Design, synthesis and bioactivity evaluation of novel β-carboline 1,3,4-oxadiazole derivatives

Article abstract of DOI:10.3390/molecules22111811

A series of novel β-carboline 1,3,4-oxadiazole derivatives were designed and synthesized, and the in vitro cytotoxic activity against Sf9 cells and growth inhibitory activity against Spodoptera litura were evaluated. Bioassay results showed that most of these compounds exhibited excellent in vitro cytotoxic activity. Especially, compound 37 displayed the best efficacy in vitro (IC₅₀ = 3.93 μM), and was five-fold more potent than camptothecin (CPT) (IC₅₀ = 18.95μM). Moreover, compounds 5 and 37 could induce cell apoptosis and cell cycle arrest and stimulate Sf-caspase-1 activation in Sf9 cells. In vivo bioassay also demonstrated that compounds 5 and 37 could significantly inhibit larvae growth of S. litura with decreasing the weight of larvae and pupae. Based on these bioassay results, compounds 5 and 37 emerged as lead compounds for the development of potential insect growth inhibitions.

Full text of DOI:10.3390/molecules22111811

molecules
Article
Design, Synthesis and Bioactivity Evaluation of
Novel β-carboline 1,3,4-oxadiazole Derivatives
Zhi-Jun Zhang 1,2 ID , Jing-Jing Zhang 1,2, Zhi-Yan Jiang 1,2 and Guo-Hua Zhong 1,2,
*
1
Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education,
Guangzhou 510642, China; zhangzhijun198803@163.com (Z.-J.Z.); zhangjingjing@stu.scau.edu.cn (J.-J.Z.);
zyjiang@stu.scau.edu.cn (Z.-Y.J.)
Lab of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China
Correspondence: guohuazhong@scau.edu.cn; Tel.: +86-20-8528-0308
2
*
Received: 4 October 2017; Accepted: 19 October 2017; Published: 29 October 2017
Abstract: A series of novel -carboline 1,3,4-oxadiazole derivatives were designed and synthesized,
β
and the in vitro cytotoxic activity against Sf9 cells and growth inhibitory activity against
Spodoptera litura were evaluated. Bioassay results showed that most of these compounds exhibited
excellent in vitro cytotoxic activity. Especially, compound 37 displayed the best efficacy in vitro
(
IC50 = 3.93 µM), and was five-fold more potent than camptothecin (CPT) (IC50 = 18.95
Moreover, compounds and 37 could induce cell apoptosis and cell cycle arrest and stimulate
Sf-caspase-1 activation in Sf9 cells. In vivo bioassay also demonstrated that compounds and
37 could significantly inhibit larvae growth of S. litura with decreasing the weight of larvae and
µM).
5
5
pupae. Based on these bioassay results, compounds
development of potential insect growth inhibitions.
5 and 37 emerged as lead compounds for the
Keywords: β-carboline; cytotoxic activity; Sf9 cells; Spodoptera litura; growth inhibitory activity
1. Introduction
In recent years, potential impact of synthetic pesticides on the environment and human health
has been of great concern, which highlights the need for environmentally friendly pesticides to
protect crops from insect infestation. Accordingly, plant-derived extracts and their bioactive natural
compounds have been considered as biorational alternatives with structural diversity and complexity,
biodegradability, environmental protection, pest specificity and reduced mammalian toxicity to
replace synthetic agrochemicals for pest management [
structural optimization of novel insecticidal leads from plant origin have recently been important
methods for the research and development of new pesticides [ ]. Harmine and its analogs, such
as harmaline and harmane (Figure 1), belonging to the -carboline alkaloids, were isolated from
Peganum harmala and Eurycoma longifolia [ ]. These compounds have been garnering increasing
interest due to numerous activities [ ], including antitumor [ ], antiviral [10], analgesic [11] and
anti-inflammatory activities [12]. Although several reports have been published on the insecticidal
activities, growth inhibitory activities and/or oviposition deterrence of solvent extracts and -carboline
alkaloids of P. harmala against Tribolium castaneum [13], Rhyzopertha dominica [14], Plutella xylostella [15],
Spodoptera littoralis [16 17], Schistocerca gregaria [18] and Bactrocera oleae [19], little is known about the
bioactivity of harmine against Spodoptera litura. In our previous work [20 21], we found that high
1]. Especially, further modification and
2–5
β
6
7
8,9
β
,
,
doses of extract and total alkaloids of P. harmala displayed moderate anti-feedant activity against
Spodoptera litura. Furthermore, we demonstrated that harmine exhibited marginal to weak cytotoxicity
against Spodoptera frugiperda Sf9 cells [22]. Accordingly, in an attempt to improve toxicity and growth
inhibitory activity of
β-carboline derivatives against S. litura, while, considering that introduction of
1,3,4-oxadiazole would usually potentiate the pharmacological properties of original molecule [23
–25],
Molecules 2017, 22, 1811; doi:10.3390/molecules22111811
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1811
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we synthesized a series of novel
β-carboline 1,3,4-oxadiazole derivatives and evaluated for their
in vitro cytotoxicity against Sf9 cells and growth inhibitory activity against S. litura.
Figure 1. Structure of the major β-carboline alkaloids of P. harmala.
2. Results and Discussion
2.1. Chemistry
The synthesis of 3-(2-substituted-1,3,4-oxadiazol-5-yl)-
Briefly, precursor 1-substituted- -carboline-3-carbohydrazides (
the procedure reported previously [26]. The intermediate could be readily converted into
3-(2-thioxo-1,3,4-oxadiazol-5-yl)- -carbolines 23 on treatment with carbon disulfide and potassium
hydroxide [27], and into 3-(2-oxo-1,3,4-oxadiazol-5-yl)- -carbolines 24 27 on treatment with
β-carbolines are depicted in Figure 2.
β
1) was prepared according to
1
β
2–
β
–
triphosgene [28]. Similarly, intermediate 1 furnished desired compounds 28 and 29 by reaction with
cyanogen bromide in the presence of sodium bicarbonate at ambient temperature [29]. For preparation
of 3-(2-substituted-1,3,4-oxadiazol-5-yl)-β-carbolines 30–36, 1-phenyl-β-carboline-3-carbohydrazide
was subjected to reaction with a series of acids, followed by treatment with phosphorus oxychloride
under reflux [30]. The compound 30 could be synthesized using other methodology [31].
The 1-phenyl-β-carboline-3-carbohydrazide was reacted with triethyl orthoformate under reflux
to give the key intermediate hydrazonoformate 37, which was used for further cyclization in pyridine
under reflux to obtain the desired compound 30. All newly synthesized compounds were purified
by crystallization and their structures were confirmed on the basis of 1H NMR, 13C NMR and HRMS
data, which are given in Section 4. Compounds
3
,
9
,
10, 13 and 25 were characterized by comparison
with NMR data previously reported [27
,
32]. Compounds 24–29 were characterized by the presence
of an additional signal at δH 12.69–12.60 or 7.25–7.29, corresponding to the 30-NH or 20-NH2 on the
1,3,4-oxadiazole scaffold, respectively.
2.2. Biological Activity
2.2.1. Anti-Proliferative Activity against Sf9 Cells
All synthesized β-carboline derivatives 2–37 were evaluated for their anti-proliferative activities
against Sf9 cells and compared with harmine and camptothecin as positive controls. The biological
results presented in Table 1 show that all of the target compounds exhibited moderate to potent
in vitro cytotoxic activity against Sf9 cells, and some of them were more potent than harmine
and camptothecin. Significantly, compounds
anti-proliferative activities. The IC50 values of these compounds were 16.25, 12.80, 10.39, 13.83,
14.22, 11.10 and 3.93 M, respectively, whereas that of camptothecin was 18.95 M. Intriguingly, the
5, 6, 9, 11, 15, 22 and 37 displayed pronounced
µ
µ
intermediate hydrazonoformate 37 proved to be the most active compound in this study, and the
cytotoxic activity was approximately 30-fold more potent than that of the cyclization product 30
and five-fold higher activities than that of camptothecin, which implied further possibilities for lead
compound development.

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Figure 2. Synthesis of 37. Reagents and conditions: (a) For
β-carboline 1,3,4-oxadiazole derivatives
2–
compounds 23: i: CS2, KOH, EtOH, reflux, 24 h; ii: acidified 2N HCl; (b) For compounds 24–27:
2–
triphosgene, CH2Cl2, reflux, 2 h; (c) For compounds 28 and 29: BrCN, NaHCO3, dioxane, water, r.t.,
4 h; (d) For compounds 30–36: acid, phosphorus oxychloride, reflux, 4–6 h; (e) triethyl orthoformate,
reflux, overnight; and (f) pyridine, reflux, 12 h.
As for the 1,3,4-oxadiazole series
investigated. As shown in Table 1, changing the 1-substituent groups could lead to remarkable
change in activity. For instance, the activity of compounds with methyl group, such as compounds
and 24, were predominantly lower than those of derivatives with an aromatic or heterocycle group.
Moreover, the potency of derivatives with the aromatic group ( 18) significantly depended upon the
2–29, the effects of different substituent groups at C-1 were
2
3
–
nature of substitutes and their position at the aromatic ring. Substitution of electron withdrawing
group on the aromatic ring at para position was more potent than that of electron donating group, while
compound
For the effect of substituted position on the phenyl ring, mono-substitution on the aromatic ring at
different position ( and 10 12) showed similar potency. By contrast to mono-substituted compounds,
9 bearing 4-OCH3-Ph group displayed the highest cytotoxic activity (IC50 = 10.39 µM).
5
,
6
–
3,4-disubstituted derivatives (14 and 16–18) were much less potent, whereas 3,4,5-trisubstituted
compound (15) showed comparable potency to the mono-substituted compounds. Comparing with
phenyl ring substituted compounds, condensed ring substituted compounds (19
–
23) displayed
equivalent activity.
For the effect of different substituent groups at 2-position on 1,3,4-oxadiazole scaffold (24
the available data analysis suggested that there was no significant difference on cytotoxic activity
when changing thioxo group to oxo group (25 27) or amino group (28 and 29) except for compound
24. However, Compounds 30 34 with aliphatic groups or aromatic groups in the R2 position showed
marginal to weak cytotoxicity (IC50 66.63 to 144.66 M), even compounds 35 and 36 with 4-CH3-Ph
and 4-Cl-Ph groups, respectively, were inactive (IC50 > 1000 M), indicating that electronegativity and
size of substituents at 2-position on 1,3,4-oxadiazole scaffold are crucial.
–
36),
–
–
µ
µ

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Table 1. In vitro cytotoxicity of compound 2–37 against sf9 cell.
−1
Compound
R1
X
R2
IC50 (µmol L
)
95% Confidence Interval
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
CH3
Ph
4-CH3-Ph
4-F-Ph
4-Cl-Ph
4-Br-Ph
4-CF3-Ph
4-OCH3-Ph
2-Cl-Ph
3-Cl-Ph
3-F-Ph
3-NO2-Ph
3,4-di-OCH3-Ph
3,4,5-tri-OCH3-Ph =S
3,4-diF-Ph
3,4-diCl-Ph
=S
=S
=S
=S
=S
=S
=S
=S
=S
=S
=S
=S
=S
35.49
45.44
47.35
16.25
12.80
34.23
17.58
10.39
27.69
13.83
22.37
63.97
64.86
14.22
213.34
98.74
25.79
23.87–49.90
28.69–70.64
29.99–87.66
0.11–61.37
6.36–20.96
20.84–55.59
9.67–31.89
8.09–12.95
7.99–57.07
1.08–42.37
16.71–29.03
32.36–134.99
22.05–135.19
8.01–22.03
171.16–293.30
48.67–316.92
10.89–42.80
=S
=S
=S
3-F-4-OCH3-Ph
19
20
=S
=S
56.61
19.11
34.02–90.47
14.48–25.90
21
=S
30.84
3.78–70.23
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
2-naphthyl
6-quinolyl
CH3
Ph
4-CF3-Ph
3,4,5-tri-OCH3-Ph =O
Ph
4-CF3-Ph
=S
=S
=O
=O
=O
11.10
197.81
725.93
18.09
27.35
54.92
28.32
44.29
122.52
129.92
66.63
3.50–21.70
130.28–380.25
355.57–2562.85
5.76–37.16
10.22–88.94
35.49–104.66
18.42–41.27
18.74–86.43
105.73–144.47
110.74–165.14
45.36–97.30
102.16–167.65
87.87–463.36
-
NH2
NH2
H
CH3
CH2CH3
cyclopropyl 127.76
Ph
4-CH3-Ph
4-Cl-Ph
144.66
>1000
>1000
3.93
-
37
-
1.03–8.01
Harmine
Camptothecin
140.68
18.95
75.05–319.53
12.43–25.68
2.2.2. Compounds 5 and 37 Could Induce Cell Apoptosis in Sf9 Cells
We found that compounds and 37 had apoptosis effect on Sf9 cells through morphology
observation after treatment with the 36 -carboline derivatives. We treated Sf9 cells with various
concentrations of compound for 24 h and observed cellular morphological changes under inverted
phase contrast microscope (IPCM). As shown in Figure S1, addition of compound to culture Sf9
cells was found to induce membrane blebbing and a small amount of apoptotic bodies and floating
cells at 10
g mL−1, and apoptotic bodies increased while adherent cells decreased with increasing
drug concentrations, whereas no apoptotic bodies were observed in 20
g mL−1-treated group.
5
β
5
5
µ
µ
Apoptosis was also confirmed by staining with acridine orange (AO); the non-apoptotic cells were
stained uniformly green and the apoptotic cells had evident orange particles in their nuclei due to
nuclear condensation or nuclear DNA fragmentation, which were typical early apoptotic characteristics.

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Analogously, we treated Sf9 cells with 12.5
µ
g mL−1 harmine, compounds
5
and 37 for 24 and 48 h and
found that compounds 5 and 37 could induce apoptotic bodies, and apoptotic bodies increased with
the extension of induction time (Figure 3a). However, both harmine-treated and untreated cells did
not exhibit typical morphologic features of apoptosis, such as cell shrinkage or formation of apoptotic
bodies, until the exposure time was extended to 48 h. Similar results could be obtained by AO staining.
Additionally, DNA fragmentation was detected through agarose gel electrophoresis and DNA ladder
was observed clearly after 24 h incubation with compounds
5 and 37 (Figure 3d), while no DNA
fragmentation were observed after treatment with various concentrations of harmine (Figure S2).
Subsequently, we detected the cell apoptosis using annexin V-FITC/PI staining and flow cytometry.
As shown in Figure 3b,c, the apoptotic rate of compounds
5 and 37 treatment increased dramatically
in comparison with untreated and harmine-treated groups, and the proportion of apoptotic cells was
23.9% and 29.8%, respectively. These results suggested that compounds
in Sf9 cells and more effectively than harmine.
5 and 37 could cause apoptosis
2.2.3. Compounds 5 and 37 Could Induce Cell Cycle Arrest in Sf9 Cells
We next assessed the cell cycle distribution of Sf9 cells by flow cytometry. Compounds
5
and 37
treated groups exhibited a high percentage of cells in S phase as compared to the harmine-treated and
untreated groups with a concomitant reduction of cell numbers in G1 phase, which suggested that
compounds 5 and 37 could arrest the cell cycle at the S phase (Figure 4a,b).
2.2.4. Compounds 5 and 37 Could Simulate Sf-Caspase-1 Activation
Caspases play a crucial role in controlling cell death. Executioner caspases (including caspase-3)
were activated by initiator caspases (such as caspase-9) and subsequently coordinate their activity to
affect main structural proteins and activate other enzymes, resulting in cell apoptosis. Sf-caspase-1,
which has the same activity as the mammalian caspase-3, is the main effector caspase in Sf9 cells [33].
To identify whether Sf-caspase-1 was involved in the apoptotic mechanism of compounds
Sf9 cells, we assessed the Sf-caspase-1 activity. The results demonstrated that compound
5
and 37 in
5
displayed
modest efficacy in Sf-caspase-1 activity; notably, Sf-caspase-1 activity increased significantly and
remains almost 3-fold higher in the compound 37 treated cells than that in untreated cells, whereas
an obvious decrease in Sf-caspase-1 activity of harmine treated group was observed, suggesting that
compounds 5 and 37 could induce Sf-caspase-1 activity in Sf9 cells (Figure 4c).
2.2.5. Compounds 5 and 37 Could Inhibit Larvae Growth of S. litura
In previous studies, effects of extracts and alkaloid of P. harmal on lepidopteran insects were
investigated. El-Gengaihi et al. [16] and Shonouda et al. [17] reported that harmine alkaloid caused an
increase in larval period and larval mortality but a decrease in the percentage of pupation of S. littoralis.
Additionally, Abbasipour et al. [15] reported that the extract of P. harmal displayed pronounced effect on
larval and pupal weight, oviposition deterrence, percent pupation, egg hatching and adult emergence
of P. xylostella. These findings indicated that
larvae. Therefore, based on the anti-feedant activity against S. litura in our previous studies, we further
evaluated the larvicidal activity and growth inhibitory activity of synthesized -carboline derivatives
against S. litura. Unfortunately, there was no significant toxicity of all synthesized compounds against
S. litura at 500 and 37, with superior cytotoxicity and apoptosis
g mL−1. Subsequently, compounds
property, were chosen to evaluate the growth inhibitory activity against 3rd instar S. litura and the
results is shown in Figure 5. We assessed the effect of compounds and 37 on larvae weight at various
concentrations and showed that mean larvae weight of -treated and 37-treated groups significantly
β-carboline alkaloids might inhibit growth of lepidopteran
β
µ
5
5
5
decreased as compared to the control group, and the difference in larvae weight between treated
and untreated groups was positively correlated with drug concentrations. Interestingly, moreover,
the difference in larvae weight between
cytotoxicity against Sf9 cells (Figure 5a–c). In addition, treatment with compounds
5
-treated and 37-treated groups was consistent with their
and 37 at various
5

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concentrations led to reduction in the weight of pupae (Figure 5d), but did not significantly affect the
percentage of pupation and adult emergence, which was similar to what Jbilou et al. described [13].
Figure 3. Analysis of apoptosis in Sf9 cells treated with 12.5 µ 5 and 37. (a) Representative
g mL−1
photographs of cell morphological change induced by harmine, 5 and 37 for 24 and 48 h, respectively.
IPCM: inverted phase contrast microscopy; AO: acridine orange. Red and white arrows represent
−1
apoptosis bodies; (b,c) Apoptotic rate of cells induced by 12.5 µg mL 5 and 37 for 24 h; (d) Agarose
−1
gel electrophoresis analysis of genomic DNA of cells treated by 12.5
µg mL
5, 22 and 37 with CPT as
a positive control. CPT: camptotothecin. Note: (**) p < 0.01 in comparison to the control.

Molecules 2017, 22, 1811
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Figure 4. Compounds
cells. ( ) Cell cycle analysis by flow cytometry. Cells were treated with 12.5
37 for 24 h; (
5 and 37 induced cell cycle arrest and stimulated Sf-caspase-1 activation in Sf9
−1
a
µg mL harmine,
5
and
b
) The percentage of cells in the G1,−S1and G2 phases of the cell cycle; (
c) Sf-caspase-1
activity of Sf9 cells after treatment with 12.5 µg mL harmine, 5 and 37 for 24 h. Note: (*) p < 0.05 and
(**) p < 0.01 in comparison to the control.
Figure 5. The growth inhibitory activity of compounds
weight of S. Litura after treatment with and 37 for two weeks at the concentrations of 50, 100 and
200 ) Mean pupae weight of S. Litura after treatment with and 37 at the
g mL−1, respectively. (
g mL−1, respectively. Note: (*) p < 0.05 and (**) p < 0.01 in comparison
5 and 37 against S. litura. (a–c) Mean larvae
5
µ
d
5
concentrations of 50, 100 and 200
to the control.
µ

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3. Materials and Methods
3.1. Chemistry
All reagents and solvents were of reagent grade or purified according to standard methods
before use. Analytical thin-layer chromatography and preparative thin-layer chromatography were
performed with silica gel plates using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd.,
1
Qingdao, Shandong Province, China). H-NMR and 13C NMR spectra were recorded at 600 MHz on a
Bruker Avance-600 superconducting nuclear magnetic resonance instrument using TMS as a reference
(Bruker Company, Gemany). High-resolution mass spectrometry (HRMS) data were obtained on an
Uplc1290-6540B Q-TOF LC/MS instrument (Agilent Co., Santa Clara, CA, USA). The key intermediate
1-substituted-β-carboline-3-carbohydrazide derivatives 1 were prepared according to the procedure
reported previously [26].
3.1.1. General Synthetic Procedure for Target Compounds 2–23
A mixture of 1-substituted- -carboline-3-carbohydrazide derivatives
β
1 (1 mmol), potassium
hydroxide (84 mg, 1.5 mmol) and carbon disulfide (0.3 mL, 5 mmol) in absolute ethanol (10 mL) was
refluxed with stirring for 24 h. Then, the solvent was removed by evaporation under reduced pressure.
The residue obtained was diluted with water and acidified with 2N HCl solution. The product was
collected by filtration, washed with water, dried and recrystallized from ethanol.
1
1-Methyl-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
DMSO-d6)
8-H), 7.60 (t, J = 8.1 Hz, 1H, 7-H), 7.31 (t, J = 7.1 Hz, 1H, 6-H), 2.85 (s, 3H, 1-CH3); 13C NMR (151 MHz,
2). Yield: 78%; H NMR (600 MHz,
δ: 12.09 (s, 1H, 9-NH), 8.71 (s, 1H, 4-H), 8.40 (d, J = 7.9 Hz, 1H, 5-H), 7.66 (d, J = 8.2 Hz, 1H,
DMSO-d6) δ: 177.90, 161.71, 143.67, 141.26, 136.05, 130.53, 129.08, 127.50, 122.77, 121.52, 120.66, 113.55,
112.78, 20.82; HRMS Calcd for 283.0648 (C14H10N4OS, [M + H]+), found 283.0862.
1
1-Phenyl-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
DMSO-d6)
Ar-H), 7.71 (d, J = 8.2 Hz, 1H, 8-H), 7.67 (t, J = 7.6 Hz, 2H, Ar-H), 7.64–7.58 (m, 2H, 7-H, Ar-H), 7.35 (t,
J = 7.5 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 177.60, 161.14, 142.82, 141.61, 137.18, 134.02,
3). Yield: 72%; H NMR (600 MHz,
δ
: 11.99 (s, 1H, 9-NH), 8.88 (s, 1H, 4-H), 8.48 (d, J = 7.9 Hz, 1H, 5-H), 8.07–8.04 (m, 2H,
δ
130.93, 129.53, 129.22, 128.98, 128.90, 128.62, 122.25, 120.88, 120.50, 113.94, 112.82; HRMS Calcd for
345.0805 (C19H12N4OS, [M + H]+), found 345.1062.
1
1-(4-Methylphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
(600 MHz, DMSO-d6)
J = 7.3 Hz, 2H, Ar-H), 7.70 (d, J = 8.0 Hz, 1H, 8-H), 7.64–7.58 (m, 1H, 7-H), 7.46 (d, J = 7.3 Hz, 2H, Ar-H),
7.36–7.30 (m, 1H, 6-H), 2.46 (s, 3H, 40-CH3); 13C NMR (151 MHz, DMSO-d6)
: 177.60, 161.15, 142.86,
4). Yield: 74%; H NMR
δ: 11.92 (s, 1H, 9-NH), 8.82 (s, 1H, 4-H), 8.44 (d, J = 7.4 Hz, 1H, 5-H), 7.95 (d,
δ
141.53, 138.74, 134.37, 133.91, 130.89, 129.39, 128.85, 128.46, 122.14, 120.87, 120.40, 113.63, 112.77, 20.94;
HRMS Calcd for 359.0961 (C20H14N4OS, [M + H]+), found 359.0962.
1-(4-Fluorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
(600 MHz, DMSO-d6) : 11.99 (s, 1H, 9-NH), 8.84 (s, 1H, 4-H), 8.45 (d, J = 7.8 Hz, 1H, 5-H), 8.09
(dd, J = 8.3, 5.6 Hz, 2H, Ar-H), 7.69 (d, J = 8.1 Hz, 1H, 8-H), 7.65–7.60 (m, 1H, 7-H), 7.48 (t, J = 8.7 Hz
2H, Ar-H), 7.34 (t, J = 7.4 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 177.62, 161.10, 141.83, 141.64,
5
). Yield: 65%; 1H NMR
δ
,
δ
133.97, 133.63, 130.93, 130.91, 130.87, 129.65, 129.09, 122.31, 120.90, 120.59, 115.90, 115.76, 113.99, 112.81;
HRMS Calcd for 363.0710 (C19H11FN4OS, [M + H]+), found 363.0819.
1-(4-Chlorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
(600 MHz, DMSO-d6) : 12.03 (s, 1H, 9-NH), 8.89 (s, 1H, 4-H), 8.48 (d, J = 7.9 Hz, 1H, 5-H), 8.08
(d, J = 8.5 Hz, 2H, Ar-H), 7.75–7.68 (m, 3H, 8-H, Ar-H), 7.66–7.61 (m, 1H, 7-H), 7.35 (t, J = 7.4 Hz, 1H,
6-H). 13C NMR (151 MHz, DMSO-d6)
: 177.61, 161.02, 141.63, 141.44, 135.92, 133.99, 130.93, 130.43,
6
). Yield: 80%; 1H NMR
δ
δ
129.76, 129.12, 128.91, 122.30, 120.85, 120.60, 114.17, 112.77. HRMS Calcd for 379.0415 (C19H11ClN4OS,
[M + H]+), found 379.0604.

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1-(4-Bromophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
7
). Yield: 78%; 1H NMR
(600 MHz, DMSO-d6)
(d, J = 8.3 Hz, 2H, Ar-H), 7.85 (d, J = 8.3 Hz, 2H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.63 (t, J = 7.6 Hz,
1H, 7-H), 7.35 (t, J = 7.4 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 177.64, 161.04, 141.65, 141.55,
δ: 12.02 (s, 1H, 9-NH), 8.89 (s, 1H, 4-H), 8.48 (d, J = 7.8 Hz, 1H, 5-H), 8.01
δ
136.30, 133.99, 131.87, 131.01, 130.71, 129.80, 129.16, 122.73, 122.34, 120.87, 120.64, 114.22, 112.79. HRMS
Calcd for 422.9910 (C19H11BrN4OS, [M + H]+), found 423.0025.
1
1-(4-Trifluoromethylphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
NMR (600 MHz, DMSO-d6)
(d, J = 8.1 Hz, 2H, Ar-H), 8.01 (d, J = 8.2 Hz, 2H, Ar-H), 7.69 (d, J = 8.2 Hz, 1H, 8-H), 7.64 (t, J = 7.6 Hz,
1H, 7-H), 7.36 (t, J = 7.4 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 177.62, 160.92, 141.68, 140.97,
8). Yield: 76%; H
δ: 12.08 (s, 1H, 9-NH), 8.92 (s, 1H, 4-H), 8.49 (d, J = 7.9 Hz, 1H, 5-H), 8.25
δ
134.16, 131.03, 129.96, 129.48, 129.21, 125.72, 125.70, 122.34, 120.79, 120.65, 114.53, 112.74. HRMS Calcd
for 413.0678 (C20H11F3N4OS, [M + H]+), found 413.0940.
1
1-(4-Methoxyphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (
(600 MHz, DMSO-d6)
J = 8.6 Hz, 2H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.60 (t, J = 7.5 Hz, 1H, 7-H), 7.32 (t, J = 7.4 Hz
1H, 6-H), 7.20 (d, J = 8.6 Hz, 2H, Ar-H), 3.89 (s, 3H, 40-OCH3); 13C NMR (151 MHz, DMSO-d6)
9
). Yield: 60%; H NMR
δ
: 11.88 (s, 1H, 9-NH), 8.76 (s, 1H, 4-H), 8.43 (d, J = 7.8 Hz, 1H, 5-H), 8.02 (d,
,
:
δ
178.03, 161.34, 160.04, 142.51, 141.52, 133.55, 131.56, 130.15, 129.94, 129.74, 129.36, 128.71, 122.07, 120.94,
120.28, 114.24, 114.09, 112.89, 112.73, 55.35; HRMS Calcd for 375.0910 (C20H14N4O2S, [M + H]+),
found 375.1054.
1
1-(2-Chlorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (10). Yield: 64%; H NMR
(600 MHz, DMSO-d6)
δ: 11.81 (s, 1H, 9-NH), 8.91 (s, 1H, 4-H), 8.47 (d, J = 7.9 Hz, 1H, 5-H), 7.72
(d, J = 8.0 Hz, 1H, 8-H), 7.67–7.57 (m, 5H, 7-H, Ar-H), 7.35–7.31 (m, 1H, 6-H); 13C NMR (151 MHz,
DMSO-d6) δ: 177.83, 161.13, 141.83, 141.49, 136.11, 134.87, 132.48, 131.89, 130.82, 129.80, 129.10, 128.74,
127.56, 122.45, 120.80, 120.42, 114.34, 112.53; HRMS Calcd for 379.0415 (C19H11ClN4OS, [M + H]+),
found 379.0415.
1
1-(3-Chlorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (11). Yield: 73%; H NMR
(600 MHz, DMSO-d6)
1H, Ar-H), 8.01 (d, J = 7.4 Hz, 1H, 8-H), 7.66 (m, 4H, 7-H, Ar-H), 7.35 (t, J = 7.4 Hz, 1H, 6-H); 13C
NMR (151 MHz, DMSO-d6) : 177.59, 160.95, 141.66, 141.03, 139.14, 134.00, 133.62, 130.93, 130.76,
δ: 12.06 (s, 1H, 9-NH), 8.87 (s, 1H, 4-H), 8.47 (d, J = 7.9 Hz, 1H, 5-H), 8.03 (s,
δ
129.88, 129.15, 129.09, 128.25, 127.30, 122.32, 120.82, 120.60, 114.37, 112.77; HRMS Calcd for 379.0415
(C19H11ClN4OS, [M + H]+), found 379.0415.
1
1-(3-Fluorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (12). Yield: 54%; H NMR
(600 MHz, DMSO-d6)
J = 7.7 Hz, 1H, Ar-H), 7.82 (d, J = 9.9 Hz, 1H, 8-H), 7.72–7.67 (m, 2H, Ar-H), 7.63 (t, J = 7.6 Hz, 1H, 7-H),
7.43 (dd, J = 8.6, 2.5 Hz, 1H, Ar-H), 7.35 (t, J = 7.5 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
δ: 12.03 (s, 1H, 9-NH), 8.87 (s, 1H, 4-H), 8.47 (d, J = 7.9 Hz, 1H, 5-H), 7.89 (d,
δ:
177.60, 163.16, 161.54, 141.65, 141.17, 139.43 (139.38), 133.96, 130.97, 130.92, 129.87, 129.14, 124.78, 122.29,
120.82, 120.60, 116.12, 115.98, 115.38, 115.24, 114.31, 112.79; HRMS Calcd for 363.0710 (C19H11FN4OS,
[M + H]+), found 363.0712.
1-(3-Nitrophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (13). Yield: 64%; 1H NMR
(600 MHz, DMSO-d6)
Ar-H), 8.41 (d, J = 7.7 Hz, 1H, 5-H), 7.94 (t, J = 7.4 Hz, 1H, Ar-H), 7.68 (d, J = 7.6 Hz, 1H, 8-H), 7.66–7.61
(m, 1H, 7-H), 7.35 (t, J = 6.8 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 178.00, 160.97, 148.18,
δ: 12.12 (s, 1H, 9-NH), 8.88 (s, 1H, 4-H), 8.79 (s, 1H, Ar-H), 8.47 (d, J = 6.5 Hz, 2H,
δ
141.68, 139.98, 138.65, 135.00, 133.97, 131.68, 130.50, 130.19, 129.25, 123.69, 123.31, 122.39, 120.84, 120.62,
114.35, 112.65; HRMS Calcd for 390.0655 (C19H11N5O3S, [M + H]+), found 390.0657.
1-(3,4-Dimethoxyphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (14). Yield: 59%; 1H NMR
(600 MHz, DMSO-d6)
δ: 11.95 (s, 1H, 9-NH), 8.82 (s, 1H, 4-H), 8.45 (d, J = 7.9 Hz, 1H, 5-H), 7.70 (d,
J = 8.2 Hz, 1H, 8-H), 7.61–7.59 (m, 2H, Ar-H), 7.57 (d, J = 1.9 Hz, 1H, 7-H), 7.35–7.32 (m, 1H, 6-H), 7.23

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(d, J = 8.4 Hz, 1H, Ar-H), 3.90 (s, 3H, 30-OCH3), 3.89 (s, 3H, 40-OCH3); 13C NMR (151 MHz, DMSO-d6)
: 177.56, 161.21, 149.84, 148.90, 143.13, 141.52, 133.97, 130.78, 129.78, 129.24, 128.86, 122.22, 121.36,
δ
120.97, 120.46, 113.57, 112.83, 112.02 (112.98), 55.76, 55.59; HRMS Calcd for 405.1016 (C21H16N4O3S,
[M + H]+), found 405.1013.
1-(3,4,5-Trimethoxyphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (15). Yield: 81%; 1H
NMR (600 MHz, DMSO-d6)
(d, J = 8.2 Hz, 1H, 8-H), 7.62–7.58 (m, 1H, 7-H), 7.32 (dd, J = 11.4, 4.4 Hz, 1H, 6-H), 7.22 (s, 2H, Ar-H),
3.93 (s, 6H, 30, 50-OCH3), 3.79 (s, 3H, 40-OCH3); 13C NMR (151 MHz, DMSO-d6)
: 178.99, 161.60,
δ: 11.85 (s, 1H, 9-NH), 8.78 (s, 1H, 4-H), 8.44 (d, J = 7.8 Hz, 1H, 5-H), 7.67
δ
153.08, 142.68, 141.49, 138.21, 133.41, 133.04, 129.40, 128.60, 122.03, 120.98, 120.06, 112.61, 112.43, 106.03,
60.06, 55.97; HRMS Calcd for 435.1122 (C22H18N4O4S, [M + H]+), found 435.1258.
1-(3,4-Difluorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (16). Yield: 76%; 1H NMR
(600 MHz, DMSO-d6)
(m, 1H, Ar-H), 7.89 (dd, J = 8.2, 3.7 Hz, 1H, Ar-H), 7.73–7.67 (m, 2H, 8-H, Ar-H), 7.65–7.61 (m, 1H, 7-H),
7.34 (t, J = 7.0 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 177.63, 160.94, 141.67, 133.91, 130.89,
δ: 12.04 (s, 1H, 9-NH), 8.85 (s, 1H, 4-H), 8.46 (d, J = 7.9 Hz, 1H, 5-H), 8.06–8.02
δ
129.92, 129.23, 125.85, 122.35, 120.83, 120.68, 118.11, 118.00, 117.84, 117.72, 114.34, 112.77; HRMS Calcd
for 381.0616 (C19H10F2N4OS, [M + H]+), found 381.0621.
1-(3,4-Dichlorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (17). Yield: 75%; 1H NMR
(600 MHz, DMSO-d6) δ: 12.08 (s, 1H, 9-NH), 8.85 (s, 1H, 4-H), 8.46 (d, J = 7.9 Hz, 1H, 5-H), 8.20
(d, J = 2.0 Hz, 1H, Ar-H), 8.01 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.89 (d, J = 8.3 Hz, 1H, Ar-H), 7.69
(d, J = 8.2 Hz, 1H, 8-H), 7.63 (t, J = 7.6 Hz, 1H, 7-H), 7.34 (t, J = 7.4 Hz, 1H, 6-H); 13C NMR (151
MHz, DMSO-d6) δ: 177.59, 160.84, 141.64, 139.93, 137.56, 133.95, 131.89, 131.64, 131.00, 130.94, 130.30,
130.00, 129.21, 128.81, 122.33, 120.78, 120.64, 114.49, 112.71; HRMS Calcd for 413.0025 (C19H10Cl2N4OS,
[M + H]+), found 413.0029.
1-(3-Fluoro-4-methoxyphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (18). Yield: 69%; 1H
NMR (600 MHz, DMSO-d6) δ: 11.95 (s, 1H, 9-NH), 8.80 (s, 1H, 4-H), 8.44 (d, J = 7.9 Hz, 1H, 5-H), 7.89
(s, 1H, Ar-H), 7.87 (d, J = 3.2 Hz, 1H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.62 (t, J = 7.6 Hz, 1H, 7-H),
7.43 (t, J = 8.7 Hz, 1H, Ar-H), 7.33 (t, J = 7.4 Hz, 1H, 6-H), 3.97 (s, 3H, 40-OCH3); 13C NMR (151 MHz,
DMSO-d6) δ: 178.08, 161.23, 152.30, 150.68, 148.03 (147.96), 141.60, 141.14, 133.55, 129.71, 128.93, 125.21,
122.20, 120.92, 120.44, 116.01, 115.88, 114.04, 112.75, 56.27; HRMS Calcd for 393.0816 (C20H13FN4O2S,
[M + H]+), found 393.0814.
1-(1,3-Benzodioxole-5-yl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (19). Yield: 80%; 1H NMR
(600 MHz, DMSO-d6)
J = 8.2 Hz, 1H, 8-H), 7.62–7.56 (m, 3H, 7-H, Ar-H), 7.33 (t, J = 7.5 Hz, 1H, 6-H), 7.19 (d, J = 7.9 Hz, 1H,
Ar-H), 6.18 (s, 2H, -OCH2O-); 13C NMR (151 MHz, DMSO-d6)
: 177.73, 161.18, 148.13, 147.68, 142.33,
δ: 11.92 (s, 1H, 9-NH), 8.80 (s, 1H, 4-H), 8.44 (d, J = 7.9 Hz, 1H, 5-H), 7.70 (d,
δ
141.51, 133.64, 131.22, 131.04, 129.45, 128.84, 122.85, 122.15, 120.90, 120.39, 113.39, 112.77, 108.72, 108.65,
101.47; HRMS Calcd for 389.0703 (C20H12N4O3S, [M + H]+), found 389.0709.
1-(2,3-Dihydro-benzofuran-5-yl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (20). Yield: 70%;
1H NMR (600 MHz, DMSO-d6)
δ: 11.87 (s, 1H, 9-NH), 8.76 (s, 1H, 4-H), 8.43 (d, J = 7.9 Hz, 1H, 5-H),
7.93 (s, 1H, Ar-H), 7.82 (d, J = 8.2 Hz, 1H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.62–7.58 (m, 1H, 7-H),
7.32 (t, J = 7.4 Hz, 1H, 6-H), 7.01 (d, J = 8.2 Hz, 1H, Ar-H), 4.66 (t, J = 8.7 Hz, 2H, -OCH2CH2-), 3.35 (t,
J = 8.6 Hz, 2H, -OCH2CH2-); 13C NMR (151 MHz, DMSO-d6)
δ: 177.84, 161.31, 160.68, 143.00, 141.46,
133.58, 131.24, 129.65, 129.27, 128.72, 128.12, 125.53, 122.10, 120.96, 120.30, 112.93, 112.71, 109.08, 71.49,
29.06; HRMS Calcd for 387.0910 (C21H14N4O2S, [M + H]+), found 387.0914.
1-(2,3-Dihydro-1,4-benzodioxine-6-yl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (21). Yield:
68%; 1H NMR (600 MHz, DMSO-d6)
δ
: 11.92 (s, 1H, 9-NH), 8.79 (s, 1H, 4-H), 8.44 (d, J = 7.9 Hz, 1H,
5-H), 7.71 (s, 1H, 8-H), 7.61 (t, J = 7.7 Hz, 1H, 7-H), 7.57–7.54 (m, 2H, Ar-H), 7.32 (t, J = 7.5 Hz, 1H,
6-H), 7.12 (d, J = 8.7 Hz, 1H, Ar-H), 4.36 (s, 4H, -OCH2CH2O-); 13C NMR (151 MHz, DMSO-d6)
δ:

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177.85, 161.24, 144.44, 143.47, 142.17, 141.54, 133.56, 131.21, 130.46, 129.43, 128.78, 122.09, 121.72, 120.91,
120.34, 117.41, 117.16, 113.19, 112.80, 64.36, 64.16; HRMS Calcd for 403.0859 (C21H14N4O3S, [M + H]+),
found 403.0862.
1-(2-Naphthyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (22). Yield: 78%; 1H NMR
(600 MHz, DMSO-d6)
5-H), 8.18 (s, 2H, Ar-H), 8.17–8.13 (m, 1H, Ar-H), 8.08–8.04 (m, 1H, Ar-H), 7.72 (d, J = 8.2 Hz, 1H, 8-H),
7.65–7.62 (m, 3H, 7-H, Ar-H), 7.35 (t, J = 7.5 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 177.86,
δ: 12.09 (s, 1H, 9-NH), 8.87 (s, 1H, 4-H), 8.60 (s, 1H, Ar-H), 8.48 (d, J = 7.9 Hz, 1H,
δ
161.23, 142.64, 141.63, 134.57, 134.19, 133.14, 132.80, 131.45, 129.62, 128.96, 128.83, 128.42, 128.00, 127.65,
127.00, 126.51, 126.30, 122.28, 120.96, 120.46, 113.74, 112.75. HRMS Calcd for 395.0961 (C23H14N4OS,
[M + H]+), found 395.0962.
1-(6-Quinolinyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (23). Yield: 74%; 1H NMR
(600 MHz, DMSO-d6) δ: 12.16 (s, 1H, 9-NH), 9.03 (dd, J = 4.2, 1.7 Hz, 1H, Ar-H), 8.92 (s, 1H, 4-H), 8.67
(d, J = 1.9 Hz, 1H, Ar-H), 8.61 (d, J = 8.1 Hz, 1H, Ar-H), 8.50 (d, J = 7.9 Hz, 1H, 5-H), 8.43 (dd, J = 8.7,
2.0 Hz, 1H, Ar-H), 8.27 (d, J = 8.7 Hz, 1H, Ar-H), 7.72 (d, J = 8.2 Hz, 1H, 8-H), 7.68 (dd, J = 8.2, 4.2 Hz,
1H, Ar-H), 7.65 (t, J = 7.1 Hz, 1H, 7-H), 7.37 (t, J = 7.9 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
δ:
177.60, 161.03, 151.10, 141.87, 141.64, 137.62, 135.11, 134.34, 131.09, 130.16, 129.79, 129.14, 129.03, 128.32,
127.81, 122.37, 122.02, 120.90, 120.61, 114.25, 112.73; HRMS Calcd for 396.0914 (C22H13N5OS, [M + H]+),
found 396.0916.
3.1.2. General Synthetic Procedure for Target Compounds 24–27
To the solution of corresponding hydrazine derivatives 1 (1 mmol) in dry dichloromethane (10 mL)
was added dropwise triphosgene (103.86 mg, 0.35 mmol) in dry dichloromethane (2 mL) under an ice
bath and nitrogen atmosphere. After refluxed for 2 h, the solvent was removed by evaporation under
reduced pressure, and the product was recrystallized from ethanol.
1
1-Methyl-3-(2-oxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (24). Yield: 78%; H NMR (600 MHz,
DMSO-d6)
δ: 12.69 (s, 1H, NH), 12.30 (s, 1H, 9-NH), 8.67 (s, 1H, 4-H), 8.41 (d, J = 7.6 Hz, 1H, 5-H), 7.68
(d, J = 8.2 Hz, 1H, 8-H), 7.63 (t, J = 7.6 Hz, 1H, 7-H), 7.33 (t, J = 7.4 Hz, 1H, 6-H), 2.88 (s, 3H, 1-CH3); 13
C
NMR (151 MHz, DMSO-d6) δ: 163.50, 154.37, 142.54, 141.78, 136.13, 134.98, 129.29, 127.57, 122.46, 121.01,
120.48, 113.88, 112.45, 19.51. HRMS Calcd for 267.0877 (C14H10N4O2, [M + H]+), found 267.0884.
1
1-Phenyl-3-(2-oxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (25). Yield: 87%; H NMR (600 MHz,
DMSO-d6)
8.06–8.03 (m, 2H, Ar-H), 7.69 (d, J = 8.1 Hz, 1H, 8-H), 7.66 (t, J = 7.6 Hz, 2H, Ar-H), 7.63–7.57 (m, 2H,
7-H, Ar-H), 7.33 (t, J = 7.5 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 154.85, 154.54, 142.49, 141.60,
δ: 12.60 (s, 1H, NH), 11.91 (s, 1H, 9-NH), 8.75 (s, 1H, 4-H), 8.44 (d, J = 7.8 Hz, 1H, 5-H),
δ
137.36, 133.73, 132.35, 129.57, 129.11, 128.85, 128.59, 122.14, 120.87, 120.32, 112.75; HRMS Calcd for
329.1033 (C19H12N4O2, [M + H]+), found 329.1310.
1-(4-Trifluoromethylphenyl)-3-(2-oxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (26). Yield: 83%; 1H NMR
(600 MHz, DMSO-d6)
5-H), 8.26 (d, J = 8.0 Hz, 2H, Ar-H), 8.01 (d, J = 8.1 Hz, 2H, Ar-H), 7.69 (d, J = 8.2 Hz, 1H, 8-H), 7.63
(t, J = 7.6 Hz, 1H, 7-H), 7.35 (t, J = 7.0 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
: 154.76, 154.29,
δ: 12.65 (s, 1H, NH), 12.03 (s, 1H, 9-NH), 8.81 (s, 1H, 4-H), 8.47 (d, J = 7.9 Hz, 1H,
δ
141.70, 141.13, 140.62, 133.88, 132.45, 130.02, 129.77, 129.42, 129.06, 125.62, 120.76, 120.44, 113.35, 112.70;
HRMS Calcd for 397.0907 (C20H11F3N4O2, [M + H]+), found 397.0910.
1-(3,4,5-Trimethoxyphenyl)-3-(2-oxo-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b] indole (27). Yield: 90%; 1H NMR
(600 MHz, DMSO-d6)
1H, 5-H), 7.69 (d, J = 8.2 Hz, 1H, 8-H), 7.60 (t, J = 7.6 Hz, 1H, 7-H), 7.32 (t, J = 7.5 Hz, 1H, 6-H), 7.22 (s,
2H, Ar-H), 3.92 (s, 6H, 30, 50-OCH3), 3.79 (s, 3H, 40-OCH3); 13C NMR (151 MHz, DMSO-d6)
: 154.77,
δ: 12.62 (s, 1H, NH), 11.90 (s, 1H, 9-NH), 8.71 (s, 1H, 4-H), 8.42 (d, J = 7.9 Hz,
δ
154.44, 153.09, 142.84, 141.51, 138.33, 133.75, 132.70, 132.10, 129.30, 128.76, 122.09, 120.88, 120.25, 112.72
(112.67), 106.04, 60.07, 55.97; HRMS Calcd for 419.1350 (C22H18N4O5, [M + H]+), found 419.1403.

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3.1.3. General Synthetic Procedure for Target Compounds 28–29
To a solution of corresponding hydrazine derivatives
sodium bicarbonate (84 mg, 1 mmol) in water (1.5 mL), and the mixture was stirred at ambient
temperature for 10 min, and then cyanogen bromide (81 L, 1.1 mmol) was added. After stirring for
1 (1 mmol) in dioxane (5 mL) was added
µ
further 4 h, the reaction mixture was diluted with 15 mL water, and the product was collected by
filtration, washed with water, dried and crystallized from methanol.
1-Phenyl-3-(2-amino-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (28). Yield: 84%; 1H NMR (600 MHz,
DMSO-d6)
Ar-H), 7.68 (d, J = 8.2 Hz, 1H, 8-H), 7.65 (t, J = 7.6 Hz, 2H, Ar-H), 7.61–7.56 (m, 2H, 7-H, Ar-H), 7.32
(t, J = 7.5 Hz, 1H, 6-H), 7.25 (s, 2H, 20-NH2); 13C NMR (151 MHz, DMSO-d6)
: 164.07, 158.41, 142.08,
δ: 11.79 (s, 1H, 9-NH), 8.77 (s, 1H, 4-H), 8.40 (d, J = 7.9 Hz, 1H, 5-H), 8.04 (d, J = 7.2 Hz, 2H,
δ
141.62, 137.50, 133.34, 133.24, 129.76, 128.90, 128.69, 128.53, 121.98, 120.81, 120.08, 112.61, 112.04; HRMS
Calcd for 328.1193 (C19H13N5O, [M + H]+), found 328.1205.
1-(4-Trifluoromethylphenyl)-3-(2-amino-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (29). Yield: 78%; 1H
NMR (600 MHz, DMSO-d6)
2H, Ar-H), 8.00 (d, J = 8.1 Hz, 2H, Ar-H), 7.67 (d, J = 8.0 Hz, 1H, 8-H), 7.62 (t, J = 7.4 Hz, 1H, 7-H), 7.33
(t, J = 7.5 Hz, 1H, 6-H), 7.29 (s, 2H, 20-NH2); 13C NMR (151 MHz, DMSO-d6)
: 164.15, 158.26, 141.74,
δ: 11.90 (s, 1H, 9-NH), 8.83 (s, 1H, 4-H), 8.42 (d, J = 7.8 Hz, 1H, 5-H), 8.23 (s,
δ
141.38, 140.36, 133.60, 133.43, 130.23, 129.40, 129.02, 125.60 (125.58), 122.16, 120.75, 120.28, 112.82, 112.57;
HRMS Calcd for 396.1067 (C20H12F3N5O, [M + H]+), found 396.1067.
3.1.4. General Synthetic Procedure for Target Compounds 30–36
An equimolar mixture of 1-phenyl-β-carboline-3-carbohydrazide 1 (302 mg, 1 mmol) and
various substituted carboxylic acids (1 mmol) in phosphoryl chloride (5 mL) was refluxed for 4–6 h.
The reaction mixture was then cooled, poured into crushed ice and neutralized with 5% NaOH solution.
The product was collected by filtration, washed with water, dried and recrystallized from methanol.
1-Phenyl-3-(1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (30). Yield: 76%; 1H NMR (600 MHz, DMSO-d6)
δ
: 11.97 (s, 1H, 9-NH), 9.39 (s, 1H, 20-H), 9.03 (s, 1H, 4-H), 8.48 (d, J = 7.9 Hz, 1H, 5-H), 8.09–8.07 (m,
2H, Ar-H), 7.71 (d, J = 8.2 Hz, 1H, 8-H), 7.67 (t, J = 7.6 Hz, 2H, Ar-H), 7.62 (m, 2H, 7-H, Ar-H), 7.35 (t,
J = 7.1 Hz, 1H, 6-H); 13C NMR (151 MHz, DMSO-d6)
δ: 164.44, 154.62, 142.74, 141.68, 137.30, 134.02,
131.94, 129.71, 129.19, 128.96, 128.89, 128.77, 128.65, 122.20, 120.90, 120.45, 114.39, 112.85; HRMS Calcd
for 313.1084 (C19H12N4O, [M + H]+), found 313.1087.
1-Phenyl-3-(2-methyl-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (31). Yield: 75%; 1H NMR (600 MHz,
DMSO-d6)
Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.67 (t, J = 7.6 Hz, 2H, Ar-H), 7.64–7.58 (m, 2H, 7-H, Ar-H), 7.34
(t, J = 7.2 Hz, 1H, 6-H), 2.64 (s, 3H, 20-CH3); 13C NMR (151 MHz, DMSO-d6)
: 164.75, 163.99, 142.63,
δ: 11.93 (s, 1H, 9-NH), 8.97 (s, 1H, 4-H), 8.47 (d, J = 7.8 Hz, 1H, 5-H), 8.07–8.05 (m, 2H,
δ
141.66, 137.32, 133.89, 132.24, 129.69, 129.13, 128.91, 128.86, 128.77, 128.64, 122.16, 120.89, 120.39, 113.85,
112.79, 10.78; HRMS Calcd for 327.1240 (C20H14N4O, [M + H]+), found 327.1285.
1
1-Phenyl-3-(2-ethyl-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (32). Yield: 80%; H NMR (600 MHz,
DMSO-d6)
δ: 11.91 (s, 1H, 9-NH), 8.96 (s, 1H, 4-H), 8.45 (d, J = 7.9 Hz, 1H, 5-H), 8.06 (d, J = 7.4 Hz,
2H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.66 (t, J = 7.6 Hz, 2H, Ar-H), 7.61 (m, 2H, 7-H, Ar-H), 7.33 (t,
0
0
J = 7.4 Hz, 1H, 6-H), 3.00 (q, J = 7.5 Hz, 2H, 2 -CH2CH3), 2.50 (s, 3H, 2 -CH2CH3); 13C NMR (151 MHz,
DMSO-d6) : 167.82, 164.65, 142.64, 141.63, 137.35, 133.89, 132.29, 129.65, 129.09, 128.88, 128.85, 128.74,
δ
128.62, 122.17, 120.88, 120.35, 113.92, 112.76, 18.52, 10.56; HRMS Calcd for 341.1397 (C21H16N4O,
[M + H]+), found 341.1400.
1-Phenyl-3-(2-cyclopropyl-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (33). Yield: 81%; 1H NMR (600 MHz,
DMSO-d6)
Ar-H), 7.70 (d, J = 8.1 Hz, 1H, 8-H), 7.66 (t, J = 7.6 Hz, 2H, Ar-H), 7.63–7.57 (m, 2H, 7-H, Ar-H), 7.33 (t,
J = 7.3 Hz, 1H, 6-H), 2.36 (m, 1H), 1.22–1.12 (m, 4H); 13C NMR (151 MHz, DMSO-d6)
: 153.38, 149.74,
δ: 11.91 (s, 1H, 9-NH), 8.93 (s, 1H, 4-H), 8.44 (d, J = 7.8 Hz, 1H, 5-H), 8.06 (d, J = 7.3 Hz, 2H,
δ

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130.71, 129.84, 126.07, 122.96, 121.55, 119.24, 118.73, 118.53, 118.42, 118.33, 112.59, 111.49, 110.99, 105.27,
104.30, 11.56, 9.76; HRMS Calcd for 353.1397 (C22H16N4O, [M + H]+), found 353.1420.
1-Phenyl-3-(2-phenyl-1,3,4-oxadiazol-5-yl)-9H-pyrido[3,4-b]indole (34). Yield: 91%; 1H NMR (600 MHz,
DMSO-d6)
8.11 (d, J = 7.3 Hz, 2H, Ar-H), 7.73–7.58 (m, 8H, 8-H, 7-H, Ar-H), 7.35 (t, J = 7.3 Hz, 1H, 6-H); 13C NMR
(151 MHz, DMSO-d6) : 164.78, 164.07, 142.80, 141.63, 137.36, 134.01, 131.97 (131.94), 129.64, 129.46,
δ: 11.96 (s, 1H, 9-NH), 9.08 (s, 1H, 4-H), 8.47 (d, J = 7.8 Hz, 1H, 5-H), 8.18–8.15 (m, 2H, Ar-H),
δ
129.14, 128.89, 128.67, 126.65, 123.56, 122.15, 120.93, 120.43, 114.47, 112.82; HRMS Calcd for 389.1397
(C25H16N4O, [M + H]+), found 389.1895.
1
1-Phenyl-3-[2-(4-methylphenyl)-1,3,4-oxadiazol-5-yl]-9H-pyrido[3,4-b]indole (35). Yield: 89%; H NMR
(600 MHz, DMSO-d6) δ: 11.95 (s, 1H, 9-NH), 9.07 (s, 1H, 4-H), 8.48 (d, J = 8.0 Hz, 1H, 5-H), 8.11 (d,
J = 7.4 Hz, 2H, Ar-H), 8.05 (d, J = 7.9 Hz, 2H, Ar-H), 7.72 (d, J = 8.1 Hz, 1H, 8-H), 7.68 (t, J = 7.5 Hz, 2H,
Ar-H), 7.65–7.59 (m, 3H, 7-H, Ar-H), 7.45 (d, J = 7.8 Hz, 2H, Ar-H), 7.36 (t, J = 7.4 Hz, 1H, 6-H), 2.42 (s,
3H, 400-CH3); 13C NMR (151 MHz, DMSO-d6)
δ: 164.50, 164.11, 142.75, 142.03, 141.60, 137.34, 133.94,
132.01, 129.93, 129.61, 129.06, 128.83, 128.61, 126.55, 122.08, 120.89, 120.77, 120.35, 114.32, 112.77, 21.11;
HRMS Calcd for 403.1553 (C26H18N4O, [M + H]+), found 403.1555.
1
1-Phenyl-3-[2-(4-chlorophenyl)-1,3,4-oxadiazol-5-yl]-9H-pyrido[3,4-b]indole (36). Yield: 84%; H NMR
(600 MHz, DMSO-d6) δ: 11.99 (s, 1H, 9-NH), 9.12 (s, 1H, 4-H), 8.49 (d, J = 7.9 Hz, 1H, 5-H), 8.19 (d,
J = 8.6 Hz, 2H, Ar-H), 8.11 (d, J = 7.0 Hz, 2H, Ar-H) , 7.74 (d, J = 8.7 Hz, 2H, Ar-H), 7.70 (dd, J = 15.3,
7.9 Hz, 3H, 8-H, Ar-H), 7.66–7.59 (m, 2H, 7-H, Ar-H), 7.37 (t, J = 7.1 Hz, 1H, 6-H); 13C NMR (151
MHz, DMSO-d6) δ: 164.86, 163.26, 142.80, 141.59, 137.31, 136.60, 134.00, 131.80, 129.56, 129.09, 128.84,
128.63, 128.37, 122.39, 122.07, 120.89, 120.40, 114.48, 112.79; HRMS Calcd for 423.1007 (C25H15ClN4O,
[M + H]+), found 423.1007.
3.1.5. General Synthetic Procedure for Ethyl Hydrazonoformate 37
A mixture of 1-phenyl-β-carboline-3-carbohydrazide 1 (302 mg, 1 mmol) in triethyl orthoformate
(5 mL) was refluxed overnight. Then, the formed solid was collected by filtration, washed with
petroleum ether, dried and recrystallized from methanol to give compound 37.
Ethyl 1-phenyl-
: 11.98 (s, 1H, 9-NH), 11.27 (s, 1H, -CONHN=), 8.90 (s, 1H, 4-H), 8.45 (d, J = 7.9 Hz, 1H, 5-H), 8.10 (d,
β
-carboline-3-carbonylhydrazonoformate (37). Yield: 92%; 1H NMR (600 MHz, DMSO-d6)
δ
J = 7.1 Hz, 2H, Ar-H), 7.71 (d, J = 8.2 Hz, 1H, 8-H), 7.67 (t, J = 7.6 Hz, 2H, Ar-H), 7.63–7.59 (m, 2H, 7-H,
Ar-H), 7.34 (t, J = 7.5 Hz, 1H, 6-H), 7.16 (s, 1H), 4.28 (t, J = 7.1 Hz, 2H, OCH2CH3), 1.35 (t, J = 7.1 Hz, 3H,
OCH2CH3); 13C NMR (151 MHz, DMSO-d6)
δ: 159.49, 146.47, 141.63, 140.44, 138.71, 137.71, 134.34, 130.22,
129.27, 128.83, 128.36, 122.15, 121.17, 120.42, 113.39, 112.76, 67.36, 15.19. ESI-MS: m/z 359.17 [M + H]+.
3.2. Biology
3.2.1. Cell Culture
Spodoptera frugiperda (Sf9) cells were grown in Grace0s insect medium supplemented 10% fetal
bovine serum (FBS), 0.33% yeast extract and 0.33% lactalbumin hydrolysate and cultivated at 28 ◦C
in a humidified incubator 2.5% CO2. Cells were seeded with 1
following treatment.
×
106 cells mL−1 density for the
3.2.2. Anti-Proliferation Assay
3-(4,5-Dimethylthiazole-2yl)-2,5-diphenyl (MTT) assay was utilized to evaluate the bioactivity of
-carboline derivatives. Sf9 cells were subjected to treatment with various concentrations of -carboline
derivatives. After 24 h incubation, 10
L freshly prepared MTT (5 mg mL−1) was added, followed
by incubation for 4 h in darkness. The supernatants were carefully discarded and 150 L of dimethyl
sulfoxide (DMSO) was added to dissolve the formazan crystal. The absorbance was measured at
β
β
µ
µ

Molecules 2017, 22, 1811
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490 nm by a microplate reader (BioTek, Winooski, VT, USA). The cytotoxic effect was calculated by the
following formula.
ODcontrol − ODtreatment
Cell growth inhibition rate (%) =
× 100
ODcontrol
3.2.3. Cell Morphological Observation
Sf9 cells were treated with various concentrations of harmine, and compounds
5
and 37.
Morphological characteristics of Sf9 cells were recorded using inverted phase contrast microscope
(IPCM) (Lecia, Tokyo, Japan).
3.2.4. Acridine Orange (AO) Staining Analysis
Sf9 cells were subjected to treatment with various concentrations of harmine, and compounds
5
and 37. After 24 h incubation, cells were collected and resuspended by PBS, followed by staining
with 1 M acridine orange and incubation for 15 min in darkness. The morphology of Sf9 cells was
µ
observed and photographed using fluorescence microscopy (Olympus BX51, Olympus, Japan).
3.2.5. Apoptosis Rate Analysis
Apoptosis assay was performed using Annexin V/FITC Kit (Nanjing KeyGEN Biotech. Co. Ltd.,
g mL−1 harmine, and compounds
Nanjing, China). Sf9 cells were subjected to treatment with 12.5 µ 5
and 37, respectively. After 24 h incubation, the cells were collected, dyed by Annexin V-FITC and PI,
and incubated for 15 min in darkness. Subsequently, the samples were processed in a FACSCalibur
(Becton Dickinson, Franklin Lakes, NJ, USA) and data were analyzed using ModFit LT software
(Becton Dickinson, USA).
3.2.6. DNA Ladder Assay
Genomic DNA of different treatment groups was isolated by TIANamp Genomic DNA Kit
(TIANGEN) and was subjected to agarose gel electrophoresis.
3.2.7. Sf-Caspase-1 Activity Assay
Sf9 cells were treated with 12.5
µ 5 and 37, respectively.
g mL−1 harmine, and compounds
After 24 h incubation, cells were collected and suspended in lysis buffer at 4 ◦C for 15 min.
After centrifuged at 2000 rpm for 10 min at 4 ◦C, the supernatant was transferred to another tube and
protein concentration was determined by Brandford method. The supernatant of lysate (50
µL) was
added to 50 L of Reaction Buffer and 5
µ
µ
L caspase-3 substrate and incubated at 37 ◦C for 4 h in the
darkness. The samples were measured at 405 nm on Microplate Reader (Biotex, Winooski, VT, USA).
3.2.8. Cell Cycle Analysis
Sf9 cells were treated with 12.5
µ 5 and 37, respectively.
g mL−1 harmine, and compounds
After 24 h incubation, a suspension of cells was fixed in 70% ice-cold ethyl alcohol for more than
2 h, followed by labeling with propidium iodide (PI). The labeled cells were analyzed using a Flow
Cytometer. The percentage of cells in the G1, S and G2 phases of the cell cycle were determined using
the Flowjo 7.6.1 software (FlowJo, LLC, Ashland, OR, USA).
3.2.9. Insect Growth Inhibition against Tobacco Cutworm (Spodoptera litura)
The insect growth inhibition of compounds
5 and 37 against S. litura were evaluated using our
previously reported methods [34 35]. Firstly, the compounds were prepared to various concentrations
,
by dissolving compounds in DMSO and adding distilled water. Leaf discs (5 cm in diameter) were cut
from fresh taro leaves were dipped in the test solution for 5 s and then air dried. The leaf discs were

Molecules 2017, 22, 1811
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placed in a petri dish lined with filter paper, and then 10 3rd-instar larvae were transferred to the petri
dish. Two days after treatment, larvae were provided with fresh, untreated leaves and maintained
under standard rearing conditions. Each treatment was performed 3 times. The surviving larvae were
weighed using an electron scale accurate to 0.0001 g before and after the treatment at 2, 4, 6, 8, 10, 12
and 14 days. The larvae were continued to rear until all butterflies in the control treatment emerged.
The pupation rate and mean pupae weight were calculated.
4. Conclusions
In conclusion, a series of novel
β-carboline 1,3,4-oxadiazole derivatives were designed,
synthesized and investigated for in vitro cytotoxic activity against Sf9 cells and growth inhibitory
activity against S. litura. Bioassay results indicated that all of these compounds exhibited significant
in vitro cytotoxic activity and were more potent than harmine. Notably, compounds
5 and 37 not only
displayed superior cytotoxicity and were more potent than CPT, but also induced cell apoptosis and cell
cycle arrest at S phase and stimulated Sf-caspase-1 activation in Sf9 cells, indicating that Sf-caspase-1
was involved in the apoptotic mechanism of compounds
bioassay also demonstrated that compounds and 37 could significantly inhibit larvae growth of
S. litura by decreasing the weight of larvae and pupae. From the result of bioassay, we can conclude
that compounds and 37 could be used as potential lead compounds for the development of novel
5 and 37 in Sf9 cells. Furthermore, in vivo
5
5
insect growth regulators. Further studies on whether growth inhibition of insects is related to cell
apoptosis are in progress.
Supplementary Materials: The following are available online. Figure S1. Representative photographs of cell
morphological change induced by compound
5 at various concentrations for 24 h, respectively. Figure S1.
Agarose gel electrophoresis analysis of genomic DNA of cells treated by harmine at various concentrations.
Acknowledgments: This work was financially supported by the National Natural Science Foundation of China
(Grant No. 31572335), Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant
No. 20134404110019), the Natural Science Foundation of Guangdong Province, China (Grant No. 2014A030313461)
and Public welfare industry (Agriculture) scientific research special fund project, China (Grant No. 201303017).
Author Contributions: Zhi-Jun Zhang designed and carried out the experimental and wrote the paper.
Jing-Jing Zhang and Zhi-Yan Jiang assisted in experiment. Guo-Hua Zhong provided study design and guidance.
All authors have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–37 are available from the authors.
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