Synthesis and biological evaluation of benzothiazole derivatives bearing the ortho-hydroxy-N-acylhydrazone moiety as potent antitumor agents

Article abstract of DOI:10.1002/ardp.201400230

A novel series of benzothiazole derivatives bearing the ortho-hydroxy-N-acylhydrazone moiety were designed, synthesized, and evaluated for their procaspase-3 kinase activation activities and antiproliferative activities against five cancer cell lines (NCI-H226, SK-N-SH, HT29, MKN-45, and MDA-MB-231). Most target compounds showed moderate to excellent cytotoxic activity against all five tested cancer lines. The most promising compound 18e (procaspase-3 EC₅₀- 0.31μM) with IC50 values ranging from 0.24 to 0.92 μM against all tested cell lines was 4.24-12.2 times more active than PAC-1 (procaspase-3 EC₅₀ - 0.41 μM). Structure-activity relationship studies indicated that the phenyl group on the 2-hydroxyphenyl ring (moiety A) was critical for pharmacological activity in vitro. In addition, introduction of a benzyloxyl group on moiety A and a mono-electron-withdrawing group at the 4-position of the benzyloxyl group were more favorable for antitumor activity. Moreover, reduction of the electron density in the phenyl ring of the benzyloxy group led to a dramatic decrease in the procaspase-3 kinase activation activity. -

Full text of DOI:10.1002/ardp.201400230

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
1
Full Paper
Synthesis and Biological Evaluation of Benzothiazole
Derivatives Bearing the ortho-Hydroxy-N-acylhydrazone
Moiety as Potent Antitumor Agents
Junjie Ma1, Guangyan Zhang1, Xiaoqi Han1, Guanglong Bao1, Lihui Wang2, Xin Zhai1,
and Ping Gong1
1
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenhe District, Shenyang, P. R. China
Department of Pharmacology, Shenyang Pharmaceutical University College of Life Science and
2
Biopharmaceuticals, Shenyang, P. R. China
A novel series of benzothiazole derivatives bearing the ortho-hydroxy-N-acylhydrazone moiety were
designed, synthesized, and evaluated for their procaspase-3 kinase activation activities and
antiproliferative activities against five cancer cell lines (NCI-H226, SK-N-SH, HT29, MKN-45, and
MDA-MB-231). Most target compounds showed moderate to excellent cytotoxic activity against all five
tested cancer lines. The most promising compound 18e (procaspase-3 EC50 ¼ 0.31 mM) with IC50 values
ranging from 0.24 to 0.92 mM against all tested cell lines was 4.24–12.2 times more active than PAC-1
(procaspase-3 EC50 ¼ 0.41 mM). Structure–activity relationship studies indicated that the phenyl group
on the 2-hydroxyphenyl ring (moiety A) was critical for pharmacological activity in vitro. In addition,
introduction of a benzyloxyl group on moiety A and a mono-electron-withdrawing group at the
4-position of the benzyloxyl group were more favorable for antitumor activity. Moreover, reduction of
the electron density in the phenyl ring of the benzyloxy group led to a dramatic decrease in the
procaspase-3 kinase activation activity.
Keywords: Antitumor / Benzothiazole / ortho-Hydroxy-N-acylhydrazone moiety / Procaspase-3
Received: June 6, 2014; Revised: July 16, 2014; Accepted: July 21, 2014
DOI 10.1002/ardp.201400230
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
human tumors, including colon cancer [1], lung cancer [2],
melanoma [3], hepatoma [4], breast cancer [5], lymphoma [6],
and neuroblastoma [7]. Thus, procaspase-3 kinase is a
promising therapeutic target for cancer, and procaspase-3
kinase activators display better superiority than other
apoptosis induction compounds, such as PAC-1 (1, Fig. 1)
and its derivatives (2, Fig. 1) [8, 9].
One of the most significant characters of cancer is the ability
to avoid apoptosis, which is a process of programmed cell
death (PCD) that occurs in higher organisms to maintain
homeostasis. Interestingly, numerous studies have demon-
strated that procaspase-3 is overexpressed in a variety of
Structure–activity relationships (SARs) studies revealed that
the activity of PAC-1 and its derivatives is dependent on the
presence of the ortho-hydroxy-N-acylhydrazone moiety [9],
which was known to participate in metal chelation,
procaspase-3 activation, and cancer cell death. Introduction
of 3,5-di-tert-butyl group on the 2-hydroxyphenyl ring (moiety
A) could enhance the antitumor activity [8]. Our laboratory
have reported a series of ortho-hydroxy-N-acylhydrazone
derivatives (3, Fig. 2) as procaspase-3 kinase activators [10]
Correspondence: Prof. Ping Gong, Key Laboratory of Structure-Based
Drug Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
E-mail: gongpinggp@126.com
Fax: þ86 24 2398 6429
Abbreviations: EWG, electron-withdrawing group; NOESY, nuclear
Overhauser effect spectroscopy; PAC-1, first procaspase activating
compound; SAR, structure–activity relationship.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Figure 1. Structures of procaspase-3 PAC-1 and its derivatives.
3 [1], and may directly interact with the triaspartic acid “safety
catch” of procaspase-3, thereby inducing the autoactivation of
procaspase-3.
Benzothiazoles exhibit a variety of biological properties
including antifungal [11], anticancer [12, 13], antiamyloid
[14], and antirheumatic [15], and have attracted considerable
attention. They have been widely used as important
pharmacophores for exploration of novel and highly active
antitumor compounds in medicinal chemistry (4–6,
Fig. 3) [16–18].
Figure 2. Structures of ortho-hydroxy-N-acylhydrazone derivatives.
and their SARs showed that introduction of benzyloxy group
on the moiety A was beneficial for improvement of the
cytotoxicity in vitro. In addition, substituents containing
nitrogen atoms that are the positively charged at
physiological pH (such as the piperazine nitrogens in PAC-
1) play a significant role in the autoactivation of procaspase-
These observations prompted us to discover more potent
anticancer agents as novel procaspase-3 activator. Therefore,
we designed a series of compounds, in which benzothiazole
was linked with ortho-hydroxy-N-acylhydrazone moiety at
2-position based on a hybrid pharmacophore design (Fig. 4).
Some secondary amino groups (e.g., 4-methylpiperidino,
Figure 3. Structures of benzothiazole derivatives.
Figure 4. The design of target compounds.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Benzothiazoles with ortho-Hydroxy-N-acylhydrazone Moiety
3
dimethylamino, and diethylamino), which are positively
charged (R1) at physiological pH, were introduced at the 6-
position of benzothiazole to avoid metabolic pathway and
improve the polarity and water-solubility. Substituted benz-
yloxy groups and some special alkyl groups (3-allyl and 3,5-di-
tert-butyl) were introduced into the 4-position of moiety A to
investigate cytotoxicity. Furthermore, the phenyl group of
moiety A was replaced with a substituted chromenonyl group
to investigate its importance.
Herein, 26 target compounds were synthesized and
evaluated for in vitro procaspase-3 kinase activation activities
and cytotoxicities against two procaspase-3 overexpressing
cancer cell lines (NCI-H226 and SK-N-SH), one cancer cell
line moderately sensitive to procaspase-3 (MDA-MB-231),
as well as two procaspase-3 low-sensitive cancer cell lines
(MKN-45 and HT29) to be further evaluated for ruling out off-
target effect.
As shown in Scheme 2, the substituted benzyloxybenzalde-
hydes 12a–g were synthesized from 2,4-dihydroxybenzalde-
hyde through regioselective O-alkylation reaction with
substituted benzylchloride in acetonitrile [20]. 2-Allyloxybenz-
aldehyde 13 was prepared by the reaction of salicylaldehyde
with allyl bromide in the presence of potassium carbonate in
DMF. 2-Hydroxy-3-allylbenzaldehyde 14 was obtained by the
Claisen thermal rearrangement of 13 at 200°C for 4 h [21].
Cyclization of m-dihydroxybenzene with ethyl acetoacetate
in sulfuric acid at 10°C provided 15, which reacted with
urotropine in glacial acetic acid and sulfuric acid in sequence,
namely a Duff reaction, to yield chromenealdehyde 16 as a
white solid [22]. Finally, the target compounds 17a–f, 18a–q,
and 19a–c were generated via the condensation of 11a–c
with appropriate 2-hydroxy aromatic aldehydes in ethanol
at 78°C in the presence of catalytic amounts of acetic
acid [23].
The chemical structures of the target compounds were
confirmed by 1H NMR, 13C NMR, 2D NOESY NMR, and MS
spectra. All target compounds could exist in either the E or Z
isomeric form because of the imino bond, so compound 18e
was selected to further confirm the stereochemistry by
undergoing 2D NOESY NMR. Results (Fig. 5 and Supporting
Information) showed that an evident NOE signal was observed
Results and discussion
Chemistry
The preparation of key intermediates 11a–c is described in
Scheme 1. The commercially available starting material 4-
nitrobenzyl bromide fused with excessive secondary amines
(4-methyl piperidine, dimethylamine, and diethylamine) in
acetonitrile at room temperature for 3 h to provide inter-
mediates 7a–c, which were then reduced in the presence of
80% hydrazine, FeCl3 ꢀ 6H2O, activated carbon to obtain the
substituted anilines 8a–c. Compounds 8a–c further reacted
with potassium thiocyanate and bromine to give 9a–c in a
satisfactory yield, using a known preparation method [19].
Treatment of 9a–c with phenyl chloroformate yielded the
amides 10a–c. Subsequently, the key intermediate semi-
carbazides 11a–c were generated via hydrazinolysis of 10a–c
with 80% hydrazine monohydrate in 1,4-dioxane at 80°C for
6 h.
–
–
between the H1 (–CH N–, d ¼ 8.24 ppm) and H ( N–NH–,
–
–
2
d ¼ 11.12 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.
Pharmacology
In vitro enzymatic assays
All the target compounds were initially screened for their
procaspase-3 kinase activities at 50 mM and expressed as
activation rate. Furthermore, several potent compounds were
Scheme 1. Reagents and conditions: (i) acetonitrile, amine, rt, 3 h; (ii) 80% hydrazine monohydrate, FeCl3 ꢀ 6H2O, activated carbon, ethanol,
65–78°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; and (v) 80% hydrazine
monohydrate, 1,4-dioxane, 80°C, 6 h.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Scheme 2. Reagents and conditions: (i) Na2CO3, KI, 65°C, 30 h; (ii) K2CO3, DMF, rt, 15 h; (iii) 200°C, 4 h; (iv) ethyl acetoacetate, H2SO4,
10°C, 4 h; (v) urotropine, HOAc, 115°C, 4 h; and (vi) acetic acid, ethanol, reflux.
Figure 5. NOESY effect of the representative compound 18e.
selected to evaluate for the enzyme activity and presented as
EC50 values, PAC-1 was used as the positive control. The mean
values of experiments performed in triplicate are shown in
Table 1.
As illustrated in Table 1, the data indicated that most
compounds exhibited good to excellent activity against
procapase-3 kinase at 50 mM and compounds 17e, 17f, 18e,
18h, 18k, and 18m showed better activity than other
compounds, which were further evaluated for the enzyme
potency as EC50 values. Among which, compounds 17f and
18e exhibited more potent activity than PAC-1, with EC50
values of 0.39 and 0.31 mM, respectively.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Benzothiazoles with ortho-Hydroxy-N-acylhydrazone Moiety
5
Table 1. Procaspase-3 kinase activity of the target compounds 17a–f, 18a–q, and 19a–c.
Compd.
17a
R1
R
Procapase-3 (% activity at 50 mM)
76.67 ꢁ 0.25
Procaspase-3 EC50 (mM)
3-Allyl
ND
17b
17c
17d
17e
17f
3,5-Di-tert-butyl
43.18 ꢁ 0.24
32.92 ꢁ 0.18
39.03 ꢁ 0.67
87.31 ꢁ 0.18
116.88 ꢁ 0.11
72.89 ꢁ 0.17
72.74 ꢁ 0.28
61.49 ꢁ 0.12
70.90 ꢁ 0.58
100.64 ꢁ 0.42
65.45 ꢁ 0.32
55.24 ꢁ 0.19
90.19 ꢁ 0.15
77.45 ꢁ 0.43
72.01 ꢁ 0.10
86.23 ꢁ 0.78
49.62 ꢁ 0.77
88.09 ꢁ 0.28
77.83 ꢁ 0.19
73.67 ꢁ 0.46
77.81 ꢁ 0.36
47.08 ꢁ 0.71
37.16 ꢁ 0.44
12.99 ꢁ 0.68
66.47 ꢁ 0.31
100
ND
ND
3-Allyl
3,5-Di-tert-butyl
ND
3-Allyl
0.45
0.39b)
ND
3,5-Di-tert-butyl
18a
18b
18c
18d
18e
18f
H
4-F
ND
3-F
ND
2-F
ND
4-CF3
2,4-Di-Cl
2,3-Di-Cl
4-F
0.31
ND
18g
18h
18i
ND
0.58
ND
3-F
18j
2-F
ND
18k
18l
4-CF3
2,3-Di-Cl
4-F
0.62
ND
18m
18n
18o
18p
18q
19a
19b
19c
PAC-1a)
0.83
ND
3-F
2-F
ND
4-CF3
2,3-Di-Cl
–
ND
ND
ND
–
ND
–
ND
0.41c)
ND, not determined.
a)
Used as the positive control.
Bold values show the procapase-3 kinase activity of the target compounds higher than the positive control.
Reported value is 0.22 mM [1].
b)
c)
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Table 2. Cytotoxicity of target compounds 17a–f, 18a–q, and 19a–c against H-226, SK-N-SH, MDA-MB-231, HT-29, and MKN-45
cancer cell lines.
IC50 (mmol/L)
Compd.
17a
R1
R
H-226
ND
SK-N-SH
1.68 ꢁ 0.11
2.28 ꢁ 0.04
MDA-MB-231
3.55 ꢁ 0.17
13.9 ꢁ 0.24
HT-29
ND
MKN-45
1.01 ꢁ 0.15
2.48 ꢁ 0.08
3-Allyl
17b
3,5-Di-tert-butyl
ND
ND
17c
17d
17e
17f
3-Allyl
ND
2.10 ꢁ 0.09
2.46 ꢁ 0.21
2.58 ꢁ 0.12
2.79 ꢁ 0.13
1.14 ꢁ 0.11
0.88 ꢁ 0.03
1.29 ꢁ 0.08
1.94 ꢁ 0.15
0.60 ꢁ 0.02
0.59 ꢁ 0.01
0.87 ꢁ 0.02
0.71 ꢁ 0.06
1.34 ꢁ 0.05
0.91 ꢁ 0.15
0.85 ꢁ 0.02
1.41 ꢁ 0.13
0.40 ꢁ 0.01
0.56 ꢁ 0.09
1.38 ꢁ 0.11
0.75 ꢁ 0.08
0.55 ꢁ 0.09
6.12 ꢁ 1.12
4.37 ꢁ 0.69
2.72 ꢁ 0.15
3.06 ꢁ 0.16
5.25 ꢁ 0.19
7.23 ꢁ 1.01
3.11 ꢁ 0.31
12.1 ꢁ 0.15
1.34 ꢁ 0.11
2.02 ꢁ 0.01
2.02 ꢁ 0.12
0.66 ꢁ 0.02
0.63 ꢁ 0.03
2.02 ꢁ 013
2.94 ꢁ 0.08
0.97 ꢁ 0.08
1.12 ꢁ 0.11
0.88 ꢁ 0.02
1.06 ꢁ 0.05
1.74 ꢁ 0.02
2.37 ꢁ 0.04
0.83 ꢁ 0.05
0.42 ꢁ 0.01
0.58 ꢁ 0.03
1.00 ꢁ 0.11
16.18 ꢁ 0.55
12.7 ꢁ 0.36
10.49 ꢁ 1.05
4.58 ꢁ 0.05
ND
1.80 ꢁ 0.12
4.06 ꢁ 1.26
1.31 ꢁ 0.22
9.70 ꢁ 0.26
1.06 ꢁ 0.10
1.72 ꢁ 0.04
1.35 ꢁ 0.04
0.35 ꢁ 0.03
0.29 ꢁ 0.01
1.38 ꢁ 0.05
1.45 ꢁ 0.03
0.31 ꢁ 0.07
0.63 ꢁ 0.10
0.55 ꢁ 0.02
0.57 ꢁ 0.01
0.71 ꢁ 0.05
0.79 ꢁ 0.12
0.38 ꢁ 0.01
0.40 ꢁ 0.02
0.31 ꢁ 0.02
0.45 ꢁ 0.07
5.32 ꢁ 0.12
5.83 ꢁ 0.48
4.35 ꢁ 1.08
3.56 ꢁ 0.05
3,5-Di-tert-butyl
ND
ND
3-Allyl
ND
ND
3,5-Di-tert-butyl
ND
ND
18a
18b
18c
18d
18e
18f
H
4-F
0.91 ꢁ 0.03
0.42 ꢁ 0.05
0.51 ꢁ 0.03
0.59 ꢁ 0.01
0.24 ꢁ 0.02
0.50 ꢁ 0.04
0.81 ꢁ 0.01
0.57 ꢁ 0.07
1.05 ꢁ 0.03
0.62 ꢁ 0.06
0.54 ꢁ 0.04
0.95 ꢁ 0.02
0.31 ꢁ 0.03
0.36 ꢁ 0.04
0.45 ꢁ 0.05
0.65 ꢁ 0.11
0.33 ꢁ 0.05
ND
2.19 ꢁ 0.17
1.32 ꢁ 0.07
1.03 ꢁ 0.12
1.51 ꢁ 0.04
0.92 ꢁ 0.08
ND
3-F
2-F
4-CF3
2,4-Di-Cl
2,3-Di-Cl
4-F
18g
18h
18i
ND
1.47 ꢁ 0.03
0.76 ꢁ 0.02
1.06 ꢁ 0.12
1.59 ꢁ 0.14
4.01 ꢁ 0.27
4.01 ꢁ 0.18
1.02 ꢁ 0.11
1.65 ꢁ 0.13
1.12 ꢁ 0.06
3.34 ꢁ 0.18
ND
3-F
18j
2-F
18k
18l
4-CF3
2,3-Di-Cl
4-F
18m
18n
18o
18p
18q
19a
19b
19c
PAC-1a)
3-F
2-F
4-CF3
2,3-Di-Cl
–
–
ND
ND
–
ND
ND
1.02 ꢁ 0.08
1.36 ꢁ 0.02
ND, not determined.
a)
Used as the positive control.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Benzothiazoles with ortho-Hydroxy-N-acylhydrazone Moiety
7
According to the data shown in Table 1, different R1 groups
made a different contribution to the potency of compounds
17a–f with the following rank order: 4-methylpiperidinyl >
dimethylamino > diethylamino. In addition, the replacement
of the phenyl group of moiety A with a substituted
comparable activity with compounds with 3,5-di-tert-butyl
group against procaspase-3 kinase, compounds with 3-allyl
group showed better antitumor activity than compounds
with 3,5-di-tert-butyl group against SK-N-SH, MDA-MB-231, and
MKN-45 by comparison of IC50 values of compounds 17a–f,
which suggested that compounds with 3-allyl group could
inhibit the growth of tumor cells not only via activating
procaspase-3 kinase, but also via another unknown mecha-
nism, further indicating that compounds with 3-allyl group
might also be multi-target compounds.
Further investigations were performed to study the effect
on cytotoxic activity of different substituents on the
benzyloxyl group. The results of compounds 18b–d, 18h–j,
and 18m–o indicated that introduction of the mono-EWG (-F)
to the 4-position of benzyloxyl group did a better contribution
to the potency against NCI-H226 and SK-N-SH than other
positions. Moreover, increasing the size of mono-EWG (-CF3;
18e, 18k, and 18p) at the 4-position of benzyloxyl group had
no obvious change on the cytotoxicity, which demonstrated
that introduction of bulky group at the 4-position of
benzyloxyl group was well tolerated. Besides, incorporation
of double-EWGs such as 2,4-di-Cl or 2,3-di-Cl on the benzyloxyl
group (18f, 18g, 18l, and 18q) showed no significant difference
against NCI-H226 and SK-N-SH, suggesting that reduction of
the electron density in the phenyl ring of benzyloxy group had
no influence on cytostatic activity.
chromenonyl group yielded compounds 19a–c with
a
dramatic decrease or even loss in potency against
a
procaspase-3 kinase, suggesting that moiety A played an
important role in the activation of procaspase-3 kinase.
Furthermore, compounds with double electron-withdrawing
groups (2,4-di-Cl and 2,3-di-Cl) on the benzyloxyl group
showed weaker activity than compounds with mono electron-
withdrawing group (-F), indicating that reduction of the
electron density in the phenyl ring of benzyloxy group was
unfavorable for the procaspase-3 kinase activity.
In vitro cytotoxicity and structure activity relationships
By using MTT assay, all target compounds were evaluated for
in vitro cytotoxicity against two procaspase-3 overexpressing
cancer cells lines (NCI-H226 and SK-N-SH) and one procaspase-
3 moderately sensitive cancer cell line (MDA-MB-231); in
addition, two procaspase-3 low-sensitive cancer cell lines
(MKN-45 and HT29) were further evaluated for ruling out off-
target effect. PAC-1 was used as the positive control. Each
compound was tested in three independent experiments and
the results are summarized in Table 2 and presented as IC50
values.
As shown in Table 2, all target compounds showed Conclusions
moderate to excellent cytotoxic activity against NCI-H226,
SK-N-SH, and MDA-MB-231. It was observed that compound
18e exhibited the most promising cytotoxic activity with IC50
values ranging from 0.24 to 0.92 mM against all five tested cell
lines, which were 4.24–12.2 times more active than PAC-1.
The data of Table 2 clearly revealed that introduction of the
benzyloxy group to the 4-position of moiety A could improve
the cytotoxicity in vitro, which was consistent with the
previous report [10]. Antitumor activity of target compounds
18a–q against NCI-H226 and SK-N-SH was directly related to
the level of procaspase-3 expression (NCI-H226 > SK-N-SH), and
compounds 18a–q with the benzyloxy groups could also
inhibit the growth of tumor cells with low-sensitivity to
procaspase-3 (MKN45) via another unknown mechanism, a
possible reason for this was that introduction of benzothia-
zole, which has diverse biological properties, made these
compounds be multi-target compounds that showed similar
cytotoxic activities both in procaspase-3 overexpressing
cancer cells lines and low-sensitive cancer cell lines. In
addition, the replacement of moiety A with a substituted
chromenyl group gave rise to compounds 19a–c, which
exhibited lower antitumor activity than other compounds,
suggesting that moiety A was critical for the activity.
Furthermore, although compounds with 3-allyl group showed
In summary, a series of novel benzothiazole derivatives
bearing ortho-hydroxy-N-acylhydrazone moiety were designed,
synthesized, and evaluated for their procaspase-3 kinase
activation activities as well as cytotoxic activities in five
human cancer cell lines (NCI-H226, SK-N-SH, MKN-45, HT29,
and MDA-MB-231). The results revealed that most compounds
showed moderate to excellent activity against procaspase-3
kinase and all tested cancer cell lines. Compound 18e
(procaspase-3 EC50 ¼ 0.31 mM) showed the most promising
antitumor activity with IC50 values ranging from 0.24 to
0.92 mM against all tested cancer cell lines, which were 4.24–
12.2 times more active than PAC-1 (procaspase-3 EC50 ¼ 0.41
mM). The SARs indicated that the phenyl group of 2-
hydroxyphenyl ring (moiety A) was critical for pharmacologi-
cal activity in vitro. Introduction of benzyloxy group to the
moiety A could enhance the antitumor activity in vitro, and
mono-EWG at the 4-position was more favorable for cytotoxic
activity than other positions of the benzyloxyl group. In
addition, reduction of the electron density in the phenyl ring
of the benzyloxyl group led to a dramatic drop in activation
activity against procaspase-3 kinase in vitro. Further studies on
their structural optimization are still underway in our
laboratory.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Experimental
4-((Diethylamino)methyl)aniline (8b)
Colorless oil; yield: 74.1%; MS (ESI) m/z (%): 178.6 [MþH]þ; 1H NMR
(400 MHz, CDCl3) d 7.13 (d, J ¼ 8.2 Hz, 2H), 6.66 (d, J ¼ 8.3 Hz, 2H),
3.63 (s, 2H), 3.50 (s, 2H), 2.53 (q, J¼ 7.1 Hz, 4H), 1.06 (t, J ¼ 7.1Hz, 6H).
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,
300 MHz; Bruker ARX-400, 400 MHz; or Bruker ARX-600, 600 MHz
spectrometers (Bruker Bioscience, Billerica, MA, USA) with tetrame-
thylsilane (TMS) as an internal standard. Column chromatography
was run on silica gel (200e300 mesh) from Qingdao Ocean Chemicals
(Qingdao, Shandong, China). All melting points were obtained on a
Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil,
Switzerland) and were uncorrected. The IR spectra were recorded by
means of the KBr pellet technique on a Bruker FTS 135 spectrometer.
Elemental analysis was determined on a Carlo-Erba 1106 elemental
analysis instrument (Carlo Erba, Milan, Italy). Unless otherwise
noted, all materials were obtained from commercially available
sources and were used without further purification.
4-((4-Methylpiperidin-1-yl)methyl)aniline (8c)
Light oil; yield: 83.5%; MS (ESI) m/z (%): 204.5 [MþH]þ; 1H NMR
(400 MHz, CDCl3) d 7.08 (d, J ¼ 8.1 Hz, 2H), 6.63 (d, J ¼ 8.1 Hz, 2H),
3.60 (s, 2H), 3.38 (s, 2H), 2.84 (d, J ¼ 11.5 Hz, 2H), 1.89 (t, J ¼ 11.7 Hz,
2H), 1.58 (d, J ¼ 12.9 Hz, 2H), 1.32 (d, J ¼ 4.7 Hz, 1H), 1.29–1.19 (m,
2H), 0.90 (d, J ¼ 6.2 Hz, 3H).
General procedure for preparation of compounds 9a–c
A mixture of compounds 8a–c (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 9a–c.
General procedure for preparation of compounds 7a–c
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, 100 mL water was added, and extracted
two times with dichloromethane (DCM). The organic extracts
were combined, dried over sodium sulfate, filtered, and
concentrated to obtain the compounds 7a–c.
6-((Dimethylamino)methyl)benzo[d]thiazol-2-amine (9a)
Light yellow solid; yield: 65.4%; m.p.: 141–143°C; MS (ESI) m/z (%):
1
208.03 [MþH]þ; H NMR (400 MHz, DMSO) d 9.51 (s, 2H), 7.99 (d,
J ¼ 1.2 Hz, 1H), 7.61 (dd, J ¼ 8.3, 1.5 Hz, 1H), 7.55 (d, J ¼ 8.3 Hz, 1H),
4.32 (d, J ¼ 4.8 Hz, 2H), 2.70 (d, J ¼ 4.4 Hz, 6H).
N,N-Dimethyl-1-(4-nitrophenyl)methanamine (7a)
Yellow oil; yield: 85.5%; MS (ESI) m/z (%): 180.5 [MþH]þ; H NMR
1
(400 MHz, CDCl3) d 8.21–8.15 (m, 2H), 7.52 (d, J ¼ 8.7 Hz, 2H), 3.55
(s, 2H), 2.28 (s, 6H).
6-((Diethylamino)methyl)benzo[d]thiazol-2-amine (9b)
Light yellow solid; yield: 88.6%; m.p.: 136–138°C; MS (ESI) m/z (%):
1
236.07 [MþH]þ; H NMR (400 MHz, DMSO) d 7.55 (s, 1H), 7.37 (s,
N-Ethyl-N-(4-nitrobenzyl)ethanamine (7b)
Yellow oil; yield: 82.7%; MS (ESI) m/z (%): 208.4 [MþH]þ; H NMR
1
2H), 7.26 (d, J ¼ 8.1 Hz, 1H), 7.14 (dd, J ¼ 8.2, 1.4 Hz, 1H), 3.52 (s,
2H), 2.45 (q, J ¼ 6.9 Hz, 4H), 0.97 (t, J ¼ 7.1 Hz, 6H).
(400 MHz, CDCl3) d 8.21–8.13 (m, 2H), 7.56 (d, J ¼ 8.6 Hz, 2H), 3.69
(s, 2H), 2.57 (q, J ¼ 7.1 Hz, 4H), 1.07 (t, J ¼ 7.1 Hz, 6H).
6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-amine
4-Methyl-1-(4-nitrobenzyl)piperidine (7c)
(9c)
Light yellow solid; yield: 92.5%; m.p.: 53–55°C; MS (ESI) m/z (%):
235.5 [MþH]þ; 1H NMR (400 MHz, CDCl3) d 8.22 (d, J ¼ 8.7 Hz, 2H),
7.77 (d, J ¼ 8.7 Hz, 2H), 3.92 (s, 2H), 3.11 (d, J ¼ 11.0 Hz, 2H), 2.38 (s,
2H), 1.73 (d, J ¼ 12.3 Hz, 2H), 1.57 (m, 3H), 0.98 (d, J ¼ 6.2 Hz, 3H).
Yellow solid; yield: 85.3%; m.p.: 201–203°C; MS (ESI) m/z (%):
262.13 [MþH]þ; H NMR (400 MHz, DMSO) d 7.75 (s, 1H), 7.65 (s,
1
2H), 7.38 (d, J ¼ 8.2 Hz, 1H), 7.30 (d, J ¼ 7.9 Hz, 1H), 4.22 (s, 2H), 3.33
(s, 2H), 2.77 (d, J ¼ 75.7 Hz, 2H), 1.77 (d, J ¼ 13.2 Hz, 2H), 1.56 (s,
1H), 1.29 (d, J ¼ 11.1 Hz, 2H), 0.90 (d, J ¼ 5.3 Hz, 3H).
General procedure for preparation of compounds 8a–c
A mixture of compounds 7a–c (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) was added, and 80% hydrazine hydrate (25 g, 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 8a–c.
General procedure for preparation of compounds 10a–c
A mixture of compounds 9a–c (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, and 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 4–6h and filtered. The white solid to light yellow solid was
collected and washed by using water to obtain the compounds 10a–c.
Phenyl-6-((dimethylamino)methyl)benzo[d]thiazol-2-
4-((Dimethylamino)methyl)aniline (8a)
ylcarbamate (10a)
Colorless oil; yield: 78.2%; MS (ESI) m/z (%): 150.6 [MþH]þ; 1H NMR
(400 MHz, CDCl3) d 7.07 (d, J ¼ 8.3 Hz, 2H), 6.63 (d, J ¼ 8.4 Hz, 2H),
3.63 (s, 2H), 3.32 (s, 2H), 2.21 (s, 6H).
White solid; yield: 94.6%; m.p.: 178–180°C; MS (ESI) m/z (%): 328.29
[MþH]þ; 1H NMR (400 MHz, DMSO) d 10.60 (s, 1H), 8.15 (s, 1H), 7.82
(d, J ¼ 8.3 Hz, 1H), 7.64 (d, J ¼ 8.4 Hz, 1H), 7.48 (t, J ¼ 7.9 Hz, 2H),
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Benzothiazoles with ortho-Hydroxy-N-acylhydrazone Moiety
9
7.33 (dd, J ¼ 9.6, 8.6 Hz, 3H), 4.36 (d, J ¼ 5.0 Hz, 2H), 2.71 (d,
J ¼ 4.6 Hz, 6H).
4-(4-Fluorobenzyloxy)-2-hydroxybenzaldehyde (12a)
White solid; yield: 77.8%; m.p.: 100–102°C; MS (ESI) m/z (%): 245.09
[MꢂH]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.49 (s, 1H), 9.75 (s, 1H), 7.47
(d, J ¼ 8.7 Hz, 1H), 7.42 (dd, J ¼ 8.7, 5.4 Hz, 2H), 7.14–7.07 (m, 2H),
6.62 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.52 (d, J ¼ 2.3 Hz, 1H), 5.09 (s, 2H).
Phenyl-6-((diethylamino)methyl)benzo[d]thiazol-2-
ylcarbamate (10b)
White solid; yield: 92.3%; m.p.: 207–209°C; MS (ESI) m/z (%): 356.16
[MþH]þ; 1H NMR (400 MHz, DMSO) d 10.41 (s, 1H), 8.22 (d,
J ¼ 1.3 Hz, 1H), 7.82 (d, J ¼ 8.3 Hz, 1H), 7.70 (dd, J ¼ 8.4, 1.7 Hz, 1H),
7.48 (dd, J ¼ 11.0, 4.8 Hz, 2H), 7.37–7.29 (m, 3H), 4.37 (d, J ¼ 5.1 Hz,
2H), 3.06 (dd, J ¼ 12.3, 7.7 Hz, 4H), 1.27 (t, J ¼ 7.2 Hz, 6H).
4-(3-Fluorobenzyloxy)-2-hydroxybenzaldehyde (12b)
Yellow solid; yield: 75.4%; m.p.: 71–73°C; MS (ESI) m/z (%): 245.09
[MꢂH]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.47 (s, 1H), 9.73 (s, 1H), 7.45
(d, J ¼ 8.7 Hz, 1H), 7.37 (td, J ¼ 7.9, 5.8 Hz, 1H), 7.18 (d, J ¼ 7.7 Hz,
1H), 7.14 (d, J ¼ 9.4 Hz, 1H), 7.07–7.01 (m, 1H), 6.61 (dd, J ¼ 8.7,
2.3 Hz, 1H), 6.49 (d, J ¼ 2.3 Hz, 1H), 5.11 (s, 2H).
Phenyl-6-((4-methylpiperidin-1-yl)methyl)benzo[d]thiazol-
2-ylcarbamate (10c)
4-(2-Fluorobenzyloxy)-2-hydroxybenzaldehyde (12c)
Yellow solid; yield: 85.7%; m.p.: 86–88°C; MS (ESI) m/z (%): 245.12
[MꢂH]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.48 (s, 1H), 9.73 (s, 1H), 7.46
(d, J ¼ 8.7 Hz, 1H), 7.42 (s, 1H), 7.32 (dt, J ¼ 10.3, 3.9 Hz, 3H), 6.61
(dd, J ¼ 8.7, 2.3 Hz, 1H), 6.49 (d, J ¼ 2.3 Hz, 1H), 5.09 (s, 2H).
White solid; yield: 97.6%; m.p.: 159–161°C; MS (ESI) m/z (%): 382.21
[MþH]þ; 1H NMR (400 MHz, DMSO) d 9.92 (s, 1H), 8.14 (s, 1H), 7.82
(d, J ¼ 8.3 Hz, 1H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.48 (t, J ¼ 7.7 Hz, 2H),
7.33 (t, J ¼ 9.4 Hz, 3H), 4.35 (d, J ¼ 4.2 Hz, 2H), 3.33 (d, J ¼ 11.7 Hz,
2H), 2.91 (dd, J ¼ 22.4, 10.9 Hz, 2H), 1.77 (d, J ¼ 13.8 Hz, 2H), 1.59 (s,
1H), 1.40 (dd, J ¼ 24.5, 11.2 Hz, 2H), 0.90 (d, J ¼ 6.3 Hz, 3H).
4-(4-(Trifluoromethyl)benzyloxy)-2-hydroxybenzaldehyde
(12d)
General procedure for preparation of compounds 11a–c
A mixture of the compounds 10a–c (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
temperature and concentrated, diethyl ether was added and
stirred for 0.5 h and filtered, a white solid to gray solid was
collected and washed with a small amount of water to get the
compounds 11a–c.
White solid; yield: 78.2%; m.p.: 99–101°C; MS (ESI) m/z (%): 295.15
[MꢂH]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.47 (s, 1H), 9.74 (s, 1H), 7.67
(d, J ¼ 8.1 Hz, 2H), 7.54 (d, J ¼ 8.1 Hz, 2H), 7.46 (d, J ¼ 8.7 Hz, 1H),
6.62 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.50 (d, J ¼ 2.3 Hz, 1H), 5.18 (s, 2H).
4-(2,3-Dichlorobenzyloxy)-2-hydroxybenzaldehyde (12e)
White solid; yield: 82.6%; m.p.: 150–152°C; MS (ESI) m/z (%): 295.09
[MꢂH]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.46 (s, 1H), 9.74 (s, 1H),
7.49–7.45 (m, 2H), 7.43 (d, J ¼ 7.6 Hz, 1H), 7.27 (s, 1H), 6.63 (dd,
J ¼ 8.6, 2.2 Hz, 1H), 6.51 (d, J ¼ 2.1 Hz, 1H), 5.22 (s, 2H).
N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-
hydrazinecarboxamide (11a)
White solid; yield: 82.6%; m.p.: 183–185°C; MS (ESI) m/z (%): 265.7
[MþH]þ. 1H NMR (400 MHz, DMSO) d 8.37 (s, 1H), 7.77 (s, 1H), 7.56
(d, J ¼ 8.1 Hz, 1H), 7.28 (d, J ¼ 8.2 Hz, 1H), 3.44 (s, 2H), 2.15 (s, 6H).
4-Benzyloxy-2-hydroxybenzaldehyde (12f)
White solid; yield: 77.8%; m.p.: 78–80°C; MS (ESI) m/z (%): 226.1 [Mꢂ
H]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.48 (s, 1H), 9.72 (s, 1H), 7.47–7.33 (m,
6H), 6.62 (dd, J¼ 8.7, 2.3 Hz, 1H), 6.51 (d, J¼ 2.3 Hz, 1H), 5.11 (s, 2H).
N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-
hydrazinecarboxamide (11b)
4-(2,4-Dichlorobenzyloxy)-2-hydroxybenzaldehyde (12g)
White solid; yield: 82.6%; m.p.: 138–140°C; MS (ESI) m/z (%): 295.09
[MꢂH]ꢂ; 1H NMR (400 MHz, CDCl3) d 11.47 (s, 1H), 9.74 (s, 1H),
7.49–7.43 (m, 3H), 7.29 (dd, J ¼ 8.3, 2.0 Hz, 1H), 6.62 (dd, J ¼ 8.7,
2.3 Hz, 1H), 6.51 (d, J ¼ 2.3 Hz, 1H), 5.17 (s, 2H).
White solid; yield: 86.3%; m.p.: 128–130°C; MS (ESI) m/z (%): 607.9
[2MþH]þ. 1H NMR (400 MHz, DMSO) d 9.34 (s, 1H), 8.49 (s, 1H), 8.03
(s, 1H), 7.70 (d, J ¼ 8.0 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 4.31 (s, 2H),
3.04 (s, 4H), 1.22 (s, 6H).
N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-yl)-
hydrazinecarboxamide (11c)
Preparation of 2-allyloxybenzaldehyde (13)
To the mixture of 2-hydroxybenzaldehyde (29 g, 0.238 mol) and
potassium carbonate (49.3g, 0.357mol) in 200mL DMF, allyl
bromide (34.6g, 0.286mol) was added drop wise. Then the mixture
was heated to 60°C for 15 h, and cooled to room temperature. One
liter of water was added and extracted two times with DCM. The
organic extracts were combined and washed two times by 10%
lithium chloride aqueous solution, and washed by 5% sodium
hydroxide solution and saturated sodium chloride aqueous
solution, dried over sodium sulfate, filtered, and concentrated to
obtain the compound 13 as a light yellow oil (31.7 g, 82.3%), boiling
point (b.p.): 273°C, MS (ESI) m/z (%): 163.2 [MþH]þ.
White solid; yield: 76.8%; m.p.: 142–144°C; MS (ESI) m/z (%): 319.6
[MþH]þ. 1H NMR (400 MHz, DMSO) d 9.33 (s, 1H), 7.79 (s, 1H), 7.57
(d, J ¼ 8.0 Hz, 1H), 7.31 (d, J ¼ 7.4 Hz, 1H), 7.16 (t, J ¼ 7.7 Hz, 1H),
6.75 (d, J ¼ 8.2 Hz, 2H), 3.54 (s, 2H), 2.81 (s, 2H), 1.94 (s, 2H), 1.58 (d,
J ¼ 11.3 Hz, 2H), 1.35 (s, 1H), 1.15 (d, J ¼ 9.6 Hz, 2H), 0.88 (d,
J ¼ 6.4 Hz, 3H).
General procedure for preparation of compounds 12a–g
A mixture of 2,4-dihydroxybenzaldehyde (1.38 g, 0.01 mol), substi-
tuted benzyl chloride (0.011mol), NaHCO3 (1.26 g, 0.015mol), and
catalytic amount of potassium iodide in 20mL acetonitrile was
heated to 60°C for 36h and hot filtered, the filtrate was cooled to
10°C and filtered, the filtrated cake was collected, and recrystallized
with EtOH to obtain compounds 12a–e.
Preparation of 3-allyl-2-hydroxybenzaldehyde (14)
The compound 13 (31.7 g, 0.196 mol) was heated to 200°C for 4 h,
and distilled under reduced pressure to get compound 14 as a
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
light yellow liquid (10.2 g, 72.8%), b.p.: 243°C, MS (ESI) m/z (%):
(101 MHz, DMSO) d 172.70, 161.73, 154.42, 153.62, 148.78, 140.89,
136.04, 132.04, 131.10, 127.93, 126.00, 125.55, 122.72, 118.58,
117.87, 62.77, 44.53 (2C), 35.12, 34.32, 31.75 (3C), 29.83 (3C). Anal.
calcd. for C26H35N5O2S (%): C, 64.83; H, 7.32; N, 14.54; Found (%):
C, 64.91; H, 7.25; N, 14.45.
1
161.1 [MꢂH]ꢂ; H NMR (400 MHz, CDCl3) d 11.32–11.28 (m, 1H),
9.88 (s, 1H), 7.46–7.38 (m, 2H), 6.97 (t, J ¼ 7.6 Hz, 1H), 6.05–5.93 (m,
1H), 5.13–5.09 (m, 1H), 5.09–5.06 (m, 1H), 3.43 (d, J ¼ 6.7 Hz, 2H).
Preparation of 7-hydroxy-4-methyl-2H-chromen-2-one (15)
Sulfuric acid (500 mL) was cooled below 10°C in an ice-salt bath,
and to the cooled acid was slowly added a solution of benzene-1,3-
diol (55 g, 0.500 mol) in ethyl acetoacetate (65 g, 0.500 mol). The
reaction mixture was stirred for 4 h. The resulting mixture was
poured onto cracked ice and stirred. The precipitates were filtered
off, washed with water and dried to afford compound 15 (67.0 g,
76.1%) as white solid. MS (ESI) m/z (%): 175.1 [MꢂH]ꢂ.
(E)-N-(6-(Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(3-
allyl-2-hydroxybenzylidene)hydrazinecarboxamide (17c)
Yield: 51%; m.p.: 183–185°C; MS (ESI) m/z (%): 436.4 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.79 (s, 1H), 11.36 (s, 1H), 8.32 (s, 1H),
8.13 (s, 1H), 7.74 (s, 1H), 7.62 (d, J ¼ 7.7 Hz, 1H), 7.38 (s, 1H), 7.17 (d,
J ¼ 7.3 Hz, 1H), 6.89 (t, J ¼ 7.5 Hz, 1H), 6.00 (m, 1H), 5.13–5.01 (m,
2H), 4.37 (s, 2H), 3.40 (d, J ¼ 6.5 Hz, 2H), 3.07 (s, 4H), 1.26 (t,
J ¼ 7.2 Hz, 6H). Anal. calcd. for C23H27N5O2S (%): C, 63.13; H, 6.22;
N, 16.01; Found (%): C, 63.23; H, 6.15; N, 16.09.
Preparation of 7-hydroxy-4-methyl-2-oxo-2H-chromene-6-
carbaldehyde (16)
(E)-N-(6-(Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(3,5-di-tert-butyl-2-hydroxybenzylidene)-
A suspension of urotropine (20 g, 0.143 mol) in glacial acetic acid
(80 mL) was heated to 40°C and stirred to form a clear solution. To
this solution compound 15 (5 g, 0.028 mol) was added in portions.
The reaction mixture was stirred for 20min and then heated to
115°C and stirred for another 2h. Finally, the reaction mixture was
cooled to 95°C, 30% sulfuric acid (15mL) was added and the
reaction continued for 1.5h. The resulting mixture was evaporated
to dryness and the dry residue obtained was extracted with ether.
The organic layer was then washed with saturated sodium
bicarbonate solution, dried over anhydrous sodium sulfate, and
evaporated to dryness to afford 16 (2.2g, 38.0%) as white solid. m.p.:
165–167°C; MS (ESI) m/z (%): 203.0 [MꢂH]ꢂ; 1H NMR (400MHz,
CDCl3) d 12.22 (s, 1H), 10.62 (s, 1H), 7.73 (d, J ¼ 9.0Hz, 1H), 6.91 (d,
J ¼ 9.0Hz, 1H), 6.21 (d, J ¼ 1.0Hz, 1H), 2.43 (d, J ¼ 1.0 Hz, 3H).
hydrazinecarboxamide (17d)
Yield: 66%; m.p.: 156–158°C; MS (ESI) m/z (%): 508.2 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.64 (s, 2H), 8.32 (s, 1H), 7.87 (s, 1H), 7.59
(d, J ¼ 7.9 Hz, 1H), 7.40 (d, J ¼ 7.6 Hz, 1H), 7.28 (s, 1H), 7.22 (s, 1H),
3.75 (s, 2H), 2.59 (s, 4H), 1.42 (s, 9H), 1.28 (s, 9H), 1.09–1.00 (m, 6H).
Anal. calcd. for C28H39N5O2S (%): C, 65.98; H, 7.71; N, 13.74; Found
(%): C, 65.90; H, 7.68; N, 13.81.
(E)-N-(6-(4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-
yl)-2-(3-allyl-2-hydroxybenzylidene)-
hydrazinecarboxamide (17e)
Yield: 54%; m.p.: 186–188°C; MS (ESI) m/z (%): 462.3 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3419.7, 3200.2, 2924.4, 2759.6, 2049.1, 1709.5,
1609.9, 1569.3, 1453.2, 1362.7, 1266.5, 1239.9, 1147.9, 1073.9,
912.4, 841.1, 748.8, 648.9; 1H NMR (400 MHz, DMSO) d 11.33 (s,
2H), 8.31 (s, 1H), 7.91 (s, 1H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.43 (d,
J ¼ 8.0 Hz, 1H), 7.33 (s, 1H), 7.16 (d, J ¼ 7.0 Hz, 1H), 6.87 (t,
J ¼ 7.5 Hz, 1H), 6.00 (m, 1H), 5.06 (t, J ¼ 13.1 Hz, 2H), 3.90 (s, 2H),
3.40 (d, J ¼ 6.5 Hz, 2H), 3.03 (s, 2H), 2.41 (s, 2H), 1.66 (d, J ¼ 12.6 Hz,
2H), 1.46 (s, 1H), 1.26 (d, J ¼ 10.9 Hz, 2H), 0.90 (d, J ¼ 6.4 Hz, 3H).
Anal. calcd. for C25H29N5O2S (%): C, 64.77; H, 6.31; N, 15.11; Found
(%): C, 64.70; H, 6.28; N, 15.19.
General procedure for preparation of compounds 17a–f,
18a–q, and 19a–c
The compounds 11a–c (0.001 mol), appropriate 2-hydroxy aro-
matic aldehydes (0.0011 mol) and a drop of glacial acetic acid
were added in 10 mL ethanol. The reaction mixture was heated at
reflux for 6 h and cooled to room temperature, separated by
filtration, and washed with diethyl ether to afford a white to light
yellow solid. The crude product was purified by chromatography
on silica gel using MeOH/CH2Cl2 to afford the compounds 17a–f,
18a–q, and 19a–c.
(E)-N-(6-(4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-2-
yl)-2-(3,5-di-tert-butyl-2-hydroxybenzylidene)-
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(3-allyl-2-hydroxybenzylidene)hydrazinecarboxamide (17a)
Yield: 57%; m.p.: 178–180°C; MS (ESI) m/z (%): 408.1 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.45 (s, 2H), 8.29 (s, 1H), 7.80 (s, 1H), 7.56
(d, J ¼ 7.9 Hz, 1H), 7.33 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 6.3 Hz, 1H),
6.88 (t, J ¼ 7.5 Hz, 1H), 6.00 (m, 1H), 5.07 (dd, J ¼ 22.4, 5.4 Hz, 2H),
3.49 (s, 2H), 3.40 (d, J ¼ 6.5 Hz, 2H), 2.18 (s, 6H). Anal. calcd. for
C21H23N5O2S (%): C, 61.59; H, 5.66; N, 17.10; Found (%): C, 61.65; H,
5.68; N, 17.17.
hydrazinecarboxamide (17f)
Yield: 74%; m.p.: 152–154°C; MS (ESI) m/z (%): 534.5 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.28 (s, 2H), 8.32 (s, 1H), 7.84 (s, 1H), 7.58
(s, 1H), 7.37 (s, 1H), 7.29 (s, 1H), 7.20 (s, 1H), 3.66 (s, 2H), 2.89 (s, 2H),
2.10 (s, 2H), 1.59 (s, 2H), 1.35 (d, J ¼ 58.6 Hz, 21H), 0.89 (s, 3H). Anal.
calcd. for C30H41N5O2S (%): C, 67.26; H, 7.71; N, 13.07; Found (%):
C, 67.35; H, 7.66; N, 13.15.
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(4-benzyloxy-2-hydroxybenzylidene)-
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(3,5-di-tert-butyl-2-hydroxybenzylidene)-
hydrazinecarboxamide (18a)
hydrazinecarboxamide (17b)
Yield: 68%; m.p.: 186–188°C; MS (ESI) m/z (%): 474.4 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.15 (s, 2H), 8.24 (s, 1H), 7.97 (s, 1H), 7.80
(s, 1H), 7.60 (d, J ¼ 7.9 Hz, 1H), 7.46 (d, J ¼ 7.2 Hz, 2H), 7.41 (t,
J ¼ 7.3 Hz, 2H), 7.34 (dd, J ¼ 12.2, 7.6 Hz, 2H), 6.57 (d, J ¼ 8.7 Hz,
Yield: 67%; m.p.: 150–152°C; MS (ESI) m/z (%): 480.2 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.23 (s, 2H), 8.31 (s, 1H), 7.83 (s, 1H), 7.57
(d, J ¼ 8.1 Hz, 1H), 7.35 (d, J ¼ 8.2 Hz, 1H), 7.29 (s, 1H), 7.20 (s, 1H),
3.59 (s, 2H), 2.25 (s, 6H), 1.42 (s, 9H), 1.28 (s, 9H); 13C NMR
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Benzothiazoles with ortho-Hydroxy-N-acylhydrazone Moiety
11
1H), 6.53 (s, 1H), 5.11 (s, 2H), 3.47 (s, 2H), 2.17 (s, 6H). Anal. calcd.
for C25H25N5O3S (%): C, 63.14; H, 5.30; N, 14.73; Found (%): C,
63.19; H, 5.35; N, 14.81.
2H), 2.17 (s, 6H); 13C NMR (101 MHz, DMSO) d 173.08, 162.55,
161.09, 160.94, 158.41, 153.35, 142.23, 134.16, 131.83, 129.96,
128.99, 128.45 (2C), 127.39, 2C (125.86, 125.82, 125.78, 125.75),
123.35, 121.88, 119.32, 113.85, 107.34, 102.43, 68.82, 63.66, 45.29
(2C). Anal. calcd. for C26H24F3N5O3S (%): C, 57.45; H, 4.45; N, 12.88.
Found (%): C, 57.40; H, 4.51; N, 12.82.
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(4-(4-fluorobenzyloxy)-2-hydroxybenzylidene)-
hydrazinecarboxamide (18b)
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(2-hydroxy-4-(4-(2,4-dichlorobenzyloxy)benzyloxy)-
benzylidene)hydrazinecarboxamide (18f)
Yield: 64%; m.p.: 186–188°C; MS (ESI) m/z (%): 492.1 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.14 (s, 2H), 8.24 (s, 1H), 7.95 (s, 1H), 7.81
(s, 1H), 7.60 (d, J ¼ 8.1 Hz, 1H), 7.51 (dd, J ¼ 8.1, 5.8 Hz, 2H), 7.33 (d,
J ¼ 8.2 Hz, 1H), 7.24 (t, J ¼ 8.8 Hz, 2H), 6.56 (dd, J ¼ 8.7, 2.0 Hz, 1H),
6.52 (s, 1H), 5.09 (s, 2H), 3.49 (s, 2H), 2.18 (s, 6H). Anal. calcd. for
C25H24FN5O3S (%): C, 60.84; H, 4.90; N, 14.19. Found (%): C, 60.91;
H, 4.97; N, 14.09.
Yield: 73%; m.p.: 194–196°C; MS (ESI) m/z (%): 542.1 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3369.6, 3172.8, 3076.7, 2936.3, 2855.2, 2762.3,
1691.6, 1677.9, 1628.1, 1608.6, 1538.1, 1502.4, 1465.7, 1360.9,
1325.0, 1292.5, 1278.5, 1181.1, 1128.3, 1115.6, 1043.7, 845.7,
812.7, 731.2, 694.7; 1H NMR (400 MHz, DMSO) d 11.13 (s, 2H), 8.24
(s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.72 (d, J ¼ 2.0 Hz, 1H), 7.61 (t,
J ¼ 8.6 Hz, 2H), 7.50 (dd, J ¼ 8.3, 2.1 Hz, 1H), 7.33 (d, J ¼ 8.1 Hz, 1H),
6.58 (dd, J ¼ 8.7, 2.2 Hz, 1H), 6.53 (s, 1H), 5.16 (s, 2H), 3.47 (s, 2H),
2.17 (s, 6H); 13C NMR (101 MHz, DMSO) d 160.84, 158.41, 158.00,
153.33, 153.06, 134.22, 134.03, 133.98, 133.80, 132.07, 131.95,
131.73, 129.94, 129.39, 128.05, 127.38, 121.87, 119.28, 114.00,
107.29, 102.29, 66.76, 63.68, 45.32 (2C). Anal. calcd. for
C25H23Cl2N5O3S (%): C, 55.15; H, 4.26; N, 12.86. Found (%): C,
55.17; H, 4.20; N, 12.91.
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(4-(3-fluorobenzyloxy)-2-hydroxybenzylidene)-
hydrazinecarboxamide (18c)
Yield: 64%; m.p.: 188–190°C; MS (ESI) m/z (%): 492.1 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3424.9, 3173.3, 3078.3, 2937.8, 2814.9, 2764.3,
1691.3, 1678.9, 1628.7, 1609.5, 1571.6, 1556.1, 1537.6, 1457.5,
1359.3, 1322.3, 1220.4, 1176.8, 1132.4, 1116.0, 1019.3, 953.2,
814.4, 783.7; 1H NMR (400 MHz, DMSO) d 11.15 (s, 2H), 8.24 (s, 1H),
7.96 (s, 1H), 7.80 (s, 1H), 7.60 (d, J ¼ 8.1 Hz, 1H), 7.50–7.42 (m, 1H),
7.35–7.26 (m, 3H), 7.22–7.14 (m, 1H), 6.57 (dd, J ¼ 8.7, 2.2 Hz, 1H),
6.53 (s, 1H), 5.14 (s, 2H), 3.47 (s, 2H), 2.17 (s, 6H). Anal. calcd. for
C25H24FN5O3S (%): C, 60.84; H, 4.90; N, 14.19. Found (%): C, 60.80;
H, 4.81; N, 14.11.
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(2-hydroxy-4-(4-(2,3-dichlorobenzyloxy))benzylidene)-
hydrazinecarboxamide (18g)
Yield: 71%; m.p.: 187–189°C; MS (ESI) m/z (%): 542.2 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.16 (s, 2H), 8.25 (s, 1H), 8.01 (s, 1H), 7.81
(s, 1H), 7.67 (d, J ¼ 7.8 Hz, 1H), 7.63–7.55 (m, 2H), 7.43 (t, J ¼ 7.5 Hz,
1H), 7.32 (d, J ¼ 8.1 Hz, 1H), 6.59 (d, J ¼ 8.6 Hz, 1H), 6.53 (s, 1H),
5.22 (s, 2H), 3.47 (s, 2H), 2.17 (s, 6H). Anal. calcd. for
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(4-(2-fluorobenzyloxy)-2-hyroxybenzylidene)-
hydrazinecarboxamide (18d)
Yield: 57%; m.p.: 190–192°C; MS (ESI) m/z (%): 492.2 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 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, 1384.2, 1360.5, 1318.4, 1280.3, 1115.8, 1037.9,
1021.5, 845.7, 835.6, 814.3, 804.1, 752.5; 1H NMR (400 MHz,
DMSO) d 11.10 (s, 2H), 8.88 (s, 1H), 8.25 (s, 1H), 7.94 (s, 1H), 7.81 (s,
1H), 7.64–7.53 (m, 2H), 7.48–7.40 (m, 1H), 7.33 (d, J ¼ 8.3 Hz, 1H),
7.26 (dd, J ¼ 15.1, 8.0 Hz, 2H), 6.62–6.51 (m, 2H), 5.15 (s, 2H), 3.51
(s, 2H), 2.19 (s, 6H); 13C NMR (400 MHz, DMSO) d 162.67, 162.49, 1C
(162.08, 158.48), 161.06, 160.33, 159.63, 153.24, 133.52, 131.74, 1C
(131.19, 131.16), 1C (130.98, 130.90), 129.83, 127.56, 1C (125.02,
124.99), 1C (124.08, 123.93), 122.08, 119.45, 1C (115.96, 115.75),
113.71, 107.21, 102.30, 1C (64.07, 64.04), 63.39, 45.03 (2C). Anal.
calcd. for C25H24FN5O3S (%): C, 60.84; H, 4.90; N, 14.19. Found (%):
C, 60.78; H, 4.95; N, 14.22.
C
25H23Cl2N5O3S (%): C, 55.15; H, 4.26; N, 12.86. Found (%): C,
55.25; H, 4.32; N, 12.81.
(E)-N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-
(4-fluorobenzyloxy)-2-hydroxybenzylidene)-
hydrazinecarboxamide (18h)
Yield: 56%; m.p.: 228–230°C MS (ESI) m/z (%): 520.1 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.16 (s, 2H), 8.25 (s, 1H), 7.94 (s, 1H), 7.83
(s, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.54–7.48 (m, 2H), 7.36 (d, J ¼ 8.1 Hz,
1H), 7.24 (t, J ¼ 8.6 Hz, 2H), 6.56 (d, J ¼ 8.8 Hz, 1H), 6.53 (s, 1H), 5.10
(s, 2H), 3.66 (s, 2H), 2.53 (s, 4H), 1.01 (t, J ¼ 6.8 Hz, 6H). Anal. calcd.
for C27H28FN5O3S (%): C, 62.17; H, 5.41; N, 13.43. Found (%): C,
62.20; H, 5.45; N, 13.50.
(E)-N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-
(3-fluorobenzyloxy)-2-hydroxybenzylidene)-
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
(2-hydroxy-4-(4-(trifluoromethyl)benzyloxy)benzylidene)-
hydrazinecarboxamide (18i)
Yield: 69%; m.p.: 229–231°C; MS (ESI) m/z (%): 520.3 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.13 (s, 2H), 8.25 (s, 1H), 8.02 (s, 1H), 7.84
(s, 1H), 7.60 (d, J ¼ 6.9 Hz, 1H), 7.46 (d, J ¼ 7.2 Hz, 1H), 7.37 (d,
J ¼ 8.1 Hz, 1H), 7.30 (d, J ¼ 6.7 Hz, 2H), 7.18 (t, J ¼ 8.4 Hz, 1H), 6.57
(d, J ¼ 8.8 Hz, 1H), 6.53 (s, 1H), 5.14 (s, 2H), 3.68 (s, 2H), 2.54 (d,
J ¼ 6.1 Hz, 4H), 1.02 (t, J ¼ 6.6 Hz, 6H). Anal. calcd. for
C27H28FN5O3S (%): C, 62.17; H, 5.41; N, 13.43. Found (%): C,
62.19; H, 5.35; N, 13.48.
hydrazinecarboxamide (18e)
Yield: 63%; m.p.: 189–191°C; MS (ESI) m/z (%): 542.3 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3421.1, 3200.6, 2919.4, 2700.0, 1695.5, 1633.2,
1611.0, 1573.7, 1510.5, 1456.0, 1384.0, 1357.9, 1259.8, 1170.5,
1127.2, 1066.9, 1018.7, 944.9, 841.7, 822.6, 810.7, 747.0, 701.1; 1H
NMR (400 MHz, DMSO) d 11.12 (s, 2H), 8.24 (s, 1H), 7.93 (s, 1H),
7.83–7.74 (m, 3H), 7.68 (d, J ¼ 8.1 Hz, 2H), 7.60 (d, J ¼ 8.4 Hz, 1H),
7.32 (dd, J ¼ 8.3, 1.3 Hz, 1H), 6.62–6.50 (m, 2H), 5.24 (s, 2H), 3.48 (s,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

12
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
(E)-N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(4-
(E)-N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-
(2-fluorobenzyloxy)-2-hydroxybenzylidene)-
2-yl)-2-(4-(2-fluorobenzyloxy)-2-hydroxybenzylidene)-
hydrazinecarboxamide (18j)
hydrazinecarboxamide (18o)
Yield: 65%; m.p.: 233–235°C; MS (ESI) m/z (%): 520.3 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.19 (s, 2H), 8.26 (s, 1H), 8.04 (s, 1H),
7.99–7.76 (m, 1H), 7.69 (d, J ¼ 7.9 Hz, 1H), 7.57 (d, J ¼ 8.7 Hz, 1H),
7.49–7.41 (m, 1H), 7.33–7.27 (m, 2H), 7.22–7.13 (m, 1H), 6.61–6.52
(m, 2H), 5.15 (s, 2H), 4.15 (s, 2H), 2.90 (s, 4H), 1.18 (s, 6H). Anal.
calcd. for C27H28FN5O3S (%): C, 62.17; H, 5.41; N, 13.43. Found (%):
C, 62.09; H, 5.45; N, 13.38.
Yield: 67%; m.p.: 218–220°C; MS (ESI) m/z (%): 546.3 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.09 (s, 2H), 8.25 (s, 1H), 7.92 (s, 1H), 7.81
(s, 1H), 7.63–7.56 (m, 1H), 7.45 (dd, J ¼ 14.1, 7.9 Hz, 1H), 7.33 (d,
J ¼ 8.5 Hz, 1H), 7.31–7.26 (m, 2H), 7.18 (s, 1H), 6.57 (dd, J ¼ 8.7,
2.2 Hz, 1H), 6.53 (s, 1H), 5.14 (s, 2H), 3.58 (s, 2H), 2.83 (d, J ¼ 10.7 Hz,
2H), 2.01 (s, 2H), 1.58 (d, J ¼ 11.4 Hz, 2H), 1.34 (s, 1H), 1.15 (dd,
J ¼ 21.2, 11.6 Hz, 2H), 0.89 (d, J ¼ 6.4 Hz, 3H). Anal. calcd. for
C29H30FN5O3S (%): C, 63.60; H, 5.52; N, 12.79; Found (%): C, 63.55;
H, 5.58; N, 12.71.
(E)-N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-
hydroxy-4-(4-(trifluoromethyl)benzyloxy)benzylidene)-
(E)-N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-
2-yl)-2-(2-hydroxy-4-(4-(trifluoromethyl)benzyloxy)-
benzylidene)hydrazinecarboxamide (18p)
hydrazinecarboxamide (18k)
Yield: 68%; m.p.: 238–240°C; MS (ESI) m/z (%): 570.1 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.17 (s, 2H), 8.26 (s, 1H), 8.01 (s, 2H), 7.78
(d, J ¼ 8.2 Hz, 2H), 7.68 (d, J ¼ 8.1 Hz, 3H), 7.52 (s, 1H), 6.61–6.54 (m,
2H), 5.25 (s, 2H), 4.11 (s, 2H), 2.84 (s, 4H), 1.15 (s, 6H). Anal. calcd.
for C28H28F3N5O3S (%): C, 58.83; H, 4.94; N, 12.25. Found (%): C,
58.90; H, 4.88; N, 12.31.
Yield: 62%; m.p.: 210–212°C; MS (ESI) m/z (%): 596.2 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.61 (s, 1H), 11.23 (s, 1H), 8.37 (s, 1H),
7.99 (s, 1H), 7.96–7.86 (m, 4H), 7.80 (d, J ¼ 8.1 Hz, 3H), 7.71 (d,
J ¼ 10.4 Hz, 1H), 7.45 (d, J ¼ 8.3 Hz, 1H), 6.70 (dd, J ¼ 8.7, 2.2 Hz,
1H), 6.66 (s, 1H), 5.37 (s, 2H), 3.68 (s, 2H), 2.94 (d, J ¼ 11.0 Hz, 2H),
2.11 (t, J ¼ 10.6 Hz, 2H), 1.69 (d, J ¼ 12.0 Hz, 2H), 1.46 (s, 1H), 1.27
(dd, J ¼ 21.1, 11.5 Hz, 2H), 1.01 (d, J ¼ 6.4 Hz, 3H). Anal. calcd. for
C30H30F3N5O3S (%): C, 60.29; H, 5.06; N, 11.72; Found (%): C, 60.35;
H, 5.11; N, 11.79.
(E)-N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-(2-
hydroxy-4-(4-(2,3-dichlorobenzyloxy))benzylidene)-
hydrazinecarboxamide (18l)
Yield: 63%; m.p.: 246–248°C; MS (ESI) m/z (%): 570.1 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.16 (s, 2H), 8.26 (s, 1H), 8.02 (s, 1H), 7.84
(s, 1H), 7.73–7.52 (m, 4H), 7.43 (t, J ¼ 7.6 Hz, 1H), 7.37 (d, J ¼ 7.9 Hz,
1H), 6.59 (d, J ¼ 8.7 Hz, 1H), 6.54 (s, 1H), 5.22 (s, 2H), 3.67 (s, 2H),
2.54 (s, 4H), 1.01 (t, J ¼ 6.5 Hz, 6H). Anal. calcd. for C27H27Cl2N5O3S
(%): C, 56.64; H, 4.75; N, 12.23; Found (%): C, 56.71; H, 4.82; N,
12.29.
(E)-N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-
2-yl)-2-(2-hydroxy-4-(4-(2,3-dichlorobenzyloxy))-
benzylidene)hydrazinecarboxamide (18q)
Yield: 68%; m.p.: 197–199°C; MS (ESI) m/z (%): 598.1 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3392.3, 3192.5, 3077.5, 2925.3, 2865.8, 1692.4,
1628.7, 1607.2, 1570.3, 1551.5, 1503.1, 1466.5, 1445.5, 1429.0,
1293.0, 1183.2, 1169.4, 1133.1, 1063.1, 1043.3, 852.5, 790.9, 775.2,
698.3; 1H NMR (400 MHz, DMSO) d 11.12 (s, 2H), 8.25 (s, 1H), 8.01 (s,
1H), 7.80 (s, 1H), 7.67 (d, J ¼ 7.6 Hz, 1H), 7.63–7.54 (m, 2H), 7.43 (t,
J ¼ 7.3 Hz, 1H), 7.33 (d, J ¼ 8.1 Hz, 1H), 6.59 (d, J ¼ 8.6 Hz, 1H), 6.53
(s, 1H), 5.22 (s, 2H), 3.53 (s, 2H), 2.80 (d, J ¼ 9.8 Hz, 2H), 1.94 (s, 2H),
1.56 (d, J ¼ 11.9 Hz, 2H), 1.33 (s, 1H), 1.21–1.07 (m, 2H), 0.88 (d,
J ¼ 6.0 Hz, 3H). Anal. calcd. for C29H29Cl2N5O3S (%): C, 58.19; H,
4.88; N, 11.70; Found (%): C, 58.26; H, 4.79; N, 11.78.
(E)-N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-
2-yl)-2-(4-(4-fluorobezyloxy)-2-hydroxybenzylidene)-
hydrazinecarboxamide (18m)
Yield: 69%; m.p.: 195–197°C; MS (ESI) m/z (%): 546.2 [MꢂH]ꢂ; H
1
NMR (400 MHz, DMSO) d 11.12 (s, 2H), 8.24 (s, 1H), 7.82 (s, 1H),
7.79 (s, 1H), 7.62–7.56 (m, 1H), 7.53–7.49 (m, 2H), 7.32 (d,
J ¼ 8.2 Hz, 1H), 7.24 (t, J ¼ 8.7 Hz, 2H), 6.56 (d, J ¼ 8.7 Hz, 1H), 6.52
(s, 1H), 5.09 (s, 2H), 3.51 (s, 2H), 2.79 (d, J ¼ 11.0 Hz, 2H), 1.92 (t,
J ¼ 11.2 Hz, 2H), 1.56 (d, J ¼ 12.3 Hz, 2H), 1.32 (s, 1H), 1.13 (dd,
J ¼ 21.3, 11.5 Hz, 2H), 0.88 (d, J ¼ 6.4 Hz, 3H). Anal. calcd. for
C29H30FN5O3S (%): C, 63.60; H, 5.52; N, 12.79; Found (%): C, 63.65;
H, 5.50; N, 12.70.
(E)-N-(6-((Dimethylamino)methyl)benzo[d]thiazol-2-yl)-2-
((7-hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)methylene)-
hydrazinecarboxamide (19a)
Yield: 80%; m.p.: 219–221°C; MS (ESI) m/z (%): 450.1 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.57 (s, 2H), 8.82 (s, 1H), 7.78 (s, 1H), 7.69
(d, J ¼ 8.9 Hz, 1H), 7.51 (d, J ¼ 8.2 Hz, 1H), 7.32 (d, J ¼ 8.3 Hz, 1H),
6.97 (d, J ¼ 8.8 Hz, 1H), 6.25 (s, 1H), 3.50 (s, 2H), 2.41 (s, 3H), 2.19 (s,
6H). Anal. calcd. for C22H21N5O4S (%): C, 58.52; H, 4.69; N, 15.51;
Found (%): C, 58.61; H, 4.72; N, 15.58.
(E)-N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-
2-yl)-2-(4-(3-fluorobenzyloxy)-2-hydroxybenzylidene)-
hydrazinecarboxamide (18n)
Yield: 68%; m.p.: 209–211°C; MS (ESI) m/z (%): 546.2 [MꢂH]ꢂ; 1H
NMR (400 MHz, DMSO) d 11.10 (s, 2H), 8.24 (s, 1H), 7.97 (s, 1H), 7.81
(s, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.46 (d, 1H), 7.34 (d, J ¼ 8.5 Hz, 1H),
7.32–7.29 (m, 2H), 7.18 (t, J ¼ 8.6 Hz, 1H), 6.57 (dd, J ¼ 8.7, 2.0 Hz,
1H), 6.53 (s, 1H), 5.14 (s, 2H), 3.58 (s, 2H), 2.83 (d, J ¼ 8.7 Hz, 2H),
2.01 (s, 2H), 1.58 (d, J ¼ 11.8 Hz, 2H), 1.35 (s, 1H), 1.15 (dd, J ¼ 21.6,
11.4 Hz, 2H), 0.89 (d, J ¼ 6.4 Hz, 3H). Anal. calcd. for C29H30FN5O3S
(%): C, 63.60; H, 5.52; N, 12.79; Found (%): C, 63.65; H, 5.50; N,
12.88.
(E)-N-(6-((Diethylamino)methyl)benzo[d]thiazol-2-yl)-2-
((7-hydroxy-4-methyl-2-oxo-2-H-chromen-6-yl)methylene)-
hydrazinecarboxamide (19b)
Yield: 75%; m.p.: 230–232°C; MS (ESI) m/z (%): 478.2 [MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3429.1, 3199.6, 2981.3, 2696.1, 2068.8, 1712.1,
1614.5, 1587.7, 1543.4, 1466.6, 1385.2, 1370.1, 1294.6, 1188.3,
1066.7, 1054.5, 946.4, 842.0, 806.3, 712.9, 684.0, 623.0, 597.4; 1H
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
Benzothiazoles with ortho-Hydroxy-N-acylhydrazone Moiety
13
NMR (400 MHz, DMSO) d 11.50 (s, 2H), 8.84 (s, 1H), 8.03 (s, 1H), 7.70
(d, J ¼ 8.9 Hz, 1H), 7.66 (d, J ¼ 7.3 Hz, 1H), 7.53 (d, J ¼ 8.2 Hz, 1H),
6.97 (d, J ¼ 8.9 Hz, 1H), 6.25 (s, 1H), 4.31 (s, 2H), 3.05 (d, J ¼ 6.7 Hz,
4H), 2.41 (s, 3H), 1.23 (t, J ¼ 7.1 Hz, 6H). Anal. calcd. for
C24H25N5O4S (%): C, 60.11; H, 5.25; N, 14.60; Found (%): C,
60.18; H, 5.18; N, 14.55.
MTT formazan) and 630 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 inhibitory
concentration 50% (IC50) were the averages of three determi-
nations and calculated by using the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software.
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
(IRT1073).
(E)-N-(6-((4-Methylpiperidin-1-yl)methyl)benzo[d]thiazol-
2-yl)-2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-6-yl)-
methylene)hydrazinecarboxamide (19c)
Yield: 77%; m.p.: 217–219°C; MS (ESI) m/z (%): 504.1[MꢂH]ꢂ; IR
(KBr, cmꢂ1): 3322.7, 2920.8, 2792.2, 1724.3, 1676.9, 1611.1,
1590.1, 1544.6, 1384.9, 1295.7, 1268.3, 1211.5, 1172.4, 1063.8,
945.1, 845.4, 811.2, 773.2; 1H NMR (400 MHz, DMSO) d 11.57 (s,
2H), 8.81 (s, 1H), 7.78 (s, 1H), 7.69 (d, J ¼ 8.9 Hz, 1H), 7.51 (d,
J ¼ 6.7 Hz, 1H), 7.33 (d, J ¼ 8.1 Hz, 1H), 6.97 (d, J ¼ 8.7 Hz, 1H), 6.24
(s, 1H), 3.59 (s, 2H), 2.84 (d, J ¼ 10.1 Hz, 2H), 2.40 (s, 3H), 2.03 (s, 2H),
1.58 (d, J ¼ 11.9 Hz, 2H), 1.35 (s, 1H), 1.23–1.09 (m, 2H), 0.88 (d,
J ¼ 6.1 Hz, 3H). Anal. calcd. for C26H27N5O4S (%): C, 61.77; H, 5.38;
N, 13.85; Found (%): C, 61.85; H, 5.43; N, 13.89.
The authors have declared no conflict of interest.
References
[1] K. S. Putt, G. W. Chen, J. M. Pearson, J. S. Sandhorst,
M. S. Hoagland, J. T. Kwon, S. K. Hwang, H. Jin,
M. I. Churchwell, M.-H. Cho, Nat. Chem. Biol. 2006, 2, 543–550.
[2] J. Procházka, X. Liu, P. Fiala, Z. Kinkor, Biol. Chem. 2004, 385,
153–168.
Pharmacology
Procaspase-3 kinase assay
[3] D. Fink, H. Schlagbauer-Wadl, E. Selzer, T. Lucas, K. Wolff,
H. Pehamberger, H. G. Eichler, B. Jansen, Melanoma Res. 2001,
11, 385–393.
The procaspase-3 activation activity was evaluated as previously
reported method [1]. (a) One hundred micromolar of target
compounds and PAC-1 were dissolved in DMSO and the resulted
solutions were stored at ꢂ20°C as mother liquor, which were
diluted to corresponding concentration by using corresponding
buffer before used. (b) Forty microliters of 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. Ten microliters, five times the final concentration of
target compounds were added, the final concentration of
procaspase-3 was 1 mM, 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 2min for 1h, 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, and were presented
as mean ꢁ SE. The activation rate (%) was calculated using the
following equation: (Atested compound ꢂ ADMSO)/(Apac-1 ꢂ ADMSO)ꢃ 100.
EC50 was calculated from the activation curves with probability
regression method.
[4] R. Persad, C. Liu, T.-T. Wu, P. S. Houlihan, S. R. Hamilton,
A. M. Diehl, A. Rashid, Mod. Pathol. 2004, 17, 861–867.
[5] N. O’Donovan, J. Crown, H. Stunell, A. D. Hill, E. McDermott,
N. O’Higgins, M. J. Duffy, Clin. Cancer Res. 2003, 9, 738–742.
[6] K. F. Izban, T. Wrone-Smith, E. D. Hsi, B. Schnitzer,
M. E. Quevedo, S. Alkan, Am. J. Pathol. 1999, 154, 1439–1447.
[7] A. Nakagawara, Y. Nakamura, H. Ikeda, T. Hiwasa, K. Kuida,
M. S. Su, H. Zhao, A. Cnaan, S. Sakiyama, Cancer Res. 1997, 57,
4578–4584.
[8] D. C. Hsu, H. S. Roth, D. C. West, R. C. Botham, C. J. Novotny,
S. C. Schmid, P. J. Hergenrother, ACS Comb. Sci. 2011, 14, 44–
50.
[9] Q. P. Peterson, D. C. Hsu, D. R. Goode, C. J. Novotny,
R. K. Totten, P. J. Hergenrother, J. Med. Chem. 2009, 52, 5721–
5731.
[10] B. Zhang, Y. Zhao, X. Zhai, L. Wang, J. Yang, Z. Tan, P. Gong,
Chem. Pharm. Bull. 2011, 60, 1046–1054.
[11] K. Yamazaki, Y. Kaneko, K. Suwa, S. Ebara, K. Nakazawa,
K. Yasuno, Biorg. Med. Chem. 2005, 13, 2509–2522.
Cytotoxic activities against tumor cells assay
[12] I. Hutchinson, M.-S. Chua, H. L. Browne, V. Trapani,
T. D. Bradshaw, A. D. Westwell, M. F. Stevens, J. Med. Chem.
2001, 44, 1446–1455.
The anti-proliferative activities of compounds 17a–f, 18a–q, and
19a–c 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% fetal bovine serum (FBS). Approximate 4 ꢃ 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 absorbency at 492 nm (for absorbance of
[13] S. Saeed, N. Rashid, P. G. Jones, M. Ali, R. Hussain, Eur. J. Med.
Chem. 2010, 45, 1323–1331.
[14] G. Henriksen, A. I. Hauser, A. D. Westwell, B. H. Yousefi,
M. Schwaiger, A. Drzezga, H. J. Wester, J. Med. Chem. 2007, 50,
1087–1089.
[15] J. Das, R. V. Moquin, J. Lin, C. Liu, A. M. Doweyko, H. F. DeFex,
Q. Fang, S. Pang, S. Pitt, D. R. Shen, Bioorg. Med. Chem. Lett.
2003, 13, 2587–2590.
[16] M. Yoshida, I. Hayakawa, N. Hayashi, T. Agatsuma, Y. Oda,
F. Tanzawa, S. Iwasaki, K. Koyama, H. Furukawa, S. Kurakata,
Bioorg. Med. Chem. Lett. 2005, 15, 3328–3332.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

14
J. Ma et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–14
[17] E. Kashiyama, I. Hutchinson, M. S. Chua, S. F. Stinson,
L. R. Phillips, G. Kaur, E. A. Sausville, T. D. Bradshaw,
A. D. Westwell, M. F. Stevens, J. Med. Chem. 1999, 42, 4172–4184.
[20] B. R. Bhattarai, B. Kafle, J.-S. Hwang, S. W. Ham, K. H. Lee,
H. Park, I. O. Han, H. Cho, Bioorg. Med. Chem. Lett. 2010, 20,
6758–6763.
[18] A. Kamal, J. R. Tamboli, V. L. Nayak, S. Adil, M. Vishnuvardhan,
S. Ramakrishna, Bioorg. Med. Chem. Lett. 2013, 23, 3208–
3215.
[21] T. N. Doan, D. T. Tran, Pharmacol. Pharm. 2011, 2, 282–
288.
[22] J. Duff, E. Bills, J. Chem. Soc. (Resumed) 1932, 1, 1987–1988.
[19] R. V. Patel, P. K. Patel, P. Kumari, D. P. Rajani, K. H. Chikhalia,
[23] R. Thirumurugan, D. Sriram, A. Saxena, J. Stables, P. Yogeeswari,
Eur. J. Med. Chem. 2012, 53, 41–51.
Bioorg. Med. Chem. 2006, 14, 3106–3112.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com