Synthesis and evaluation of 5-aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2- (3H)-thiones as P-glycoprotein inhibitors

Article abstract of DOI:10.1248/cpb.51.728

5-Aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 3 were prepared by cyclocondensation of 1-(4-hydroxyphenyl)-2-aroylhydrazines with thiophosgene. All compounds exhibited antiproliferation activity in K562, IC₅₀ ranging from 24 to 94 μM comparable efficacy with apigenin and genistein and showed more potent antiproliferation of K562/adr cells, highly expressing P-glycoprotein. Compounds 3g, 3e and 3a inhibited the function of P-glycoprotein with the α_0.5 equal to 10±3 μM, 21±5 μM and 34±7 μM, respectively.

Full text of DOI:10.1248/cpb.51.728

728
Notes
Chem. Pharm. Bull. 51(6) 728—730 (2003)
Vol. 51, No. 6
Synthesis and Evaluation of 5-Aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-
2-(3H)-thiones as P-Glycoprotein Inhibitors
a
b
,a
*
Chatchanok LOETCHUTINAT, François CHAU, and Samlee MANKHETKORN
a Laboratory of Physical Chemistry Molecular and Cellular Biology, Faculty of Science, Burapha University; Bangsaen,
b
Chonburi 20131 Thailand: and Laboratoire de Chimie organique médicale, Université Paris 7, Faculté de Médecine
Xavier Bichat 16; rue Henri Huchard 75018 Paris France. Received December 24, 2002; accepted March 14, 2003
5-Aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 3 were prepared by cyclocondensation of 1-(4-
hydroxyphenyl)-2-aroylhydrazines with thiophosgene. All compounds exhibited antiproliferation activity in
K562, IC50 ranging from 24 to 94 mM comparable efficacy with apigenin and genistein and showed more potent
antiproliferation of K562/adr cells, highly expressing P-glycoprotein. Compounds 3g, 3e and 3a inhibited the
function of P-glycoprotein with the a0.5 equal to 10؎3 mM, 21؎5 mM and 34؎7 mM, respectively.
Key words oxadiazolethione; multidrug resistance (MDR); P-glycoprotein; inhibitor
Multidrug resistance (MDR) is a main reason for the can- 2(3H)-thiones 3 were synthesized and screened for their anti-
cer chemotherapy failure. One widely studied mechanism of cancer and P-glycoprotein inhibitor activities. It was found
this phenomenon involves the ability of an ATP dependent that all synthesized oxadiazolethiones 3 exhibited anticancer
170 kDa plasma membrane protein, P-glycoprotein, to act as activity against K562 cells with IC50 ranging from 24 to
an efficient efflux pump against anticancer drug molecules.1) 94 mM, comparable efficacy with apigenin and genistein
Some compounds such as cyclosporin A,2—4) verapamil,5,6) (Table 1). The antiproliferative effects of several symmetri-
estrogen7) and genistein8) have been reported to reverse cally substituted 5-phenyl-3-(4-hydroxyphenyl)-1,3,4-oxadia-
MDR, resulting in an increased accumulation of antitumor zole-2(3H)-thione 3a derivatives were studied. We intro-
agents in MDR cells.9,10)
duced various substituents on the C4 position of the phenyl
Among different series of oxadiazolethione synthesized in group: electron donating group, CH3 (3b) and OCH3 (3c),
our laboratory, we found that 5-aryl-3-(4-hydroxyphenyl)- electron withdrawing group, NO2 (3d) and halogen (3e, f).
1,3,4-oxadiazole-2(3H)-thiones 3 potently inhibited the func- (Table 1). As compared with unsubstituted phenyl lead com-
tionality of P-glycoprotein. Interestingly, they also showed pound 3a, electron donating substituted phenyl oxadiazo-
growth inhibitory properties in the K562 human leukemia lethiones (3b, c) were less cytotoxic. Electron withdrawing
cell line. Herein, we reported the synthesis of 5-aryl-3-(4-hy- (3d) or halogen (3e, f) substitutions did not increase cytotoxi-
droxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones and prelimi- city. The trimethoxy substituted phenyl oxadiazolethione 3g
nary in vitro results.
showed more potent cytotoxicity (IC50ϭ24Ϯ4 mM) than the
The synthesis of the compounds investigated in this work monomethoxy substituted analogue 3c (IC50ϭ94Ϯ10 mM). It
is outlined in Fig. 1. Condensation of substituted benzoic has been reported that trimethoxylated chalcone showed
acid hydrazide with 1,4-benzoquinone in acid conditions al- greater cytotoxicity against K562 cell line than the corre-
lowed to obtain benzoic acid (cyclohexadien-1-ylidene)hy- sponding mono and dimethoxylated derivatives.13) Interest-
drazide substituted derivatives 1a—g which were reduced ingly, oxadiazolethiones 3 exhibited potent antiproliferation
with phenylhydrazine11) in 1-butanol to afford the corre- of multidrug resistant K562/adr cells with resistance factor
sponding substituted benzoic acid 2-(4-hydroxyphenyl)hy- (RF) ranging from 0.5 to 1.0. It should be noted that 3a, 3b,
drazide 2a—g. Cyclocondensation of 2a—g with thiophos- 3e and 3g were at least 2 times more toxic in MDR tumor
gene12) in water yielded the 5-aryl-3-(4-hydroxyphenyl)- cells than the parental K562 cells.
1,3,4-oxadiazole-2(3H)-thiones 3a—g (Caution: reactions
involving thiophosgene were achived in a hood).
All oxadiazolethiones 3 used in our experiments inhibited
the functionality of P-glycoprotein except 3f. Compounds 3g,
3e and 3a showed a0.5 equal to 10Ϯ3mM, 21Ϯ5 mM and
34Ϯ7 mM, respectively. Compounds 3b, 3c, and 3d gave
Results and Discussion
Seven new 5-aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole- maximal inhibition only about 20% of the functionality of P-
Fig. 1. Synthetic Route to 1,3,4-Oxadiazole-2(3H)-thione Derivatives 3a—g
* To whom correspondence should be addressed. e-mail: samlee@bucc4.buu.ac.th
© 2003 Pharmaceutical Society of Japan

June 2003
729
3260, 1639. 1H-NMR (DMSO-d6, 200 MHz) d: 3.69, 3.82 (2S, 9H), 6.58,
6.64 (2m, 3H), 7.23, 7.38 (2d, 3H), 8.69 (br s, 1H), 10.27 (br s, 1H). (2e)
Yield: 41.50%; mp 202 °C (EtOH–H2O). IR (KBr) cmϪ1: 3274, 1642. 1H-
NMR (DMSO-d6, 200 MHz) d: 6.64 (m, 4H), 7.56, 7.92 (2d, 4H), 8.69 (br s,
1H), 10.40 (br s, 1H). (2f) Yield: 60%; mp 194 °C (EtOH–H2O) (191—
192 °C).14) IR (KBr) cmϪ1: 3245, 1664. 1H-NMR (DMSO-d6, 200 MHz) d:
6.64 (m, 4H), 7.71, 7.84 (2d, 4H), 8.70 (br s, 1H), 10.40 (br s, 1H). (2g)
Yield: 67.50%; mp 203 °C (AcOEt–petroleum ether). IR (KBr) cmϪ1: 3242,
3109, 1668. 1H-NMR (DMSO-d6, 200 MHz) d: 6.60 (m, 4H), 7.52 (br s,
1H), 8.12, 8.33 (2d, 4H), 8.72 (br s, 1H), 10.63 (br s, 1H).
General Procedure for the Preparation of 5-Aryl-3-(4-hydroxy-
phenyl)-1,3,4-oxadiazole-2(3H)-thione (3) To a stirred suspension of (2)
(10 mmol) in water (100 ml) was added at room temperature pure thiophos-
gene (1.53 ml, 20 mmol). The mixture was stirred 30 min and filtered. The
precipitate was washed with water, dried and recristallized using suitable
solvent.
Table 1. In Vitro Cytotoxicity against K562 and K562/adr Cells and In-
hibitory P-Glycoprotein Activity of Oxadiazolethiones 3
Substitutions
Compounds
IC50(S) mM
RF
a0.5 mM
X
Y
Z
3a
3b
3c
3d
3e
H
H
H
H
H
H
Me
OMe
NO2
Cl
H
H
H
H
H
34.0Ϯ2.0
48.0Ϯ1.0
94.0Ϯ10.0 0.7
37.0Ϯ 7.0
29.0Ϯ 6.0
35.0Ϯ2.0
24.0Ϯ4.0
50.0Ϯ9.0
27.0Ϯ2.0
0.6 34.0Ϯ7.0
0.6
A
A
A
0.8
0.6 21.0Ϯ5.0
1.0
0.5 10.0Ϯ3.0
3f
3g
H
OMe
Br
OMe
H
OMe
B
Genistein
Apigenin
0.9
0.9
B
B
A: 20% of maximum inhibition and B: not active
5-Phenyl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thione (3a): Yield:
72%; mp 232 °C (EtOH). IR (KBr) cmϪ1: 3300, 1590, 1517. 1H-NMR
glycoprotein. Trimethoxylated oxadiazolethione 3g also (DMSO-d6, 200 MHz) d: 6.93, 7.93 (2d, 4H), 7.65 (m, 5H), 10.03 (br s, 1H).
Anal. Calcd for C14H10N2O2S: C, 62.20; H, 3.72; N, 10.36. Found: C, 62.25;
showed more potent P-glycoprotein inhibition than the
H, 3.68; N, 10.42.
monomethoxylated analogue 3c. The results seem to suggest
5-(4-Methylphenyl)-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
that electronic effect do not play an important role. However,
they suggest that 3, 4, 5 trilipophilic substituents on the
phenyl ring are important for demonstrating cytotoxicity and
P-glycoprotein inhibition. In fact that apigenin and genistein,
up to 100 mM, did not inhibit the function of P-glycoprotein.
The present study clearly indicated that: (a) oxadiazol-
ethiones 3 exhibited anticancer properties and were more cy-
totoxic in multidrug resistant K562/adr cells than the
parental K562 cells, (b) they inhibited the functionality of P-
glycoprotein and (c) both activities were improved by trisub-
stitution of lipophilic methoxy group on the 3, 4 and 5 posi-
tions of the phenyl ring.
(3b): Yield: 71.50%; mp 210 °C (n-PrOH). IR (KBr) cmϪ1: 3420, 1606,
1569. 1H-NMR (DMSO-d6, 200 MHz) d: 2.40 (s, 3H), 6.92, 7.42, 7.71, 7.82
(4d, 8H), 10.02 (br s, 1H). Anal. Calcd for C15H12N2O2S: C, 63.36; H, 4.25;
N, 9.85. Found: C, 63.40; H, 4.31; N, 9.90.
5-(4-Methoxyphenyl)-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thi-
one (3c): Yield: 52%; mp 249 °C (EtOH–H2O). IR (KBr) cmϪ1: 3321, 1614,
1621. 1H-NMR (DMSO-d6, 200 MHz) d: 3.85 (s, 3H), 6.91, 7.15, 7.71, 7.88
(4d, 8H), 10.01 (br s, 1H). Anal. Calcd for C15H12N2O3S: C, 59.99; H, 4.02;
N, 9.32. Found: C, 60.08; H, 4.10; N, 9.40.
5-(3,4,5-Trimethoxyphenyl)-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2
(3H)-thione (3d): Yield: 49.50%; mp 212 °C (EtOH–H2O). IR (KBr) cmϪ1
:
3323, 1577, 1502. 1H-NMR (DMSO-d6, 200 MHz) d: 3.75, 3.87 (2s, 9H),
6.92, 7.71 (2d, 4H), 7.16 (s, 2H), 10.02 (br s, 1H). Anal. Calcd for
C17H16N2O5S: C, 56.65; H, 4.47; N, 7.77. Found: C, 56.70; H, 4.52; N, 7.85.
5-(4-Chlorophenyl)-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
(3e): Yield: 54.50%; mp 226 °C (n-PrOH). IR (KBr) cmϪ1: 3413, 1610,
Experimental
Chemistry 1H nuclear magnetic resonance spectra were recorded with a
Bruker AC200 spectrometer. Chemical shifts (d) are reported as ppm using
tetramethylsilane as internal standard. IR spectra were recorded in KBr pel-
lets on a Perkin-Elmer Spectrum BX I spectrophotometer. Elemental analy-
ses (C, H, N) were performed by the Service de microanalyse at the Univer-
sité Pierre et Marie Curie and were within Ϯ0.4% of the theoretical values.
Melting points were determined using a Kofler hotstage apparatus and are
uncorrected.
1
1515. H-NMR (DMSO-d6, 200 MHz) d: 6.92, 7.93 (2d, 4H), 7.68 (m, 4H),
10.03 (br s, 1H). Anal. Calcd for C14H9ClN2O2S: C, 55.17; H, 2.97; N, 9.19.
Found: C, 55.20; H, 3.02; N, 9.25.
5-(4-Bromophenyl)-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
(3f): Yield: 29%; mp 226 °C (n-PrOH). IR (KBr) cmϪ1: 3414, 1608, 1587.
1H-NMR (DMSO-d6, 200 MHz) d: 6.91, 7.70 (2d, 4H), 7.84 (m, 4H), 10.03
(br s, 1H). Anal. Calcd for C14H9BrN2O2S: C, 48.15; H, 2.59; N, 8.02.
Found: C, 48.17; H, 2.63; N, 8.10.
General Procedure for the Preparation of Benzoic Acid (Cyclohexa-
dien-1-ylidene)hydrazide Substituted Derivatives (1) 1,4-Benzoquinone
(5.40 g, 50 mmol) was suspended in water (350 ml) at room temperature and
benzoic acid hydrazide derivative (55 mmol) dissolved in HCl 10% (35 ml)
was added. The mixture was then stirred 15 min. The precipitate was col-
lected, washed and dried. It was used without further purification. (1a) mp
148 °C (143—144 °C).11) IR (KBr) cmϪ1: 3280, 1675, 1620, 1595. (1b) mp
124 °C. IR (KBr) cmϪ1: 3320, 1706, 1672, 1630. (1c) mp 80 °C. IR (KBr)
cmϪ1: 3445, 1665, 1620, 1590. (1d) mp 90 °C. IR (KBr) cmϪ1: 3430, 1683,
1607, 1582. (1e) mp 165 °C. IR (KBr) cmϪ1: 3270, 1699, 1681, 1638. (1f)
mp 172 °C. IR (KBr) cmϪ1: 3305, 1681, 1633, 1586. (1g) mp 240—242 °C.
IR (KBr) cmϪ1: 3108, 1670, 1643, 1602.
5-(4-Nitrophenyl)-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
(3g): Yield: 49.50%; mp 246 °C (n-PrOH). IR (KBr) cmϪ1: 3438, 1593,
1513. 1H-NMR (DMSO-d6, 200 MHz) d: 6.93, 7.71, 8.18, 8.41 (4d, 8H),
10.06 (br s, 1H). Anal. Calcd for C14H9N3O4S: C, 53.33; H, 2.87; N, 13.32.
Found: C, 53.38; H, 2.98; N, 13.38.
Cytotoxicity Assay Anticancer and P-glycoprotein inhibitor activities of
oxadiazolethiones 3a—g were performed by using human myelogenous
leukemia K562 and K562/adr cell line with overexpression of the P-glyco-
protein.9,10) In vitro cytotoxic activity was measured by incubation of 5ϫ104
cells/ml in the presence of various concentrations of compounds 3a—g for
72 h in 24-well plates. IC50 (50% inhibitory drug concentration) was deter-
mined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) colorimetric assay. RF represents the ratio of the IC50 in the resistant
cells to the IC50 level in the corresponding sensitive cells (IC50(S)).
Inhibition of P-Glycoprotein Functionality The efficacy of molecules
to inhibit P-glycoprotein activity was evaluated by following an increase in
intracellular concentration of pirarubicin in K562/adr cells in the presence
of inhibitor, has been extensively described.15—17) Briefly, 2ϫ106 cells were
incubated in 2 ml of HEPES-Naϩ in 1 cm quartz cuvettes, vigorously stirred
at 37 °C, then 1 mM pirarubicin was added to the cells. The fluorescence in-
tensity of pirarubicin at 590 nm (excited at 480 nm) was followed as a func-
tion of time (Perkin-Elmer model LS 50B spectrofluorometer). At the steady
state, the addition of MDR modulators verapamil or compound 3 yielded a
decrease in fluorescence intensity to reach a new steady state Fin. The ability
of compound 3 (a) to inhibit the P-gp-mediated efflux of a drug can be de-
termined using the ratio
General Procedure for the Preparation of Benzoic Acid 2-(4-Hydroxy-
phenyl)hydrazide Substituted Derivatives (2) To a stirred suspension of
(1) (35 mmol) in 1-butanol (75 ml) was added dropwise at 0 °C pure phenyl-
hydrazine (7.57 g, 70 mmol). The mixture was then allowed to rise to room
temperature and remained for 1 h. Excess of petroleum ether was added and
after 15 min stirring, the resulting precipitate was collected by filtration and
washed with petroleum ether. (2a) Yield: 48%; mp 172 °C (AcOEt–petro-
leum ether) (170—171 °C).14) IR (KBr) cmϪ1: 3225, 1730, 1630. 1H-NMR
(DMSO-d6, 200 MHz) d: 6.64, 7.52, 7.90 (3m, 9H), 8.68 (br s, 1H), 10.32
(br s, 1H). (2b) Yield: 47.50%; mp 184 °C (AcOEt–petroleum ether) (178—
179 °C).14) IR (KBr) cmϪ1: 3220, 1610. 1H-NMR (DMSO-d6, 200 MHz) d:
2.35 (s, 3H), 6.57, 6.64, 7.28, 7.36, 7.78 (5d, 8H), 8.68 (br s, 1H), 10.22
(br s, 1H). (2c) Yield: 57%; mp 202 °C (AcOEt–petroleum ether) (195—
196 °C).14) IR (KBr) cmϪ1: 3220, 3230, 1620, 1600. 1H-NMR (DMSO-d6,
200 MHz) d: 3.81 (s, 1H), 6.65 (m, 4H), 7.01, 7.89 (2d, 4H), 8.67 (br s, 1H),
kai/ka0
10.19 (br s, 1H). (2d) Yield: 71%; mp 204 °C (EtOH–H2O). IR (KBr) cmϪ1
:

730
Vol. 51, No. 6
where kai and ka0 stand for the P-gp-mediated active efflux of drug in the
presence and absence of modulator, respectively. kai/ka0 is equal to 1 when
there is no inhibition of the active efflux, and to 0 when the P-gp-mediated
active efflux is completely blocked. a0.5 was defined as a concentration of 5-
aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 3 required to in-
hibit the P-glycoprotein function 50%.
7) Zalcberg J. R., Hu X. F., Ching M., Wakeling A., Wall D. M.,
Marschner I. C., de Luise M., Cancer Chemother. Pharmacol., 33,
123—129 (1993).
8) Castro A. F., Altenberg G. A., Biochem. Pharmacol., 53, 89—93
(1997).
9) Mankhetkorn S., Garnier-Suillerot A., Eur. J. Pharmacol., 343, 313—
321 (1998).
10) Mankhetkorn S., Teodori E., Scapecchi S., Garnier-Suillerot A.,
Biochem. Pharmacol., 52, 213—217 (1996).
11) Borsche W., Ockinga K. A., Lieb. Ann. der Chem., 340, 85—109
(1905).
Acknowledgments We thank the Ministère de l’Education Nationale,
de la Recherche et des Technologies, France and the Royal Golden Jubilee
Program (RGJ), Thai Research Fund, Thailand for their financial support.
C.L. thanks the Royal Golden Jubilee Ph.D. Program for financial support.
12) Sherman W. R., J. Org. Chem., 26, 88—95 (1961).
13) Ducki S., Hadfield J. A., Hepworth L. A., Lawrence N. J., Liu C. Y.,
McGown A. T., Bioorg. Med. Chem. Lett., 24, 3091—3094 (1997).
14) Burmistrov K. S., Belov V. V., Burmistrov S. I., Ukr Khim. Zh., 49,
761—763 (1983).
References
1) Gottesman M. M., Pastan I., Annu. Biochem., 62, 385—427 (1993).
2) Slater L. M., Sweet P., Stupecky M., Gupta S., J. Clin. Invest., 77,
1405—1408 (1986).
15) Frezard F., Garnier-Suillerot A., Eur. J. Biochem., 196, 483—491
(1991).
16) Mankhetkorn S., Teodori E., Garnier-Suillerot A., Chemico-Biological
Interactions, 121, 125—140 (1999).
3) Safa A. R., Cancer Invest., 10, 295—305 (1992).
4) Beck W. T., Qian, X. D., Biochem. Pharmacol., 43, 89—93 (1992).
5) Tsuruo T., Lida H., Tsukagoshi S., Sakurai Y., Cancer Res., 41,
1967—1972 (1981).
17) Frézard F., Garnier-Suillerot A., Biochem. Biophys. Acta, 1091, 29—
35 (1991).
6) Watanabe T., Tsuge H., Oh-Hara T., Naito M., Tsuruo T., Acta Oncol.,
34, 235—241 (1995).