5, 1H), 8.07–8.01 (m, 2H), 7.95 (d, J = 5.0 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H),
7.56 (dd, J = 8.8, 2.3 Hz, 1H), 7.43 (dd, J = 8.4, 2.0 Hz, 1H), 7.00 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz,
CDCl3)
δ 163.2, 159.4, 148.2, 146.0, 139.8, 139.6, 137.2, 136.6, 135.9, 133.1, 132.6, 131.5, 129.7, 129.0, 127.4,
127.0, 125.6, 124.3, 118.1, 116.7, 116.1. ESI-MS m/z: [M + H]+(found) = 410.34, [M + H]+(calcd) = 410.22;
Elemental analysis calculated for C21H10Cl2N2O3 (%): C, 61.64; H, 2.46; N, 6.85 (found: C, 61.61; H,
2.38; N, 6.77).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 4-methoxybenzoate (7j): Yield: 72%; yellow powder;
m.p. > 250 ˝C; IR,
(3.98) nm, 382 (3.96) nm. H-NMR (500 MHz, CDCl3)
υ
max (KBr) cm´1: 1726 (C=O), 1673 (ester), 1419 (C=N). UV
λmax (lg
ε
) (DCM) 364
1
δ 8.84 (d, J = 5.0 Hz, 1H), 8.73 (d, J = 8.8 Hz, 1H),
8.24–8.18 (m, 2H), 8.05 (d, J = 9.8 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.94 (d, J = 5.0 Hz, 1H), 7.55 (dd,
J = 8.8, 2.3 Hz, 1H), 7.05–6.98 (m, 3H), 3.92 (s, 3H). 13C-NMR (125 MHz, CDCl3)
165.0, 164.2, 159.4,
148.8, 146.0, 139.7, 137.0, 136.5, 132.6, 132.4, 129.9, 129.0, 129.5, 124.6, 121.4, 118.0, 116.6, 116.3, 114.0,
55.6. ESI-MS m/z: [M + H]+ = 371.34, [M + H]+
= 371.36; Elemental analysis calculated for
δ
(found)
(calcd)
C22H14N2O4 (%): C, 71.35; H, 3.81; N, 7.56 (found: C, 71.29; H, 3.92; N, 7.49).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 2-fluorobenzoate (7k): Yield: 65%; pale yellow powder;
˝
m.p. 245.3–246.2 C; IR,
υ
(KBr) cm´1: 1744 (C=O), 1675 (ester), 1420 (C=N). UV
λ
max
(lg
ε
) (DCM)
max
1
364 (4.36) nm, 382 (4.34) nm. H-NMR (500 MHz, CDCl3)
δ 8.84 (d, J = 5.0 Hz, 1H), 8.72 (d, J = 8.8 Hz,
1H), 8.16 (td, J = 7.6, 1.9 Hz, 1H), 8.08–8.00 (m, 2H), 7.94 (d, J = 5.0 Hz, 1H), 7.68–7.62 (m, 1H), 7.57 (dd,
J = 8.8, 2.3 Hz, 1H), 7.32 (td, J = 7.6, 1.1 Hz, 1H), 7.29–7.22 (m, 1H), 7.00 (d, J = 9.8 Hz, 1H). 13C-NMR
(125 MHz, CDCl3)
135.6 (d, J = 262.7 Hz), 132.6, 129.8, 129.0, 125.5, 124.5, 124.3, 124.3, 118.0, 117.7 (d, J = 9.7 Hz), 117.4 (d,
J = 22.4 Hz), 116.6, 116.2. ESI-MS m/z: [M + H]+ = 359.34, [M + H]+
= 359.33; Elemental
δ 162.9 (d, J = 4.0 Hz), 162.4 (d, J = 262.7 Hz), 159.4, 148.3, 146.0, 139.7, 137.1, 136.5,
(found)
(calcd)
analysis calculated for C21H11FN2O3 (%): C, 70.39; H, 3.09; N, 7.82 (found: C, 70.41; H, 3.06; N, 7.81).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 3-fluorobenzoate (7l): Yield: 62%; pale yellow powder;
˝
m.p. 233.4–234.2 C; IR,
υ
(KBr) cm´1: 1747 (C=O), 1675 (ester), 1422 (C=N). UV
λ
(lg
ε
) (DCM)
8.84 (d, J = 5.0 Hz, 1H), 8.73 (d, J = 8.8 Hz,
1H), 8.07–8.02 (m, 2H), 8.01 (d, J = 2.3 Hz, 1H), 7.95–7.90 (m, 2H), 7.57–7.51 (m, 2H), 7.41–7.36 (m, 1H),
7.00 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, CDCl3)
164.2 (d, J = 3.2 Hz), 163.7 (d, J = 247.0 Hz),
max
max
1
364 (4.16) nm, 382 (4.15) nm. H-NMR (500 MHz, CDCl3)
δ
δ
159.4, 148.4, 146.0, 139.7, 137.1, 136.6, 132.6, 131.3 (d, J = 7.7 Hz), 130.4 (d, J = 7.8 Hz), 129.7, 129.0, 126.0
(d, J = 3.1 Hz), 125.6, 124.4, 121.1 (d, J = 21.6 Hz), 118.1, 117.2 (d, J = 22.2 Hz), 116.6, 116.1. ESI-MS m/z:
[M + H]+
= 359.33, [M + H]+
= 359.33; Elemental analysis calculated for C21H11FN2O3 (%):
(calcd)
(found)
C, 70.39; H, 3.09; N, 7.82 (found: C, 70.44; H, 3.12; N, 7.79).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 4-fluorobenzoate (7m): Yield: 66%; pale yellow powder;
˝
m.p. 226.2–227.4 C; IR,
υ
(KBr) cm´1: 1736 (C=O), 1687 (ester), 1417 (C=N). UV
λ
max
(lg ε)
max

Molecules 2016, 21, 390
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1
(DCM) 364 (4.15) nm, 382 (4.12) nm. H-NMR (500 MHz, CDCl3)
δ
8.83 (d, J = 5.0 Hz, 1H), 8.72 (d,
J = 8.8 Hz, 1H), 8.30–8.24 (m, 2H), 8.03 (d, J = 9.8 Hz, 1H), 8.00 (d, J = 2.3 Hz, 1H), 7.92 (d, J = 5.0 Hz,
1H), 7.54 (dd, J = 8.8, 2.3 Hz, 1H), 7.25–7.20 (m, 2H), 6.99 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz,
CDCl3)
129.7, 129.0, 125.6, 125.4 (d, J = 3.0 Hz), 124.4, 118.0, 116.6, 116.2, 116.0 (d, J = 23.1 Hz). ESI-MS m/z:
[M + H]+ = 359.34, [M + H]+
= 359.33; Elemental analysis calculated for C21H11FN2O3 (%):
δ 167.4 (d, J = 257.0 Hz), 164.3, 159.4, 148.5, 146.0, 139.7, 137.1, 136.5, 132.6, 132.4 (d, J = 9.6),
(found)
(calcd)
C, 70.39; H, 3.09; N, 7.82 (found: C, 70.26; H, 2.98; N, 7.87).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 2-(trifluoromethyl) benzoate (7n): Yield: 61%; pale
yellow powder; m.p. 191.3–192.1 ˝C; IR,
υ
max (KBr) cm´1: 1754 (C=O), 1681 (ester), 1420 (C=N). UV
1
λ
max
(lg ε) (DCM) 363 (4.26) nm, 382 (4.24) nm. H-NMR (500 MHz, CDCl ) δ 8.85 (d, J = 5.0 Hz, 1H),
3
8.72 (d, J = 8.8 Hz, 1H), 8.08–8.00 (m, 3H), 7.97 (d, J = 5.0 Hz, 1H), 7.90–7.86 (m, 1H), 7.76–7.71 (m,
2H), 7.57 (dd, J = 8.8, 2.3 Hz, 1H), 6.99 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, CDCl3)
δ
165.2, 159.4,
148.2, 146.0, 139.8, 137.2, 136.6, 132.6, 132.1, 132.0, 130.8, 130.0, 129.7, 129.2 (d, J = 42.5 Hz), 127.1 (d,
J = 5.4 Hz), 127.1 (d, J = 5.5 Hz), 125.6, 124.1, 123.5 (d, J = 273.8 Hz), 118.1, 116.7, 115.8. ESI-MS m/z:
[M + H]+
= 409.38, [M + H]+
= 409.34; Elemental analysis calculated for C22H11F3N2O3 (%):
(found)
(calcd)
C, 64.71; H, 2.72; N, 6.86 (found: C, 64.66; H, 2.73; N, 6.89).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 3-(trifluoromethyl) benzoate (7o): Yield: 62%; pale
yellow powder; m.p. 199.9–200.8 ˝C; IR,
υ
max (KBr) cm´1: 1742 (C=O), 1687 (ester), 1422 (C=N). UV
1
λ
max
(lg ε) (DCM) 364 (4.31) nm, 382 (4.33) nm. H-NMR (500 MHz, CDCl ) δ 8.84 (d, J = 5.0 Hz, 1H),
3
8.74 (d, J = 8.8 Hz, 1H), 8.52 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.07–8.00 (m, 2H), 7.96–7.91 (m, 2H), 7.71
(t, J = 7.8 Hz, 1H), 7.57 (dd, J = 8.8, 2.3 Hz, 1H), 7.00 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz, CDCl3)
δ
164.0, 159.4, 148.3, 146.0, 139.8, 137.2, 136.6, 133.4, 132.6, 131.6 (d, J = 33.4 Hz), 130.4 (d, J = 3.8 Hz), 130.1,
129.6, 129.5, 129.0, 127.2 (d, J = 3.9 Hz), 125.6, 124.6 (d, J = 272.7 Hz), 124.3, 118.1, 116.6, 116.0. ESI-MS
m/z: [M + H]+(found) = 409.40, [M + H]+(calcd) = 409.34; Elemental analysis calculated for C22H11F3N2O3
(%): C, 64.71; H, 2.72; N, 6.86 (found: C, 64.80; H, 2.74; N, 6.90).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 4-(trifluoromethyl) benzoate (7p): Yield: 62%; pale
yellow powder; m.p. 223.8–224.9 ˝C; IR, max (KBr) cm´1: 1743 (C=O), 1677 (ester), 1415 (C=N). UV
υ
λ
max
(lg ε δ 8.85 (d, J = 5.0 Hz,
) (DCM) 364 (4.21) nm, 382 (4.22) nm. 1H-NMR (500 MHz, CDCl3)
1H), 8.74 (d, J = 8.8 Hz, 1H), 8.37 (d, J = 8.2 Hz, 2H), 8.07–7.99 (m, 2H), 7.94 (d, J = 5.0 Hz, 1H),
7.83 (d, J = 8.3 Hz, 2H), 7.56 (dd, J = 8.8, 2.3 Hz, 1H), 7.00 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz,
CDCl3)
129.0, 125.8 (d, J = 4.0 Hz), 125.6, 124.5 (d, J = 273.6 Hz), 124.3, 118.1, 116.6, 116.0. ESI-MS m/z:
[M + H]+ = 409.37, [M + H]+
= 409.34; Elemental analysis calculated for C22H11F3N2O3 (%):
δ 164.1, 159.4, 148.3, 146.0, 139.8, 137.2, 136.6, 135.4 (d, J = 33.0 Hz), 132.6, 132.4, 130.7, 129.6,
(found)
(calcd)
C, 64.71; H, 2.72; N, 6.86 (found: C, 64.79; H, 2.77; N, 6.81).
6-Oxo-6H-In˝dolo [3,2,1-de] [1,5] naphthyridin-10-yl 1-naphthoate (7q): Yield: 83%; white powder; m.p.
221.3–222.4 C; IR,
υ
(KBr) cm´1: 1732 (C=O), 1678 (ester), 1420 (C=N). UV
λ
max
(lg ε) (DCM)
max
364 (4.57) nm, 382 (4.55) nm. 1H-NMR (500 MHz, CDCl3)
δ 9.06 (dd, J = 8.6, 1.1 Hz, 1H), 8.83 (d,
J = 5.0 Hz, 1H), 8.74 (d, J = 8.7 Hz, 1H), 8.55 (dd, J = 7.3, 1.3 Hz, 1H), 8.14 (dt, J = 8.2, 1.1 Hz, 1H), 8.07
(d, J = 2.3 Hz, 1H), 8.03 (d, J = 9.8 Hz, 1H), 7.97–7.92 (m, 2H), 7.68–7.55 (m, 4H), 7.00 (d, J = 9.8 Hz, 1H).
13C-NMR (125 MHz, CDCl3)
δ 165.8, 159.4, 148.7, 146.0, 139.7, 137.1, 136.5, 134.8, 134.0, 132.6, 131.8,
131.5, 129.8, 129.0, 128.8, 128.4, 126.6, 125.6, 125.6, 125.3, 124.7, 124.6, 118.1, 116.6, 116.4. ESI-MS m/z:
[M + H]+(found) = 391.39, [M + H]+(calcd) = 391.40; Elemental analysis calculated for C25H14N2O3 (%):
C, 76.92; H, 3.61; N, 7.18 (found: C, 76.99; H, 3.57; N, 7.24).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl 2-naphthoate (7r): Yield: 80%; white powder; m.p.
˝
234.0–235.1 C; IR, υ
(KBr) cm´1: 1735 (C=O), 1685 (ester), 1419 (C=N). UV λ
(lg ε) (DCM) 364
max
max
1
(4.59) nm, 382 (4.58) nm. H-NMR (500 MHz, CDCl3)
δ 8.86–8.79 (m, 2H), 8.73 (d, J = 8.8 Hz, 1H),
8.22 (dd, J = 8.6, 1.7 Hz, 1H), 8.06 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 9.8 Hz, 1H), 8.01 (d, J = 8.2, 1H), 7.96
(d, J = 8.6, 1H), 7.94–7.89 (m, 2H), 7.67–7.57 (m, 3H), 6.99 (d, J = 9.8 Hz, 1H). 13C-NMR (125 MHz,

Molecules 2016, 21, 390
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CDCl3)
δ 165.4, 159.4, 148.8, 146.0, 139.7, 137.0, 136.5, 136.0, 132.6, 132.5, 132.2, 129.8, 129.5, 129.0, 128.9,
128.6, 127.9, 127.0, 126.3, 125.6, 125.4, 124.6, 118.0, 116.6, 116.3. ESI-MS m/z: [M + H]+(found) = 391.38,
[M + H]+(calcd) = 391.40; Elemental analysis calculated for C25H14N2O3 (%): C, 76.92; H, 3.61; N, 7.18
(found: C, 76.85; H, 3.57; N, 7.12).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl acetate (7s): Yield: 52%; yellow solid; m.p.
198.5–199.3 ˝C; IR,
(3.87) nm, 382 (3.85) nm. H-NMR (500 MHz, CDCl3)
υ
max (KBr) cm´1: 1758 (C=O), 1675 (ester), 1420 (C=N). UV
λmax (lg
ε
) (DCM) 364
1
δ 8.80 (d, J = 5.0 Hz, 1H), 8.63 (d, J = 8.8 Hz, 1H),
8.00 (d, J = 9.8 Hz, 1H), 7.88 (d, J = 5.0 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.40 (dd, J = 8.8, 2.3 Hz, 1H), 6.96
(d, J = 9.8 Hz, 1H), 2.38 (s, 3H). 13C-NMR (125 MHz, CDCl3)
169.5, 159.3, 148.4, 145.9, 139.7, 136.9,
136.5, 132.5, 129.7, 129.0, 125.4, 124.3, 117.9, 116.6, 116.0, 21.2. ESI-MS m/z: [M + H]+(found) = 279.26,
[M + H]+
= 279.27; Elemental analysis calculated for C16H10N2O3 (%): C, 69.06; H, 3.62; N, 10.07
δ
(calcd)
(found: C, 69.01; H, 3.58; N, 10.13).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl propionate (7t): Yield: 51%; yellow solid; m.p.
166.7–168.1 ˝C; IR,
(3.89) nm, 382 (3.85) nm. H-NMR (500 MHz, CDCl3)
υ
max (KBr) cm´1: 1753 (C=O), 1681 (ester), 1414 (C=N). UV
λmax (lg
ε
) (DCM) 364
1
δ 8.81 (d, J = 5.0 Hz, 1H), 8.65 (d, J = 8.8 Hz, 1H),
8.01 (d, J = 9.8 Hz, 1H), 7.89 (d, J = 5.0 Hz, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.40 (dd, J = 8.8, 2.3 Hz, 1H),
6.97 (d, J = 9.8 Hz, 1H), 2.68 (q, J = 7.6 Hz, 2H), 1.32 (t, J = 7.6 Hz, 3H). 13C-NMR (125 MHz, CDCl3)
δ
173.0, 159.3, 148.5, 145.9, 139.7, 136.9, 136.5, 132.5, 129.8, 129.0, 125.4, 124.4, 117.9, 116.6, 116.0, 27.8,
9.1. ESI-MS m/z: [M + H]+(found) = 293.27, [M + H]+(calcd) = 293.29; Elemental analysis calculated for
C17H12N2O3 (%): C, 69.86; H, 4.14; N, 9.58 (found: C, 69.79; H, 4.17; N, 9.63).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl butyrate (7u): Yield: 55%; yellow solid; m.p.
138.2–139.5 ˝C; IR,
(3.87) nm, 382 (3.86) nm. H-NMR (500 MHz, CDCl3)
υ
max (KBr) cm´1: 1753 (C=O), 1674 (ester), 1418 (C=N). UV
λmax (lg
ε
) (DCM) 364
1
δ 8.83 (d, J = 5.0 Hz, 1H), 8.67 (d, J = 8.8 Hz, 1H),
8.02 (d, J = 9.8 Hz, 1H), 7.92 (d, J = 5.0 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 8.8, 2.3 Hz, 1H),
6.98 (d, J = 9.8 Hz, 1H), 2.63 (t, J = 7.4 Hz, 2H), 1.89–1.81 (m, 2H), 1.10 (t, J = 7.4 Hz, 3H). 13C-NMR
(125 MHz, CDCl3)
δ
172.2, 159.4, 148.5, 145.9, 139.7, 136.9, 136.5, 132.6, 129.8, 129.0, 125.5, 124.4, 117.9,
= 307.28, [M + H]+
= 307.32; Elemental
116.6, 116.0, 36.3, 18.5, 13.7. ESI-MS m/z: [M + H]+
(found)
(calcd)
analysis calculated for C18H14N2O3 (%): C, 70.58; H, 4.61; N, 9.15 (found: C, 70.55; H, 4.66; N, 9.18).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl pentanoate (7v): Yield: 56%; yellow solid; m.p.
141.6–142.7 ˝C; IR,
(3.87) nm, 382 (3.85) nm. H-NMR (500 MHz, CDCl3)
υ
max (KBr) cm´1: 1755 (C=O), 1675 (ester), 1417 (C=N). UV
λmax (lg
ε
) (DCM) 364
1
δ 8.81 (d, J = 5.0 Hz, 1H), 8.64 (d, J = 8.8 Hz, 1H),
8.00 (d, J = 9.8 Hz, 1H), 7.90 (d, J = 5.0 Hz, 1H), 7.84 (d, J = 2.3 Hz, 1H), 7.39 (dd, J = 8.8, 2.3 Hz, 1H), 6.96
(d, J = 9.8 Hz, 1H), 2.64 (t, J = 7.5 Hz, 2H), 1.83–1.76 (m, 2H), 1.54–1.45 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).
13C NMR (125 MHz, CDCl3) 172.4, 159.3, 148.5, 145.9, 139.6, 136.9, 136.5, 132.5, 129.7, 129.0, 125.4, 124.4,
117.9, 116.6, 116.0, 34.1, 27.0, 22.3, 13.8. ESI-MS m/z: [M + H]+
= 321.29, [M + H]+
= 321.35;
(calcd)
(found)
Elemental analysis calculated for C19H16N2O3 (%): C, 71.24; H, 5.03; N, 8.74 (found: C, 71.16; H, 5.11;
N, 8.69).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl hexanoate (7w): Yield: 53%; yellow solid; m.p.
112.8–113.7 ˝C; IR,
(3.85) nm, 382 (3.86) nm. H-NMR (500 MHz, CDCl3)
υ
max (KBr) cm´1: 1761 (C=O), 1675 (ester), 1417 (C=N). UV
λmax (lg
ε
) (DCM) 364
1
δ 8.81 (d, J = 5.0 Hz, 1H), 8.64 (d, J = 8.8 Hz,
1H), 8.01 (d, J = 9.8 Hz, 1H), 7.90 (d, J = 5.0 Hz, 1H), 7.85 (d, J = 2.3 Hz, 1H), 7.39 (dd, J = 8.8, 2.3 Hz,
1H), 6.97 (d, J = 9.8 Hz, 1H), 2.63 (t, J = 7.5 Hz, 2H), 1.85–1.78 (m, 2H), 1.48–1.38 (m, 4H), 0.96 (t,
J = 7.1 Hz, 3H). 13C-NMR (126 MHz, CDCl3)
δ 172.4, 159.3, 148.5, 145.9, 139.7, 136.9, 136.5, 132.5, 129.8,
129.0, 125.4, 124.4, 117.9, 116.6, 116.0, 34.4, 31.3, 24.6, 22.4, 13.9. ESI-MS m/z: [M + H]+(found) = 335.35,
[M + H]+(calcd) = 335.38; Elemental analysis calculated for C20H18N2O3 (%): C, 71.84; H, 5.43; N, 8.38
(found: C, 71.85; H, 5.46; N, 8.43).
6-Oxo-6H-Indolo [3,2,1-de] [1,5] naphthyridin-10-yl dodecanoate (7x): Yield: 49%; yellow solid; m.p.
65.4–66.6 ˝C; IR,
υ
(KBr) cm´1: 1757 (C=O), 1682 (ester), 1420 (C=N). UV
λ
max
(lg ε) (DCM) 364
max

Molecules 2016, 21, 390
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1
(3.83) nm, 383 (3.82) nm. H-NMR (500 MHz, CDCl3)
δ
8.84 (d, J = 5.0 Hz, 1H), 8.67 (d, J = 8.8 Hz, 1H),
8.07 (d, J = 9.8 Hz, 1H),7.94 (d, J = 5.0 Hz, 1H), 7.87 (d, J = 2.3 Hz, 1H), 7.41 (dd, J = 8.8, 2.3 Hz, 1H),
7.00 (d, J = 9.8 Hz, 1H), 2.64 (t, J = 7.5 Hz, 2H), 2.34 (t, J = 7.5 Hz, 4H), 1.85–1.76 (m, 2H), 1.69–1.57
(m, 4H), 1.53–1.41 (m, 2H), 0.90–0.86 (m, 9H). 13C-NMR (125 MHz, CDCl3) δ 172.4, 159.4, 148.5, 145.7,
139.5, 137.0, 136.4, 132.6, 130.0, 129.1, 125.4, 124.5, 118.0, 116.7, 116.1, 34.4, 34.1, 31.9, 29.6, 29.5, 29.3,
29.3, 29.1, 25.0, 24.8, 22.7. ESI-MS m/z: [M + H]+
= 419.53, [M + H]+
= 419.54; Elemental
(calcd)
(found)
analysis calculated for C26H30N2O3 (%): C, 74.61; H, 7.23; N, 6.69 (found: C, 74.58; H, 7.19; N, 6.67).
3.3. Procedure for Determining Antifungal Activity
The in vitro antifungal activities of compounds 4–6 and 7a–7x against three phytopathogenic
fungi (A. solani, F. graminearum, and F. solani) were investigated using the mycelium linear growth rate
method [24]. The test compounds and TBZ were completely dissolved in 0.5 mL of DMSO and the
solution added to 9.5 mL of sterile water. The resulting solution was added to 90 mL of melted potato
˝
dextrose agar (PDA) medium at a temperature below 55 C. After quickly and completely mixing, the
medium containing the compounds at a concentration of 50 g/mL was poured into a sterilized Petri
µ
dish for screening. DMSO without any compounds mixed with PDA served as the negative control.
When the medium in the plates was partially solidified, a 4 mm thick, 5 mm diameter disc of fungus
cut from previously subcultured Petri dishes was picked up and inoculated in the center of the PDA
dishes. The dishes were kept in an incubator at 28 ˝C for 72 h. Three replicates were performed for
each experiment. The inhibitory rate was calculated according to the following formula:
inhibition rate p%q “ rpdc ´ d0q ´ pds ´ d0qs{pdc ´ d0q ˆ 100%
(1)
where d0 is the diameter of the fungus cut, dc is the diameter of the untreated control fungus, and ds is
the diameter of the sample-treated fungus.
3.4. Procedure for Determining Antibacterial Activity
The test compounds were dissolved in dichloromethane at a concentration of 2 mg/mL, and
then 5
µL of each solution was transferred onto sterile paper discs (6.0 mm diameter), so that the disc
contained 10
µ
g of the compound. Melted LB agar medium (10 mL) was then mixed with 100 L of
µ
˝
bacterial suspension at 45 C and poured into a sterilized Petri dish. The discs were placed gently on
the surface of the media inoculated with the microorganism. Penicillin sodium was used as a positive
control and dichloromethane as a negative control. The plates were allowed to stand for 2 h at 4 C to
˝
˝
allow for diffusion and then incubated at 37 C for 12 h. The diameter of the zone of growth inhibition
around the disc was measured. Experiments were repeated in triplicate.
The MICs of some synthesized compounds against four bacterial strains were evaluated using
the micro-broth dilution method using Muller-Hinton broth in 96-well plates. The test compounds
and penicillin sodium were first dissolved in DMSO at a concentration of 10 mg/mL, and then diluted
20-fold with sterile water to give the stock solution. An aliquot (50 µL) of this solution was pipetted
into the first well of each line in a 96-well plate. The stock solution was then serially diluted to
give two-fold serial dilutions of the test compounds and positive control in the subsequent wells.
Then 50 µ
L of bacterial suspension (106 cfu/mL) was added to each well and the plate set aside for
incubation. One well containing bacterial cells and 2.5% DMSO was used as growth control, and
another well containing only growth medium was used as a blank control. After incubation for 12 h at
˝
37 C, MICs were measured. Three replicates were performed for each experiment.
4. Conclusions
In this study, two naturally occurring canthine-6-ones and 24 novel ester derivatives of
10-hydroxycanthin-6-one were synthesized and their in vitro antimicrobial activity was evaluated.
Most of the synthesized compounds showed potential antibacterial and antifungal activity. Compound

Molecules 2016, 21, 390
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7s represents a promising lead compound for further study, as it had significant antimicrobial
activity against all the fungi and bacterial strains tested. Further research is required into more
extensive structural modification, the structure-activity relationship and the mechanism of action
of canthin-6-ones.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/3/390/s1.
Acknowledgments: This project was financially supported by the National Natural Science Foundation of China
(Grant No. 31270388) and the Fundamental Research Funds for the Central Universities of China (Grant No.
QN 2011066).
Author Contributions: Jun-Ru Wang, Fei Zhao, and Jiang-Kun Dai conceived and designed the experiments;
Fei Zhao performed the experiments; Jiang-Kun Dai, Dan Liu, and Shi-Jun Wang analyzed the data; Fei Zhao
wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5, 6, and 7a–7x are available from the authors.
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