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Ten cytoselective compounds have been identified from 372 thiazolidinone analogues by applying iterative
library approaches. These compounds selectively killed both non-small cell lung cancer cell line H460 and
its paclitaxel-resistant variant H460taxR at an IC50 between 0.21 and 2.93 M while showing much less
toxicity to normal human fibroblasts at concentrations up to 195 M. Structureactivity relationship studies
revealed that (1) the nitrogen atom on the 4-thiazolidinone ring (ring B in Figure 1) cannot be substituted,
(2) several substitutions on ring A are tolerated at various positions, and (3) the substitution on ring C is
restricted to the -NMe2 group at the 4-position. A pharmacophore derived from active molecules suggested
that two hydrogen bond acceptors and three hydrophobic regions were common features. Activities against
P-gp-overexpressing and paclitaxel-resistant cell line H460taxR and modeling using a previously validated
P-gp substrate pharmacophore suggested that active compounds were not likely P-gp substrates.
Introduction
Lung cancer is the number one cause of cancer-related deaths
worldwide and in the United States (160,000 deaths annually).
Despite advances in cancer research, the overall five-year
survival of lung cancer patients remains a dismal 15% in contrast
to most other solid organ tumors according to the American
Association for Cancer Research (http://www.aacr.org/home/
public--media/for-the-media/fact-sheets/organ-site-fact-sheets/
lung-cancer.aspx). More than 80% of bronchogenic malignancy
is from non-small cell lung cancer (NSCLC).a Among other
factors, inherent and acquired resistance to treatment1 and the
dose-limiting toxicity caused by the narrow therapeutic window
of many cancer drugs2 are recognized as major obstacles for
effective cancer therapy. Multidrug resistance (MDR) is a
phenotype of cross-resistance to multiple drugs with diverse
chemical structures. One of the well-documented MDR mechanisms is the overexpression of the MDR-1 gene that encodes
the transmembrane, ATP-dependent drug efflux transporter
P-glycoprotein (P-gp) in response to chemotherapy.35 P-gp
prevents the intracellular accumulation of many cancer drugs
by increasing their efflux out of cancer cells as well as through
hepatic, renal, or intestinal clearance pathways.4 Attempts to
* Author to whom correspondence should be addressed (telephone
+9014952797; fax +9014955715; e-mail bing.yan@stjude.org).
Shandong University.
Zhou et al.
a
Reagents and conditions: (i) NH4SCN (2), 6 N HCl, 80 ; (ii) NH3 H2O
(4), rt; (iii) ethyl chloroacetate (6); NaOAc, EtOH, 60 C; (iv) aldehydes
(8), piperidine, EtOH, 60 C.
a
Reagents and conditions: (i) R1, -NH2, (10), rt; (ii) ethyl chloroacetate
(6), NaOAc, EtOH, 60 C; (iii) aldehydes (8), piperideine, EtOH, 60 C.
as low as 300 nM. Except for compound I-25 (IC50 for NHFB
is 195 M), NHFBs did not reach 50% cell inhibition at the
highest compound concentration used (100 M) for active
compounds. Higher compound concentrations could not be used
due to the solubility limitation and the limited allowable DMSO
(1% for cells used here) in cell cultures. We tentatively took
>100 M as the IC50 of the compounds tested against NHFB
cells. It was noted that compounds such as I-27-, I-47-, III-
Zhou et al.
Figure 5. Time-dependent morphology changes of H460, H460taxR, and NHFB cells in the presence of compound I-7 at 1 M. The magnification
is 200.
Table 1. P-gp Pharmacophore Modeling for Group 1
compd no.
R1
R2
P-gp
substrate fit
I-18
I-36
I-48
I-69
I-94
I-98
I-114
I-118
I-126
I-132
I-196
I-213
I-233
H
2-Me
4-Me
2-Cl
4-Cl
4-Cl
2-OMe
2-OMe
3-NO2
3-NO2
4-OH
4-phenoxy
4-ethoxycarbonyl
4-SMe
9H-fluoren-2-yl
2-Cl
4-formyl
2,4,5-trimethoxy
4-SMe
2,4,5-trimethoxy
4-SMe
2-OH
4-naphthalen-2-yl
9H-fluoren-2-yl
4-COOH
4-COOH
no mapa
2.64b
no map
no map
3.86
1.75
3.62
0.716
no map
3.21
0.702
2.6
2.61
features
mapped
4H, 1HBAc
4H,
3H,
4H,
3H,
1HBA
2HBA
1HBA
2HBA
3H,
4H,
3H,
3H,
2HBA
2HBA
2HBA
2HBA
a
If two or more features were missed, the compound was designated
no map. b Higher values suggested better map. c H, hydrophobe; HBA,
hydrogen bond acceptor.
R1
R2
I-7
I-25
I-27
I-47
I-67
I-86
I-87
I-166
I-187
III-289
III-324
H
2-Me
2-Me
4-Me
2-Cl
4-Cl
4-Cl
3-CF3
4-OH
3-Cl
4-OMe
4-NMe2
4-OMe
4-NMe2
4-NMe2
4-NMe2
2-OH
4-NMe2
2-OH
4-NMe2
4-NHAc
4-NMe2
no mapa
no map
no map
no map
no map
1.88b
1
no map
no map
3.24
1.4
features mapped
3H, 2HBAc
3H, 2HBA
3H, 2HBA
3H, 2HBA
a
If two or more features were missed, the compound was designated
no map. b Higher values suggested better map. c H, hydrophobe; HBA,
hydrogen bond acceptor.
Figure 6. P-gp substrates mapped to the substrate pharmacophore.36 Green spheres represent hydrogen bond acceptors, and cyan features represent
hydrophobic features.
Table 3. Cytoselective Anticancer Activity
IC50(M)
R1
R2
H460
H460taxR
NHFB
I-7(95%)
I-25(95%)
I-27(94%)
I-47(96%)
I-67(93%)
I-86(90%)
I-87(85%)
I-187(93%)
III-289(95%)
III-324(93%)
H
2-Me
2-Me
4-Me
2-Cl
4-Cl
4-Cl
4-OH
3-Cl
4-OMe
4-NMe2
4-OMe
4-NMe2
4-NMe2
4-NMe2
2-OH
4-NMe2
4-NMe2
4-NHAc
4-NMe2
0.50 ( 0.15
1.78 ( 0.60
0.65 ( 0.25
0.94 ( 0.20
0.73 ( 0.02
1.85 ( 0.65
2.89 ( 0.03
1.32 ( 0.25
1.19 ( 0.02
2.52 ( 0.12
0.21 ( 0.03
1.70 ( 0.50
0.54 ( 0.10
0.82 ( 0.07
0.88 ( 0.01
1.18 ( 0.03
2.93 ( 0.12
1.08 ( 0.02
1.25 ( 0.10
1.60 ( 0.10
>100
195
>100
>100
>100
>100
>100
>100
>100
>100
Zhou et al.
5-(4-Dimethamino-benzylidene)-2-phenylimino-3-(2-hydroethyl)-thiazolidin-4-one (II-18). II-18 was prepared from 3-(2hydroxyethyl)-2-phenylimino-thiazolidin-4-one and 4-dimethaminobenzaldehyde (AL007) according to the procedure described above:
yield, 71.0%, yellow powder; 1H NMR (400 MHz, MeOD), 7.52
(s, 1H), 7.26 (m, 4H), 7.08 (t, 1H, J ) 7.5 Hz), 6.91 (t, 2H, J )
9.6 Hz), 6.64 (d, 2H, J ) 9.0 Hz), 4.02 (t, 2H, J ) 6.0 Hz), 3.78
(t, 2H, J ) 5.9 Hz), 2.92 (d, 6H, J ) 10.0 Hz); ESI-MS, m/z 368.7
(M + 1). HR-MS calculated for C20H21N3O2S (M + H)+: 368.1433;
found 368.1447.
5-(4-Hydroxy-3-methoxybenzylidene)-2-phenylimino-3-(2-hydroethyl)-thiazolidin-4-one (II-20). II-18 was prepared from 3-(2hydroxyethyl)-2-phenylimino-thiazolidin-4-one and 4-hydroxy-3methoxybenzaldehyde (AL003) according to the procedure described
above: yield, 70.0%, yellow powder; 1H NMR (400 MHz, MeOD),
8.05 (s, 1H), 7.40 (t, 2H, J ) 7.9 Hz), 7.19 (t, 1H, J ) 7.5 Hz),
7.05 (d, 2H, J ) 8.4 Hz), 6.93 (s, 1H), 6.73 (s, 1H), 4.15 (t, 2H, J
) 5.9 Hz), 3.89 (m, 8H), 3.71 (s, 3H); ESI-MS, m/z 415.8 (M +
1). HR-MS calculated for C21H22N2O5S (M + H)+: 415.1328: found
415.1324.
5-(2,4,5-Trimethoxy-benzylidene)-2-phenylimino-3-(3-methoxy-propyl-thiazolidin-4-one (II-25). II-25 was prepared from
3-(3-methoxy-propyl)-2-phenylimino-thiazolidin-4-one and 2,4,5trimethoxy-benzaldehyde (AL014) according to the procedure
described above: yield, 77.0%, yellow powder; 1H NMR (400 MHz,
MeOD), 7.91 (s, 1H), 7.27 (t, 2H, J ) 7.7 Hz), 7.07 (t, 1H, J )
7.5 Hz), 6.92 (d, 2H, J ) 8.4 Hz), 6.78 (s, 1H), 6.61 (d, 1H, J )
13.2 Hz), 3.96 (t, 2H, J ) 6.9 Hz), 3.79 (d, 6H, J ) 4.5 Hz), 3.58
(s, 3H), 3.41 (t, 2H, J ) 6.0 Hz), 3.23 (s, 3H), 1.94 (p, 2H, J ) 6.5
Hz); ESI-MS, m/z 443.7 (M + 1). HR-MS calculated for
C23H26N2O5S (M +H)+: 443.1641; found 443.1648.
General Procedure for the Library Synthesis. Using a 10
10 library as an example, 10 intermediates 7 or 12 (5 mmol) were
dissolved in 10 mL of absolute ethanol. Ultrasonic and heating baths
were used to make sure they were all dissolved. Each solution was
divided into 10 parts. Next, 10 different benzylaldehydes (6 mmol)
were dissolved in 10 mL of absolute ethanol and divided into 10
parts following the same procedure. Each of the 100 reaction vessels
contained a solution of intermediate 7 or 12 (0.5 mmol) and different
benzylaldehydes (0.6 mmol) in 2 mL of absolute ethanol. After
piperidine was added (50 L, 0.5 mmol), all reaction vessels were
put into a Zhicheng ZHMY-113H parallel synthesizer and shaken
overnight. The conversion of the reactions was monitored by HPLC/
MS. After reaction completion, the reaction vessels were cooled
to room temperature, and the solutions were simultaneously filtered
and washed with absolute ethanol repeatedly to yield the target
products. All of the residues were dried in a vacuum at 60 C
overnight and analyzed by an HPLC/MS system.
Cells and Culture Conditions. The human NSCLC H460 cell
line and paclitaxel-resistant and P-gp-overexpressing H460taxR cell
lines were routinely propagated in monolayer culture in RPMI 1640
medium supplemented with 10% heat-inactivated fetal calf serum,
100 units/mL penicillin, and 100 mg/mL streptomycin. NHFBs were
maintained in Dulbeccos modified Eagles medium with the same
supplements. All cells were maintained in the presence of 5% CO2
at 37 C.
Cytotoxicity Assays. The inhibitory effects of thiazolidinone
analogues on cell growth were determined by the sulforhodamine
B assay. Cells [(28) 103 cells in 100 L of culture medium/
well] were seeded in 96-well flat-bottom plates and treated the next
day with the drugs at the indicated concentrations. The IC50 was
determined by sulforhodamine B assay 72 h after treatment, as
described previously.40,41 DMSO solvent was used to dissolve
compounds, and its volume was <1% in the assay medium. An
equal volume of DMSO was used as a control. The experiments
were performed at least three times for each cell line. Cell viability
was calculated using the following formula: cell viability ) 100
Atreatment/Acontrol (percentage). The IC50 was determined by the
SigmaPlot 10.0 program (Systat Software, Inc., San Jose, CA) using
a four-parameter logistic function for the sigmoid doseresponse
curves.
Zhou et al.
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