Molecular hybrid design, synthesis, in vitro and in vivo anticancer evaluation, and mechanism of action of N-acylhydrazone linked, heterobivalent β-carbolines

Article abstract of DOI:10.1016/j.bioorg.2020.103612

A series of N-acylhydrazone-linked, heterobivalent β-carboline derivatives was designed and synthesized from L-tryptophan in a nine-step reaction sequence. The effort resulted in the heterobivalent β-carbolines 10a–t in good yields. The target compounds were characterized by ¹H NMR, ¹³C NMR and high-resolution mass spectrometry (HRMS). The in vitro cytotoxic activity of the synthesized compounds was evaluated against normal EA.HY926 cells and five cancer cell lines: LLC (Lewis lung carcinoma), BGC-823 (gastric carcinoma), CT-26 (murine colon carcinoma), Bel-7402 (liver carcinoma), and MCF-7 (breast carcinoma). Compound 10e, with an IC₅₀ value of 2.41 μM against EA.HY926 cells, was the most potent inhibitor. It showed cytotoxicity against all five cancer cell lines of different origin – murine and human, with IC₅₀ values ranging from 4.2 ± 0.7 to 18.5 ± 3.1 μM. A study of structure-activity relationships indicated that the influence on cytotoxic activities of the substituent in the R₉′-position followed the tendency, 2,3,4,5,6-perfluorophenylmethyl > 4-fluorobenzyl > 3-phenylpropyl group. The antitumor efficacies of the selected compounds were also evaluated in mice. Compound 10e exhibited potent antitumor activity, with tumor inhibition of more than 40% for Sarcoma 180 and 36.7% for Lewis lung cancer. Furthermore, the pharmacological mechanisms showed that compound 10e has a certain impairment in the motility of LLC cells, which suggests the anti-metastatic potential. And compound 10e inhibited angiogenesis in chicken chorioallantoic membrane assay, and the anti-angiogenetic potency was more potent than the reference drug combretastatin A4-phosphate (CA4P) at a concentration 50 μM.

Full text of DOI:10.1016/j.bioorg.2020.103612

BioorganicChemistry96(2020)103612
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Molecular hybrid design, synthesis, in vitro and in vivo anticancer evaluation,
and mechanism of action of N-acylhydrazone linked, heterobivalent β-
carbolines
⁎
⁎
Liang Guoa, Xiaofei Chena, Wei Chenb, Qin Mab, Wenxi Fanb, Jie Zhanga, , Bin Daia,
a School of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi 832003,
PR China
b Xinjiang Huashidan Pharmaceutical Research Co. Ltd., 175 He Nan East Road, Urumqi 830011, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of N-acylhydrazone-linked, heterobivalent β-carboline derivatives was designed and synthesized from L-
tryptophan in a nine-step reaction sequence. The effort resulted in the heterobivalent β-carbolines 10a–t in good
yields. The target compounds were characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry
(HRMS). The in vitro cytotoxic activity of the synthesized compounds was evaluated against normal EA.HY926
cells and five cancer cell lines: LLC (Lewis lung carcinoma), BGC-823 (gastric carcinoma), CT-26 (murine colon
carcinoma), Bel-7402 (liver carcinoma), and MCF-7 (breast carcinoma). Compound 10e, with an IC50 value of
2.41 μM against EA.HY926 cells, was the most potent inhibitor. It showed cytotoxicity against all five cancer cell
Heterobivalent β-carboline
Acylhydrazone group
Cytotoxic activity
Structure-activity relationships
lines of different origin – murine and human, with IC50 values ranging from 4.2
0.7 to 18.5
3.1 μM. A
study of structure-activity relationships indicated that the influence on cytotoxic activities of the substituent in
the R9′-position followed the tendency, 2,3,4,5,6-perfluorophenylmethyl > 4-fluorobenzyl > 3-phenylpropyl
group. The antitumor efficacies of the selected compounds were also evaluated in mice. Compound 10e ex-
hibited potent antitumor activity, with tumor inhibition of more than 40% for Sarcoma 180 and 36.7% for Lewis
lung cancer. Furthermore, the pharmacological mechanisms showed that compound 10e has a certain impair-
ment in the motility of LLC cells, which suggests the anti-metastatic potential. And compound 10e inhibited
angiogenesis in chicken chorioallantoic membrane assay, and the anti-angiogenetic potency was more potent
than the reference drug combretastatin A4-phosphate (CA4P) at a concentration 50 μM.
1. Introduction
antitumor agents, and a large number of β-carboline derivatives have
been prepared in search of agents that are more potent [7–11]. The
Cancer is a serious clinical problem affecting millions of patients
[1]. The occurrence of cancer is increasing due to the increase and
aging of the world population. The World Health Organization has es-
timated that, by 2030, there will be 15 million deaths due to cancer
worldwide [2]. Although extensive efforts have been made to deal with
this situation, the methods for management of cancer remain limited. In
pharmaceutical research, the search for new, effective, and safe che-
motherapeutic agents for treatment of this disease remains a challenge.
β-Carboline alkaloids are a class of natural and synthetic products
that have a broad spectrum of biochemical effects and pharmacological
properties, including nervous system [3], antimicrobial [4], and anti-
neoplasmic treatments [5]. They are also effective in the treatment of
dermatoses [6]. In recent decades, there have been intense research
efforts in the design and development of β-carbolines as a new class of
results indicate that this class of compounds exert antitumor effects
through various mechanisms of action, including intercalating with
DNA [12,13], and by inhibition of cyclin-dependent kinase (CDK) [14],
topoisomerases I and II [7,12,15], polo-like kinase 1 (PLK1) [16,17],
and IκB kinase [18].
The bioactive acylhydrazone (eCONHN]) scaffold has been widely
applied in medicinal chemistry, and it is often used in the design of
drugs with heterocyclic moieties. Relative to amide groups, it endows
molecules with better thermal stability as well as with hydrolytic and
chemical stability [19,20]. The acylhydrazone group is present in nu-
merous compounds that act on various types of molecular targets
[21–23]. Among the pharmacological effects of acylhydrazone-derived
compounds are anti-inflammatory and anticancer activities [24,25]. For
example, aldoxorubicin (Fig. 1) is currently in a phase III trial for the
⁎ Corresponding authors.
E-mail addresses: zhangjie-xj@163.com (J. Zhang), db_tea@shzu.edu.cn (B. Dai).
https://doi.org/10.1016/j.bioorg.2020.103612
Received 6 June 2019; Received in revised form 25 December 2019; Accepted 20 January 2020
Availableonline23January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

L. Guo, et al.
BioorganicChemistry96(2020)103612
NH2
OH
O
O
O
OH
OH
O
O
CH3
O
O
O
NO2
O
N
N
H
N
N
N
H
HO
HO
HO
O
Aldoxorubicin
Nifuroxazide
Fig. 1. Structures of N-acylhydrazone derivatives Aldoxorubicin and Nifuroxazide.
treatment of metastatic, locally advanced, or unresectable soft tissue
sarcoma [26]. This (maleinimidoalkanoyl)hydrazone derivative of
doxorubicin covalently binds to serum albumin through its maleimide
moiety. Nifuroxazide (Fig. 1), another N-acylhydrazone derivative, is a
nitrofuran antibiotic commonly used as an intestinal anti-infective
agent [27].
activities against cultured cells. In the present study, we reported
synthesis, in vitro evaluation, in vivo efficacies and preliminary struc-
ture-activity relationships for the new N-acylhydrazone-linked, het-
erobivalent β-carboline derivatives.
2. Results and discussion
Our research group [28–31] has focused on incorporating sub-
stituents into positions 1, 2, 3, 7, and 9 of the β-carboline nucleus as
antitumor agents. Structure-activity relationship (SAR) analysis in-
dicated that (1) the β-carboline moiety was associated with their po-
tential antitumor activities; and (2) the introduction of appropriate
substituents into positions 1, 3, and 9 of the β-carboline nucleus en-
hanced their antitumor potencies. Recently, we reported the SAR for
the homobivalent β-carbolines and heterobivalent β-carbolines with an
alkyl or alkylamino spacer in positions 1, 3, 7, and 9 of the β-carboline
nucleus [32–37] (Fig. 2). Of these synthesized bivalent compounds, 1-
2.1. Chemistry
The target heterobivalent β-carboline derivatives 10a–t were syn-
thesized according to the procedures depicted in Schemes 1 and 2. The
1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2a) and 1-methyl-
1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2b) were prepared by
the condensation of L-tryptophan with appropriate aldehydes via the
Pictet-Spengler condensation. Esterification of the intermediates 2a
with ethanol in the presence of thionylchloride (SOCl2) allowed pre-
paration of ethyl 1,2,3,4-tetrahydro-β-carboline-3-carboxylates 3a–b;
followed by dehydrogenation with sulfur in refluxing xylene, the
monovalent β-carbolines 4a–b were obtained. The N9 of 4a–b was al-
kylated by the action of sodium hydride (NaH) in anhydrous N,N-di-
methylformamide (DMF), followed by the addition of 1-iodobutane or
benzyl bromide to obtain compounds 5a–d. The key intermediates 6a–d
were synthesized by the reaction of compounds 5a–d with hydrazine
monohydrate.
Methyl-9-[4-(1-methyl-β-carboline-9-yl)butyl]-β-carboline
(B-9-3)
[36,38], which was a symmetric dimeric β-carboline compound that
contains two molecules of harman bound to each other by a tetra-
methylene group, exhibited potent antitumor activity. Compounds B-1
[32], B-2 [37], and B-3 [33] exhibited significant angiogenesis in-
hibitory effects in CAM assay, and the anti-angiogenetic potency was
comparable or more potent with the drug Endostar. The pharmacolo-
gical mechanisms showed that B-9-3 selectively induces apoptosis of
endothelial cells, in part through disruption of VEGF-A/VEGFR2 sig-
naling [39], and also acts on the TGF-β signaling pathway [40].
Since molecular hybridization has been an effective approach for
the development of new drugs, we utilized the tactic of combining the
acylhydrazone and β-carboline moieties to develop a library of het-
erobivalent β-carboline derivatives, and to test their antitumor
Subsequently, we focused on synthesis of intermediates 9a–e. 1-
Methyl-β-carboline 7 was obtained by the oxidation and decarboxyla-
tion of 2b in a single step through the action of active manganese di-
oxide (MnO2). From compound 7, compounds 8a–e were afforded ac-
cording to a synthetic procedure similar to that used for compounds
5a–d. The methyl group in position 1 of compounds 8a–e was oxidized
N
CH
NHCH2
2NH(CH2)5
CH3
N
CH3
N
N
N
N
(CH2)4
N
R2
N
R2
R1
N
R1
N
(CH2)4
N
B-1 R1 C H4 -OCH
R2 CH3
=
=
p
H
(
3),
3C
6
N
N
N
B-2 R1
R2 CH2C6H4
=
=
m
H,
(
-Cl)
B-5
B-9-3
N
OCH3
N
N
N
O
O
6
N
N
N
O
(CH2)4
N
N
N
CH3
H
3C
CH
NHCH2
2NH(CH2)5
N
CH3
(CH2)3
B-3
B-4
B-6
Heterobivalent -carbolines
Homobivalent -carbolines
Fig. 2. The chemical structure of the representative reported bivalent β-carbolines.
2

L. Guo, et al.
BioorganicChemistry96(2020)103612
COOH
NH
H5
COOC2
NH
R1
H5
COOC2
N
iii
iv
N
H
N
H
N
H
R1
i
2a
4a-b
3a-b
=
=
H
4a R
3a,
3b,
1
v
CH3
4b R
1
H5
COOC2
COOH
COOH
NH2
ii
vii
NH
CH3
N
N
N
H
N
N
N
H
R1
H
CH3
R9
2b
L-tryptophan
7
v
5a-d
vi
CONHNH2
viii
N
CHO
N
N
N
N
CH3
N
R9'
R1
R9'
8a-e
R9
9a-e
6a-d
=
=
=
=
=
n
9a R '
H9
-C4
8a,
8b,
8c,
8d,
8e,
9
=
=
=
n
9b R '
H5
H
p
4( -F)
-CH2C6
-CH2C6
-CH2CH2CH2C6
2,3,4,5,6-
6a R
6b R
6c R
6d R
R
H9
-C4
5a,
5b,
5c,
5d,
-H,
-H,
9
1
9
=
=
=
9c R '
R
H5
H9
-CH2C6
=
=
9
1
9
,
n
9d R '
H5
R9
R9
-CH3
-C4
9
1
,
9e R '
H5
-CH2C6
-CH3
perfluorophenyl
9
1
Scheme 1. Synthesis of the key intermediate 6a-d, 9a-e. Reagents and conditions: (i) NaOH, H2O, formaldehyde, reflux, 3 h; (ii) H2SO4, H2O, acetaldehyde, room
temperature, 3 h; (iii) ethanol, SOCl2, reflux, 4 h; (iv) xylene, S8, reflux, 8 h; (v) DMF, NaH, alkyl halogenide, stirred at RT; (vi) hydrazine hydrate, ethanol, reflux,
4 h. (vii) H2SO4, MnO2; (viii) SeO2, dioxane, reflux, 2 h.
by selenium dioxide (SeO2) in anhydrous dioxane to provide the β-
carboline-1-carboxaldehydes 9a–e.
2.2. Biological evaluation
Finally, the title compounds 10a–t were prepared in good yields
from β-carboline-3-carbohydrazides 6a–d with β-carboline-1-carbox-
aldehydes 9a–e; the reactions were accomplished in ethanol under re-
flux conditions. The chemical structures of the synthesized compounds
were characterized by 1H NMR, 13C NMR, and high-resolution mass
spectrometry (HRMS).
2.2.1. Inhibitory effect on EA.HY926s proliferation
The 20 synthesized heterobivalent β-carbolines were evaluated
against EA.HY926 cells for anti-proliferative effects by 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), with
CA4P and Endostar (rh-endostatin) as reference drugs.
As shown in Table 1, half of heterobivalent β-carbolines exhibited
good anti-proliferative effects, with the half maximal inhibitory con-
centration (IC50) values of less than 10.0 μM against EA.HY926 cells.
Compounds 10c, 10e, 10n, and 10o displayed anti-proliferative
CONHNH2
N
O
C
CH3OH
reflux
H
N
R9'
N
N
HC N N
+
N
N
CHO
R1
N
R9'
9a-e
R9
N
R9
6a-d
R1
10a-t
=
=
=
R1
CH3
n
=
=
=
R1
n
10k R9' R9
10a R9' R9
H
=
-butyl,
=
-butyl,
,
=
=
n
=
=
n
10l R9'
R
R9
CH3
10b R9'
R
R
9
benzyl,
=
-butyl
H,
1
benzyl,
-butyl
1
,
=
=
=
=
n
n
=
=
=
=
=
R
9
n
10m R9'
10n R9'
10o R9'
R
R
R9
R9
R
CH3
10c R9'
10d R9'
10e R9'
10f R9'
R
4-fluorobenzyl,
3-phenylpropyl,
2,3,4,5,6-
-butyl
H,
1
1
4-fluorobenzyl,
3-phenylpropyl,
2,3,4,5,6-
-butyl
1
,
=
=
=
=
n
CH3
R
R
9
-butyl
,
H,
-butyl
1
=
=
=
=
=
n
=
=
=
n
R9
CH3
R
R
perfluorophenyl,
-butyl
H,
1
perfluorophenyl,
-butyl
1
9
9
,
=
=
n
=
=
=
R
9
=
R
9
n
R9'
R1
R9
CH3
R1
10p
-butyl,
benzyl
H,
H,
-butyl,
benzyl
,
=
=
=
=
=
R9'
R1
R9
=
CH3
R1
R9'
R1
10q
benzyl,
benzyl
10g
benzyl,
benzyl
H,
H,
,
=
=
R9
=
=
10r R9'
10s R9'
10t R9'
CH3
10h R9'
R1
R1
R
4-fluorobenzyl,
3-phenylpropyl,
benzyl
=
4-fluorobenzyl,
benzyl
9
,
=
=
=
=
=
R1
R9
CH3
10i R9'
R
benzyl
3-phenylpropyl,
2,3,4,5,6-
benzyl
=
9
,
=
2,3,4,5,6-
perfluorophenyl,
=
CH3
=
R1
R9
R9'
R1
R
benzyl
H,
10j
benzyl
perfluorophenyl,
Scheme 2. Synthetic route for the heterobivalent β-carbolines (10a-t) preparation.
3

L. Guo, et al.
BioorganicChemistry96(2020)103612
Table 1
compound 10g (except for the BGC823 cell line), and the activity of
compound 10e was superior to that of compound 10j (except for the
MCF-7 cell line). Compounds 10d, 10k, 10l, and 10o, with n-butyl
groups in the R9-position of the β-carboline nucleus, showed the same
tendency, suggesting that the R9 substituent affected the cytotoxic ac-
tivities and that a n-butyl substitution was more favorable than a benzyl
group.
Inhibitory effect of novel heterobivalent β-carbolines on EA.HY926 cell pro-
liferation.
Compd.
IC50 (μM)
SD
Compd.
IC50 (μM)
SD
CA4P
Endostar
10a
7.6
1.3
0.5
3.3
2.7
10j
6.2
0.8
1.6
10k
10l
16.2
24.3
11.2
4.5
2.9
4.6
2.3
14.6
16.8
4.7
The effect of various substituents on the R9′-position of the β-car-
boline core was examined, and SAR studies revealed that the in-
troduction of aromatic substituents was necessary for potent cytotoxi-
city. Compounds 10a–e contained n-butyl in R9-position of the β-
carboline core, and, in addition, n-butyl (10a), benzyl (10b), 4-fluor-
10b
10m
10n
10o
10p
10q
10r
10s
10t
10c
0.6
0.7
10d
9.3
2.3
0.8
3.2
0.4
1.6
3.2
10e
2.4
10.5
17.2
5.7
10f
16.2
10.6
8.3
3.2
10g
2.6
2.8
3.17
0.7
obenzyl
(10c),
3-phenylpropyl
(10d),
or
2,3,4,5,6-per-
10h
7.9
0.9
1.2
10i
11.7
5.4
fluorophenylmethyl (10e) in the R9′-position of another β-carboline
ring. Of these five heterobivalent β-carbolines, compound 10e dis-
played higher cytotoxic activities against LLC, BGC-823, CT-26, and
potencies, with IC50 values of 4.7
3.2
0.6, 2.4
0.8, 4.5
0.7, and
Bel-7402 cells, with IC50 values of 7.6
0.8, 7.7
1.2, 8.4
1.1,
0.4 against these cells, respectively. Compounds 10a–b, 10d,
and 4.2
0.7 μM, respectively. Additionally, compounds 10f–j,
10f–k, 10m, and 10p–t showed only moderate anti-proliferative po-
bearing benzyl in the R9-position of the β-carboline core, and additional
n-butyl, benzyl, 4-fluorobenzyl, 3-phenylpropyl, or 2,3,4,5,6-per-
fluorophenylmethyl groups in the R9′-position of another β-carboline
tencies, with IC50 values ranging from 5.4
1.2 to 17.2
3.2 μM.
4.6 μM,
Unfortunately, compound 10l, with an IC50 value of 24.3
had marginal anti-proliferative effects. However, compound 10e, with
ring, and compound 10j, showed IC50 values ranging from 9.2
0.4 to
an IC50 value of 2.4
potent inhibitory agent; its potency was greater than that for the po-
sitive control drug, CA4P (7.6 1.3 μM), but less than that of another
positive drug control, Endostar (1.6 0.5 μM).
0.8 μM against EA.HY926 cells, was the most
15.3
0.9 μM.
Compared to cisplatin, some of these compounds displayed greater
cytotoxicity against these tumor cell lines. In the cytotoxicity assay with
the LLC cell line, compounds 10b–e, 10h–j, 10m–p, and 10r–t ex-
Of heterobivalent β-carboline derivatives 10a-j, all bearing a n-
butyl or benzyl substituent in R9-position of β-carboline nucleus, while
in another β-carboline core, the substituted group of the R9′-position
were n-butyl (10a, 10f), benzyl (10b, 10g), 4-fluorobenzyl (10c, 10h),
3-phenylpropyl (10d, 10i), and 2,3,4,5,6-perfluorophenylmethyl (10e,
10j), respectively. The influence of substituent in R9′-position on anti-
proliferative effects followed the tendency of 2,3,4,5,6-per-
hibited greater cytotoxicity than cisplatin (IC50 = 21.3
1.1 μM),
with IC50 values in the range of 6.7
0.4 to 18.6
2.7 μM. In the
cytotoxicity assay with Bel-7402 cells, compounds 10c, 10e, 10h–j,
10m, 10o, and 10q–t showed higher cytotoxicity than cisplatin
(IC50 = 15.4
13.3
the BGC823 cell line was 7.7
1.9 μM), with IC50 values in the range of 4.2
1.9 to
1.2 μM. In contrast, the IC50 value for compound 10e against
1.2 μM, as compared to that of cis-
fluorophenylmethyl
> 4-fluorobenzyl > 3-phenylpropyl group.
platin (IC50 = 8.4
0.7 μM), but most compounds exhibited activities
Similarly, the anti-proliferative potencies of compounds 10p-t with a
same substituent group followed the same tendency.
against this cell line, with IC50 values lower than 20 μM (except for 10a,
10l, and 10q). Similarly, the IC50 value of compound 10j against the
We then found the following anti-proliferative effects trend based
on the R9′-position of the β-carboline nucleus: 2,3,4,5,6-per-
MCF-7 cell line was 9.5
1.1 μM, as compared to that of cisplatin,
IC50 = 10.5
2.3 μM. Compounds 10e and 10j both bear 2,3,4,5,6-
fluorophenylmethyl
> 4-fluorobenzyl > 3-phenylpropyl group.
perfluorophenylmethyl in the R9′-position of the β-carboline ring.
There is no obvious difference between the R1-position substituent
group, which indicates that this position is not tolerated for modifica-
tion.
2.3. Acute toxicity studies with healthy mice and in vivo antitumor activity
Since compounds 10e, 10j, 10o, and 10t showed the most cytotoxic
activity, the antitumor activities of these compounds were evaluated
against mice bearing Sarcoma 180 or Lewis lung cancer, and the results
were compared with that for cyclophosphamide (CTX).
2.2.2. In vitro cytotoxic screening
The cytotoxic potencies of the heterobivalent β-carbolines against a
panel of tumor cell lines were determined and compared with that of a
reference drug, cisplatin. The tumor cell panel included LLC (Lewis lung
carcinoma), BGC-823 (gastric carcinoma), CT-26 (murine colon carci-
noma), Bel-7402 (liver carcinoma) and MCF-7 (breast carcinoma) of
different origin – murine and human. The results are summarized in
Table 2.
The LD50 (median lethal dose) values of the selected compounds
after intraperitoneal (i.p.) administration to mice are listed in Table 3.
Of these four investigated heterobivalent β-carboline derivatives, all
had a 2,3,4,5,6-perfluorophenylmethyl group in the R9-position of the
β-carboline ring, and they displayed acute toxicity with LD50 values in
The cytotoxic activity results revealed that most of the newly de-
signed compounds were active against the tested cell lines. Compounds
10b–e, 10h–j, 10m, 10o, and 10r–t displayed a broad spectrum of
cytotoxic activities, with IC50 values of less than 20 μM against four or
five tumor cell lines; compounds 10a, 10f–g, 10l, and 10q showed
cytotoxicities with IC50 values of less than 20 μM against one or two
tumor cell lines.
the range of 50–85 mg kg−1
.
Our previous investigation [34] demonstrated that, in mice, Lewis
lung cancer cells were more susceptible to β-carbolines than other an-
imal models; therefore, this animal model was selected and evaluated.
The tumor inhibition rates of all of the compounds are presented in
Table 3. The highest tumor inhibition was 46.8% for the group with
Sarcoma 180 and treated with compound 10e; the tumor inhibitions of
compounds 10j, 10o, and 10t were 37.9%, 33.5%, and 39.2%, re-
spectively. The tested compounds showed moderate antitumor activ-
ities for Lewis lung cancers with tumor inhibitions ranging from 21.2 to
36.7% at doses ranging from 10 to 17 mg/kg.
Introduction of methyl groups on the R1-position of the β-carboline
ring of compounds 10a–j yielded the compounds 10k–t. A comparison
of the cytotoxicities of compounds 10a–j and 10k–t revealed that
omission of the methyl group in the R1-position did not have a strong
influence on cytotoxicity (10a vs. 10k, 10e vs. 10o, 10h vs. 10r, and
10i vs. 10s). Introduction of a n-butyl substituent into the R9-position of
the β-carboline nucleus enhanced antitumor activities, as seen for
compound 10b, for which the antitumor activity was better than that of
2.4. Inhibitory effect of 10e on tumor cell migration
In the present study, to explore whether compound 10e could
4

L. Guo, et al.
BioorganicChemistry96(2020)103612
Table 2
Cytotoxic activities of derivatives in vitro (IC50, µM).
a
Compd.
R9′
R1
R9
IC50 (μM)
LLCb
SD
BGC823
CT-26
Bel-7402
MCF-7
10a
10b
n-butyl
H
H
n-butyl
n-butyl
39.3
17.7
4.2
32.7
18.8
3.7
2.7
15.1
13.3
2.5
1.3
44.2
30.1
3.3
4.6
24.5
19.1
4.6
3.2
3.1
2.4
2.2
10c
10d
10e
H
H
H
n-butyl
n-butyl
n-butyl
14.0
16.3
7.6
9.3
9.5
7.7
0.8
0.6
1.2
19.6
17.1
8.4
2.1
2.5
12.9
32.3
1.4
2.7
32.9
16.5
18.5
1.9
3.1
3.1
0.8
1.1
4.2
0.7
10f
10g
10h
10i
10j
n-butyl
H
H
H
H
H
28.1
29.6
6.7
2.9
3.8
19.6
11.7
12.3
11.7
15.3
2.7
19.8
19.2
21.7
19.6
10.5
1.3
3.4
3.3
5.2
1.2
28.2
33.4
9.9
4.1
6.4
96.1
34.3
12.8
20.6
9.5
13.2
3.9
2.2
0.7
3.2
0.9
0.4
1.7
2.5
8.7
0.9
0.4
14.1
11.6
4.3
2.3
2.7
9.2
1.1
10k
10l
n-butyl
n-butyl
CH3
CH3
n-butyl
n-butyl
33.8
56.7
5.2
8.2
18.6
41.4
3.6
5.1
24.5
14.9
2.3
2.1
18.8
3.1
16.5
40.3
3.4
3.3
> 100
10m
10n
10o
CH3
CH3
CH3
n-butyl
n-butyl
n-butyl
8.4
1.2
1.7
0.7
10.1
14.1
11.2
1.7
0.8
0.4
7.6
0.8
5.1
1.1
13.3
62.6
4.7
1.2
17.2
17.9
12.5
3.4
3.8
2.3
12.2
7.8
61.7
9.8
10.9
0.7
10p
10q
10r
10s
10t
CH3
CH3
CH3
CH3
CH3
18.6
> 100
6.9
2.7
19.5
24.4
14.6
9.3
4.2
4.1
0.7
> 100
18.6
74.6
8.3
9.7
0.7
1.7
2.2
0.6
19.3
> 100
17.2
32.0
16.2
4.4
2.5
1.2
0.7
2.3
0.9
1.6
0.5
13.6
9.5
2.4
4.7
1.4
11.7
7.4
0.6
12.2
11.5
7.2
8.7
1.4
0.7
13.5
Cisplatin
21.3
1.1
8.4
4.2
0.7
15.4
1.9
10.5
2.3
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of treated cells 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.
b
Cell lines include Lewis lung carcinoma (LLC), gastric carcinoma (BGC), murine colon carcinoma (CT-26), liver carcinoma (Bel-7402), and breast carcinoma
(MCF-7).
inhibit HT-29 and LLC cells migration, we performed cell migration
angiogenic activity is the highly vascularized chorioallantoic mem-
brane (CAM) of chicken embryos. The aim was to assess the inhibitory
effect of compound 10e on neovascularization. In this experiment,
CA4P was used as a positive control (Fig. 4A). At a concentration
0.5 μM, the reference anti-angiogenic drug, CA4P, elicited 21% in-
hibition of angiogenesis, but, at this concentration, compound 10e
showed less anti-angiogenic activity (17% inhibition). The anti-angio-
genic activity of compound 10e, however, was comparable with that of
CA4P at 5 μM. In this assay, 10e inhibited blood vessel formation by
51%, compared to 47% inhibition induced by CA4P. At 50 μM, com-
pound 10e inhibited chicken CAM angiogenesis by 89%, whereas CA4P
caused 78% inhibition. Thus, the results showed dose-dependent anti-
angiogenic activity for compound 10e (Fig. 4B).
assays. Cell migration plays an important role in tumor formation and
cancer metastasis. It is relevant for angiogenesis to ensure tumor nu-
trition as well as for the formation of metastases, in which tumor cells
leave the primary tumor site and spread to other tissues. The two cancer
cell lines were treated with different concentrations of positive control
CA4P and 10e after 24 h were shown in Fig. 3. Compound 10e inhibited
cell migration of the two cancer cell lines dose-dependently.
2.5. Inhibition of angiogenesis in the chicken chorioallantoic membrane
assay
A model system that has been used extensively to evaluate anti-
5

L. Guo, et al.
BioorganicChemistry96(2020)103612
Table 3
antitumor activity, with tumor inhibition of more than 40% for
Sarcoma 180. Furthermore, the pharmacological mechanisms showed
that compound 10e has a certain impairment in the motility of LLC
cells, which suggests the anti-metastatic potential, and it showed ob-
vious angiogenesis inhibitory effects in CAM assay, and the anti-an-
giogenetic potency was more potent than the reference drug CA4P at a
concentration 50 μM. Compared with the prototype B-9-3, the target
compounds didn’t exhibit better antitumor activity. So the further
pharmacological mechanisms and pharmacological target of this class
of compounds are not underway in our laboratory. Preliminary SARs
analysis indicated that: the influence on cytotoxic activities of the
substituent in the R9′-position followed the tendency, 2,3,4,5,6-per-
fluorophenylmethyl > 4-fluorobenzyl > 3-phenylpropyl group.
Acute toxic effects of heterobivalent β-carbolines in mice and antitumor ac-
tivities of these compounds against mice bearing Sarcoma 180 and Lewis lung
cancer.
Compd. Mice number
Acute
Dosage
Tumor inhibition rate (%)a
toxicity
LD50
(mg/kg)
Begin
End
Sarcoma 180 Lewis lung
cancer
(mg/kg)
10e
9
9
9
9
9
9
8
9
50
85
75
55
200
–
10
17
15
11
40
30
46.8
37.9
33.5
39.2
56.2
84.2
4.7
3.9
6.7
5.5
36.7
25.6
31.4
21.2
40.4
81.4
5.4
4.6
3.9
4.3
10j
10o
10t
B-9-3b
CTX
9
8
3.7
2.3
a
b
Data are expressed as mean
See Ref. [36].
standard deviation.
4. Experimental section
4.1. General information
3. Conclusion
Column chromatography was performed with silica gel from
Huanghai Chemical Reagent (200–300 mesh) and analytical TLC on
silica gel 60-F254. Melting points was measured on a WRS-1B melting
point apparatus and were uncorrected. 1H NMR and 13C NMR spectra
were recorded on a Bruker Avance III HD 400 spectrometer at ambient
temperature. Chemical shifts (δ) are reported in ppm relatively to the
residual solvent peak. All chemical shifts for 1H and 13C NMR spec-
troscopy were assigned to residual signals from CDCl3 (1H) at 7.26 ppm
and (13C) at 77.16 ppm, or DMSO‑d6 (1H) at 2.50 ppm and (13C) at
39.52 ppm. The multiplicity of each signal is designated by the
In conclusion, a series of new N-acylhydrazone-linked, hetero-
bivalent β-carboline derivatives were synthesized, and their in vitro
anti-proliferative activities were investigated. SAR studies showed that
compound 10e, containing a 2,3,4,5,6-perfluorophenylmethyl group in
the R9′-position of the β-carboline ring had anti-proliferative activity
against EA.HY926 cells, with an IC50 value of 2.4
0.8 μM. In ad-
dition, this compound exhibited the most potent cytotoxic activity
against the tested cancer cell lines, with IC50 values ranging from
4.2
0.7 to 18.5
3.1 μM. In mice, compound 10e exhibited potent
0 h
24 h
A
B
Control
160
140
120
100
80
**
**
**
**
**
CA4P
60
40
20
0
10e
0.5
5
50
CA4P
10e
0 h
24 h
D
C
Control
CA4P
80
70
60
50
40
30
20
10
0
*
*
*
10e
0.5
5
50
CA4P
10e
Fig. 3. Wound healing migration assay of HT-29 (A and B) and LLC (C and D) cells after 24 h of treatment with 10e.
6

L. Guo, et al.
BioorganicChemistry96(2020)103612
Fig. 4. Inhibitory effects of compound 10e on the angiogenesis of CAM.
following abbreviations: s = singlet, d = doublet, t = triplet,
q = quartet; m = multiplet. Coupling constants (J) are quoted in Hz.
High resolution mass spectra (HRMS) were recorded on Bruker
ultrafleXtreme MALDI-TOF/TOF-MS and Thermo Scientific LTQ
Orbitrap XL mass spectrometer.
4.2.3. 9-benzyl-1-methyl-β-carboline-3-carbohydrazide (6d)
A white solid, yield: 93.8%, 1H NMR (400 MHz, DMSO‑d6) δ 9.57 (s,
1H, NH), 8.73 (s, 1H, ArH), 8.46 (d, J = 8.0 Hz, 1H, ArH), 7.72 (d,
J = 8.4 Hz, 1H, ArH), 7.62–7.58 (m, 1H, ArH), 7.44–7.16 (m, 4H, ArH),
6.93 (d, J = 7.2 Hz, 2H, ArH), 5.98 (s, 2H, ArCH2), 4.54 (s, 2H NH2),
2.85 (s, 3H, CH3). 13C NMR (100 MHz, DMSO‑d6) δ 164.17, 142.41,
140.86, 139.46, 139.13, 136.45, 129.36, 129.25, 127.72, 125.72,
122.54, 121.40, 121.01, 112.51, 111.16, 47.84, 23.24.
All reagents and solvents were purchased from commercial sup-
pliers (Adamas-beta, and J&K) and used without further purification.
The following intermediates, 9-benzyl-β-carboline-3-carbohydrazide
(6b) [41], 9-n-butyl-1-methyl-β-carboline (8a) [41], 9-benzyl-1-me-
thyl-β-carboline (8b) [41], 9-(4-fluorobenzyl)-1-methyl-β-carboline
(8c) [42], 9-(3-phenylpropyl)-1-methyl-β-carboline (8d) [41], 9-
(2,3,4,5,6-perfluorophenylmethyl)-1-methyl-β-carboline (8e) [41], 9-n-
butyl-β-carboline-1-carboxaldehyde (9a) [42], 9-benzyl-β-carboline-1-
carboxaldehyde (9b) [42], 9-(4-fluorobenzyl)-β-carboline-1-carbox-
aldehyde (9c) [42], 9-(3-phenylpropyl)-β-carboline-1-carboxaldehyde
(9d) [42] are known compounds.
4.3. General procedure for the preparation of compounds 9a–e
To a stirring solution of 8a–e (5 mmol) in dioxane (100 mL), SeO2
(10 mmol) was added at room temperature. The reaction medium was
kept under stirring and reflux and was monitored by TLC for develop-
ment of the reactions. After consumption of the starting material, the
mixture was cooled and filtered through Celite. The filtrate was eva-
porated in a vacuum, and the residue was dissolved in EtOAc. The or-
ganic layer was washed with water, dried over Na2SO4, and evaporated
to dryness. The residue was purified by column chromatography
(hexane/EtOAc) to afford the compounds 9a–e.
4.2. General procedure for the preparation of compounds 6a–d
To a solution of compound 5a–d (10 mmol) in ethanol (100 mL) was
added 85% hydrazine hydrate (10 mL), and then the mixture was re-
fluxed for 8 h. After completion of the reaction as indicated by TLC, the
resulting mixture was cooled to 5 °C, and the precipitate was collected
by filtration. The crude product was further purified by washing with
ethanol, and by recrystallization in ethanol to obtain compound 6a–d
with yield 80–85%.
4.3.1. 9-(2,3,4,5,6-perfluorophenylmethyl)-β-carboline-1-carboxaldehyde
(9e)
The compound was obtained as a white solid in 89% yield. 1H NMR
(400 MHz, CDCl3) δ 10.33 (s, 1H, CHO), 8.71 (d, J = 4.8 Hz, 1H, ArH),
8.20–8.18 (m, 1H, ArH), 8.15 (d, J = 8.0 Hz, 1H, ArH), 7.62 (t,
J = 8.0 Hz, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.38 (t,
J = 7.6 Hz, 1H, ArH), 6.54 (s, 2H, ArCH2). 13C NMR (100 MHz, CDCl3)
δ 195.4, 146.5 (m), 144.0 (m), 142.1 (m), 141.4, 139.2, 138.8 (m),
138.2, 136.3 (m), 136.2, 132. 6, 129. 8, 121.6, 121.6, 121.4 118.8,
110.8 (m), 109. 9 (t, J = 2.1 Hz), 39.6.
4.2.1. 9-n-butyl-β-carboline-3-carbohydrazide (6a)
A yellow solid, yield: 94.5%, 1H NMR (400 MHz, DMSO‑d6) δ 9.70
(s, 1H, CONH), 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, eCH2CH2CH2CH3), 1.85–1.76 (m, 2H, eCH2CH2CH2CH3),
1.35–1.24 (m, 2H, eCH2CH2CH2CH3), 0.88 (t, J = 7.2 Hz, 3H,
eCH2CH2CH2CH3). 13C 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.
4.4. General procedure for the preparation of heterobivalent β-carbolines
10a-t
To a solution of β-carboline-3-carbohydrazide 6a–d (1 mmol) in
ethanol (30 mL) was added the corresponding β-carboline-1-carbalde-
hyde 9a–e (1 mmol), then reaction mixture was refluxed for 5 h. After
completion of the reaction as indicated by TLC, the solution was al-
lowed to cool to room temperature. Then the precipitates formed and
filtered, the crude product was recrystallized with ethanol to afford the
target compounds 10a–t.
4.2.2. 9-n-butyl-1-methyl-β-carboline-3-carbohydrazide (6c)
A light yellow solid, yield: 91.7%, 1H NMR (400 MHz, DMSO‑d6) δ
9.59 (s, 1H, NH), 8.67 (s, 1H, ArH), 8.39 (d, J = 7.6 Hz, 1H, ArH), 7.76
(d, J = 8.3 Hz, 1H, ArH), 7.66–7.61 (m, 1H, ArH), 7.34–7.29 (m, 1H,
ArH), 4.62 (t, J = 7.6 Hz, 2H, eCH2CH2CH2CH3), 4.57 (s, 2H, NH2),
3.04 (s, 3H, CH3), 1.79–1.70 (m, 2H, eCH2CH2CH2CH3), 1.44–1.34 (m,
4.4.1. N'-((9-n-butyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-n-butyl-
9H-pyrido[3,4-b]indole-3-carbohydrazide (10a)
2H, eCH2CH2CH2CH3), 0.92 (t, J = 7.6 Hz, 3H, eCH2CH2CH2CH3). 13
C
NMR (100 MHz, DMSO‑d6) δ 164.25, 141.91, 140.62, 138.97, 136.01,
128.98, 128.94, 122.36, 121.30, 120.60, 112.36, 111.11, 44.51, 33.10,
23.64, 20.00, 14.16.
A light yellow solid, yield: 92.4%, m.p. 213.2–214.4℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.61 (s, 1H, NH), 9.18 (d, J = 3.2 Hz, 2H,
ArH), 9.06 (d, J = 0.8 Hz, 1H, ArH), 8.52–8.49 (m, 2H, ArH), 8.35 (d,
7

L. Guo, et al.
BioorganicChemistry96(2020)103612
J = 8.0 Hz, 1H, ArH), 8.29 (d, J = 4.8 Hz, 1H, CH = N), 7.86 (d,
J = 8.8 Hz, 1H, ArH), 7.83 (d, J = 8.4 Hz, 1H, ArH), 7.72–7.65 (m, 2H,
123.05, 122.15, 122.11, 121.19, 120.98, 120.86, 120.55, 115.84,
115.72, 111.22, 111.18, 43.14, 32.53, 31.42, 31.09, 20.22, 14.18.
HRMS calcd for C37H35N6O [M+H]+ 579.2867, found 579.2864.
ArH), 7.41–7.32 (m, 2H, ArH), 5.05 (t,
J = 7.6 Hz, 2H,
eCH2CH2CH2CH3), 4.65 (t, J = 8.2 Hz, 2H, eCH2CH2CH2CH3),
1.89–1.81 (m, 2H, eCH2CH2CH2CH3), 1.56–1.48 (m, 2H,
eCH2CH2CH2CH3), 1.36–1.27 (m, 2H, eCH2CH2CH2CH3), 1.16–1.06
(m, 2H, eCH2CH2CH2CH3), 0.90 (t, J = 7.2 Hz, 3H, eCH2CH2CH2CH3),
0.75 (t, J = 7.2 Hz, 3H, eCH2CH2CH2CH3). 13C NMR (100 MHz,
DMSO‑d6) δ 162.25, 148.15, 142.71, 141.76, 139.43, 138.43, 138.40,
138.03, 133.85, 131.66, 131.28, 129.40, 129.24, 128.44, 123.03,
122.07, 121.18, 120.96, 120.84, 120.47, 115.79, 115.69, 111.38,
111.16, 44.67, 43.14, 31.42, 31.09, 20.22, 19.73, 14.18, 14.08. HRMS
calcd for C32H33N6O [M+H]+ 517.2710, found 517.2712.
4.4.5. N'-((9-((perfluorophenyl)methyl)-9H-pyrido[3,4-b]indol-1-yl)
methylene)-9-n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10e)
A light yellow solid, yield: 88.1%, m.p. 258.6–260.9℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.61 (s, 1H, NH), 9.16 (d, J = 1.2 Hz, 1H,
ArH), 9.05 (s, 1H, ArH), 9.02 (d, J = 0.8 Hz, 1H, ArH), 8.55 (d,
J = 4.8 Hz, 1H, ArH), 8.49 (d, J = 8.0 Hz, 1H, ArH), 8.37 (d,
J = 7.6 Hz, 1H, ArH), 8.31 (d, J = 4.8 Hz, 1H, CH = N), 7.82 (d,
J = 8.4 Hz, 1H, ArH), 7.79 (d, J = 8.4 Hz, 1H, ArH), 7.72–7.66 (m, 2H,
ArH), 7.41–7.35 (m, 2H, ArH), 6.76 (s, 2H, ArCH2), 4.64 (t, J = 6.8 Hz,
2H, eCH2CH2CH2CH3), 1.89–1.81 (m, 2H, eCH2CH2CH2CH3),
1.36–1.26 (m, 2H, eCH2CH2CH2CH3), 0.89 (t, J = 7.2 Hz, 3H,
eCH2CH2CH2CH3). 13C NMR (100 MHz, DMSO‑d6) δ 162.36, 149.16,
146.39 (m), 143.94 (m), 142.58, 142.58, 141.74, 139.35, 139.29,
139.07, 138.02, 136.08 (m), 134.56, 131.85, 131.66, 129.59, 129.40,
128.39, 123.00, 122.20, 121.58, 121.36, 121.16, 120.84, 115.90,
115.69, 111.80 (m), 111.17, 43.13, 31.41, 20.21, 14.16. HRMS calcd
for C35H26F5N6O [M+H]+ 641.2083, found 641.2081.
4.4.2. N'-((9-benzyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-n-butyl-9H-
pyrido[3,4-b]indole-3-carbohydrazide (10b)
A light yellow solid, yield: 90.3%, m.p. 211.2–213.5℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.59 (s, 1H, NH), 9.18 (d, J = 0.8 Hz, 1H,
ArH), 9.10 (d, J = 0.8 Hz, 1H, ArH), 8.94 (s, 1H, ArH), 8.55–8.49 (m,
2H, ArH), 8.41 (d, J = 7.6 Hz, 1H, ArH), 8.34 (d, J = 4.8 Hz, 1H,
CH = N), 8.00 (d, J = 8.4 Hz, 1H, ArH), 7.83 (d, J = 8.4 Hz, 1H, ArH),
7.73–7.67 (m, 2H, ArH), 7.39 (t, J = 7.6 Hz, 2H, ArH), 6.97–6.91 (m,
2H, ArH), 6.77–6.72 (m, 2H, ArH), 6.52 (s, 2H, ArCH2), 4.65 (t,
4.4.6. N'-((9-n-butyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-benzyl-9H-
pyrido[3,4-b]indole-3-carbohydrazide (10f)
J
=
6.8 Hz, 2H, eCH2CH2CH2CH3), 1.89–1.81 (m, 2H,
eCH2CH2CH2CH3), 1.37–1.26 (m, 2H, eCH2CH2CH2CH3), 0.90 (t,
J = 7.2 Hz, 3H, eCH2CH2CH2CH3). 13C NMR (100 MHz, DMSO‑d6) δ
162.51, 148.50, 143.14, 141.77, 139.33, 138.95, 138.70, 138.06,
135.12, 135.09, 133.92, 131.75, 131.71, 129.60, 129.41, 128.43,
128.18, 128.10, 123.02, 122.20, 121.19, 121.02, 120.87, 115.96,
115.84, 115.80, 115.63, 111.64, 111.18, 47.68, 43.14, 31.42, 20.21,
A light yellow solid, yield: 89.7%, m.p. 237.2–238.9℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.60 (s, 1H, NH), 9.21 (d, J = 0.8 Hz, 1H,
ArH), 9.17 (s, 1H, ArH), 9.08 (d, J = 0.8 Hz, 1H, ArH), 8.53 (d,
J = 7.6 Hz, 1H, ArH), 8.50 (d, J = 4.8 Hz, 1H, ArH), 8.34 (d,
J = 7.6 Hz, 1H, ArH), 8.28 (d, J = 4.8 Hz, 1H, CH = N), 7.85 (dd,
J = 8.4, 4.0 Hz, 2H, ArH), 7.71–7.65 (m, 2H, ArH), 7.42–7.38 (m, 1H,
ArH), 7.34–7.26 (m, 7H, ArH), 5.93 (s, 2H, ArCH2), 5.04 (t, J = 7.2 Hz,
2H, eCH2CH2CH2CH3), 1.55–1.47 (m, 2H, eCH2CH2CH2CH3),
1.15–1.05 (m, 2H, eCH2CH2CH2CH3), 0.74 (t, J = 7.2 Hz, 3H,
eCH2CH2CH2CH3). 13C NMR (100 MHz, DMSO‑d6) δ 162.19, 148.19,
142.71, 141.90, 139.84, 138.43, 138.37, 138.13, 137.59, 133.85,
131.95, 131.28, 129.58, 129.24, 128.82, 128.10, 127.42, 127.29,
123.13, 122.07, 121.39, 121.16, 120.96, 120.47, 115.80, 115.76,
111.40, 46.74, 44.66, 31.08, 19.73, 14.08. HRMS calcd for C35H31N6O
[M+H]+ 551.2554, found 551.2553.
14.17. HRMS calcd for
551.2553.
C
35H31N6O [M+H]+ 551.2554, found
4.4.3. N'-((9-(4-fluorobenzyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-
n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10c)
A light yellow solid, yield: 84.9%, m.p. 250.6–252.3℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.57 (d, J = 5.6 Hz, 1H, NH), 9.18 (s, 1H,
ArH), 9.09 (s, 1H, ArH), 8.93 (s, 1H, ArH), 8.55–8.49 (m, 2H, ArH),
8.40 (d, J = 7.6 Hz, 1H, ArH), 8.34 (d, J = 4.8 Hz, 1H, CH = N), 7.99
(d, J = 8.4 Hz, 1H, ArH), 7.83 (d, J = 8.4 Hz, 1H, ArH), 7.73–7.67 (m,
2H, ArH), 7.39 (t, J = 7.6 Hz, 2H, ArH), 7.11–7.06 (m, 1H, ArH),
6.97–6.89 (m, 2H, ArH), 6.76–6.69 (m, 2H, ArH), 6.52 (s, 2H, ArCH2),
4.65 (t, J = 6.8 Hz, 2H, eCH2CH2CH2CH3), 1.89–1.81 (m, 2H,
eCH2CH2CH2CH3), 1.37–1.26 (m, 2H, eCH2CH2CH2CH3), 0.90 (t,
J = 7.6 Hz, 3H, eCH2CH2CH2CH3). 13C NMR (100 MHz, DMSO‑d6) δ
162.50, 161.45 (d, J = 245 Hz), 148.48, 143.14, 141.77, 139.32,
138.95, 138.69, 138.06, 135.11, 133.92, 131.76 (d, J = 4.4 Hz),
129.61, 129.42, 128.89, 128.43, 128.18 (d, J = 8.1 Hz), 126.22,
123.02, 122.21, 121.19, 121.03, 120.87, 115.97, 115.80, 115.63 (d,
J = 21.2 Hz), 111.64, 111.19, 47.68, 43.15, 31.43, 20.22, 14.18. HRMS
calcd for C35H30FN6O [M+H]+ 569.2460, found 569.2459.
4.4.7. N'-((9-benzyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-benzyl-9H-
pyrido[3,4-b]indole-3-carbohydrazide (10g)
A light yellow solid, yield: 88.8%, m.p. 250.3–251.3℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.56 (s, 1H, NH), 9.21 (d, J = 1.2 Hz, 1H,
ArH), 9.12 (d, J = 1.2 Hz, 1H, ArH), 8.91 (s, 1H, ArH), 8.54 (d,
J = 8.0 Hz, 1H, ArH), 8.51 (d, J = 4.8 Hz, 1H, ArH), 8.40 (d,
J = 7.6 Hz, 1H, ArH), 8.33 (d, J = 4.8 Hz, 1H, CH = N), 7.99 (d,
J = 8.4 Hz, 1H, ArH), 7.85 (d, J = 8.4 Hz, 1H, ArH), 7.71–7.66 (m, 2H,
ArH), 7.40 (q, J = 7.2 Hz, 2H, ArH), 7.32–7.25 (m, 5H, ArH),
7.10–7.05 (m, 3H, ArH), 6.71–6.68 (m, 2H, ArH), 6.51 (s, 2H, ArCH2),
5.93 (s, 2H, ArCH2). 13C NMR (100 MHz, DMSO‑d6) δ 162.40, 148.41,
143.27, 141.90, 139.77, 138.92, 138.86, 138.70, 138.16, 137.59,
134.05, 131.98, 131.68, 129.59, 129.55, 129.24, 128.89, 128.82,
128.11, 127.42, 127.27, 126.22, 123.12, 122.18, 121.40, 121.18,
121.15, 120.95, 115.93, 115.85, 111.67, 111.42, 48.28, 46.74. HRMS
calcd for C38H29N6O [M+H]+ 585.2397, found 585.2399.
4.4.4. N'-((9-(3-phenylpropyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-
n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10d)
A light yellow solid, yield: 83.6%, m.p. 233.4–235.1℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.62 (s, 1H, NH), 9.18 (d, J = 2.4 Hz, 2H,
ArH), 9.07 (s, 1H, ArH), 8.52–8.49 (m, 2H, ArH), 8.35 (d, J = 7.6 Hz,
1H, ArH), 8.29 (d, J = 5.2 Hz, 1H, CH = N), 7.83 (d, J = 8.4 Hz, 1H,
ArH), 7.75–7.63 (m, 3H, ArH), 7.40–7.32 (m, 2H, ArH), 7.15–7.10 (m,
4.4.8. N'-((9-(4-fluorobenzyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-
benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10h)
2H, ArH), 7.06–7.02 (m, 3H, ArH), 5.11 (t,
J
=
7.6 Hz, 2H,
A light yellow solid, yield: 82.4%, m.p. 270.6–272.2℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.58 (s, 1H, NH), 9.21 (d, J = 1.2 Hz, 1H,
ArH), 9.12 (d, J = 1.2 Hz, 1H, ArH), 8.91 (s, 1H, ArH), 8.54 (d,
J = 8.0 Hz, 1H, ArH), 8.52 (d, J = 4.8 Hz, 1H, ArH), 8.40 (d,
J = 7.2 Hz, 1H, ArH), 8.33 (d, J = 4.8 Hz, 1H, CH = N), 7.99 (d,
J = 8.4 Hz, 1H, ArH), 7.86 (d, J = 8.4 Hz, 1H, ArH), 7.72–7.66 (m, 2H,
ArH), 7.43–7.37 (m, 2H, ArH), 7.34–7.25 (m, 5H, ArH), 6.96–6.91 (m,
ArCH2CH2CH2), 4.65 (t, J = 6.8 Hz, 2H, eCH2CH2CH2CH3), 2.51–2.46
(m, 2H, eCH2CH2CH2CH3), 1.90–1.81 (m, 4H, eCH2CH2CH2CH3,
ArCH2CH2CH2), 1.36–1.26 (m, 2H, ArCH2CH2CH2), 0.89 (t, J = 7.2 Hz,
3H, eCH2CH2CH2CH3). 13C NMR (100 MHz, DMSO‑d6) δ 162.30,
148.40, 142.55, 141.76, 139.46, 138.51, 138.35, 138.05, 133.86,
131.67, 131.30, 129.40, 129.27, 128.65, 128.47, 128.44, 126.14,
8

L. Guo, et al.
BioorganicChemistry96(2020)103612
2H, ArH), 6.75–6.71 (m, 2H, ArH), 6.51 (s, 2H, ArCH2), 5.93 (s, 2H,
(400 MHz, CDCl3) δ 11.29 (s, 1H, NH), 8.92 (d, J = 2.0 Hz, 1H, ArH),
8.53 (s, 1H, ArH), 8.51 (dd, J = 5.2, 1.6 Hz, 1H, ArH), 8.19 (d,
J = 7.6 Hz, 2H, ArH, CH = N), 8.04–8.01 (m, 1H, ArH), 7.62–7.58 (m,
3H, ArH), 7.48–7.45 (m, 1H, ArH), 7.37–7.30 (m, 3H, ArH), 7.09–7.06
(m, 3H, ArH), 6.82–6.79 (m, 2H, ArH), 6.43 (s, 2H, ArCH2), 4.51 (t,
J = 7.2 Hz, 2H, eCH2CH2CH2CH3), 3.05 (s, 3H, CH3), 1.87–1.80 (m,
2H, eCH2CH2CH2CH3), 1.50–1.41 (m, 2H, eCH2CH2CH2CH3), 0.99 (t,
J = 7.2 Hz, 3H, eCH2CH2CH2CH3). 13C NMR (100 MHz, CDCl3) δ
161.54, 147.15, 143.22, 141.79, 139.77, 138.45, 138.10, 137.49,
137.43, 136.62, 134.49, 131.86, 129.29, 129.06, 128.60, 128.46,
126.83, 125.96, 121.84, 121.61, 121.34, 121.12, 120.61, 120.41,
115.25, 113.73, 110.56, 110.03, 48.69, 44.82, 33.01, 23.67, 20.19,
13.86. 19F NMR (376 MHz, CDCl3) δ −115.94. HRMS calcd for
ArCH2). 13C NMR (100 MHz, DMSO‑d6)
δ 162.44, 161.44 (d,
J = 241.1 Hz), 148.54, 143.15, 141.90, 139.74, 138.95, 138.68,
138.16, 137.59, 135.08 (d, J = 2.9 Hz), 133.91, 131.99, 131.76,
129.61, 129.24, 128.81, 128.18 (d, J = 7.7 Hz), 127.42, 123.12,
122.21, 121.40, 121.18, 121.03, 115.97, 115.87, 115.84 (d,
J
= 21.3 Hz), 111.64, 111.43, 47.67, 46.74. HRMS calcd for
C
38H28FN6O [M+H]+ 603.2303, found 603.2309.
4.4.9. N'-((9-(3-phenylpropyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-9-
benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10i)
A light yellow solid, yield: 83.1%, m.p. 233.8–235.9℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.62 (s, 1H, NH), 9.22 (d, J = 0.8 Hz, 1H,
ArH), 9.16 (s, 1H, ArH), 9.09 (d, J = 0.8 Hz, 1H, ArH), 8.54 (d,
J = 7.6 Hz, 1H, ArH), 8.51 (d, J = 4.8 Hz, 1H, ArH), 8.34 (d,
J = 7.6 Hz, 1H, ArH), 8.29 (d, J = 5.2 Hz, 1H, CH = N), 7.85 (d,
J = 8.4 Hz, 1H, ArH), 7.73 (d, J = 8.4 Hz, 1H, ArH), 7.70–7.64 (m, 2H,
ArH), 7.42–7.37 (m, 1H, ArH), 7.34–7.25 (m, 6H, ArH), 7.14–7.09 (m,
2H, ArH), 7.06–7.01 (m, 3H, ArH), 5.93 (s, 2H, ArCH2), 5.10 (t,
C
36H33N6O [M+H]+ 565.2710, found 565.2703.
4.4.13. N'-((9-(4-fluorobenzyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-
methyl-9-n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10m)
A light yellow solid, yield: 95.7%, m.p. 264.8–267.1℃. 1H NMR
(400 MHz, CDCl3) δ 11.31 (s, 1H, NH), 8.91 (s, 1H, ArH), 8.55 (s, 1H,
ArH), 8.51 (d, J = 5.2 Hz, 1H, ArH), 8.19 (dd, J = 8.0, 4.8 Hz, 2H,
ArH), 8.02 (d, J = 5.2 Hz, 1H, CH = N), 7.64–7.57 (m, 3H, ArH), 7.47
(d, J = 8.4 Hz, 1H, ArH), 7.38–7.32 (m, 2H, ArH), 6.81–6.73 (m, 4H,
ArH), 6.44 (s, 2H, ArCH2), 4.51 (t, J = 7.6 Hz, 2H, eCH2CH2CH2CH3),
3.06 (s, 3H, CH3), 1.88–1.80 (m, 2H, eCH2CH2CH2CH3), 1.51–1.41 (m,
J
= 7.6 Hz, 2H, ArCH2CH2CH2), 2.47 (t, J = 8.0 Hz, 2H,
ArCH2CH2CH2), 1.89–1.80 (m, 2H, ArCH2CH2CH2). 13C NMR
(100 MHz, DMSO‑d6) δ 162.24, 148.43, 142.54, 141.91, 141.74,
139.87, 138.50, 138.31, 138.14, 137.59, 133.86, 131.96, 131.30,
129.58, 129.27, 129.24, 128.83, 128.64, 128.47, 128.10, 127.41,
126.14, 123.14, 122.11, 121.40, 121.17, 120.98, 120.54, 115.85,
115.77, 111.41, 111.21, 46.74, 44.84, 32.53, 31.08. HRMS calcd for
2H, eCH2CH2CH2CH3), 0.99 (t, J = 7.2 Hz, 3H, eCH2CH2CH2CH3). 13
C
NMR (100 MHz, CDCl3) δ 161.60, 160.46 (d, J = 243.1 Hz), 147.47,
143.09, 141.80, 139.83, 138.58, 137.42, 136.64, 134.32, 133.89 (d,
J = 3.0 Hz), 131.91, 129.51, 129.11, 128.63, 127.65 (d, J = 8.0 Hz),
121.83, 121.60, 121.37, 121.16, 120.64, 120.52, 115.39 (d,
J = 21.3 Hz), 115.29, 113.72, 110.49, 110.05, 48.12, 44.82, 33.01,
23.68, 20.19, 13.85. HRMS calcd for C36H32FN6O [M+H]+ 583.2616,
found 583.2621.
C
40H33N6O [M+H]+ 613.2710, found 613.2712.
4.4.10. N'-((9-((perfluorophenyl)methyl)-9H-pyrido[3,4-b]indol-1-yl)
methylene)-9-benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10j)
A light yellow solid, yield: 84.7%, m.p. 258.4–260.1℃. 1H NMR
(400 MHz, DMSO‑d6) δ 12.60 (s, 1H, NH), 9.20 (d, J = 0.8 Hz, 1H,
ArH), 9.04 (d, J = 0.8 Hz, 1H, ArH), 9.03 (s, 1H, ArH), 8.54 (d,
J = 5.2 Hz, 1H, ArH), 8.52 (d, J = 8.0 Hz, 1H, ArH), 8.36 (d,
J = 7.6 Hz, 1H, ArH), 8.30 (d, J = 4.8 Hz, 1H, CH = N), 7.84 (d,
J = 8.4 Hz, 1H, ArH), 7.78 (d, J = 8.4 Hz, 1H, ArH), 7.71–7.65 (m, 2H,
ArH), 7.39 (td, J = 7.6, 2.4 Hz, 2H, ArH), 7.34–7.29 (m, 2H, ArH),
7.29–7.24 (m, 3H, ArH), 6.75 (s, 2H, ArCH2), 5.92 (s, 2H, ArCH2). 13C
NMR (100 MHz, DMSO‑d6) δ 162.30, 149.21, 146.49 (m), 143.92 (m),
142.57, 141.88, 139.70, 139.35, 139.04, 138.13, 137.57, 136.10 (m),
134.56, 131.95, 131.85, 129.59, 129.23, 128.78, 128.10, 127.41,
123.10, 122.20, 121.58, 121.37, 121.16, 115.91, 115.76, 111.78 (m),
4.4.14. N'-((9-(3-phenylpropyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-
methyl-9-n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10n)
A light yellow solid, yield: 88.4%, m.p. 203.2–205.5℃. 1H NMR
(400 MHz, CDCl3) δ 11.41 (s, 1H, NH), 8.98 (s, 1H, ArH), 8.91 (s, 1H,
ArH), 8.54 (d, J = 5.2 Hz, 1H, ArH), 8.20 (d, J = 8.0 Hz, 1H, ArH),
8.13 (d, J = 8.0 Hz, 1H, ArH), 8.01 (d, J = 5.2 Hz, 1H, CH = N),
7.64–7.56 (m, 2H, ArH), 7.49 (d, J = 8.4 Hz, 1H, ArH), 7.42 (d,
J = 8.4 Hz, 1H, ArH), 7.36–7.27 (m, 2H, ArH), 7.19–7.14 (m, 2H, ArH),
7.09–7.05 (m, 3H, ArH), 5.14 (t, J = 7.6 Hz, 2H, ArCH2CH2CH2), 4.56
(t, J = 7.6 Hz, 2H, eCH2CH2CH2CH3), 3.10 (s, 3H, CH3), 2.60 (t,
J = 7.6 Hz, 2H, ArCH2CH2CH2), 2.08–2.01 (m, 2H, eCH2CH2CH2CH3),
111.41, 111.18, 46.74. HRMS calcd for
675.1926, found 675.1924.
C
38H24F5N6O [M+H]+
1.90–1.82
(m,
2H,
ArCH2CH2CH2),
1.52–1.42
(m,
2H,
4.4.11. N'-((9-n-butyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-methyl-9-
n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10k)
eCH2CH2CH2CH3), 1.00 (t, J = 7.6 Hz, 3H, eCH2CH2CH2CH3). 13C
NMR (100 MHz, CDCl3) δ 161.54, 147.89, 142.62, 141.83, 141.41,
139.77, 138.21, 137.64, 137.11, 136.65, 134.48, 131.76, 129.61,
128.70, 128.61, 128.35, 128.25, 125.77, 121.89, 121.65, 121.23,
121.12, 120.62, 120.02, 115.30, 113.72, 110.55, 110.03, 44.96, 44.87,
33.02, 32.64, 30.72, 23.73, 20.21, 13.87. HRMS calcd for C38H37N6O
[M+H]+ 593.3023, found 593.3019.
A light yellow solid, yield: 86.3%, m.p. 199.7–201.3℃. 1H NMR
(400 MHz, CDCl3) δ 11.46 (s, 1H, NH), 9.08 (s, 1H, ArH), 8.90 (s, 1H,
ArH), 8.54 (d, J = 4.8 Hz, 1H, ArH), 8.21–8.14 (m, 2H, ArH), 8.03 (d,
J = 4.8 Hz, 1H, CH = N), 7.65–7.57 (m, 3H, ArH), 7.49 (d, J = 8.4 Hz,
1H, ArH), 7.36–7.29 (m, 2H, ArH), 5.08 (t,
J = 7.2 Hz, 2H,
eCH2CH2CH2CH3), 4.57 (t, J = 7.6 Hz, 2H, eCH2CH2CH2CH3), 3.10 (s,
3H, CH3), 1.90–1.82 (m, 2H, eCH2CH2CH2CH3), 1.74–1.63 (m, 2H,
eCH2CH2CH2CH3), 1.53–1.43 (m, 2H, eCH2CH2CH2CH3), 1.31–1.20
(m, 2H, eCH2CH2CH2CH3), 1.00 (t, J = 7.2 Hz, 3H, eCH2CH2CH2CH3),
0.84 (t, J = 7.2 Hz, 3H, eCH2CH2CH2CH3). 13C NMR (100 MHz, CDCl3)
δ 161.56, 147.73, 142.79, 141.84, 139.79, 138.13, 137.65, 137.10,
136.67, 134.52, 131.72, 129.62, 128.67, 128.62, 121.90, 121.66,
121.26, 121.10, 120.63, 119.95, 115.30, 113.72, 110.58, 110.03,
45.22, 44.88, 33.03, 31.40, 23.75, 20.22, 19.96, 13.92, 13.88. HRMS
calcd for C33H35N6O [M+H]+ 531.2867, found 531.2873.
4.4.15. N'-((9-((perfluorophenyl)methyl)-9H-pyrido[3,4-b]indol-1-yl)
methylene)-1-methyl-9-n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide
(10o)
A
white solid, yield: 85.9%, m.p. 253.6–255.1℃. 1H NMR
(400 MHz, CDCl3) δ 11.43 (s, 1H, NH), 8.85 (s, 1H, ArH), 8.78 (s, 1H,
ArH), 8.56 (d, J = 5.2 Hz, 1H, ArH), 8.17 (d, J = 8.0 Hz, 1H, ArH),
8.12 (d, J = 7.6 Hz, 1H, ArH), 7.99 (d, J = 4.8 Hz, 1H, CH = N),
7.63–7.55 (m, 3H, ArH), 7.48 (d, J = 8.4 Hz, 1H, ArH), 7.33 (t,
J = 7.2 Hz, 2H, ArH), 6.89 (s, 2H, ArCH2), 4.54 (t, J = 7.6 Hz, 2H,
eCH2CH2CH2CH3), 3.08 (s, 3H, CH3), 1.88–1.80 (m, 2H,
eCH2CH2CH2CH3), 1.52–1.42 (m, 2H, eCH2CH2CH2CH3), 0.99 (t,
J = 7.6 Hz, 3H, eCH2CH2CH2CH3). 13C NMR (100 MHz, CDCl3) δ
161.58, 149.27, 146.64 (m), 144.19 (m), 142.00, 141.82, 139.88,
4.4.12. N'-((9-benzyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-methyl-9-
n-butyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10l)
A light yellow solid, yield: 90.8%, m.p. 246.8–248.2℃. 1H NMR
9

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BioorganicChemistry96(2020)103612
138.85, 138.71 (m), 138.04, 137.33, 136.68, 136.20 (m), 135.44,
132.03, 129.55, 129.15, 128.66, 121.85, 121.80, 121.61, 121.28,
120.99, 120.67, 115.34, 113.75, 111.47 (m), 110.27, 110.04, 44.87,
39.06, 33.02, 23.69, 20.20, 13.86. HRMS calcd for C36H28F5N6O [M
+H]+ 655.2239, found 655.2244.
ArCH2CH2CH2). 13C NMR (100 MHz, CDCl3) δ 161.46, 147.91, 142.63,
142.28, 141.39, 140.27, 138.16, 137.44, 137.10, 137.02, 134.46,
131.79, 129.80, 129.14, 128.99, 128.73, 128.34, 128.26, 127.74,
125.77, 125.30, 121.95, 121.69, 121.25, 121.11, 121.07, 120.05,
115.32, 113.77, 110.55, 110.16, 48.31, 44.96, 32.64, 30.70, 23.33.
HRMS calcd for C41H35N6O [M+H]+ 627.2867, found 627.2873.
4.4.16. N'-((9-n-butyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-methyl-9-
benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10p)
4.4.20. N'-((9-((perfluorophenyl)methyl)-9H-pyrido[3,4-b]indol-1-yl)
methylene)-1-methyl-9-benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide
(10t)
A light yellow solid, yield: 83.3%, m.p. 213.5–215.1℃. 1H NMR
(400 MHz, CDCl3) δ 11.42 (s, 1H, NH), 9.04 (s, 1H, ArH), 8.94 (s, 1H,
ArH), 8.53 (d, J = 5.2 Hz, 1H, ArH), 8.22 (d, J = 7.6 Hz, 1H, ArH),
8.13 (d, J = 7.6 Hz, 1H, ArH), 8.00 (d, J = 5.2 Hz, 1H, CH), 7.63–7.53
(m, 3H, ArH), 7.41–7.23 (m, 6H, ArH), 6.98–6.95 (m, 2H, ArH), 5.81 (s,
2H, ArCH2), 5.07 (t, J = 7.6 Hz, 2H, eCH2CH2CH2CH3), 2.91 (s, 3H,
CH3), 1.71–1.63 (m, 2H, eCH2CH2CH2CH3), 1.30–1.20 (m, 2H,
eCH2CH2CH2CH3), 0.83 (t, J = 7.6 Hz, 3H, eCH2CH2CH2CH3). 13C
NMR (100 MHz, CDCl3) δ 161.44, 147.79, 142.75, 142.24, 140.26,
138.12, 137.44, 137.05, 137.01, 134.49, 131.69, 129.74, 129.11,
129.06, 128.96, 128.68, 127.72, 125.29, 121.88, 121.64, 121.25,
121.06, 121.04, 119.95, 115.29, 113.70, 110.55, 110.15, 48.27, 45.20,
31.39, 23.31, 19.94, 13.91. HRMS calcd for C36H33N6O [M+H]+
565.2710, found 565.2704.
A off white solid, yield: 92.3%, m.p. > 280℃. 1H NMR (400 MHz,
CDCl3) δ 11.40 (s, 1H, NH), 8.91 (s, 1H, ArH), 8.76 (s, 1H, ArH), 8.55
(d, J = 5.2 Hz, 1H, ArH), 8.23 (d, J = 7.6 Hz, 1H, ArH), 8.13 (d,
J = 8.0 Hz, 1H, ArH), 8.01 (d, J = 4.8 Hz, 1H, CH = N), 7.63–7.55 (m,
3H, ArH), 7.42–7.32 (m, 3H, ArH), 7.30–7.27 (m, 2H, ArH), 6.99–6.96
(m, 2H, ArH), 6.89 (s, 2H, ArCH2), 5.83 (s, 2H, ArCH2), 2.93 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3) δ 161.54, 146.65 (m), 144.10 (m),
142.31, 142.18, 140.44, 138.63 (m), 138.44, 137.82, 137.76, 137.38,
137.20, 135.37, 132.34, 129.79, 129.37, 129.16, 129.12, 129.07,
127.79, 125.35, 125.30, 121.94, 121.74, 121.67, 121.55, 121.38,
121.15, 121.13, 115.43, 114.38, 113.87, 111.43 (m), 110.33, 110.20,
48.36, 39.11, 23.31. RMS calcd for C39H26F5N6O [M+H]+ 689.2083,
found 689.2079.
4.4.17. N'-((9-benzyl-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-methyl-9-
benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10q)
4.5. MTT assay
A light yellow solid, yield: 89.5%, m.p. 247.7–249.5℃. 1H NMR
(400 MHz, CDCl3) δ 11.27 (s, 1H, NH), 9.00 (s, 1H, ArH), 8.52 (d,
J = 5.2 Hz, 2H, ArH), 8.27 (d, J = 7.2 Hz, 1H, ArH), 8.21 (d,
J = 8.0 Hz, 1H, ArH), 8.06 (d, J = 5.2 Hz, 1H, CH = N), 7.64–7.56 (m,
3H, ArH), 7.44–7.35 (m, 3H, ArH), 7.32–7.26 (m, 3H, ArH), 7.08– 7.06
(m, 3H, ArH), 7.01–6.97 (m, 2H, ArH), 6.81–6.77 (m, 2H, ArH), 6.44 (s,
2H, ArCH2), 5.85 (s, 2H, ArCH2), 2.91 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 161.48, 147.13, 143.31, 142.30, 140.30, 138.33, 138.06,
138.04, 137.43, 137.36, 137.16, 134.48, 132.00, 129.80, 129.15,
129.10, 129.02, 128.47, 127.76, 126.85, 125.95, 125.32, 121.97,
121.71, 121.39, 121.12, 120.48, 115.32, 113.86, 110.61, 110.19,
48.71, 48.33, 23.31. HRMS calcd for C39H31N6O [M+H]+ 599.2554,
found 599.2563.
Target compounds were assayed by the MTT method for de-
termining cytotoxic activity as described previously [34]. 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 Institute of Bio-
chemistry and Cell Biology, Chinese Academy of Science. Growth in-
hibition rates were calculated with the following equitation: Inhibition
OD
OD
OD
OD
compd
blank
ratio (%) =
×100%. The half-maximal inhibitory con-
DMSO
blank
centration (IC50) of each compound was calculated using GraphPad
Prism software (version 6.0).
4.6. Evaluation of the antitumor activity of heterobivalent β-carbolines in
mice
4.4.18. N'-((9-(4-fluorobenzyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-
methyl-9-benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10r)
A light yellow solid, yield: 91.6%, m.p.
>
280℃. 1H NMR
Sarcoma 180 and Lewis lung cancer cells were provided by the
Shanghai Institute of Pharmaceutical Industry. Mice received sub-
cutaneous injections of viable tumor cells (2 × 106 cells/mouse) in the
armpit. At 7 days after mice were inoculated with these cells, the tu-
mors were removed, and the cells were harvested. Each test compound
was administered at a dosage of about one-fifth of the LD50 via i.p.
injection to groups containing 9 female mice. Dosing was performed at
24 h after the inoculation and once a day for seven consecutive days.
For most of the compounds, the amount was the maximum tolerated
dose based on our preliminary studies. CTX (30 mg kg−1) was used as
the positive control and the vehicle as the negative control. The weights
of the animals were recorded every three days. All of the animals were
killed on the 21st day after tumor inoculation, and the tumors were
excised and weighed. Tumor inhibition was calculated as follows:
(400 MHz, CDCl3) δ 11.29 (s, 1H, NH), 8.98 (s, 1H, ArH), 8.53 (s, 1H,
ArH), 8.51 (d, J = 5.2 Hz, 1H, ArH), 8.26 (d, J = 7.6 Hz, 1H, ArH),
8.20 (d, J = 8.0 Hz, 1H, ArH), 8.04 (d, J = 5.2 Hz, 1H, CH = N),
7.66–7.55 (m, 3H, ArH), 7.42–7.34 (m, 3H, ArH), 7.31–7.27 (m, 3H,
ArH), 6.99–6.96 (m, 2H, ArH), 6.79–6.72 (m, 4H, ArH), 6.44 (s, 2H,
ArCH2), 5.82 (s, 2H, ArCH2), 2.90 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 161.53, 160.47 (d, J = 243.4 Hz), 147.44, 143.15, 142.28,
140.35, 138.48, 137.95, 137.40, 137.36, 137.13, 134.31, 133.86 (d,
J = 2.9 Hz), 132.02, 129.75, 129.19, 129.14, 129.03, 127.76, 127.63
(d, J = 8.0 Hz), 125.30, 121.93, 121.66, 121.41, 121.15, 121.11,
120.57, 115.41 (d, J = 21.3 Hz), 115.34, 113.81, 110.52, 110.20,
48.30, 48.13, 23.30. 19F NMR (376 MHz, CDCl3) δ −115.92. HRMS
calcd for C39H30FN6O [M+H]+ 617.2460, found 617.2455.
4.4.19. N'-((9-(3-phenylpropyl)-9H-pyrido[3,4-b]indol-1-yl)methylene)-1-
(C T)/C × 100,
methyl-9-benzyl-9H-pyrido[3,4-b]indole-3-carbohydrazide (10s)
A
yellow solid, yield: 87.2%, m.p. 214.8–216.2℃. 1H NMR
where T is the average tumor weight of the treated group and C is the
average tumor weight of the negative control group.
(400 MHz, CDCl3) δ 11.38 (s, 1H, NH), 8.96 (s, 2H, ArH), 8.53 (d,
J = 4.8 Hz, 1H, ArH), 8.25 (d, J = 7.6 Hz, 1H, ArH), 8.13 (d,
J = 7.6 Hz, 1H, ArH), 8.01 (d, J = 5.2 Hz, 1H, CH = N), 7.61–7.54 (m,
2H, ArH), 7.44–7.35 (m, 3H, ArH), 7.32–7.26 (m, 4H, ArH), 7.18–7.14
(m, 2H, ArH), 7.08–7.04 (m, 3H, ArH), 7.00–6.96 (m, 2H, ArH), 5.84 (s,
2H, ArCH2), 5.13 (t, J = 7.6 Hz, 2H, ArCH2CH2CH2), 2.93 (s, 3H, CH3),
4.7. Wound healing assay in vitro
HT-29 and LLC cells (about 1 × 106/mL) were seeded in a 24-well
plate at a density that after 24 h of growth, and they were allowed to
reach 90% confluence in complete medium. A single scratch wound was
2.59 (t,
J = 7.6 Hz, 2H, ArCH2CH2CH2), 2.07–2.00 (m, 2H,
10

L. Guo, et al.
BioorganicChemistry96(2020)103612
created on the confluent monolayers using a micropipette tip across the
center of the well and a straight line was scratched in one direction.
Then, wounded monolayers were washed with phosphate buffer saline
(PBS) to remove the detached cells, and each assay was carried out
three times. After washing, fresh media with FBS was added, various
concentrations of 10e were added to their respective wells, and then
they were incubated for 24 h. The medium in each well was discarded
and washed several times with PBS. Cells migrated to the wound sur-
face and the average distance of migrating cells was determined under
an inverted microscope at designated time points. Pictures of three
different regions of each wound were taken. The experiment was per-
formed three times.
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Acknowledgements
This research was supported by grants from 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.
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