Design, synthesis and biological evaluation of benzimidazole-rhodanine conjugates as potent topoisomerase II inhibitors

Article abstract of DOI:10.1039/c8md00278a

In this study, a series of benzimidazole-rhodanine conjugates were designed, synthesized and investigated for their topoisomerase II (Topo II) inhibitory and cytotoxic activities. The results from Topo II-mediated pBR322 DNA relaxation and cleavage assays showed that the synthesized compounds might act as Topo II catalytic inhibitors. Certain compounds displayed potent Topo II inhibition at 10 μM. The cytotoxic activities of these compounds against HeLa, A549, Raji, PC-3, MDA-MB-201, and HL-60 cancer cell lines were evaluated. The results indicated that these compounds exhibited strong antiproliferative activity. A good relationship was observed between the Topo II inhibitory potency and the cytotoxicity of these compounds. The structure-activity relationship revealed that the electronic effects, the phenyl group, and the rhodanine moiety were particularly important for the Topo II inhibitory potency and cytotoxicity.

Full text of DOI:10.1039/c8md00278a

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Table of contents

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Title page
Design, synthesis and biological evaluation of benzimidazole-rhodanine
conjugates as potent topoisomerase II inhibitors
Penghui Lia, Wenjin Zhanga, Hong Jianga, Yongliang Lia, Changzhi Donga,b, Huixiong Chena,c,
Kun Zhanga,d, Zhiyun Dua*
aInstitute of Natural Medicine & Green Chemistry, School of Chemical Engineering and Light
Industry, Guandong University of Technology, Guangzhou 510006, China
bUniversite Paris Diderot, Sorbonne Paris Cite, ITODYS, UMR 7086 CNRS, 15 rue JꢀA de
Baif, 75270 Cedex 13 Pairs, France
cCNRS, UMR8601, Laboratoire de Chimine et Biochimie Pharmacologiques et
Toxicologiques, CBNIT, Universite Paris Descartes PRES Sorbonne Paris Cite, UFR
Biomedicale, 45 rue des SaintsꢀPeres, 75270 Cedex 06 Paris, France.
dWuyi University, Jiangmen 529020, China.
*Corresponding authors:
Eꢀmail addresses: zhiyundu@gdut.edu.cn (Z. Y. Du).
Abstract
In this study, a series of benzimidazoleꢀrhodanine conjugates were designed, synthesized and
investigated for their topoisomerase II (Topo II) inhibition and cytotoxic activities. The results
from Topo IIꢀmediated pBR322 DNA relaxation and cleavage assay showed that the
synthesized compounds might act as Topo II catalytic inhibitors. Certain compounds
displayed potent Topo II inhibition at 10 ꢁM. The cytotoxic activities against Hela, A549, Raji,
PCꢀ3, MDAꢀMBꢀ201, and HLꢀ60 cancer cell lines of these compounds were evaluated. The
results indicated that these compounds exhibited strong antiproliferation activity. A good
relationship was observed between the Topo II inhibition potency and the cytotoxicity of
these compounds. The structureꢀactivity relationship revealed that the electronic effects, the
phenyl group, and the rhodanine moiety were particularly important for the Topo II inhibition
potency and cytotoxicity.
Keywords
Benzimidazole
Rhodanine
Topoisomerase
Anticancer
Pharmacophore hybridization strategy
1

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1. Introduction
DNA topoisomerases are important cellular enzymes found in almost all types of
living cells. These enzymes mediated the topological adjustments required for DNA
replication, transcription, recombination, repair, and chromatin assembly1ꢀ3. These
enzymes are important molecular drug targets and inhibitors of these enzymes are widely
used as effective anticancer agents. Topoisomerase inhibitors are used in clinical treatment of
cancer for more than 30 years. About 50% of chemotherapeutic regimens use at least one drug
that targets these enzymes4. There are two types of topoisomerases in human: type I
topoisomerase (Topo I) and type II topoisomerase (Topo II). Topo II has been reported
as the specific target of certain most active anticancer drugs such as etoposide, doxorubicin
and amscrine5. However, Topo II inhibitors have some therapeutic limitations beacuse of
theirserious side effects during cancer chemotherapy. Thus, development of novel anticancer
Topo II inhibitors is necessary for improving cancer treatment6ꢀ10
.
Benzimidazole is found in many clinically useful drugs11and represents a precious scaffold
in the preparation of a wide variety of biological active compounds such as antibacterial,
antiꢀinflammatory and anticancer agents12. Several benzimidazole derivatives were reported
as novel Topo II inhibitors (1 and 2, Fig.1). Structureꢀactivity relationship (SAR) studies
showed that the benzimidazole rings as a fused system in the structures is significant for Topo
II inhibition potency and the phenyl group linked to benzimidazole is particularly
important13,14. Mechanism studies and molecular docking revealed that these compounds
function as Topo II catalytic inhibitors by blocking the ATPꢀbinding site of the enzyme15,16
.
Rhodanineis recognized as a privileged heterocycle in medicinal chemistry17. The
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molecules containing rhodanine moiety are known for their rich pharmacological profile,
including antibacterial, antiꢀparasitic, antiꢀmicrobial, antiꢀtubercular and anticancer18ꢀ21
Rhodanines possess good activity against different types of cancer including relatively simple
derivatives as well as complex or hybrids/conjugates bearing nonꢀfused rhodanine core22–24
.
.
For example, commercial GSK1059615 (3, Fig. 1) is a reversible, ATPꢀcompetitive, inhibitor
of PI3Kα, which shows potential anticancer activity. Compound 4 (Fig. 1) displays a potent
cytotoxicity toward several cancer cell lines.
Pharmacophore hybridization strategy is often used for the design of new drugs and a
certain number successful examples are reported in drug discovery and developmen25ꢀ28. We
have recently designed and synthesized various Topo II inhibitors through pharmacophore
hybridization strategy and the results demonstrated that these conjugates possessed generally
higher cytotoxicity and Topo II inhibition potency than the individual agents alone16,20,29. In an
effort to discover novel anticancer agents that target Topo II more efficiently,
benzimidazoleꢀrhodanine conjugates (Fig.1) were synthesized in this work and the Topo I and
II inhibition, the DNA interaction, and the cytotoxicity properties of these newly synthesized
compounds were evaluated. It was found that these compounds displayed significantly Topo
II inhibition potency and showed effective cytotoxicity against six human cancer cell lines.
2. Results and Discussion
2.1. Chemistry
A series of benzimidazoleꢀrhodanine conjugates (8aꢀ8o, 9aꢀ9d, 10aꢀ10h and 11aꢀ11h)
were synthesized as outlined in Scheme 1. The synthetic strategy consisted of the
benzimidazole and rhodanine moieties and then the condensation of them to provide the target
3

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compounds (Fig. 2). Substitutedꢀ(benzo[d]ꢀimidazoleꢀ2ꢀyl)methanols (1aꢀ1d) were prepared
by refluxing substitutedꢀ1,2ꢀdiaminobenzene with glycolic acid in hydrochloric acid.
Substitutedꢀ(1ꢀalkylꢀ1Hꢀbenzo[d]imidazoleꢀ2yl)methanols (2aꢀ2s, 3aꢀ3f and 4aꢀ4f) were
achieved by Nꢀalkylation of 1aꢀ1d with appropriate benzyl bromide, bromoethane or
2ꢀbromoꢀ1ꢀphenylethanone
using
K2CO3
as
base
in
27%–88%
yield.
Substitutedꢀ1ꢀalkylꢀ1Hꢀbenzo[d]imidazoleꢀ2ꢀcarbaldehydes (5aꢀ5s, 6aꢀ6f and 7aꢀ7f) were
obtained by oxidation of the corresponding primary alcohols with DessꢀMartin reagent in
36%–68% yield30. The target compounds (8aꢀ8o, 9aꢀ9d, 10aꢀ10h and 11aꢀ11h) were
synthesized through the HornorꢀWadsworthꢀEmmons reaction of rhodanine moiety with the
prepared aldehydes (5aꢀ5s, 6aꢀ6f and 7aꢀ7f) in 69%–91% yield. Since the reaction was not
regionꢀselective in the preparation of 3aꢀ3f and 4aꢀ4f, the R1 group could be either in C5 or
C6 position of benzimidazole scaffold in the process of substitution reaction31. 2D NOESY
spectrum was performed to determine the isomer of 3a and 3b (Fig. S1 and S2). The
synthesized compounds were characterized by 1H NMR, 13C NMR, and HRMS (ESI), which
was in full accordance with their depicted structures and the purities were confirmed by
analytical HPLC.
2.2. Cytotoxicity
The synthesized compounds were evaluated for their cytotoxic activities using the MTT
assay against six human cancer cell lines including: the human acute leukemia cell line
(HLꢀ60), human cervical cancer cell line (Hela), human breast cancer cell line
(MDAꢀMBꢀ201), adenocarcinomic human alveolar basal epithelial cancer cell line (A549),
human lymphoma cancer cell line (Raji), and human prostate cancer cells line (PCꢀ3).
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Etoposide and camptothecin were chosen as positive controls. The inhibitory activities (IC50)
of the tested compounds are listed in Table 1.
Most of the compounds displayed significant cytotoxic activities with low micromolar IC50
values. In addition, a good correlation was found between the Topo II inhibition potency and
the cytotoxic activities. Compounds 8g, 8j, 8n, 9a, 9c, 10a, and 10cꢀ10e, which showed
strong Topo II inhibition potency, displayed promising cytotoxic activity against six cancer
cell lines. Specially, 8g and 8j, which exhibited excellent Topo II inhibition activity at 10 ꢁM,
showed more potent cytotoxic activity, with an IC50 values ranging from 0.54 to 3.22 ꢁM and
from 0.21 to 2.67 ꢁM, respectively. Whereas, others compounds, revealed to be poor Topo II
inhibitors showed low or moderate cytotoxic activities. It is worth to mention that HLꢀ60 was
revealed more sensitive to these compounds, as a lower IC50 value was obtained compared to
other cancer cell lines (Table 1).
2.3. Topo II inhibition and SAR study
Topo IIꢀmediated DNA relaxation, cleavage of the complex and unwinding assays as well
as molecule docking were performed to evaluate the ability of the synthesized compounds to
inhibit Topo II and to investigate their mode of action.
Topo IIꢀmediated DNA relaxation assay was first employed to determine the capacity of
these compounds, using etoposide as positive control and pBR322 DNA plasmid as substrate8.
Compounds 8aꢀ8o and 9aꢀ9d (Fig. 2) were first synthesized and submitted to this assay. As
shown in Fig. 3A and 3B, most of the tested compounds exhibited significantly Topo II
inhibition potency at 50 ꢁM and displayed a similar effect with etoposide. Compounds 8g, 8j,
8n, 9a, and 9c showed the best inhibition potency at 10 ꢁM (Fig. 3D). It was found that the
benzyl group is necessary for the activity (8f, Fig. 3A), since its replacement with ethyl group
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(8o, Fig. 3A) led to a decrease of inhibitory activity. Moreover, compounds 8g and 8n with
electron donor groups showed better Topo II inhibition than those with electron withdrawing
groups (8a, 8d, 8e, and 8h, Fig. 3A, 3B and 3D). Along with this observation, 8b, 8k, and 8m
with two withdrawing substitution groups on benzyl showed weaker activity than those with
one withdrawing substitutions group (8j, Fig. 3A, 3B and 3D). The results indicated that
benzyl group and the electronic effects of substitutions have significant impact on Topo II
inhibitory activity and should be maintained in further structure modification.
In order to investigate the effect of the rhodanineꢀ3ꢀacetic acid moiety on the inhibition of
Topo II activity, it was changed to rhodanine (10g and 11g) or rhodanineꢀ3ꢀ(3ꢀpropionic acid)
(10h and 11h). It was found that, the acetic acid moiety was crucial, since 10g, 11g, 10h, and
11h did not show better Topo II inhibition at 20 ꢁM (Fig. 3C). These results revealed that the
acetic acid group linked to rhodanine was also optimal for the activity.
Next, the benzimidazole scaffold was modified with various substitutions (–F, –Cl, and
–OCH3) on the benzene ring. 5 or 6ꢀMonosubstituted isomers can be obtained (10a-10f and
11a-11f, Scheme 2) and no obvious difference in Topo II inhibition potency was observed
between the isomers at 50 ꢁM (Fig. 3C). The results showed that 10a, 10c, 10d, and 10e also
displayed strong Topo II inhibition activity even at 10 ꢁM (Fig. 3C and 3D). However,
modified 8j with the same substitutions (11aꢀ11f) led to a decrease of Topo II inhibition
potency (Fig. 3C and 3D).
Topo Iꢀmediated DNA relaxation assay was employed to study whether this class of
compounds also targeted Topo I. The results are presented in Fig. 4. None of the tested
compounds exhibited Topo I inhibition potency even at 50 ꢁM, indicating that these
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compounds displayed selective inhibition against Topo II.
As compounds 8g and 8j showed the best Topo II inhibition and cytotoxic activity, they
were selected for the further mechanism studies.
2.4. Compounds 8g and 8j are non-intercalative Topo II catalytic inhibitors
Topo II inhibitors have different mechanism of actions. Topo II poisons stabilize the
cleavage complex of DNA and promote the formation of linear DNA, whereas Topo II
catalytic inhibitors block the activity of the enzyme to perform catalysis1ꢀ3. In addition, Topo
II catalytic inhibitors can antagonize Topo II poisonꢀmediated DNA damage16. Topo
IIꢀmediated DNA cleavage assay was employed for investigating the mode of action of 8g
and 8j. As shown in Fig. 5A, etoposide, a classical Topo II poison, produced the linear DNA,
while the linear form of the DNA was not observed when 8g and 8j were used up to 50 ꢁM.
In addition, pretreatment with 8g and 8j could reduce the amount of linear DNA trapped by
etopoisde. These results provide evidence that 8g and 8j act as Topo II catalytic inhibitors.
There are two types of Topo II catalytic inhibitors: DNA intercalators and
nonꢀintercalators5. Topo Iꢀmediated DNA unwinding assay was carried out to determine
whether 8g and 8j function as intercalators. In this assay, the supercoiled DNA, relaxed by
excessive Topo I, could be regenerated if the test compound is a DNA intercalator. As shown
in Fig. 5B, ethidium bromide (EB), a classic intercalator of DNA did help the regeneration of
the supercoiled DNA at 5 µM, while 8g and 8j didn’t even at 50 ꢁM. These findings suggest
that 8g and 8j are nonꢀintercalating Topo II catalytic inhibitors.
2.5. Molecular docking
Molecular docking was carried out to identify the potential interactions of these
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compounds with Topo II by using the Surflexꢀdock program incorporated with the SYBYL
software package (Tripos. Inc. St. Louis. MO) 32,33. We first docked 8g and 8j into various
binding pockets in Topo II. The best result was obtained when the compounds were docked
into the ATPꢀbinding pocket (PDB code: 1ZXM). As shown in Fig. 6, for both compounds,
the nitrogen group (N3) of benzimidazole formed a special hydrogen bond with the amino
group of the residues Asn91, and the carboxylic group formed a hydrogen bond with the
amino groups of Arg98 and Asn95. These hydrogen bonds may provide stability to the
enzymeꢀligand interactions. The benzimidazole group showed hydrophobic interactions with
the hydrophobic part of the enzyme, whereas the phenyl group formed hydrophobic
interactions with residues Asn150 and Thr159. These results showed that 8g and 8j are
catalytic inhibitors, whose mode of action seems to occupy the ATPꢀbinding pocket of the
ATPase domain of Topo II and make favorable interactions with its key residues.
3. Conclusion
In this study, a series of benzimidazoleꢀrhodanine conjugates were designed and
synthesized as potential Topo IIꢀtargeting anticancer agents. The results revealed that these
compounds displayed strong Topo II inhibition potency and nine of them, namely, 8g, 8j, 8n,
9a, 9c, 10a, 10c, 10d, and 10e almost completely suppressed the Topo II activity at 10 ꢁM.
The SAR study revealed that the benzyl group and the electronic effects of substitutions as
well as the rhodanine moiety have significant impact on Topo II inhibition potency.
Mechanism studies demonstrated that the leading compounds 8g and 8j function as
nonintercalative Topo II catalytic inhibitors. The molecular docking analysis suggested that
the inhibition mode of 8g and 8j may be through blocking the ATPꢀbinding site of the enzyme.
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These compounds displayed potent cytotoxic activities with low micromolar IC50 values
toward six cancer cell lines. Meanwhile, most of the compounds showed good relationship
between their Topo II inhibition potency and the cytotoxicity. In conclusion, the
pharmacophore hybridization strategy used in this work on the basis of benzimidazole with
rhodanine moiety represents a feasible way to discover Topo IIꢀtargeting anticancer agents.
Further work is ongoing to optimize the bioactivity of these compounds.
4. Experimental section
4.1. Chemistry
All reagents were commercially available and purchased from SigmaꢀAldrich, TCI, AlfaAesar,
and Aladdin. They were used without further purification. HPLC grade methanol was order
from Sinopharm (China) and silica gel (200ꢀ300 mesh, Qingdao Haiyang Chemical Co., Ltd).
1H and 13C NMR spectrum were recorded in DMSOꢀd6 or CDCl3 with a Bruker BioSpin
GmbH spectrometer at 400 and 101 MHz, respectively. The chemical shifts are reported in
parts per million (ppm) relative to residual CDCl3 (δ = 7.26, 1H; δ = 77.0, 13C) and DMSOꢀd6
1
(δ = 2.50, H; δ = 39.5, 13C) in the corresponding deuterium agents. Highꢀresolution mass
spectra (HRMS) were recorded on Shimadzu LCMSꢀITꢀTO. The melting point (mp) was
determined using an SRSꢀOptiMelt automated melting point instrument without correction.
The purities of synthesized compounds were confirmed by analytical HPLC performed with a
dual pump Shimadzu LCꢀ20AB system equipped with an Ultimate XBꢀC18 column and
eluted with methanol/water (80%) at a flow rate of 0.5 mL·minꢀ1, and the purities were proved
to be higher than 95%. The chemical structure and the general method for the synthesis of the
intermediates are list in Supporting Information.
4.2. General method for synthesis of the target compounds (8a–8o, 9a–9d, 10a–10h and
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11a–11h).
A mixture of the appropriate aldehydes (5a–5s, 6a–6f and 7a–7f, 0.1 mmol), the
rhodanine moiety (0.1 mmol), and NaOAc (0.3 mmol) in acetic acid (6 mL) heated to 110 °C
for 4 h. Then, it was cooled to room temperature and poured into water. The product was then
filtered through the suction pump, washed with water/EtOH (1/1, v/v) to remove the excess
acetic acid and recrystallized from EtOH.
4.2.1.
(Z)-2-(5-((1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8a). Rhodanineꢀ3ꢀacetic acid and 5a were used as
reactants to give 8a. Yellow solid, yield: 73%, mp: 297.1–299.2 °C.1H NMR (400 MHz,
DMSOꢀd6) δ 13.45 (s, 1H), 7.97 (s, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H),
7.53 (d, J = 8.1 Hz, 2H), 7.41 – 7.33 (m, 2H), 7.10 (d, J = 8.0 Hz, 2H), 5.88 (s, 2H), 4.74 (s,
2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.3, 167.8, 166.3, 147.8, 143.6, 136.9, 135.9, 132.3,
130.3, 129.2, 125.5, 124.3, 121.4, 120.6, 116.4, 112.0, 46.1, 45.2. HRMS (ESI):
C20H14BrN3O3S2 [M + H]+487.9717, found 487.9711.
4.2.2.
(Z)-2-(5-((1-(4-bromo-2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (8b). Rhodanineꢀ3ꢀacetic acid and 5b were used as
1
reactants to give 8b. Yellow solid, yield: 86%, mp: 283.7–285.4 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.46 (s, 1H), 8.03 (s, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H),
7.60 (d, J = 9.9 Hz, 1H), 7.39 – 7.33 (m, 3H), 7.05 (t, J = 8.2 Hz, 1H), 5.91 (s, 2H), 4.75 (s,
2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.3, 167.8, 166.3 (d, J = 251.0 Hz), 161.6, 159.1,
147.9, 143.5, 135.7, 131.1 (d, J = 4.5 Hz), 130.2, 128.5 (d, J = 3.5 Hz), 125.5, 124.3, 123.7 (d,
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J = 14.7 Hz), 122.0 (d, J = 9.6 Hz), 120.6, 119.6 (d, J = 24.5 Hz), 116.6, 111.9, 45.2, 41.4 (d,
J = 2.6 Hz). HRMS (ESI): calcd for C20H13BrFN3O3S2 [M + H]+505.9663, found 505.9658.
4.2.3
(Z)-2-(5-((1-(4-cyanobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl) acetic acid (8c). Rhodanineꢀ3ꢀacetic acid and 5c were used as
1
reactants to give 8c. Yellow solid, yield: 91%, mp: 284.6–286.3 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.47 (s, 1H), 7.96 (s, 1H), 7.87 (d, J = 7.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H),
7.66 (d, J = 8.0 Hz, 1H), 7.41 – 7.34 (m, 2H), 7.28 (d, J = 8.0 Hz, 2H), 6.02 (s, 2H), 4.74 (s,
2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.2, 167.8, 166.3, 147.9, 143.56, 143.0, 135.9,
133.3, 130.5, 127.8, 125.6, 124.4, 120.6, 119.0, 116.2, 111.9, 111.1, 46.3, 45.2. HRMS (ESI):
calcd for C20H14FN3O3S2 [M + H]+435.0531, found 435.0542.
4.2.4.
(Z)-2-(5-((1-(4-nitrobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8d). Rhodanineꢀ3ꢀacetic acid and 5d were used as
1
reactants to give 8d. Yellow solid, yield: 79%, mp: 290.5–292.3 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.44 (s, 1H), 8.20 (d, J = 8.4 Hz, 2H), 7.97 (s, 1H), 7.88 (d, J = 6.5 Hz, 1H),
7.67 (d, J = 8.1 Hz, 1H), 7.41 – 7.33 (m, 4H), 6.08 (s, 2H), 4.74 (s, 2H). 13C NMR (101 MHz,
DMSOꢀd6) δ 198.2, 167.7, 166.2, 147.8, 147.5, 144.9, 143.6, 135.8, 130.5, 128.1, 125.6,
124.5, 124.4, 120.7, 116.2, 111.9, 46.2, 45.2. HRMS (ESI): calcd for C20H14N4O5S2 [M +
H]+455.0439, found 455.0427.
4.2.5. (Z)-2-(5-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8e). Rhodanineꢀ3ꢀacetic acid and 5e were used as reactants
to give 8e. Yellow solid, yield: 83%, mp: 287.7–289.2 °C. 1H NMR (400 MHz, DMSOꢀd6) δ
13.46 (s, 1H), 7.99 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.41 – 7.33 (m,
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2H), 7.23 – 7.15 (m, 4H), 5.88 (s, 2H), 4.74 (s, 2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.3,
167.8, 166.3, 162.1(d, J = 244.0 Hz), 147.7, 143.6, 135.9, 133.6 (d, J = 3.0 Hz), 130.3, 129.2
(d, J = 8.4 Hz), 125.5, 124.3, 120.6, 116.4, 116.1 (d, J = 21.6 Hz), 112.0, 46.0, 45.2. HRMS
(ESI): calcd for C21H14N4O3S2 [M + H]+428.0508, found 428.0524.
4.2.6.
(Z)-2-(5-((1-benzyl-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8f). Rhodanineꢀ3ꢀacetic acid and 5f were used as reactants
to give 8f. Yellow solid, yield: 77%, mp: 293.4–295.1 °C. 1H NMR (400 MHz, DMSOꢀd6) δ
13.45 (s, 1H), 7.97 (s, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.40 – 7.32 (m,
4H), 7.29 – 7.24 (m, 1H), 7.15 (d, J = 7.5 Hz, 2H), 5.89 (s, 2H), 4.74 (s, 2H). 13C NMR (101
MHz, DMSOꢀd6) δ 198.3, 167.8, 166.3, 147.8, 143.6, 137.4, 136.0, 130.1, 129.4, 128.2, 127.0,
125.4, 124.2, 120.6, 116.5, 112.0, 46.7, 45.2. HRMS (ESI): calcd for C20H15N3O3S2 [M +
H]+409.0619, found 409.0627.
4.2.7.
(Z)-2-(5-((1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8g). Rhodanineꢀ3ꢀacetic acid and 5g were used as
reactants to give 8g. Yellow solid, yield: 87%, mp:＞300 °C. 1H NMR (400 MHz, DMSOꢀd6)
δ 13.47 (s, 1H), 7.95 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.40 – 7.32
(m, 2H), 7.16 –7.10 (m, 2H), 7.08 – 7.02 (m, 2H), 5.82 (s, 2H), 4.74 (s, 2H), 2.23 (s, 3H). 13
C
NMR (101 MHz, DMSOꢀd6) δ 198.3, 167.8, 166.3, 147.7, 143.6, 137.6, 136.0, 134.4, 130.1,
129.9, 127.0, 125.4, 124.2, 120.5, 116.6, 112.1, 46.5, 45.3, 21.1. HRMS (ESI): calcd for
C21H17N3O3S2 [M + H]+424.0753, found 424.0739.
4.2.8.
(Z)-2-(5-((1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8h). Rhodanineꢀ3ꢀacetic acid and 5h were used as
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reactants to give 8h. Yellow solid, yield: 90%, mp:＞300 °C. 1H NMR (400 MHz, DMSOꢀd6)
δ 13.46 (s, 1H), 7.97 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.42 – 7.33
(m, 4H), 7.19 – 7.15 (m, 2H), 5.89 (s, 2H), 4.74 (s, 2H). 13C NMR (101 MHz, DMSOꢀd6) δ
198.3, 167.8, 166.3, 147.8, 143.6, 136.5, 135.9, 132.9, 130.3, 129.4, 128.9, 125.5, 124.3,
120.6, 116.4, 112.0, 46.0, 45.2. HRMS (ESI): calcd for C20H14ClN3O3S2 [M + H]+ 444.0212,
found 444.0226.
4.2.9.
(Z)-2-(5-((1-(4-trifluoromethylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
(4-oxo-2-thioxo thiazolidin-3-yl)acetic acid (8i). Rhodanineꢀ3ꢀacetic acid and 5i were used as
1
reactants to give 8i. Yellow solid, yield: 81%, mp: 281.8–283.4 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.44 (s, 1H), 7.98 (s, 1H), 7.87 (d, J = 7.1 Hz, 1H), 7.70 (m, 3H), 7.42 – 7.35
(m, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.02 (s, 2H), 4.74 (s, 2H). 13C NMR (101 MHz, DMSOꢀd6)
δ 198.3, 167.7, 166.3, 147.9, 143.6, 142.1, 135.9, 130.5, 128.8 (q, J = 32.0 Hz), 127.7, 126.3,
(q, J = 3.7 Hz), 125.6, 124.6 (q, J = 272.0 Hz), 120.6, 116.3, 111.2, 46.2, 45.2. HRMS (ESI):
calcd for C21H14F3N3O3S2 [M + H]+478.0471, found 478.0462.
4.2.10.
(Z)-2-(5-((1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-
2-thioxothiazolidin-3-yl)acetic acid (8j). Rhodanineꢀ3ꢀacetic acid and 5j were used as
reactants to give 8j. Yellow solid, yield: 82%, mp: 272.6–274.2 °C.1H NMR (400 MHz,
DMSOꢀd6) δ 13.44 (s, 1H), 8.03 (s, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.69 (d, J = 7.3 Hz, 1H),
7.42 – 7.31 (m, 3H), 7.29 – 7.20 (m, 1H), 7.19 – 7.07 (m, 2H), 5.93 (s, 2H), 4.75 (s, 2H). 13
C
NMR (101 MHz, DMSOꢀd6) δ 198.4, 167.8, 166.3, 160.5 (d, J = 245.7 Hz),, 147.9, 143.5,
135.8, 130.7 (d, J = 8.2 Hz), 130.1, 129.6 (d, J = 3.8 Hz), 125.4 (d, J = 2.3 Hz), 125.4, 124.2,
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124.0(d, J = 14.5 Hz), 120.6, 116.7, 116.2 (d, J = 20.9 Hz), 112.0, 45.2, 41.7 (d, J = 3.4 Hz)
.
HRMS (ESI): calcd for C20H14FN3O3S2 [M + H]+428.0516, found 428.0523.
4.2.11.
(Z)-2-(5-((1-(2,4-difluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-
2-thioxothiazolidin-3-yl)acetic acid (8k). Rhodanineꢀ3ꢀacetic acid and 5k were used as
1
reactants to give 8k. Yellow solid, yield: 77%, mp: 261.8–263.3 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.48 (s, 1H), 8.05 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H),
7.44 – 7.19 (m, 4H), 7.06 (t, J = 8.5 Hz, 1H), 5.90 (s, 2H), 4.75 (s, 2H). 13C NMR (101 MHz,
DMSOꢀd6) δ 198.4, 167.8, 166.3, 162.5 (dd, J = 247.2, 12.2 Hz), 160.64 (dd, J = 248.9, 12.5
Hz), 147.8, 143.5, 135.7, 131.1 (dd, J = 10.0, 5.5 Hz), 130.1, 125.4, 124.3, 120.6, 120.5 (dd, J
= 14.8, 3.6 Hz), 116.6, 112.4 (dd, J = 21.3, 3.5 Hz), 111.95, 104.9 (t, J = 25.8 Hz), 45.2, 41.3
(d, J = 2.4 Hz). HRMS (ESI): calcd for C20H13F2N3O3S2 [M + H]+446.0427, found 446.0438.
4.2.12.
(Z)-2-(5-((1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (8l). Rhodanineꢀ3ꢀacetic acid and 5l were used as reactants
1
to give 8l. Yellow solid, yield: 71%, mp: 288.2–290.6 °C. H NMR (400 MHz, DMSOꢀd6) δ
13.55 (s, 1H), 7.97 (s, 1H), 7.86 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.45 – 7.32 (m,
3H), 7.12 (t, J = 8.5 Hz, 1H), 7.05 (d, J = 9.8 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 5.91 (s, 2H),
4.74 (s, 2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.3, 167.8, 166.3, 162.7 (d, J = 244.7 Hz),
147.8, 143.6, 140.2 (d, J = 7.2 Hz), 135.9, 131.5 (d, J = 8.4 Hz), 130.4, 125.56, 124.3, 122.9
(d, J = 2.6 Hz), 120.6, 116.3, 115.1 (d, J = 20.9 Hz), 114.0 (d, J = 22.1 Hz), 111.9, 46.2 (d, J
= 0.6 Hz), 45.2. HRMS (ESI): calcd for C20H14FN3O3S2 [M + H]+428.0516, found 428.0522.
4.2.13.
(Z)-2-(5-((1-(3,4-difluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-
2-thioxothiazolidin-3-yl)acetic acid (8m). Rhodanineꢀ3ꢀacetic acid and 5m were used as
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1
reactants to give 8m. Yellow solid, yield: 76%, mp: 267.6–269.2 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.46 (s, 1H), 8.00 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H),
7.48 – 7.28 (m, 4H), 6.93 (s, 1H), 5.88 (s, 2H), 4.75 (s, 2H). 13C NMR (101 MHz, DMSOꢀd6)
δ 198.3, 167.8, 166.3, 149.9 (dd, J = 246.8, 12.8 Hz), 149.4 (dd, J = 246.1, 12.4 Hz), 147.8,
143.6, 135.8, 135.1 (dd, J = 5.4, 3.9 Hz), 130.4, 125.6, 124.3, 123.9 (dd, J = 6.7, 3.5 Hz),
120.6, 118.5 (d, J = 17.4 Hz), 116.6 (d, J = 17.8 Hz), 116.3, 111.9, 45.7, 45.2. HRMS (ESI):
calcd for C20H13F2N3O3S2 [M + H]+ 446.0527, found 446.0532.
4.2.14.
(Z)-2-(5-((1-(3,5-dimethoxybenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-
oxo-2-thioxothiazolidin-3-yl)acetic acid (8n). Rhodanineꢀ3ꢀacetic acid and 5n were used as
reactants to give 8n. Yellow solid, yield: 78%, mp: 259.8–261.3 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 7.95 (s, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.44 – 7.33 (m,
2H), 6.41 (t, J = 2.1 Hz, 1H), 6.27 (d, J = 2.1 Hz, 2H), 5.79 (s, 2H), 4.74 (s, 2H), 3.66 (s, 6H).
13C NMR (101 MHz, DMSOꢀd6) δ 198.3, 167.7, 166.3, 161.3, 147.8, 143.5, 139.7, 136.1,
130.1, 125.5, 124.2, 120.6, 116.5, 112.0, 105.4, 99.3, 55.7, 46.6, 45.2. HRMS (ESI): calcd for
C22H19N3O5S2[M + H]+ 470.0882, found 470.0871.
4.2.15.
(Z)-2-(5-((1-ethyl-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3- yl)acetic acid (8o). Rhodanineꢀ3ꢀacetic acid and 5o were used as
1
reactants to give 8o. Yellow solid, yield: 81%, mp: 226.5–227.8 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.45 (s, 1H), 7.93 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H),
7.40 (t, J = 7.2 Hz, 1H), 7.34 (t, J = 7.2 Hz, 1H), 4.76 (s, 2H), 4.60 (q, J = 7.1 Hz, 2H), 1.36 (t,
J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.4, 167.8, 166.4, 147.0, 143.6, 135.4,
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129.7, 125.2, 124.0, 120.5, 116.6, 111.7, 45.2, 38.7, 16.3. HRMS (ESI): calcd for
C15H13N3O3S2 [M + H]+348.0428, found 348.0422.
4.2.16. (Z)-2-(5-((1-(2-(4-fluorophenyl)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (9a). Rhodanineꢀ3ꢀacetic acid and 5p were used as
1
reactants to give 9a. Yellow solid, yield: 81%, mp: 247.7–249.3 °C. H NMR (400 MHz,
DMSOꢀd6) δ 8.27 – 8.21 (m, 2H), 7.97 (s, 1H), 7.89 –7.85 (m, 1H), 7.68 – 7.64 (m, 1H), 7.51
(t, J = 8.7 Hz, 2H), 7.38 – 7.32 (m, 2H), 6.42 (s, 2H), 4.75 (s, 2H). 13C NMR (101 MHz,
DMSO) δ 198.5, 192.3, 167.8, 166.3, 166.1(d, J = 253.2 Hz), 148.8, 143.4, 136.5, 132.1 (d, J
= 9.7 Hz), 131.6 (d, J = 2.7 Hz), 129.8, 125.2, 124.1, 120.4, 117.4, 116.4 (d, J = 22.1 Hz),
111.89, 50.6, 45.2. HRMS (ESI): calcd for C21H14FN3O4S2 [M + H]+456.0429, found
456.0431.
4.2.17.
(Z)-2-(5-((1-(2-(4-methoxyphenyl)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl)
methylene)- 4-oxo-2-thioxothiazolidin-3-yl)acetic acid (9b). Rhodanineꢀ3ꢀacetic acid and 5q
were used as reactants to give 9b. Yellow solid, yield: 72%, mp: 256.4–258.1 °C. δ 13.36 (s,
1H), 8.12 (d, J = 8.7 Hz, 2H), 7.90 (s, 1H), 7.88 – 7.82 (m, 1H), 7.67 – 7.61 (m, 1H), 7.38 –
7.32 (m, 2H), 7.17 (d, J = 8.7 Hz, 2H), 6.37 (s, 2H), 4.74 (s, 2H), 3.90 (s, 3H).13C NMR (101
MHz, DMSOꢀd6) δ 198.5, 191.8, 167.8, 166.3, 164.5, 148.8, 143.5, 136.5, 131.4, 129.7, 127.7,
125.2, 124.0, 120.4, 117.4, 114.6, 111.8, 56.2, 50.2, 45.2. HRMS (ESI): calcd for
C22H17N3O5S2 [M + H]+468.0653, found 468.0641.
4.2.18. (Z)-2-(5-((1-(2-(4-chlorophenyl)-2-oxoethyl)-1H-benzo[d]imidazol-2-yl) methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (9c). Rhodanineꢀ3ꢀacetic acid and 5r were used
1
as reactants to give 9c. Yellow solid, yield: 83%, mp: 256.9–258.2 °C. H NMR (400 MHz,
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DMSOꢀd6) δ 8.16 (d, J = 8.5 Hz, 2H), 7.98 (s, 1H), 7.90 – 7.83 (m, 1H), 7.76 (d, J = 8.5 Hz,
2H), 7.69 – 7.63 (m, 1H), 7.38 – 7.32 (m, 2H), 6.44 (d, J = 19.5 Hz, 2H), 4.74 (s, 2H). 13C
NMR (101 MHz, DMSOꢀd6) δ 198.5, 192.7, 167.8, 166.3, 148.8, 143.4, 139.6, 136.5, 133.5,
130.9, 129.8, 129.4, 125.2, 124.1, 120.4, 117.4, 111.9, 50.6, 45.2. HRMS (ESI): calcd for
C22H17ClN3O4S2 [M + H]+472.0193, found 472.0172.
4.2.19. (Z)-2-(4-oxo-5-((1-(2-oxo-2-(p-tolyl)ethyl)-1H-benzo[d]imidazol-2-yl)methylene)-2-
thioxothiazolidin-3-yl)acetic acid (9d). Rhodanineꢀ3ꢀacetic acid and 5s were used as reactants
to give 9d. Yellow solid, yield: 76%, mp: 263.1–265.3 °C. 1H NMR (400 MHz, DMSOꢀd6) δ
8.06 (d, J = 7.7 Hz, 2H), 7.93 (s, 1H), 7.88 – 7.84 (m, 1H), 7.67 – 7.63 (m, 1H), 7.47 (d, J =
7.6 Hz, 2H), 7.40 – 7.34 (s, 2H), 6.39 (s, 2H), 4.74 (s, 2H), 2.46 (s, 3H). 13C NMR (101 MHz,
DMSOꢀd6) δ 198.5, 193.1, 167.8, 166.3, 148.8, 145.3, 143.5, 136.5, 132.3, 129.9, 129.7,
129.1, 125.2, 124.1, 120.4, 117.4, 111.8, 50.4, 45.2, 21.8. HRMS (ESI): calcd for
C22H17N3O4S2 [M + H]+452.0727, found 452.0731.
4.2.20.
(Z)-2-(5-((5-chloro-1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-
oxo-2-thioxothiazolidin-3-yl)acetic acid (10a). Rhodanineꢀ3ꢀacetic acid and 6a were used as
1
reactants to give 10a. Yellow solid, yield: 84%, mp: 297.9–299.3 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.48 (s, 1H), 7.95 – 7.91 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.42 (dd, J = 8.7,
1.8 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 5.83 (s, 2H), 4.74 (s, 2H), 2.24
(s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.0, 167.8, 166.3, 148.8, 142.2, 137.6, 136.7,
134.2, 130.8, 123.0, 129.9, 127.0, 124.7, 121.9, 116.1, 111.9, 46.6, 45.3, 21.1. HRMS (ESI):
calcd for C21H16ClN3O3S2 [M + H]+ 458.0334, found 458.0329.
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4.2.21.
(Z)-2-(5-((6-chloro-1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (10b). Rhodanineꢀ3ꢀacetic acid and 6b were used
1
as reactants to give 10b. Yellow solid, yield: 69%, mp:＞300 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.48 (s, 1H), 7.94 – 7.91 (m, 2H), 7.86 (d, J = 8.7 Hz, 1H), 7.37 (dd, J = 8.7,
1.9 Hz, 1H), 7.15 (d, J = 7.9 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 5.83 (s, 2H), 4.74 (s, 2H), 2.25
(s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.1, 167.8, 166.3, 149.1, 144.1, 137.6, 134.8,
134.2, 131.1, 129.9, 128.7, 127.0, 125.5, 119.8, 116.9, 113.5, 46.7, 45.2, 21.1. HRMS (ESI):
calcd for C21H16ClN3O3S2 [M + H]+458.0334, found 458.0343.
4.2.22.
(Z)-2-(5-((5-fluoro-1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (10c). Rhodanineꢀ3ꢀacetic acid and 6c were used
as reactants to give 10c. Yellow solid, yield: 77%, mp: 291.5–292.9 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 13.44 (s, 1H), 7.96 (s, 1H), 7.78 – 7.74 (m, 1H), 7.68 (dd, J = 9.5, 2.4 Hz, 1H),
7.32 – 7.24 (m, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 5.85 (s, 2H), 4.76 (s,
2H), 2.25 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.1, 167.8, 166.3, 159.9 (d, J = 238.1
Hz), 149.1, 143.7 (d, J = 13.3 Hz), 137.6, 134.3, 132.8, 130.7, 129.9, 127.0, 116.2, 114.0 (d, J
= 26.7 Hz), 113.2 (d, J = 10.5 Hz), 105.7 (d, J = 24.1 Hz), 46.7, 45.3, 21.1. HRMS (ESI):
calcd for C21H16FN3O3S2 [M + H]+442.0651, found 442.0641.
4.2.23.
(Z)-2-(5-((6-fluoro-1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (10d). Rhodanineꢀ3ꢀacetic acid and 6d were used
1
as reactants to give 10d. Yellow solid, yield: 73%, mp:＞300 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.48 (s, 1H), 7.93 (s, 1H), 7.91 – 7.87 (m, 1H), 7.70 (dd, J = 9.2, 2.4 Hz, 1H),
7.27 – 7.21 (m, 1H), 7.16 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 5.82 (s, 2H), 4.75 (s,
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2H), 2.26 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.1, 167.8, 166.38, 160.6 (d, J = 241.1
Hz), 148.7 (d, J = 3.2 Hz), 140.2, 137.6, 136.5, 134.2, 129.9, 127.1, 127.0, 121.9 (d, J = 10.4
Hz), 116.3, 112.9 (d, J = 26.0 Hz), 98.5 (d, J = 27.9 Hz), 46.6, 45.2, 21.1. HRMS (ESI): calcd
for C21H16FN3O3S2 [M + H]+442.0651, found 442.0647.
4.2.24.
(Z)-2-(5-((5-methoxy-1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (10e). Rhodanineꢀ3ꢀacetic acid and 6e were used
as reactants to give 10e. Yellow solid, yield: 85%, mp: 295.3–296.6 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 13.44 (s, 1H), 7.92 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.32 (d, J = 2.3 Hz, 1H),
7.13 (d, J = 8.0 Hz, 2H), 7.05 – 7.00 (m, 3H), 5.78 (s, 2H), 4.73 (s, 2H), 3.83 (s, 3H), 2.23 (s,
3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.3, 167.8, 166.3, 157.4, 147.5, 144.6, 137.5, 134.5,
130.8, 129.9, 129.1, 127.0, 116.6, 116.5, 112.6, 101.5, 56.1, 46.5, 45.2, 21.1. HRMS (ESI):
calcd for C22H19N3O4S2 [M + H]+454.0821, found 454.0826.
4.2.25.
(Z)-2-(5-((6-methoxy-1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (10f). Rhodanineꢀ3ꢀacetic acid and 6f were used as
1
reactants to give 10f. Yellow solid, yield: 84%, mp: 294.2–295.7 °C. H NMR (400 MHz,
DMSOꢀd6) δ 13.43 (s, 1H), 7.84 (s, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H),
7.14 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 6.98 (dd, J = 8.9, 2.3 Hz, 1H), 5.79 (s, 2H),
4.72 (s, 2H), 3.82 (s, 3H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.2, 167.8, 166.3,
158.6, 146.8, 138.5, 137.5, 137.2, 134.5, 129.9, 127.8, 127.0, 121.4, 116.7, 115.0, 94.2, 56.3,
46.2, 45.2, 21.1. HRMS (ESI): calcd for C22H19N3O4S2 [M + H]+ 454.0821, found 454.0812.
4.2.26.
(Z)-5-((1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-2-
thioxothiazolidin- 4-one (10g). Rhodanine and 5g were used as reactants to give 10g. Yellow
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solid, yield: 86%, mp:＞300 °C. 1H NMR (400 MHz, DMSOꢀd6) δ 13.77 (s, 1H), 7.80 (d, J =
7.5 Hz, 1H), 7.70 (d, J = 6.8 Hz, 2H), 7.38 – 7.30 (m, 2H), 7.12 (d, J = 7.9 Hz, 2H), 7.03 (d, J
= 7.9 Hz, 2H), 5.78 (s, 2H), 2.23 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ 200.7, 169.2,
147.8, 143.5, 137.5, 136.0, 134.5, 133.8, 129.9, 127.0, 125.1, 124.0, 120.4, 114.5, 111.9, 46.4,
21.1. HRMS (ESI): calcd for C19H15N3OS2 [M + H]+366.0752, found 366.0743.
4.2.27.
(Z)-3-(5-((1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)propanoic acid (10h). Rhodanineꢀ3ꢀpropanoic acid and 5g were used
as reactants to give 10h. Yellow solid, yield: 92%, mp: 267.1–269.6 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 12.49 (s, 1H), 7.86 (s, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H),
7.35 (dq, J = 13.5, 6.6 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 5.81 (s, 2H),
4.25 – 4.20 (m, 2H), 2.67 – 2.62 (m, 2H), 2.23 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ
198.4, 172.2, 166.7, 147.8, 143.6, 137.5, 136.0, 134.5, 130.8, 129.9, 127.0, 125.3, 124.1,
120.5, 115.5, 112.0, 46.4, 31.4, 21.1. HRMS (ESI): calcd for C22H19N3O3S2 [M +
H]+438.0934, found 438.0926.
4.2.28.
(Z)-2-(5-((5-chloro-1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (11a). Rhodanineꢀ3ꢀacetic acid and 7a were used
as reactants to give 11a. Yellow solid, yield: 71%, mp: 296.4–298.1 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 13.50 (s, 1H), 8.02 (s, 1H), 7.94 (d, J = 1.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H),
7.42 (dd, J = 8.8, 2.0 Hz, 1H), 7.40 – 7.34 (m, 1H), 7.27 – 7.22 (m, 1H), 7.19 – 7.10 (m, 2H),
5.95 (s, 2H), 4.75 (s, 2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.1, 167.8, 166.3, 160.49 (d, J
= 245.9 Hz), 149.2, 144.1, 134.7, 131.1, 130.8 (d, J = 8.2 Hz), 129.6 (d, J = 3.8 Hz), 128.7,
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125.5 (d, J = 3.4 Hz), 123.9 (d, J = 14.5 Hz), 119.8, 116.4, 116.2, 113.5, 45.3, 42.0 (d, J = 3.4
Hz). HRMS (ESI): calcd for C20H13ClFN3O3S2 [M + H]+ 462.0142, found 462.0131.
4.2.29.
(Z)-2-(5-((6-chloro-1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (11b). Rhodanineꢀ3ꢀacetic acid and 7b were used
as reactants to give 11b. Yellow solid, yield: 76%, mp: 288.7–290.1 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 13.48 (s, 1H), 7.98 (s, 1H), 7.90 (d, J = 1.8 Hz, 1H), 7.87 (d, J = 8.7 Hz, 1H),
7.41 – 7.36 (m, 2H), 7.28 – 7.22 (m, 1H), 7.20 – 7.10 (m, 2H), 5.94 (s, 2H), 4.75 (s, 2H). 13
C
NMR (101 MHz, DMSOꢀd6) δ 198.1, 167.8, 166.3, 160.48 (d, J = 246.0 Hz), 148.9, 142.1,
136.6, 130.8, 129.9, 129.5 (d, J = 3.8 Hz), 125.4 (d, J = 3.3 Hz), 124.4, 123.8 (d, J = 14.6 Hz),
121.9, 116.2, 111.9, 45.3, 41.9 (d, J = 2.9 Hz). HRMS (ESI): calcd for C20H13ClFN3O3S2 [M
+ H]+462.0142, found 462.0137.
4.2.30.
(Z)-2-(5-((5-fluoro-1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (11c). Rhodanineꢀ3ꢀacetic acid and 7c were used
as reactants to give 11c. Yellow solid, yield: 77%, mp: 292.4–293.6 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 13.47 (s, 1H), 8.01 (s, 1H), 7.73 (dd, J = 9.0, 4.7 Hz, 1H), 7.68 (dd, J = 9.5, 2.3
Hz, 1H), 7.39 – 7.35 (m, 1H), 7.31 – 7.21 (m, 2H), 7.18 – 7.10 (m, 2H), 5.94 (s, 2H), 4.75 (s,
2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.1, 167.8, 166.3, 160.6 (d, J = 240.6 Hz), 160.5 (d,
J = 246.0 Hz), 148.8 (d, J = 3.1 Hz), 140.2, 136.4 (d, J = 14.0 Hz), 130.9 (d, J = 8.1 Hz),
123.0, 129.6 (d, J = 3.8 Hz), 125.4 (d, J = 3.4 Hz), 123.7 (d, J = 14.6 Hz), 122.0 (d, J = 10.3
Hz), 116.4, 116.2, 112.9 (d, J = 25.7 Hz), 98.4 (d, J = 28.1 Hz), 45.2, 41.9 (d, J = 2.6 Hz).
HRMS (ESI): calcd for C20H13F2N3O3S2 [M + H]+446.0471, found 446.0438.
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4.2.31.
(Z)-2-(5-((6-fluoro-1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (11d). Rhodanineꢀ3ꢀacetic acid and 7d were used
as reactants to give 11d. Yellow solid, yield: 83%, mp: 275.4–277.3 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 13.46 (s, 1H), 7.97 (s, 1H), 7.90 – 7.86 (m, 1H), 7.64 (dd, J = 9.2, 2.3 Hz, 1H),
7.39 – 7.34 (m, 1H), 7.28 – 7.20 (m, 2H), 7.18 – 7.11 (m, 2H), 5.91 (s, 2H), 4.74 (s, 2H). 13
C
NMR (101 MHz, DMSOꢀd6) δ 198.2, 167.8, 166.3, 160.5 (d, J = 245.9 Hz),159.9 (d, J =
237.9 Hz), 149.3, 143.6 (d, J = 13.3 Hz), 132.6, 130.8 (d, J = 8.4 Hz), 130.6 129.6 (d, J = 3.8
Hz), 125.4 (d, J = 3.3 Hz), 123.8 (d, J = 14.5 Hz), 116.4, 116.2, 113.9 (d, J = 26.6 Hz), 113.1
(d, J = 10.1 Hz), 105.9 (d, J = 24.2 Hz), 45.2, 41.9 (d, J = 3.1 Hz). HRMS (ESI): calcd for
C20H13F2N3O3S2 [M + H]+ 446.0451, found 446.0442.
4.2.32.
(Z)-2-(5-((1-(2-fluorobenzyl)-5-methoxy-1H-benzo[d]imidazol-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (11e). Rhodanineꢀ3ꢀacetic acid and 7e were used
as reactants to give 11e.Yellow solid, yield: 80%, mp: 278.8–280.2 °C.1H NMR (400 MHz,
DMSOꢀd6) δ 13.46 (s, 1H), 7.99 (s, 1H), 7.58 (d, J = 9.0 Hz, 1H), 7.38 – 7.32 (m, 2H), 7.26 –
7.21 (m, 1H), 7.15 (t, J = 7.4 Hz, 1H), 7.09 – 7.05 (m, 1H), 7.01 (dd, J = 9.0, 2.3 Hz, 1H),
5.89 (s, 2H), 4.74 (s, 2H), 3.83 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ198.4, 167.8, 166.4,
160.4 (d, J = 245.8 Hz), 157.4, 147.7, 144.5, 130.7 (d, J = 8.2 Hz), 129.5 (d, J = 3.8 Hz),
129.1, 125.4 (d, J = 3.4 Hz), 124.1 (d, J = 14.5 Hz), 116.7, 116.5, 116.1 (d, J = 20.9 Hz),
112.6, 101.6, 56.1, 45.2, 41.7 (d, J = 3.2 Hz). HRMS (ESI): calcd for C21H16FN3O4S2 [M +
H]+ 458.0619, found 458.0623.
4.2.33. (Z)-2-(5-((1-(2-fluorobenzyl)-6-methoxy-1H-benzo[d]imidazol-2-yl) methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid (11f). Rhodanineꢀ3ꢀacetic acid and 7f were used as
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reactants to give 11f. Yellow solid, yield: 91%, mp: 283.4–285.1 °C.1H NMR (400 MHz,
DMSOꢀd6) δ 13.44 (s, 1H), 7.91 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.38 – 7.34 (m, 1H), 7.29 –
7.23 (m, 2H), 7.15 (t, J = 7.6, 0.8 Hz, 1H), 7.03 (t, J = 7.7, 1.2 Hz, 1H), 6.99 (dd, J = 8.9, 2.3
Hz, 1H), 5.90 (s, 2H), 4.73 (s, 2H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSOꢀd6) δ198.2,
167.8, 166.3, 160.4 (d, J = 245.6 Hz), 158.5, 147.0, 138.5, 137.0, 130.7 (d, J = 8.0 Hz), 128.0,
125.4 (d, J = 3.4 Hz), 124.1 (d, J = 14.6 Hz), 121.4, 116.8, 116.1 (d, J = 20.7 Hz), 114.9, 94.3,
56.2, 45.2, 41.4 (d, J = 3.5 Hz). HRMS (ESI): calcd for C21H16FN3O4S2 [M + H]+ 458.0619,
found 458.0626.
4.2.34.
(Z)-5-((1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-2-
thioxothiazolidin-4-one (11g). Rhodanine and 5j were used as reactants to give 11g. Yellow
solid, yield: 90%, mp: 258.4–260.1 °C.1H NMR (400 MHz, DMSOꢀd6) δ 13.81 (s, 1H), 7.89
– 7.61 (m, 3H), 7.27 (m, 4H), 7.19 – 6.98 (m, 2H), 5.88 (s, 2H). 13C NMR (101 MHz,
DMSOꢀd6) δ 200.7, 169.2, 160.4 (d, J = 245.8 Hz), 148.0, 143.5, 135.8, 133.9, 130.7 (d, J =
8.1 Hz), 129.5 (d, J = 3.6 Hz), 125.8.6, 125.3 (d, J = 3.2 Hz), 124.1 (d, J = 14.5 Hz), 120.4,
116.1 (d, J = 20.9 Hz), 114.6, 111.8, 41.6 (d, J = 3.3 Hz). HRMS (ESI): calcd for
C14H12FN3OS2 [M + H]+370.0418, found 370.0432.
4.2.35.
(Z)-3-(5-((1-(2-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)methylene)-4-
oxo-2-thioxothiazolidin-3-yl)propanoic acid (11h). Rhodanineꢀ3ꢀpropanoic and 5j were used
as reactants to give 11h. Yellow solid, yield: 86%, mp: 258.4–260.1 °C. 1H NMR (400 MHz,
DMSOꢀd6) δ 12.51 (s, 1H), 7.94 (s, 1H), 7.85 – 7.81 (m, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.39 –
7.31 (m, 3H), 7.27 – 7.22 (m, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.06 (t, J = 7.0 Hz, 1H), 5.92 (s,
2H), 4.26 – 4.20 (m, 2H), 2.68 – 2.62 (m, 2H). 13C NMR (101 MHz, DMSOꢀd6) δ 198.4,
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172.2, 166.7, 160.4 (d, J = 245.9 Hz), 148.0, 143.5, 135.8, 131.00, 130.8 (d, J = 8.2 Hz),
129.6 (d, J = 3.5 Hz), 125.4 (d, J = 7.2 Hz), 124.1, 120.5, 116.1 (d, J = 20.9 Hz), 115.6, 111.9,
41.6, 41.5, 31.4. HRMS (ESI): calcd for C21H16FN3O3S2 [M + H]+ 442.0617, found 442.0623.
4. 3. Biological evaluation
4.3.1. Topo I inhibitory activity
In this relaxation assay, pBR322 plasmid (TaKaRa, Kyoto, Japan) was used to determine the
effects of the synthesized compounds on DNA relaxation catalytic by Topo I (TaKaRa, Kyoto,
Japan) by using camptothecin as a positive control. The reaction mixture was prepared
according to the literature with minor modifications, and incubated at 37 °C for 30 min. The
reactions were terminated by the addition of dye solution containing 1% SDS, 0.02%
bromophenol blue and 50% glycerol. The mixtures were applied to 1% agarose gel and
subjected to electrophoresis for 1.5 h, in 1×TAE buffer (40 mM Trisꢀacetate, 2 mM EDTA).
Gels were stained for 30 min in an aqueous solution of Ged Red (0.5 ꢁg·ml−1). DNA bands
were visualized by transillumination with UV light and then photographed with an Alpha
Innotech digital imaging system6.
4.3.2. Topo I-mediated DNA unwinding assay
Relaxed pBR322 DNA plasmid utilized in this unwinding assay was generated by treating
pBR322 with Topo I in the reaction buffer (50 mM TrisꢀHCl, pH 7.5, 50 mM KCl, 10 mM
MgCl2, 0.5 mM DTT, 0.1 mM EDTA, and 30 ꢁg.mlꢀ1 BSA) prior to the addition of other
components. Assay mixtures is composed of 0.1 ꢁg relaxed pBR322 DNA, Topo I (4 U), and
the tested compounds in 20 ꢁL Topo I reaction buffer. Following a 10 min incubation of DNA
and drugs at room temperature, Topo I (1 U) was added, and the reaction were incubated at
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37 °C for 30 min. An equal volume of phenol chloroform was added to stop the reaction.
Aqueous samples (20 ꢁL) were removed from the reaction, and 3 ꢁL of stop solution (0.77%
SDS, 77 mmol NaEDTA, pH 8.0) followed by 2 ꢁL of agarose gel loading buffer was added
to each sample. Samples were subjected to electrophoresis in 1×TAE buffer (40 mM
Trisꢀacetate, 2 mM EDTA). DNA bands were visualized by transillumination with UV light
and then photographed with an Alpha Innotech digital imaging system8.
4.3.3. Topo II inhibitory activity
Relaxation assay were carried out according to the manufacturer’s instructions with minor
modifications. The reaction mixture containing 200 ng of pBR322 DNA plasmid and 1 U of
Topo II (TopoGEN) was incubated in the presence or absence of the synthesized compounds
in a final volume of 20 ꢁL in Topo II reaction buffer (50 mM TrisꢀHCl, pH 8.0, 150 mM NaCl,
10 mM MgCl2, 2 mM ATP, 0.5 mM DTT, and 30 ꢁg·mLꢀ1 BSA). The mixture was incubated
for 30 min at 37 °C and was terminated with 6× stop buffer (5 ꢁL per 20 ꢁL reaction volume).
Stop buffer contained 5% sarcosyl, 0.02% bromophenol blue, and 25% glycerol. Reaction
products were analyzed on a 1% agarose gel and electrophoresis in TAE buffer (40 mM
Trisꢀacetate and 2 mM EDTA) for 1.5 h at 75 V. Gels were stained for 30 min in an aqueous
solution of Ged Red (0.5 ꢁg·mLꢀ1). DNA bands were visualized through transillumination
with UV light and then photographed with an Alpha Innotech digital imaging system16.
4.3.4. Topo II-mediated DNA cleavage reaction assay
In brief, Topo II (6 U), 0.1 ꢁg of pBR322 DNA, and 50 ꢁM synthesized compounds (or
etoposide, 100 ꢁM) were employed in a total of 20 ꢁL of Topo II buffer (50 mM TrisꢀHCl, pH
8.0, 150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM DTT, and 30 ꢁg·mLꢀ1 BSA). After
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incubating for 6 min at 37 °C to reach the cleavage religation equilibrium, cleavage
intermediates were trapped by adding 2 ꢁL of 1% SDS, followed by 2 ꢁL of 250 mM
NaEDTA at pH 8.0. Proteinase K was added (2 ꢁL of 0.8 mg·mLꢀ1), and reactions were
incubated for 30 min at 45 °C to digest the Topo II. Samples were mixed with 2 ꢁL of agarose
gel loading buffer (30% sucrose, 0.5% bromophenol blue, and 0.5% xylene cyanole FF in 10
mM TrisꢀHCl, pH 7.9), heated at 72 °C for 2 min, and subjected to electrophoresis in a 1%
agarose gel in TAE buffer (40 mM Trisꢀacetate and 2 mM EDTA) for 1 h at 75 V. Gels were
stained for 30 min in an aqueous solution of Ged Red (0.5 ꢁg· mLꢀ1) and kept on
electrophoresis for 30ꢀ45 min at 75 V. Cleavage was monitored by the conversion of
negatively supercoiled plasmid to nicked DNA. DNA bands were visualized by UV light and
photographed with an Alpha Innotech digital imagingsystem16.
4.3.5. MTT assay
Briefly, the cells were plated at a density of 5000 per well in 96ꢀwell microplates and allowed
to incubate over night. The compounds were added to the wells at increasing concentrations
(0ꢀ50 ꢁM). After 48 h, each well was treated with 20 ꢁL MTT (2.5 mg·mLꢀ1) solution, and the
cells were further incubated at 37 °C for 4 h. At the end of the incubation, the untransformed
MTT was removed, and 100 ꢁL of DMSO was added. The microplates were well shaken to
dissolve the formazan dye, and the absorbance at 570 nm was measured using a microplate
reader (BioꢀTek)29.
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Figure and Scheme Captions
Fig. 1. Design of novel agents as potential Topo IIꢀtargeting anticancer agents.
Fig. 2. Chemical structure of the target compounds.
Fig. 3. Agarose gel assay for Topo II inhibition by the synthesized compounds. (A, B, C, and
D), lane D, pBR322 DNA; lane T, pBR322 DNA + Topo II; lane E, pBR322 DNA+ Topo II +
etoposide (100 ꢁM); other lanes, pBR322 DNA + Topo II + the synthesized compounds.
Fig. 4. Topo I inhibitory activity of the synthesized compounds. (A), (B), (C) and (D) lane D:
pBR322 DNA; lane T: pBR322 DNA + Topo I; lane C: pBR322 DNA + Topo I +
camptothecin (100 ꢁM); other lanes: pBR322 DNA + Topo I + the synthesized compounds
(50 ꢁM).
Fig. 5. (A) Effects of compounds 8g and 8j on Topo II mediated DNA cleavage complexes
formation. Lanes 1ꢀ2: control group of supercoiled pBR322 DNA without or with Topo II;
lanes 3ꢀ5, effects of etoposide (100 ꢁM) and tested compound (50 ꢁM) on Topo II with DNA.
Lanes 6ꢀ7, pretreatment of tested compounds (50 ꢁM) antagonizes the etoposide (100
ꢁM)ꢀenhanced DNA cleavage. The positions of supercoiled DNA (S), relaxed DNA (R),
28