Accepted Manuscript

Discovery of novel β-carboline/acylhydrazone hybrids as potent antitumor agents and

overcome drug resistance

Yong Li, Wei Yan, Jianhong Yang, Zhuang Yang, Mengshi Hu, Peng Bai, Minghai
Tang, Lijuan Chen

PII: S0223-5234(18)30407-0
DOI: 10.1016/j.ejmech.2018.05.003
Reference: EJMECH 10413

To appear in: European Journal of Medicinal Chemistry

Received Date: 17 March 2018

Revised Date: 23 April 2018
Accepted Date: 2 May 2018

Please cite this article as: Y. Li, W. Yan, J. Yang, Z. Yang, M. Hu, P. Bai, M. Tang, L. Chen, Discovery
of novel β-carboline/acylhydrazone hybrids as potent antitumor agents and overcome drug resistance,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.003.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

1 Discovery of novel β-carboline/acylhydrazone hybrids as potent antitumor agents and

2 overcome drug resistance
3 Yong Li, Wei Yan, Jianhong Yang, Zhuang Yang, Mengshi Hu, Peng Bai, Minghai
4 Tang, Lijuan Chen∗
5 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan

PT
6 University and Collaborative Innovation Center, Chengdu, Sichuan, 610041, China.
7 Abstract

RI
8 Twenty-four novel β-carboline/acylhydrazone hybrids were synthesized and

SC
9 evaluated for their in vitro antiproliferative activity. Among them, 12r exhibited the
10 most potent activity, with IC50 values of 1-2 µM against panel of cancer cell lines and

U
11 retained significant activity in multidrug resistant cancer cells. Treated cells were not
arrested in any phase of cell cycle but resulted in late cellular apoptosis on both
AN
12

13 MCF-7 and MCF-7/ADR cancer cells. Importantly, 12r showed certain antitumor
14 effect on inhibiting the tumor growth with low toxic and side effects and without
M

15 significant body weight loss.

D

16 Keywords: β-carboline; antitumor activity; multidrug resistance; cellular

17 apoptosis; H460 xenograft model
TE
C EP
AC

∗
Corresponding author. State Key Laboratory of Biotherapy and Cancer Center, West China

Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, Sichuan 610041,

China.
Tel: +86-28-85164103; Fax: +86-28-85164060.
E-mail address: chenlijuan125@163.com, chenlj125@163.com.
 ACCEPTED MANUSCRIPT

18 1. Introduction

19 Natural products have been a rich source of compounds for drug discovery, and
20 the majority of anticancer drugs currently used in clinical settings are derived from
21 natural product scaffolds such as paclitaxel and vinblastine. β-Carboline is made up of
22 planar tricyclic pyrido-[3,4-b]indole ring structures, and its derivatives are widely

PT
23 distributed in plants, marine creatures, insects, and mammals such as human tissues
24 and bodily fluids [1]. The best-known members of β-carboline compounds are

RI
25 harmine (Fig. 1 1), harman (Fig. 1 2) and norharman (Fig. 1 3). Naturally occurring

SC
26 and synthetic β-carboline alkaloids are widely studied due to their large spectrum of
27 important pharmacological and biological properties, such as antitrypanosomal and

U
28 antileishmanial [2-5], anti-Alzheimer [6, 7], anti-platelet aggregation and
29 anti-thrombotic [8, 9], anti-Parkinson [10], and as DYRK1A inhibitors [11, 12]. It's
AN
30 worth mentioning the potent antitumor activity of β-carboline alkaloids [13, 14].
31 Most of these compounds are endowed with inhibiting cancer cell growth and
M

32 leading to apoptosis through multiple mechanisms such as inhibition of DNA

D

33 topoisomerases I and II [15-20], IkappaB kinase (IKK) [21], CDK [22], PLK [23],
34 and MAO [24]. In particular, β-carbolines can intercalate DNA, alter DNA replication
TE

35 fidelity, and influence enzymatic activities in DNA repair processes because of the
36 presence of polycyclic aromatic planar pharmacophore, which is capable of stacking
EP

37 between DNA base pairs [25, 26]. In fact, several β-carbolines including harmane,
38 norharman, and β-carboline-benzimidazole conjugates have been reported to
C

39 intercalate DNA, leading to altering DNA replication fidelity or influencing

AC

40 enzymatic activities in DNA repair processes [25-27].

MeO N N N
N N N
H Me H Me H

41 1 harmine 2 harmane 3 norharman

42 Figure 1. Chemical structures of representative β-carboline derivatives.

43 Meanwhile, acylhydrazone scaffold (-CONHN=) has attracted considerable
 ACCEPTED MANUSCRIPT

44 attention for decades due to their broad applications ranging from medicinal agents,
45 agrochemicals to functional materials. Many compounds containing this moiety have
46 been reported, and which demonstrates that the introduction of this pharmacophore
47 may result in highly potential antitumor activity [28-34]. One of the anticancer agent
48 SP2509 (Fig. 2 4) with a benzo-hydrazone moiety exhibited potent antitumor activity

PT
49 against several tumor cell lines by inhibiting LSD1 [35]. Compound 5 (Fig. 2 5), a
50 N-acylhydrazone derivative, was reported as a potent HDAC6/8 dual inhibitor [36]. It

RI
51 was particularly notable that two antibody-drug conjugates Mylotarg (used to treat
52 relapsed acute myeloid leukemia, marketed) and Inotuzumab ozogamicin (used to

SC
53 treat relapsed or refractory B-cell precursor acute lymphoblastic leukemia, teminated
54 in phase III) containing an acylhydrazone group which made a great difference on

U
55 effenciency [37, 38].
AN
M

56
D

57 Figure 2. Chemical structures of representative hydrazone-contained compounds.

TE

58 Recent results from in vitro and in vivo studies have demonstrated that both
59 β-carboline and acylhydrazone scaffolds possess antitumor activity [28]. There are
EP

60 reasonable grounds to believe that β-carboline derivatives may possess synergistic or

61 additive antitumor effects in combination with acylhydrazone. Therefore,
β-carboline/acylhydrazone hybrids may represent a promising strategy to achieve
C

62

63 increased anticancer efficacy. The synthesized title compounds were evaluated for
AC

64 their in vitro and in vivo antitumor activity.

65 2. Results and discussion
66 2.1. Chemistry

67 The synthetic routes employed in the preparation of the hybrids

68 N'-arylidene-9H-pyrido[3,4-b]indole-3-carbohydrazide (12a-e),
69 N'-(3-(substituted-benzyloxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
 ACCEPTED MANUSCRIPT

70 (12f-x) are shown in Scheme 1.

71 To obtain the hybrids β-carboline/acylhydrazone hybrids proposed, firstly
72 β-carboline-3-carbohydrazide (10), the common intermediate for the synthetic route,
73 was synthesized. For this, the commercial L-tryptophan (6) was esterified with
74 methanol in the presence of SOCl2 to afford L-tryptophan methyl ester (7), which was

PT
75 subjected to Mannich reaction with formaldehyde, in acidic media, to provide the
76 tetrahydro-β-carboline-3-carboxylates (8). The oxidation of 8 with

RI
77 trichloroisocyanuric acid (TCCA) in N,N-dimethylformamide (DMF) at -20 – 0 ℃
78 afforded methyl 9H-pyrido[3,4-b]indole-3-carboxylate （9）. Then intermediate 10

SC
79 was obtained by nucleophilic substitution reaction of 9 with hydrazine hydrate in
80 methanol. Secondly, aryl-aldehyde (11a-e) are commercially available, and

U
81 3-(substituted-benzyloxy)benzaldehyde (11f-x) were obtained from
AN
82 3-hydroxybenzaldehyde and substituted benzyl chloride or bromide. Finally,
83 β-carboline-3-carbohydrazide reacted with aromatic aldehyde to give the title
M

84 compounds (12a-x).
85
To evaluate the antitumor activity effect of acylhydrazone which acts as a linker
86
D

of β-carboline and aromatic substituent, the 2-(O or

87
TE

N-substitued-methyl)-5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazole
88
carbohydrazides (15a-d) were synthesized (Scheme 2). Firstly, intermediate 10
89
EP

reacted with chloroacetyl chloride to give

90
N'-(2-chloroacetyl)-9H-pyrido[3,4-b]indole-3-carbohydrazide (13). Then, compound
91
refluxed in POCl3 to obtain the cyclization product
C

13
92
2-(chloromethyl)-5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazole (14). Finally, title
AC

93
compounds 15a-d were easily prepared from 14 by reacting with substituted phenol or
94
N-methylbenzylamine.
 ACCEPTED MANUSCRIPT
95

PT
96
Scheme 1. Reagents and conditions: a) SOCl2, MeOH, 0 ℃ - rt for 4h, then
97

RI
reflux for 3h, 95% yield; b) 37% HCHO, MeOH, reflux for 3h, 89% yield; c) TCCA,
98
Et3N, DMF, -20 - 0 ℃, then 0 ℃ for 2h, 80% yield; d) 85% NH2NH2·H2O, MeOH,

SC
99
reflux for 6h, 85% yield; e) aromatic aldehyde 11, cat. conc. HCl, MeOH, reflux for 4
100
– 6h.

U
101
AN
M
D
TE

102
EP

Scheme 2. Reagents and conditions: a) chloroacetyl chloride, 4-Methylmorpholine

103
N-oxide monohydrate, cyanuric chloride, CH2Cl2, rt for 1.5h, 62% yield; b) POCl3,
104
reflux for 3h, 56% yield; c) K2CO3, cat. KI, DMF, 65 - 80 ℃ for 4h.
C
AC

105 2.2 Biological activities

106 2.2.1 Antitumor activity

107 The in vitro antitumor activities of the synthesized β-carboline/acylhydrazone

108 hybrids were firstly evaluated against the human tumor cell lines MCF-7 (breast),
109 MDA-MB-231 (breast), Raji (lymphoma) and H460 (lung). The preliminary
110 screening of growth inhibition rate of the above tumor cell lines were summarized in
 ACCEPTED MANUSCRIPT

111 Table 1. The results showed that compound 12r, 12t and 12u exhibited good
112 inhibition activities on MCF-7, Raji and H460 cells at the concentration of 1 µM.
113 However, none of the title compounds were sensitive to MDA-MB-231 cells. Careful
114 examination and establishment of a significant regular structure activity relationship
115 (SAR) for compounds 12f-r indicates that a slightly bulk and electron-withdrawing

PT
116 substituent at the 4 position of the benzyl group, will lead to more active compounds.
117 In this, 12s-w were synthesized to verify the SAR analysis, and fortunately, it turned

RI
118 out to be correct.

U SC
119
120 Table 1.
AN
121 Antitumor activity of the synthesized β-carboline/acylhydrazone hybrids in vitro

inhibition ratea (1 µM, %)

M

compd. R
MCF-7 MDA-MB-231 Raji H460

12a -13.75 8.13 9.69 5.08

D

OH
TE

12b 35.72 21.76 51.75 40.74

NH

12c 6.31 7.10 8.66 16.24

EP

12d 36.57 29.26 62.47 44.26

C
AC

12e
O
-18.61 6.76 2.06 22.10

12f O 23.14 25.57 50.16 29.70

12g O 46.24 24.72 54.33 43.19

F
12h O 17.11 16.48 40.36 21.69
 ACCEPTED MANUSCRIPT

12i O 23.65 27.25 47.97 29.67

F

Cl

12j O 36.17 28.64 56.31 45.40

Cl
12k O 22.37 15.63 48.22 31.93

PT
12l O 24.26 30.73 56.74 40.44

RI
Cl

Me

18.18 24.83 36.03 29.39

SC
12m O

Me
12n 26.34 22.69 33.21 27.39

U
O AN
12o O 53.48 28.69 44.95 29.24
Me
M

CN

12p O 15.09 20.74 37.39 15.48

D

CN
12q O 17.92 21.88 24.05 12.81
TE

12r O 59.63 29.55 42.69 51.00

EP

CN

12s O 36.98 23.60 52.03 38.60

C

OMe
AC

12t O 50.36 31.76 58.70 52.78

Br

12u O 56.43 31.82 63.79 25.89

COOMe

12v O 38.39 24.38 51.22 23.37

CF3
 ACCEPTED MANUSCRIPT

12w O 32.80 20.17 56.69 38.36

NO2

OMe

12x O -6.31 22.16 25.84 20.67

4 59.32 41.28 51.66 43.25

PT
a
122 % inhibition rate after drug treatment for 72h.

The inhibition rate of the synthesized compounds greater than 50% at 1 µM were

RI
123

124 chosen to proceed to examine the half maximal inhibitory concentration (IC50). The

SC
125 results were shown in Table 2. For relatively lack of activity on MDA-MB-231 cells,
126 further in vitro antitumor activity screening on this cell line was not performed. All

U
127 the chosen compounds showed moderate to good antitumor activities in the low
micromolar range with IC50 values from 1 µM to 10 µM, except 12b (MCF-7
AN
128

129 IC50=14.92 ± 1.02 µM). It is worth mentioning that 12r owning a cyanogroup, a
130 strong electron-withdrawing substituent with proper size, exhibited the best active on
M

131 almost the three tested tumor cells with IC50 values of 1.73 ± 0.10 µM (H460), 0.93 ±
D

132 0.03 µM (MCF-7), 1.20 ± 0.07 µM (Raji).

133 Table 2.
TE

134 Antiproliferative activities of the chosen compounds against H460, MCF-7 and Raji
135 cells in vitro
EP

IC50a (µM) IC50a (µM)

compd. compd.
H460 MCF-7 Raji H460 MCF-7 Raji
C

12b 5.71±0.61 14.92±1.02 2.04±0.10 12r 1.73±0.10 0.93±0.03 1.20±0.07

AC

12d 3.64±0.32 4.56±0.36 1.04±0.13 12s 2.84±0.25 9.37±0.88 2.34±0.19

12f 6.52±0.56 3.56±0.30 2.62±0.21 12t 2.42±0.14 7.22±0.46 6.74±0.55

12g 1.36±0.11 6.50±0.44 2.94±0.12 12u 1.17±0.06 1.64±0.16 3.88±0.15

12j 1.27±0.08 2.32±0.13 3.02±0.18 12v 7.49±0.32 6.60±0.43 2.04±0.17

12l 5.02±0.43 8.62±0.76 5.02±0.59 12w 1.04±0.08 8.30±0.69 3.02±0.22

12o 2.18±0.18 5.24±0.54 4.06±0.23 4 0.47±0.03 0.62±0.02 0.98±0.06

 ACCEPTED MANUSCRIPT
a
136 50% inhibitory concentration after drug treatment for 72h. Data are expressed as the
137 mean ± SD from the dose-response curves of at least three independent experiment.

138 To evaluate the influence made by acylhydrazone scaffold to efficiency, 15a-d

139 were synthesized (Scheme 2) and tested for their in vitro antitumor activity (Table 3).
140 1,3,4-oxadiazole was a cyclization product of diacylhydrazone. None of the title

PT
141 compounds showed any activity to the tested tumor cells, thus proving that
142 acylhydrazone scaffold was essential to antitumor activity.

RI
143 Table 3.

SC
144 In vitro antitumor activity of the synthesized β-carboline/1,3,4-oxadiazole hybrids

U
AN
145

inhibition ratea （1 µM, %）

M

Entry R
MCF-7 MDA-MB-231 Raji H460
O
D

15a -5.47 7.42 1.69 4.84

TE

15b -2.52 2.98 9.17 6.63

CHO

O CHO
15c 26.76 9.83 17.71 13.94
EP

15d N -1.84 -3.26 16.28 3.74

C

4 59.32 41.28 51.66 43.25

a
AC

146 % inhibition rate after drug treatment for 72h.

147 2.2.2 Effect on multidrug resistant cells

148 Drug resistance is a major obstacle for the first-line chemotherapy [39, 40]. The
149 common mechanism of resistance identified in preclinical or clinical study involves
150 two main categories: the overexpression of a cellular membrane protein called
151 P-glycoprotein (P-gp) and changing in the levels of expression of different β-tubulin
 ACCEPTED MANUSCRIPT

152 isotypes (β-III gene) [41-45]. As it has been known, paclitaxel resistant A549/T
153 (human lung cancer cell line), vinblastine resistant HCT-8/V (human colon cancer cell
154 lines), and adriamycin resistant MCF-7/ADR (human breast cancer cell line) are all
155 P-gp overexpressed, and paclitaxel resistant A2780/T (human ovarian cancer cell lines)
156 are β-tubulin III overexpressed [46-48]. Hence, the activities of 12r in these resistant

PT
157 and their related sensitive cancer cells were evaluated and compared, with vinblastine,
158 paclitaxel, colchicine and adriamycin as reference compounds. As shown in Table 4,

RI
159 12r exhibited potent cytotoxic activity against A549T (IC50 = 3.13 µM), A2780/T
160 (IC50 = 3.99 µM), and MCF-7/ADR (IC50 = 0.98 µM). Compared with vinblastine,

SC
161 paclitaxel, colchicine and adriamycin, 12r had much lower drug resistance indexes
162 (2.28 for A549/T, 1.32 for A2780/T, 1.06 for MCF-7/ADR) than vinblastine (12.72 for

U
163 A549/T, 1073.76 for A2780/T), paclitaxel (32.76 for A549/T, 3852.53 for A2780/T),
AN
164 colchicine (60.41 for A549/T, 1630.02 for A2780T) and adriamycin (1221.53 for
165 MCF-7/ADR). These results indicated that 12r might be useful in the treatment of
M

166 tumors with resistance to other anticancer drugs.

167 Table 4.
D

168 Drug tolerances of 12r against different drug resistant cancer cells.
TE

IC50a (nM)
compd. R.R.b
A549 A549T
EP

12r 1373.25±84.85 3132.38±532.44 2.28

vinblastine 19.51±0.53 248.27±3.88 12.72

C

paclitaxel 23.18±0.69 759.39±5.54 32.76

AC

colchicine 32.76±0.91 167.34±2.77 60.41

IC50a (nM)

A2780S A2780T

12r 3023.37±66.47 3992.89±165.58 1.32

vinblastine 15.73±0.55 16890.22±5.61 1073.76

paclitaxel 18.94±0.68 72967.46±8.12 3852.53

 ACCEPTED MANUSCRIPT

colchicine 21.86±0.71 35632.33±4.38 1630.02

IC50a (nM)

MCF-7 MCF-7/ADR

12r 924.33±15.30 982.36±16.76 1.06

adriamycin 44.82±0.10 54749.19±6.56 1221.53

PT
a
169 50% inhibitory concentration after drug treatment for 72h. Data are expressed as the
170 mean ± SD from the dose-response curves of at least three independent experiments.

RI
b
171 Resistance ratio: (IC50 value of drug resistant cancer cell) / (IC50 value of drug
172 parental cancer cell)

SC
173 2.2.3 Effect on non-cancerous HUVEC cell line

U
174 The previous investigations have verified that 12r had good antiproliferative
AN
175 activity on the tested tumor cell lines and different drug resistant cancer cells. It was
176 necessary to find out whether 12r was toxic for normal cells. Thus, the
M

177 antiproliferative activity of 12r on Human Umbilical Vein Endothelial Cells (HUVEC,
178 a non-cancerous cell line) was performed. As depicted in Table 5, 12r exhibited
D

179 potent activity on 3 tested tumor cell lines but very weak activity on HUVEC cell line
TE

180 (IC50 = 88.63 µM). The antiproliferative activity of 12r on cancerous cell lines was
181 more than 50-fold than the non-cancerous HUVEC cell line. These results indicated
EP

182 that 12r might have good antitumor activity but non-toxic to normal cells when
183 treated tumors in vivo.
C

184 Table 5.
185 In vitro antiproliferative activity of 12r on HUVEC.
AC

IC50a (µM)
H460 MCF-7 Raji HUVEC
12r 1.64±0.08 1.02±0.02 1.06±0.05 88.63±4.33
a
186 50% inhibitory concentration after drug treatment for 72h. Data are expressed as the
187 mean ± SD from the dose-response curves of at least three independent experiment.
188 2.2.4 Cell cycle effects and induced apoptosis

189 To explore whether the cytotoxicity of 12r was due to the cell cycle arrest, the
 ACCEPTED MANUSCRIPT

190 effects on cell cycle progression was detected using propidium iodide (PI) staining by
191 flow cytometry in MCF-7 cells. As depicted in Fig. 3 (A, B), no interference on the
192 cell cycle profile was observed with compound 12r at the concentration of 3 µM, but
193 the treatment with 10 -100 µM promoted slightly reduction of cells in G2/M phase in
194 a concentration-dependant manner, which was accompanied by increased percentages

PT
195 of cells in the G0/G1 phase. That is to say there is a sign of sub-G1 arrest, which
196 indicates DNA fragmentation, typical of cell death. Evidences have showed that

RI
197 β-carboline scaffold can intercalate with DNA, alter DNA replication fidelity, and
198 influence enzymatic activities in DNA repair processes [25-27]. Though 12r resulted

SC
199 in unconspicuous sub-G1 arrest, the character of interacting with DNA still remained
200 more or less, because of the presence of polycyclic aromatic planar pharmacophore.

U
201 Overall, the results suggested that the cytotoxicity in MCF-7 of 12r made nearly no
AN
202 difference with cell cycle arrest.
M
D
TE
C EP
AC

203

204 Figure 3. A) Effects of 12r on cell cycle phase arrest in MCF-7 cells. Cells were
205 treated with 3, 10, 30 and 100 µM for 18 h. B) Percentages of MCF-7 cells in the
206 different phases of the cell cycle.
207 Next, we evaluated the ability of 12r to induce MCF-7 cell apoptosis by a
208 biparametric cytofluorimetric analysis using fluorescent immunolabeling of the
 ACCEPTED MANUSCRIPT

209 protein annexin-V (V-FITC) and propidium iodide (PI). Dual staining for annexin-V
210 and with PI can provide discrimination between live cells (annexin-V-/PI-), early
211 apoptotic cells (annexin-V+/PI-), late apoptotic cells (annexin-V+/PI+), and necrotic
212 cells (annexin-V-/PI+). As depicted in Fig. 4 (A, C), treatment with 12r for 48 h
213 induced a remarkable accumulation of late apoptotic cells (annexin-V+/PI+) in

PT
214 comparison with control in a concentration-dependent manner. To understand whether
215 the cytotoxicity of 12r on drug resistant cancer cells was due to cell apoptosis, the

RI
216 effects of 12r induced MCF-7/ADR cancer cell apoptosis were detected as the same
217 method as above. As shown in Fig. 4 (B, D), likewise, 12r induced a remarkable

SC
218 accumulation of late apoptotic cells (annexin-V+/PI+).

U
AN
M
D
TE
C EP
AC

219
220 Figure 4. A) Representative flow cytometric histograms of apoptotic MCF-7 cells
221 after 48 h treatment with 12r. B) Representative flow cytometric histograms of
222 apoptotic MCF-7/ADR cells after 48 h treatment with 12r.The cells were harvested
223 and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. C)
224 Percentage of MCF-7 cells found in the different regions of the biparametric
225 histograms after incubation with 12r for 48 h as indicated. D) Percentage of
 ACCEPTED MANUSCRIPT

226 MCF-7/ADR cells found in the different regions of the biparametric histograms after
227 incubation with 12r for 48 h as indicated.
228 2.2.5 Antitumor activity in vivo

229 H460 xenograft model was established in nude mice to determine whether the
230 antitumor ability of 12r could also be reflected in vivo. H460 cells were inoculated by

PT
231 sc injection of 5 × 106 cells in the right flank of the mice. When the tumor volume
232 once reached a size of 100 mm3, the mouse were selected and randomized into

RI
233 treatment groups (6 mice per group). Then 12r was given orally (poorly soluble to ip)

SC
234 at dose of 100 mg/kg three times a week. Taxol, a positive control drug, was
235 administrated once a week with 30 mg/kg ip for comparison. As shown in Fig. 5, 12r

U
236 caused a slightly suppression of tumor growth and the average inhibitory rate reached
237 17.18% at day 18, while taxol at a dosage of 30 mg/kg caused 65.21% tumor
AN
238 reduction. Taxol has been in the clinic for about 20 years, rare antitumor candidate
239 could surpass it. The antitumor activities of 12r were far from taxol both in vitro and
M

240 in vivo. The low in vivo antitumor activity might partly because first pass effect which
D

241 will lead to low pharmacological bioavailability. During the therapy, only slight
242 weight losses (<5%) were observed in both of the two drug treatment groups. Serious
TE

243 toxic and side effects including feeding and abnormal behavior were not observed in
244 12r-treatment group.
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
245
246 Figure 5. Antitumor effect of 12r and taxol on the H460 xenograft models. dosing
M

247 schedules (3 times a week for 12r; 1 time a week for taxol) A) 12r inhibited tumor
248 growth on the H460 xenograft model. Tumor volumes were measured every 2 days
D

249 when administrated with a caliper (calculated volume (mm3)) = (π/6) × length × width
TE

250 × width). B) 12r caused mice body weight loss. Body weights were measured every 2
251 days. C, D) 12r inhibited tumor weight loss.
EP

252 3. Conclusion

In this research, 24 novel β-carboline/acylhydrazone hybrids were synthesized

C

253

254 and evaluated for their antiproliferative activities. 4 β-carboline/1,3,4-oxadiazole

AC

255 hybrids were designed and synthesized to verify that acylhydrazone scaffold was vital
256 important for biological activity. Among the 24 title compounds, compound 12r
257 exhibited potent antitumor abilities in several human tumor cell lines including
258 multidrug resistant human tumor cell lines. 12r made no differences in cell cycle
259 arrest but induced remarkable accumulation of late apoptotic cells (annexin-V+/PI+)
260 on both MCF-7 and MCF-7/ADR cancer cells in a concentration-dependent manner.
261 12r exhibited potent cytotoxic activity against different drug resistant cancer cells
 ACCEPTED MANUSCRIPT

262 A549T, A2780/T, and MCF-7/ADR. In vivo activity of 12r evaluated on H460
263 xenografts models showed certain antitumor effect on inhibiting the tumor growth
264 with low toxic and side effects and without significant body weight loss. Therefore,
265 12r represented by the β-carboline/acylhydrazone hybrids was proved to be active but
266 less than ideal situation. Efforts must be tried to find out the target for this kind of

PT
267 compounds and improve their solubility and biological activity.
268 4. Experimental

RI
269 4.1 Chemistry

SC
270 All the chemical solvents and reagents used in this study were analytically pure
271 without further purification and commercially available. TLC was performed on 0.20

U
272 mm silica gel 60 F254 plates (Qingdao Ocean Chemical Factory, Shandong, China).
273 Visualization of spots on TLC plates was done by UV light. Melting points were
AN
274 recorded with a micro melting point tester (Neware Technology Ltd., Guangdong,
275 China) and are uncorrected. NMR data were measured for 1H at 400 MHz and for 13C
M

276 at 101 MHz on a Bruker Avance 400 spectrometer (Bruker Company, Germany) using
D

277 TMS as an internal standard. High Resolution Mass Spectra (HRMS) were recorded
278 on a Q-TOF Bruker Daltonics model IMPACT II massspectrometer (Micromass,
TE

279 Manchester, UK) in a positive mode.

280 4.1.1 General procedure for the synthesis of Methyl L-tryptophanate hydrochloride
EP

281 (7)
282 To a suspension of 2.04 g (10 mmol) of commercial L-tryptophan (6) in 50 mL of
C

283 MeOH was stirred at 0 ℃. Thionyl chloride (0.88 mL, 12 mmol) was added dropwise
AC

284 over 10 mins. The reaction mixture was then allowed to warm to room temperature
285 before being heated under reflux for 3h. The solvent and volatiles were evaporated
286 under reduced pressure and the product was triturated with ethyl acetate to give the
287 methyl ester hydrochloride salt as a colorless solid. The pure product was obtained in
288 95% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.82 (s, 3H), 7.54 (d, J =
289 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 7.13 – 7.05 (m, 1H),
290 7.04 – 6.94 (m, 1H), 4.19 (dd, J = 7.0, 5.4 Hz, 1H), 3.62 (s, 3H), 3.44 – 3.28 (m, 2H).
 ACCEPTED MANUSCRIPT

291 4.1.2 General procedure for the synthesis of (S)-methyl

292 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate hydrochloride (8)
293 To a stirring solution of methyl L-tryptophen ester hydrochloride (24.5 g, 96.1
294 mmol) in methanol (250mL) was added 37% aqueous formaldehyde solution (8.65g,
295 55 mmol) at room temperature. The resulting mixture was stirred at reflux for 3 h.

PT
296 The total volume was reduced in vacuo to approximately 50 mL, where upon a white
297 precipitate formed. Methyl-tert-butyl ester (100 mL) was added, and the white

RI
298 suspension was stirred for 15 min. The resulting solid was received through filtration
299 and then dried on high vacuum to afford pure compound 8 (22.3 g, 89% yield) as a

SC
300 fluffy white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 7.48 (d, J = 7.8 Hz,
301 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.15 – 7.06 (m, 1H), 7.05 – 6.96 (m, 1H), 4.63 (dd, J =

U
302 10.0, 5.3 Hz, 1H), 4.40 (s, 2H), 3.82 (s, 3H), 3.44 – 3.23 (m, 3H), 3.08 (dd, J = 16.0,
AN
303 9.9 Hz, 1H).
304 4.1.3 General procedure for the synthesis of methyl
M

305 9H-pyrido[3,4-b]indole-3-carboxylate (9)

306 To a stirred solution of compound 8 (7.95 g, 34.5 mmol) and triethyl amine (9.09
D

307 g, 89.8 mmol) in 50 mL DMF, a DMF solution of trichloroiscyanuric acid (TCCA,

TE

308 8.0 g, 34.5 mmol) was added slowly at -20 ℃. After the addition was completed, the
309 reaction was allowed to warm slowly up to 0 ℃ with stirring for 2 h. The resulting
EP

310 product was precipitated from ice water, washed with ice water and dried in vacuo.
311 The crude product yielding 80% as a gray solid and was used without further
purification. 1H NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H), 8.97 (d, J = 0.9 Hz, 1H),
C

312

313 8.92 (s, 1H), 8.40 (d, J = 7.9 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.60 (ddd, J = 8.3, 6.9,
AC

314 1.1 Hz, 1H), 7.32 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 3.91 (s, 3H).
315 4.1.4 General procedure for the synthesis of 9H-pyrido[3,4-b]indole-3-carbohydrazide
316 (10)
317 A solution of compound 9 (12.0 g, 50 mmol) in methanol (120 mL) containing
318 hydrazine hydrate (85%, 20 mL) was refluxed for 6 h until there were no starting
319 materials (TLC control). The resulting mixture was cooled, and the precipitate that
320 formed was collected by filtration, washed with methanol (1 × 20 mL) and dried
 ACCEPTED MANUSCRIPT

321 under vacuum to afford compound 10 (10.3 g, 85% yield). 1H NMR (400 MHz,
322 DMSO-d6) δ 11.93 (s, 1H), 9.66 (s, 1H), 8.89 (d, J = 0.8 Hz, 1H), 8.83 (d, J = 0.8 Hz,
323 1H), 8.41 (d, J = 7.9 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.60 (ddd, J = 8.1, 6.8, 1.0 Hz,
324 1H), 7.30 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 4.56 (s, 2H).
325 4.1.5 General procedure for the synthesis of β-carboline/acylhydrazone hybrids

PT
326 (12a-x)
327 To a solution of compound 10 (1.0 mmol) in methanol (20 mL) was added the

RI
328 corresponding aromatic aldehyde (1.0 mmol). The reaction mixture was catalyzed by
329 concentrated hydrochloric acid and refluxed for 4 -6 h, and monitored by TLC. After

SC
330 consumption of starting material the mixture was cooled and the precipitate formed
331 was collected by filtration and washed with methanol to afford the title compounds

U
332 12a-x.
AN
333 N'-(3-hydroxybenzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (12a)
334 White solid, yield: 90%. M.P. > 300 ℃. 1H NMR (400 MHz, DMSO-d6) δ 12.02 (s,
M

335 2H), 9.63 (s, 1H), 8.98 (s, 1H), 8.96 (s, 1H), 8.58 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H),
336 7.69 (d, J = 8.2 Hz, 1H), 7.66 – 7.58 (m, 1H), 7.38 – 7.19 (m, 3H), 7.11 (d, J = 7.6 Hz,
D

337 1H), 6.84 (dd, J = 8.0, 1.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 161.63, 158.15,
TE

338 151.63, 148.57, 141.54, 139.36, 137.86, 136.35, 132.82, 130.33, 128.76, 122.85,
339 121.39, 120.62, 119.18, 117.75, 115.47, 113.17, 112.81. HRMS-ESI: calcd for
C19H15N4O2 [M+H]+ 331.1196, found: 331.1192.
EP

340

341 N'-(naphthalen-2-ylmethylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (12b)

White solid, yield: 91%. M.P. >300 ℃. 1H NMR (400 MHz, DMSO-d6) δ 12.20 (s,
C

342

343 1H), 12.04 (s, 1H), 9.01 (s, 1H), 8.99 (s, 1H), 8.84 (s, 1H), 8.48 (d, J = 7.9 Hz, 1H),
AC

344 8.14 (s, 1H), 8.05 (q, J = 3.1 Hz, 1H), 8.04 – 7.99 (m, 2H), 7.96 (dt, J = 6.8, 3.4 Hz,
345 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.63 (ddd, J = 8.2, 6.9, 1.0 Hz, 1H), 7.58 (dq, J = 6.8,
346 3.6 Hz, 2H), 7.34 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
347 161.75, 148.42, 141.54, 139.40, 137.88, 134.17, 133.37, 132.87, 132.82, 129.25,
348 128.97, 128.95, 128.80, 128.24, 127.51, 127.20, 123.31, 122.89, 121.42, 120.65,
349 115.58, 112.80. HRMS-ESI: calcd for C23H16N4ONa [M+Na]+ 387.1222, found:
350 387.1222.
 ACCEPTED MANUSCRIPT

351 N'-((1H-indol-3-yl)methylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (12c)

352 White solid, yield: 86%. M.P. 202.9 - 204.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ
353 12.00 (s, 1H), 11.72 (s, 1H), 11.58 (s, 1H), 8.98 – 8.92 (m, 2H), 8.80 (s, 1H), 8.46 (d,
354 J = 7.9 Hz, 1H), 8.35 (d, J = 7.3 Hz, 1H), 7.79 (d, J = 2.7 Hz, 1H), 7.69 (d, J = 8.3 Hz,
355 1H), 7.66 – 7.58 (m, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.35 – 7.32 (m, 1H), 7.24 -7.19 (m,

PT
13
356 2H). C NMR (101 MHz, DMSO-d6) δ 160.99, 145.52, 141.53, 139.99, 137.71,
357 137.48, 132.75, 130.36, 129.18, 128.78, 124.96, 123.06, 122.82, 122.47, 121.41,

RI
358 120.80, 120.55, 115.01, 112.77, 112.37, 112.29. HRMS-ESI: calcd for C21H15N5ONa
359 [M+Na]+ 376.1175, found: 376.1173.

SC
360 N'-([1,1'-biphenyl]-4-ylmethylene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (12d)
361 White solid, yield: 90%. M.P. >300 ℃. 1H NMR (400 MHz, DMSO-d6) δ 12.12 (s,

U
362 1H), 12.04 (s, 1H), 9.0 (s, 1H), 8.98 (s, 1H), 8.73 (s, 1H), 8.47 (d, J = 7.8 Hz, 1H),
AN
363 7.82 (dd, J = 18.9, 8.3 Hz, 4H), 7.72 (dd, J = 17.0, 7.9 Hz, 3H), 7.63 (t, J = 7.6 Hz,
13
364 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.3 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H). C
M

365 NMR (101 MHz, DMSO-d6) δ 161.68, 148.05, 141.87, 141.54, 139.83, 139.38,
366 137.88, 134.20, 132.82, 129.48, 129.25, 128.80, 128.31, 128.14, 127.50, 127.12,
D

367 122.87, 121.42, 120.64, 115.54, 112.80. HRMS-ESI: calcd for C25H18N4ONa
[M+Na]+ 413.1379, found: 413.1373.
TE

368

369 N'-(3-phenoxybenzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (12e)

White solid, M.P. 253.6 - 254.1 ℃. 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H),
EP

370

371 12.01 (s, 1H), 8.95 (s, 2H), 8.64 (s, 1H), 8.45 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 8.2 Hz,
1H), 7.62 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.57 – 7.39 (m, 4H), 7.37 – 7.26 (m, 2H),
C

372
13
373 7.24 – 7.16 (m, 1H), 7.15 – 7.00 (m, 3H). C NMR (101 MHz, DMSO-d6) δ 161.68,
AC

374 157.73, 156.82, 147.77, 141.52, 139.24, 137.87, 137.09, 132.81, 131.07, 130.67,
375 129.26, 128.76, 124.30, 123.17, 122.86, 121.38, 120.65, 119.44, 116.17, 115.53,
376 112.80. HRMS-ESI: calcd for C25H18N4O2Na [M+Na]+ 429.1328, found: 429.1327.
377 N'-(3-(benzyloxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide (12f)
378 White solid, yield: 93%. M.P. 238.8 - 240.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ
379 12.10 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.47 (d, J = 7.9
380 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.3 Hz, 2H),
 ACCEPTED MANUSCRIPT

381 7.46 – 7.30 (m, 7H), 7.11 (dd, J = 8.0, 1.9 Hz, 1H), 5.18 (s, 2H). 13C NMR (101 MHz,
382 DMSO-d6) δ 161.69, 159.16, 148.34, 141.53, 139.34, 137.87, 137.40, 136.56, 132.81,
383 130.45, 129.25, 128.90, 128.78, 128.32, 128.16, 122.87, 121.40, 120.64, 117.19,
384 115.53, 112.89, 112.80, 69.77. HRMS-ESI: calcd for C26H20N4O2Na [M+Na]+
385 443.1484, found: 443.1485.

PT
386 N'-(3-((2-fluorobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
387 (12g)

RI
388 White solid, yield: 92%. M.P. 256.9 - 257.4 ℃. 1H NMR (400 MHz, DMSO-d6) δ
389 12.09 (s, 1H), 12.03 (s, 1H), 8.98 (s, 1H), 8.96 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9

SC
390 Hz, 1H), 7.69 (d, J=8.3 Hz, 1H), 7.65 - 7.59 (m, 1H), 7.50 – 7.38 (m, 3H), 7.37 – 7.28
391 (m, 4H), 7.21 – 7.14 (m, 1H), 7.11 (dd, J = 8.0, 2.1 Hz, 1H), 5.22 (s, 2H). 13C NMR

U
392 (101 MHz, DMSO-d6) δ 162.10, 161.69, 159.65, 158.98, 148.26, 141.53, 139.33,
AN
393 137.86, 136.62, 132.80, 131.17, 131.13, 130.92, 130.84, 130.51, 129.25, 128.77,
394 125.03, 125.00, 124.27, 124.12, 122.87, 121.40, 120.77, 120.64, 117.08, 115.97,
M

19
395 115.76, 115.53, 112.93, 112.80, 64.17, 64.13. F NMR (376 MHz, DMSO-d6) δ
396 -118.26. HRMS-ESI: calcd for C26H19FN4O2Na [M+Na]+ 461.1390, found: 461.1390.
D

397 N'-(3-((3-fluorobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
TE

398 (12h)
399 White solid, yield: 87%. M.P. 217.5 - 217.9 ℃. 1H NMR (400 MHz, DMSO-d6) δ
EP

400 12.10 (s, 1H), 12.03 (s, 1H), 8.98 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9
401 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.66 - 7.56 (m, 2H), 7.48 – 7.37 (m, 3H), 7.37 –
13
7.21 (m, 4H), 7.13 (dd, J = 8.2, 2.6 Hz, 1H), 5.23 (s, 2H). C NMR (101 MHz,
C

402

403 DMSO-d6) δ 163.89, 161.70, 161.47, 158.94, 148.29, 141.53, 140.41, 140.34, 139.33,
AC

404 137.87, 136.60, 132.80, 130.97, 130.89, 130.48, 129.24, 128.77, 123.97, 122.86,
405 121.40, 120.84, 120.63, 117.19, 115.54, 115.16, 114.95, 114.79, 114.57, 112.84,
19
406 68.90. F NMR (376 MHz, DMSO-d6) δ -113.13. HRMS-ESI: calcd for
407 C26H19FN4O2Na [M+Na]+ 461.1390, found: 461.1388.
408 N'-(3-((4-fluorobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
409 (12i)
 ACCEPTED MANUSCRIPT

410 White solid, yield: 91%. M.P. 258.6 - 260.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ
411 12.10 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9
412 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.66 - 7.59 (m, 1 H), 7.59 – 7.52 (m, 2H), 7.44 –
413 7.37 (m, 2H), 7.37 - 7.29 (m, 2H), 7.28 – 7.20 (m, 2H), 7.10 (dd, J = 8.1, 1.8 Hz, 1H),
13
414 5.17 (s, 2H). C NMR (101 MHz, DMSO-d6) δ 163.46, 161.68, 161.04, 159.06,

PT
415 148.30, 141.52, 139.33, 137.86, 136.57, 133.65, 132.80, 130.48, 130.40, 129.25,
416 128.77, 122.88, 121.39, 120.72, 120.64, 117.21, 115.83, 115.62, 115.54, 112.83,

RI
19
417 112.80, 69.06. F NMR (376 MHz, DMSO-d6) δ -114.42. HRMS-ESI: calcd for
418 C26H19FN4O2Na [M+Na]+ 461.1390, found: 461.1384.

SC
419 N'-(3-((2-chlorobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
420 (12j)

U
421 White solid, yield: 89%. M.P. 254.2 - 255.1 ℃. 1H NMR (400 MHz, DMSO-d6) δ
AN
422 12.09 (s, 1H), 12.04 (s, 1H), 8.98 (s, 1H), 8.96 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9
423 Hz, 1H), 7.71 – 7.59 (m, 3H), 7.57 – 7.51 (m, 1H), 7.46 – 7.37 (m, 4H), 7.37 – 7.29
M

424 (m, 2H), 7.13 (dd, J = 8.0, 1.9 Hz, 1H), 5.24 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
425 δ 161.69, 158.98, 148.26, 141.53, 139.33, 137.87, 136.64, 134.67, 133.12, 132.81,
D

426 130.64, 130.56, 130.38, 129.87, 129.26, 128.77, 127.87, 122.87, 121.40, 120.90,
TE

427 120.64, 117.11, 115.53, 112.83, 112.80, 67.43. HRMS-ESI: calcd for
428 C26H19ClN4O2Na [M+Na]+ 477.1095, found: 477.1096.
EP

429 N'-(3-(3-chlorophenoxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
430 (12k)
White solid, yield: 92%. M.P. 264.3 - 265.0 ℃. 1H NMR (400 MHz, DMSO-d6) δ
C

431

432 12.08 (s, 1H), 12.02 (s, 1H), 8.97 (d, J = 5.0 Hz, 2H), 8.65 (s, 1H), 8.46 (d, J = 7.9 Hz,
AC

433 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.57 (s, 1H), 7.51 – 7.37 (m,
13
434 5H), 7.33 (dt, J = 7.2, 3.5 Hz, 2H), 7.11 (dd, J = 8.1, 2.2 Hz, 1H), 5.21 (s, 2H). C
435 NMR (101 MHz, DMSO-d6) δ 161.70, 158.92, 148.28, 141.53, 140.01, 139.34,
436 137.87, 136.61, 133.59, 132.80, 130.82, 130.48, 129.24, 128.78, 128.23, 127.77,
437 126.63, 122.87, 121.40, 120.85, 120.63, 117.19, 115.54, 112.82, 112.80, 68.84.
438 HRMS-ESI: calcd for C26H19ClN4O2Na [M+Na]+ 477.1095, found: 477.1093.
 ACCEPTED MANUSCRIPT

439 N'-(3-((4-chlorobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
440 (12l)
441 White solid, yield: 94%. M.P. 277.5 - 278.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ
442 12.10 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9
443 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H),

PT
444 7.48 (d, J = 8.5 Hz, 2H), 7.44 – 7.37 (m, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.10 (dd, J =
13
445 8.0, 2.0 Hz, 1H), 5.19 (s, 2H). C NMR (101 MHz, DMSO-d6) δ 161.68, 158.98,

RI
446 148.28, 141.53, 139.33, 137.86, 136.59, 136.49, 132.89, 132.80, 130.48, 129.97,
447 129.25, 128.91, 128.77, 122.87, 121.40, 120.79, 120.64, 117.19, 115.53, 112.88,

SC
448 112.80, 68.93. HRMS-ESI: calcd for C26H20ClN4O2 [M+H]+ 455.1276, found:
449 455.1278.

U
450 N'-(3-((2-methylbenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
AN
451 (12m)
452 White solid, yield: 89%. M.P. 252.9 - 253.5 ℃. 1H NMR (400 MHz, DMSO-d6) δ
M

453 12.10 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.66 (s, 1H), 8.46 (d, J = 7.9
454 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.50 – 7.38 (m, 3H), 7.36 –
D

455 7.29 (m, 2H), 7.29 - 7.18 (m, 3H), 7.13 (dd, J = 8.1, 2.5 Hz, 1H), 5.16 (s, 2H), 2.37 (s,
13
TE

456 3H). C NMR (101 MHz, DMSO-d6) δ 161.69, 159.28, 148.36, 141.54, 139.33,
457 137.87, 137.15, 136.56, 135.28, 132.80, 130.61, 130.46, 129.25, 129.12, 128.78,
EP

458 128.59, 126.26, 122.87, 121.40, 120.64, 117.15, 115.53, 112.79, 68.53, 18.98.
459 HRMS-ESI: calcd for C27H22N4O2Na [M+Na]+ 457.1641, found: 457.1644.
N'-(3-(3-methylbenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
C

460

461 (12n)
AC

462 White solid, yield: 91%. M.P. 266.3 - 267.6 ℃. 1H NMR (400 MHz, DMSO-d6) δ
463 12.08 (s, 1H), 12.03 (s, 1H), 8.98 (d, J = 7.2 Hz, 2H), 8.65 (s, 1H), 8.46 (d, J = 7.9 Hz,
464 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.44 – 7.36 (m, 2H), 7.36 –
465 7.22 (m, 5H), 7.20 – 7.13 (m, 1H), 7.10 (dd, J = 8.1, 1.8 Hz, 1H), 5.13 (s, 2H), 2.34 (s,
13
466 3H). C NMR (101 MHz, DMSO-d6) δ 161.72, 159.19, 148.36, 141.54, 139.35,
467 138.06, 137.88, 137.29, 136.55, 132.81, 130.43, 129.24, 128.95, 128.79, 128.71,
468 125.24, 122.86, 121.41, 120.63, 117.16, 115.55, 112.85, 112.80, 69.79, 21.47.
 ACCEPTED MANUSCRIPT

469 HRMS-ESI: calcd for C27H23N4O2 [M+H]+ 435.1822 found: 435.1821.

470 C27H22N4O2Na [M+Na]+ 457.1641, found: 457.1640.
471 N'-(3-((4-methylbenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
472 (12o)
473 White solid, yield: 84%. M.P. 276.4 - 278.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ

PT
474 12.09 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9
475 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.66 – 7.58 (m, 1H), 7.42 – 7.29 (m, 6H), 7.22 (d, J

RI
13
476 = 7.8 Hz, 2H), 7.09 (dd, J = 8.1, 1.9 Hz, 1H), 5.13 (s, 2H), 2.32 (s, 3H). C NMR
477 (101 MHz, DMSO-d6) δ 161.70, 159.17, 148.37, 141.54, 139.33, 137.87, 137.54,

SC
478 136.53, 134.34, 132.80, 130.41, 129.44, 129.24, 128.78, 128.24, 122.86, 121.40,
479 120.63, 120.57, 117.20, 115.53, 112.92, 112.80, 69.67, 21.25. HRMS-ESI: calcd for

U
480 C27H22N4O2Na [M+Na]+ 457.1641, found: 457.1633.
AN
481 N'-(3-((2-cyanobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
482 (12p)
M

483 White solid, yield: 90%. M.P. 257.4 - 258.6 ℃. 1H NMR (400 MHz, DMSO-d6) δ
484 12.11 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.67 (s, 1H), 8.47 (d, J = 7.9
D

485 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.66
TE

486 – 7.56 (m, 2H), 7.48 – 7.40 (m, 2H), 7.39 – 7.28 (m, 2H), 7.15 (dd, J = 7.9, 1.9 Hz,
487 2H), 5.33 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.70, 158.88, 148.20, 141.53,
EP

488 140.37, 139.32, 137.87, 136.67, 133.91, 133.78, 132.80, 130.57, 130.16, 129.60,
489 129.25, 128.78, 122.86, 121.39, 121.10, 120.64, 117.70, 117.10, 115.54, 112.92,
112.80, 111.84, 68.17. HRMS-ESI: calcd for C27H19N5O2Na [M+Na]+ 468.1437,
C

490

491 found: 468.1434.

AC

492 N'-(3-((3-cyanobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
493 (12q)
494 White solid, yield: 88%. M.P. 222.8 - 224.1 ℃. 1H NMR (400 MHz, DMSO-d6) δ
495 12.10 (s, 1H), 12.03 (s, 1H), 8.98 (s, 1H), 8.97 (s, 1H), 8.66 (s, 1H), 8.47 (d, J = 7.9
496 Hz, 1H), 7.98 (s, 1H), 7.90 – 7.79 (m, 2H), 7.72 – 7.58 (m, 3H), 7.47 – 7.37 (m, 2H),
497 7.37 - 7.27 (m, 2H), 7.13 (dd, J = 7.8, 2.1 Hz, 1H), 5.26 (s, 2H). 13C NMR (101 MHz,
498 DMSO-d6) δ 161.69, 158.85, 148.25, 141.53, 139.33, 139.19, 137.87, 136.63, 132.88,
 ACCEPTED MANUSCRIPT

499 132.80, 132.13, 131.49, 130.52, 130.20, 129.25, 128.77, 122.87, 121.40, 120.97,
500 120.64, 119.16, 117.21, 115.54, 112.80, 111.93, 68.60. HRMS-ESI: calcd for
501 C27H19N5O2Na [M+Na]+ 468.1437, found: 468.1438.
502 N'-(3-((4-cyanobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
503 (12r)

PT
504 White solid, yield: 91%. M.P. 296.8 - 298.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ
505 12.10 (s, 1H), 12.04 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.47 (d, J = 7.9

RI
506 Hz, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.70 (dd, J = 8.2, 4.2 Hz, 3H), 7.66 - 7.58 (m, 1H),
507 7.45 – 7.38 (m, 2H), 7.37 - 7.28 (m, 2H), 7.12 (dd, J = 8.1, 1.9 Hz, 1H), 5.31 (s, 2H).

SC
13
508 C NMR (101 MHz, DMSO-d6) δ 161.70, 158.80, 148.23, 143.26, 141.53, 139.31,
509 137.87, 136.65, 132.88, 132.81, 130.53, 129.25, 128.77, 128.54, 122.86, 121.39,

U
510 121.02, 120.64, 119.23, 117.18, 115.54, 112.80, 110.97, 68.82. HRMS-ESI: calcd for
AN
511 C27H19N5O2Na [M+Na]+ 468.1437, found: 468.1437.
512 N'-(3-((4-methoxybenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
M

513 (12s)
514 White solid, yield: 92%. M.P. 266.3 - 266.9 ℃. 1H NMR (400 MHz, DMSO-d6) δ
D

515 12.09 (s, 1H), 12.05 (s, 1H), 8.99 (s, 1H), 8.97 (s, 1H), 8.65 (s, 1H), 8.47 (d, J = 7.9
TE

516 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.65 – 7.59 (m, 1H), 7.46 – 7.28 (m, 6H), 7.09 (dd,
13
517 J = 8.1, 1.8 Hz, 1H), 6.97 (d, J = 8.6 Hz, 2H), 5.09 (s, 2H), 3.77 (s, 3H). C NMR
EP

518 (101 MHz, DMSO-d6) δ 161.67, 159.49, 159.20, 148.37, 141.53, 139.34, 137.86,
519 136.52, 132.80, 130.41, 129.97, 129.26, 128.77, 122.87, 121.39, 120.64, 120.50,
117.23, 115.52, 114.30, 112.94, 112.80, 69.57, 55.57. HRMS-ESI: calcd for
C

520

521 C27H23N4O3 [M+H]+ 451.1771, found: 451.1772.

AC

522 N'-(3-((4-bromobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
523 (12t)
524 White solid, yield: 94%. M.P. 278.4 - 279.7 ℃. 1H NMR (400 MHz, DMSO-d6) δ
525 12.09 (s, 1H), 12.03 (s, 1H), 8.98 (s, 1H), 8.96 (s, 1H), 8.64 (s, 1H), 8.46 (d, J = 7.9
526 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.67 – 7.57 (m, 3H), 7.47 (d, J = 8.3 Hz, 2H), 7.45
527 – 7.36 (m, 2H), 7.36 – 7.28 (m, 2H), 7.10 (dd, J = 8.1, 1.9 Hz, 1H), 5.17 (s, 2H). 13C
528 NMR (101 MHz, DMSO-d6) δ 161.70, 158.95, 148.29, 141.53, 139.32, 137.87,
 ACCEPTED MANUSCRIPT

529 136.90, 136.59, 132.81, 131.83, 130.47, 130.26, 129.25, 128.77, 122.86, 121.42,
530 121.39, 120.81, 120.64, 117.19, 115.53, 112.88, 112.80, 68.96. HRMS-ESI: calcd for
531 C26H19BrN4O2Na[M+Na]+ 521.0589, 523.0569, found: 521.0591, 523.0577.
532 methyl-4-((3-((2-(9H-pyrido[3,4-b]indole-3-carbonyl)hydrazono)methyl)phenoxy)
533 methyl)benzoate (12u)

PT
534 White solid, yield: 93%. M.P. 266.8 - 267.4 ℃. 1H NMR (400 MHz, DMSO-d6) δ
535 12.09 (s, 1H), 12.03 (s, 1H), 9.01 – 8.93 (m, 2H), 8.64 (s, 1H), 8.46 (d, J = 7.9 Hz,

RI
536 1H), 8.06 – 7.96 (m, 2H), 7.72 – 7.58 (m, 4H), 7.45 – 7.38 (m, 2H), 7.37 – 7.28 (m,
537 2H), 7.12 (dd, J = 7.9, 2.0 Hz, 1H), 5.30 (s, 2H), 3.87 (s, 3H). 13C NMR (101 MHz,

SC
538 DMSO-d6) δ 166.49, 161.68, 158.94, 148.25, 143.02, 141.53, 139.33, 137.86, 136.62,
539 132.80, 130.52, 129.80, 129.47, 128.77, 127.97, 122.88, 121.39, 120.87, 120.64,

U
540 117.15, 115.53, 112.92, 112.80, 69.10, 52.62. HRMS-ESI: calcd for C28H22N4O4Na
AN
541 [M+Na]+ 501.1539, found: 501.1537.
542 N'-(3-((4-(trifluoromethyl)benzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carboh
M

543 ydrazide (12v)

544 White solid, yield: 84%. M.P. 268.4 - 268.9 ℃. 1H NMR (400 MHz, DMSO-d6) δ
D

545 12.09 (s, 1H), 12.03 (s, 1H), 8.98 (s, 1H), 8.96 (s, 1H), 8.65 (s, 1H), 8.46 (d, J = 7.9
TE

546 Hz, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.76 – 7.66 (m, 3H), 7.65 – 7.59 (m, 1H), 7.45 –
13
547 7.38 (m, 2H), 7.37 – 7.28 (m, 2H), 7.12 (dd, J = 7.8, 2.2 Hz, 1H), 5.32 (s, 2H). C
EP

548 NMR (101 MHz, DMSO-d6) δ 161.68, 158.89, 148.24, 142.36, 141.53, 139.33,
549 137.86, 136.65, 132.80, 130.53, 129.25, 128.77, 128.48, 125.82, 125.78, 122.87,
121.39, 120.92, 120.64, 117.14, 115.53, 112.88, 112.80, 68.87. 19F NMR (376 MHz,
C

550

551 DMSO-d6) δ -60.93. HRMS-ESI: calcd for C27H19F3N4O2Na [M+Na]+ 511.1358,

AC

552 found: 511.1360.

553 N'-(3-((4-nitrobenzyl)oxy)benzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide
554 (12w)
555 White solid, yield: 91%. M.P. 281.3 - 282.4 ℃. 1H NMR (400 MHz, DMSO-d6) δ
556 12.13 (s, 1H), 12.07 (s, 1H), 9.01 (s, 1H), 8.99 (s, 1H), 8.68 (s, 1H), 8.47 (d, J = 7.9
557 Hz, 1H), 8.28 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.2 Hz, 1H),
558 7.67 – 7.58 (m, 1H), 7.47 – 7.39 (m, 2H), 7.38 – 7.29 (m, 2H), 7.13 (dd, J = 8.1, 1.8
 ACCEPTED MANUSCRIPT
13
559 Hz, 1H), 5.35 (s, 2H). C NMR (101 MHz, DMSO-d6) δ 161.71, 158.75, 148.22,
560 147.48, 145.37, 141.53, 139.30, 137.87, 136.67, 132.81, 130.55, 129.25, 128.77,
561 128.69, 124.06, 122.86, 121.39, 121.08, 120.64, 117.16, 115.54, 112.84, 112.80,
562 68.58. HRMS-ESI: calcd for C26H20N5O4 [M+H]+ 466.1516, found: 466.1519.
563 N'-(3-(benzyloxy)-4-methoxybenzylidene)-9H-pyrido[3,4-b]indole-3-carbohydrazide

PT
564 (12x)
565 White solid, yield: 89%. M.P. 261.0 - 262.2 ℃. 1H NMR (400 MHz, DMSO-d6) δ

RI
566 12.03 (s, 1H), 11.95 (s, 1H), 8.97 (d, J = 2.2 Hz, 2H), 8.59 (s, 1H), 8.46 (d, J = 7.9 Hz,
567 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.55 – 7.46 (m, 3H), 7.43 (t, J

SC
568 = 7.4 Hz, 2H), 7.39 - 7.29 (m, 2H), 7.25 (dd, J = 8.3, 1.4 Hz, 1H), 7.09 (d, J = 8.4 Hz,
13
569 1H), 5.16 (s, 2H), 3.84 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 161.48, 151.46,

U
570 148.67, 148.62, 141.53, 139.49, 137.82, 137.36, 132.78, 129.22, 128.86, 128.77,
AN
571 128.39, 127.76, 122.85, 122.56, 121.40, 120.61, 115.38, 112.79, 112.31, 110.73,
572 70.46, 56.14. HRMS-ESI: calcd for C27H22N4O3Na [M+Na]+ 473.1590, found:
M

573 473.1591.
574 4.1.6 General procedure for the synthesis of
D

575 N'-(2-chloroacetyl)-9H-pyrido[3,4-b]indole-3-carbohydrazide (13)

TE

576 4-methylmorpholine N-oxide (1.76 g, 15.0 mmol) and chloroacetyl chloride

577 (1.13 g, 10 mmol) were added to a suspension of compound 10 (2.26 g, 10 mmol) in
EP

578 CH2Cl2 (35 mL) and the mixture was stirred at room temperature for 1.5 h. After
579 completion of the reaction, the mixture was filtered and the residue was extracted with
CH2Cl2.Then concentrated under reduced pressure, the residue was purified by silica
C

580

581 gel column chromatography to afford a white solid 13 as a white solid, yielding 62%.
AC

582 4.1.7 General procedure for the synthesis of

583 2-(chloromethyl)-5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazole (14)
584 Phosphorus oxychloride (POCl3, 20 mL) was slowly added to a 50 mL
585 round-bottom flask containing compound 13 (3.01 g, 10 mmol). The resulting mixture
586 was refluxed for 3 h. Then excess POCl3 was removed under vacuum. The residue
587 was dissolved in saturated sodium carbonate (30 mL), extracted with ethyl acetate (3
588 × 20 mL), washed with brine, dried with anhydrous sodium sulfate and evaporated in
 ACCEPTED MANUSCRIPT

589 vacuo. The crude product was purified by chromatograph on silica gel with petroleum
590 ether and ethyl acetate (1/1, v/v) as eluent. Yielding 56%. 1H NMR (400 MHz,
591 DMSO-d6) δ 12.11 (s, 1H), 9.06 (s, 1H), 9.05 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H), 7.69 (d,
592 J = 8.3 Hz, 1H), 7.66 – 7.60 (m, 1H), 7.34 (ddd, J = 7.9, 6.9, 1.1 Hz, 1H), 5.19 (s,
593 2H).

PT
594 4.1.8 General procedure for the synthesis of β-carboline/1,3,4-oxadiazole hybrids
595 (15a-d)

RI
596 To a stirred solution of 14 (0.28 g, 0.1 mmol), K2CO3 (0.21 g, 1.5 mmol), KI
597 (0.02 g, 0.01 mmol) in DMF was added substituted phenol or N-methyl benzylamine

SC
598 (0.1 mmol). The resulting mixture was stirred at 65 - 80 ℃ for 4 h and monitored by
599 TLC. After completion of the reaction, the resulting mixture was poured into water

U
600 and extracted with ethyl acetate (3 × 20 mL), washed with brine, dried with
AN
601 anhydrous sodium sulfate and evaporated in vacuo. The crude product was purified by
602 chromatograph on silica gel with petroleum ether and ethyl acetate (1/2, v/v) as
M

603 eluent.
604 2-(phenoxymethyl)-5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazole (15a)
D

605 White solid, yield: 84%. M.P. 245.7 - 246.9 ℃. 1H NMR (400 MHz, DMSO-d6) δ
TE

606 12.10 (s, 1H), 9.05 (s, 1H), 9.04 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 8.3 Hz,
607 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.42 – 7.31 (m, 3H), 7.14 (d, J = 8.1 Hz, 2H), 7.03 (t, J
= 7.3 Hz, 1H), 5.53 (s, 2H). HRMS-ESI: calcd for C20H15N4O2 [M+H]+ 343.1196,
EP

608

609 found: 343.1190.

C

610 4-((5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazol-2-yl)methoxy)benzaldehyde (15b)

611 Pale yellow solid, yield: 84%. M.P. 253.2 - 254.9 ℃. 1H NMR (400 MHz, DMSO-d6)
AC

612 δ 12.10 (s, 1H), 9.92 (s, 1H), 9.05 (d, J = 1.4 Hz, 2H), 8.46 (d, J = 7.9 Hz, 1H), 7.99 –
613 7.90 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.65 – 7.60 (m, 1H), 7.39 – 7.30 (m, 3H), 5.69
614 (s, 2H). HRMS-ESI: calcd for C21H15N4O3 [M+H]+ 371.1145, found: 371.1141.
615 3-((5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazol-2-yl)methoxy)benzaldehyde (15c)
616 Pale yellow solid, yield: 78%. M.P. 229.2 - 229.7 ℃. 1H NMR (400 MHz, DMSO-d6)
617 δ 12.10 (s, 1H), 10.02 (s, 1H), 9.06 (s, 1H), 9.04 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H),
 ACCEPTED MANUSCRIPT

618 7.73 – 7.57 (m, 5H), 7.52-7.47 (m, 1H), 7.38 – 7.31 (m, 1H), 5.65 (s, 2H). HRMS-ESI:
619 calcd for C21H15N4O3 [M+H]+ 371.1145, found: 371.1146.
620 1-(5-(9H-pyrido[3,4-b]indol-3-yl)-1,3,4-oxadiazol-2-yl)-N-benzyl-N-methylmethana
621 mine (15d)
622 White solid, yield: 82%. M.P. 181.7 - 182.5 ℃. 1H NMR (400 MHz, DMSO-d6) δ

PT
623 12.09 (s, 1H), 9.06 (s, 1H), 8.98 (s, 1H), 8.44 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 8.1 Hz,
624 1H), 7.66 – 7.57 (m, 1H), 7.39 – 7.29 (m, 5H), 7.29 – 7.23 (m, 1H), 3.96 (s, 2H), 3.67

RI
13
625 (s, 2H), 2.28 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 165.65, 164.40, 141.56,
626 138.55, 137.32, 134.85, 132.38, 129.36, 128.71, 128.41, 127.62, 122.89, 121.11,

SC
627 120.65, 115.48, 112.85, 60.77, 56.49, 41.99. HRMS-ESI: calcd for C22H20N5O
628 [M+H]+ 370.1669, found: 370.1673.

U
629 4.2 Biological assay methods
AN
630 4.2.1 Cell proliferation assay
631 The anti-proliferation activities of the compounds were tested in MCF-7 cells,
M

632 H460 cells, Raji cells, MCF-7/ADR cells, MDA-MB-231 cells, A549 cells, A549T
633 cells, A2780S cells and A2780T cells. Cells in logarithmic phase were seeded in
D

634 96-well plates and allowed to adhere. Then the cells were incubated with indicated
TE

635 concentrations of the compounds for 48 h or 72 h. MTT was subsequently added for
636 an extra 2-3 h of incubation. The MTT formazan precipitate was dissolved in DMSO,
EP

637 and the absorbance was measured at a wavelength of 570 nm by a Spectramax M5

638 microtiter plate luminometer (Molecular Devices, Sunnyvale, CA, USA).
4.2.2 Flow cytometry
C

639

640 MCF-7 cells were incubated with various concentrations of selected compound
AC

641 or DMSO vehicle for 18 h at 37 ℃. The cells were washed by PBS, and then the cell
642 DNA was stained with 50 mg/mL PI containing 1 mg/mL of DNase-free RNaseA for a
643 minimum of 9 min. The samples were analyzed by a flow cytometer (BD FACS
644 Calibur, Franklin Lakes, NJ, USA).
645 4.2.3 Animal tumor models and treatment
646 H460 xenograft model in vivo was established in a similar method as described.
647 5-to-6-week-old female Balb/C and athymic nude mice were used, respectively, and
 ACCEPTED MANUSCRIPT

648 the indicated number of cells suspended in 90 CE HBSS was implanted in the right
649 flank of mice. When tumor volumes reached 100 mm3, the animals were treated with
650 vehicle, taxol (30 mg/kg, once a week), or 12r (100 mg/kg, 3 times a week). Signs of
651 toxicity and mortality were observed daily. Tumor volumes and body weights were
652 measured every 2 days when administrated with a caliper (calculated volume (mm3))

PT
653 = (π/6) × length × width × width). The antitumor activity of compound was evaluated
654 by tumor inhibitory rate = (1 - tumor weight of treated group) / (tumor weight of

RI
655 control group) × 100%.
656 Acknowledgements

SC
657 The authors greatly appreciate the financial support from National Natural Science
658 Foundation of China (Grant U1402222 and 81703344) and China Postdoctoral

U
659 Science Foundation (2017M612980).
AN
660 References
661 [1] K. Patel, M. Gadewar, R. Tripathi, S.K. Prasad, D.K. Patel, A review on medicinal
M

662 importance, pharmacological activity and bioanalytical aspects of β-carboline alkaloid

663 “Harmine”, Asian Pacific Journal of Tropical Biomedicine, 2 (2012) 660-664.
D

664 [2] L.T. Tonin, M.R. Panice, C.V. Nakamura, K.J. Rocha, A.O. dos Santos, T.
TE

665 Ueda-Nakamura, W.F. da Costa, M.H. Sarragiotto, Antitrypanosomal and

666 antileishmanial activities of novel
EP

667 N-alkyl-(1-phenylsubstituted-β-carboline)-3-carboxamides, Biomed. Pharmacother.,

668 64 (2010) 386-389.
[3] R.H. Valdez, L.T. Tonin, T. Ueda-Nakamura, S.O. Silva, B.P. Dias Filho, E.N.
C

669

670 Kaneshima, S.F. Yamada-Ogatta, L.M. Yamauchi, M.H. Sarragiotto, C.V. Nakamura,
AC

671 In vitro and in vivo trypanocidal synergistic activity of

672 N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxamide
673 associated with benznidazole, Antimicrob. Agents Chemother., 56 (2012) 507-512.
674 [4] V.M. Gohil, K.G. Brahmbhatt, P.M. Loiseau, K.K. Bhutani, Synthesis and
675 anti-leishmanial activity of 1-aryl-β-carboline derivatives against Leishmania
676 donovani, Bioorg. Med. Chem. Lett., 22 (2012) 3905-3907.
 ACCEPTED MANUSCRIPT

677 [5] S.S. Chauhan, S. Pandey, R. Shivahare, K. Ramalingam, S. Krishna, P.

678 Vishwakarma, M.I. Siddiqi, S. Gupta, N. Goyal, P.M.S. Chauhan, Novel
679 β-carboline–quinazolinone hybrid as an inhibitor of Leishmania donovani
680 trypanothione reductase: Synthesis, molecular docking and bioevaluation,
681 MedChemComm, 6 (2015) 351-356.

PT
682 [6] Y. Rook, K.U. Schmidtke, F. Gaube, D. Schepmann, B. Wunsch, J. Heilmann, J.
683 Lehmann, T. Winckler, Bivalent beta-carbolines as potential multitarget

RI
684 anti-Alzheimer agents, J. Med. Chem., 53 (2010) 3611-3617.
685 [7] J.S. Lan, S.S. Xie, S.Y. Li, L.F. Pan, X.B. Wang, L.Y. Kong, Design, synthesis and

SC
686 evaluation of novel tacrine-(beta-carboline) hybrids as multifunctional agents for the
687 treatment of Alzheimer's disease, Bioorg. Med. Chem., 22 (2014) 6089-6104.

U
688 [8] J. Liu, X. Jiang, M. Zhao, X. Zhang, M. Zheng, L. Peng, S. Peng, A class of
AN
689 3S-2-aminoacyltetrahydro-beta-carboline-3-carboxylic acids: their facile synthesis,
690 inhibition for platelet activation, and high in vivo anti-thrombotic potency, J. Med.
M

691 Chem., 53 (2010) 3106-3116.

692 [9] K. Yao, M. Zhao, X. Zhang, Y. Wang, L. Li, M. Zheng, S. Peng, A class of oral
D

693 N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carbonyl]-
TE

694 N'-(amino-acid-acyl)hydrazine: discovery, synthesis, in vitro anti-platelet

695 aggregation/in vivo anti-thrombotic evaluation and 3D QSAR analysis, Eur. J. Med.
EP

696 Chem., 46 (2011) 3237-3249.

697 [10] W. Polanski, H. Reichmann, G. Gille, Stimulation, protection and regeneration of
dopaminergic neurons by 9-methyl-beta-carboline: a new anti-Parkinson drug?.
C

698

699 Expert Rev. Neurother., 11 (2011) 845-860.

AC

700 [11] B. Drung, C. Scholz, V.A. Barbosa, A. Nazari, M.H. Sarragiotto, B. Schmidt,
701 Computational & experimental evaluation of the structure/activity relationship of
702 beta-carbolines as DYRK1A inhibitors, Bioorg. Med. Chem. Lett., 24 (2014)
703 4854-4860.
704 [12] K. Ruben, A. Wurzlbauer, A. Walte, W. Sippl, F. Bracher, W. Becker, Selectivity
705 Profiling and Biological Activity of Novel beta-Carbolines as Potent and Selective
706 DYRK1 Kinase Inhibitors, PLoS One, 10 (2015) e0132453.
 ACCEPTED MANUSCRIPT

707 [13] Y. Song, D. Kesuma, J. Wang, Y. Deng, J. Duan, J.H. Wang, R.Z. Qi, Specific
708 inhibition of cyclin-dependent kinases and cell proliferation by harmine, Biochem.
709 Biophys. Res. Commun., 317 (2004) 128-132.
710 [14] R. Frederick, C. Bruyere, C. Vancraeynest, J. Reniers, C. Meinguet, L. Pochet, A.
711 Backlund, B. Masereel, R. Kiss, J. Wouters, Novel trisubstituted harmine derivatives

PT
712 with original in vitro anticancer activity, J. Med. Chem., 55 (2012) 6489-6501.
713 [15] M. Zhao, L. Bi, W. Wang, C. Wang, M. Baudy-Floc'h, J. Ju, S. Peng, Synthesis

RI
714 and cytotoxic activities of beta-carboline amino acid ester conjugates, Bioorg. Med.
715 Chem., 14 (2006) 6998-7010.

SC
716 [16] M. Sathish, B. Kavitha, V.L. Nayak, Y. Tangella, A. Ajitha, S. Nekkanti, A.
717 Alarifi, N. Shankaraiah, N. Nagesh, A. Kamal, Synthesis of podophyllotoxin linked

U
718 beta-carboline congeners as potential anticancer agents and DNA topoisomerase II
AN
719 inhibitors, Eur. J. Med. Chem., 144 (2018) 557-571.
720 [17] A. Kamal, M. Sathish, V.L. Nayak, V. Srinivasulu, B. Kavitha, Y. Tangella, D.
M

721 Thummuri, C. Bagul, N. Shankaraiah, N. Nagesh, Design and synthesis of

722 dithiocarbamate linked beta-carboline derivatives: DNA topoisomerase II inhibition
D

723 with DNA binding and apoptosis inducing ability, Bioorg. Med. Chem., 23 (2015)
TE

724 5511-5526.
725 [18] N. Shankaraiah, C. Jadala, S. Nekkanti, K.R. Senwar, N. Nagesh, S. Shrivastava,
EP

726 V.G. Naidu, M. Sathish, A. Kamal, Design and synthesis of C3-tethered

727 1,2,3-triazolo-beta-carboline derivatives: Anticancer activity, DNA-binding ability,
viscosity and molecular modeling studies, Bioorg. Chem., 64 (2016) 42-50.
C

728

729 [19] N. Shankaraiah, K.P. Siraj, S. Nekkanti, V. Srinivasulu, P. Sharma, K.R. Senwar,
AC

730 M. Sathish, M.V. Vishnuvardhan, S. Ramakrishna, C. Jadala, N. Nagesh, A. Kamal,

731 DNA-binding affinity and anticancer activity of beta-carboline-chalcone conjugates as
732 potential DNA intercalators: Molecular modelling and synthesis, Bioorg. Chem., 59
733 (2015) 130-139.
734 [20] A. Kamal, V. Srinivasulu, V.L. Nayak, M. Sathish, N. Shankaraiah, C. Bagul,
735 N.V. Reddy, N. Rangaraj, N. Nagesh, Design and synthesis of
736 C3-pyrazole/chalcone-linked beta-carboline hybrids: antitopoisomerase I,
 ACCEPTED MANUSCRIPT

737 DNA-interactive, and apoptosis-inducing anticancer agents, ChemMedChem., 9

738 (2014) 2084-2098.
739 [21] A.C. Castro, L.C. Dang, F. Soucy, L. Grenier, H. Mazdiyasni, M. Hottelet, L.
740 Parent, C. Pien, V. Palombella, J. Adams, Novel IKK inhibitors: β-carbolines, Bioorg.
741 Med. Chem. Lett., 13 (2003) 2419-2422.

PT
742 [22] L. He, S.Y. Liao, C.P. Tan, R.R. Ye, Y.W. Xu, M. Zhao, L.N. Ji, Z.W. Mao,
743 Ruthenium-Arene-beta-Carboline Complexes as Potent Inhibitors of

RI
744 Cyclin-Dependent Kinase 1: Synthesis, Characterization and Anticancer Mechanism
745 Studies, Chem. Eur. J., 19 (2013) 12152-12160.

SC
746 [23] X. Han, J. Zhang, L. Guo, R. Cao, Y. Li, N. Li, Q. Ma, J. Wu, Y. Wang, S. Si, A
747 series of beta-carboline derivatives inhibit the kinase activity of PLKs, PLoS One, 7

U
748 (2012) e46546.
AN
749 [24] T. Herraiz, D. Gonzalez, C. Ancin-Azpilicueta, V.J. Aran, H. Guillen,
750 beta-Carboline alkaloids in Peganum harmala and inhibition of human monoamine
M

751 oxidase (MAO), Food Chem. Toxicol., 48 (2010) 839-845.

752 [25] A. Kamal, M.P. Rao, P. Swapna, V. Srinivasulu, C. Bagul, A.B. Shaik, K.
D

753 Mullagiri, J. Kovvuri, V.S. Reddy, K. Vidyasagar, N. Nagesh, Synthesis of

TE

754 beta-carboline-benzimidazole conjugates using lanthanum nitrate as a catalyst and

755 their biological evaluation, Org. Biomol. Chem., 12 (2014) 2370-2387.
EP

756 [26] J. Wu, M. Zhao, K. Qian, K.H. Lee, S. Morris-Natschke, S. Peng, Novel
757 N-(3-carboxyl-9-benzyl-beta-carboline-1-yl)ethylamino acids: synthesis, anti-tumor
evaluation, intercalating determination, 3D QSAR analysis and docking investigation,
C

758

759 Eur. J. Med. Chem., 44 (2009) 4153-4161.

AC

760 [27] A.M. Sobhani, S.A. Ebrahimi, M.J. Mahmoudian, An In Vitro Evaluation of
761 Human DNA Topoisomerase I Inhibition by Peganum harmala L. Seeds Extract and
762 Its β-Carboline Alkaloids, J. Pharm. Pharm. Sci., 5 (2002) 19-23.
763 [28] V.A. Barbosa, P. Barea, R.S. Mazia, T. Ueda-Nakamura, W.F.D. Costa, M.A.
764 Foglio, A.L.T. Goes Ruiz, J.E. Carvalho, D.B. Vendramini-Costa, C.V. Nakamura,
765 M.H. Sarragiotto, Synthesis and evaluation of novel hybrids
 ACCEPTED MANUSCRIPT

766 beta-carboline-4-thiazolidinones as potential antitumor and antiviral agents, Eur. J.

767 Med. Chem., 124 (2016) 1093-1104.
768 [29] S. Misra, S. Ghatak, N. Patil, P. Dandawate, V. Ambike, S. Adsule, D. Unni, K.
769 Venkateswara Swamy, S. Padhye, Novel dual cyclooxygenase and lipoxygenase
770 inhibitors targeting hyaluronan-CD44v6 pathway and inducing cytotoxicity in colon

PT
771 cancer cells, Bioorg. Med. Chem., 21 (2013) 2551-2559.
772 [30] T. Liu, C. Sun, X. Xing, L. Jing, R. Tan, Y. Luo, W. Huang, H. Song, Z. Li, Y.

RI
773 Zhao, Synthesis and evaluation of 2-[2-(phenylthiomethyl)-1H-benzo[d]
774 imidazol-1-yl)acetohydrazide derivatives as antitumor agents, Bioorg. Med. Chem.

SC
775 Lett., 22 (2012) 3122-3125.
776 [31] L.N.F. Cardoso, T.C.M. Nogueira, F.A.R. Rodrigues, A.C.A. Oliveira, M.C.d.S.

U
777 Luciano, C. Pessoa, M.V.N. de Souza, N-acylhydrazones containing thiophene
AN
778 nucleus: a new anticancer class, Med. Chem. Res., 26 (2017) 1605-1608.
779 [32] K. Sun, J.D. Peng, F.Z. Suo, T. Zhang, Y.D. Fu, Y.C. Zheng, H.M. Liu, Discovery
M

780 of tranylcypromine analogs with an acylhydrazone substituent as LSD1 inactivators:

781 Design, synthesis and their biological evaluation, Bioorg. Med. Chem. Lett., 27
D

782 (2017) 5036-5039.

TE

783 [33] Y. Zheng, J. Ren, Y. Wu, X. Meng, Y. Zhao, C. Wu, Proteolytic Unlocking of
784 Ultrastable Twin-Acylhydrazone Linkers for Lysosomal Acid-Triggered Release of
EP

785 Anticancer Drugs, Bioconjug. Chem., 28 (2017) 2620-2626.

786 [34] F.Y. Li, X. Wang, W.G. Duan, G.S. Lin, Synthesis and In Vitro Anticancer
Activity of Novel Dehydroabietic Acid-Based Acylhydrazones, Molecules, 22 (2017)
C

787

788 1087-1098.
AC

789 [35] V. Sorna, E.R. Theisen, B. Stephens, S.L. Warner, D.J. Bearss, H. Vankayalapati,
790 S. Sharma, High-throughput virtual screening identifies novel
791 N'-(1-phenylethylidene)-benzohydrazides as potent, specific, and reversible LSD1
792 inhibitors, J. Med. Chem., 56 (2013) 9496-9508.
793 [36] D.A. Rodrigues, G.A. Ferreira-Silva, A.C. Ferreira, R.A. Fernandes, J.K. Kwee,
794 C.M. Sant'Anna, M. Ionta, C.A. Fraga, Design, Synthesis, and Pharmacological
 ACCEPTED MANUSCRIPT

795 Evaluation of Novel N-Acylhydrazone Derivatives as Potent Histone Deacetylase 6/8

796 Dual Inhibitors, J. Med. Chem., 59 (2016) 655-670.
797 [37] R.V. Chari, M.L. Miller, W.C. Widdison, Antibody-drug conjugates: an emerging
798 concept in cancer therapy, Angew. Chem. Int. Ed., 53 (2014) 3796-3827.
799 [38] H.L. Perez, P.M. Cardarelli, S. Deshpande, S. Gangwar, G.M. Schroeder, G.D.

PT
800 Vite, R.M. Borzilleri, Antibody-drug conjugates: current status and future directions,
801 Drug Discov. Today, 19 (2014) 869-881.

RI
802 [39] G.D. Lenoard, T. Fojo, S.E. Bates, The role of ABC transporters in cilcial
803 practice, Oncologist, 8 (2003) 411-424.

SC
804 [40] J.J. Yeh, W.H. Hsu, J.J. Wang, S.T. Ho, A. Kao, Predicting chemotherapy
805 response to paclitaxel-based therapy in advanced non-small-cell lung cancer with

U
806 P-glycoprotein expression, Respiration, 70 (2003) 32-35.
AN
807 [41] B.C. Baguley, Multiple drug resistance mechanisms in cancer, Mol. Biotechnol.,
808 46 (2010) 308-316.
M

809 [42] A. Ganguly, F. Cabral, New insights into mechanisms of resistance to

810 microtubule inhibitors, Biochim. Biophys. Acta, 1816 (2011) 164-171.
D

811 [43] Z. Yang, W. Wu, J. Wang, L. Liu, L. Li, J. Yang, G. Wang, D. Cao, R. Zhang, M.
TE

812 Tang, J. Wen, J. Zhu, W. Xiang, F. Wang, L. Ma, M. Xiang, J. You, L. Chen, Synthesis
813 and biological evaluation of novel millepachine derivatives as a new class of tubulin
EP

814 polymerization inhibitors, J. Med. Chem., 57 (2014) 7977-7989.

815 [44] G. Szakacs, J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, M.M. Gottesman,
Targeting multidrug resistance in cancer, Nat. Rev. Drug Discov., 5 (2006) 219-234.
C

816

817 [45] M. Kavallaris, Microtubules and resistance to tubulin-binding agents, Nat. Rev.
AC

818 Cancer, 10 (2010) 194-204.

819 [46] Z. Duan, C. Chen, J. Qin, Q. Liu, Q. Wang, X. Xu, J. Wang, Cell-penetrating
820 peptide conjugates to enhance the antitumor effect of paclitaxel on drug-resistant lung
821 cancer, Drug Deliv., 24 (2017) 752-764.
822 [47] A. Pepe, L. Sun, I. Zanardi, X. Wu, C. Ferlini, G. Fontana, E. Bombardelli, I.
823 Ojima, Novel C-seco-taxoids possessing high potency against paclitaxel-resistant
 ACCEPTED MANUSCRIPT

824 cancer cell lines overexpressing class III beta-tubulin, Bioorg. Med. Chem. Lett., 19
825 (2009) 3300-3304.
826 [48] F. Liu, Z. Xie, G. Cai, Y. Jiang, The effect of survivin on multidrug resistance
827 mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells, Biol. Pharm.
828 Bull., 30 (2007) 2279-2283.

PT
829

RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

> 24 novel β-carboline/acylhydrazone hybrids were synthesized;

> 12r exhibited significant activity in multidrug resistant cancer cells;
> 12r showed certain antitumor effect on the H460 xenograft models.

PT
RI
U SC
AN
M
D
TE
C EP
AC