Design, synthesis, and biological evaluation of novel N-acylhydrazone bond linked heterobivalent β-carbolines as potential anticancer agents

Article abstract of DOI:10.3390/molecules24162950

Utilizing a pharmacophore hybridization approach, we have designed and synthesized a novel series of 28 new heterobivalent β-carbolines. The in vitro cytotoxic potential of each compound was evaluated against the five cancer cell lines (LLC, BGC-823, CT-26, Bel-7402, and MCF-7) of different origin—murine and human, with the aim of determining the potency and selectivity of the compounds. Compound 8z showed antitumor activities with half-maximal inhibitory concentration (IC₅₀) values of 9.9 ± 0.9, 8.6 ± 1.4, 6.2 ± 2.5, 9.9 ± 0.5, and 5.7 ± 1.2 μM against the tested five cancer cell lines. Moreover, the effect of compound 8z on the angiogenesis process was investigated using a chicken chorioallantoic membrane (CAM) in vivo model. At a concentration of 5 μM, compound 8z showed a positive effect on angiogenesis. The results of this study contribute to the further elucidation of the biological regulatory role of heterobivalent β-carbolines and provide helpful information on the development of vascular targeting antitumor drugs.

Full text of DOI:10.3390/molecules24162950

Article
Design, Synthesis, and Biological Evaluation of
Novel N-Acylhydrazone Bond Linked Heterobivalent
β-Carbolines as Potential Anticancer Agents
1,†
1,†
2
2
2
1,
Xiaofei Chen , Liang Guo , Qin Ma , Wei Chen , Wenxi Fan and Jie Zhang *
1
School of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical
Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi 832003, China
Xinjiang Huashidan Pharmaceutical Research Co. Ltd., 175 He Nan East Road, Urumqi 830011, China
These authors contributed equally to the work.
2
†
* Correspondence: zhangjie-xj@163.com; Tel.: +86-993-205-7215
Received: 21 July 2019; Accepted: 8 August 2019; Published: 14 August 2019
Abstract: Utilizing a pharmacophore hybridization approach, we have designed and synthesized a
novel series of 28 new heterobivalent β-carbolines. The in vitro cytotoxic potential of each
compound was evaluated against the five cancer cell lines (LLC, BGC-823, CT-26, Bel-7402, and
MCF-7) of different origin—murine and human, with the aim of determining the potency and
selectivity of the compounds. Compound 8z showed antitumor activities with half-maximal
inhibitory concentration (IC50) values of 9.9 ± 0.9, 8.6 ± 1.4, 6.2 ± 2.5, 9.9 ± 0.5, and 5.7 ± 1.2 µM against
the tested five cancer cell lines. Moreover, the effect of compound 8z on the angiogenesis process
was investigated using a chicken chorioallantoic membrane (CAM) in vivo model. At a
concentration of 5 µM, compound 8z showed a positive effect on angiogenesis. The results of this
study contribute to the further elucidation of the biological regulatory role of heterobivalent β-
carbolines and provide helpful information on the development of vascular targeting antitumor
drugs.
Keywords: asymmetric dimeric β-carboline; acylhydrazone group; cytotoxic; antitumor; structure–
activity relationship
1. Introduction
Cancer remains a leading cause of death in developed and developing countries, although much
significant progress has been achieved recently [1]. Cancer resistance to therapy is becoming a
common phenomenon that threatens the current strategies against this disease. For that reason, we
need to discover new anticancer agents. One of the successful and effective methods for the discovery
of new anticancer drugs from natural products is synthesis of novel compounds through chemical
structural modifications on the basis of leading compounds.
β-Carbolines are a large group of heterocyclic compounds with a 9H-pyrido[3,4-b]indole
structural unit. They compose a class of alkaloids that are widely distributed in nature, including
plants, foodstuffs, marine creatures, insects, mammals, human tissues, and body fluids [2]. In the last
few decades, there have been intense research efforts in the design and development of β-carbolines
as a new class of antitumor agents. A large number of β-carboline derivatives have been prepared in
search of more potent antitumor agents. The structure−activity relationships (SARs) of these β-
carbolines have been extensively investigated [3–10]. Research has indicated that this class of
compounds exert their antitumor effects through multiple mechanisms of action, including
intercalating into DNA [11–13] and inhibiting topoisomerases I and II [14,15], cyclin-dependent
Molecules 2019, 24, 2950; doi:10.3390/molecules24162950
www.mdpi.com/journal/molecules

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kinase (CDK) [16,17], polo-like kinase 1 (PLK1) [18], kinesin-like protein Eg5 [19], and IκB kinases
[20].
Among frequently studied novel bioactive chemical entities, the acylhydrazone scaffold
(−CONHN=) has attracted considerable attention for decades due to its broad applications ranging
from medicinal agents to agrochemicals to functional materials. Many compounds containing this
moiety have been reported, and many reports demonstrate that the introduction of this
pharmacophore may have high potential for antitumor activity [21–26]. The acylhydrazone moiety is
able to act as pharmacophore or auxophore subunit in different pharmaceutic classes, with a variety
of action profiles, depending on the other functionalities present in the molecular structure [27]. For
TM
example, Mylotarg (gemtuzumab ozogamicin; Pfizer) [28] (Figure 1) is a humanized anti-CD33
monoclonal antibody linked covalently to the cytotoxic agent N-acetyl gamma calicheamicin.
Peterson reported PAC-1 (Figure 1), another N-acylhydrazone small-molecule, induces apoptotic
death in cancer cells via the chelation of inhibitory zinc from procaspase-3, which leads to
autocatalytic activation and subsequent generation of caspase-3 [29]. Carbazochrome (Figure 1), a
semicarbazone-related compound, has been used as a hemostatic agent and is specifically indicated
for capillary and parenchymal hemorrhage [30].
Figure 1. Structures of Mylotarg, PAC-1, and carbazochrome.
Our research group [31–34] has focused on incorporating substituents into positions 1, 2, 3, 7,
and 9 of the β-carboline nucleus as antitumor agents. Structure–activity relationship (SAR) analysis
indicated that (1) the β-carboline moiety was associated with their potential antitumor activities, and
(2) the introduction of appropriate substituents into positions 1, 3, and 9 of the β-carboline nucleus
enhanced their antitumor potencies. Previous research has shown that some antitumor agents when
dimerized via an appropriate linker can lead to significantly improved antitumor effects, giving 100-
to 500-fold improvement over the corresponding monomers [35–38].
So our group reported the synthesis, in vitro evaluation, in vivo efficacies, and SARs of the new
homobivalent β-carbolines and heterobivalent β-carbolines with alkyl or alkylamino spacers in
positions 1, 3, 7, and 9 of the β-carboline nucleus (Figure 2) [39–42]. In these homobivalent β-
carbolines, 1-Methyl-9-[4-(1-methyl-β-carboline-9-yl)butyl]-β-carboline (B-9-3) [43,44] exhibited
potent antitumor activity. The pharmacological mechanisms showed that B-9-3 selectively induces
apoptosis of endothelial cells, in part through disruption of VEGF-A/VEGFR2 signaling [45], and also
acts on the TGF-β signaling pathway [46]. Compounds B-3 [39] and B-4 [40] exhibited significant
angiogenesis inhibitory effects in chicken chorioallantoic membrane (CAM) assay, and the anti-
angiogenetic potency was comparable or more potent with the drug Endostar.

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Figure 2. The chemical structure of the representative reported diremic β-carbolines.
Continuing our studies to develop effective cytotoxic agents, the objective of this study was to
synthesize potential anticancer compounds that are hybrids of β-carboline and acylhydrazone
fragments (Figure 3). We have evaluated their cytotoxic activities for the first time, and the study also
includes an investigation of the mechanism of action of these compounds for angiogenesis inhibition.
These findings as well as our study of the SARs of the new compounds are discussed.
Figure 3. Hybrids of β-carboline and acylhydrazone fragments giving the target heterobivalent β-
carbolines.
2. Results and Discussion
2.1. Chemistry
The syntheses of compounds 8a–ab are depicted in Schemes 1−3. Monovalent β-carbolines 6a–l
and 7a–l were synthesized according to previously published methods [39,47,48]. Using L-
tryptophan as starting material, the tetrahydro-β-carboline skeleton (2a–g) was constructed via
Pictet−Spengler cyclization. Then, the obtained carboxylic acid 2 reacted with thionyl chloride and
ethanol to form ethyl ester 3, which subsequently reacted with sulfur in xylene to afford compounds
4a–g. Then compounds 4a–g were reduced to their corresponding alcohols by lithium borohydride
(LiBH4) in dry THF to provide compounds 5a–g, and further oxidized by MnO2 in CH3CN to afford
the key intermediates, the 3-carboxaldehyde derivatives 6a–g [39]. Alternatively, refluxing of
compounds 4a–g with 80% hydrazine hydrate in ethanol gave the other key intermediates,
hydrazides 7a–g [39] (see Scheme 1).

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COOC2H5
COOH
NH
COOC2H5
NH
R1
N
iv
v
N
H
N
H
N
H
R1
i
4a-g
2a
3a-g
viii
CONHNH2
vi
CH2OH
COOH
COOH
N
ii
N
NH2
NH
CH3
N
H
R1
N
H
N
N
H
R1'
H
7a-g
2b
5a-g
L-tryptophan
vii
iii
COOH
CHO
NH
R1
N
N
H
N
H
R1'
2c-g
6a-g
CH3
CH3
a R1=
H
b R1=
CH3
d R1=
c R1=
Cl
OCH3
e R1=
f R1=
g R1=
Scheme 1. Synthesis of the key intermediates 6a–g, 7a–g. Reagents and conditions: (i) NaOH, H2O,
formaldehyde, reflux, 3 h; (ii) H2SO4, H2O, acetaldehyde, room temperature, 3 h; (iii) acetic acid,
R1CHO, reflux, 3 h; (iv) ethanol, SOCl2, reflux, 4 h; (v) xylene, S8, reflux, 8 h; (vi) THF, LiBH4, stirred
at RT; (vii) CH3CN, MnO2, reflux, 2 h. (viii) hydrazine hydrate, ethanol, reflux, 4 h.
9
The N -alkylated derivative of compound 4a was prepared by the action of sodium hydride
(NaH) in anhydrous N,N-dimethylformamide (DMF) followed by the addition of alkyl halide to
afford compounds 4h–l, and following this, the intermediates 6h–l and 7h–l were prepared according
to the same method for compounds 6a–g and 7a–g (see Scheme 2). Finally, the synthesis of
compounds 8a–ac (see Scheme 3) containing the acylhydrazone fragment was accomplished by the
condensation of compounds 7a–l with the corresponding aldehydes 6a–l. We obtained the products
easily in moderate to good yields when the aldehyde was 1 equiv and when the reactions were carried
1
out in ethanol under reflux conditions. The structures of all compounds were confirmed by H-NMR,
13
C-NMR (see Supplementary Materials), and high-resolution mass spectra (HRMS).

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Scheme 2. Synthesis of the key intermediates 6h–l, 7h–l. Reagents and conditions: (i) DMF, NaH, alkyl
halogenide, stirred at RT; (ii) hydrazine hydrate, ethanol, reflux, 4 h. (iii) THF, LiBH4, stirred at RT;
(iv) CH3CN, MnO2, reflux, 2 h.
Scheme 3. Synthesis of the asymmetric dimeric β-carboline derivatives 8a–ab. Reagents and
conditions: (i) methanol, reflux, 4–6 h.
2.2. MTT Assay of Compounds 8a-8ab
From the synthetic route mentioned above, we obtained a series of novel heterobivalent β-
carbolines. All of the target compounds were assayed for anticancer activity in various cancer cell
lines including LLC (Lewis lung carcinoma), BGC-823 (gastric carcinoma), CT-26 (murine colon
carcinoma), Bel-7402 (liver carcinoma), and MCF-7 (breast carcinoma), using the MTT method. The
half-maximal inhibitory concentration (IC50) values for each compound with respect to the five cancer
cell lines were calculated, and the results are summarized in Table 1. These values represent the
concentrations at which a 50% decrease in cell growth is observed after 72 h of incubation in the
presence of the drug compared with control cells treated with DMSO or positive control Cisplatin
(DDP) under similar conditions.
Table 1. Cytotoxic activity of acylhydrazone linked heterobivalent β-carbolines 8a–ab in vitroa (IC50,
µMb).
IC50(μM) ± SD
CT-26
Comp.
R1’
R9’
R1
R9
c
LLC
BGC823
Bel-7402
86.3 ± 9.4
45.7 ± 3.2
14.6 ± 3.2
20.7 ± 3.3
MCF-7
8a
8b
8c
H
CH3
CH(CH3)2
H
H
H
H
H
H
H
H
H
H
H
H
91.9 ± 6.8 68.4 ± 6.2
63.6 ± 7.5
94.7 ± 11.8
38.5 ± 10.4
57.2 ± 4.1
56.6 ± 3.5
40.6 ± 5.4
15.3 ± 2.7
11.5 ± 2.1
81.2 ± 7.5
76.7 ± 6.3
61.3 ± 5.5
3.2 ± 0.7
9.7 ± 0.9
6.3 ± 1.6
8d
H
H
H
H
H
H
58.5 ± 5.7 10.8 ± 1.4
70.3 ± 6.8 10.2 ± 2.3
68.4 ± 4.8
22.2 ± 3.2
54.3 ± 6.9
11.7 ± 0.9
34.2 ± 5.2
17.6 ± 2.6
8e
8f
H
H
H
48.8 ± 3.8 61.4 ± 7.6
57.4 ± 4.5
52.6 ± 5.8
30.9 ± 4.2
8g

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71.3 ±
10.8
8h
H
CH3
H
H
78.6 ± 6.5
43.4 ± 4.2
41.2 ± 3.1
21.4 ± 5.3
8i
8j
H
H
n-butyl
H
H
H
H
30.7 ± 2.9 27.4 ± 3.2
40.7 ± 4.7 25.6 ± 2.1
57.1 ± 5.6
23.4 ± 2.8
15.3 ± 3.5
68.9 ± 5.4
14.0 ± 3.5
21.6 ± 3.7
8k
8l
H
H
H
H
H
H
17.4 ± 4.6 52.9 ± 5.8
29.2 ± 5.1 69.9 ± 8.4
15.6 ± 4.1
15.8 ± 3.2
16.3 ± 4.7
15.5 ± 2.9
18.5 ± 5.1
17.3 ± 3.4
8m
8n
8o
H
H
H
H
H
H
CH3
CH(CH3)2
H
H
H
24.7 ± 3.9 17.5 ± 2.1
38.2 ± 5.7 19.9 ± 2.9
13.8 ± 2.7
22.0 ± 3.7
56.3 ± 4.2
15.6 ± 2.9
41.1 ± 7.8
73.7 ± 9.8
16.0 ± 4.3
14.5 ± 2.1
14.4 ± 4.2
72.1 ± 6.5
9.1 ± 1.4
8p
H
H
H
68.4 ± 9.4 76.6 ± 5.2
69.5 ± 11.7
70.2 ± 8.5
>100
8q
8r
H
H
H
H
H
CH3
20.3 ± 1.3 24.5 ± 5.1
88.7 ± 5.6 34.8 ± 4.6
86.5 ±
2.4 ± 0.6
10.7
13.3 ± 2.3 14.7 ± 2.1
83.2 ±
5.3 ± 0.9
14.6
13.4 ± 3.7
14.0 ± 3.3
13.3 ± 2.1
12.4 ± 4.2
14.7 ± 4.2
68.9 ± 7.5
H
H
8s
8t
H
H
H
H
H
H
H
H
n-butyl
3.1 ± 1.2
12.6 ± 1.7
2.7 ± 0.3
33.5 ± 2.7
21.5 ± 4.2
20.1 ± 5.2
8.7 ± 1.5
16.8 ± 1.9
2.5 ± 0.4
2.8 ± 0.7
76.8 ± 5.2
H
H
H
8u
8v
41.5 ± 5.1 43.4 ± 3.6
46.7 ± 8.3
n-butyl
n-butyl
8w
8x
H
H
n-butyl
n-butyl
H
CH3
58.6 ± 4.4 28.2 ± 2.4
70.2 ± 6.4 66.2 ± 5.8
33.3 ± 6.2
55.8 ± 4.7
25.3 ± 7.5
72.2 ± 11.4
73.0 ± 12.9
49.5 ± 9.8
8y
H
n-butyl
CH3
57.1 ± 4.6 39.2 ± 3.4
41.8 ± 5.3
60.4 ± 4.2
>100
n-butyl
n-butyl
8z
CH3
H
9.9 ± 0.9
8.6 ± 1.4
6.2 ± 2.5
9.9 ± 0.5
5.7 ± 1.2
8aa
CH3
CH3
CH3
CH3
44.5 ± 4.9 54.8 ± 3.2
18.4 ± 2.7 61.6 ± 4.3
44.3 ± 5.5
58.6 ± 4.3
28.2 ± 3.2
8ab
19.9 ± 3.5
4.2 ± 0.7
44.7 ± 5.7
15.4 ± 1.9
30.1 ± 4.2
10.5 ± 2.3
DDP
21.3 ± 1.1
8.4 ± 0.7
a Data represent the mean values of three independent determinations; b cytotoxicity as IC50 for each
cell line is the concentration of compound which reduced the optical density of treated cells by 50%
with respect to untreated cells using the MTT assay. The data represent the mean values ± SD of at
least three independent determinations. Values > 100 µM indicate less than 50% growth inhibition at
> 100 µM; c cell lines include Lewis lung carcinoma (LLC), gastric carcinoma (BGC), murine colon
carcinoma (CT-26), liver carcinoma (Bel-7402), and breast carcinoma (MCF-7).
For the first experiment, we examined the influence of the substituents in position 1 of the β-
carboline core on cytotoxic activities. In order to enhance the range of substituents, we designed 11
novel compounds with methyl and isopropyl substitutions and different patterns of aryl rings
substituted by electron withdrawing (Cl) and donating (OCH3) groups in the C-1 position of β-
carboline. Of these 11 compounds, most of them showed medium or marginal cytotoxic activities in
all cell lines. Interestingly, compounds 8b, 8c, 8d, and 8o were selectively active against BGC823 cells
with IC50 values lower than 10 µM. In particular, compound 8b was more potent against BGC823 cells
than against the four other cell lines, with potencies in the double-digit µM range. Compounds 8m
(R1 = CH3) and 8q (R1 = benzyl) were exceptional; they showed no distinct difference between each
other, and their IC50 values were in the ranges of 13.8–24.7 µM and 13.3–24.5 µM, respectively. Next,
we examined the influence of the substituents in position 9 of the β-carboline ring on antiproliferative
effects. Compound 8s, with an n-butyl group, was found to be the most potent agent among the
heterobivalent β-carbolines, with IC50 values of 2.4 ± 0.6 µM (for BGC823) and 3.1 ± 1.2 µM (for CT26).
Introduction of benzyl to the R9 position on β-carboline yielded compound 8u, and it demonstrated
higher cytotoxic activity than other compounds against all tested tumor cell lines, except for the
BGC823 cell line.
Among all these novel molecules, the cytotoxic potencies of most heterobivalent β-carbolines
(8a–ab) showed no distinct differences, and the IC50 values of this class of compounds ranged from
10 to 100 µM. 8a and 8p had poor inhibitory activities with IC50 values above 50 µM. Compound 8z
exhibited the most potent anticancer activity against the LLC, BGC-823, CT-26, Bel-7402, and MCF-7
cell lines, with IC50 values of 9.9 ± 0.9, 8.6 ± 1.4, 6.2 ± 2.5, 9.9 ± 0.5, and 5.7 ± 1.2 µM, respectively.

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2.3. Inhibition of Angiogenesis in the Chicken Chorioallantoic Membrane (CAM) Assay
The CAM assay was deployed to assess the inhibitory effect of compound 8z on
neovascularization. In this experiment, we used Combretastatin A4 disodium phosphate (CA4P) as
a positive control. The inhibitory effects of compound 8z on angiogenesis in CAM are shown in
Figure 4A. At the dose 0.5 µM, the reference anti-angiogenic drug CA4P elicited 21% inhibition of
angiogenesis, and compound 8z did not show a significant anti-angiogenic activity at this
concentration (13% inhibition). The anti-angiogenetic activity of compound 8z was comparable with
CA4P in an in vivo CAM assay at the 5 µM level. In this assay, 8z inhibited blood vessel formation
by 45%, compared to 47% inhibition induced by CA4P (P < 0.05). At 50 µM, CA4P significantly
inhibited blood vessel formation, eliciting 78% inhibition (Figure 4B).
B
A
Figure 4. In vivo anti-angiogenic effect of compounds 8z in CAM assay. A. The representative photos
of the experiments. B. The anti-angiogenetic activity of compound 8z was semi-quantitatively
analyzed using Graph Pad Prism 5.0. The data represent the mean values ± SD of at least three
independent determinations. (**, P < 0.05; *, P < 0.5).
3. Materials and Methods
3.1. Reagents and General Methods
MTT was obtained from Sigma-Aldrich (Darmstadt, Germany) and Cisplatin from Qilu
pharmaceutical (Jinan, China). Other commercially available starting materials and solvents were
reagent grade and were purchased from Adamas-beta and used without further purification.
Reactions and products were routinely monitored by thin-layer chromatography (TLC) on silica gel
1
13
F254 plates (Qingdao Haiyang Inc., Qingdao, China). H-NMR and C-NMR spectra were recorded
at room temperature on a Bruker Avance III HD 400 instrument (Bruker Company, Bremen, Gemany)
using tetramethylsilane as the internal reference. Chemical shifts (δ) were reported in ppm relative
to the residual solvent peak, and the multiplicity of each signal was designated by the following
abbreviations—s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Coupling constants (J) were
quoted in Hz. HRMS were recorded on a Bruker ultrafleXtreme MALDI-TOF/TOF-MS and Thermo
Scientific LTQ Orbitrap XL (Thermo Fisher Scientific Inc, Waltham, USA). Column chromatography
was performed with silica gel (200–300 mesh, Qingdao Haiyang Inc., Qingdao, China).
3.2. General Procedure for the Preparation of 6a–lc
A mixture of compound 5a (1.98 g, 10 mmol) and activated MnO
2
(30 mmol) in CH CN (60 mL)
3
was stirred under reflux for 2 h. After completion of the reaction (monitored by TLC), the products
were cooled to room temperature and filtered through Celite. The filtrate was passed through silica
gel and washed with dichloromethane, and the solvent was removed under reduced pressure. The
residue was crystallized from acetone or acetone−petroleum ether to give the corresponding
compound 6a. Products 6b–l were prepared according to the same method as 6a.

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1-benzyl-β-carboline-3-carbaldehyde (6f): The compound was obtained as a white solid with 81% yield.
1
H-NMR (400 MHz, CDCl3) δ 10.24 (s, 1H, CHO), 8.68 (s, 1H, ArH), 8.64 (s, 1H, ArH), 8.14 (d, J = 8.0
Hz, 1H, ArH), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.43 (d, J = 8.4 Hz, 1H, ArH), 7.35–7.30 (m, 4H, ArH), 7.25–
13
7.23 (m, 2H, ArH), 4.61 (s, 2H, ArCH2). C-NMR (100 MHz, CDCl3) δ 193.41, 144.21, 143.97, 137.68,
129.04, 129.00, 128.94, 128.81, 128.77, 127.08, 127.01, 121.92, 121.80, 121.20, 114.09, 112.11, 41.56.
1-(2-chlorophenyl)-β-carboline-3-carbaldehyde (6g): The compound was obtained as a light yellow solid
1
with 72% yield. H-NMR (400 MHz, CDCl3) δ 10.28 (s, 1H, CHO), 8.79 (s, 1H, ArH), 8.39 (s, 1H, ArH),
8.25 (d, J = 8.0 Hz, 1H, ArH), 7.69 – 7.66 (m, 1H, ArH), 7.64–7.59 (m, 2H, ArH), 7.56–7.49 (m, 3H, ArH),
13
7.40 (t, J = 7.6 Hz, 1H, ArH). C-NMR (100 MHz, CDCl3) δ 193.33, 144.43, 141.54, 140.61, 136.39, 136.02,
132.89, 132.03, 130.79, 130.32, 129.56, 129.42, 127.71, 122.22, 122.16, 121.48, 114.17, 111.97.
3.3. General Procedure for the Preparation of Compounds 7a–l
We added 85% hydrazine hydrate (10 mL) to a solution of compound 4a (2.40 g, 10 mmol) in
ethanol (100 mL), and then the mixture was refluxed for 8 h. Following the completion of reaction (as
demonstrated by TLC), the resulting mixture was cooled to 5 °C and the precipitate was collected by
filtration. The crude product was further purified first by washing with ethanol and then by
recrystallization in ethanol to obtain compound 7a with a yield of 85%. Product 7b–l was prepared
according to the same method as 7a.
1-isopropyl-β-carboline-3-carbohydrazide (7c): The compound was obtained as a white solid with 91%
1
yield. H-NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H, NH), 8.65 (s, 1H, ArH), 8.35 (d, J = 7.6 Hz, 1H,
ArH), 7.65 (d, J = 8.0 Hz, 1H, ArH), 7.60–7.55 (m, 1H, ArH), 7.30–7.26 (m, 1H, ArH), 4.58 (s, 2H, NH2),
13
3.75–3.64 (m, 1H, CH), 1.43 (d, J = 6.8 Hz, 6H, CH3). C-NMR (100 MHz, DMSO-d6) δ 164.65, 149.72,
141.16, 138.99, 134.81, 128.67, 128.22, 122.44, 121.92, 120.27, 112.62, 112.19, 31.21, 21.81.
1-benzyl-β-carboline-3-carbohydrazide (7f): The compound was obtained as a white solid with 94% yield.
1
H-NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H, NH), 8.69 (s, 1H, ArH), 8.37 (d, J = 8.0 Hz, 1H, ArH), 7.67
(d, J = 8.4 Hz, 1H, ArH), 7.63–7.57 (m, 1H, ArH), 7.51 (d, J = 7.2 Hz, 2H, ArH), 7.34–7.25 (m, 4H, ArH),
13
7.20–7.15 (m, 1H, ArH), 4.60 (s, 2H, NH2), 4.51 (s, 2H, ArCH2). C-NMR (100 MHz, DMSO-d6) δ 164.43,
143.63, 141.29, 139.36, 139.33, 135.69, 129.36, 129.05, 128.92, 128.82, 128.76, 126.65, 122.60, 121.88,
120.44, 112.68, 112.59, 61.00.
9-n-butyl-β-carboline-3-carbohydrazide (7i): The compound was obtained as a light yellow solid with
1
80% yield. H-NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H, NH), 9.05 (d, J = 1.2 Hz, 1H, ArH), 8.84 (d, J =
0.8 Hz, 1H, ArH), 8.44 (d, J = 8.0 Hz, 1H, ArH), 7.77 (d, J = 8.4 Hz, 1H, ArH), 7.68–7.63 (m, 1H, ArH),
7.36–7.31 (m, 1H, ArH), 4.59–4.54 (m, 4H, NH2, −CH2CH2CH2CH3), 1.85–1.76 (m, 2H,
−CH2CH2CH2CH3), 1.35–1.24 (m, 2H, −CH2CH2CH2CH3), 0.88 (t, J = 7.2 Hz, 3H, −CH2CH2CH2CH3).
13
C-NMR (100 MHz, DMSO-d6) δ 164.37, 141.62, 140.00, 137.58, 131.70, 129.16, 128.14, 122.87, 121.16,
120.55, 114.14, 110.97, 43.05, 31.38, 20.20, 14.14.
9-(3-phenylpropyl)-β-carboline-3-carbohydrazide (7j): The compound was obtained as a gray powder
1
with 86% yield. H-NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H, NH), 9.00 (d, J = 1.2 Hz, 1H, ArH), 8.83
(d, J = 0.8 Hz, 1H, ArH), 8.44 (d, J = 8.0 Hz, 1H, ArH), 7.72 (d, J = 8.4 Hz, 1H, ArH), 7.68–7.62 (m, 1H,
ArH), 7.37–7.31 (m, 1H, ArH), 7.29–7.22 (m, 2H, ArH), 7.21–7.13 (m, 3H, ArH), 4.60 (t, J = 7.2 Hz, 2H,
ArCH2CH2CH2), 4.56 (s, 2H, NH2), 2.67 (t, J = 7.6 Hz, 2H, ArCH2CH2CH2), 2.19–2.10 (m, 2H,
13
ArCH2CH2CH2). C-NMR (101 MHz, DMSO-d6) δ 164.34, 141.59, 141.55, 140.08, 137.52, 131.62, 129.20,
128.80, 128.58, 128.24, 126.37, 122.92, 121.22, 120.62, 114.16, 110.89, 43.03, 32.85, 30.84.
9-(4-fluorobenzyl)-β-carboline-3-carbohydrazide (7l): The compound was obtained as a white solid with
1
96% yield. H-NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H, NH), 9.11 (d, J = 1.2 Hz, 1H, ArH), 8.89 (d, J =
1.2 Hz, 1H, ArH), 8.48 (d, J = 7.6 Hz, 1H, ArH), 7.83 (d, J = 8.4 Hz, 1H, ArH), 7.68–7.63 (m, 1H, ArH),
7.39–7.34 (m, 1H, ArH), 7.33–7.29 (m, 2H, ArH), 7.17–7.11 (m, 2H, ArH), 5.85 (s, 2H, ArCH2), 4.61 (s,
13
2H, NH2). C-NMR (100 MHz, DMSO-d6) δ 164.30, 162.52 (d, J = 242.1 Hz), 141.65, 140.50, 137.58,

Molecules 2019, 24, 2950
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133.86 (d, J = 3.2 Hz), 131.90, 129.51, 129.43, 129.39, 128.59, 122.98, 121.17 (d, J = 46.3 Hz), 115.99 (d, J =
21.4 Hz), 114.25, 111.18, 45.88.
3.4. General Procedure for the Preparation of Heterobivalent β-Carbolines 8a–ab
We added β-carboline-3-carbaldehyde 6a–l (1 mmol) to a solution of β-carboline-3-
carbohydrazide 7a–l (1 mmol) in EtOH (50 mL), and then the reaction mixture was refluxed for 5 h.
The solution was allowed to cool to room temperature. Then, the precipitates formed and were
filtered, and the crude product was recrystallized with ethanol to afford compounds 8a–ab.
N′-((9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8a): The compound
1
was obtained as a yellow solid with 93% yield. H-NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H, NH),
12.11 (s, 1H, NH), 9.28 (s, 1H, ArH), 9.16 (d, J = 0.8 Hz, 1H, ArH), 9.02 (s, 1H, ArH), 8.63 (s, 1H, ArH),
8.46 (d, J = 8.0 Hz, 1H, CH), 8.34 (d, J = 8.0 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.73 (d, J = 8.0 Hz, 1H,
13
ArH), 7.70 (d, J = 8.0 Hz, 1H, ArH), 7.68–7.61 (m, 2H, ArH), 7.39–7.32 (m, 2H, ArH). C-NMR (100
MHz, DMSO-d6) δ 162.68, 142.36, 141.65, 141.50, 140.77, 139.78, 137.85, 135.54, 133.55, 133.31, 129.49,
129.16, 128.80, 128.66, 122.81, 122.53, 121.47, 121.24, 120.74, 120.59, 118.99, 115.46, 112.99, 112.78.
+
+
HRMS m/z calculated for C24H17N6O (M + H) : 405.1458; found 405.1459.
N'-((1-methyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8b): The
1
compound was obtained as a yellow solid with 84% yield. H-NMR (400 MHz, DMSO-d6) δ 12.15 (s,
1H, NH), 12.02 (s, 1H, NH), 9.08 (d, J = 0.8 Hz, 1H, ArH), 9.03 (s, 1H, ArH), 8.46 (d, J = 8.0 Hz, 1H,
CH), 8.44 (s, 1H, ArH), 8.29 (d, J = 8.0 Hz, 1H, ArH), 7.74 (s, 1H, ArH), 7.73–7.69 (m, 2H, ArH), 7.66–
13
7.60 (m, 2H, ArH), 7.34 (t, J = 7.6 Hz, 2H, ArH), 3.20 (s, 3H, CH3). C-NMR (100 MHz, DMSO-d6) δ
162.54, 141.96, 141.86, 141.48, 141.36, 140.92, 139.96, 137.89, 134.27, 133.02, 129.17, 129.13, 128.75,
128.02, 122.80, 122.45, 121.71, 121.48, 120.66, 120.60, 117.15, 115.48, 112.88, 112.81, 20.96. HRMS m/z
+
+
calculated for C25H19N6O (M + H) : 419.1615; found 419.1620.
N'-((1-isopropyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8c): The
1
compound was obtained as a yellow solid with 89% yield. H-NMR (400 MHz, DMSO-d6) δ 15.64 (s,
1H, CONH), 12.10 (s, 1H, NH), 12.05 (s, 1H, NH), 9.07 (s, 1H, ArH), 9.02 (s, 1H, ArH), 8.49 (d, J = 8.0
Hz, 1H, CH), 8.46 (s, 1H, ArH), 8.30 (d, J = 8.0 Hz, 1H, ArH), 7.78 (s, 1H, ArH), 7.75–7.69 (m, 2H, ArH),
7.67–7.62 (m, 2H, ArH), 7.38–7.33 (m, 2H, ArH), 4.02–3.91 (m, 1H, CH(CH3)2), 1.76 (d, J = 7.2 Hz, 6H,
13
CH(CH3)2). C-NMR (100 MHz, DMSO-d6) δ 162.76, 151.03, 142.16, 141.51, 141.38, 141.17, 139.54,
138.00, 132.90, 132.63, 129.25, 129.20, 128.88, 128.60, 122.87, 122.35, 121.72, 121.45, 120.66, 120.62,
+
+
117.59, 115.71, 112.87, 112.82, 31.57, 21.63. HRMS m/z calculated for C27H23N6O (M + H) : 447.1928;
found 447.1932.
N'-((1-phenyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8d): The
1
compound was obtained as a yellow solid with 96% yield. H-NMR (400 MHz, DMSO-d6) δ 15.58 (s,
1H, CONH), 12.06 (s, 1H, NH), 11.95 (s, 1H, NH), 8.99 (s, 1H, ArH), 8.65 (s, 1H, ArH), 8.44 (d, J = 8.4
Hz, 2H, CH, ArH), 8.35 (d, J = 8.0 Hz, 1H, ArH), 8.26–8.23 (m, 2H, ArH), 7.85 (s, 1H, ArH), 7.82–7.73
13
(m, 4H, ArH), 7.69–7.59 (m, 3H, ArH), 7.41–7.35 (m, 1H, ArH), 7.35–7.29 (m, 1H, ArH). C-NMR (100
MHz, DMSO-d6) δ 162.83, 142.56, 142.51, 142.15, 141.43, 140.85, 139.47, 137.75, 137.64, 133.10, 132.78,
130.10, 129.75, 129.47, 129.44, 129.32, 129.16, 128.59, 122.81, 122.36, 121.46, 121.41, 120.91, 120.57,
+
+
118.56, 115.52, 113.36, 112.74. HRMS m/z calculated for C30H21N6O (M + H) : 481.1771; found 481.1777.
N'-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8e): The compound was obtained as a yellow solid with 91% yield. H-NMR (400 MHz, DMSO-d6) δ
15.53 (s, 1H, CONH), 12.12 (s, 1H, NH), 11.90 (s, 1H, NH), 9.01 (s, 1H, ArH), 8.59–8.57 (m, 2H, ArH),
8.45 (d, J = 7.6 Hz, 1H, CH), 8.33 (d, J = 7.6 Hz, 1H, ArH), 8.23 (d, J = 8.8 Hz, 2H, ArH), 7.83 (s, 1H,
ArH), 7.74 (d, J = 8.4 Hz, 1H, ArH), 7.70 (d, J = 8.4 Hz, 1H, ArH), 7.68–7.60 (m, 2H, ArH), 7.39–7.31 (m,
13
4H, ArH), 4.02 (s, 3H, OCH3). C-NMR (100 MHz, DMSO-d6) δ 162.86, 160.53, 142.53, 142.45, 142.07,
141.48, 140.97, 139.51, 137.76, 132.92, 132.80, 131.10, 130.00, 129.88, 129.30, 129.18, 128.65, 122.82,
122.29, 121.50, 121.42, 120.84, 120.58, 118.19, 115.63, 114.72, 113.36, 112.75, 56.05. HRMS m/z calculated
+
+
for C31H23N6O2 (M + H) : 511.1877; found 511.1878.

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N'-((1-benzyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8f): The
1
compound was obtained as a yellow solid with 82% yield. H-NMR (400 MHz, DMSO-d6) δ 12.19 (s,
1H, NH), 11.95 (s, 1H, NH), 9.06 (s, 1H, ArH), 8.88 (s, 1H, ArH), 8.50 (s, 1H, ArH), 8.47 (d, J = 7.6 Hz,
1H, CH), 8.29 (d, J = 7.6 Hz, 1H, ArH), 7.78–7.72 (m, 4H, ArH), 7.69–7.59 (m, 4H, ArH), 7.37–7.32 (m,
13
2H, ArH), 7.23 (t, J = 7.6 Hz, 2H, ArH), 7.13 (t, J = 7.2 Hz, 1H, ArH), 4.90 (s, 2H, ArCH2). C-NMR (100
MHz, DMSO-d6) δ 162.61, 144.08, 142.15, 141.48, 141.44, 140.83, 139.92, 139.26, 137.88, 133.58, 132.82,
131.50129.39, 129.23, 129.11, 128.95, 128.82, 126.84, 122.85, 122.44, 121.68, 121.45, 120.80, 120.64, 117.69,
+
+
115.64, 112.96, 112.78. HRMS m/z calculated for C31H23N6O (M + H) : 495.1928; found 495.1932.
N'-((1-(2-chlorophenyl)-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8g): The compound was obtained as a yellow solid with 81% yield. H-NMR (400 MHz, DMSO-d6) δ
12.04 (s, 1H, NH), 11.85 (s, 1H, NH), 8.92 (s, 1H, ArH), 8.70 (s, 1H, ArH), 8.41 (d, J = 8.0 Hz, 1H, CH),
8.37 (d, J = 8.0 Hz, 1H, ArH), 7.96 (d, J = 1.2 Hz, 1H, ArH), 7.90–7.85 (m, 3H, ArH), 7.84–7.79 (m, 1H,
ArH), 7.77–7.72 (m, 1H, ArH), 7.70–7.55 (m, 5H, ArH), 7.40–7.35 (m, 1H, ArH), 7.31 (t, J = 7.2 Hz, 1H,
13
ArH). C-NMR (100 MHz, DMSO-d6) δ 162.71, 142.01, 141.37, 140.58, 140.53, 139.50, 137.61, 136.59,
133.76, 133.32, 132.75, 132.47, 131.09, 130.30, 129.58, 129.55, 129.09, 128.46, 127.95, 122.75, 122.50,
+
121.40, 121.38, 120.86, 120.52, 118.77, 115.25, 113.09, 112.70. HRMS m/z calculated for C30H20ClN6O (M
+
+ H) : 515.1382; found 515.1386.
N'-((9-methyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8h): The
1
compound was obtained as an ivory solid with 84% yield. H-NMR (400 MHz, DMSO-d6) δ 12.14 (s,
1H, NH), 9.45 (s, 1H, ArH), 9.15 (s, 1H, ArH), 9.01 (s, 1H, ArH), 8.64 (s, 1H, ArH), 8.46 (d, J = 8.0 Hz,
1H, CH), 8.36 (d, J = 8.0 Hz, 1H, ArH), 7.83 (d, J = 8.4 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.74 (d, J = 7.6
Hz, 1H, ArH), 7.70 (d, J = 8.4 Hz, 1H, ArH), 7.63 (t, J = 7.6 Hz, 1H, ArH), 7.41 (t, J = 7.6 Hz, 1H, ArH),
13
7.34 (t, J = 7.4 Hz, 1H, ArH), 4.15 (s, 3H, CH3). C-NMR (100 MHz, DMSO-d6) δ 162.68, 142.51, 142.47,
141.50, 140.63, 139.79, 137.86, 136.16, 133.45, 132.20, 129.61, 129.18, 128.68, 128.32, 122.82, 122.60,
+
121.47, 120.92, 120.60, 118.70, 115.47, 112.77, 111.11, 30.24. HRMS calculated for C25H19N6O [M + H] :
419.1615; found 419.1615.
N'-((9-n-butyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8i): The
1
compound was obtained as an ivory solid with 79% yield. H-NMR (400 MHz, DMSO-d6) δ 12.12 (s,
1H, NH), 9.46 (s, 1H, ArH), 9.17 (s, 1H, ArH), 9.02 (s, 1H, ArH), 8.64 (s, 1H, ArH), 8.47 (d, J = 7.6 Hz,
1H, CH), 8.36 (d, J = 8.0 Hz, 1H, ArH), 7.84 (d, J = 8.4 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.75–7.68 (m,
2H, ArH), 7.63 (t, J = 7.6 Hz, 1H, ArH), 7.40 (t, J = 7.6 Hz, 1H, ArH), 7.34 (t, J = 7.6 Hz, 1H, ArH), 4.67
(t, J = 7.2 Hz, 2H, −CH2CH2CH2CH3), 1.92–1.84 (m, 2H, −CH2CH2CH2CH3), 1.42–1.31 (m, 2H,
13
−CH2CH2CH2CH3), 0.92 (t, J = 7.6 Hz, 3H, −CH2CH2CH2CH3). C-NMR (100 MHz, DMSO-d6) δ 162.70,
142.48, 141.84, 141.51, 140.60, 139.77, 137.85, 135.67, 133.51, 132.16, 129.61, 129.19, 128.69, 128.42,
122.82, 122.69, 121.48, 121.00, 120.90, 120.60, 118.83, 115.47, 112.78, 111.30, 43.26, 31.50, 20.27, 14.19.
+
HRMS calculated for C28H25N6O [M + H] : 461.2084; found 461.2088.
N'-((9-(3-phenylpropyl)-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8j): The compound was obtained as a yellow solid with 84% yield. H-NMR (400 MHz, DMSO-d6) δ
12.15 (s, 1H, NH), 9.35 (s, 1H, ArH), 9.15 (d, J = 1.2 Hz, 1H, ArH), 9.03 (s, 1H, ArH), 8.64 (s, 1H, ArH),
8.47 (d, J = 7.6 Hz, 1H, CH), 8.36 (d, J = 8.0 Hz, 1H, ArH), 7.81–7.78 (m, 2H, ArH), 7.73–7.68 (m, 2H,
ArH), 7.66–7.63 (m, 1H, ArH), 7.42–7.38 (m, 1H, ArH), 7.37–7.34 (m, 1H, ArH), 7.32–7.27 (m, 3H, ArH),
7.25–7.20 (m, 3H, ArH), 4.69 (t, J = 7.2 Hz, 2H, ArCH2CH2CH2), 2.74 (t, J = 7.2 Hz, 2H, ArCH2CH2CH2),
13
2.27–2.18 (m, 2H, ArCH2CH2CH2). C-NMR (100 MHz, DMSO-d6) δ 162.70, 142.55, 141.79, 141.57,
141.51, 140.59, 139.77, 137.85, 135.59, 133.44, 131.99, 129.63, 129.20, 128.84, 128.82, 128.69, 128.62,
126.43, 122.83, 122.72, 121.48, 121.06, 120.96, 120.61, 118.85, 115.50, 112.78, 111.19, 43.12, 32.84, 30.87.
+
HRMS calculated for C33H27N6O [M + H] : 523.2241; found 523.2242.
N'-((9-benzyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (8k): The
1
compound was obtained as a yellow solid with 86% yield. H-NMR (400 MHz, DMSO-d6) δ 12.32 (s,
1H, NH), 12.06 (s, 1H, NH), 9.13 (d, J = 0.8 Hz, 1H, ArH), 9.04 (s, 1H, ArH), 9.01 (d, J = 1.2 Hz, 1H,
ArH), 8.92 (s, 1H, ArH), 8.83 (d, J = 0.8 Hz, 1H, ArH), 8.48 (t, J = 8.0 Hz, 2H, ArH, CH), 7.79 (d, J = 8.4

Molecules 2019, 24, 2950
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Hz, 1H, ArH), 7.71 (d, J = 8.4 Hz, 1H, ArH), 7.67–7.63 (m, 1H, ArH), 7.38–7.23 (m, 8H, ArH), 5.83 (s,
13
2H, ArCH2). C-NMR (100 MHz, DMSO-d6) δ 161.86, 150.06, 144.04, 141.98, 141.53, 139.51, 137.86,
137.69, 137.05, 132.81, 129.36, 129.26, 129.19, 128.79, 128.76, 128.00, 127.35, 127.31, 122.96, 122.90,
121.43, 121.30, 120.74, 120.64, 115.62, 112.80, 111.71, 111.15, 46.53. HRMS calculated for C31H23N6O [M
+
+ H] : 495.1928; found 495.1922.
N'-((9-(4-fluorobenzyl)-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8l): The compound was obtained as a light gray solid with 92% yield. H-NMR (400 MHz, DMSO-
d6) δ 12.31 (s, 1H, NH), 12.04 (s, 1H, NH), 9.15 (d, J = 0.8 Hz, 1H, ArH), 9.03 (s, 1H, ArH), 9.00 (d, J =
1.2 Hz, 1H, ArH), 8.91 (s, 1H, ArH), 8.82 (d, J = 1.2 Hz, 1H, ArH), 8.48 (dd, J = 8.0, 4.8 Hz, 2H, CH,
ArH), 7.81 (d, J = 8.4 Hz, 1H, ArH), 7.71 (d, J = 8.4 Hz, 1H, ArH), 7.68–7.61 (m, 2H, ArH), 7.38–7.30 (m,
13
4H, ArH), 7.17–7.11 (m, 2H, ArH), 5.82 (s, 2H, ArCH2). C-NMR (100 MHz, DMSO-d6) δ 161.96 (d, J =
241.8 Hz), 161.84, 150.05, 144.12, 141.86, 141.53, 139.51, 137.85, 136.94, 133.92 (d, J = 3.1 Hz), 132.80,
129.53, 129.45, 129.40, 129.24, 128.81, 128.79, 123.00 (d, J = 9.6 Hz), 121.43, 121.34, 120.80, 120.64, 116.11
+
(d, J = 21.3 Hz), 115.62, 112.79, 111.70, 111.12, 45.79. HRMS calculated for C31H22FN6O [M + H] :
513.1834; found 513.1832.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-1-methyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8m): The
1
compound was obtained as a yellow solid with 85% yield. H-NMR (400 MHz, DMSO-d6) δ 12.05 (s,
1H, NH), 12.02 (s, 1H, NH), 11.83 (s, 1H, NH), 8.95 (d, J = 0.8 Hz, 1H, ArH), 8.90 (s, 1H, ArH), 8.85 (s,
1H, ArH), 8.78 (s, 1H, ArH), 8.42 (t, J = 8.4 Hz, 2H, CH, ArH), 7.69–7.57 (m, 4H, ArH), 7.35–7.28 (m,
13
2H, ArH), 2.94 (s, 3H, CH3). C-NMR (100 MHz, DMSO-d6) δ 161.84, 150.09, 143.33, 141.66, 141.61,
141.28, 138.85, 136.87, 136.60, 133.94, 129.05, 128.88, 128.76, 127.95, 122.77, 122.70, 121.90, 121.33,
+
120.55, 120.26, 113.68, 112.71, 112.63, 111.84, 20.87. HRMS calculated for C25H19N6O [M + H] : 419.1615;
found 419.1617.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-1-isopropyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8n): The
1
compound was obtained as a yellow solid with 91% yield. H-NMR (400 MHz, DMSO-d6) δ 15.65 (s,
1H, CONH), 12.17 (s, 1H, NH), 12.06 (s, 1H, NH), 9.22 (s, 1H, ArH), 8.88 (s, 1H, ArH), 8.65 (s, 1H,
ArH), 8.42 (d, J = 7.6 Hz, 1H, CH), 8.34 (d, J = 7.6 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.74–7.60 (m, 4H,
13
ArH), 7.40–7.30 (m, 2H, ArH), 3.92–3.82 (m, 1H, CH(CH3)2), 1.68 (d, J = 6.8 Hz, 6H, CH(CH3)2). C-
NMR (100 MHz, DMSO-d6) δ 162.96, 149.74, 142.52, 141.59, 141.29, 140.56, 139.03, 135.63, 135.24,
132.99, 129.52, 128.88, 128.79, 128.53, 122.59, 121.98, 121.19, 120.75, 120.50, 119.37, 113.80, 112.96,
+
112.71, 31.12, 22.22. HRMS calculated for C27H23N6O [M + H] : 447.1928; found 447.1926.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-1-phenyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8o): The
1
compound was obtained as a yellow solid with 91% yield. H-NMR (400 MHz, DMSO-d6) δ 15.97 (s,
1H, CONH), 12.27 (s, 1H, NH), 12.03 (s, 1H, NH), 9.05 (d, J = 5.2 Hz, 2H, ArH), 8.63 (s, 1H, ArH), 8.50
(d, J = 8.0 Hz, 1H, CH), 8.39 (d, J = 6.8 Hz, 2H, ArH), 8.33 (d, J = 7.6 Hz, 1H, ArH), 7.91 (t, J = 7.6 Hz,
2H, ArH), 7.81 (s, 1H, ArH), 7.79–7.72 (m, 3H, ArH), 7.68–7.62 (m, 2H, ArH), 7.39–7.34 (m, 2H, ArH).
13
C-NMR (100 MHz, DMSO-d6) δ 162.62, 142.40, 142.07, 141.63, 141.12, 140.82, 139.66, 138.23, 135.57,
134.93, 132.94, 130.58, 129.75, 129.51, 129.20, 128.74, 122.61, 122.56, 121.71, 121.18, 120.84, 120.72,
+
119.28, 114.68, 113.22, 112.93. HRMS calculated for C30H21N6O [M + H] : 481.1771; found 481.1772.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8p): The compound was obtained as a yellow solid with 87% yield. H-NMR (400 MHz, DMSO-d6) δ
15.89 (s, 1H, CONH), 12.27 (s, 1H, NH), 11.96 (s, 1H, NH), 9.07 (s, 1H, ArH), 9.00 (s, 1H, ArH), 8.64 (s,
1H, ArH), 8.47 (d, J = 7.6 Hz, 1H, CH), 8.37–8.32 (m, 2H, ArH), 7.81 (s, 1H, ArH), 7.75 (dd, J = 8.0, 4.4
Hz, 2H, ArH), 7.69–7.61 (m, 2H, ArH), 7.44 (d, J = 8.8 Hz, 2H, ArH), 7.39–7.32 (m, 2H, ArH), 4.04 (s,
13
3H, OCH3). C-NMR (100 MHz, DMSO-d6) δ 162.69, 160.72, 142.41, 142.01, 141.63, 141.10, 140.77,
139.53, 135.60, 134.70, 133.00, 130.57, 130.49, 130.34, 129.52, 129.08, 128.77, 122.58, 122.54, 121.75,
121.20, 120.78, 120.73, 119.33, 114.92, 114.17, 113.22, 112.94, 56.01. HRMS calculated for C31H23N6O2 [M
+
+ H] : 511.1877; found 511.1874.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-1-benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8q): The
1
compound was obtained as an ivory solid with 83% yield. H-NMR (400 MHz, DMSO-d6) δ 12.14 (s,

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1H, CONH), 11.99 (s, 1H, NH), 11.86 (s, 1H, NH), 8.98 (s, 1H, ArH), 8.91 (d, J = 2.4 Hz, 2H, ArH), 8.81
(s, 1H, ArH), 8.45 (d, J = 8.0 Hz, 1H, CH), 8.40 (d, J = 7.6 Hz, 1H, ArH), 7.72–7.66 (m, 2H, ArH), 7.65–
13
7.55 (m, 4H, ArH), 7.35–7.28 (m, 4H, ArH), 7.21 (t, J = 7.2 Hz, 1H, ArH), 4.65 (s, 2H, ArCH2). C-NMR
(100 MHz, DMSO-d6) δ 161.65, 150.29, 143.62, 143.18, 141.67, 141.39, 139.26, 138.89, 136.90, 136.13,
133.99, 129.39, 129.08, 129.04, 128.96, 128.83, 128.78, 126.71, 122.77, 122.70, 121.89, 121.32, 120.66,
+
120.27, 113.96, 112.77, 112.64, 111.94. HRMS calculated for C31H23N6O [M + H] : 495.1928; found
495.1929.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-9-methyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8r): The
1
compound was obtained as a light yellow solid with 81% yield. H-NMR (400 MHz, DMSO-d6) δ 12.31
(s, 1H, CONH), 11.82 (s, 1H, NH), 9.14 (s, 1H, ArH), 9.03 (s, 1H, ArH), 8.94 (d, J = 0.8 Hz, 1H, ArH),
8.92 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.52 (d, J = 7.6 Hz, 1H, CH), 8.41 (d, J = 8.0 Hz, 1H, ArH), 7.79 (d,
J = 8.4 Hz, 1H, ArH), 7.73–7.68 (m, 1H, ArH), 7.64 (d, J = 8.4 Hz, 1H, ArH), 7.62–7.57 (m, 1H, ArH),
13
7.41 – 7.36 (m, 1H, ArH), 7.32–7.28 (m, 1H, ArH), 4.10 (s, 3H, CH3). C-NMR (100 MHz, DMSO-d6) δ
161.77, 150.35, 143.37, 142.39, 141.65, 139.73, 138.38, 136.86, 133.88, 131.51, 129.35, 129.04, 128.75,
128.36, 122.96, 122.70, 121.34, 121.09, 120.81, 120.25, 115.37, 112.62, 111.79, 110.90, 30.18. HRMS
+
calculated for C25H19N6O [M + H] : 419.1615; found 419.1618.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-9-n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8s): The
1
compound was obtained as a light yellow solid with 92% yield. H-NMR (400 MHz, DMSO-d6) δ 12.28
(s, 1H, CONH), 11.82 (s, 1H, NH), 9.15 (d, J = 0.8 Hz, 1H, ArH), 9.04 (d, J = 0.8 Hz, 1H, ArH), 8.95 (d,
J = 0.8 Hz, 1H, ArH), 8.92 (s, 1H, ArH), 8.79 (s, 1H, ArH), 8.52 (d, J = 7.6 Hz, 1H, CH), 8.41 (d, J = 8.0
Hz, 1H, ArH), 7.81 (d, J = 8.4 Hz, 1H, ArH), 7.71–7.69 (m, 1H, ArH), 7.65 (d, J = 8.0 Hz, 1H, ArH), 7.62–
7.57 (m, 1H, ArH), 7.40–7.35 (m, 1H, ArH), 7.32–7.28 (m, 1H, ArH), 4.63 (t, J = 6.8 Hz, 2H,
−CH2CH2CH2CH3), 1.88–1.80 (m, 2H, −CH2CH2CH2CH3), 1.36–1.26 (m, 2H, −CH2CH2CH2CH3), 0.89
13
(t, J = 7.2 Hz, 3H, −CH2CH2CH2CH3). C-NMR (100 MHz, DMSO-d6) δ 161.76, 150.35, 143.36, 141.75,
141.65, 139.72, 137.94, 136.87, 133.89, 131.55, 129.35, 129.04, 128.75, 128.44, 123.07, 122.69, 121.34,
121.19, 120.80, 120.25, 115.45, 112.62, 111.80, 111.11, 49.07, 31.41, 20.21, 14.17. HRMS calculated for
+
C28H25N6O [M + H] : 461.2084; found 461.2085.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-9-(3-phenylpropyl)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8t): The compound was obtained as a yellow solid with 88% yield. H-NMR (400 MHz, DMSO-d6) δ
12.32 (s, 1H, CONH), 11.85 (s, 1H, NH), 9.10 (s, 1H, ArH), 9.06 (s, 1H, ArH), 8.97 (s, 1H, ArH), 8.94 (s,
1H, ArH), 8.80 (s, 1H, ArH), 8.52 (d, J = 7.6 Hz, 1H, CH), 8.39 (d, J = 8.0 Hz, 1H, ArH), 7.76 (d, J = 8.0
Hz, 1H, ArH), 7.71–7.64 (m, 2H, ArH), 7.62–7.58 (m, 1H, ArH), 7.38 (t, J = 7.6 Hz, 1H, ArH), 7.32–7.25
(m, 3H, ArH), 7.19–7.16 (m, 3H, ArH), 4.65 (t, J = 7.2 Hz, 2H, ArCH2CH2CH2), 2.69 (t, J = 7.6 Hz, 2H,
13
ArCH2CH2CH2), 2.22–2.14 (m, 2H, ArCH2CH2CH2). C-NMR (100 MHz, DMSO-d6) δ 161.81, 150.40,
143.37, 141.70, 141.64, 141.50, 139.79, 137.86, 136.88, 133.94, 131.43, 129.37, 129.03, 128.81, 128.73,
128.58, 128.55, 126.39, 123.09, 122.67, 121.34, 121.25, 120.85, 120.24, 115.49, 112.63, 111.84, 110.99, 43.06,
+
32.85, 30.82. HRMS calculated for C33H27N6O [M + H] : 523.2241; found 523.2242.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-9-benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (8u): The
1
compound was obtained as a light yellow solid with 80% yield. H-NMR (400 MHz, DMSO-d6) δ 12.28
(s, 1H, CONH), 11.82 (s, 1H, NH), 9.19 (s, 1H, ArH), 9.07 (s, 1H, ArH), 8.95 (s, 1H, ArH), 8.90 (s, 1H,
ArH), 8.78 (s, 1H, ArH), 8.55 (d, J = 8.0 Hz, 1H, CH), 8.41 (d, J = 8.0 Hz, 1H, ArH), 7.84 (d, J = 8.0 Hz,
1H, ArH), 7.70–7.63 (m, 2H, ArH, NH), 7.62–7.57 (m, 1H, ArH), 7.39 (t, J = 7.5 Hz, 1H, ArH), 7.34–7.24
13
(m, 6H, ArH), 5.91 (s, 2H, ArCH2). C-NMR (100 MHz, DMSO-d6) δ 161.70, 150.39, 143.33, 141.89,
141.64, 140.14, 138.04, 137.60, 136.87, 133.89, 131.84, 129.54, 129.23, 129.04, 128.84, 128.75, 128.09,
127.41, 123.17, 122.70, 121.40, 121.34, 121.11, 120.25, 115.52, 112.63, 111.80, 111.35, 46.73. HRMS
+
calculated for C31H23N6O [M + H] : 495.1928; found 495.1927.
N'-((9H-pyrido[3,4-b]indol-3-yl)methylene)-9-(4-fluorobenzyl)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8v): The compound was obtained as a light yellow solid with 90% yield. H-NMR (400 MHz, DMSO-
d6) δ 12.27 (s, 1H, CONH), 11.82 (s, 1H, NH), 9.20 (d, J = 1.2 Hz, 1H, ArH), 9.06 (d, J = 1.2 Hz, 1H, ArH),
8.94 (d, J = 1.2 Hz, 1H, ArH), 8.90 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.55 (d, J = 7.6 Hz, 1H, CH), 8.41 (d,

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J = 8.0 Hz, 1H, ArH), 7.86 (d, J = 8.4 Hz, 1H, ArH), 7.71–7.57 (m, 3H, ArH), 7.39 (t, J = 7.6 Hz, 1H, ArH),
13
7.35–7.28 (m, 3H, ArH), 7.19–7.12 (m, 2H, ArH), 5.90 (s, 2H, ArCH2). C-NMR (100 MHz, DMSO-d6)
δ 162.00 (d, J = 242.1 Hz), 161.68, 150.43, 143.34, 141.77, 141.64, 140.21, 137.94, 136.88, 133.90, 133.82 (d,
J = 3 Hz), 131.81, 129.59, 129.51, 129.04, 128.89, 128.74, 123.20, 122.70, 121.39 (d, J = 10.2 Hz), 121.17,
120.25, 116.05 (d, J = 21.3 Hz), 115.52, 112.62, 111.80, 111.32, 49.07. HRMS calculated for C31H22FN6O
+
[M + H] : 513.1834; found 513.1835.
9-n-butyl-N'-((9-n-butyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
1
(8w): The compound was obtained as a yellow solid with 91% yield. H-NMR (400 MHz, DMSO-d6)
δ 12.28 (s, 1H, CONH), 9.15 (s, 1H, ArH), 9.10 (s, 1H, ArH), 9.03 (s, 1H, ArH), 8.91 (s, 1H, ArH), 8.80
(s, 1H, ArH), 8.52 (d, J = 8.0 Hz, 1H, CH), 8.45 (d, J = 8.0 Hz, 1H, ArH), 7.82 (d, J = 8.4 Hz, 1H, ArH),
7.76 (d, J = 8.4 Hz, 1H, ArH), 7.71–7.64 (m, 2H, ArH), 7.40–7.32 (m, 2H, ArH), 4.63 (t, J = 6.8 Hz, 2H,
−CH2CH2CH2CH3), 4.55 (t, J = 6.8 Hz, 2H, −CH2CH2CH2CH3), 1.88–1.79 (m, 4H, −CH2CH2CH2CH3),
13
1.37–1.27 (m, 4H, −CH2CH2CH2CH3), 0.92–0.88 (m, 6H, −CH2CH2CH2CH3). C-NMR (100 MHz,
DMSO-d6) δ 161.75, 150.25, 143.59, 141.85, 141.75, 139.70, 137.95, 136.96, 132.61, 131.57, 129.36, 129.17,
128.44, 128.40, 123.08, 122.88, 121.19, 121.09, 120.80, 120.40, 115.45, 111.64, 111.12, 110.90, 43.12, 42.99,
+
31.42, 31.34, 20.24, 20.22, 14.18. HRMS calculated for C32H33N6O [M + H] : 517.2710; found 517.2706.
9-n-butyl-N'-((9-n-butyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-1-methyl-9H-pyrido[3,4-b]indole-3-
1
carbohydrazide (8x): The compound was obtained as a yellow solid with 88% yield. H-NMR (400 MHz,
CDCl3) δ 11.32 (s, 1H, CONH), 9.04 (s, 1H, ArH), 8.92 (s, 1H, ArH), 8.84 (s, 1H, ArH), 8.59 (s, 1H, CH),
8.25 (d, J = 8.0 Hz, 1H, ArH), 8.22 (d, J = 8.0 Hz, 1H, ArH), 7.64–7.59 (m, 2H, ArH), 7.47 (dd, J = 8.4, 2.8
Hz, 2H, ArH), 7.35 (t, J = 7.6 Hz, 2H, ArH), 4.54 (t, J = 7.6 Hz, 2H, −CH2CH2CH2CH3), 4.36 (t, J = 7.2
Hz, 2H, −CH2CH2CH2CH3), 3.09 (s, 3H, CH3), 1.94–1.81 (m, 4H, −CH2CH2CH2CH3), 1.53–1.36 (m, 4H,
−CH2CH2CH2CH3), 1.00 (t, J = 7.6 Hz, 3H, −CH2CH2CH2CH3), 0.97 (t, J = 7.2 Hz, 3H,
13
−CH2CH2CH2CH3). C-NMR (100 MHz, CDCl3) δ 161.29, 148.92, 142.53, 141.84, 141.81, 139.67, 137.77,
137.00, 136.59, 130.81, 129.58, 129.19, 128.72, 128.53, 122.59, 121.98, 121.69, 121.48, 120.56, 120.27,
113.75, 113.00, 109.97, 109.60, 44.84, 43.34, 33.02, 31.27, 23.71, 20.52, 20.21, 13.87, 13.81. HRMS
+
calculated for C33H35N6O [M + H] : 531.2867; found 531.2873.
9-benzyl-N'-((9-n-butyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-1-methyl-9H-pyrido[3,4-b]indole-3-
1
carbohydrazide (8y): The compound was obtained as a yellow solid with 84% yield. H-NMR (400 MHz,
CDCl3) δ 11.27 (s, 1H, CONH), 9.03 (s, 1H, ArH), 8.99 (s, 1H, ArH), 8.84 (s, 1H, ArH), 8.55 (s, 1H, CH),
8.26 (dd, J = 10.4, 8.0 Hz, 2H, ArH), 7.64–7.55 (m, 2H, ArH), 7.47 (d, J = 8.4 Hz, 1H, ArH), 7.41–7.34 (m,
3H, ArH), 7.33–7.23 (m, 3H, ArH), 7.01–6.95 (m, 2H, ArH), 5.83 (s, 2H, ArCH2), 4.37 (t, J = 7.2 Hz, 2H,
−CH2CH2CH2CH3), 2.93 (s, 3H, CH3), 1.94–1.87 (m, 2H, −CH2CH2CH2CH3), 1.47–1.37 (m, 2H,
13
−CH2CH2CH2CH3), 0.96 (t, J = 7.2 Hz, 3H, −CH2CH2CH2CH3). C-NMR (100 MHz, CDCl3) δ 161.19,
149.29, 142.63, 142.26, 141.78, 140.15, 138.33, 137.49, 137.06, 131.05, 129.80, 129.13, 129.07, 128.91,
128.64, 127.72, 125.32, 122.56, 122.05, 121.75, 121.51, 121.03, 120.23, 113.81, 113.00, 110.11, 109.59, 48.30,
+
43.32, 31.27, 23.32, 20.52, 13.82. HRMS calculated for C36H33N6O [M + H] : 565.2710; found 565.2715.
N'-((9-benzyl-1-methyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9-n-butyl-9H-pyrido[3,4-b]indole-3-
1
carbohydrazide (8z): The compound was obtained as a yellow solid with 85% yield. H-NMR (400 MHz,
CDCl3) δ 11.22 (s, 1H, CONH), 9.06 (s, 1H, ArH), 8.92 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.50 (s, 1H, CH),
8.25 (t, J = 7.2 Hz, 2H, ArH), 7.65–7.61 (m, 1H, ArH), 7.56–7.48 (m, 2H, ArH), 7.39–7.32 (m, 3H, ArH),
7.31 – 7.23 (m, 3H, ArH), 7.00 (d, J = 6.4 Hz, 2H, ArH), 5.76 (s, 2H, ArCH2), 4.39 (t, J = 7.2 Hz, 2H,
−CH2CH2CH2CH3), 2.88 (s, 3H, CH3), 1.94–1.87 (m, 2H, −CH2CH2CH2CH3), 1.46–1.36 (m, 2H,
13
−CH2CH2CH2CH3), 0.97 (t, J = 7.2 Hz, 3H, −CH2CH2CH2CH3). C-NMR (100 MHz, CDCl3) δ 161.36,
149.11, 142.47, 142.18, 141.57, 140.82, 138.81, 137.87, 137.70, 136.08, 130.00, 129.88, 129.05, 128.95,
128.81, 128.75, 127.59, 125.40, 122.41, 122.16, 121.76, 121.48, 120.69, 120.57, 115.31, 111.46, 109.91,
+
109.81, 48.26, 43.48, 31.32, 23.00, 20.52, 13.81. HRMS calculated for C36H33N6O [M + H] : 565.2710;
found 565.2703.
N'-((9-benzyl-1-methyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-9-n-butyl-1-methyl-9H-pyrido[3,4-b]indole-
1
3-carbohydrazide (8aa): The compound was obtained as a yellow solid with 83% yield. H-NMR (400

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MHz, CDCl3) δ 11.30 (s, 1H, CONH), 8.90 (d, J = 4.4 Hz, 2H, ArH), 8.51 (s, 1H, CH, ArH), 8.26 (d, J =
7.6 Hz, 1H, ArH), 8.22 (d, J = 7.6 Hz, 1H, ArH), 7.63–7.59 (m, 1H, ArH), 7.56–7.52 (m, 1H, ArH), 7.46
(d, J = 8.4 Hz, 1H, ArH), 7.38–7.29 (m, 4H, ArH), 7.29–7.22 (m, 2H, ArH), 7.01–6.98 (m, 2H, ArH), 5.74
(s, 2H, ArCH2), 4.51 (t, J = 7.6 Hz, 2H, −CH2CH2CH2CH3), 3.06 (s, 3H, CH3), 2.88 (s, 3H, CH3), 1.88–
1.80 (m, 2H, −CH2CH2CH2CH3), 1.51–1.42 (m, 2H, −CH2CH2CH2CH3), 1.00 (t, J = 7.6 Hz, 3H,
13
−CH2CH2CH2CH3). C-NMR (100 MHz, CDCl3) δ 161.26, 142.48, 142.10, 141.79, 140.81, 139.64, 137.73,
137.68, 136.55, 136.02, 129.90, 129.54, 129.06, 128.78, 128.52, 127.60, 125.40, 125.32, 122.17, 121.97,
121.73, 121.68, 120.71, 120.55, 113.67, 111.45, 109.97, 109.91, 48.24, 44.82, 33.01, 23.71, 22.93, 20.21, 13.86.
+
HRMS calculated for C37H35N6O [M + H] : 579.2867; found 579.2861.
9-benzyl-N'-((9-benzyl-1-methyl-9H-pyrido[3,4-b]indol-3-yl)methylene)-1-methyl-9H-pyrido[3,4-b]indole-3-
1
carbohydrazide (8ab): The compound was obtained as a yellow solid with 85% yield. H-NMR (400
MHz, CDCl3) δ 11.25 (s, 1H, CONH), 8.96 (s, 1H, ArH), 8.90 (s, 1H, ArH), 8.48 (s, 1H, ArH), 8.28–8.25
(m, 2H, ArH, CH), 7.58–7.52 (m, 2H, ArH), 7.39–7.32 (m, 4H, ArH), 7.30–7.26 (m, 3H, ArH), 7.25–7.21
(m, 2H, ArH), 7.00–6.95 (m, 5H, ArH), 5.78 (s, 2H, ArCH2), 5.74 (s, 2H, ArCH2), 2.88 (s, 3H, CH3), 2.86
13
(s, 3H, CH3). C-NMR (100 MHz, CDCl3) δ 161.08, 142.44, 142.22, 142.14, 140.82, 140.09, 138.28, 137.70,
137.49, 136.98, 136.05, 129.83, 129.73, 129.11, 129.04, 128.88, 128.72, 127.70, 127.58, 125.39, 125.30,
122.14, 122.01, 121.75, 121.72, 120.99, 120.68, 113.67, 111.34, 110.09, 109.90, 48.24, 23.31. HRMS
+
calculated for C40H33N6O [M + H] : 613.2710; found 613.2719.
3.5. MTT Assay
Target compounds were assayed by the MTT method for determining cytotoxic activity as
described previously [41]. The panel of cell lines included the human umbilical vein cell line
EA.HY926, Lewis lung carcinoma (LLC), gastric carcinoma (BGC-823), murine colon carcinoma (CT-
26), liver carcinoma (Bel-7402), and breast carcinoma (MCF-7). Cell lines were obtained from
Shanghai Cell Institute, Chinese Academy of Science. Growth inhibition rates were calculated with
ꢀꢁ
ꢂꢃꢄꢅꢆ
ꢇꢀꢁ
the following equitation: Inhibition ratio (%) =
ꢈꢉꢊꢋꢌ × 100%. The half-maximal inhibitory
ꢀꢁ ꢇꢀꢁ
ꢍꢎꢏꢐ
ꢈꢉꢊꢋꢌ
concentration (IC50) of each compound was calculated using GraphPad Prism software (version 6.0).
3.6. CAM Assay in Vivo
To determine in vivo anti-angiogenic activity of heterobivalent β-carbolines, a CAM assay was
performed as previously described [39]. In brief, five-day-old fertilized chicken eggs were purchased
from a local hatchery. All the eggs were incubated at 37 °C in an incubator. We injected 0.5 mL of
saline, and the eggs were incubated horizontally to allow the CAM to detach from the shell to make
a bogus chamber. Compound 8z was prepared in gelatin sponge discs at concentrations of 0.5, 5.0,
and 50 µM/disc. CA4P was used as a positive control drug. Discs containing the vehicle only (DMSO)
were used as negative controls. A small window opening was made in the shell, and the discs were
directly applied onto the CAM. The square opening was covered with sterilized surgical tape, and
the embryos were incubated for 48 h at 38.5 °C. The CAMs were photographed under a dissecting
microscope, and blood vessels in each CAM were counted. The results are presented as a mean
percentage of inhibition compared to the control ± SD, n = 3.
4. Conclusions
On the basis of our previous work, this study has focused on the synthesis of a series of
heterobivalent β-carbolines bearing an acylhydrazone bond (8a–ab). All of the target compounds
were investigated for their in vitro antiproliferative activity using the MTT-based assay against five
cancer cell lines (LLC, BGC-823, CT-26, Bel-7402, and MCF-7). Most compounds showed medium
antiproliferative activities against the tested cancer cell lines. In particular, compound 8z showed
antitumor activities with IC50 values of 9.9, 8.6, 6.2, 9.9, and 5.7 µM against the LLC, BGC-823, CT-26,
Bel-7402, and MCF-7 cell lines, respectively. The anti-angiogenic activity of compound 8z was
comparable with CA4P in an in vivo CAM assay at the 5 µM level.

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Supplementary Materials: The following are available online, 1H and 13C-NMR spectra for the target
compounds are available online.
Author Contributions: Conceptualization, J.Z.; methodology, L.G., X.-F.C., Q.M., and W.-X. F.; formal analysis,
L.G. and W.C.; investigation, X.-F.C. and L.G.; writing—original draft preparation, X.-F.C. and L.G.; writing—
review and editing, J.Z. and L.G.; project administration, J.Z.
Funding: We gratefully thank the Scientific Research Innovation Project in Shihezi University (No. SHYL-
YB201804), the Xinjiang Science and Technology Major Project (No. 2016A03005-1), the Program for Changjiang
Scholars and Innovative Research Team in University (No. IRT15R46), and Xinjiang Huashidan Pharmaceutical
Research Co. Ltd.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8a–ab are available from the authors.
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