Synthesis and Antineoplastic Evaluation of Novel Unsymmetrical 1,3,4-Oxadiazoles

Article abstract of DOI:10.1021/acs.jmedchem.6b00468

A series of novel 1,3,4-oxadiazoles was synthesized and evaluated for their cytotoxic activity in in vitro tumor models. Four of the new compounds (2d, 2j, 2k, and 2n) showed growth inhibition in the XTT dye assay. The most active agent, 2j, showed high potency against human cancer cells with IC₅₀s ranging from 0.05 to 1.7 μM. Preliminary SAR correlations suggested that the nature of chains on the oxadiazole is important for antitumor potency in vitro. Compound 2j determined a G₂/M arrest of the cell cycle and also activated a strong apoptotic response. The β-tubulin immunofluorescence analysis indicated that compound 2j effectively inhibited the microtubule organization in all cancer cell lines, causing the formation of abnormal spindle, which did not affect the normal human fibroblast cells NB1, Mrc-5 and IBR3. For all these reasons, compound 2j could be a good candidate in chemopreventive or chemotherapeutic strategies.

Full text of DOI:10.1021/acs.jmedchem.6b00468

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Article
Synthesis and antineoplastic evaluation
of novel unsymmetrical 1,3,4-oxadiazoles.
Valentina Nieddu, Giansalvo Pinna, Irene Marchesi, Luca Sanna, Battistina
Asproni, Gérard Aimè Pinna, Luigi Marco Bagella, and Gabriele Murineddu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00468 • Publication Date (Web): 01 Nov 2016
Downloaded from http://pubs.acs.org on November 1, 2016
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Synthesis and antineoplastic evaluation of novel unsymmetrical 1,3,4-oxadiazoles.
a§
b§
a,c
a
b
Valentina Nieddu, Giansalvo Pinna, Irene Marchesi, Luca Sanna, Battistina Asproni, Gerard
b
*,a,d
*,b
A. Pinna, Luigi Marco Bagella,
and Gabriele Murineddu
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a) Department of Biomedical Sciences, and National Institute of Biostructures and Biosystems,
University of Sassari, 07100 Sassari, Italy.
b) Department of Chemistry and Pharmacy, University of Sassari, 07100 Sassari, Italy.
c) Kitos biotech, Porto Conte Ricerche, 07041 Alghero, Italy
d) Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College
of Science and Technology, Temple University, Philadelphia, PA 19122, USA.
ABSTRACT
A series of novel 1,3,4ꢀoxadiazoles was synthesized and evaluated for their cytotoxic activity in in
vitro tumor models. Four of the new compounds (2d, 2j, 2k and 2n) showed growth inhibition in
the XTT dye assay. The most active agent, 2j showed high potency against human cancer cells with
IC50 ranging from 0.05 to 1.7 ꢁM. Preliminary SAR correlations suggested that the nature of chains
2
on the oxadiazole is important for antitumor potency in vitro. Compound 2j determined a G /M
arrest of the cell cycle and also activated a strong apoptotic response. The βꢀtubulin
immunofluorescence analysis indicated that compound 2j effectively inhibited the microtubule
organization in all cancer cell lines causing the formation of abnormal spindle, while did not affect
the normal human fibroblast cells, NB1, Mrcꢀ5 and IBR3. For all these reasons, compound 2j could
be a good candidate in chemopreventive or chemotherapeutic strategies.
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INTRODUCTION
Among diseases, cancer is not a simple pathological state, but it can be identified as a broad group
of diseases characterized by high proliferative index and the spreading of aberrant cell from their
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site of origin. This malignant disease is considered the second major leading cause of death
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worldwide after cardiovascular desease, to be noted that cancer mortality began decreasing in
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,4
1992. Clinically, the therapeutic treatment of cancer is usually a combination of surgery and/or
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radiotherapy with chemotherapy.
Current chemotherapy consists of drugs that are placed into three major periods, each with
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distinctive antitumor activity levels of action and toxicity.
The first period (classical chemotherapeutics) contains drugs, which mainly blocked DNA
replication and cell division, such as DNA crossꢀlinking agents, mitosis inhibitors or topoisomerase
poisons.
The second period is characterized by drugs, which directly interact with signaling intermediates
contributing to cancer growth, most notably tyrosine kinase and related inhibitors.
In the third period the drugs are categorized to target broad cellular machineries with a mode of
action that is not directly associated with DNA replication or cell division, but they are involved in
the interaction with nucleosome, proteasome, protein chaperones or DNA repair process such as
histone deacetylase inhibitors, DNA methyltransferase inhibitors, proteasome inhibitors or PARP
inhibitors (Figure 1).
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a) First period antitumor agents
NH3 Cl
N
O
O
OH
OH
O
OH
Cl
N
O
O
OH
F
HO
O
O
NH
O
N
O
O
P
Cl
O
Cl
Cl
NH2
Pt
N
H
O
F
NH
2OH
NH
NH2
OH F
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Cyclophosphamide7
Cisplatin7
5-Fluorouracil8
Gemcitabine9
Doxorubicin10
(anthracyclines,
(alkylating agents,
nitrogen mustard)
(organoplatinum complex)
(antimetabolites,
pyrimidine antagonist)
(nucleoside analogues
DNA polymerase inhibitor)
topoisomerase inhibitors)
HO
O
O
HO
N
N
O
O
O
O
N
HO
O
H
O
O
N
HN
O
O
OHO
N
OH
O
O
NH
O
OH O
O
O
O
N
O
HO
O
O
Topotecan11
Paclitaxel11
(taxanes,
Vincristine11
(camptothecins,
(vinca alkaloids
mitosis inhibitor)
topoisomerase poison)
mitosis inhibitors)
b) Second period antitumor agents
H
N
N
F
O
N
HN
N
O
N
N
HN
O
N
O
O
N
HN
H
N
O
N
O
N
H
Erlotinib12,13
Sunitinib12,13
Imatinib12,13
(EGFR inhibitor)
(VEGFR inhibitor)
(BcrꢀAbl inhibitor)
c) Third period antitumor agents
NH
2
NH
2
O
N
N
N
N
H
N
HN
O
N
O
N
O
O
S
S
HO
HO
NH
O
OH
NH
O
O
HN
N
H
O
OH OH
OH
O
O
O
-Azacytidine14
(Chromatin modification agent
Decitabine14
(Chromatin modification agent
Romidepsin14
Vorinostat14
5
(Chromatin modification agent
HDAC inhibitor)
(Chromatin modification agent
HDAC inhibitor)
DNA methyltrasferase inhibitor) DNA methyltrasferase inhibitor)
O
NH
N
N
O
OH
B
H
N
N
N
N
N
OH
H
H
N
NH
O
N
HO
OH
Ganetespib15
Protein folding agent
HSP90 inhibitor)
Bortezomib16
Nirafarib17
(DNA repair agent
PARP inhibitor)
(
(Proteasome function agent
Proteasome inhibitor)
Figure 1. Selected drugs belonging to the three periods.
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The therapeutic use of chemotherapy agents can lead to common complications arising from
clinical systemic toxicity and development of resistance. Systemic toxicity associated with
anticancer drugs is usually observed in the bone marrow, the gastroꢀintestinal (GI) tract and hair.
Therefore, the search for novel chemical structures, with broader therapeutic windows and
acceptable resistance profiles, are being actively pursued.
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In a previous paper, we reported the preparation and the antiproliferative effect of a series of 2,5ꢀ
1
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disubstituted 1,3,4ꢀoxadiazole. This study indicated that these compounds, 1a,b (Figure 2), with
one nitrogen tricyclic heterocycle at position 2 and a phenyl moiety at position 5 exhibited
considerable cytotoxicity in vitro.
Q
X
N N
O
G
X = CH (a); GI = 2.9 ꢀM
2
50
2
5
X = CH=CH (b); GI50 = 1.28 ꢀM
HN
1
Figure 2. Compounds 1a,b.
Although the mechanism of action of the oxadiazoles 1a,b is probably multimodal in nature, our
previous study indicated that an antimitotic activity with microtubule polymerization inhibition may
be a major cellular event. In continuation of our research in this field, we wondered whether the
introduction of different sideꢀarm substitution patterns at the 2 and 5 positions of the 1,3,4ꢀ
oxadiazole pharmacophore might provide compounds with interesting anticellular features.
In the report, we described novel unsymmetrically disubstituted 1,3,4ꢀoxadiazole of general
structure 2 (see Table 1). The biological evaluations of the antitumor activities of the unsymmetrical
analogues 2a-q have been performed and the results will be discussed.
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Table 1. 1,3,4-Oxadiazoles 2,5 disubstituted 2a-l
N N
Q
G
O
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Compounds
Q
G
2
2
a
b
2c
2
d
e
2
2f
2g
2
h
2
2
i
j
2
k
2l
2m
2n
2o
2
p
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2
q
CHEMISTRY
Scheme 1. Retrosynthesis of disubstituted oxadiazoles 2
0
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NH2
N
N
X
OEt
HN
+
or
EtO
EtO
Q
G
O
O
G
G
Q
O
2
A
B
B'
O
H
N
G
Q
N
H
O
C
The oxadiazoles described in this report were prepared classically by the straightforward and
operationally simple method shown in the retrosynthetic sense in Scheme 1. 1,3,4ꢀOxadiazoles 2
were synthesized by direct coupling of the appropriately substituted hydrazide A to the necessary
acid derivative B or triethylorthoformiate B’. Alternatively, the hydrazide A were converted to the
acyl hydrazide C with acid derivative B and then cyclized to provide 1,3,4ꢀoxadiazoles 2. The
hydrazides 8a, 11b and acyl hydrazides 4a-i, 9b-d and 12a, used in preparation of the 1,3,4ꢀ
oxadiazoles in Table 1 were synthesized as illustrated in Scheme 2.
For example, treatment of hydrazide 3 in combination with an acid in the presence of appropriate
coupling reagent such as an acid chloride and pyridine or EDCI and HOBt provided 1,4ꢀ
dihydroindeno[1,2ꢀb]pyrrole acyl hydrazides 4a-i (Scheme 2, equat 1).
Equation 2 afforded the synthesis of intermediates 8a-d and 9b-d. These molecules were easily
obtained starting from ketones 5a-d that, by a click reaction, were converted into the corresponding
tricyclic scaffolds passing through the acyl derivatives 6a-d. 8a-d were achieved with different time
of reaction from 7a (24 h) and 7b-d (72 h) using hydrazine hydrate. 8a was directly used for the
next step (see scheme 3), whereas 8b-d didn’t react with ethyl orthobenzoate in the same manner of
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8a so were transformed in the corresponding acyl hydrazides 9b-d, as previously described in
equation 1.
a
Scheme 2. Preparation of hydrazides and acylhydrazides
4a : G = 4ꢀClPh
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4b : G = 3ꢀClPh
O
H
N
O
4c : G = 3,4ꢀCl Ph
NH2
NH
O
2
i
N
4d :
G = 4ꢀtert-butylPh
G
(
equat 1)
H
4e : G =3,4,5ꢀtrimethoxyPh
N
H
N
H
4f : G = Cy
3
4a-i
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4h : G = 4ꢀClBn
4i
: G = 4ꢀMethylBn
O
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O
5
6
O
ii
iii
R
(equat 2)
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5a-d
6a-d
7a-d
O
H
N
NH2
O
NH
iv
i
N
O
H
6
6
R 7
N
H
R 7
N
H
8a-d
9b-d
R
a
b
c
d
5
5ꢀH
5ꢀBr
6ꢀBr
5ꢀCl
6ꢀOCH
3
3
6-9 6ꢀH
6ꢀCl 7ꢀOCH
H
O
O
O
NH2
NH
O
N
N
O
iv
v
N
H
(equat 3)
N
N
N
R
N
N
R
R
10a : R = CH3
0b : R = Ph
11a : R = CH3
11b : R = Ph
12a : R = CH3
1
a
Reagents and conditions: (i) ArCOCl, NMP, rt, 12ꢀ48 h (for 4a, 4c, 4f, 4g, 8b-d) or ArCOCl, Py,
DCM, rt, 24ꢀ30 h (for 4b, 4d, 4e) or ArCH COOH, EDCI, HOBt, NMM, DMF/DCM, rt, 24 h for
2
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(4h, 4i); (ii) EAA, NaH, THF anhydrous, rt, overnight; (iii) AcONH , EtOH, 80 °C, 12 h; (iv) NH ꢀ
4
2
NH ·H O, dry EtOH, reflux, 24ꢀ72 h; (v) PhCOCl, Py, DCM, rt, 2 h.
2
2
Reaction of 1ꢀmethyl and 1ꢀphenylꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazole, 10a and 10b, (Scheme 2,
equat 3) with hydrazine hydrate in ethanol furnished the desired hydrazides 11a,b. 11a on further
reaction with benzoyl chloride yielded the required acylhydrazide 12a.
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Scheme 3. Preparation of 1,3,4-oxadiazoles 2a-q in Table 1
a
Reagents and conditions: (i) POCl
, DMF, reflux, 3ꢀ24 h; (iv) 2k, alkylboronic acid, Pd(OAc)
MW, 130 °C, 5 h.
3
, 110 °C, 2 h; (ii) CH
3
CN, TEA, CCl
4
, PPh
3
, rt, 6ꢀ48 h; (iii)
PhC(OEt)
3
2
, Py P, K
3
3
PO
4
, toluene, H O,
2
The 1,3,4ꢀoxadiazoles in Table 1 were prepared according to the methods in Scheme 3. 1,3,4ꢀ
oxadiazoles 2a, 2c, 2h, 2k and 2l (Scheme 3, equat 1) were prepared by heating acyl hydrazides
4
a,c,h and 9b,c with phosphorous oxychloride. Compounds 2m,n were obtained from 2k by a
Suzuki reaction in presence methylboronic acid (for 2m) or cyclopropylboronic acid (for 2n),
tricyclohexylphosphine and Pd(OAc)
2
.
Cyclization of compounds 4b,d-g,i, 9d and 12a (Scheme 3, equat 2) was performed using
triphenylphosphine and triethylamine in dichloromethane affording 1,3,4ꢀoxadiazoles 2b,d-g,i,o
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and 2q. The desired 1,3,4ꢀoxadiazoles 2j and 2p (Scheme 3, equat 3) were obtained directly from
hydrazides 8a and 11b by reaction with triethyl orthobenzoate in refluxing dimethylformamide.
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BIOLOGY
Antiproliferative activity against different human cancer cell lines.
Preliminary screening of the synthesized compounds (2a-q) was performed to evaluate cytotoxic
activity against selected human cancer cell lines, Mfc7 (breast cancer) and HeLa (cervix
adenocarcinoma) by employing a colorimetric assay (XTT) for the quantification of cell
proliferation and viability. The cells were incubated for 72 hours with 10ꢀ5ꢀ2 ꢀM of individual
compounds, or doxorubicin (referenced drug), or DMSO (molecules’ solvent, used as control). The
results of this cytotoxicity data at three different doses in comparison with positive controls, such as
1
a and doxorubicin, are reported in Figure 3.
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Figure 3. Inhibition of tumor cell proliferation in vitro as determined by XTT assay. All the
compounds were dissolved in dimethylsulphoxide (DMSO). Mcf7 and HeLa were incubated with
DMSO (vehicle control), or different concentrations (10ꢀ5ꢀ2 ꢀM) of individual compounds: 1a, or
distinctive compound 2a to 2q, or doxorubicin. Cells were seeded in 96 well plates at a density of
3
1
,5/2×10 cells/well. After 24 hours of cultivation, the adhered cells were treated with the
compounds and incubated for 72 hours. The percentages of viable cell were calculated by
comparing treated and control cells. Cell viability is represented in relation to DMSO and XTT
assays were repeated 3 times.
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To evaluate the effect of the bioisosteric replacement of the pyrazole nucleous of 1a, first we
synthesized compound 2a and analogues 2b-i. Among the nine compounds 2a-i, derivative 2d,
bearing a 1,4ꢀdihydroindenopyrrole scaffold at C
2
of the central oxadiazole core together with a
phenyl side chain substituent attached to the C , showed significant activity against the two tumor
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cell lines (IC50 = 3.4 ꢀM for HeLa and IC50 = 9.5 ꢀM for Mcf7) (Figure 3).
To further evaluate the effect of substitution on the 1,4ꢀdihydroindenopyrazole system of 1a,
derivatives 2j-o were synthetized. Interestingly, only the 2’ꢀmethyldihydroindenopyrrole compound
2
j, at 2 ꢀM, had better activity in both tumor cells (IC = 0.05 ꢀM for HeLa and IC = 1.7 ꢀM for
50
50
Mcf7) showing higher antiproliferative activity than 1a and reference drug doxorubicin.
Their calculated IC values are listed in Table 2. The bromine substitution at the C ꢀposition (2k)
50
6
gave comparable potency (IC50 = 0.51 ꢀM for HeLa and IC50 = 4.26 ꢀM for Mcf7) to compound 2j,
whereas the less lipophilic chlorine derivative 2l appeared less favorable (IC = 45 ꢀM for HeLa
5
0
and IC50 >10 ꢀM for Mcf7). Replacement of the bromine with less and more lipophilic alkyl
groups such as methyl (2m) or cyclopropyl (2n) moieties provided the cyclopropyl analogue 2n that
was equipotent (IC50 = 1.58 ꢀM for HeLa and IC50 = 2.31 ꢀM for Mcf7) respect to 2j. Introduction
7
of a hydrophilic methoxy group at the C ꢀposition of the tricyclic ring system lowered the potency.
Among these compounds the degree of anticellular potency increases in the sequence
OCH <Cl<CH <Br<cyclopropyl<H.
3
3
The above findings suggest that lipophilic substituents on the benzyl ring of tricyclic moiety of 2j
may favorably influence in vitro activity.
We also briefly explored the effect that tricyclic ring system replacement had on cytotoxic activity.
Compounds 2p,q had a 1,4ꢀdihydroindenopyrazole moiety as 1a, but they displayed a minor to
significant decrease in cell viability when compared both to 1a and to the 2ꢀmethylꢀ1,4ꢀ
dihydroindenopyrrole analogue 2j (Figure 3).
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As a result, an interesting and large impact on cytotoxic activity was found for 2 and 5 substitution
of the central 1,3,4ꢀoxadiazole core. Whereas 5ꢀsubstitution with different groups as 4ꢀClꢀphenyl, 4ꢀ
tertbutylphenyl, trimethoxyphenyl, cycloexyl or benzyl had minimal effects on cytotoxic activity,
2
ꢀsubstitution of dihydroindenopyrrole system with a dihydroindenopyrazole moiety markedly
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reduced the cytotoxic activity. Thus, compound 2j exhibited the highest anticellular activity among
all the new compounds tested in this study.
a
Table 2. Cytotoxic activities of 1a and 2a-q
Compound
IC50 Mcf7
IC50 HeLa
1
2a
2b
2
a
2.1 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
9.5 ꢁM
0.5 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
3.4 ꢁM
c
2
d
2
2
e
f
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
4.25 ꢁM
1.7 ꢁM
4.26 ꢁM
> 10 ꢁM
> 10 ꢁM
2,31 ꢁM
> 10 ꢁM
> 10 ꢁM
7.59 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
0.05 ꢁM
0.51 ꢁM
4.5 ꢁM
2
g
2h
2i
2
j
2
k
2
l
2
m
5.3 ꢁM
2
n
1.58 ꢁM
> 10 ꢁM
> 10 ꢁM
> 10 ꢁM
2o
2
2
p
q
a
Cytotoxic activities of the new compounds as given by IC50 values in selected cancer cell lines
HeLa and Mcf7.
Compound 2j was found to be the most interesting and potent of the series; thus, its IC50 value was
determined and its cytotoxicity evaluated by comparing its activities in a panel of 11 different
cancer cell lines as summarized in Table 3. Compound 2j was considered highly cytotoxic, with
IC50 values of < 1.7 ꢀM. The observed activity pattern was quite variable among different cancer
cell lines.
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a
Table 3. Cytotoxic activities of 1a and 2j
Cell line
Cervix Adenocarcinoma
IC50 1a
IC50 2j
HeLa
RD
0.5 ± 0.02 ꢁM
0.4 ± 0.05 ꢁM
0.5 ± 0.03 ꢁM
0.69 ± 0.06 ꢁM
0.9 ± 0.04 ꢁM
0.76 ± 0.04 ꢁM
1 ± 0.07 ꢁM
0.05 ± 0.03 ꢁM
0.06 ± 0.04 ꢁM
0.06 ± 0.02 ꢁM
0.07 ± 0.04 ꢁM
0.12 ± 0.05 ꢁM
0.19 ± 0.06 ꢁM
0.24 ± 0.08 ꢁM
0.25 ± 0.04 ꢁM
0.38 ± 0.07 ꢁM
0.67 ± 0.04 ꢁM
1.7 ± 0.09 ꢁM
Rhabdomyosarcoma
Gliobastoma multiforme
Neuroblastoma
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T98G
SH-SY5Y
H1299
SKNAS
A549
Non small lung cancer
Neuroblastoma
Non small lung cancer
Neuroblastoma
Kelly
1.5 ± 0.09 ꢁM
0.78 ± 0.05 ꢁM
1.9 ± 0.08 ꢁM
2.1 ± 0.12 ꢁM
LAN1
PC3
Neuroblastoma
Prostate cancer
Mcf7
Breast cancer
a
Cytotoxic activities of 2j as given by IC values in selected cell lines of a panel of 11 different
5
0
cancers. Molecule 2j inhibited tumor cell proliferation in all cell lines with IC50 values between
0.05 and 1.7 ꢁM.
Compound 2j was selective as it was 28ꢀ34 times more active against HeLa, RD and T98G cell than
against Mcf7 cells. The activity of 2j against H1299, SHꢀSY5Y and SKNAS cells was high with
IC50 ranging from 0.12 to 0.19 ꢀM. Moreover, also A549, Kelly, Lanꢀ1 and PC3 cells were
sensitive to 2j with IC values of 0.24, 0.25, 0.38 and 0.67 ꢀM, respectively.
5
0
Since nonspecific site of action and toxicity of drugs represent two of the main problems in
chemotherapy, comparable studies were carried on human dermal and lung fibroblast cells (NB1,
Mrcꢀ5, IBR3) used as control, and on the corresponding immortalized cell lines (NB1 hꢀTERT,
Mrc5 SV1 S40, IBR3ꢀG S40).
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As shown in Table 4, it is interesting to note that active derivative 2j exhibited selectively
cytotoxicity to cancer cells, with a weak effect on human fibroblast cells, used as control. Therefore
in all normal analyzed cell lines, compared to the corresponding immortalized nonꢀtumorigenic
cells, compound 2j revealed less sensitivity and a higher IC50
.
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Table 4. Cytotoxic activities of 1a and 2j
Cell line
IC50 1a
IC50 2j
NB1
Primary human dermal
Immortalized human
Primary human dermal
Immortalized human
Primary human lung
Immortalized human
1.8 ± 0.08 ꢁM
1.6 ± 0.05 ꢁM
1.6 ± 0.07 ꢁM
1.8 ± 0.08 ꢁM
1.9 ± 0.05 ꢁM
1.7 ± 0.07 ꢁM
1.3 ± 0.06 ꢁM
0.13 ± 0.03 ꢁM
0.77 ± 0.06 ꢁM
0.21 ± 0.04 ꢁM
0.19 ± 0.05 ꢁM
0.13 ± 0.03 ꢁM
NB1 h-TERT
IBR3-G
IBR3-G SV40
Mrc5
Mrc5-SV1 S40
Table 4: The IC50 values for the synthesized compounds 1a and 2j against human normal and
immortalized cell lines show that primary cells are less sensitive than the same immortalized cell
lines.
Compound 2j inhibit colony formation in cancer cells.
The cellular longꢀterm effects of 1a and 2j were tested in all the cell lines by determining their
ability to inhibit colony formations after drug washout. This experimental paradigm displays a
cancer cell ability to produce a viable colony after treatment and it is a mean that may help to
1
9,20
predict efficacy in vivo.
The cellular persistence of 1a and 2j and the number of colonies formed after two weeks of
exposition in a medium with the drugs were determined using a clonogenic assay. Clonal growth of
all cell lines was inhibited by 2j in a dose dependent manner. Representative pictures of colonies
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following treatment with 0.3 ꢁM 1a or 0.03 ꢁM 2j are shown in Figure 4. At low concentration, like
0
.03 ꢀM, only compound 2j was able to potently inhibit up to 50% of colony formation inducing
cytotoxicity and suggesting a high level of cellular persistence. Conversely, 1a was not able to
inhibit clonogenic growth under the same conditions, but only at the highest concentration. These
results are consistent with the data obtained in the in vitro antiproliferative activity and the IC50
values of compounds 1a and 2j. The data also confirm the greater potency and activity of
compound 2j compared to 1a.
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Figure 4. Representative photographs from one of the triplicate demonstrating the effects of
exposure to 1a and 2j on colony formation. HeLa were treated with DMSO, 1a and 2j at 0.3 and
0
.03 ꢁM. Colonies have been fixed and stained with glutaraldehyde and crystal violet, two weeks
after cell treatment.
Inhibitor agents of ABC transporters improve 2j effects on resistance cell lines
The phenomenon of multidrug resistance (MDR) in the treatment of malignant tumors is a
distinguished problem in oncology whereby cancers become resistant to various agents,
2
1
consequently requires careful consideration in cancer therapy.
Tumor cells adopt several mechanisms to evade death induced by antiꢀtumor agents. These include
changes in apoptotic pathways and activation of cellꢀcycle checkpoints to increase DNA repair.
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Alternatively, cancer cells develop resistance by the upꢀregulation of a family of transmembrane
2
2
ATP binding cassette (ABC) transporter proteins that are present in all living organisms and
altered anticancer drug transport mechanisms. These specific proteins are able to trigger
chemotherapy resistance carrying out in an active manner the extrusion of a large collection of
therapeutic compounds from cancer cells. They bind ATP in their ATP binding domain and use the
energy to transport various molecules across the cell. The ABC transporter family has been divided
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into seven subfamilies based on the sequence similarity as well as structural organization. Among
these proteins, Pꢀglycoprotein (Pgp, ABCB1), multidrug resistanceꢀassociated protein (MRP1,
ABCC1) and breast cancer resistance protein (BCRP, ABCG2) are chiefly responsible for drug
resistance in tumor cells.
In literature it is just known that the betaꢀcarboline alkaloid Harmine reduced resistance to some
24
anticancer drugs mediated by BCRP and might be an interesting new reversal agent. Furthermore,
25, 26
Reversan inhibits MRP1 and Pꢀglycoprotein (Pgp) very electively.
In this study, we show that the addition of small molecule inhibitors of MRP1 and BCRP had a
significant effect on cancer cell response to 2j.
Figure 5. Effects of the inhibitors of MRP1 and BCRP on HeLa and Mcf7. Cell lines were treated
with 2j (0.5  M), Harmine (1ꢀ3ꢀ5  M) or Reversan (5ꢀ15ꢀ25  M) or coꢀtreated with 2j + Harmine
or 2j + Reversan for 72 hours, and tested for chemosensitivity using XTT assay.
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As illustrated in Figure 5, we treated the cell lines with different nonꢀtoxic concentrations of
24,27
Harmine or Reversan as previously suggested.
Considering the different 2j IC50 values for HeLa
and Mcf7, cell lines were also treated with 0.5  M of 2j, a concentration that allowed a clear
inhibitory effect in both cell lines.
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Evaluation of XTT assay indicated that in Mcf7, the cells more resistance to 2j, the coꢀtreatment of
Harmine or Reversan with 2j, significantly inhibited cell proliferation with higher activity than 2j
alone. Specifically, the most notably enhanced cell death was evident when Mcf7 cells were treated
with a combination of 2j and Harmine (5  M). This coꢀtreatment was able to potently inhibit until
40% of proliferation improving response to cell death comparing than 2j alone.
However, in HeLa, the most sensitive cells to 2j treatment, inhibition of ABC transporters by
Reversan and Harmine did not lead to a significant change in drugꢀinduced cell death on 2j drug
response.
These results possibly suggested that increased MRP1 and BCRP expression in some cell lines,
such as Mcf7, contributes to chemoresistance through increased drug efflux and reduced
bioavailability of the derivative 2j within the cancerous cells. Improving intracellular exposure to
efficient chemotherapeutics, through Harmine and Reversan, would significantly increase Mcf7 cell
death when used in combination with compound 2j.
2
2
j induces G /M phase cell cycle arrest
In order to investigate the cellular mechanisms of prototype 1a and compound 2j, a cell cycle
distribution of all cell lines was analyzed by flow cytometry. From FACS analysis, a significant
increase in G
2
/M phase was observed in comparison to untreated control after 2j treatment.
/M
Compound 2j showed various effects on the different cell lines. After 2 hours treatment, G
2
phase cell cycle arrest was observed in HeLa, RD and T98G, followed by gradual increase during
the time point. This effect changed after 24 hours of treatment and the rapid arrest of cell growth
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was followed by cell death. Also 1a led to a G
A).
In Mcf7, PC3 and SHꢀSY5Y, 2j induced a remarkable increase in G /M phase (from 24.2% to
2
/M arrest but with less activity than 2j (Figure
6
2
7
8.1%) and a dramatic decrease in G
1
phase (from 54.1% to 15.7%) and S phase (from 21.7% to
/M arrest persisted almost unchanged until 144
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.3%) after 24 hours treatment. Interestingly, the G
2
hours (Figure 6B).
In further cell lines, such as H1299, A549 and Lanꢀ1 cell lines in comparison with the DMSO
control group, 24 hours treatment of 2j led to G /M arrest with 74.6% of cell lines in this phase
2
compared with 22.2% in DMSO; 48 hours of treatment induced a decrease in G /M (42.9% with 2j
2
in comparison to 11.8% DMSO), a reduction in G
1
phase (34.3% with 2j compared to 81% with
(Figure 6C).
DMSO) and an increase (13.4% of cell population) in subꢀG
1
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Figure 6.
distribution of HeLa (A), Mcf7 (B) and H1299 (C) cancer cell lines after treatment with prototype
a and 2j. Profiles were obtained by flow cytometry analysis using the DNA intercalating dye,
Cell
cycle
1
propidium iodide (PI) to monitor cell cycle progression on the basis of DNA quantitation.
As shown in Table 5, the results obtained with the FACS analysis specify that the promising
2
anticancer activity of 2j may be ascribed to the G /M arrest.
Table 5. Distribution of cells in every phases of cell cycle after 1a and 2j treatment.
Cell line
HeLa
Compound Sub G
1
G
1
S
2
G /M
CTR
DMSO
1a
59.1±2.12 21.7±1.67 19.2±1.09
62.2±1.34 21.4±1.94 16.4±2.15
35.5±1.63 16.9±0.96 47.6±1.28
2j
RD
CTR
DMSO
55.1±1.93 15.9±2.18 29.0±1.37
56.7±1.48 14.4±0.92 28.9±1.93
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1a
2.4±0.93 42.9±1.79 18.6±1.05 36.1±1.57
2
j
6.1±0.58 36.9±2.06 10.6±0.89 46.4±1.28
83.0±1.53 6.1±0.79 10.9±1.20
86.1±1.29 5.1±0.82 8.8±0.93
3.3±0.63 38.7±1.69 9.6±1.03 48.4±0.79
16.8±0.94 11.1±1.37 6.2±0.88 65.9±0.69
59.0±1.95 20.5±1.34 20.5±1.14
61.2±1.69 16.4±1.76 22.2±1.74
67.8±1.49 13.4±1.37 18.8±0.93
17.8±1.42 7.6±1.57 74.6±0.64
62.8±2.04 13.7±1.03 23.5±1.32
59.6±1.94 14.3±1.52 26.2±1.27
81.3±1.37 3.0±0.77 15.7±1.05
15.7±0.91 24.8±1.04 15.6±1.01 43.9±1.36
52.5±1.66 22.1±0.99 25.4±1.13
51.5±1.39 21.3±1.14 25.2±1.32
22.1±1.02 26.5±1.32 51.4±1.28
6.3±0.73 10.8±0.63 9.9±0.84 73.0±2.06
64.8±2.03 11.7±1.14 23.5±1.26
65.4±1.84 7.6±0.63 27.0±1.32
70.1±2.16 9.4±0.77 20.5±1.18
13.8±0.86 11.4±0.93 9.5±0.82 65.3±1.66
62.7±1.63 17.5±1.34 19.8±1.36
65.9±1.87 16.1±1.28 18.0±1.28
54.7±1.49 3.8±0.74 31.5±1.74
13.4±0.94 34.3±1.42 9.7±0.91 42.9±1.19
49.8±1.15 22.8±1.12 27.4±1.21
60.3±1.82 23.5±1.32 26.2±1.92
33.6±1.64 29.7±1.15 34.7±1.64
11.8±1.06 5.6±0.95 82.6±2.27
66.0±1.85 4.8±0.73 29.2±1.73
67.0±1.47 6.2±0.94 26.8±1.69
58.6±1.38 8.1±1.02 33.3±1.45
23.3±1.19 4.7±1.07 72.0±1.99
54.2±1.60 20.4±1.28 25.4±1.48
54.1±1.52 21.7±1.42 24.2±1.62
34.3±1.29 10.6±1.69 55.1±1.73
15.7±1.17 6.3±0.95 78.1±2.16
T98G
H1299
SH-SY5Y
SKNAS
A549
CTR
DMSO
1
a
2
j
CTR
DMSO
1a
2j
CTR
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DMSO
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a
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j
CTR
DMSO
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2j
CTR
DMSO
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Kelly
CTR
DMSO
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Lan-1
PC3
CTR
DMSO
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CTR
DMSO
1
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Mcf7
CTR
DMSO
1a
2
j
Table 5. FACS analysis of untreated and treated (DMSO, 1a, 2j) cancer cell lines showing the
distribution of cells in various phases of cell cycle. The table shows the percentage of cells in each
phase after 24 hours exposure to the compound at a concentration 1 ꢀM.
Moreover, in order to evaluate whether 2j effect is reversible or irreversible, cells were exposed to
2
j for 24 hours followed by replacement with normal cultured media (Figure 6A). The results
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shown in Figure 6B, suggested that the G
2
/M arrest caused by 2j is reversible and its effect is
cytostatic and not cytotoxic. After being changed with fresh medium, the cell cycle showed a
comparable distribution among normal and control cycle (G /M phase: 59.5% after 24 hours 2j
2
treatment and 31.7% after 24 hours treatment + 24 hours change medium; 28.1% after DMSO
treatment and 26.4% with DMSO treatment + 24 hours change medium).
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Figure 7. Effects of 1a and 2j on cell cycle distribution of Mcf7 cells after 24 hours of treatment
with vehicle, 1a or 2j (A), and after 24 hours of treatment with compounds, followed by 24 hours of
treatment with normal medium (B).
2
Furthermore, it is interesting that 2j does not appear to be involved in G /M arrest on primary cell
lines as shown in Figure 8A. Cells treated with 2j from 24 to 72 hours were not significantly
influenced by the treatment and the distribution of population among all the phases of cell cycle
appeared similar to the control and DMSO (Figure 8A). When immortalized cells were treated with
2
j under the same conditions, a reduction of the percentages of G
1
and G
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/M arrested cells was
observed. Interestingly, 25% of immortalized cells displayed a subꢀG
1
peak after 48 and 72 hours
exposure to the treatment of 2j (Figure 8B).
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Figure 8. Flow cytometric analysis in primary NB1 (A) and immortalized NB1 hꢀTERT (B) cell
lines after treatment with 2j on cell cycle.
Compound 2j induce alteration on tubulin organization
Suggested by the evident G /M cell cycle arrest, the effect of 1a and 2j in a mitotic spindle
2
microtubules, and overall cellular morphology were evaluated by immunofluorescence analyses
using βꢀtubulin antibody.
Cells were treated with the compounds for 24 hours at the concentration of 1 ꢀM, subsequently
were permeabilized, fixed, and stained with βꢀtubulin and DAPI for DNA stain.
Deregulation of tubulin network after 2j treatment was observed whereas no particular changes in
βꢀtubulin organization were highlighted in both DMSO and in the control.
Different phenotypes were observed in cancer cell lines: Mcf7 showed an aberrant dissemination
of βꢀtubulin, distinguished by uneven distribution around the nuclei, induction of micronucleated
cells by 2j, smaller and more compact nuclei as compared to the control.
In HeLa cell, low concentrations of 2j (1 ꢀM for 1a and 0.1 ꢀM for 2j) induced abnormal cell
division resulting in the formation of new daughter cells with tubulin structure but without nuclei
or with incorrect βꢀtubulin organization and two nuclei.
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Finally, SHꢀSY5Y revealed an irregular microtubule network with an atypical βꢀtubulin
distribution. Figure 9 depicts the loss of its elongated structure with shorter microtubule bundles
distributed around the nuclei and with apoptotic and pyknotic nuclei.
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Figure 9. Representative images of immunofluorescence analysis showing βꢀtubulin expression
(green) and DNA (blue) in various cancer cells Mcf7 (A), HeLa (B) and SHꢀSY5Y (C) after
incubation with DMSO, 1a and 2j.
Molecule 2j increase p53 protein level in cancer cell lines.
In order to determine whether the antiꢀproliferative effects and the mechanisms associated with cell
cycle arrest suggesting the involvement of p53, cells were treated with 1 ꢁM of 2j for 24 hours,
lysed, and p53 protein levels were evaluated by immunoblotting and quantified by densitometric
analysis using GAPDH as control. As shown in Figure 10, 2j treatment enhances a substantial
increase of p53 expression levels in all cancer cell lines, respectively 2 or 3 times higher compared
to the untreated controls. Interestingly, no modulation in p53 expression was observed in HeLa
because these cells contain no detectable p53 protein. Moreover, the data showed unaltered p53
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levels for fibroblasts and a p53 deregulation, even low, for immortalized cells, confirming that 2j
preferentially inhibits the growth of cancer cells
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Figure 10. Overexpression of p53 protein in cancer (HeLa, Mcf7, A549), primary (NB1) and
immortalized (NB1 hꢀTERT) cells after 24 hours of 2j treatment. GAPDH is shown as a control of
protein loading. The overexpression of p53 has been measured and the data displayed in a
histogram. Columns are means of three different experiments.
Molecule 2j induced an apoptotic response in cancer cell lines.
To determine whether 2j induces apoptosis, Annexin VꢀFITC/PI assay was performed. After
treatment with 5 ꢀM of 2j for 48 or 72 hours, cells were double stained with Annexin V and PI and
assayed by flow cytometry.
In Figure 11, the data show that compound 2j induced apoptosis of cancer cells: in HeLa cells, after
4
8 hours, the rate of apoptosis in the control was 3.6% and in DMSO was 6.5%, while the
percentage of apoptotic cells was 89.7% after treatment with 5 ꢀM of 2j.
In other cell lines such as A549, 2j needs 72 hours of treatment to induce apoptosis with percentage
of 52.7% respect to 0.9% in the control and in 6.5% in DMSO.
Interestingly, at the same doses and after 72 hours, only 6% of fibroblast and 11.8% of
immortalized cells showed apoptotic effect respect to DMSO.
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These results suggest that 2j induced apoptosis and had a more potent effect in cancer cell lines; on
the contrary, primary cells displayed a low sensitivity to the compound.
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Figure 11. HeLa (A), A549 (B), NB1 (C), NB1 hꢀTERT (D) were cultured with 2j at 5  M for 48
(HeLa) or 72 hours. Cells were stained by Annexin V FITC/PI and apoptosis was analyzed by flow
cytometry. The lower left quadrants show the vital cells; in the upper right quadrants the late
apoptotic cells are indicated; the lower right quadrants show the early apoptotic cells and the upper
left quadrants display the dead and necrotic cells.
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DISCUSSION AND CONCLUSIONS
Seventeen new unsymmetrical 2,5ꢀdisubstituted 1,3,4ꢀoxadiazoles were synthesized and screened as
new potential anticancer candidates.
The antiproliferative effects of substituted 1a derivatives were studied and it was found that
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compound
2ꢀ(2ꢀmethylꢀ1,4ꢀdihydroindeno[1,2ꢀb]pyrrolꢀ3ꢀyl)ꢀ5ꢀphenylꢀ1,3,4ꢀoxadiazole,
2j,
exhibited the strongest anticancer activity against a panel of different human cancer cell lines with
IC50 ranging from 0.05 to 1.7 ꢀM, more effectively than 1a and doxorubicin in inhibiting cancer
cell growth.
The different potency of compound 2j against several cell lines studied could be due to the ABC
transporters, in fact they can increase the efflux of 2j from cancer cells, decreasing the intracellular
drug concentration. Modulators of ABC transporters, like Harmine and Reversan, have the potential
to increase the efficacy of 2j in the resistance cell lines.
Together, our data suggest that the designed molecule 2j could be a prospective anticancer drug,
thus, clearly display its ability to induce a solid antiproliferative effect, especially targeting cancer
cells. Moreover our results demonstrate that all the fibroblasts were less sensitive to 2j when treated
at the same concentrations compared to immortalized cell lines, suggesting that this compound
shows low systemic toxicity and preferential cytotoxic activity on cancer cells.
Usually, cells, grown in monolayer, are more sensitive to cytotoxic agents than cells grown in
colonies due to the greater surface available to the chemotherapy agents, compared to the limited
28
drug penetration in the colonies. Interestingly, colony formation assay indicate that 2j can abolish
proliferation of cancer cells in vitro, in both monolayer and colony cell culture conditions.
Moreover, our data suggest that 2j could perhaps show anticancer activity in vivo, without affecting
2
9
the proliferation of normal cells, a common limit of many anticancer drugs.
Since deregulations of cell cycle progression are typical of cancer, many of the new drugs used as
anticancer agents work at multiple steps in the cell cycle and their effects may be cytostatic or
30,31
cytotoxic, depending on the cell cycle status of the target cells.
The knowledge of the molecular
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interactions involved may suggest ways to alert cells to the effects of these compounds, hence the
molecular and cellular target mechanisms utilized by 2j were explored through flow cytometry
2
analysis and immunofluorescence. FACS analysis highlighted a considerable G /M phase arrest,
indicating an important role of 2j during cell cycle distribution. On the contrary, normal fibroblasts
treated with 2j under the same conditions, did not show significant effects in terms of blocking the
cell cycle, again notifying the favorite activity of 2j on cancer cells.
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Various studies have highlighted that various cancer cells possess irregular G /S checkpoint
1
controls, which induce these cells to an abnormal dependence of G
cell cycle. Moreover, the regulation of G /M transition is more unpredictable in cancer cells than in
In the last year, various studies led to believe G ꢀphase transition as a crucial
2
checkpoint mechanisms during
2
32,33
normal cells.
2
objective for the development of new antitumor drugs. Our data, gathered after 2j treatments,
suggested that this molecule could belong to this class of anticancer compounds.
Microtubules are a major component of the cytoskeleton involved in maintaining cell structure and
cell division, including the formation of mitotic spindles, chromatid alignment and their division
during mitosis. They are assembled by polymerization of alphaꢀbeta dimers of tubulin. The
polymerization is a reversible process, which is in a dynamic equilibrium. This phenomenon,
known as microtubule dynamic instability, is a result of periods of rapid microtubule
polymerization alternate with periods of shrinkage. Interference with the dynamics of microtubules
polymerization and depolymerization, and consequently with cell division, has been proven to be
34
clinically useful for anticancer drugs. These drugs can either stabilize the polymerization process
or destabilize it; in both cases the microtubule dynamics is disturbed, leading cell cycle to arrest and
to apoptosis. Therefore, owing to their essential role in forming dynamic spindle apparatus in
mitosis, microtubules are considered an ideal target for anticancer drug development.
Our experiments demonstrated a modification of βꢀtubulin filamentous and tubulin organization
with abnormal distribution of tubulin around the nuclei, micronucleated cells and apoptotic and
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pyknotic nuclei. Based on our data it is possible to assume that 2j plays a key role in the
microtubule assembly and dynamics.
One noteworthy feature occurring in cancer cells upon 2j treatment, as evidenced by western blot, is
an upregulation of p53. The p53 tumor suppressor protein is a transcription factor that responds to a
variety of cellular stress conditions and controls key cellular processes, such as DNA damage and
repair, hypoxia, cell cycle progression, differentiation, senescence, angiogenesis, metastasis and
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apoptosis. p53 plays an important role in the arrest of damaged cells, favoring their repair and
36,37
survival, or inducing cell cycle arrest and apoptosis when DNA is damaged irreparably.
In our model, upꢀregulation of p53 seems to have a key role in mediating both antiꢀproliferative and
2
proꢀapoptotic effects of 2j, suggesting that the deregulation of p53 and the G /M arrest could be
linked to apoptosis by activation of apoptotic pathways or inhibition of cell cycle regulators via
p53ꢀdependent pathways.
All these data deliver interesting evidences and useful suggestion to speculate on the mode of action
of 2j focusing on targeted experiments in order to verify its molecular mechanisms.
Moreover, p53 could also induce cyclinꢀdependent kinase inhibitors, particularly Cdk1/Cyclin B
complex, which plays a crucial role during G phase. p53 transcriptionally represses Cyclin B and
2
1,
Cdk1, but also Cyclin B , and Cyclin A inducing cell growth inhibition and G /M cell cycle
2
2
3
8, 39
arrest.
Furthermore, p53 could inhibit Topoisomerase, which plays a key role on chromosome segregation
40
2
and progression into mitosis. Topoisomerase II has a crucial function during the G /M transition,
41
where it helps to form highly condensed mitotic chromosomes. Moreover 2j could induce a
downregulation of topoisomerase II promoter activity, p53 dependent, blocking entry into mitosis.
Our findings underscore that oxadiazole derivatives could be interesting candidates for potential
anticancer drugs and distinctly demonstrate that 2j has robust antiproliferative activity, inhibits
2
drastically growth and colony formation, and cause G /M cell cycle arrest and apoptosis,
preferentially targeting cancer cells. Although further researches are ongoing to more fully elucidate
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molecular targets affected by 2j, additional investigations are fundamental to verify clinical
evidences and explore its anticancer activity in vivo. In conclusion, based on our data, we affirm
that 2j is a prospective compound that could be evaluated as a promising candidate for new
therapeutic strategies against cancer.
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EXPERIMENTAL SECTION
Chemistry: All reactions involving air or moistureꢀsensitive compounds were performed under
argon atmosphere. Solvents and reagents were obtained from commercial suppliers and were used
without further purification. Microwave irradiation experiments were carried out in a Biotage®
Microwave Initiator Eight 2.5 in the standard configuration as delivered, including proprietary
software. All experiments were carried out in sealed microwave process vials under normal or low
absorption. After completion of the reaction, the vial was cooled down to 25 °C via air jet cooling
before opening. Reaction temperatures were monitored by an IR sensor on the outside wall of the
reaction. Hydrogenations were carried out in the 4560 Parr Apparatus using a H2PEMꢀ100 Parker
Balston Hydrogen Generator. Flash column chromatography (FC) was performed automatically on
Flashꢀmaster (Biotage®) with preꢀpacked Biotage® SNAP silica gel cartridges or manually on
silica gel (Kieselgel 60, 0.040e0.063 mm, Merck®). The progress of all reactions was monitored by
thin layer chromatography (TLC) performed with Polygram SIL NꢀHR/HV254 preꢀcoated plastic
sheets (0.2 mm) on aluminum sheets (Kieselgel 60 F254, Merck®). Melting points were obtained
on a Kofler melting point apparatus and are uncorrected. IR spectra were recorded as nujol mulls on
NaCl plates with a Jasco FT/IR 460 plus spectrophotometer and are expressed in n (cm 1). NMR
experiments were run on a Varian Unity 200 spectrometer (200.07 MHz for 1H, and 50.31 MHz for
1
3C) and Bruker Advance III spectrometer (400 MHz for 1H, and 101 MHz for 13C). Spectra were
acquired using deuterated chloroform (chloroformꢀd) as solvent. Chemical shifts (d) for 1H and
3C NMR spectra are reported in parts per million (ppm) using the residual nonꢀdeuterated solvent
1
resonance as the internal standard (for chloroformꢀd: 7.26 ppm, 1H and 77.16 ppm, 13C; for
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