Design, synthesis, and structure-activity relationships of novel benzothiazole derivatives bearing the ortho-hydroxy N-carbamoylhydrazone moiety as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.08.058

A series of novel benzothiazole derivatives bearing the ortho-hydroxy N-carbamoylhydrazone moiety were designed and synthesized and their cytotoxic activities against five cancer cell lines (NCI-H226, SK-N-SH, HT29, MKN45, and MDA-MB-231) were screened in vitro. Most of them showed moderate to excellent activity against all the tested cell lines. Among them, compounds 15g (procaspase-3 EC₅₀ = 1.42 μM) and 16b (procaspase-3 EC ₅₀ = 0.25 μM) exhibited excellent antitumor activity with IC ₅₀ values ranging from 0.14 μM to 0.98 μM against all cancer cell lines, which were 1.8-8.7 times more active than the first procaspase activating compound (PAC-1) (procaspase-3 EC₅₀ = 4.08 μM). The structure-activity relationship (SAR) analyses indicated that the introduction of a lipophilic group (a benzyloxy or heteroaryloxy group) at the 4-position of the 2-hydroxy phenyl ring was beneficial to antitumor activity, and the presence of substituents containing nitrogen that are positively charged at physiological pH could also improve antitumor activity. It was also confirmed that the steric effect of the 4-position substituent of the benzyloxy group had a significant influence on cytotoxic activity.

Full text of DOI:10.1016/j.ejmech.2014.08.058

Accepted Manuscript
Design, synthesis, and structure-activity relationships of novel benzothiazole
derivatives bearing the ortho-hydroxy N-carbamoylhydrazone moiety as potent
antitumor agents
Junjie Ma, Dong Chen, Kuan Lu, Lihui Wang, Xiaoqi Han, Yanfang Zhao, Ping Gong
PII:
S0223-5234(14)00793-4
10.1016/j.ejmech.2014.08.058
EJMECH 7291
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2014
Revised Date: 15 August 2014
Accepted Date: 16 August 2014
Please cite this article as: J. Ma, D. Chen, K. Lu, L. Wang, X. Han, Y. Zhao, P. Gong, Design, synthesis,
and structure-activity relationships of novel benzothiazole derivatives bearing the ortho-hydroxy N-
carbamoylhydrazone moiety as potent antitumor agents, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.08.058.
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ACCEPTED MANUSCRIPT
A series of novel benzothiazole derivatives bearing ortho-hydroxy N-carbamoylhydrazone moiety
were synthesized and evaluated for their cytotoxic activities. Six potent compounds were further
examined for their procaspase-3 kinase activity

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationships of novel benzothiazole
derivatives bearing the ortho-hydroxy N-carbamoylhydrazone moiety as potent
antitumor agents
Junjie Maa, Dong Chenb, Kuan Lua, Lihui Wangc, Xiaoqi Hana, Yanfang Zhaoa, Ping Gonga,
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, 103 Wenhua Road, Shenhe District, 110016, P. R. China.
bQilu Pharmaceutical Research Institute, Qilu Pharmaceutical Co., Ltd., Ji'nan 250100.
cDepartment of Pharmacology, Shenyang Pharmaceutical University College of life science and
biopharmaceutical, Shenyang, 110016
Corresponding author.
Tel: +86 24 2398 6429; Fax: +86 24 2398 6429;
E-mail address: gongpinggp@126.com
Abstract
A
series of novel benzothiazole derivatives bearing the ortho-hydroxy
N-carbamoylhydrazone moiety were designed and synthesized and their cytotoxic activities
against five cancer cell lines (NCI-H226, SK-N-SH, HT29, MKN45, and MDA-MB-231) were
screened in vitro. Most of them showed moderate to excellent activity against all the tested cell
lines. Among them, compounds 15g (procaspase-3 EC50 =1.42 ꢀM) and 16b (procaspase-3 EC50 =
0.25 ꢀM) exhibited excellent antitumor activity with IC50 values ranging from 0.14 ꢀM to 0.98
ꢀM against all cancer cell lines, which were 1.8–8.7 times more active than the first procaspase
activating compound (PAC-1) (procaspase-3 EC50 = 4.08 ꢀM). The structure-activity relationship
(SAR) analyses indicated that the introduction of a lipophilic group (a benzyloxy or heteroaryloxy
group) at the 4-position of the 2-hydroxy phenyl ring was beneficial to antitumor activity, and the
presence of substituents containing nitrogen that are positively charged at physiological pH could
also improve antitumor activity. It was also confirmed that the steric effect of the 4-position
substituent of the benzyloxy group had a significant influence on cytotoxic activity.
Keywords: antitumor; benzothiazole; ortho-hydroxy N-carbamoylhydrazone moiety; procaspase-3
1. Introduction
The primary cause of cancer development and progression is the dysregulation of
apoptosis[1]. Thus, it would be an effective approach to explore novel apoptosis-inducing
compounds for the treatment of cancer. Compounds such as p53 disruptors (tenovin-1)[2] and
inhibitors of XIAP (GDC-0152)[3] or Bcl-2 (GDC-0199)[4] act directly on proteins in the
apoptotic pathway, to induce apoptosis and lead to the death of cancer cells. However, direct
activation of procaspase-3 by small molecules (PAC-1 and S-PAC-1, Fig. 1) could have
advantages over the above, because numerous studies have demonstrated that procaspase-3 is
overexpressed in a variety of human tumors, including those of colon cancer[5], lung cancer[6],
melanoma[7], hepatoma[8], breast cancer[9], lymphoma [10], and neuroblastoma[11].
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Structure−activity relationship (SAR) studies revealed that the activity of PAC-1 and
S-PAC-1 in vitro and in cell culture is dependent on the presence of the ortho-hydroxy
N-acylhydrazone moiety, which is known to participate in metal chelation[12]. In addition, studies
have also revealed that the piperazine nitrogens in PAC-1 and S-PAC-1 may play a very
important role in their activity[5], because the autoactivation mechanism provides another channel
for the activation of procaspase-3 to caspase-3[13]. It is possible that the piperazine nitrogens in
PAC-1 and S-PAC-1 are positively charged at physiological pH, which may directly interact with
the triaspartic acid “safety catch” of procaspase-3, as such inducing the autoactivation of
procaspase-3 and catalyze the hydrolysis of hundreds of protein substrates, leading to cell death.
(Fig. 1. should be listed here)
Our group have reported a series of derivatives bearing an ortho-hydroxy N-acylhydrazone
moiety (3, 4, Fig. 2), the SAR of which exhibited that the introduction of either a benzyloxy or
heteroaryloxy group at the 2-hydroxy phenyl ring could enhance activity in vitro[14-16].
Benzothiazoles have attracted much attention over many years due to their diverse biological
properties, including antifungal[17], anticancer[18, 19], antiamyloid[20], and antirheumatic[21]
utility, and have been widely used as important structural components in modern drug design for
the treatment of cancer[22-24].
(Fig. 2. should be listed here)
In our continued exploration for potent and novel procaspase-3 activators as potential
anticancer agents with multi-targeted molecular mechanisms, we combined benzothiazole with the
ortho-hydroxy carbamoylhydrazone moiety in target compounds based on
a hybrid
pharmacophore design. Some groups (R1) that are positively charged at physiological pH (e.g.,
4-methylpiperidine, morpholine, dimethylamine, and diethylamine), were introduced at the
6-position of benzothiazole to avoid the metabolic pathway, and the effect of positively charged
groups on the activity was examined by changing the number of carbon atoms (n). Substituted
benzyloxy and heteroaryloxy groups (R), which were beneficial to the antitumor activity in our
previous study, were also introduced into the 4-position of the 2-hydroxy phenyl ring to heighten
the activity.
Herein, we describe a series of novel target compounds and their in vitro antitumor activities
against two procaspase-3 over-expression cancer cell lines (NCI-H226, Lung cancer; and
SK-N-SH, neuroblastoma) and one cancer cell line moderately sensitive to procaspase-3
(MDA-MB-231, breast cancer). Furthermore, two cancer cell lines with low-sensitivity to
procaspase-3 (MKN45, human gastric cancer cell; and HT29, human colorectal cancer cell) were
evaluated to rule out off-target effects[25]. Several compounds with potent activity were selected
for the enzymatic assays to determine procaspase-3 kinase activation.
(Fig. 3. should be listed here)
2. Chemistry
The preparation of key intermediates 9a-e is described in Scheme 1. The commercially
available starting materials 4-nitrobenzyl bromide or 4-chloronitrobenzene combined with
excessive secondary amines (4-methylpiperidine, morpholine, dimethylamine and diethylamine)
in acetonitrile at room temperature for 3 h underwent a nucleophilic substitution reaction to
provide intermediates 5a-e. These were then reduced in the presence of 80% hydrazine, FeCl3·6
H2O, and activated carbon to obtain the aryl amines 6a-e, which were further reacted with
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potassium thiocyanate and bromine to give 7a-e in a satisfactory yield, using a known preparation
method[26]. Further treatment with phenyl chloroformate was used to convert 7a-e into the
amides 8a-e. Subsequently, the key intermediate semicarbazides 9a-e were generated via
hydrazinolysis of 8a-e with 80% hydrazine monohydrate in 1,4-dioxane at 80°C for 6 h.
(Scheme 1. should be listed here)
As shown in Scheme 2, the substituted benzyloxy 2-hydroxybenzaldehydes 10a–g were
synthesized from 2,4-dihydroxy benzaldehyde through a reaction with substituted benzylchloride
in acetonitrile in the presence of sodium bicarbonate and potassium iodide[27].
3,4-(methylenedioxy)phenylacetonitrile was reacted with sodium hydrosulfide to obtain 11[28],
which was condensed with 1,3-dichloro-2-propanone at 50 °C for 4 h in acetonitrile to produce 12.
Compound 13 was prepared via
a
regioselective O-alkylation reaction of
2,4-dihydroxybenzaldehyde with 12. Finally, the target compounds 14a-h, 15a-x, and 16a-d were
generated via the condensation of 9a-d with appropriate 2-hydroxy aromatic aldehydes
(substituted 2-hydroxy benzaldehydes, 10a-g, and 13) in ethanol at 78 °C with catalytic amounts
of acetic acid[29].
(Scheme 2. should be listed here)
1
The chemical structures of the target compounds were confirmed by H NMR, 13C NMR,
NOESY NMR, and MS spectra. All target compounds could exist in either the E or Z isomeric
form due to the imino bond, so compound 15v was selected to further confirm the stereochemistry
by undergoing NOESY NMR. Results (Fig. 4 and supplementary information) showed that an
evident NOE signal was observed between the H1 (–CH=N–, δ=8.23 ppm) and H2 (=N–NH–,
δ=10.98 ppm), which existed only in the E isomer due to the appropriate intramolecular H–H
distance; no NOE signals were observed if the target compounds existed in the Z isomer. Thus,
there was no doubt that the configurations of target compounds were the E isomers.
(Fig. 4. should be listed here)
3. Results and discussion
3.1. In vitro cytotoxicity and structure-activity relationships
All target compounds were evaluated for their cytotoxicity in vitro against two procaspase-3
over-expression cancer cell lines (NCI-H226, Lung cancer cells and SK-N-SH, neuroblastoma
cells and one cancer cell line moderately sensitive to procaspase-3 (MDA-MB-231,breast cancer
cells by using an MTT assay. In addition, two cancer cell lines with low-sensitivity to
procaspase-3 (MKN45, human gastric cancer cells and HT29, human colorectal cancer cells) were
further evaluated to rule out off-target effects. Taking PAC-1 as the reference drug, each
compound was tested in three independent experiments and the results are summarized in Table 1
and presented as IC50 values.
As illustrated in Table 1, all the synthesized target compounds showed moderate to excellent
cytotoxic activity against all tested cancer cell lines. It was observed that compounds 15g, 15h,
15n, 15p, 15u, and 16b showed more potent antitumor activity against all five cancer cell lines
than the other compounds, and compound 15v displayed higher selectivity against HT29 with an
IC50 value of 0.0018 ꢀM, which was 760 times that of the reference drug.
By comparison of the IC50 values of compounds 15a-x, 16a-d, and compounds 14a-h, it was
clearly revealed that the introduction of a lipophilic group (e.g., a benzyloxy or heteroaryloxy
group) at the 4-position of the 2-hydroxy phenyl ring could improve cytotoxicity in vitro, which
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was consistent with the previous study in our laboratory. However, introduction of –OH at the
4-position of the 2-hydroxy phenyl ring reduced the antitumor activity against NCI-H226 and
SK-N-SH. In addition, there was an obvious negative correlation between cellular concentration of
procaspase-3 (NCI-H226 > SK-N-SH) and the IC50 values of the target compounds (NCI-H226 <
SK-N-SH). This suggested that the antitumor effect of the target compounds in cultured cells was
directly related to the level of procaspase-3 expression. Meanwhile, compounds 15a–x and 16a–d
also exhibited high activity against the MKN45 cell line, indicating that compounds 15a–x and
16a–d with a lipophilic group might be multi-target compounds. Furthermore, compound 14g with
a morpholinyl group that is positively charged at physiological pH showed better activity against
all five cancer cell lines than compound 14h with a morpholinyl group that is not positively
charged at physiological pH due to a p-π conjugation effect, thereby demonstrating that the
existence of substituents containing nitrogen that are positively charged at physiological pH, could
also enhance the pharmacological activity.
Further investigations were performed to study the effect on cytotoxic activity of different
substituents on the benzyloxyl group (compounds 15a-x). The results revealed that introduction of
electron-withdrawing groups or electron-donating groups caused no remarkable alteration in
cytotoxic activity, furthermore, introduction of a mono-chlorine or 2,4-dichlorine on the
benzyloxyl group was well tolerated. It was worth noting that increasing the size of substituents at
the 4-position of the benzyloxyl group could apparently enhance cytotoxic activity (15a vs. 15b,
15f vs. 15g, 15m vs. 15n, and 15t vs. 15u). The above findings indicated that the electronic effect
of the benzyloxyl group is not the main factor contributing to cytotoxicity, but rather the steric
effect of the group at the 4-position of the benzyloxyl group had a significant impact.
(Table 1. should be listed here)
3.2. In vitro enzymatic assays
As shown in Table 2, the six tested compounds displayed moderate to excellent procaspase-3
enzymatic potency, suggesting that the activation of procaspase-3 may be a mechanism for the
antitumor effect of these target compounds. Compound 15u showed lower activity against
procaspase-3 kinase than the other compounds; a possible reason for this is that there is no
positively charged group in compound 15u. It is worthy to mention that compounds 15g and 16b,
with positively charged groups, showed greater enzyme activities, with EC50 values of 1.42ꢀM
and 0.25ꢀM, which were 2.9 times and 16 times more active than PAC-1, respectively, indicating
that the two compounds deserve further research.
(Table 2. should be listed here)
4. Conclusions
In summary, we designed and synthesized a series of novel benzothiazole derivatives bearing
an ortho-hydroxy N-carbamoylhydrazone. The prepared compounds were evaluated for in vitro
cytotoxic activity in five human cancer cell lines (NCI-H226, SK-N-SH, MKN45, HT29, and
MDA-MB-231). Most of them had moderate to excellent activity against all tested cancer cell
lines and were sensitive to the level of cellular concentration of procaspase-3. Compounds 15g
and 16b exhibited more potent antitumor activity against all tested cancer cell lines than PAC-1,
with IC50 values ranging from 0.14 to 0.98 ꢀM. Furthermore, their strong procaspase-3 activation
potency (15g, EC50 = 1.42 ꢀM; 16b, EC50 = 0.25 ꢀM) suggested that they might inhibit the growth
of tumor cells by activating procaspase-3 kinase. The analysis of SARs indicated that introduction
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of a lipophilic group (e.g., a benzyloxy group or heteroaryloxy group) at the 4-position of the
2-hydroxy phenyl ring could enhance in vitro cytotoxic activity, and the introduction of
substituents containing nitrogen that are positively charged at physiological pH, was beneficial to
antitumor activity. In addition, the electronic effect of the benzyloxyl group showed no
remarkable effect on cytotoxic activity, whereas the steric effect of a group at the 4-position of the
benzyloxyl group had a major influence. Further optimization of the structure to improve the
bioavailability and solubility are ongoing.
5. Experimental
5.1. Chemistry
All the mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo
Alto, CA, USA). Proton (1H) nuclear magnetic resonance spectroscopy was performed using
Bruker ARX-300, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as
an internal standard. Column chromatography was run on silica gel (200-300 mesh) from Qingdao
Ocean Chemicals (Qingdao, Shandong, China). The IR spectra were recorded by means of the
KBr pellet technique on a Bruker FTS 135 spectrometer. Unless otherwise noted, all materials
were obtained from commercially available sources and were used without further purification.
5.2. General procedure for preparation of compounds 5a-e
4-Nitrobenzyl bromide (10.8 g, 0.05 mol) or 4-chloronitrobenzene (7.9 g, 0.05 mol) was
dissolved in acetonitrile (50 mL), excessive secondary amine (50 ml) was added, the reaction was
stirred at room temperature for 3 h, water (100 mL) was added, and extracted two times with
DCM. The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated
to obtain the compounds 5a-e.
5.2.1. N,N-dimethyl-1-(4-nitrophenyl)methanamine (5a)
Yellow oil; Yield: 85.5%; MS (ESI) m/z: 181.4 [M+H]+.
5.2.2. N-ethyl-N-(4-nitrobenzyl)ethanamine (5b)
Yellow oil; Yield: 82.7%; MS (ESI) m/z: 209.3 [M+H]+.
5.2.3. 4-methyl-1-(4-nitrobenzyl)piperidine (5c)
Yellow oil; Yield: 92.5%; MS (ESI) m/z: 235.5 [M+H]+.
5.2.4. 4-(4-nitrophenyl)morpholine (5d)
Yellow solid; Yield: 88.5%; MS (ESI) m/z: 209.5 [M+H]+.
5.2.5. 4-(4-nitrobenzyl)morpholine (5e)
Yellow oil; Yield: 87.9%; MS (ESI) m/z: 223.1 [M+H]+.
5.3. General procedure for preparation of compounds 6a-e
A mixture of compounds 5a-e (0.05 mol) in ethanol was heated to 65 °C, FeCl3·6H2O (2.8 g,
0.001 mol) and activated carbon (0.18 g, 0.015 mol) were added, and 80% hydrazine hydrate (25g,
0.5 mol) was added drop wise at such a rate to keep the temperature below 70 °C, the reaction was
heated at reflux for 5 h and then cooled to room temperature and concentrated. Water (100 mL)
was added, the reaction solution was extracted three times with DCM. The organic extracts were
combined, dried over sodium sulfate, filtered, and concentrated to obtain the compounds 6a-e.
5.3.1. 4-((dimethylamino)methyl)aniline (6a)
Colorless oil; Yield: 78.2%; MS (ESI) m/z: 151.2 [M+H]+.
5.3.2. 4-((diethylamino)methyl)aniline (6b)
Colorless oil; Yield: 74.1%; MS (ESI) m/z: 179.3 [M+H]+.
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5.3.3. 4-((4-methylpiperidin-1-yl)methyl)aniline (6c)
Colorless oil; Yield: 83.5%; MS (ESI) m/z: 205.3 [M+H]+.
5.3.4. 4-morpholinoaniline (6d)
White solid; Yield: 74.1%; MS (ESI) m/z: 179.2 [M+H]+.
5.3.5. 4-(morpholinomethyl)aniline (6e)
White solid; Yield: 80.9%; MS (ESI) m/z: 193.2 [M+H]+.
5.4. General procedure for preparation of compounds 7a-e
A mixture of compounds 6a-e (0.05 mol) and NH4SCN (19.03 g, 0.25 mol) in 100 mL glacial
acetic acid was cooled to 10 °C in an ice bath and stirred for 10-20 min, and then bromine (2.82
ml, 0.055 mol) in glacial acetic acid was added drop wise at such a rate to keep the temperature
below 10°C throughout the addition. The reaction mixture was stirred at room temperature for 4-6
h and then poured into hot water, and basified to pH 11.0 with ammonia solution (NH4OH). The
resulting precipitate was filtered, washed with water and dried to get a light yellow to brown solid.
The crude product was purified by chromatography on silica gel using MeOH/CH2Cl2 to afford
the compounds 7a-e.
5.4.1. 6-((dimethylamino)methyl)benzo[d]thiazol-2-amine (7a)
Light yellow solid; Yield: 65.4%; MS (ESI) m/z: 208.4 [M+H]+.
5.4.2. 6-((diethylamino)methyl)benzo[d]thiazol-2-amine (7b)
Light yellow solid; Yield: 88.6%; MS (ESI) m/z: 236.5 [M+H]+.
5.4.3. 6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-amine (7c)
Yellow solid; Yield: 85.3%; MS (ESI) m/z: 262.5 [M+H]+.
5.4.4. 6-morpholinobenzo[d]thiazol-2-amine (7d)
Brown solid; Yield: 57.3%; MS (ESI) m/z: 236.4 [M+H]+.
5.4.5. 6-(morpholinomethyl)benzo[d]thiazol-2-amine (7e)
Yellow solid; Yield: 71.2%; MS (ESI) m/z: 250.1 [M+H]+.
5.5. General procedure for preparation of compounds 8a-e
A mixture of compounds 7a-e (0.01 mol) and pyridine (0.8 ml,0.02 mol) in 20 mL DCM was
cooled to 0 °C in an ice bath and stirred for 0.5 h, phenyl chloroformate was added drop wise at
such a rate to keep the temperature below 10 °C throughout the addition. 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 8a-e.
5.5.1. Phenyl-6-((dimethylamino)methyl)benzo[d]thiazol-2-ylcarbamate (8a)
White solid; Yield: 94.6%; MS (ESI) m/z: 328.5 [M+H]+.
5.5.2. Phenyl-6-((diethylamino)methyl)benzo[d]thiazol-2-ylcarbamate (8b)
White solid; Yield: 92.3%; MS (ESI) m/z: 356.4[M+H]+.
5.5.3. Phenyl-6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-ylcarbamate (8c)
White solid; Yield: 97.6%; MS (ESI) m/z: 382.5[M+H]+.
5.5.4. Phenyl-6-morpholinobenzo[d]thiazol-2-ylcarbamate (8d)
Light yellow solid;Yield: 88.3%; MS (ESI) m/z: 356.4[M+H]+.
5.5.5. Phenyl 6-(morpholinomethyl)benzo[d]thiazol-2-ylcarbamate (8e)
White solid; Yield: 88.3%; MS (ESI) m/z: 370.1[M+H]+.
5.6. General procedure for preparation of compounds 9a-e
A mixture of the compounds 8a-e (0.01 mol) and 80% hydrazine hydrate (1.29 ml, 0.02 mol)
in 20 mL 1,4-dioxane was heated to 80 °C for 6 h, then the reaction mixture was cooled to room
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temperature and concentrated, diethyl ether was added and stirred for 0.5 h and filtered, a white to
gray solid was collected and washed with a small amount of water. The crude product was
purified by chromatography on silica gel using MeOH/CH2Cl2 to get the compounds 9a-e.
5.6.1. N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)hydrazinecarboxamide (9a)
White solid; Yield: 82.6%; MS (ESI) m/z: 265.7 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ
8.37 (s, 1H), 7.77 (s, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H), 3.44 (s, 2H), 2.15 (s,
6H).
5.6.2. N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)hydrazinecarboxamide (9b)
White solid; Yield: 86.3%; MS (ESI) m/z: 607.9 [2M+Na]+; 1H NMR (400 MHz, DMSO-d6)
δ 9.34 (s, 1H), 8.49 (s, 1H), 8.03 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 4.31 (s,
2H), 3.04 (s, 4H), 1.22 (s, 6H).
5.6.3. N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)hydrazinecarboxamide (9c)
White solid; Yield: 76.8%; MS (ESI) m/z: 319.6 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ
9.33 (s, 1H), 7.79 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.16 (t, J = 7.6 Hz,
1H), 6.76 (s, 1H), 6.74 (s, 1H), 3.54 (s, 2H), 2.81 (s, 2H), 1.94 (s, 2H), 1.58 (d, J = 11.6 Hz, 2H),
1.35 (s, 1H), 1.15 (m, 2H), 0.88 (d, J = 6.4 Hz, 3H).
5.6.4. N-(6-morpholinobenzo[d]thiazol-2-yl)hydrazinecarboxamide (9d)
Gray solid; Yield: 90.2%; MS (ESI) m/z: 608.0 [2M+Na]+; 1H NMR (400 MHz, DMSO-d6) δ
8.27 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.05 (dd, J = 8.8, 1.6 Hz, 1H), 3.76
(t, J = 4.4 Hz, 4H), 3.10 (t, J = 4.4 Hz, 4H).
5.6.5 N-(6-(morpholinomethyl)benzo[d]thiazol-2-yl)hydrazinecarboxamide (9e)
White solid; Yield: 80.8%; MS (ESI) m/z: 308.1 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ
8.39 (s, 1H), 7.80 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 3.57 (s, 4H), 3.54 (s,
2H), 2.38 (s, 4H).
5.7. General procedure for preparation of compounds 10a-g
A mixture of 2,4-dihydroxybenzaldehyde (1.38 g, 0.01 mol), substituted benzyl chloride
(0.011 mol), NaHCO3 (1.26 g, 0.015 mol) and catalytic amount of potassium iodide in 20 mL
acetonitrile was heated to 60 °C for 36 h and hot filtered, the filtrate was cooled to 10 °C and
filtered, the filtrated cake was collected and recrystallized with EtOH to obtain compounds 10a-g.
5.7.1. 4-benzyloxy-2-hydroxybenzaldehyde (10a)
1
White solid; Yield: 77.8%; MS (ESI) m/z: 227.2 [M-H]-; H NMR (400 MHz, CDCl3) δ
11.48 (s, 1H), 9.72 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.43 – 7.39 (m, 4H), 7.39 – 7.33 (m, 1H),
6.62 (dd, J = 8.8, 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 5.11 (s, 2H).
5.7.2. 4-(4-methylbenzyloxy)-2-hydroxybenzaldehyde (10b)
1
Yellow solid; Yield: 75.4%; MS (ESI) m/z: 241.2 [M-H]-; H NMR (400 MHz, CDCl3) δ
11.48 (s, 1H), 9.71 (s, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz,
2H), 6.60 (dd, J = 8.8, 2.4 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 5.06 (s, 2H), 2.37 (s, 3H).
5.7.3. 4-(4-tert-butylbenzyloxy)-2-hydroxybenzaldehyde (10c)
1
Yellow solid; Yield: 85.7%; MS (ESI) m/z: 282.6 [M-H]-; H NMR (400 MHz, CDCl3) δ
11.49 (s, 1H), 9.72 (s, 1H), 7.45 – 7.41 (m, 3H), 7.42 (s, 1H), 7.36 (s, 1H), 7.34 (s, 1H), 6.61 (dd, J
= 8.8, 2.4 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H), 5.07 (s, 2H), 1.33 (s, 9H).
5.7.4. 4-(4-chlorobenzyloxy)-2-hydroxybenzaldehyde (10d)
1
White solid; Yield: 78.2%; MS (ESI) m/z: 261.8 [M-H]-; H NMR (400 MHz, CDCl3) δ
11.48 (s, 1H), 9.72 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.40-7.32 (m, 4H), 6.60 (dd, J = 8.8, 2.4 Hz,
7

ACCEPTED MANUSCRIPT
1H), 6.48 (d, J = 2.4 Hz, 1H), 5.07 (s, 2H).
5.7.5. 4-(3-chlorobenzyloxy)-2-hydroxybenzaldehyde (10e)
White solid; Yield: 80.9%; MS (ESI) m/z: 261.2 [M-H]-; H NMR (400 MHz, CDCl3) δ
11.47 (s, 1H), 9.73 (s, 1H), 7.50 – 7.43 (m, 2H), 7.35 (m, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.11 (t, J =
7.6 Hz, 1H), 6.62 (dd, J = 8.4, 2.0 Hz, 1H), 6.53 (d, J = 2.0 Hz, 1H), 5.18 (s, 2H).
5.7.6. 4-(2-chlorobenzyloxy)-2-hydroxybenzaldehyde (10f)
1
White solid; Yield: 75.8%; MS (ESI) m/z: 261.9 [M-H]-; H NMR (400 MHz, CDCl3) δ
1
11.48 (s, 1H), 9.73 (s, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.42 (s, 1H), 7.32 (m, 3H), 6.61 (dd, J = 8.8,
2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 5.09 (s, 2H).
5.7.7. 4-(2,4-dichlorobenzyloxy)-2-hydroxybenzaldehyde (10g)
Gray solid; Yield: 82.6%; MS (ESI) m/z: 295.1 [M-H]-; 1H NMR (400 MHz, CDCl3) δ 11.47
(s, 1H), 9.74 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.45 – 7.43 (m, 2H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H),
6.62 (dd, J = 8.8, 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 5.17 (s, 2H).
5.8. Preparation of 2-(benzo[d][1,3]dioxol-5-yl)ethanethioamide (11)
.
A mixture of the sodium hydrosulfide (14 g, 0.25 mol), MgCl2 6H2O (25.4 g, 0.125 mol) and
2-(benzo[d][1,3]dioxol-5-yl)acetonitrile (16.1 g, 0.1 mol) in 140 mL DMF and 30 mL water was
stirred for 15 h at room temperature, and then poured into ice-water with acutely stirring, The
solution was treated with concentrated hydrochloric acid to achieve pH to 5.0. The product was
separated by filtration to obtain compound 11 as a white solid (10.3 g, 53%), MS (ESI) m/z:
196.1[M+H]+.
5.9. Preparation of 2-(benzo[d][1,3]dioxol-5-ylmethyl)-4-(chloromethyl)thiazole (12)
The compound 11 (10.3 g, 0.05 mol) and 1,3-dichloroacetone (7 g, 0.05 mol) were added in
100 mL acetonitrile and stirred for 4 h at 50 °C and then filtered. The filter cake was collected and
washed with water to get the compound 12 as a light yellow solid (8.7 g, 61%). MS (ESI) m/z:
268.8 [M+H]+.
5.10. Preparation of 4-((2-(benzo[d][1,3]dioxol-5-ylmethyl)thiazol-4-yl)methoxy)-2-hydroxyben-
zaldehyde (16)
A mixture of the compound 12 (8.7 g, 0.03 mol) and 2,4-dihydroxybenzaldehyde (4.5 g,
0.03mol), NaHCO3 (3.2 g, 0.035 mol) and catalytic amount of potassium iodide in 50 mL
acetonitrile was heated to 80 °C in an oil-bath for approximate 2 h. After cooling to ambient
temperature, the contents were concentrated under reduced pressure, 30 mL methanol was added,
the solution was decolorized with active carbon and hot filtered, the filtrate was cooled to room
temperature and filtered, a tan solid was collected as compound 13 (7.6 g, 64%). MS (ESI) m/z:
367.2 [M-H]-; 1H NMR (400 MHz, CDCl3) δ 11.47 (s, 1H), 9.73 (s, 1H), 7.45 (d, J = 8.8 Hz, 1H),
7.21 (s, 1H), 6.80 (s, 1H), 6.79 (s, 2H), 6.64 (dd, J = 8.8, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H),
5.96 (s, 2H), 5.21 (s, 2H), 4.25 (s, 2H).
5.11 General procedure for preparation of compounds 14a-h, 15a-x, 16a-d
A mixture of the compounds 9a-e (0.001 mol), appropriate 2-hydroxy aromatic aldehydes
(0.0011 mol) and a drop of glacial acetic acid in 10 mL ethanol was heated at reflux for 6 h and
cooled to room temperature, separated by filtration, and the cake was washed with diethyl ether to
get a white to light yellow solid. The crude product was purified by chromatography on silica gel
using MeOH/CH2Cl2 to afford the compounds 14a-h, 15a-x, 16a-d.
5.11.1. (E)-N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxybenzylidene)hydraz-
inecarboxamide (14a)
8

ACCEPTED MANUSCRIPT
Yield: 58%; MS (ESI) m/z: 368.2 [M-H]-; IR (KBr, cm-1): 3424.1, 2921.9, 1725.3, 1705.8,
1630.3, 1609.1, 1565.8, 1543.6, 1501.9, 1487.9, 1465.1, 1442.7, 1384.3, 1276.5, 1246.1, 1128.1,
1036.0, 924.9, 809.1, 779.3, 619.1; 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 2H), 8.34 (s, 1H),
8.03 (s, 1H), 7.83 (s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.25 (t, J = 7.6 Hz,
1H), 6.89 (m, 2H), 3.52 (s, 2H), 2.20 (s, 6H); Anal. Calcd, for C18H19N5O2S (%): C, 58.52; H,
5.18; N.18.96. Found (%): 58.53; H, 5.17; N.18.94.
5.11.2. (E)-N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2,4-dihydroxybenzylidene)hyd-
razinecarboxamide (14b)
Yield: 54%; MS (ESI) m/z: 384.1 [M-H]-; IR (KBr, cm-1): 3187.6, 3093.8, 2950.2,
2553.5,1695.9, 1679.2, 1629.8, 1604.0, 1564.2, 1549.3, 1501.7, 1478.0, 1460.0, 1320.2, 1288.9,
1
1166.5, 1123.4, 978.1, 947.6, 840.5, 795.6, 695.2, 671.3, 645.2, 608.9; H NMR (400 MHz,
DMSO-d6) δ 11.05 (s, 2H), 9.82 (s, 2H), 8.21 (s, 1H), 7.81 (s, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.32
(d, J = 8.0 Hz, 1H), 6.32 (s, 2H), 3.48 (s, 2H), 2.17 (s, 6H); Anal. Calcd, for C18H19N5O3S (%): C,
56.09; H, 4.97; N, 18.17. Found (%): C, 56.11; H, 4.96; N, 18.18.
5.11.3. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxybenzylidene)hydrazine
carboxamide (14c)
1
Yield: 62%; MS (ESI) m/z: 396.4 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 2H),
8.34 (s, 1H), 8.04 (s, 1H), 7.84 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.25 (t, J
= 7.6 Hz, 1H), 6.88 (m, 2H), 3.66 (s, 2H), 2.52 (d, J = 6.8 Hz, 4H), 1.01 (t, J = 6.8 Hz, 6H); Anal.
Calcd, for C20H23N5O2S (%): C, 60.43; H, 5.83; N, 17.62. Found (%): C, 60.45; H, 5.82; N, 17.64.
5.11.4. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2,4-dihydroxybenzylidene)hydr-
azinecarboxamide (14d)
1
Yield: 56%; MS (ESI) m/z: 412.4 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 2H),
9.93 (s, 1H), 8.21 (s, 1H), 7.84 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 6.32 (m,
2H), 3.67 (s, 2H), 2.55 (d, J = 6.8 Hz, 4H), 1.01 (t, J = 6.8 Hz, 6H); Anal. Calcd, for C20H23N5O3S
(%): C, 58.09; H, 5.61; N, 16.94. Found (%): C, 58.10; H, 5.63; N, 16.92.
5.11.5. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxybenzylidene)
hydrazinecarboxamide (14e)
1
Yield: 64%; MS (ESI) m/z: 422.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 2H),
8.34 (s, 1H), 8.03 (s, 1H), 7.80 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H),
7.29-7.20 (m, 1H), 6.94-6.84 (m, 2H), 3.52 (s, 2H), 2.79 (d, J = 11.2 Hz, 2H), 1.93 (t, J = 10.8 Hz,
2H), 1.56 (d, J = 11.2 Hz, 2H), 1.32 (m, 1H), 1.23-1.06 (m, 2H), 0.88 (d, J = 6.4 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 160.24, 156.87, 153.43, 147.76, 141.87, 134.01, 131.75, 131.44,
128.46, 127.49, 121.92, 120.32, 119.69, 119.35, 116.57, 62.80, 53.68(2C), 34.34(2C), 30.73,
22.27; Anal. Calcd, for C22H25N5O2S (%): C, 62.39; H, 5.95; N, 16.54. Found (%): C, 62.37; H,
5.96; N, 16.52.
5.11.6. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(2,4-dihydroxybenzyli-
dene)hydrazinecarboxamide (14f)
Yield: 71%; MS (ESI) m/z: 438.3 [M-H]-; 1H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 2H),
10.57 (s, 2H), 8.23 (s, 1H), 8.12 (s, 1H), 7.94 (s, 1H), 7.70 (d, J = 8.0 Hz,, 1H), 7.64 (d, J = 8.0 Hz,
1H), 6.35 (m, 2H), 4.31 (s, 2H), 3.29 (d, J = 11.2 Hz, 2H), 2.88 (d, J = 11.2 Hz, 2H), 1.73 (d, J =
12.4 Hz, 2H), 1.59-1.41 (m, 3H), 0.88 (d, J = 4.8 Hz, 3H); Anal. Calcd, for C22H25N5O3S (%): C,
60.12; H, 5.73; N, 15.93. Found (%): C, 60.14; H, 5.74; N, 15.91.
5.11.7. (E)-N-(6-(morpholinomethyl)benzo[d]thiazol-2-yl)-2-(2-hydroxybenzylidene) hydrazinec-
9

ACCEPTED MANUSCRIPT
arboxamide (14g)
Yield: 76%; MS (ESI) m/z: 410.1 [M-H]-; IR (KBr, cm-1): 3421.7, 3200.2, 3074.0, 2925.1,
2855.6, 2806.3, 1695.0, 1679.3, 1608.8, 1571.7, 1541.7, 1488.4, 1461.3, 1332.8, 1311.4, 1274.3,
1115.0, 1007.2, 864.5, 808.4, 760.8, 684.7; 1H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 2H), 10.02
(s, 1H), 8.34 (s, 1H), 8.09 (s, 1H), 7.84 (s, 1H), 7.62 (d, J = 5.6 Hz, 1H), 7.36 (d, J = 5.6 Hz, 1H),
7.25 (t, J = 7.6 Hz,1H), 6.88 (m, 2H), 3.58 (s, 4H), 3.54 (s, 2H), 2.37 (s, 4H); Anal. Calcd, for
C20H21N5O3S (%): C, 58.38; H, 5.14; N, 17.02. Found (%): C, 58.38; H, 5.15; N, 17.01.
5.11.8. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(2-hydroxybenzylidene)hydrazinecarboxami-
de (14h)
1
Yield: 82%; MS (ESI) m/z: 396.1 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 2H),
8.34 (s, 1H), 8.03 (s, 1H), 7.83 (s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.25 (t, J
= 7.6 Hz, 1H), 6.89 (m, 2H), 3.52 (s, 2H), 2.20 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 156.80,
153.18, 148.33, 133.16, 131.45, 128.33, 120.35, 119.71, 116.54, 116.14, 107.57, 66.62(2C),
49.99(2C); Anal. Calcd, for C19H19N5O3S (%): C, 57.42; H, 4.82; N, 17.62. Found (%): C, 57.41;
H, 4.83; N, 17.60.
5.11.9. (E)-N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxy-4-(4-methylbenzyl-
oxy)benzylidene)hydrazinecarboxamide (15a)
1
Yield: 71%; MS (ESI) m/z: 488.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 2H),
8.23 (s, 1H), 7.94 (s, 1H), 7.81 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.31 (s,
1H), 7.21 (d, J = 7.6 Hz, 2H), 6.55 (dd, J = 8.4, 2.0 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 5.06 (s, 2H),
3.47 (s, 2H), 2.31 (s, 3H), 2.17 (s, 6H).; Anal. Calcd, for C26H27N5O3S (%): C, 63.78; H, 5.56; N,
14.30. Found (%): C, 63.80; H, 5.55; N, 14.31.
5.11.10. (E)-N-(6-((dimethylamino)methyl)-benzo[d]thiazol-2-yl)-2-(4-(4-tert-butylbenzyloxy)-2-
hydroxybenzylidene)hydrazinecarboxamide (15b)
Yield: 69%; MS (ESI) m/z: 530.6 [M-H]-, 566.6 [M+Cl]-; 1H NMR (400 MHz, DMSO-d6) δ
11.38 (s, 2H), 8.26 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.42
(d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 6.56 (s, 1H), 6.54 (s, 1H), 5.07 (s, 2H), 4.36 (s, 2H),
2.72 (s, 6H), 1.29 (s, 9H); Anal. Calcd, for C29H33N5O3S (%): C, 65.51; H, 6.26; N, 13.17. Found
(%): C, 65.54; H, 6.25; N, 13.16.
5.11.11. (E)-N-(6-((dimethylamino)methyl)ben-zo[d]thiazol-2-yl)-2-(4-(4-chlorobenzyloxy)-2-hy-
droxybenzylidene)hydrazinecarboxamide (15c)
1
Yield: 65%; MS (ESI) m/z: 508.1 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 2H),
8.24 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.48 (s, 4H), 7.32 (d, J = 8.0 Hz,
1H), 6.56 (dd, J = 8.4, 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 5.12 (s, 2H), 3.47 (s, 2H), 2.17 (s,
6H); Anal. Calcd, for C25H24ClN5O3S (%): C, 58.88; H, 4.74; N, 13.73. Found (%): C, 58.89; H,
4.75; N, 13.70.
5.11.12. (E)-N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(3-chlorobenzyloxy)-2-hyd-
roxybenzylidene)hydrazinecarboxamide (15d)
1
Yield: 69%; MS (ESI) m/z: 508.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 2H),
8.24 (s, 1H), 7.98 (s, 1H), 7.81 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.46 – 7.39 (m, 3H),
7.32 (d, J = 8.0 Hz, 1H), 6.57 (dd, J = 8.8, 2.4 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H), 5.14 (s, 2H), 3.47
(s, 2H), 2.17 (s, 6H); Anal. Calcd, for C25H24ClN5O3S (%): C, 58.88; H, 4.74; N, 13.73. Found
(%): C, 58.85; H, 4.77; N, 13.72.
5.11.13. (E)-N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(2-chlorobenzyloxy)-2hydr-
10

ACCEPTED MANUSCRIPT
oxybenzylidene)hydrazinecarboxamide (15e)
Yield: 75%; MS (ESI) m/z: 508.2 [M-H]-; IR (KBr, cm-1): 3178.0, 3077.6, 2976.5, 2940.0,
2856.4, 2814.6, 2766.8, 1690.7, 1679.0, 1628.3, 1609.5, 1571.9, 1556.5, 1538.5, 1492.3, 1457.2,
1
1384.2, 1360.5, 1318.4, 1280.3, 1115.8, 1037.9, 1021.5, 845.7, 835.6, 814.3, 804.1, 752.5; H
NMR (400 MHz, DMSO-d6) δ 11.10 (s, 2H), 8.25 (s, 1H), 7.85 (s, 1H), 7.81 (s, 1H), 7.64 – 7.53
(m, 2H), 7.48 – 7.40 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.30 – 7.21 (m, 2H), 6.58 (dd, J = 8.4, 2.4
Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 5.15 (s, 2H), 3.51 (s, 2H), 2.19 (s, 6H); Anal. Calcd, for
C25H24ClN5O3S (%): C, 58.88; H, 4.74; N, 13.73. Found (%): C, 58.86; H, 4.78; N, 13.71.
5.11.14. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxy-4-(4-methylbenzylo-
xy)benzylidene)hydrazinecarboxamide (15f)
1
Yield: 55%; MS (ESI) m/z: 516.4 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 2H),
8.24 (s, 1H), 7.94 (s, 1H), 7.85 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.34 (d,
J = 7.6 Hz, 2H), 7.21 (d, J = 7.6 Hz, 2H), 6.55 (dd, J = 8.4, 2.0 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H),
5.05 (s, 2H), 3.70 (s, 2H), 2.55 (q, J = 7.2 Hz, 4H), 2.31 (s, 3H), 1.02 (t, J = 7.2 Hz, 6H).; Anal.
Calcd, for C28H31N5O3S (%): C, 64.97; H, 6.04; N, 13.53; Found (%): C, 64.99; H, 6.03; N, 13.52.
5.11.15. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(4-tert-butylbenzyloxy)-2-hy-
droxybenzylidene)hydrazinecarboxamide (15g)
1
Yield: 75%; MS (ESI) m/z: 558.6 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H),
11.19 (s, 1H), 8.26 (s, 1H), 8.13 (s, 1H), 8.00 (s, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.8 Hz,
1H), 7.42 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 6.56 (dd, J = 8.4, 2.0 Hz, 1H), 6.53 (d, J =
2.0 Hz, 1H), 5.07 (s, 2H), 4.38 (s, 2H), 3.08 (q, J = 7.2 Hz, 4H), 1.29 (s, 9H), 1.26 (t, J = 7.2 Hz,
6H); 13C NMR (100 MHz, DMSO-d6) δ 161.41, 161.05, 158.50, 152.99, 150.86, 150.09, 141.72,
134.25, 132.23, 129.89, 129.47, 128.11(2C), 125.65(2C), 125.20, 124.78, 120.26, 113.35, 107.36,
102.34, 69.55, 55.16, 46.03(2C), 34.76, 31.59(3C), 8.79(2C); Anal. Calcd, for C31H37N5O3S (%):
C, 66.52; H, 6.66; N, 12.51; Found (%): C, 66.55; H, 6.63; N, 12.50.
5.11.16. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(4-chlorobenzyloxy)-2-hydr-
oxybenzylidene)hydrazinecarboxamide (15h)
1
Yield: 72%; MS (ESI) m/z: 536.3 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 2H),
8.25 (s, 1H), 7.96 (s, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.48 (s, 5H), 6.56 (dd, J = 8.8, 2.0 Hz, 1H),
6.53 (d, J = 2.0 Hz, 1H), 5.12 (s, 2H), 3.96 (s, 2H), 2.75 (s, 4H), 1.11 (s, 6H); Anal. Calcd, for
C27H28ClN5O3S (%): C, 60.27; H, 5.25; N, 13.02; Found (%): C, 60.27; H, 5.27; N, 13.03.
5.11.17. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(3-chlorobenzyloxy)-2-hydr-
oxybenzylidene) hydrazinecarboxamide (15i)
1
Yield: 81%; MS (ESI) m/z: 536.4 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 2H),
8.25 (s, 1H), 7.96 (s, 2H), 7.69 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.50-7.37 (m, 4H), 6.57 (dd, J =
8.4, 2.0 Hz, 1H), 6.54 (d, J = 2.0 Hz, 1H), 5.14 (s, 2H), 3.96 (s, 2H), 2.76 (s, 4H), 1.12 (s, 6H);
Anal. Calcd, for C27H28ClN5O3S (%): C, 60.27; H, 5.25; N, 13.02; Found (%): C, 60.24; H, 5.26;
N, 13.04.
5.11.18. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(2-chlorobenzyloxy)-2-hydr-
oxybenzylidene) hydrazinecarboxamide (15j)
1
Yield: 78%; MS (ESI) m/z: 536.3 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 2H),
8.88 (s, 1H), 8.27 (s, 1H), 8.11 (s, 1H), 7.96 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.56 (d,
J = 8.0 Hz, 1H), 7.48 – 7.41 (m, 1H), 7.29 – 7.22 (m, 2H), 6.61 – 6.55 (m, 2H), 5.15 (s, 2H), 4.31
(s, 2H), 3.02 (s, 4H), 1.24 (s, 6H); Anal. Calcd, for C27H28ClN5O3S (%): C, 60.27; H, 5.25; N,
11

ACCEPTED MANUSCRIPT
13.02; Found (%): C, 60.28; H, 5.26; N, 13.01.
5.11.19. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-(2,4-dichlorobenzyloxy)-2-hy-
droxybenzylidene)hydrazinecarboxamide (15k)
1
Yield: 85%; MS (ESI) m/z: 570.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 2H),
8.25 (s, 1H), 8.04 (s, 1H), 7.85 (s, 1H), 7.72 (d, J = 1.2 Hz, 1H), 7.61 (m, 2H), 7.50 (dd, J = 8.0,
1.2 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 6.53 (s, 1H), 5.16 (s, 2H), 3.68 (s,
2H), 2.54 (d, J = 7.2 Hz, 4H), 1.02 (t, J = 7.2 Hz, 6H); Anal. Calcd, for C27H27Cl2N5O3S (%): C,
56.64; H, 4.75; N, 12.23; Found (%): C, 56.66; H, 4.74; N, 12.22.
5.11.20. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxy-4-benzyloxybenzyl-
idene)hydrazinecarboxamide (15l)
Yield: 84%; MS (ESI) m/z: 504.1 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H),
11.16 (s, 1H), 8.26 (s, 1H), 8.09 (s, 2H), 7.76 (d, J = 7.2 Hz, 1H), 7.55 (m, 1H), 7.46 (d, J = 7.2
Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.36 (d, J = 7.2 Hz, 1H), 6.57 (dd, J = 8.8, 2.4 Hz, 1H), 6.53 (d,
J = 2.4 Hz, 1H), 5.12 (s, 2H), 4.40 (s, 2H), 3.11 (q, J = 7.2 Hz, 4H), 1.25 (t, J = 7.2 Hz, 6H); Anal.
Calcd, for C27H29N5O3S (%): C, 64.39; H, 5.80; N, 13.91; Found (%): C, 64.41; H, 5.81; N, 13.90.
5.11.21. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(2-hydroxy-4-(4-meth-
ylbenzyloxy)benzylidene) hydrazinecarboxamide (15m)
1
Yield: 67%; MS (ESI) m/z: 542.3 [M-H]-; 578.3[M+Cl]-; H NMR (400 MHz, DMSO-d6) δ
11.11 (s, 2H), 8.24 (s, 1H), 7.98 (s, 1H), 7.82 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.0 Hz,
3H), 7.21 (d, J = 8.0 Hz, 2H), 6.55 (dd, J = 8.4, 2.0 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 5.06 (s, 2H),
3.61 (s, 2H), 2.85 (d, J = 9.2 Hz, 2H), 2.31 (s, 3H), 2.04 (s, 2H), 1.58 (d, J = 11.2 Hz, 2H), 1.35 (s,
1H), 1.16 (m, 2H), 0.89 (d, J = 6.4 Hz, 3H); Anal. Calcd, for C30H33N5O3S (%): C, 66.27; H, 6.12;
N, 12.88; Found (%): C, 66.24; H, 6.14; N, 12.89.
5.11.21. (E)-N-(6-((4-methylpiperidin-1-yl)-methyl)benzo[d]thiazol-2-yl)-2-(4-(4-tert-butylbenzy-
loxy)-2-hydroxybenzylidene) hydrazinecarboxamide (15n)
Yield: 71%; MS (ESI) m/z: 584.6 [M-H]-; 620.6[M+Cl]-; IR (KBr, cm-1): 3198.0, 3088.5,
2947.0, 2928.1, 2866.7, 2791.1, 2753.5, 1693.2, 1629.7, 1608.5, 1569.6, 1549.7, 1503.6, 1465.3,
1294.1, 1165.2, 1131.9, 1119.4, 1045.2, 1017.6, 865.0, 817.5, 789.8, 617.5, 545.7; 1H NMR (400
MHz, DMSO-d6) δ 11.10 (s, 2H), 8.24 (s, 1H), 8.00 (s, 1H), 7.80 (s, 1H), 7.59 (d, J = 7.2 Hz, 1H),
7.42 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 6.55 (dd, J = 8.4, 2.0
Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 5.07 (s, 2H), 3.52 (s, 2H), 2.79 (d, J = 10.4 Hz, 2H), 1.93 (s,
2H), 1.56 (d, J = 11.2 Hz, 2H), 1.29 (s, 10H), 1.13 (m, 2H), 0.88 (d, J = 6.4 Hz, 3H); Anal. Calcd,
for C33H39N5O3S (%): C, 67.66; H, 6.71; N, 11.96; Found (%): C, 67.63; H, 6.72; N, 11.94.
5.11.22. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(4-(4-chlorobenzylox-
y)-2-hydroxybenzylidene) hydrazinecarboxamide (15o)
1
Yield: 78%; MS (ESI) m/z: 562.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 2H),
8.25 (s, 1H), 7.96 (s, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.49-7.46 (m, 5H), 6.56 (dd, J = 8.0, 2.4 Hz,
1H), 6.52 (d, J = 2.4 Hz, 1H), 5.12 (s, 2H), 4.10 (s, 2H), 3.17 (s, 2H), 2.67 (s, 2H), 1.71 (d, J =
12.4 Hz, 2H), 1.52 (s, 1H), 1.28 (s, 2H), 0.90 (d, J = 6.4 Hz, 3H); Anal. Calcd, for C29H30ClN5O3S
(%): C, 61.75; H, 5.36; N, 12.42; Found (%): C, 61.78; H, 5.35; N, 12.40.
5.11.23. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(4-(3-chlorobenzylox-
y)-2-hydroxybenzylidene) hydrazinecarboxamide (15p)
1
Yield: 75%; MS (ESI) m/z: 562.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 2H),
8.24 (s, 1H), 8.04 (s, 1H), 7.82 (s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.53 (s, 1H), 7.43 (m, 3H), 7.34
12

ACCEPTED MANUSCRIPT
(d, J = 8.4 Hz, 1H), 6.57 (dd, J = 8.4, 2.0 Hz, 1H), 6.52 (J = 2.0 Hz, 1H), 5.14 (s, 2H), 3.62 (s, 2H),
2.85 (d, J = 10.0 Hz, 2H), 2.05 (s, 2H), 1.59 (d, J = 12.4 Hz, 2H), 1.35 (s, 1H), 1.16 (m, 2H), 0.88
(d, J = 6.4 Hz, 3H); Anal. Calcd, for C29H30ClN5O3S (%): C, 61.75; H, 5.36; N, 12.42; Found (%):
C, 61.79; H, 5.34; N, 12.43.
5.11.24. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(4-(2-chlorobenzylox-
y)-2-hydroxybenzylidene) hydrazinecarboxamide (15q)
Yield: 71%; MS (ESI) m/z: 562.2 [M-H]-; IR (KBr, cm-1): 3420.5, 2922.8, 1698.9, 1631.2,
1572.6, 1557.4, 1510.2, 1461.7, 1384.8, 1250.1, 1212.4, 1182.2, 1129.0, 1113.9, 1011.2, 975.0,
1
824.6, 797.4, 651.3; H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 2H), 8.25 (s, 1H), 8.00 (s, 1H),
7.80 (s, 1H), 7.62-7.54 (m, 2H), 7.49 – 7.40 (m, 1H), 7.35-7.29 (m, 1H), 7.29-7.23 (m, 2H), 6.58
(dd, J = 8.4, 2.0 Hz, 1H), 6.54 (d, J = 2.0 Hz, 1H), 5.15 (s, 2H), 3.52 (s, 2H), 2.79 (d, J = 10.0 Hz,
2H), 1.93 (s, 2H), 1.56 (d, J = 12.0 Hz, 2H), 1.33 (s, 1H), 1.14 (m, 2H), 0.88 (d, J = 6.4 Hz, 3H);
Anal. Calcd, for C29H30ClN5O3S(%): C, 61.75; H, 5.36; N, 12.42; Found (%): C, 61.72; H, 5.37; N,
12.45.
5.11.25. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(4-(2,4-dichlorobenzy-
loxy)-2-hydroxybenzylidene) hydrazinecarboxamide (15r)
1
Yield: 88%; MS (ESI) m/z: 596.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 2H),
8.25 (s, 1H), 7.99 (s, 1H), 7.79 (s, 1H), 7.71 (s, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.59 (s, 1H), 7.50 (d,
J = 8.4 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.16 (s, 2H), 3.51
(s, 2H), 2.79 (d, J = 10.8 Hz, 2H), 1.92 (t, J = 10.8 Hz, 2H), 1.56 (d, J = 12.0 Hz, 2H), 1.32 (s, 1H),
1.15 (m, 2H), 0.88 (d, J = 6.4 Hz, 3H); Anal. Calcd, for C29H29Cl2N5O3S(%): C, 58.19; H, 4.88; N,
11.70; Found (%): C, 58.21; H, 4.86; N, 11.69.
5.11.26. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(4-benzyloxy-2-hydro-
xybenzylidene) hydrazinecarboxamide (15s)
1
Yield: 58%; MS (ESI) m/z: 528.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 2H),
8.24 (s, 1H), 7.97 (s, 1H), 7.83 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 7.2 Hz, 2H), 7.41 (t, J
= 7.2 Hz, 2H), 7.37 – 7.32 (m, 2H), 6.57 (dd, J = 8.4, 2.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 5.11 (s,
2H), 3.61 (s, 2H), 2.85 (J = 6.8 Hz, 2H), 2.05 (s, 2H), 1.58 (d, J = 10.8 Hz, 2H), 1.35 (s, 1H), 1.16
(m, 2H), 0.88 (d, J = 6.4 Hz, 3H); Anal. Calcd, for C29H31N5O3S (%): C, 65.76; H, 5.90; N, 13.22;
Found (%): C, 65.78; H, 5.91; N, 13.20.
5.11.27. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(2-hydroxy-4-(4-methylbenzyloxy)benzyli-
dene)hydrazinecarboxamide (15t)
1
Yield: 65%; MS (ESI) m/z: 516.3 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 2H),
8.23 (s, 1H), 7.86 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.33 (d, J = 8.0 Hz,
2H), 7.21 (d, J = 8.0 Hz, 2H), 7.09 (dd, J = 8.8, 2.4 Hz, 1H), 6.55 (dd, J = 8.8, 2.4 Hz, 1H), 6.51
(d, J = 2.4 Hz, 1H), 5.06 (s, 2H), 3.76 (t, J = 4.4 Hz, 4H), 3.12 (t, J = 4.4 Hz, 4H), 2.31 (s, 3H);
Anal. Calcd, for C27H27N5O4S (%): C, 62.65; H, 5.26; N, 13.53; Found (%): C, 62.68; H, 5.24; N,
13.52.
5.11.28. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(4-(4-tert-butylbenzyloxy)-2-hydroxybenzy-
lidene) hydrazinecarboxamide (15u)
1
Yield: 73%; MS (ESI) m/z: 558.6 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 2H),
8.23 (s, 1H), 7.87 (s, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.42 (d, J = 8.4 Hz,
2H), 7.37 (d, J = 8.4 Hz, 2H), 7.09 (dd, J = 8.8, 2.4 Hz, 1H), 6.55 (dd, J = 8.8, 2.4 Hz, 1H), 6.51
(d, J = 2.4 Hz, 1H), 5.07 (s, 2H), 3.76 (t, J = 4.4 Hz, 4H), 3.12 (t, J = 4.4 Hz, 4H), 1.29 (s, 9H) ;
13

ACCEPTED MANUSCRIPT
Anal. Calcd, for C30H33N5O4S (%): C, 64.38; H, 5.94; N, 12.51; Found (%): C, 64.39; H, 5.95; N,
12.49.
5.11.29. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(4-(4-chlorobenzyloxy)-2-hydroxybenzylid-
ene) hydrazinecarboxamide (15v)
Yield: 70%; MS (ESI) m/z: 536.3 [M-H]-; IR (KBr, cm-1): 3421.7, 3183.4, 3072.2, 2960.5,
2924.8, 2857.1, 1897.5, 1693.1, 1679.7, 1628.0, 1607.9, 1555.2, 1492.5, 1467.2, 1449.3, 1313.1,
1
1279.3, 1221.6, 1184.4, 1118.0, 1039.9, 1014.9, 946.6, 825.9, 805.1, 747.6; H NMR (400 MHz,
DMSO-d6) δ 10.98 (s, 2H), 8.23 (s, 1H), 7.92 (s, 1H), 7.60-7.40 (m, 6H), 7.09 (d, J = 8.8 Hz, 1H),
6.56 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 5.11 (s, 2H), 3.76 (t, J = 4.4 Hz, 4H), 3.12 (t, J = 4.4 Hz,
4H); 13C NMR (100 MHz, DMSO-d6) δ 161.06, 158.32, 157.52, 153.06, 148.33, 142.78, 136.36,
133.31, 132.94, 129.98(2C), 129.84, 128.92(2C), 120.38, 116.10, 113.72, 107.56, 107.38, 102.38,
68.87, 66.63(2C), 50.00(2C); Anal. Calcd, for C26H23ClN5O4S (%): C, 58.04; H, 4.50; N, 13.02;
Found (%): C, 58.06; H, 4.52; N, 13.01.
5.11.30. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(4-(3-chlorobenzyloxy)-2-hydroxybenzylid-
ene) hydrazinecarboxamide (15w)
Yield: 74%; MS (ESI) m/z: 536.3 [M-H]-; IR (KBr, cm-1): 3414.3, 3183.9, 3067.5, 2958.4,
2919.9, 2852.5, 1690.8, 1679.4, 1628.6, 1607.9, 1554.2, 1499.6, 1473.4, 1448.4, 1383.9, 1298.6
1
1223.0, 1171.5, 1120.7, 1041.5, 947.5, 828.0, 781.6, 746.2, 680.8, 617.1; H NMR (400 MHz,
DMSO-d6) δ 10.98 (s, 2H), 8.23 (s, 1H), 7.81 (s, 1H), 7.52 (s, 2H), 7.43 (m, 4H), 7.09 (d, J = 8.8
Hz, 1H), 6.57 (d, J = 8.8 Hz, 1H), 6.52 (s, 1H), 5.13 (s, 2H), 3.76 (s, 4H), 3.12 (s, 4H); Anal.
Calcd, for C26H23ClN5O4S (%): C, 58.04; H, 4.50; N, 13.02; Found (%): C, 58.02; H, 4.51; N,
13.03.
5.11.31. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(4-(2,4-dichlorobenzyloxy)-2-hydroxybenz-
ylidene) hydrazinecarboxamide (15x)
1
Yield: 75%; MS (ESI) m/z: 570.2 [M-H]-; H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 2H),
8.24 (s, 1H), 7.92 (s, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.55-7.47 (m, 2H),
7.44 (d, J = 2.0 Hz, 1H), 7.09 (dd, J = 8.8, 2.4 Hz, 1H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.53 (d, J =
2.4 Hz, 1H), 5.16 (s, 2H), 3.76 (t, J = 4.4 Hz, 4H), 3.12 (t, J = 4.4 Hz, 4H); Anal. Calcd, for
C26H23Cl2N5O4S (%): C, 54.55; H, 4.05; N, 12.23; Found (%): C, 54.58; H, 4.05; N, 12.21.
5.11.32. (E)-N-(6-((dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-((2-(benzo[d][1,3]dioxol-5-
ylmethyl)thiazol-4-yl)methoxy)-2-hydroxybenzylidene) hydrazinecarboxamide (16a)
1
Yield: 81%; MS (ESI) m/z: 615.1 [M-H]-, 651.1[M+Cl]-; H NMR (400 MHz, DMSO-d6) δ
11.11 (s, 2H), 8.25 (s, 1H), 8.04 (s, 1H), 7.85 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.58 (s, 1H), 7.37
(d, J = 8.4 Hz, 1H), 6.93 (d, J = 1.2 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.83 (dd, J = 8.0, 1.2 Hz,
1H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.55 (d, J =2.4 Hz, 1H), 6.01 (s, 2H), 5.12 (s, 2H), 4.25 (s, 2H),
3.63 (s, 2H), 2.27 (s, 6H); Anal. Calcd, for C30H28N6O5S2 (%): C, 58.43; H, 4.58; N, 13.63; Found
(%): C, 58.46; H, 4.57; N, 13.61.
5.11.33. (E)-N-(6-((diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-((2-(benzo[d][1,3]dioxol-5-y-
lmethyl)thiazol-4-yl)methoxy)-2-hydroxybenzylidene) hydrazinecarboxamide (16b)
Yield: 79%; MS (ESI) m/z: 643.2 [M-H]-, 679.2[M+Cl]-; IR (KBr, cm-1): 3339.7, 2961.7,
2919.9, 1703.6, 1613.2, 1578.0, 1557.7, 1484.1, 1462.6, 1416.3, 1404.2, 1384.8, 1277.5, 1236.8,
1
1151.0, 1047.1, 942.9, 879.6, 847.7, 808.8, 756.1, 734.1,658.6, 644.5; H NMR (400 MHz,
DMSO-d6) δ 11.38 (s, 1H), 11.21 (s, 1H), 8.26 (s, 1H), 8.14 (s, 1H), 8.01 (s, 1H), 7.74 (d, J = 8.8
Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.59 (s, 1H), 6.93 (s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.83 (d, J =
14

ACCEPTED MANUSCRIPT
8.0 Hz, 1H), 6.58 (m, 1H), 6.56 (s, 1H), 6.01 (s, 2H), 5.12 (s, 2H), 4.38 (s, 2H), 4.25 (s, 2H), 3.08
(q, J = 7.2 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 171.31, 165.17,
163.48, 161.29, 161.12, 152.98, 151.57, 150.99, 147.87, 146.67, 132.13, 129.49, 125.22, 124.79,
122.68, 119.98, 119.35, 118.87, 116.94, 109.91, 108.84, 108.24, 107.23, 102.30, 101.42, 65.70,
55.13, 46.02(2C), 38.58, 8.79(2C); Anal. Calcd, for C32H32N6O5S2 (%): C, 59.61; H, 5.00; N,
13.03; Found (%): C, 59.63; H, 5.01; N, 13.01.
5.11.34. (E)-N-(6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-2-(4-((2-(benzo[d][1,3]-
dioxol-5-ylmethyl)thiazol-4-yl)methoxy)-2-hydroxybenzylidene)hydrazinecarboxamide (16c)
1
Yield: 82%; MS (ESI) m/z: 669.3 [M-H]-,705.2[M+Cl]-; H NMR (400 MHz, DMSO-d6) δ
11.11 (s, 2H), 8.88 (s, 1H), 8.25 (s, 1H), 7.95 (s, 1H), 7.83 (s, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.58
(s, 1H), 7.35 (d, J = 8.0 Hz, 1H), 6.93 (s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H),
6.58 (d, J = 8.8 Hz, 1H), 6.55 (s, 1H), 6.00 (s, 2H), 5.12 (s, 2H), 4.25 (s, 2H), 3.64 (s, 2H), 2.86 (s,
2H), 2.07 (s, 2H), 1.59 (d, J = 12.0 Hz, 2H), 1.36 (s, 1H), 1.17 (m, 2H), 0.89 (d, J = 6.4 Hz, 3H);
Anal. Calcd, for C34H34N6O5S2 (%): C, 60.88; H, 5.11; N, 12.53; Found (%): C, 60.86; H, 5.10; N,
12.52.
5.11.35. (E)-N-(6-morpholinobenzo[d]thiazol-2-yl)-2-(4-((2-(benzo[d][1,3]dioxol-5-ylmethyl)thi-
azol-4-yl)methoxy)-2-hydroxybenzylidene)hydrazinecarboxamide (16d)
Yield: 85%; MS (ESI) m/z: 643.3 [M-H]-; IR (KBr, cm-1): 3186.4, 3075.9, 2957.1, 1691.4,
1626.7, 1607.3, 1570.0, 1543.1, 1501.5, 1489.0, 1444.6, 1277.6, 1246.3, 1223.6, 1185.4, 1116.6,
1
1038.3, 947.2, 925.8, 809.6, 743.2, 705.7, 650.5, 534.7; H NMR (400 MHz, DMSO-d6) δ 11.00
(s, 2H), 8.24 (s, 1H), 7.95 (s, 1H), 7.58 (s, 1H), 7.53 (m, 1H), 7.45 (s, 1H), 7.10 (d, J = 8.8 Hz,
1H), 6.93 (s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.58 (dd, J = 8.8, 2.0 Hz, 1H),
6.54 (d, J = 2.0 Hz, 1H), 6.01 (s, 2H), 5.12 (s, 2H), 4.25 (s, 2H), 3.76 (t, J = 4.4 Hz, 4H), 3.12 (t, J
= 4.4 Hz, 4H); Anal. Calcd, for C31H28N6O6S2 (%): C, 57.75; H, 4.38; N, 13.04; Found (%): C,
57.73; H, 4.39; N, 13.05.
5.12. Pharmacology
5.12.1. MTT assay in vitro
The anti-proliferative activities of compounds 14a-h, 15a-x and 16a-d were evaluated against
NCI-H226, SK-N-SH, HT29, MKN-45 and MDA-MB-231 cell lines using the standard MTT
assay in vitro, with PAC-1 as the positive control. The cancer cell lines were cultured in minimum
essential medium (MEM) supplemented with 10% foetal 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 aterminal 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.
5.12.2. Procaspase-3 kinase assay
The procaspase-3 activity was evaluated using this method as previously reported[5]. (a)
100ꢀM target compounds and PAC-1 were dissolved in DMSO and stored at -20 °C as mother
liquor, which were diluted to corresponding concentration by using corresponding buffer when
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used. (b) 40ꢀL procaspase-3, which was dissolved in buffer (50 mM HEPES, 0.1% CHAPS, 10%
glycerol, 100 mM NaCl, 0.1 mM EDTA, 10 mM DTT, pH 7.4), was added in 384-well plates.
10ꢀL five times the final concentration of target compounds were added, the final concentration of
procaspase-3 was 1ꢀM, and procaspase-3 was incubated in buffer at 37 °C for 2 h. (c) 0.4 mM
Ac-DEVD-pNa was added in the buffer (50 mM HEPES, 0.1% CHAPS, 10% glycerol, 100 mM
NaCl, 0.1 mM EDTA, 10 mM DTT, pH 7.4) of every plates, absorbance values were read in every
two minute for 1 h, and absorbance values of per minute were calculated as enzyme activity of
detection index. (d) all data were detected and analyzed by using SPSS 16.0, each data was
presented as mean±S.E. The activation rate (%) was calculated using the following equation:
(Asample-Admso)/(Apac-1-Admso)×100. EC50 was calculated from the activation curves with probability
regression method.
5.12.3. Explanation about anticancer activity and enzymatic activity of PAC-1
There were many factors that affected the anticancer activity and enzymatic activity of
PAC-1, which were different of reported data. Of them, the main reason about biological assay
difference was the difference in test methods. In our work, the anti-proliferative activity of PAC-1
was evaluated by using the standard MTT assay, however, PAC-1 was evaluated in the literature
(Nature chemical biology 2006, 2 (10): 543-50) by using the MTS-PMS-assay. In addition,
although the test method of enzymatic activity was mainly refer to this method reported in the
literature (Nature chemical biology 2006, 2 (10): 543-50), there were some differences in the
details, for example, concentration of procaspase-3 and Ac-DEVD-pNa were different, absorbance
values were read at different time frequency, detected and analyzed software and calculation
method were different. All above could be a possible reason that made our data be different of
reported data.
Acknowledgements
The work was supported by Program for Innovative Research Team of the Ministry of
Education of the People’s Republic of China and Program for Liaoning Innovative Research Team
in University.
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Legends
Fig. 1. Structures of procaspase-3 PAC-1 and SPAC-1
Fig. 2. Structures of derivatives bearing an ortho-hydroxy N-acylhydrazone moiety
Fig. 3. The design of target compounds
Scheme 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) Br2, NH4SCN,
HOAC, 10°C to rt, 4-6 h; (iv) phenyl chloroformate, pyridine, CH2Cl2, 0°C to rt, 4-6 h; (v)
80%hydrazine monohydrate, 1,4-dioxane, 80°C, 6 h.
Scheme 2. Reagents and conditions: (i) Na2CO3, KI, 65 °C,30 h; (ii) NaHS, MgCl2·6H2O, DMF,
H2O, rt, 15 h; (iii) 1,3-dichloro-2-propanon, acetonitrile, 50 °C, 4 h; (iv) 2,4-dihydroxy
benzaldehyde, NaCO3, KI, acetonitrile, 80 °C, 2 h; (v) acetic acid, ethanol, reflux.
Fig. 4. NOE of the representative compound 15v
Table 1. cytotoxicity of target compounds 14a-h, 15a-x, 16a-d against H226, SK-N-SH, HT-29,
MKN45 and MDA-MB-231 cancer cell lines.
Table 2. procaspase-3 activity of selected compounds 15g, 15h, 15n, 15p, 15u, 16b and PAC-1 in
vitro.
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Table 1. cytotoxicity of target compounds 14a-h, 15a-x, 16a-d against H226, SK-N-SH, HT-29, MKN45 and MDA-MB-231 cancer cell
lines.
IC50 (µmol/L) ±SDa
Compd.
R1
n
R
H226
SK-N-SH
5.14±1.02
HT29
MKN45
MDA-MB-231
5.95±0.21
14a
14b
14c
14d
14e
14f
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
H
4-OH
H
2.03 ±0.38
5.96±0.52
1.54±1.01
N
N
2.98±0.34
1.81±0.11
5.16±0.19
2.96±0.28
4.61±0.15
2.71±0.18
4.26±0.11
1.08±0.05
0.83±0.17
1.17±0.10
0.36±0.07
0.58±0.01
1.01±0.02
0.16±0.01
0.43±0.01
1.30±0.14
0.56±0.10
14.31±1.35
5.79±1.01
6.05±0.17
4.36±1.52
7.74±1.26
7.05±0.76
11.68±0.78
2.58±0.11
1.66±0.15
2.74±0.12
1.19±0.18
2.94±0.15
1.43±0.09
0.48±0.02
0.74±0.05
1.83±0.13
2.01±0.01
77.92±5.40
4.03±0.54
23.73±1.67
10.40±2.62
2.73±0.05
10.32±1.06
67.93±2.11
2.13±0.04
1.51±0.11
2.15±0.18
2.94±0.11
1.61±0.25
1.33±0.13
0.77±0.01
1.02±0.02
1.88±0.01
2.23±0.16
2.85±0.13
1.71±0.08
1.74±0.26
0.68±0.06
4.09±0.16
2.34±0.32
9.31±0.51
1.27±0.02
1.04±0.01
0.70±0.09
0.19±0.01
0.33±0.01
0.58±0.01
0.50±0.02
0.54±0.08
0.42±0.02
0.42±0.01
>100
11.33±0.23
4.11±0.16
12.29±0.03
1.66±0.31
12.04±1.21
>100
4-OH
H
4-OH
H
14g
14h
15a
15b
15c
15d
15e
15f
H
4-CH3
4-C(CH3)3
4-Cl
2.65±0.02
1.14±0.01
0.60±0.05
0.54±0.04
0.68±0.12
0.71±0.03
0.98±0.03
0.61±0.01
0.57±0.02
0.74±0.03
N
N
N
N
N
3-Cl
2-Cl
4-CH3
4-C(CH3)3
4-Cl
15g
15h
15i
3-Cl
15j
2-Cl

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15k
15l
1
1
1
1
1
1
1
1
1
0
0
0
0
0
1
1
1
0
2,4-(Cl)2
0.70±0.02
0.88±0.01
1.06±0.05
0.18±0.02
0.41±0.06
0.36±0.03
0.79±0.06
0.39±0.06
0.61±0.11
0.53±0.05
0.24±0.03
0.26±0.01
0.79±0.11
1.22±0.23
0.72±0.03
0.14±0.01
0.81±0.02
1.04±0.05
1.67±0.07
1.56±0.02
0.27±0.03
1.06±0.07
0.34±0.01
0.85±0.07
1.03±0.19
1.32±0.05
2.51±0.15
0.92±0.33
1.37±0.03
1.48±0.13
3.14±0.08
0.79±0.01
0.48±0.03
1.25±0.01
2.43±0.03
1.44±0.23
1.04±0.03
0.87±0.03
0.95±0.10
1.72±0.04
1.03±0.14
1.00±0.09
1.57±0.02
1.58±0.17
0.92±0.15
0.52±0.03
ND
1.52±0.03
ND
H
4-CH3
4-C(CH3)3
4-Cl
15m
15n
15o
15p
15q
15r
15s
0.31±0.02
0.17±0.02
0.21±0.01
0.67±0.09
0.76±0.06
0.28±0.03
ND
1.51±0.02
0.56±0.01
0.88±0.03
1.12±0.02
2.3±0.05
0.30±0.03
ND
3-Cl
2-Cl
2,4-(Cl)2
H
15t
4-CH3
4-C(CH3)3
4-Cl
0.75±0.03
0.46±0.04
0.29±0.01
0.28±0.02
1.75±0.10
0.62±0.01
0.51±0.01
0.55±0.03
19.34±0.31
1.04±0.05
4.46±0.03
1.18±0.01
15.06±1.01
1.04±0.11
0.55±0.01
0.88±0.02
15u
15v
15w
15x
16a
16b
16c
0.0018±0.000
2
3-Cl
0.59±0.04
4.72±0.13
1.88±0.03
0.72±0.02
1.12±0.15
2,4-(Cl)2
-
N
-
-
16d
-
2.14±0.11
1.02±0.01
6.21±0.23
3.06±0.04
2.31±0.16
1.36±0.02
1.01±0.01
2.61±0.05
4.03±0.04
4.8±0.02
PAC-1b
Bold values show the IC50 values of the target compounds lower than the values of the positive control. ND: Not determined.
a IC50: concentration of the compound (µM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the MTT
assay. Each experiment was carried out in triplicate.
b Used as the positive control.

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Table 2. procaspase-3 activity of selected compounds 15g, 15h, 15n, 15p, 15u, 16b and PAC-1 in vitro.
Compd.
EC50 on procaspase-3 (µM)
15g
1.42
4.96
2.42
4.26
18.23
0.25
4.08b
15h
15n
15p
15u
16b
PAC-1a
a Used as the positive control.
b Reported value is 0.22 µM [5].

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