Design, synthesis and biological evaluation of 3,5-diaryl isoxazole derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2020.127427

The present study was carried out in the attempt to synthesize a new class of potential anticancer agents comprising eleven compounds (24–34) sharing the 3,5-diarylisoxazole as a core. The chemical structure of the new synthesized compounds was established by IR, ¹H NMR, ¹³C NMR and elemental analysis. Their biological potential towards prostate cancer was evaluated by using cancer PC3 cells and non-tumorigenic PNT1a cells. Interestingly, compound 26 distinguished from others with a quite high selectivity value that is comparable to 5-FU. The binding mode of 26 towards Ribosomal protein S6 kinase beta-1 (S6K1) was investigated at a molecular level of detail by employing docking simulations based on GLIDE standard precision as well as MM-GBSA calculations.

Full text of DOI:10.1016/j.bmcl.2020.127427

Journal Pre-proofs
Design, synthesis and biological evaluation of 3,5-diaryl isoxazole derivatives
as potential anticancer agents
Derya Anıl Aktaş, Gökçen Akinalp, Fatma Sanli, Mehmet Ali Yucel, Nicola
Gambacorta, Orazio Nicolotti, Omer Faruk Karatas, Oztekin Algul, Serdar
Burmaoglu
PII:
S0960-894X(20)30538-2
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127427
BMCL 127427
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 April 2020
17 July 2020
19 July 2020
Please cite this article as: Anıl Aktaş, D., Akinalp, G., Sanli, F., Ali Yucel, M., Gambacorta, N., Nicolotti, O.,
Faruk Karatas, O., Algul, O., Burmaoglu, S., Design, synthesis and biological evaluation of 3,5-diaryl isoxazole
derivatives as potential anticancer agents, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/
10.1016/j.bmcl.2020.127427
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Design, synthesis and biological evaluation of
3,5-diaryl isoxazole derivatives as potential
anticancer agents
Leave this area blank for abstract info.
Derya Anıl Aktaşa, Gökçen Akinalpb, Fatma Sanlic,d, Mehmet Ali Yucele,f, Nicola Gambacortae, Orazio
Nicolottie, Omer Faruk Karatasc,d, Oztekin Algulg* and Serdar Burmaoglub*
F
O
OMe N
MeO
OMe
26
IC50 :15.893 (μM) against PC3 cancer cell line
Selectivity: 7.51

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and biological evaluation of 3,5-diaryl isoxazole derivatives as
potential anticancer agents
Derya Anıl Aktaşa, Gökçen Akinalpb, Fatma Sanlic,d, Mehmet Ali Yucele,f, Nicola Gambacortae, Orazio
Nicolottie, Omer Faruk Karatasc,d, Oztekin Algulf and Serdar Burmaoglub
aDepartment of Chemistry and Chemical Process Technologies, Erzurum Vocational High School, Atatürk University, 25240, Erzurum, Turkey
bDepartment of Chemistry, Faculty of Science, Atatürk University, 25240, Erzurum, Turkey
cDepartment of Molecular Biology and Genetics, Erzurum Technical University, 25050, Erzurum, Turkey
dMolecular Cancer Biology Laboratory, High Technology Application and Research Center, Erzurum Technical University, Erzurum, Turkey
eDipartimento di Farmacia—Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, Via E. Orabona, 4, I-70125 Bari, Italy
fDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin, 33169, Turkey
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The present study was carried out in the attempt to synthesize a new class of potential anticancer
agents comprising eleven compounds (24-34) sharing the 3,5-diarylisoxazole as a core. The
chemical structure of the new synthesized compounds was established by IR, 1H NMR, 13C NMR
and elemental analysis. Their biological potential towards prostate cancer was evaluated by using
cancer PC3 cells and non-tumorigenic PNT1a cells. Interestingly, compound 26 distinguished
from others with a quite high selectivity value that is comparable to 5-FU. The binding mode of
26 towards Ribosomal protein S6 kinase beta-1 (S6K1) was investigated at a molecular level of
detail by employing docking simulations based on GLIDE standard precision as well as MM-
GBSA calculations.
Keywords:
Synthesis
Isoxazole
Anticancer agent
Molecular modeling
Proliferation
2009 Elsevier Ltd. All rights reserved.
———
 Serdar Burmaoglu. Tel.: +90-442-231-4434; fax: +90-442-231-4109; e-mail: sboglu@atauni.edu.tr
 Oztekin Algul. Tel.: +90-324-341-2815/12122; fax: +90 324 341 3022; e-mail: oztekinalgul@mersin.edu.tr

Prostate cancer is the most widespread non-cutaneous cancer
among men worldwide and it is the second leading cause of cancer
deaths within the male population in western countries.1,2 174,650
new prostate cancer cases were estimated to be diagnosed in 2019,
representing approximately 10% of new cancer cases within the
same period.3 Various approaches such as radical prostatectomy,
radical radiotherapy, chemotherapy, and hormone ablation therapy
are considered as the first line treatment option to contrast the
disease in early stage clinically localized prostate tumors.4
However, modern medical approaches remains to be ineffective in
the treatment of advanced prostate cancer.5 Therefore, novel
treatment strategies and tools are needed to be developed in order
to enhance the success of the current diagnostic and therapeutic
methods.6
N
OH
O
Cl
N
O
O
N
N
O
OMe
O
S
MeO
MeO
NH
N
N
NH2
O
N
H
O
O
O
OMe
F
Sulfamehoxazole
antiproliferative
nicotinic receptor modulators
antiviral
antibiotic
Figure 1. Some 3,5-disubstituted isoxazole based compounds with a spectrum of biological properties.
Recent developments in organic chemistry have contributed
significantly to progress in many fields of science, particularly
medicinal chemistry. The common characteristic of the most
newly synthesized organic compounds is the presence of at least
one heterocyclic ring in their structure. Thus, heterocyclic
chemistry is an important tool for the search of new active
substances in biological applications. One of the most interesting
heterocyclic rings is isoxazole, which is a five-membered ring
containing oxygen and nitrogen atoms. The synthesis of the
isoxazole ring, which has a wide range of applications, is 1,3-
dipolar cycloaddition to the alkenes and alkynes with nitrile oxide
and the reaction of a 1,3-diketone or α,β-unsaturated ketone
containing three carbons with hydroxyl amine.7 Isoxazole
derivatives, a special unified class of compounds, exhibit
antibiotic, antiproliferative, and antiviral properties and act as
nicotinic receptor modulators (Fig. 1).8
about the combination of heterocyclic pharmacophores in a hybrid
molecule.
In this study, we aimed at preparing 3,5-diarylsubstituted
isoxazoles for the construction of new drug-like molecules and at
evaluating their biological activities against different cancer cell
lines along with an immortalized normal prostate epithelial cell.
The general structure of the synthesized compounds is shown in
Figure 2.
N
O
R1
R2
R1 = OMe
R2 = H, F, Cl, NO2
Figure 2. General structure of synthesized compounds.
In our previous paper, fluoro and hydroxyl substituted chalcone
derivatives exhibited high antiproliferative activities against
A549, A498, HeLa, A375, HepG2 cancer cell lines.9 These results
encouraged and enabled us to plan the development of new
chalcone derivatives. In addition, we investigated the role of the
two aromatic rings of the chalcone structure for the activity and
the role of the linker group (the enone moiety) to design more rigid
compounds by incorporating heterocyclic fragments. The
discovery of some 3,5-diarylsubstituted isoxazole-based
compounds with a spectrum of antiproliferative properties in the
literature has led to better address our studies towards chalcone-
like systems whose enone bridge was replaced by isoxazole linker.
The preliminary anticancer screenings on prostate cancer cell lines
demonstrated these new chalcone-like systems show the best
cytotoxic effect on PC3 cell line. Based on this cytotoxicity
screening, further studies are required to provide more insights
The synthetic procedure for the preparation of 3,5-
diarylsubstituted isoxazoles is depicted in Scheme 1. The
condensation of trimethoxyacetophenone (1) with related
benzaldehydes 2-12 in an aqueous solution of KOH afforded
chalcone derivatives 13-23 in 70-90% yields through the adoption
10
of a general synthesis protocol.9 Then, reaction of chalcone
derivatives 13-23 with tosylhydroxylamine in the presence of
K2CO3 and H2O in MeOH afforded the isoxazole derivatives 24-
34 in 60-78% yields.11 The newly synthesized target compounds
1
24-34 were characterized using IR, H and 13C NMR as well as
elemental analysis. The characterization data of the target
molecules were found to be compatible with their structure. The
details of spectral data are given in the experimental section and
spectra are provided in supplementary file.
OMe O
OMe O
OMe N
O
O
i
ii
H
R
R
R
MeO
OMe
MeO
OMe
MeO
OMe
2: R = H, 3: R = 2-F
4: R = 3-F, 5: R = 4-F
6: R = 3,4-diF, 7: R = 3,5-diF
8: R = 3-Br, 9: R = 4-Br
10: R = 3-Cl, 11: R = 4-Cl
12: R = NO2
13: R = H, 14: R = 2-F
15: R = 3-F, 16: R = 4-F
17: R = 3,4-diF, 18: R = 3,5-diF
19: R = 3-Br, 20: R = 4-Br
21: R = 3-Cl, 22: R = 4-Cl
23: R = NO2
24: R = H, 25: R = 2-F
26: R = 3-F, 27: R = 4-F
28: R = 3,4-diF, 29: R = 3,5-diF
30: R = 3-Br, 31: R = 4-Br
32: R = 3-Cl, 33: R = 4-Cl
34: R = NO2
1
Scheme 1. General synthetic procedure. Reagents and conditions: (i) 1, 2-12, KOH, (ii) 13-23, tosylhydroxylamine, K2CO3, H2O, MeOH

We initially investigated the effects of the compounds on the
proliferation capacity of PC3 cells by employing in vitro cancer
model and PNT1a cells as corresponding normal control. Sorted in
the order of the selectivity values from higher to smaller,
compounds 26, 30, 27, 29, 25, 34, 28, and 24 proved to be effective
and selective against cancer cells compared to normal prostate
epithelial cells (Fig. 3A-D). Disappointingly, compounds 31, 32,
and 33 lack such selectivity against cancer cells being their
observed selectivity values lower than 1. Interestingly, compound
26 distinguished from other compounds with a quite high
selectivity value that is even comparable to 5-FU. Table 1 reports
the selectivity of the entire pool of our 3,5-diarylsubstituted
isoxazole derivatives along with 5-FU as a reference towards both
cancerous PC3 and non-tumorigenic PNT1a cells. Importantly, all
the structures listed in Table 1 comply the Lipinski's rule of five as
shown in Supporting Information.
Figure 3. Relative viability of PC3 and PNT1a cells treated with diarylsubstituted isoxazole derivatives or 5-FU as a reference.
To predict potential drug targets for these compounds, we used
the PPB2 Polypharmacology Browser, which indicate several key
proteins with a likely mechanistic role to explain at a molecular
level the observed selective inhibitory potential against cancer
cells (see Supplementary File for the list of predicted targets). To
minimize the occurrence of false positive results and thus to
properly prioritize the predicted targets, we excluded 1) those
shared indistinctly by all the eleven compounds experimentally
tested, 2) those shared by compounds with discordant selectivity
values (that are greater or lower than 1 at the same time), or 3)
those related to irrelevant genes considering prostate cancer
pathogenesis. Interestingly, CYP1A1, CYP1A2 and CYP1B1
were predicted as putative potential targets for those compounds
having experimental selectivity values greater than 1. More
interestingly, compound 26, which is provided with the highest
observed selectivity value, matched 2 unique targets that are CDC
like kinase 4 (CLK4) and Ribosomal protein S6 kinase beta-1
(S6K1), which did not occur in the other remaining compounds.
Considering their oncogenic potential in prostate cancer as well as
in other cancers, selective targeting of these 2 genes might serve
as a potential tool for selective elimination of cancer cells that
exclusively have higher expression for these genes compared to
normal cells.
Table 1. IC50 and selectivity values of compounds tested on PC3 and PNT1a cells
IC50
Selectivity
PC3
1.28
Compounds
PNT1a
PC3
24
25
3.40 ± 0.52
5.21 ± 1.11
117.33 ± 3.78
11.89 ± 0.96
9.16 ± 1.21
9.51 ± 2.89
21.81 ± 1.32
6.89 ± 1.58
4.49 ± 0.25
15.74 ± 4.36
5.58 ± 0.79
9.70 ± 0.57
2.66 ± 0.22
3.58 ± 0.15
16.79 ± 6.24
5.06 ± 0.47
7.25 ± 2.54
4.77 ± 1.54
6.68 ± 1.36
5.19 ± 0.96
14.38 ± 5.27
26.44 ± 7.15
13.09 ± 2.85
1.64 ± 0.87
1.46
26
6.99
27
2.35
28
1.26
29
1.99
30
3.27
31
1.33
32
0.31
33
0.60
34
0.43
5-FU
5.90

In order to study the binding mode of compound 26 towards
S6K1, docking studies were performed through GLIDE standard
precision docking and MM-GBSA calculations. The redocking
analysis of the cognate ligand F177 returned docking score and G
binding values equal to -10.439 kcal/mol and to -99.25 kcal/mol,
respectively, as well as RMSD (Root Mean Square Deviation)
value as small as 0.305 Å compared to the X-ray posing. As shown
in Table 2, docking analysis of 26 was carried out, by returning
docking score and G binding values equal to -6.419 kcal/mol and
-59.07 kcal/mol, respectively.
proliferation test results, selective inhibitory potential of the
compounds tested might be associated with expression of those
genes exclusively in cancer cells.
CLK4 is one of the critical components of pre-mRNA splicing,
which is quite important for cellular functions.18 In a recent study,
inhibition of CLK4 proteins was postulated as a novel anticancer
strategy, which aimed at selective depletion of cancer-relevant
proteins after turnover.19 Interestingly, its inhibition resulted in the
depletion of another putative target for 26, that is S6K1.19
S6K1 is one of the downstream targets of PI3K/Akt/mTOR
signaling pathway, which is particularly involved in mRNA
translation, ribosome biogenesis, proliferation, metabolism,
development, aging and malignancies like cancer.20 One of the
critical negative regulators of this signaling pathway PTEN, is lost
in almost 80% of prostate cancers, resulting in constitutive
activation of PI3K/Akt/mTOR signaling pathway.21 PC3 cells
used in our tests are known to be null for PTEN, therefore have
constitutively active PI3K/Akt/mTOR signaling, which is not the
case for PNT1a cells.22 Recent studies also pointed the hyperactive
status of S6K1 in PC3 cells due to active PI3K/Akt/mTOR
signaling.20,22,23 Most importantly, 26 did not kill DU-145 prostate
cancer cells, which are wild type for PTEN (Supplementary Figure
1). This might explain selective targeting of cancer cells with
active PI3K/Akt/mTOR signaling by 26. Interestingly, in a recent
study, a S6K1 inhibitor PF-4708671 was demonstrated to
particularly reduce migration and proliferation of PC3 cell line, but
not DU-145 prostate cancer cells that carry wild type PTEN
alleles.24 Docking studies conducted on 26 proved its potential as
S6K1 inhibitor being able to interact at the level of the hinge
region of S6K1 protein likewise F177 cognate ligand and other
inhibitors.13
Table 2. Docking score and G binding values of F177 cognate
ligand and 26.
Compound
F177 cognate-ligand
26
Docking Score
-10.439 kcal/mol
-6.419 kcal/mol
G binding
-99.25 kcal/mol
-59.07 kcal/mol
Such a discrepancy in scoring when comparing 26 and F177
could be due to the different interactions established at the binding
site. However, the estimation of the ligand efficiency (LE)12,
which represents a size-dependent measure for effective binding,
disclosed that 26 would be a promising starting point for molecular
optimization based on the observation that a smaller gap existed
when comparing the LE values of 26 vs F177 that was -0.267
kcal/mol vs -0.316 kcal/mol.
In summary, the novel (2,4,6-trimethoxyphenyl)isoxazole
compounds were synthesized in two steps, formation of the
corresponding chalcones by a well-known standard method and
condensation with TsNHOH under strongly basic conditions in
good yields and purities. The preliminary anticancer screenings on
prostate cancer cell lines demonstrated that the chalcone with a
fluoro substituted show the best cytotoxic effect on PC3 cell line.
Based on this preliminary cytotoxicity screening, further studies
are required to provide more insights about the combination of
heterocyclic pharmacophores in a hybrid molecule and their
structure-activity-relationship in prostate cancer therapy. Also,
Multi-fingerprint Similarity Search algorithm (MuSSel)25,26 was
used to find other potential targets. In this respect, protein tyrosine
phosphatase 1B (PTP1B) is the first target for 10 out of 11
molecules. PTP1B is related with obesity and type 2 diabetus
mellitus and PTP1B was also found involved with breast, pancreas
gastric, ovarium cancer and prostate cancer in recent years.27
These result may be very helpful us for forward research. Further
synthetic work on similar structures containing heterocyclic
hybrids are currently in progress for generating new targeted
libraries for biological screenings.
Figure 4. Zoomed in view at the binding pocket of S6K1 (PDB entry: 3WF9)
whose residues are rendered in gray sticks and labeled. Green sticks and yellow
wireframes represent 26 and F177 cognate ligand, respectively. Red arrows
indicate hydrogen bonds.
As shown in Figure 4, the aromatic rings of 26 are nicely
superimposed to the sulfomoylphenylamino ring and
methyltetrahydroacridine ring of F177, which faces the hinge
region of the protein.13
Acknowledgments
In addition, F177 and 26 share the same hydrophobic contacts
with the following residues: L97, G98, K99, G100, V105, A121,
L172, E173, Y174, L175 and M225. Furthermore, 26 established
a hydrogen bond through its para-methoxyl engaging the
backbone nitrogen atom of L175 in the hinge region, likewise
F177 cognate ligand.
We thank the Scientific and Technological Research Council of
Turkey (TUBITAK, Grant number: 119S045) and The Scientific
Research Projects of Erzurum Technical University (Grant
number: 2019/12)
CYP1A1 and CYP1A2 demonstrated to not be present in
normal prostate samples.14 However, they were reported to be
expressed in PC3 cells.15 Besides, CYP1B1 has been shown to
have oncogenic properties in prostate cancer.16,17 Considering our
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