Synthesis and anti-proliferative activity of a small library of 7-substituted 5H-pyrrole [1,2-a][3,1]benzoxazin-5-one derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.05.046

In this study, we investigate the anti-proliferative activity of a small library of 7-substituted 5H-pyrrolo[1,2-a][3,1]benzoxazin-5-one derivatives, against a panel of human cancer cell lines. We reported the synthesis of these compounds in a previous work. 7-Bromo-5H-benzo[d]pyrrolo[2,1-b][1,3]oxazin-5-one showed a promising anti-proliferative effect. As starting material for Suzuki-Miyaura cross coupling reaction, it was selected for the design and the synthesis of six further derivatives, with the aim to better define structure-activity relationships. The anti-proliferative MTT assay revealed a dose-dependent reduction of cell viability, especially for 7-([1,1′-biphenyl]-4-yl)-5H-benzo[d]pyrrolo[2,1-b][1,3]oxazin-5-one. Cell cycle and western blotting analysis suggested apoptosis as possible mechanism for its anti-proliferative activity. These preliminary results encourage our interest for further optimizations.

Full text of DOI:10.1016/j.bmcl.2017.05.046

Accepted Manuscript
Synthesis and anti-proliferative activity of a small library of 7-substituted 5H-
pyrrole [1,2-a] [3,1] benzoxazin-5-one derivatives
Mariateresa Badolato, Gabriele Carullo, Biagio Armentano, Salvatore Panza,
Rocco Malivindi, Francesca Aiello
PII:
S0960-894X(17)30527-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.046
BMCL 24991
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
3 April 2017
9 May 2017
13 May 2017
Please cite this article as: Badolato, M., Carullo, G., Armentano, B., Panza, S., Malivindi, R., Aiello, F., Synthesis
and anti-proliferative activity of a small library of 7-substituted 5H-pyrrole [1,2-a] [3,1] benzoxazin-5-one
derivatives, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.046
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Synthesis and anti-proliferative activity of a
small library of 7-substituted 5H-pyrrole [1,2-a]
[3,1] benzoxazin-5-one derivatives
Leave this area blank for abstract info.
Mariateresa Badolatoa, Gabriele Carulloa, Biagio Armentanoa, Salvatore Panzaa, Rocco Malivindia and Francesca
Aiello*a

Bioorganic & Medicinal Chemistry Letters
Synthesis and anti-proliferative activity of a small library of 7-substituted 5H-
pyrrole [1,2-a] [3,1] benzoxazin-5-one derivatives
Mariateresa Badolatoa, Gabriele Carulloa, Biagio Armentanoa, Salvatore Panzaa, Rocco Malivindia and
Francesca Aiello*a
a Dipartimento di Farmacia e Scienze della Salute e della Nutrizione,, Edificio Polifunzionale, Università della Calabria, Arcavacata di Rende, 87036, Rende
(CS), Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
In this study, we investigate the anti-proliferative activity of a small library of 7-substituted 5H-
pyrrolo[1, 2-a][3, 1]benzoxazin-5-one derivatives, against a panel of human cancer cell lines.
Revised
We reported the synthesis of these compounds in
a previous work. 7-bromo-5H-
Accepted
benzo[d]pyrrolo[2,1-b][1,3]oxazin-5-one showed promising anti-proliferative effect. As
a
Available online
starting material for Suzuki-Miyaura cross coupling reaction, it was selected for the design and
the synthesis of six further derivatives, with the aim to better define structure-activity
relationships. The anti-proliferative MTT assay revealed a dose-dependent reduction of cell
viability, especially for 7-([1,1'-biphenyl]-4-yl)-5H-benzo[d]pyrrolo[2,1-b][1,3]oxazin-5-one.
Cell cycle and western blotting analysis suggested apoptosis as possible mechanism for its anti-
proliferative activity. These preliminary results encourage our interest for further optimizations.
Keywords:
benzoxazin-5-one
anti-proliferative activity
Suzuki-Miyaura cross coupling
Cell cycle analysis
Cancer cell lines
2009 Elsevier Ltd. All rights reserved.
Heterocycles containing benzoxazinone skeleton have been
always attracting scaffolds in the field of medicinal chemistry
(Fig. 1).
Moreover, it has been demonstrated that benzoxazinoids could
be useful for the treatment of cardiovascular diseases and
cancer.7,8
The benzoxazinone skeleton contains three different potential
areas for functionalization, the carbon atom in position 3 (C3),
the nitrogen in position 4 (N4) and the aromatic ring. One of the
modification that involved both the C3 and N4 atoms was their
introducing in a fused pyrrole ring. A number of methods have
been reported for the synthesis of 4H-pyrrolo[2,1-
c][1,4]benzoxazin-4-one frame.9,10
Two derivatives of this class of compounds were identified as
selective antagonists of G-protein coupled estrogen receptor
(GPER), inhibiting the proliferation of SkBr3 cells and the
migration of cancer-associated fibroblasts (CAFs) induced by
17β-estradiol and G-1.11 Thus, heterocycles containing
benzopyrroloxazinone frame can be considered as privileged
scaffolds for the development of potential new drugs, including
the treatment of cancer. The isomer 5H-pyrrolo[1,2-
a][3,1]benzoxazin-5-one is not very studied yet. Recently, we
reported the synthesis of a small library of 5H-pyrrolo[1,2-
a][3,1]benzoxazine-5-one derivatives.12
Figure 1. General structure of heterocycle containing benzoxazinone
skeleton
Since their discovery as secondary metabolites of the grasses
over 50 years ago, benzoxazinoids have been extensively studied.
Different aspects of their chemistry and a wide range of their
diverse biological activities have been published. Originally, they
were found playing an important role in the chemical defense of
plants, acting as natural pesticides and exhibiting allelopathic
properties.1,2 They also could function as genotoxins for human
cells.3 Inhibiting the bacterial type IIa topoisomerase, a series of
In this study, our interest was to investigate about the anti-
proliferative activity of those compounds against different human
breast cancer cell lines with the aim to further develop an
understanding of the SAR study of this class of compounds.
From a preliminary screening, 5H-naphtho[2,3-d]pyrrolo[2,1-
b][1,3]oxazin-5-one (2), and 7-bromo-5H-benzo[d]pyrrolo[2,1-
6-substituted benzoxazinone derivatives showed
a
rapid
bactericidal activity.4 Furthermore, benzoxazinoids showed
thrombin and cyclooxygenase inhibitory properties, as well as
antimicrobial and anti-inflammatory activities.5,6

b][1,3]oxazin-5-one (3), bearing a bromine atom on C7, showed
a good anti-proliferative activity against all used cell lines. On
was performed using activated MnO2, leading to compound 3
(Scheme 1).12
the
contrary,
the
unsubstituted
5H-pyrrolo[1,2-
a][3,1]benzoxazin-5-one (1) and 5H-pyrido[2,3-d]pyrrolo[2,1-
b][1,3]oxazin-5-one (4) didn’t show the same effect. Since
compound 4 moderately inhibited the growth of the used cancer
cells, the presence of benzene ring in the derivatives may be
necessary for their anti-proliferative activity. Furthermore, the
presence of substituents on the aromatic ring of compound 1 may
improve the cytotoxicity of this class of compounds (Fig. 2).
Scheme 1. Reagents and conditions: (i) 4-Chloropyridine hydrochloride
(1 equiv.), 2,5-dimethoxytetrahydrofuran (1.2 equiv.), 1,4-dioxane,
reflux, 24 h; (ii) MnO2 (5 equiv.), dry toluene, reflux, 24 h, 3 30%.
Compounds 3a - 3f were obtained in good yields and with a
fast purification procedure, through crystallization, using a
similar approach (Scheme 2). We adjusted the Suzuki-Miyaura
cross coupling reaction, to optimize the synthesis of our
derivatives. Since all the reactions were performed in water,
TBAB was added to improve the solubility of the reactants and
arylboronic acids were used as alcoholic solution. The quick
reaction lasted about four hours and led to appreciable quantities
of all the derivatives, except in the case of compound 3f, due to
the poor solubility of the starting (2-methoxypyridin-3-yl)boronic
acid in the alcoholic solution and in others organic media.
Figure 2. Some of 5H-pyrrolo[1,2-a][3,1]benzoxazin-5-one analogs
previously prepared and their respective cytotoxicity at 50 μM
Continuously to our interest in the synthesis of 5H-
pyrrolo[1,2-a][3,1]benzoxazin-5-one
derivatives,
having
biological and pharmacological activities, we designed and
synthesized a new series of 7-subsituted analogs and their in vitro
anti-proliferative activity was evaluated, with the aim to better
define the SAR, determining which moieties are essential for the
anti-proliferative effect (Fig. 3).
Scheme 2. Reagents and conditions: (i) alcoholic solution of
arylboronic acid (1.5 equiv.), 3 (1 equiv.), PPh3 (0.3 equiv.), Pd(OAc)2
(0.1 equiv.), TBAB (0.1 equiv.), 1M Na2CO3 (0.2 mL), water, 130°C, 4
h, 3a 60%, 3b 80%, 3c 86%, 3d 51%, 3e 30%, 3f 5%.
To establish the biological profile of all compounds, cell
viability and proliferation were evaluated in MCF-7, MDA-MB
231, and SkBr3 breast cancer cell lines. To evaluate the possible
toxicity of the derivatives, a mammary epithelial cell line (MCF-
10A) was used.
Figure 3. Compounds of interest to further investigate the SAR of 7-
substituted 5H-pyrrolo[1,2-a][3,1]benzoxazine-5-one analogs
Due to its promising anti-proliferative activity, compound 3
was chosen as starting material, suitable substrate for coupling
reaction. In particular, we performed Suzuki-Miyaura cross
coupling, adjusting the reaction conditions for the synthesis of
our derivatives. 13 To validate the importance of the substitutions
on the aromatic ring, variously substituted phenyl, phenol and
pyridines moieties were selected to decorate the new compounds.
Then, a preliminary cytotoxicity was evaluated and the possible
mechanism of action of this class of compounds was
investigated.
Figure 4. Biological profile of the synthesized derivatives; the titled
compound dox is Doxorubicin, which was used as positive control.
Firstly, 5-bromo-2-(1H-pyrrol-1-yl)benzoic acid (II) was
prepared by the reaction of I with 4-chloropyridine hydrochloride
and 2,5-dimethoxytetrahydrofuran. The subsequent cyclization

As shown in Figure 4, most of the new synthesized
compounds demonstrated moderately improved anti-
involved in cell cycle, such as cyclin D1 (CD1), were detected.
CD1 is a critical modulator in the cycle G1/S transition and its
overexpression is one of the most commonly observed alterations
in human endometrial cancer. Thus, the potential ability of
compound 3b to modulate CD1 level in MCF-7 breast cancer
cell line was evaluated.
a
proliferative effect compared to the parent compound 3. The
presence of the unsubstituted phenyl ring in 3a and the ethanone
moiety in para in 3d, did not affect the cell viability prominently.
Compounds 3e and 3f, both presenting a pyridine ring, reflect the
same behavior of the previous molecules. Compound 3c, with a
hydroxyl group in para position of the phenyl ring, showed a
good anti-proliferative effect, and in all used cancer cell lines.
However, it also demonstrated a cytotoxic activity against MCF-
10A cells. On the contrary, compound 3b, bearing a biphenyl
ring able to confer a total planarity to the molecule, showed the
best biological profile. It is interesting to note that, the biphenyl
derivative, although demonstrated to be the most cytotoxic
against the human cancer cells, it also was the safest compound
against the mammary epithelial cells, with an IC50 value over 200
μM.
Figure 6. Immunoblots of PARP cleavage, p53, CD1, p21WAF1/Cip1, from
extracts of MCF-7 cells, treated with vehicle (ctrl), or compound 3b (1,
5, 10 μM) for 24 hr. β-Actin was used as loading control.
Based on these promising results, further in vitro studies are
ongoing, to evaluate the exact mode of action of selected
compound 3b. Additional decorations in the 5H-pyrrolo [1,2-
a][3,1]benzoxazin-5-one structure have been performed in our
laboratory, to obtain derivatives with better biological activity
and lend them useful anticancer agents.
The mechanisms by which drugs induce death of cancer cells
are different and include necrosis, apoptosis and autophagy.
Therefore, an understanding of these mechanisms could be
helpful to discover more convenient therapeutic strategies. To
further examine the effect of compound 3b on cell proliferation
and survival, we analyzed the cell cycle phase distribution by
flow-cytometric analysis of propidium iodide-stained cells after
treatment with the compound. Figure 5 clearly displays that 24 h
treatment with 3b reduced the percentage of cells in the S-phase
of the cell cycle, compared with control, in a dose-dependent
manner. During the S-phase of the cell cycle, the genetic
information of the DNA is synthesized and duplicated.
References and notes
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Figure 5. Cell cycle phase distribution in control and treatments samples.
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Cancer Res. 1991; 23:6304.
Furthermore, the proteolysis of Poly (ADP-ribose) polymerase
(PARP), crucial target indicating the presence of DNA damage
and facilitating DNA repair, was estimated by immunoblotting
analysis (Fig. 6). Increased levels of the proteolytic form of
PARP (86 kDa) were detected in MCF-7 cells after 24 h
treatment with compound 3b, compared to the control. Increasing
evidence has suggested the straightforward role of p53 signaling
in both growth inhibition and apoptotic cascades.14,15 p53 acts as
a tumor suppressor depending on its physical and functional
interaction with diverse cellular proteins, including some nuclear
receptors that, in turn, exert an inhibitory activity on p53
biological outcomes. Activation of p53 by UV damage or other
agents/signals results in p53-mediated transcription or up-
regulation of genes such as the cyclin-dependent kinase inhibitor
p21WAF1/Cip1 to induce the apoptotic process, inhibiting the growth
of cells with damaged DNA or cancer cells.16-18
20. Levine AJ. Cell 1997; 3:323.
A. Supplementary Material
The ability of compound 3b to modulate the expression of
p53,19,20 along with its natural target gene p21WAF1/Cip1 was also
examined. The treatment with compound 3b induced
a
significant increase in both p53 and p21WAF1/Cip1 protein content in
MCF-7 cells. Finally, changes in the expression of genes





5H-pyrrolo [1, 2-a][3, 1] benzoxazin-5-one is an
interesting scaffold
Various decorations have been performed to
find an anti-proliferative agents
Preliminary studies identified 3b a good
candidate
The mechanism of action will be investigated