Semicarbazone derivatives bearing phenyl moiety: Synthesis, anticancer activity, cell cycle, apoptosis-inducing and metabolic stability study

Article abstract of DOI:10.1248/cpb.c18-00738

A series of semicarbazone derivatives bearing phenyl moiety were synthesized and evaluated for the vitro anticancer activities in four human cancer cell lines (human colon cancer (HT29), human neuroblastoma (SK-N-SH), human breast cancer (MDA-MB-231), and human gastric cancer (MKN45)). Biological evaluation led to the identification of 11q and 11s, which showed excellent anticancer activities against tested cancer cell lines with IC₅₀ values ranging from 0.32 to 1.57μM, respectively, while exhibiting weak cytotoxicity on the normal cells (human umbilical vein endothelial cell (HUVEC)). Flow cytometric assay for cell cycle and apoptosis revealed that 11q and 11s caused an arrest in the Sub-G1 cell cycle and inhibited proliferation of cancer cells by inducing apoptosis in a dose-dependent manner. Further enzymatic assay suggested that 11q and 11s could significantly activated procaspase-3 to caspase-3. Metabolic stability study indicated that 11q and 11s showed moderate stability in vitro in human and rat liver microsomes. In view of promising pharmacological activities of 11q and 11s, which had emerged as the valuable lead for further development in the treatment for cancer.

Full text of DOI:10.1248/cpb.c18-00738

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00738
Advance Publication
January 24, 2019
Regular Article
Semicarbazone derivatives bearing phenyl moiety: synthesis, anticancer activity, cell
cycle, apoptosis-inducing and metabolic stability study
Junjie Ma,a Xin Ni,a Yali Gao,c Kun Huang,a Yu Wang,a Jiaan Liu,d Guowei Gongb*
a Medical college, Huaqiao University, Quanzhou, 362021, China.
b Department of Pharmaceutical Sciences, Zunyi Medical College Zhuhai Campus, Zhuhai,
Guangdong, 519000, China.
c
Pharmacy Department, the Second Affiliated Hospital of Fujian Medical University.
Quanzhou, 362000, China.
d Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts
01003, United States.
 To whom correspondence should be addressed. E-mail: majunjie3612@hqu.edu.cn, amygong90@hotmail.com.
Ⓒ 2019 The Pharmaceutical Society of Japan

A series of semicarbazone derivatives bearing phenyl moiety were synthesized and
evaluated for the vitro anticancer activities in four human cancer cell lines (HT29,
SK-N-SH, MDA-MB-231 and MKN45). Biological evaluation led to the identification of
11q and 11s, which showed excellent anticancer activities against tested cancer cell lines
with IC50 values ranging from 0.32 to 1.57 μM, respectively, while exhibiting weak
cytotoxicity on the normal cells (HUVEC). Flow cytometric assay for cell cycle and
apoptosis revealed that 11q and 11s caused an arrest in the Sub-G1 cell cycle and
inhibited proliferation of cancer cells by inducing apoptosis in a dose-dependent
manner. Further enzymatic assay suggested that 11q and 11s could significantly
activated procaspase-3 to caspase-3. Metabolic stability study indicated that 11q and
11s showed moderate stability in vitro in human and rat liver microsomes. In view of
promising pharmacological activities of 11q and 11s, which had emerged as the valuable
lead for further development in the treatment for cancer.
Key words anticancer activity; semicarbazone; sub-G1; apoptosis; metabolic stability.
Chemical and Pharmaceutical Bulletin Advance Publication

Cancer is one of the most death-defying health hazards distressing a greater part of the
world population. As evasion of apoptosis is a hallmark of cancer1, 2), thus, it has been an
effective approach to explore novel apoptosis-inducing compounds for the treatment of
cancer. Procaspase-3 is the most common and key proapoptotic protein in the downstream
apoptotic cascade, playing a significant role in the cancer development and progression.
Procaspase-3 activators, which can directly induce apoptosis by activating over-expression
procaspase-3 to caspase-33-8), show greater advantages over anticancer agents targeting early
or intermediate positions in the apoptotic cascade, such as p53 disruptors (CBL-0137 and
COTI-2)9-11), XIAP inhibitors (AZD5582 and GDC-0152)12, 13), MDM2 inhibitors(HDM201
and RG7112) 14, 15), and Bcl-2 inhibitors (GDC-0199 and ABT-737)16, 17), which likely resist
the anticancer effects due to mutations of downstream apoptotic proteins.
PAC-1 (Fig. 1) was reported as the first procaspase-3 activator that induced apoptosis by
selectively activating procaspase-3 to caspase-3. Structure−activity relationships (SARs)
revealed that ortho-hydroxy N-acylhydrazone moiety of PAC-1 was responsible for strong
caspase-3 activation activity. In our previous study, based on the SARs, inspired by
semicarbazone moiety widely used as building block in design of different potential
anticancer agents due to the presence of several hydrogen donors and acceptors as well as its
flexible skeleton18-20), a series of semicarbazone derivatives containing benzothiazole had
been independently reported as procaspase-3 activators21, 22), and the compounds 1 and 2
(Fig. 1) showed promising procaspase-3 activation activities and anticancer activities, further
SARs of which indicated that introduction of lipophilic groups (eg. substituted benzyloxy or
heteroaryloxy groups) in the 2-hydroxy phenyl ring could enhance the anticancer activity.
(Fig. 1 should be listed here)
In this manuscript, we described a series of optimized semicarbazone derivatives
(Fig. 1), in which we reserved these lipophilic groups that were beneficial for anticancer
activity, while we replaced benzothiazole with phenyl to reduce lipophilicity. Furthermore, all
the designed compounds were salified with hydrogen chloride (HCl) to increase the solubility.
All the target compounds were evaluated for their in vitro anticancer activities against four
cancer cell lines (HT29, SK-N-SH, MDA-MB-231 and MKN45). Compounds with
promising anticancer activities were selected to further determine the cytotoxicity on the
normal cells (HUVEC). Cell cycle and apoptosis studies were carried out to explore their
mechanism of action. Metabolic stability studies were also performed to assess T1/2 in vitro
and provided a reliable guide for further PK/PD in vivo.
Results and Discussion
Chemistry The synthesis of target compounds is illustrated in Chart 1. 4-nitrobenzyl
bromide reacted with excessive secondary amines (dimethylamine and diethylamine) in
acetonitrile via a nucleophilic substitution reaction for 3 h to produce intermediates 3a-b,
which were further reduced in the presence of FeCl3·6H2O and 80 % hydrazine to obtain the
substituted anilines 4a-b. Treatment of 4a-b with phenyl chloroformate though N-acylation
reaction yielded 5a-b. The key semicarbazides 6a-b were synthesized via hydrazinolysis of
5a-b with 80% hydrazine in 1,4-dioxane at 80 °C for 6 h. In addition, the key intermediates
7a–j and 10 were prepared by using known preparation methods in our previous work21).
Subsequently, 6a-b reacted with appropriate 2-hydroxy aromatic aldehydes (7a–j and 10) via
Chemical and Pharmaceutical Bulletin Advance Publication

condensation reaction in ethanol at 78 °C in the presence of catalytic amounts of acetic acid,
then reaction mixtures were further salified with hydrogen chloride (HCl) in ethanol to get
target compounds 11a-v. Target compounds 11a-v might exist as either the E or Z isomeric
form due to presence of imino bond. Accordingly, compound 11s was selected to determine
the stereochemistry via undergoing NOESY-NMR. Our results (Fig. 2) indicated that an
obvious NOE signal was observed between the H1 (-NH-N=, 10.99 ppm) and H2 (-N=CH-,
8.24 ppm), which existed only in the E isomer due to the appropriate intramolecular H1–H2
distance. No NOE signal would be observed when compound existed in the Z isomer. Thus,
based on the above results, target compounds 11a-v were confirmed as the E isomer.
(Chart 1 should be listed here)
(Fig. 2 should be listed here)
In vitro anticancer activity All the target compounds (11a-v) were evaluated for their
in vitro anticancer activities against four human cancer cell lines, including human colon
cancer(HT29), human neuroblastoma(SK-N-SH), human breast cancer(MDA-MB-231) and
human gastric cancer(MKN45). The results were summarized in Table 1. As shown in Table
1, compared to the positive control PAC-1, most compounds displayed moderate to
prominent anticancer activities against these tested cancer cell lines. Among them, the
promising compounds 11q and 11s showed excellent activities against four cancer cell lines
with IC50 values ranging from 0.32 to 1.57 μM.
Further investigations were carried out to study the effect of different substituents of
benzyloxyl group on the activity (compounds 11a-p). The results revealed that incorporation
of bulk substituent at the 4-position of the benzyloxyl group could enhance anticancer
activity (11b vs. 11c, 11h vs. 11i), indicating that increasing steric hindrance of the group at
this region of benzyloxyl group exhibited a positive effect on the activity. Moreover, chlorine
(11d, 11j, R3=4-Cl) at the 4-position of benzyloxyl group was more preferable than the
fluorine (11e, 11m, R3=4-F), and the anticancer activities of compounds 11j, 11k and 11l
revealed that the 4-position of benzyl group did a better contribution to the potency than the 2
or 3-position. In addition, introduction of mono-electron-withdrawing group (11d, 11j,
R3=4-Cl) or weak mono-electron-donating group (11b, 11h, R=4-CH3) caused no remarkable
and regular alteration in activity, whereas introduction of two-EWGs such as 2,4-di-Cl or
2,3-di-Cl (11f, 11g, 11n and 11o) on the benzyl group result in a dramatic decrease in activity,
implying that greatly reducing the electron density of benzyl group was not beneficial for the
anticancer activity.
(Table 1 should be listed here)
To exclude the effect of cytotoxicity, compounds (11q and 11s) with promising
anticancer activities were carried out to evaluate their inhibitory activities for 48 h against
normal cells derived from Human Umbilical Vein Endothelial Cells (HUVEC). The results
(Fig. 3) revealed that no significant inhibition of proliferation was observed for treatment
with different concentrations (0.25, 0.5, 1, 2, 4, 8 μM) of 11q and 11s, suggesting that 11q
and 11s exhibited weak cytotoxicity on the normal cells. In addition, we also tested the
anticancer activities of 11q and 11s against three other tumor cell lines (U937, MCF-7 and
H226) in our laboratory, as show in Table 2, 11q and 11s showed potent activities against
these three cancer cell lines with IC50 values ranging from 1.60 to 3.85 μM, which were more
Chemical and Pharmaceutical Bulletin Advance Publication

active than PAC-1. Taken together, these above findings identified compound 11q and 11s as
the promising lead compounds for subsequent biological assessment.
(Fig.3 should be listed here)
(Table 2 should be listed here)
Cell cycle Encouraged by the anticancer activities of compounds 11q and 11s, we
conducted a series of cell-based assays to study their mechanism in depth. The assessment of
cell cycle of HT-29 cells treated with different concentrations (0, 2, 4, 8 μM) of 11q and 11s
was performed. Fig. 4 and Fig. 5 showed the distribution of HT-29 cells in different phases
of the cell cycle. It was observed that the treatment with 11q lead to a remarkable increase in
the sub-G1 area from 10.9% to 26.7% and 94.1% in the concentrations of 2, 4 and 8 μM,
respectively, while the treatment with 11s lead to a more obvious increase in the sub-G1 area
from 19.6% to 35.0% and 94.3% in the same concentrations. These results indicated that 11q
and 11s inhibited cancer cells proliferation by arresting the Sub-G1 phase (indicative of
apoptosis) in a dose-dependent manner.
(Fig. 4 should be listed here)
(Fig. 5 should be listed here)
Apoptosis study In order to determine whether the inhibitory effects of 11q and 11s
were dependent on inducing cancer cells apoptosis, HT29 cells were incubated with different
concentrations (0, 2, 4, and 8 μM) of 11q and 11s for 48 h and the percentages of apoptotic
cells were determined by FITC-Annexin V/PI staining and flow cytometry. As shown Fig. 6,
treatment HT-29 cells with different concentrations of 11q and 11s for 48ꢀh caused a
significant dose-dependent increase in the population of both early and late apoptotic cells
compared to the control cells, suggesting that 11q and 11s could induce cancer cells
apoptosis in a dose-dependent manner.
(Fig. 6 should be listed here)
In vitro procaspase-3 activation assay In order to explore further mechanism,
compounds 11q and 11s were selected for the enzymatic assay to determine procaspase-3
activation activity. As shown in Table 3, compared to PAC-1, compounds 11q and 11s
displayed potent procaspase-3 activation activities at 10 μM with degrees of 68.4% and
76.3%, respectively, suggesting that apoptosis of cancer cells induced by activation of
procaspase-3 might be a potential mechanism for antitumor effects of these target
compounds.
(Table 3 should be listed here)
Metabolic stability study Compounds 11q and 11s were submitted for in vitro
metabolic stability study in the presence of human, rat and mouse liver microsomes and
NADPH. The metabolic half-time(T1/2) and intrinsic clearance(Clint) were determined by
using LC-MS analysis. As shown in Table 4, compounds 11q and 11s showed moderate
metabolism in human and rat liver microsomes in vitro with suitable T1/2 values of 72.19 min,
57.17 min and 57.01 min, 47.69 min(T1/2 = 30-120 min), respectively. However, compounds
11q and 11s were susceptible to metabolism in mouse microsomes, with T1/2 values of 16.72
and 4.04 min(T1/2 < 30 min). These above findings provided a reliable guide for further
PK/PD study in vivo.
(Table 4. should be listed here)
Chemical and Pharmaceutical Bulletin Advance Publication

Conclusions
In conclusion, we synthesized a series of semicarbazone derivatives bearing phenyl
moiety. All the target compounds were evaluated for their in vitro anticancer activities in four
human cancer cell lines (HT29, SK-N-SH, MDA-MB-231 and MKN45). Most of them
exhibited moderate to prominent activities against all the tested cancer cell lines. We
identified 2 promising compounds (11q and 11s), which showed excellent anticancer
activities with IC50 values ranging from 0.32 to 1.57 μM, and exhibited weak cytotoxicity on
the normal cells (HUVEC). Cell cycle analysis revealed that 11q and 11s caused an arrest in
the Sub-G1 (indicative of apoptosis) cell cycle. Apoptosis induction study indicated that 11q
and 11s could inhibit proliferation of HT29 cells by inducing apoptosis in a dose-dependent
manner. Procaspase-3 activation assay suggested that 11q and 11s could activate
procaspase-3 by 68.4% and 76.3% at a concentration of 10 μM comparing to PAC-1.
Metabolic stability study revealed that 11q and 11s showed moderate stability in vitro in
human and rat liver microsomes. All these findings demonstrated that compounds 11q and
11s have the potential to be developed as valuable lead compounds. Studies on the
mechanism of action and in vivo PK/PD are in progress and will be reported in future.
Experimental section
Chemistry Reagents and solvents were obtained from commercial sources and used
without further purification. All the reactions were monitored by TLC using silica gel GF/UV
1
13
254. Flash chromatography was performed using silica gel (300–400mesh). The H and C
NMR spectra were recorded on Bruker AV-400 spectrometer, with TMS as an internal
standard. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo
Alto, CA, U.S.A.). The elemental analysis of compounds were performed on a Perkin Elmer
2400 Elemental Analyser.
General procedure for preparation of compounds 3a–b and 4a-b The preparation
methods of 3a–b and 4a-b has been illustrated in detail in previous study21, 22), and so the
synthesis method would not be listed here.
General procedure for preparation of compounds 5a-b To the mixture of compounds
4a-b (0.01 mol), DCM (20 mL) and pyridine (0.8 ml,0.02 mol) was added at room
temperature, then the mixture was cooled to 0 °C in an ice-salt bath, phenyl chloroformate
was added drop wise at such a rate to keep the temperature below 10 °C. The reaction
mixture was stirred at room temperature for 4-6 h and filtered. The white to light yellow solid
was collected and washed with DCM to obtain the compounds 5a-b.
Phenyl (4-((dimethylamino)methyl)phenyl)carbamate (5a) White solid; Yield: 82 %;
MS (ESI) m/z: 270.7 [M+H]+.
Phenyl (4-((diethylamino)methyl)phenyl)carbamate (5b) White solid; Yield: 88 %;
MS (ESI) m/z: 298.8 [M+H]+.
General procedure for preparation of 6a-b To the mixture of the 5a-b (0.01 mol),
1,4-dioxane (20 mL) and 80% hydrazine hydrate (1.29 ml, 0.02 mol)was added at room
temperature. The mixture was heated to 80 °C for 6 h, then the reaction mixture was cooled
to room temperature and concentrated, diethyl ether was added and stirred for 0.5 h and
filtered, a white solid was collected to get the compounds 6a-b.
N-(4-((dimethylamino)methyl)phenyl)hydrazinecarboxamide (6a) White solid; Yield:
Chemical and Pharmaceutical Bulletin Advance Publication

54 %; MS (ESI) m/z: 208.4 [M+H]+.
N-(4-((diethylamino)methyl)phenyl)hydrazinecarboxamide (6b) White solid; Yield:
62%; MS (ESI) m/z: 236.7 [M+H]+.
General procedure for preparation of 7a-j, 8, 9 and 10 The intermidiates 7a-j, 8, 9
and 10 were prepared according to a known procedure21, 22)
.
General procedure for preparation of 11a-v A mixture of the compounds 6a-b (0.001
mol), appropriate 2-hydroxy aromatic aldehydes or 7a-j or 10 (0.0011 mol) and a drop of
glacial acetic acid in 10 mL absolute ethanol was heated at reflux for 6 h and cooled to room
temperature, HCl in absolute ethanol (3 mL) was added, and then stirred at room temperature
for 4-6 h and filtered to get a light yellow solid, the crude product was washed with 5 mL
diethyl ether to afford compounds 11a-v.
(E)-2-(4-(benzyloxy)-2-hydroxybenzylidene)-N-(4-((dimethylamino)methyl)phenyl)
hydrazine-1-carboxamide hydrochloride (11a) Yellow solid; yield: 47%; MS (ESI) m/z:
418.8 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 10.64 (s, 1H), 10.44 (s, 1H),
9.14 (s, 1H), 8.19 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.50 – 7.43 (m,
4H), 7.40 (t, J = 7.2 Hz, 2H), 7.35 (d, J = 7.2 Hz, 1H), 6.59–6.52 (m, 2H), 5.10 (s, 2H), 4.19
(d, J = 4.4 Hz, 2H), 2.67 (d, J = 4.4 Hz, 6H); Anal. Calcd. for C24H27ClN4O3 (%):C, 63.36; H,
5.98; N, 12.32. Found (%):C, 63.44; H, 5.92; N, 12.44.
(E)-2-(2-hydroxy-4-((4-methylbenzyl)oxy)benzylidene)-N-(4-((dimethylamino)meth
yl)phenyl)hydrazine-1-carboxamide hydrochloride (11b) Yellow solid; yield: 52%; MS
1
(ESI) m/z: 432.5 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 10.36 (s, 2H),
9.10 (s, 1H), 8.18 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4
Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 6.57 – 6.48 (m, 2H), 5.04 (s, 2H),
4.19 (d, J = 4.8 Hz, 2H), 2.68 (d, J = 4.8 Hz, 6H), 2.31 (s, 3H); Anal. Calcd. for
C25H29ClN4O3 (%):C, 64.03; H, 6.23; N, 11.95. Found (%):C, 64.13; H, 6.28; N, 11.86.
(E)-2-(4-((4-(tert-butyl)benzyl)oxy)-2-hydroxybenzylidene)-N-(4-((dimethylamino)
methyl)phenyl)hydrazine-1-carboxamide hydrochloride (11c) Yellow solid; yield: 56%;
MS (ESI) m/z: 474.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 10.31 (s, 2H),
9.08 (s, 1H), 8.18 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.47 – 7.39 (m,
4H), 7.37 (d, J = 8.4 Hz, 2H), 6.57 – 6.49 (m, 2H), 5.05 (s, 2H), 4.19 (s, 2H), 2.68 (d, J = 4.0
Hz, 6H), 1.28 (s, 9H); Anal. Calcd. for C28H35ClN4O3 (%):C, 65.81; H, 6.90; N, 10.96. Found
(%):C, 65.88; H, 6.92; N, 10.86.
(E)-2-(4-((4-chlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((dimethylamino)methy
l)phenyl) hydrazine-1-carboxamide hydrochloride (11d) Yellow solid; yield: 54%; MS
1
(ESI) m/z: 452.1 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 10.59 (s, 1H),
10.44 (s, 1H), 9.14 (s, 1H), 8.19 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H),
7.48 (s, 5H), 7.45 (s, 1H), 6.57 – 6.51 (m, 2H), 5.10 (s, 2H), 4.19 (d, J = 4.8 Hz, 2H), 2.67 (d,
J = 4.8 Hz, 6H); 13C NMR (101 MHz, DMSO) δ 160.74, 158.23, 153.20, 140.95, 136.38,
132.92, 131.98, 130.00, 128.92, 124.07, 119.54, 114.02, 107.18, 102.42, 68.83, 59.53, 41.69.
Anal. Calcd. for C24H22Cl2N4O3 (%):C, 58.90; H, 5.36; N, 11.45. Found (%):C, 58.81; H,
5.42; N, 11.52.
(E)-2-(4-((4-fluorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((dimethylamino)methy
l)phenyl) hydrazine-1-carboxamide hydrochloride (11e) Yellow solid; yield: 54%; MS
Chemical and Pharmaceutical Bulletin Advance Publication

1
(ESI) m/z: 436.1 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.49 (s, 2H),
9.12 (s, 1H), 8.19 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.54 – 7.48 (m,
2H), 7.46 (d, J = 8.4 Hz, 2H), 7.23 (t, J = 8.8 Hz, 2H), 6.59 – 6.50 (m, 2H), 5.08 (s, 2H), 4.19
(d, J = 4.8 Hz, 2H), 2.67 (d, J = 4.8 Hz, 6H); Anal. Calcd. for C24H26ClFN4O3 (%):C, 60.95;
H, 5.54; N, 11.85. Found (%):C, 60.86; H, 5.59; N, 11.78.
(E)-2-(4-((2,4-dichlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((dimethylamino)m
ethyl)phenyl)hydrazine-1-carboxamide hydrochloride (11f) Yellow solid; yield: 59%; MS
1
(ESI) m/z: 486.0 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 10.37 (s, 1H),
9.01 (s, 1H), 8.19 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.66 (d, J = 8.4
Hz, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 8.4, 2.0 Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H),
6.58 – 6.53 (m, 2H), 5.15 (s, 2H), 3.90 (s, 2H), 2.50 (s, 6H); Anal. Calcd. for C24H25Cl3N4O3
(%):C, 55.03; H, 4.81; N, 10.70. Found (%):C, 55.22; H, 4.73; N, 10.75.
(E)-2-(4-((2,3-dichlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((dimethylamino)m
ethyl)phenyl)hydrazine-1-carboxamide hydrochloride (11g) Yellow solid; yield: 55%; MS
1
(ESI) m/z: 486.1 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 10.49 (s, 1H),
9.12 (s, 1H), 8.20 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.67 (t, J = 8.1 Hz, 3H), 7.57 (d, J = 7.2
Hz, 1H), 7.48 – 7.39 (m, 3H), 6.60 – 6.54 (m, 2H), 5.20 (s, 2H), 4.10 (s, 2H), 2.61 (s, 6H).
13C NMR (101 MHz, DMSO) δ 160.51, 158.22, 153.21, 140.65, 137.31, 132.41, 131.67,
130.82, 130.61, 129.11, 128.87, 128.77, 119.57, 114.36, 114.03, 107.15, 102.34, 67.62, 60.07,
42.20. Anal. Calcd. for C24H25Cl3N4O3 (%):C, 55.03; H, 4.81; N, 10.70. Found (%):C, 55.12;
H, 4.77; N, 10.53.
(E)-2-(2-hydroxy-4-((4-methylbenzyl)oxy)benzylidene)-N-(4-((diethylamino)methyl)
phenyl)hydrazine-1-carboxamide hydrochloride (11h) Yellow solid; yield: 62%; MS (ESI)
m/z: 460.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 10.39 (s, 1H), 10.18 (s,
1H), 9.10 (s, 1H), 8.18 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.3 Hz, 2H), 7.50 (d, J =
8.4 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 6.59 – 6.47 (m, 2H), 5.04 (s,
2H), 4.21 (d, J = 5.0 Hz, 2H), 3.03 (dd, J = 12.3, 7.4 Hz, 4H), 2.31 (s, 3H), 1.24 (t, J = 7.2 Hz,
6H); Anal. Calcd. for C26H31ClN4O3 (%):C, 64.65; H, 6.47; N, 11.60. Found (%):C, 64.72; H,
6.58; N, 11.51.
(E)-2-(4-((4-(tert-butyl)benzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)me
thyl)phenyl)hydrazine-1-carboxamide hydrochloride (11i) Yellow solid; yield: 63%; MS
1
(ESI) m/z: 502.5 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 10.39 (s, 1H),
10.18 (s, 1H), 9.10 (s, 1H), 8.18 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H),
7.50 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 6.59 – 6.47 (m,
2H), 5.04 (s, 2H), 4.21 (d, J = 5.0 Hz, 2H), 3.03 (dd, J = 12.4, 7.2 Hz, 4H), 2.31 (s, 3H), 1.24
(t, J = 7.2 Hz, 6H); Anal. Calcd. for C30H39ClN4O3 (%):C, 66.84; H, 7.29; N, 10.39. Found
(%):C, 66.92; H, 7.42; N, 10.31.
(E)-2-(4-((4-chlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)methyl)
phenyl)hydrazine-1-carboxamide hydrochloride (11j) Yellow solid; yield: 55%; MS (ESI)
m/z: 480.2 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.42 (s, 1H), 10.26 (s,
1H), 9.12 (s, 1H), 8.19 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.51 (d, J =
8.8 Hz, 2H), 7.47 (s, 4H), 6.59 – 6.50 (m, 2H), 5.10 (s, 2H), 4.21 (d, J = 4.8 Hz, 2H), 3.02 (dd,
J = 12.4, 7.2 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); Anal. Calcd. for C26H30Cl2N4O3 (%):C, 60.35;
Chemical and Pharmaceutical Bulletin Advance Publication

H, 5.84; N, 10.83. Found (%):C, 60.48; H, 5.88; N, 10.89.
(E)-2-(4-((3-chlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)methyl)
phenyl)hydrazine-1-carboxamide hydrochloride (11k) Yellow solid; yield: 58%; MS (ESI)
m/z: 480.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 10.38 (s, 1H), 10.15 (s,
1H), 9.08 (s, 1H), 8.17 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.50 (s, 2H),
7.47 (s, 1H), 7.43 – 7.36 (m, 3H), 6.56 – 6.50 (m, 2H), 5.10 (s, 2H), 4.19 (d, J = 5.2 Hz, 2H),
3.10 – 2.92 (m, 4H), 1.23 (t, J = 7.2 Hz, 6H); Anal. Calcd. for C26H30Cl2N4O3 (%):C, 60.35;
H, 5.84; N, 10.83. Found (%):C, 60.45; H, 5.83; N, 10.79.
(E)-2-(4-((2-chlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)methyl)
phenyl)hydrazine-1-carboxamide hydrochloride (11l) Yellow solid; yield: 65%; MS (ESI)
m/z: 480.2 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 10.40 (s, 1H), 10.19 (s,
1H), 9.11 (s, 1H), 8.20 (s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.71 (d, J = 8.5 Hz, 2H), 7.56 (dd, J
= 8.4, 6.8 Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.44 (ddd, J = 9.5, 7.4, 1.7 Hz, 1H), 7.26 (dd, J =
15.8, 7.9 Hz, 2H), 6.57 (dd, J = 5.7, 2.3 Hz, 2H), 5.14 (s, 2H), 4.21 (d, J = 5.3 Hz, 2H), 3.57
(s, 4H), 3.03 (dd, J = 12.5, 7.6 Hz, 4H), 1.25 (t, J = 7.2 Hz, 6H); Anal. Calcd. for
C26H30Cl2N4O3 (%):C, 60.35; H, 5.84; N, 10.83. Found (%):C, 60.30; H, 5.81; N, 10.88.
(E)-2-(4-((4-fluorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)methyl)
phenyl)hydrazine-1-carboxamide hydrochloride (11m) Yellow solid; yield: 48%; MS (ESI)
m/z: 464.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.42 (s, 1H), 10.26 (s,
1H), 9.12 (s, 1H), 8.19 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.51 (d, J =
8.8 Hz, 2H), 7.47 (s, 4H), 6.59 – 6.50 (m, 2H), 5.10 (s, 2H), 4.21 (d, J = 4.8 Hz, 2H), 3.02 (dd,
J = 12.4, 7.2 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); Anal. Calcd. for C26H30ClFN4O3 (%):C, 62.33;
H, 6.04; N, 11.18. Found (%):C, 62.38; H, 6.14; N, 11.07.
(E)-2-(4-((2,4-dichlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)met
hyl)phenyl)hydrazine-1-carboxamide hydrochloride (11n) Yellow solid; yield: 65%; MS
1
(ESI) m/z: 514.4 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 10.38 (s, 1H),
10.15 (s, 1H), 9.08 (s, 1H), 8.17 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.72 – 7.66 (m, 3H), 7.59 (d,
J = 8.4 Hz, 1H), 7.52 – 7.45 (m, 3H), 6.57 – 6.51 (m, 2H), 5.13 (s, 2H), 4.19 (d, J = 5.2 Hz,
2H), 3.10 – 2.92 (m, 4H), 1.23 (t, J = 7.2 Hz, 6H); Anal. Calcd. for C26H29Cl3N4O3 (%):C,
56.58; H, 5.30; N, 10.15. Found (%):C, 56.42; H, 5.39; N, 10.33.
(E)-2-(4-((2,3-dichlorobenzyl)oxy)-2-hydroxybenzylidene)-N-(4-((diethylamino)met
hyl)phenyl)hydrazine-1-carboxamide hydrochloride (11o) Yellow solid; yield: 55%; MS
1
(ESI) m/z: 514.3 [M+H]+; H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 10.28 (s, 1H),
8.98 (s, 1H), 8.20 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 7.9 Hz, 2H), 7.66 (d, J = 7.6
Hz, 1H), 7.56 (d, J = 7.1 Hz, 1H), 7.49 – 7.29 (m, 3H), 6.57 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H),
5.21 (s, 2H), 4.15 (s, 2H), 3.08 – 2.90 (m, 4H), 1.20 (t, J = 6.4 Hz, 6H); 13C NMR (101 MHz,
DMSO) δ 160.56, 158.04, 153.31, 137.30, 132.42, 131.53, 130.79, 130.61, 128.99, 128.89,
128.73, 119.90, 114.44, 107.25, 102.24, 67.62, 65.37, 46.32, 15.63; Anal. Calcd. for
C26H29Cl3N4O3 (%):C, 56.58; H, 5.30; N, 10.15. Found (%):C, 56.50; H, 5.41; N, 10.23.
(E)-2-(2-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzylidene)-N-(4-((diethylami
no)methyl)phenyl)hydrazine-1-carboxamide hydrochloride (11p) Yellow solid; yield:
52%; MS (ESI) m/z: 514.0 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.43 (s,
1H), 10.25 (s, 1H), 9.12 (s, 1H), 8.19 (s, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.78 (d, J = 8.4 Hz,
Chemical and Pharmaceutical Bulletin Advance Publication

2H), 7.69 (t, J = 9.2 Hz, 4H), 7.51 (d, J = 8.4 Hz, 2H), 6.59 – 6.52 (m, 2H), 5.23 (s, 2H), 4.21
(d, J = 5.2 Hz, 2H), 3.12 – 2.94 (m, 4H), 1.24 (t, J = 7.2 Hz, 6H); Anal. Calcd. for
C27H30ClF3N4O3 (%):C, 58.86; H, 5.49; N, 10.17. Found (%):C, 58.93; H, 5.41; N, 10.28.
(E)-2-(3,5-di-tert-butyl-2-hydroxybenzylidene)-N-(4-((dimethylamino)methyl)phen
yl)hydrazine-1-carboxamide hydrochloride (11q) Yellow solid; yield: 61%; MS (ESI) m/z:
424.5 [M+H]+ 1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 10.99 (s, 1H), 10.54 (s, 1H),
9.63 (s, 1H), 8.24 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 2.4
13
Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 4.20 (s, 2H), 2.68 (s, 6H), 1.41 (s, 9H), 1.28 (s, 9H); C
NMR (101 MHz, DMSO) δ 154.23, 152.37, 146.84, 141.07, 140.85, 135.89, 132.21, 125.80,
125.17, 124.03, 118.91, 118.07, 59.57, 41.74, 35.10, 34.36, 31.80, 29.83; Anal. Calcd. for
C25H37ClN4O2 (%):C, 65.13; H, 8.09; N, 12.15. Found (%):C, 65.28; H, 8.19; N, 12.10.
(E)-2-(3-allyl-2-hydroxybenzylidene)-N-(4-((dimethylamino)methyl)phenyl)hydrazi
ne-1-carboxamide hydrochloride (11r) Yellow solid; yield: 44%; MS (ESI) m/z: 352.5
[M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 10.57 (s, 1H), 9.52 (s, 1H), 8.24 (s,
1H), 7.62 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 6.4 Hz, 1H), 7.14 (d, J =
6.4 Hz, 1H), 6.87 (t, J = 7.2 Hz, 1H), 6.07 – 5.91 (m, 1H), 5.10 – 5.01 (m, 2H), 4.19 (d, J =
4.8 Hz, 2H), 3.38 (d, J = 6.8 Hz, 2H), 2.68 (s, 3H), 2.66 (s, 3H); Anal. Calcd. for
C20H25ClN4O2 (%):C, 61.77; H, 6.48; N, 14.41. Found (%):C, 61.89; H, 6.42; N, 14.55.
(E)-2-(3,5-di-tert-butyl-2-hydroxybenzylidene)-N-(4-((diethylamino)methyl)phenyl)
hydrazine-1-carboxamide hydrochloride (11s) Yellow solid; yield: 57%; MS (ESI) m/z:
452.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 10.99 (s, 1H), 10.31 (s, 1H),
9.63 (s, 1H), 8.24 (s, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 2.4
Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 4.22 (s, 2H), 3.16 – 2.92 (m, 4H), 1.41 (s, 9H), 1.28 (s, 9H),
1.27 – 1.20 (m, 6H); 13C NMR (101 MHz, DMSO) δ 154.23, 152.38, 146.84, 140.96, 140.85,
135.89, 132.24, 125.80, 125.17, 123.80, 118.94, 118.07, 54.78, 45.93, 35.10, 34.36, 31.80,
29.82, 8.79; Anal. Calcd. for C27H41ClN4O2 (%):C, 66.30; H, 8.45; N, 11.46. Found (%):C,
66.41; H, 8.40; N, 11.33.
(E)-2-(3-allyl-2-hydroxybenzylidene)-N-(4-((diethylamino)methyl)phenyl)hydrazine
-1-carboxamide hydrochloride (11t) Yellow solid; yield: 46%; MS (ESI) m/z: 380.5
[M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 10.75 (s, 1H), 9.96 (s, 1H), 9.39 (s,
1H), 8.22 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 6.4 Hz, 1H),
7.12 (d, J = 7.2 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 6.04 – 5.90 (m, 1H), 5.09 – 4.98 (m, 2H),
4.20 (d, J = 4.2 Hz, 2H), 3.36 (d, J = 6.4 Hz, 2H), 3.31 (s, 1H), 3.08 – 2.96 (m, 4H), 1.22 (t, J
= 7.2 Hz, 6H); Anal. Calcd. for C22H29ClN4O2 (%):C, 63.37; H, 7.01; N, 13.44. Found (%):C,
63.45; H, 7.09; N, 13.34.
(E)-2-(4-((2-(benzo[d][1,3]dioxol-5-ylmethyl)thiazol-4-yl)methoxy)-2-hydroxybenzy
lidene)-N-(4-((dimethylamino)methyl)phenyl)hydrazine-1-carboxamide hydrochloride
1
(11u) Yellow solid; yield: 67%; MS (ESI) m/z: 559.2 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 10.60 (s, 1H), 10.36 (s, 1H), 8.99 (s, 1H), 8.18 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H),
7.64 (d, J = 8.2 Hz, 2H), 7.58 (s, 1H), 7.33 (d, J = 8.2 Hz, 2H), 6.93 (s, 1H), 6.89 (d, J = 7.8
Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.62 – 6.51 (m, 2H), 6.00 (s, 2H), 5.11 (s, 2H), 4.24 (s, 2H),
3.80 (s, 2H), 2.43 (s, 6H); Anal. Calcd. for C28H28ClN5O5S (%):C, 57.78; H, 4.85; N, 12.03.
Found (%):C, 57.92; H, 4.89; N, 11.93.
Chemical and Pharmaceutical Bulletin Advance Publication

(E)-2-(4-((2-(benzo[d][1,3]dioxol-5-ylmethyl)thiazol-4-yl)methoxy)-2-hydroxybenzy
lidene)-N-(4-((diethylamino)methyl)phenyl)hydrazine-1-carboxamide
1
hydrochloride(11v) Yellow solid; yield: 71%; MS (ESI) m/z (%): 587.1 [M+H]+; H NMR
(400 MHz, DMSO-d6) δ 10.49 (s, 1H), 10.29 (s, 1H), 8.83 (s, 1H), 8.17 (s, 1H), 7.83 (d, J =
8.4 Hz, 1H), 7.57 (s, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.21 (d, J = 7.2 Hz, 2H), 6.93 (s, 1H),
6.89 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.60 – 6.49 (m, 2H), 6.00 (s, 2H), 5.11 (s,
2H), 4.24 (s, 2H), 3.46 (s, 2H), 2.49 – 2.34 (m, 4H), 1.07 – 0.89 (m, 6H); Anal. Calcd. for
C30H32ClN5O5S (%):C, 59.06; H, 5.29; N, 11.48. Found (%):C, 59.16; H, 5.23; 11.35.
MTT assay The anticancer activities of compounds 11a-v were evaluated against tested
cell lines using the standard MTT assay in vitro, with PAC-1 as the positive control. Cancer
cell lines were cultured in minimum essential medium (MEM) supplemented with 10% fetal
bovine serum (FBS). Approximate 4 × 103 cells, suspended in MEM medium, were plated
onto each well of a 96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The compounds
tested at the indicated final concentrations were added to the culture medium and the cell
cultures were continued for 72 h. Fresh MTT was added to each well at a terminal
concentration of 5 mg/mL, and incubated with cells at 37 °C for 4 h. The formazan crystals
were dissolved in 100 mL of DMSO each well, and the absorbance at 492 nm (for absorbance
of MTT formazan) and 490 nm (for the reference wavelength) was measured with an ELISA
reader. All compounds were tested three times in each cell line. The results expressed as IC50
(inhibitory concentration 50%) were the averages of three determinations and calculated by
using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
Cell cycle HT29ꢀcells were treated with different concentrations of 11q and 11s for
48ꢀh and were harvested by digestion with trypsin and centrifugation (1500ꢀrpm for 15ꢀmin).
The cell pellets were suspended with 70 % ethanol at −20ꢀ°C overnight. Afterwards, cells
were washed with PBS twice and incubated with RNase (180ꢀμg/mL) for 30ꢀminꢀat 37ꢀ°C, and
followed by incubation with PI solution (final concentration 50ꢀμg/mL) for 30ꢀmin in the dark.
Cells were analyzed by flow cytometry system (BD Biosciences) and results were analyzed
by FlowJo V10 software.
Apoptosis study Apoptosis of HT-29 cells was detected using a flow cytometric assay.
Briefly, cells were seeded in 6-well plates and incubated overnight. The following day, cells
were treated with different concentrations of compounds 11q and 11s for 48 hours. The cells
and supernatants were harvested and washed twice with cold PBS and then resuspended in
100µl 1× Binding Buffer. 5 µl of FITC Annexin V and 5 µl PI were added in each tube and
the cells were then gently vortexed incubated for 15 min at RT (25°C) in the dark. 400 µl of
1× Binding Buffer then added to each tube. The stained cells were analyzed by a flow
cytometer (FACS Calibur; BD).
Procaspase-3 activation assay Procaspase-3 was purchased from R&D Systems.
Procaspase-3 was incubated at 100 nM, in the presence of selected compounds at 10 μM in a
reaction buffer consisted of 20 mM Tris, 300 mM NaCl, 5 mM Dithiothreitol, 5% Sucrose
and 0.05% CHAPS, pH 8.0. The mixtures were assayed for kinetic activity by incubation
with 20 μM Ac-DEVD-AFC for 5 minutes after 4 hours of incubation at 37 °C. Fluorescence
value was read at excitation and emission wavelengths of 400 nm and 505 nm. The activation
rate (%) was calculated using the following equation: (Fcompound-Fdmso)/(FPAC-1-Fdmso)×100.
Chemical and Pharmaceutical Bulletin Advance Publication

Metabolic stability Experimental procedure: (1) Buffer A: 1.0 L of 0.1 M monobasic
Potassium Phosphate buffer containing 1.0 mM EDTA, Buffer B: 1.0 L of 0.1 M Dibasic
Potassium Phosphate buffer containing 1.0 mM EDTA, Buffer C: 0.1 M Potassium
Phosphate buffer, 1.0 mM EDTA, pH 7.4 by titrating 700 mL of buffer B with buffer A while
monitoring with the pH meter. (2) Reference compouds(Ketanserin) and test compounds
spiking solution: 500 µM spiking solution: add 10 µL of 10 mM DMSO stock solution into
190 µL ACN. 1.5 µM spiking solution in microsomes (0.75 mg/mL): add 1.5 µL of 500 µM
spiking solution and 18.75 µL of 20 mg/mL liver microsomes into 479.75 µL of Buffer C on
ice. (3) Prepare NADPH stock solution (6 mM) by dissolving NADPH into buffer C. (4)
Dispense 30 µL of 1.5 µM spiking solution containing 0.75 mg/mL microsomes solution to
the assay plates designated for different time points (0, 5, 15, 30, 45 min) on ice. (5) For
0-min, add 135 µL of ACN containing IS to the wells of 0-min plate and then add 15 µL of
NADPH stock solution (6 mM). (6) Pre-incubate all other plates at 37 °C for 5 minutes. (7)
Add 15 µL of NADPH stock solution (6 mM) to the plates to start the reaction and timing. (8)
At 5-min, 15-min, 30-min, and 45-min, add 135 µL of ACN containing IS to the wells of
corresponding plates, respectively, to stop the reaction. (9) After quenching, shake the plates
at the vibrator (IKA, MTS 2/4) for 10 min (600 rpm/min) and then centrifuge at 5594 g for 15
min (Thermo Multifuge × 3R). (10) Transfer 50 μL of the supernatant from each well into a
96-well sample plate containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for
LC/MS analysis.
Acknowledgments This work was financially supported by the National Natural
Science Foundation of China(NO. 81602970), FuJiang Province Natural Science
Foundation(NO.2017J01143), Promotion Program for Young and Middle-aged Teacher in
Science and Technology Research of Huaqiao University(NO.ZQN-PY519) and Subsidized
Project for Postgraduates’Innovative Fund in Scientific Research of Huaqiao University.
Conflicts of interest The authors declare no conflict of interest.
Supplementary Materials The online version of this article contains supplementary
materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 1. Reported procaspase-3 activators and target compounds in this work
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1. Reagents and conditions: (i) Acetonitrile, amine, rt, 3 h; (ii) 80% hydrazine
monohydrate, FeCl3·6H2O, activated carbon, ethanol, 65 °C to 78 °C, 5 h; (iii) Phenyl
chloroformate, pyridine, CH2Cl2, 0 °C to rt, 4-6 h; (iv) 80% hydrazine monohydrate,
1,4-dioxane, 80 °C,6 h; (v) Na2CO3, KI, 65 °C, 30 h; (vi) NaHS, MgCl2·6H2O, DMF, H2O, rt,
15 h; (vii) 1,3-dichloro-2-propanon, acetonitrile, 50 °C, 4 h; (viii) 2,4-dihydroxy
benzaldehyde, NaHCO3, KI, acetonitrile, 80 °C, 2 h; (ix) Acetic acid, ethanol, reflux; HCl in
ethanol, rt.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 2. NOE of the representative compound 11s.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 1. Anticancer activities for synthesized compounds against tumor cell lines.
HCl
N
O
R1
HCl
N
HCl
R3 R1
HO
S
HO
O
O
O
R1
HO
O
R2
R3
N
O
O
R2
N
N
N
N
R2
N
N
N
N
N
N
H
H
H
H
H
H
11u and 11v
11a-p
11q-t
IC50±SD(μM)a
R1
N
R2
Compd.
R3
HT29
SK-N-SH
MDA-MB-231
MKN45
N
N
11a
11b
H
9.06±0.57
1.70±0.17
8.43±0.39
>100
1.30±0.18
6.90±0.87
4-CH3
1.78±0.11
10.34±1.57
N
N
11c
11d
4-C(CH3)3
4-Cl
1.40±0.23
3.64±0.48
1.44±0.18
8.51±0.63
3.32±0.62
4.92±1.23
2.60±0.49
0.83±0.21
N
11e
4-F
4.45±0.24
8.56±0.53
8.88±0.86
1.40±0.23
N
N
11f
11g
11h
11i
2,4-(Cl)2
2,3-(Cl)2
4-CH3
4-C(CH3)3
4-Cl
8.79±1.03
3.94±0.51
1.92±0.18
1.56±0.23
1.37±0.54
2.92±0.39
2.35±0.41
5.51±0.61
6.09±1.46
8.35±1.56
4.96±1.36
13.67±1.43
4.74±1.21
2.70±0.33
0.78±0.11
1.01±0.38
2.16±0.52
4.02±0.91
3.55±0.48
5.65±1.11
6.74±0.89
6.53±1.91
10.71±1.28
9.4±1.01
35.9±1.56
4.20±1.03
1.11±0.83
1.10±0.32
0.51±0.21
1.80±0.26
2.71±0.58
0.88±0.29
4.01±0.45
6.72±1.22
2.02±1.02
N
12.36±1.73
2.28±0.37
4.40±0.93
7.22±1.24
8.94±0.72
5.19±0.38
12.77±1.88
10.21±1.63
13.33±1.78
N
N
N
N
N
N
N
N
11j
11k
11l
3-Cl
2-Cl
11m
11n
11o
11p
4-F
2,4-(Cl)2
2,3-(Cl)2
4-CF3
N
N
11q
3,5-di-tert-butyl
0.32±0.13
0.83±0.21
1.56±0.42
0.91±0.21
11r
11s
11t
3-allyl
3,5-di-tert-butyl
3-allyl
1.39±0.29
0.37±0.21
2.21±0.29
4.50±0.21
0.87±0.15
6.21±0.47
9.04±1.09
1.57±0.37
13.70±1.03
4.60±0.93
1.50±0.39
1.80±0.20
N
N
N
11u
-
2.44±0.53
2.07±0.61
1.96±0.48
2.32±0.21
N
11v
-
-
2.11±0.73
1.16±0.51
1.64±0.18
3.91±0.61
1.92±0.36
1.52±0.13
2.06±0.31
1.16±0.38
PAC-1
-
a IC50: The biological data are generated from at least three independent experiments.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 3. Effect of 11q and 11s on viability of normal cells(HUVEC).
Chemical and Pharmaceutical Bulletin Advance Publication

Table 2. Anticancer activities of 11q and 11s against three cancer cell lines.
IC50±SD(μM)a
Compd.
U937
MCF-7
3.34±0.82
3.85±0.67
>40
H226
2.57±0.51
2.68±0.71
6.01±0.58
1.60±0.31
1.62±0.38
2.08±0.18
11q
11s
PAC-1
a IC50: The biological data are generated from at least three independent experiments.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 4. Cell cycle arrest at Sub-G1 phase by 11q and 11s in HT-29 cells. HT-29 cells were
incubated with the indicated concentrations of 11q and 11s for 48 h and the cells were stained
with PI. Cellular DNA content, for cell cycle distribution analysis, was measured using a
flow cytometry. The diagrams showed the distribution of the cells according to their DNA
content. The inserts gave the percentages in different cell cycle phases.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig.5. Column graph of DNA content in different cell cycle phases
Chemical and Pharmaceutical Bulletin Advance Publication

Fig.6. Apoptosis in HT-29 cells by the treatment with 11q and 11s. HT-29 cells were
incubated with different concentrations of 11q and 11s for 48 h and the cells were stained
with annexin V-FITC and PI, followed by flow cytometry analysis.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 3. Procaspase-3 activity of selected compounds 11q, 11s and PAC-1 in vitro.
Compd.
Procapase-3 (% activity at 10μM)
11q
68.4±0.8
11s
76.3±0.6
PAC-1
100
Chemical and Pharmaceutical Bulletin Advance Publication

Table 4. In vitro metabolic half-life (T1/2) [min] and intrinsic clearance (Clint) of compounds
11q and 11s.
Species
Comp.
ketanserin
11q
Index
human
36.07
rat
mouse
20.46
T1/2 (min)
14.40
Clint (mL/min/kg)
T1/2 (min)
Clint (mL/min/kg)
48.19
72.19
24.08
172.52
57.17
43.44
266.76
8.95
609.88
T1/2 (min)
51.01
34.08
47.69
52.08
4.04
11s
Clint (mL/min/kg)
1351.68
Chemical and Pharmaceutical Bulletin Advance Publication