Synthesis of new α-Aryl-α-tetralones and α-Fluoro-α-aryl-α-tetralones, preliminary antiproliferative evaluation on drug resistant cell lines and in silico prediction of ADMETox properties

Article abstract of DOI:10.1016/j.bioorg.2021.104790

α-aryl-α-tetralones and α-fluoro-α-aryl-α-tetralones derivatives were synthesized by palladium catalyzed α-arylation reaction of α-tetralones and α-fluoro-α-tetralones, with bromoarenes in moderate to good yields. These compounds were evaluated for their

Full text of DOI:10.1016/j.bioorg.2021.104790

Bioorganic Chemistry 110 (2021) 104790
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of new α-Aryl-α-tetralones and α-Fluoro-α-aryl-α-tetralones,
preliminary antiproliferative evaluation on drug resistant cell lines and in
silico prediction of ADMETox properties
Luana G. de Souzaa, Eduardo J. Salustianob *, Kelli M. da Costab, Angela T. Costac,
,
c
d
e
a,*
´
Vivian M. Rumjanek , Jorge L.O. Domingos , Magdalena N. Renno , Paulo R.R. Costa
a
´
ˆ
˜
Laboratorio de Química Bioorganica, Instituto de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro, Ilha do Fundao, CCS, Bloco H - Sala H27,
21941-902 Rio de Janeiro, RJ, Brazil
b
´
ˆ
Laboratorio de Glicobiologia, Instituto de Biofísica Carlos Chagas Filho, Centro de Ciencias da Saúde, Bloco C sala C1-042, Universidade Federal do Rio de Janeiro, RJ
21941-590, Brazil
c
´
´
ˆ
Laboratorio de Imunologia Tumoral, Instituto de Bioquímica Medica Leopoldo de Meis, Centro de Ciencias da Saúde, Bloco H sala 003, Universidade Federal do Rio de
Janeiro, RJ 21941-590, Brazil
d
ˆ
ˆ
˜
Departamento de Química Organica, Centro de Tecnologia e Ciencias, Universidade do Estado do Rio de Janeiro, Rua Sao Francisco Xavier 524, Pav. Haroldo Lisboa da
˜
Cunha – s 406 – Maracana, 20550-900 Rio de Janeiro, RJ, Brazil
e
´
ˆ
ˆ
Laboratorio Integrado de Biologia Computacional e Pesquisa em Ciencias Farmaceuticas, Instituto de Biodiversidade e Sustentabilidade (NUPEM), Universidade Federal
˜
´
´
do Rio de Janeiro, Rua Sao Jose do Barreto 764, 27965-045 Macae, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
α
-aryl-
α
-tetralones and
α
-fluoro-
α
-aryl-
α
-tetralones derivatives were synthesized by palladium catalyzed
α-ary-
Carba-isoflavonoids
lation reaction of
α
-tetralones and
α
-fluoro-α
-tetralones, with bromoarenes in moderate to good yields. These
α
-Tetralone
compounds were evaluated for their in vitro anti-proliferative effects against human breast cancer and leukemia
cell lines with diverse profiles of drug resistance. The most promising compounds, 3b, 3c, 8a and 8c, were
effective on both neoplastic models. 3b and 8a induced higher toxicity on multidrug resistant cells and were able
to avoid efflux by ABCB1 and ABCC1 transporters. Theoretical calculations of the physicochemical descriptors to
predict ADMETox properties were favorable concerning Lipinski’s rule of five, results that reflected on the low
effects on non-tumor cells. Therefore, these compounds showed great potential for development of pharma-
ceutical agents against therapy refractory cancers.
Molecular descriptors
SAR
Multifactorial drug resistance
ABC transporters
Synthetic lethality
1. Introduction
isoflavanones were synthesized by Ma and coworkers [9,10] and eval-
uated for inhibition of aromatase activity, an enzyme that catalyzes
Cancer is a pathological condition where a series of enabling char-
acteristics such as replicative immortality and resistance to cell death
grant cells abnormal growth and invasion of healthy tissues and organs
[1]. It is a serious public health problem in many parts of the world and
considered one of the leading causes of death [2]. The main cause for
this disease is the environmental exposure to chemical, physical and
biological carcinogenic agents, and evidences indicate that age increase
positively correlates to emergence of this disease [3–5]. In addition, the
resistance developed by neoplastic cells may hamper the efficacy of
chemotherapy-based treatments [6].
estrogen biosynthesis from androgen precursors. In Fig. 1 are shown
compounds 1 and 2 which showed the best inhibitory activity [9,10].
Compound 2 also presented antiproliferative effect on MCF-7 cell line
[10]. They are interesting prototypes for the discovery of new anti-
proliferative agents, in special for pharmaceutical agents against ther-
apy refractory cancers.
In this paper we report the synthesis and the evaluation of the
antiproliferative activity of
cell lines.
α-aryl-α-tetralones 3–12 on drug resistant
Compounds 3–12 can be considered as isosteres of 1 and 2 in which
the oxygen atom at the chromanone ring is replaced by a methylene
group. This approach was successfully used by Miller and co-workers
Isoflavanones are a sub-group of isoflavonoids comprising inter-
esting biologically active compounds [7,8]. Some synthetic
* Corresponding authors.
E-mail addresses: salustiano@bioqmed.ufrj.br (E.J. Salustiano), prrcosta2011@gmail.com (P.R.R. Costa).
https://doi.org/10.1016/j.bioorg.2021.104790
Received 17 July 2020; Received in revised form 5 February 2021; Accepted 28 February 2021
Available online 4 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

L.G. de Souza et al.
Bioorganic Chemistry 110 (2021) 104790
multidrug-resistant cell lines (MDR), the study of the efflux activity
mediated by ABCB1 or ABCC1 proteins of the compounds with the
lowest relative resistance indexes (RR) and the prediction of in silico
ADMETox properties.
2. Results and discussion
Scheme 1 shows the
palladium catalyzed -arylation reaction described by Fernandes et al.
[18], using commercial -tetralones and o-MeO-bromoarenes in the
α-aryl-α-tetralones 3–7 synthesized through a
α
α
presence of Pd2(dba)3 as catalyst, tBu3PHBF4 as ligand and KOH with
heating by microwave. Compounds 3a-c were prepared in excellent
yields under these conditions, but poor yields were observed for com-
pounds 4–7. In the case of compounds 4a-c, we needed to increase the
catalytic system to 5 mol% of Pd2(dba)3 and 20 mol% of tBu3PHBF4, and
the temperature was raised to 130 ◦C. The other compounds, 5–7, better
yields were obtained when Pd2(dba)3 was changed for Pd(dba)2 in the
presence of NaOH.
Fig. 1. Bioactive synthetic isoflavanones (1 and 2) and the 1-carba-ana-
logues (3–12).
once pterocarpens and 5-carba-pterocarpens showed similar affinity for
α
and β estrogen receptors, being as potent as estradiol, the natural
The synthesis of the
with the preparation of the corresponding
rivatives 15a-c by -fluorination of 13a-c with Selectfluor in PEG-400.
α
-fluoro-
α
-aryl-
α
-tetralones (8–12) was initiated
ligand [11]. In our group a synthetic aza-pterocarpan was active but less
potent as antiproliferative on breast cancers and on leukemia cell lines
when compared to its 1-carba-analogue [12]. Another interesting
example is the substitution of the oxygen atom in prostacyclin (PGI2) by
a methylene group. The carba-analogue showed the chemical stability
greater than PGI2 and the same pharmacological activity profile. [13].
α
-fluoro- -tetralones de-
α
α
These compounds were prepared in good yield and were used in the
α
-arylation with 14a-e (Scheme 2).
The structures were determined by 1H and 13C nuclear magnetic
resonance (NMR) and mass spectroscopy, whereas a high-resolution
mass spectrum was performed for the new compounds. In general, the
To design the new α-aryl-α-tetralones 3–12 (Fig. 2) we considered
the therapeutic benefits that often result by the replacement of a
hydrogen atom for a fluorine atom [14]. It is estimated that 20–25% of
the drugs on the market have at least one fluorine atom in their struc-
tures [15]. The presence of this atom in a molecule can change bio-
absorption, binding affinity, chemical reactivity, metabolic stability
and, in some cases, can prevent the epimerization process [16–17]. In A-
ring we maintained the same pattern of substitution used by Ma and co-
workers.
1H NMR spectrum showed the
α-carbonyl proton in 3.90–4.6 ppm as dd
signals in
α-aryl-
α-tetralones (3–7) and none signal in this region for
α-fluoro-
α
-aryl-α-tetralones (8–12). The aromatic protons signals
appeared at 6.8–8.0 ppm and the aliphatic protons at 2.0–3.85 ppm as
mutiplet signals. The 13C spectrum showed duplicated signals in some
carbon signals and the C-F had a coupling constant of approximately
200 Hz.
At the B-ring we introduced different patterns of oxygenation, trying
to improve the affinity of this ring for the Fe2+ present at the catalytic
site of aromatase. The nitrile was selected considering the presence of
this group in the structure of drugs employed for breast cancer therapy
as aromatase inhibitors, such as letrozole and anastrozole. Finally, the
2.1. Biological evaluation
The synthesized compounds were evaluated for their anti-
proliferative effect on four human neoplastic cell lines with diverse
phenotypes of drug resistance, and the results compared to the controls
daunorubicin (DNR), a pro-oxidant, DNA-intercalating anthracycline
and vincristine (VCR), a tubulin-binding antimitotic alkaloid. The cell
lines employed in this study are specified as follows: MCF-7, an invasive,
endocrine therapy-sensitive breast ductal carcinoma, estrogen and
progesterone receptors positive and Her2/neu overexpression negative;
K562, a chronic myeloid leukemia from erythroid origin with constitu-
tive BCR/ABL tyrosine kinase activity, resistant to oxidative stress
presence of one fluorine atom at α-position in α-tetralone was proposed
due to reasons already discussed.
Our target compounds are shown in Fig. 2. We report their synthesis,
a preliminary evaluation of their potential antineoplastic activity on
Scheme 1. Synthesis of
α
-aryl-
α
-tetralones 3–7. 1Reactions were carried out
with ketone (0.2 mmol), bromo-aryl (0.24 mmol), Pd2(dba)3 (0.005 mmol),
tBu3PHBF4 (0.02 mmol), and KOH (0.5 mmol) in dioxane:water (2.0 mL) for 1 h
at 100 ◦C in MW. 2Optimized conditions: 5 mol% Pd2(dba)3 (0.01 mmol), 20
mol% tBu3PHBF4 (0.04 mmol) and NaOH (0.5 mmol) for 1 h at 130 ◦C in MW.
3Optimized conditions: 5 mol% Pd(dba)2 (0.01 mmol), 10 mol% tBu3PHBF4
(0.02 mmol) and NaOH (0.5 mmol) for 1 h at 100 ◦C in MW.
Fig. 2. New synthetic carba-analogues of isoflavanones with potential anti-
proliferative activity.
2

L.G. de Souza et al.
Bioorganic Chemistry 110 (2021) 104790
Table 1
Antineoplastic effect (IC50) of synthesized compounds on models of human
breast cancer and chronic myeloid leukemia.
Compound
MCF-7
MCF-
10A
SI
K562
Lucena-1
FEPS
MCF-
10A/
MCF-7
3b
66.87
± 7.45
88.37
± 6.00
60.30
± 7.41
84.23
± 4.70
35.65
± 5.56
NA
152.67
± 11.50
172.22
± 10.40
235.67
± 21.15
>320
2.43
1.95
3.91
3.67
1.73
–
72.30 ±
8.03
63.17 ±
1.87
36.37
± 2.05
34.62
3c
79.72 ±
2.57
58.02 ±
3.60
± 1.73
27.06
8a
63.42 ±
4.68
46.84 ±
2.62
± 1.28
29.50
8b
49.64 ±
3.06
39.00 ±
4.35
± 1.51
>4000
DNR*
VCR*
61.57 ±
4.78
109.68
± 3.58
23.72 ±
0.39
2066.29
± 138.82
405.61 ±
89.75
NA
>960
Results are reported as IC50 values ± SD in µM. Data represent means obtained
from three independent experiments, with each concentration evaluated in
triplicates. Assays and calculations were performed as described in the Experi-
mental Section. NA = Not analyzed. *For DNR and VCR, mean IC50 ± SD is
expressed in nM rather than in μM.
with aromatase, the last and rate-limiting step in estrogen biosynthesis
[9]. This is reasonable since we observed higher IC50 on MCF-7 than on
MCF-10A, which does not rely on estrogens for proliferation. Myeloid
leukemias express aromatase as well, and though estrogen metabolism
was linked to hematopoiesis by regulating myeloid cell differentiation in
two leukemia cell types [23–24], its role is not yet fully understood.
However, considering the roles of estrogens as master regulators of cell
bioenergetics [25], limiting its levels would disrupt mitochondrial
function and lead cells to death due to ATP loss.
Scheme 2. Synthesis of α-fluoro-tetralones (15a-c) and α-fluoro-α-aryl-α-tet-
In addition, results indicated lower IC50 values for the drug-resistant
leukemia FEPS, which reflected on the relative resistance indexes (RR).
Increased sensitivity of MDR cells compared to parental ones is a form of
synthetic lethality known as collateral sensitivity [26]. As such, RR were
calculated for Lucena-1 and FEPS (Fig. 3) and when one compound
showed RR ≤ 0.5 it was considered a collateral sensitizing agent [27].
When RR ≥ 2.0 cells were considered cross-resistant to a drug, which
was the case for both Lucena-1 and FEPS when treated with the standard
chemotherapeutics VCR or DNR.
ralones (8–12). 4Reactions were carried out with fluoro-ketone (0.2 mmol),
bromo-aryl (0.24 mmol) in dioxane:water (2.0 mL) for 1 h at 100 ◦C in MW.
5Optimized mmol), Pd2(dba)3 (0.005 mmol), tBu3PHBF4 (0.02 mmol), and KOH
(0.5 condition: 5 mol% Pd2(dba)3 (0.01 mmol), 20 mol% tBu3PHBF4 (0.04
mmol) and NaOH (0.5 mmol) for 1 h at 130 ◦C in MW. 6Optimized condition: 5
mol% Pd(dba)2 (0.01 mmol), 10 mol% tBu3PHBF4 (0.02 mmol) and NaOH (0.5
mmol) for 1 h at 100 ◦C in MW.
[19,20]; Lucena-1 and FEPS, multidrug resistant (MDR) K562 de-
rivatives. Lucena-1 was selected after continuous exposure of K562 to
VCR, resulting in ABCB1 (P-glycoprotein) overexpression and resistance
to UV radiation and hydrogen peroxide [20]. FEPS was selected after
continuous exposure of K562 to DNR, selecting cells resistant to a variety
of natural and synthetic compounds owing to its high efflux activity
mediated by the ABC transporters ABCB1 and ABCC1 (MRP1) [21]. The
compounds were assessed in triplicate measurements and the mean IC50
values are presented in Table 1. MCF-10A, a spontaneously immortal-
ized, non-tumorigenic and estrogen receptor negative human mammary
epithelial cell [22] was employed for calculation of selectivity indices
(SI) as well.
Results on Fig. 3 indicate that despite
α-aryl-α-tetralones and
Concerning breast cancer, results showed that 8a and 3b inhibited
the mitochondrial reducing activity of MCF-7 cells with the lowest IC50
,
respectively 60.30 ± 7.41 µM and 66.87 ± 7.45 µM. Of note, both
compounds present one fluorine atom in their structures, indicating that
it could contribute to toxicity for breast cancer. In addition, 8a presented
the highest selectivity index (3.91), which demonstrates that this com-
pound is more active in the tumor cell line MCF-7. Concerning chronic
myeloid leukemias, notably 8a and 8b were the most promising com-
pounds, with IC50 lower than 30 µM in the MDR FEPS cell. In agreement
Fig. 3. Relative resistance (RR) indexes for the
α-aryl-α-tetralones and α-fluoro-
α
-aryl- -tetralones evaluated on chronic myeloid leukemias. Calculations were
α
performed as described in the Experimental Section. RR = (IC50 resistant cell
line, Lucena-1 or FEPS)/(IC50 parental cell line, K562). Purple squares and red
inverted triangles respectively indicate Lucena-1 or FEPS, and the numbers
indicate the calculated RR for each compound. (For interpretation of the ref-
erences to colour in this figure legend, the reader is referred to the web version
of this article.)
with previous results, both structures present α-fluorine atoms.
Leukemic cells showed lower IC50 for most compounds than breast
cancer, suggesting differences in either drug distribution or in mecha-
nisms of action on cells from epithelial or blood origins. The new α-aryl-
α
-tetralones and α-Fluoro-α-aryl-α-tetralones were proposed to interact
3

L.G. de Souza et al.
Bioorganic Chemistry 110 (2021) 104790
α
-fluoro-
α
-aryl-
α
-tetralones presented lower IC50 on Lucena-1 when
activity by the inhibitors verapamil (VP), trifluoperazine and cyclo-
sporin A [20,21,32], or the ABCC1 inhibitors MK-571, indomethacin
and probenecid [21,32]; as such, we employed VP and MK-571 as pos-
itive controls (Fig. 4).
compared to K562, none induced collateral sensitivity. In contrast, 3b as
well as 3c and 8a produced this effect on FEPS cells. Studies with in-
hibitors of kinesin spindle proteins (KSP) have already linked the pres-
ence of halogens to higher cytotoxicity to drug-resistant cancer cells, in
which the increase in bioavailability was important for this effect
[28–29]. Moreover, inhibition of aromatase by anastrozole and letrozole
was shown to induce increases in glutathione levels in breast cancer
cells, likely to counter the oxidative stress after the depletion of estro-
gens [30]. On leukemias, and mostly on FEPS cells that overexpress
ABCC1, this would possibly explain the increased sensitivity, since both
impairment of energy production and increased efflux of glutathione (a
prime ABCC1 substrate) are two possible inducers of collateral sensi-
tivity [26,31]. Depletion of glutathione and ATP would render cells
unable to deal with oxidative stress, leading them to apoptotic cell death
[32,33].
Results on Fig. 4 show that neither compound increased intracellular
contents of Rho 123 or CF after incubations with concentrations equal to
or double their IC50. These profiles indicate that 3c and 8a are not
substrates for ABCB1 or ABCC1, which is on par with our earlier ob-
servations. This would represent an interesting scenario for patients
with endocrine therapy resistant cancers, since it has been described
that ABCB1 actively extrudes anastrozole [34] and that little informa-
tion is available concerning ABCC1. A variety of compounds exert
collateral sensitization through increases in production of endogenous
substrates of ABC transporters or directly by competitive inhibition
[31–33]. Regardless of the latter not being observed here, monitoring of
alterations associated with the onset of MDR but not directly ABC ac-
tivity may provide clues to better understand our results.
So far, the synthesized α-aryl-α-tetralones and α-fluoro-α-aryl-α-tet-
ralones exerted cytotoxicity regardless of the expression of the ABC
proteins ABCB1 and ABCC1 on FEPS. To further investigate this possi-
bility, we assessed if 3c and 8a, molecules with the lowest RR, would
modulate the efflux activity by ABCB1 or ABCC1 on those cells. ABCB1
activity was measured by the efflux of rhodamine 123 (Rho 123)
whereas ABCC1 was probed by the efflux of carboxyfluorescein (CF), the
hydrolyzed form of 5(6)-carboxyfluorescein diacetate (CFDA). The MDR
phenotype of both cells can be reversed by impairment of ABCB1
2.2. In silico analysis
The compounds were evaluated according to the Lipinski ‘‘Rule of
Five’’ [35] and polar surface area (PSA) which is also a parameter for
oral bioavailability. It was related that orally administered drugs with
high PSA (>120 Å2) were badly absorbed by passive transcellular route
while drugs with low PSA (<60 Å2) were well absorbed [36]. The values
of molecular weight (270.10 to 288.09 Da), cLogP (3.63 to 4.04), HBD
(0), HBA (2 to 3) and PSA (26.30 to 35.53 Å2) showed in Table 2 were
favorable for the evaluated compounds and may be well absorbed by
oral route. The results were compared to the chemotherapeutic drugs
daunorubicin (DNR) and vincristine (VCR). We also calculated the
druglikeness, that evaluates whether the molecule contains predomi-
nantly fragments commonly present in commercial drugs in comparison
to non-drug-like collection of Fluka compounds. All compounds showed
negative druglikeness (-7.22 to ꢀ 0.87), which demonstrated the pres-
ence of uncommon fragments compared to traded drugs as well as to
commercially available chemicals (Fluka).
It is important to freezing the changes of fluorine substituent in the
molecules and the similarities of the molecules that presented the anti-
proliferative results. These compounds have similarities in the physi-
cochemical properties which have the same values of PSA (26.30) and
HBA (2) that may indicate the same mechanism of action and the need
for low PSA and HBA values.
The analysis of theoretical potential toxicity risks of the compounds
showed similar characteristics, with no risks of mutagenic, tumorigenic
and irritating effects and high risk of reproductive effect for all com-
pounds, comparable to the standard antineoplastic drugs VCR and DNR
(data not shown). These predictions processes are based on the analysis
of fragments of the structure designed and compared to a list of frag-
ments created by the program, based on the assumption that commercial
drugs are largely free of toxic effects. Any fragment was considered a risk
Table 2
Fig. 4. Competition efflux assays of 3c and 8a for ABCB1 or ABCC1-mediated
transport in FEPS cells. ABCB1 or ABCC1-mediated transport was evaluated by
rhodamine 123 (Rho 123) or carboxyfluorescein (CF) efflux assays, as described
Calculated physicochemical properties.
Comp.
AMWa
PSAb
HBAc
HBDd
cLogPe
Druglikeness
in the Experimental Section. 10 μM verapamil (VP), 25 μM MK-571, standard
3b
270.10
282.12
270.10
288.09
527.18
824.40
26.30
35.53
26.30
26.30
185.84
171.17
2
0
0
0
0
5
3
4.04
3.87
3.63
3.73
1.09
2.98
ꢀ 2.21
ꢀ 0.87
ꢀ 7.22
ꢀ 7.22
6.16
3c
3
inhibitors for ABCB1 and ABCC1, or concentrations equal to (IC50) or two times
the IC50 (2 × IC50) of 3c (35 and 70 M) and 8a (30 and 60 M) were employed
8a
2
μ
μ
8b
2
as competitive inhibitors. Representative histograms for intracellular (A) Rho
123 or (C) CF after efflux, with the median fluorescence intensities (MFI) listed
on the right. Continuous lines indicate cells treated with diluent (CTR, free
efflux) or with standard inhibitors (VP or MK-571, inhibited efflux). Dotted or
dashed lines indicate cells, respectively, treated with 3c or 8a in the indicated
concentrations. Scatter plots for the MFI obtained for (B) Rho 123 or (D) CF
after the free, 3c– or 8a-competitive or after inhibited efflux. A.U., arbitrary
units. Inverted triangles indicate FEPS cells, with lines indicating the median of
each population. n = 3, with ***p < 0.001.
DNR
11
14
VCR
4.03
a
b
c
AMW, absolute molecular weight.
PSA, polar surface area.
HBA, hydrogen bond acceptor.
HBD, hydrogen bond donor.
d
e
cLogP, calculated logarithm of partition coefficient between n-octanol and
water.
4

L.G. de Souza et al.
Bioorganic Chemistry 110 (2021) 104790
factor if it occurred often as substructures of harmful compounds but
never or rarely in commercial drugs.
4.2.1.1. 4-methoxy-3-(1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)benzoni-
1
◦
trile (4a): Yellow solid (25 mg, 45%). Mp: 159–161 C. H NMR (400
MHz, CDCl3) δ 8.07 (d, J = 7.7 Hz, 1H), 7.59 (dd, J = 8.6, 2.0 Hz, 1H),
7.52 (t, J = 7.4 Hz, 1H), 7.41 (d, J = 1.9 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H),
7.30 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 4.07 (dd, J = 12.9, 4.5
Hz, 1H), 3.82 (s, 3H), 3.18 (ddd, J = 16.5, 12.3, 4.3 Hz, 1H), 3.05 (dt, J
= 16.6, 3.7 Hz, 1H), 2.49 (ddd, J = 25.3, 12.8, 4.3 Hz, 1H), 2.30 – 2.23
(m, 1H); 13C NMR (101 MHz, CDCl3) δ 196.75, 160.53, 143.82, 133.52,
133.14, 133.10, 132.54, 130.48, 128.78, 127.66, 126.81, 119.28,
111.26, 103.97, 55.80, 49.67, 29.69, 29.48. HRMS (ESI): m/z calcd. for
3. Conclusion
A series of
α-aryl-α-tetralones and α-fluoro-α-aryl-α-tetralones (1-
carba-isoflavanone analogues) were synthesized with moderate to good
yields, up to 92%. Some compounds were evaluated for cytotoxicity
toward human breast cancer and chronic myeloid leukemias with
diverse profiles of drug resistance. Concerning breast cancer, 8a and 3b
showed the best anti-proliferative results with the first, with a fluorine
atom in a chiral center, presenting the highest selectivity. 8a and 3c,
both with fluorine substitutions, showed the most promising results for
chronic myeloid leukemias, notably for the MDR cell FEPS when
compared to the drug-sensitive K562. This feature, known as collateral
sensitivity, has been observed in several other classes of compounds and
relates to changes in energy metabolism, membrane fluidity and
C18H15NO2: 277.1103; found: 277.1094.
4.2.1.2. 3-(7-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)-4-methox-
ybenzonitrile (4b). yellow solid (36 mg, 61%). Mp: 162–166 ◦C.1H NMR
(400 MHz, CDCl3) δ 7.69 (dd, J = 9.1, 2.4 Hz, 1H), 7.57 (dd, J = 8.5, 1.5
Hz, 1H), 7.38 (d, J = 1.4 Hz, 1H), 7.29 – 7.24 (m, 1H), 7.20 (td, J = 8.2,
2.5 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 4.00 (dd, J = 13.0, 4.4 Hz, 1H),
3.79 (s, 3H), 3.16 – 3.06 (m, 1H), 3.01 (dt, J = 16.4, 3.4 Hz, 1H), 2.51 –
2.40 (m, 1H), 2.28 – 2.20 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 195.67
(d, J = 1.8 Hz), 162.79, 160.40 (d, J = 11.2 Hz), 139.52 (d, J = 3.0 Hz),
134.10 (d, J = 6.1 Hz), δ 133.20 (d, J = 2.1 Hz), 133.18 – 133.07 (m),
130.62 (d, J = 7.1 Hz), 130.16, 120.82 (d, J = 22.2 Hz), 119.17, 113.48
(dd, J = 21.8, 4.6 Hz), 111.34, 104.05, 55.81, 49.53, 29.42, 28.79.
HRMS (ESI): m/z calcd. for C18H14FNO2: 295.1009; found: 295.1003.
mechanisms of drug efflux. In this context, the
α-aryl-α-tetralone 3b and
the -fluoro- -aryl- -tetralone 8a exerted cytotoxicity by evading the
α
α
α
two most common ABC transporters. The in silico analysis of ADMETox
properties revealed that the compounds meet the criteria to be well
absorbed following oral administration, with reduced probability of
toxic effects. Therefore, these compounds exhibit interesting properties
for the development of pharmaceutical agents aimed to chemotherapy-
refractory cancers.
4.2.1.3. 4-methoxy-3-(7-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-
1
◦
4. Experimental
yl)benzonitrile (4c). yellow solid (26.4 mg, 43%). Mp: 155–158 C. H
NMR (400 MHz, CDCl3) δ 7.59 (dd, J = 8.5, 1.8 Hz, 1H), 7.55 (d, J = 2.6
Hz, 1H), 7.40 (d, J = 1.6 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (dd, J =
8.4, 2.7 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 4.04 (dd, J = 12.8, 4.4 Hz,
1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.10 (ddd, J = 16.2, 12.2, 4.2 Hz, 1H),
2.98 (dt, J = 16.4, 3.6 Hz, 1H), 2.46 (qd, J = 12.6, 4.2 Hz, 1H), 2.28 –
2.21 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 196.69, 160.55, 158.40,
136.44, 133.32, 133.05, 130.56, 129.99, 121.79, 119.25, 111.23,
109.64, 109.62, 109.59, 103.99, 55.80, 55.49, 49.49, 29.68, 28.62.
HRMS (ESI): m/z calcd. for C19H17NO3: 307.1208; found: 307.1198.
4.1. General
Reagents and solvents were obtained commercially and used without
further purification. The microwave-assisted reactions were conducted
on a single-mode Discover System from CEM Discover and Explorer-
Coolmate accessory. NMR spectra were recorded in deuterated chloro-
form, unless otherwise stated, on a spectrometer operating at 400 MHz
1H NMR, 101 MHz 13C NMR and 470 MHz 19F (spectra were recorded
using Varian INOVA 400 MHz and Varian INOVA 500 MHz) with tet-
ramethylsilane (TMS) and chloroform as internal standards. Chemical
shifts are reported as δ values (ppm). The following abbreviations are
used for the multiplicities: s: singlet, d: doublet, dd: doublet of doublet, t:
triplet, q: quadruplet, m: multiplet. High resolution mass spectra (GC-EI)
were recorded using a QTOF Agilent 7200 instrument for the exact mass
and Agilent 7890B for the GC. The progress of reactions was monitored
by thin layer chromatography (TLC) and column chromatography were
carried out on silica gel, the flash column chromatography silica gel 60
(40–60 mm) was employed. Analytical TLC was done using ALUGRAM®
Xtra SIL G/ UV254 silica gel plates, and the spots were determined under
UV light (λ = 254 nm).
4.2.1.4. 7-methoxy-2-(3-methoxyphenyl)-3,4-dihydronaphthalen-1(2H)-
one (5c). brown oil (30.4 mg, 54%).1H NMR (300 MHz, CDCl3) δ 7.57
(d, J = 2.8 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H),
7.08 (dd, J = 8.4, 2.8 Hz, 1H), 6.81 (ddd, J = 8.3, 2.5, 0.9 Hz, 1H), 6.79 –
6.72 (m, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 3.74 (d, J = 7.8 Hz, 1H), 3.09 –
2.92 (m, 2H), 2.39 (ddd, J = 7.9, 6.3, 3.6 Hz, 2H). 13C NMR (75 MHz,
CDCl3) δ 198.01, 159.66, 158.41, 141.34, 136.69, 133.59, 130.00,
129.47, 121.81, 120.79, 114.45, 112.15, 109.66, 55.50, 55.16, 54.20,
31.40, 27.84. HRMS (ESI): m/z calcd. for C18H18O3: 282.1256; found:
282.1259.
4.2.1.5. 7-methoxy-2-(3,4,5-trimethoxyphenyl)-3,4-dihydronaphthalen-1
(2H)-one (7). green solid (29.5 mg, 43%). Mp: 176–178 ◦C.1H NMR
(300 MHz, CDCl3) δ 7.58 (d, J = 2.8 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H),
7.14–7.07 (m, 1H), 6.41 (s, 2H), 3.85 (s, 3H), 3.85 (s, 3H), 3.83 (s, 6H),
3.14–2.94 (m, 2H), 2.46–2.34 (m, 2H). 13C NMR (75 MHz, CDCl3) δ
198.09, 158.39, 153.17, 136.77, 136.65, 135.61, 133.48, 130.03,
121.83, 109.67, 105.44, 60.80, 56.01, 55.50, 54.77, 31.72, 28.24.
HRMS (ESI): m/z calcd. for C20H22O5: 342.1467; found: 342.1484.
4.2. Synthesis of α-aryl-α-tetralones
4.2.1. General procedure for the synthesis of α-aryl-α-tetralones
In a microwave tube were added a suspension of Pd(dba)2 (0.0057 g,
0.01 mmol), tBu3PHBF4 (0.0058 g, 0.02 mmol), NaOH (0.016 g, 0.4
mmol), aryl bromide (0.24 mmol) and
α-tetralone (0.2 mmol) in a
mixture of degassed dioxane/water (4:1, v/v, 2 mL) and heated under
argon atmosphere and microwave irradiation (100 W of initial power,
100–130 ◦C, 60 min, infrared probe). Then, the mixture was allowed to
cool to rt, diluted in AcOEt, washed with saturated NH4Cl solution, dried
over anhydrous NaSO4, filtered and concentrated under reduced pres-
sure. The crude material was purified by silica gel column with hexane:
AcOEt (95:5) as solvent.
4.2.2. General procedure for the synthesis of
α-fluoro-α- aryl-α-tetralones
In a microwave tube were added a suspension of Selectfluor (0.5314
g, 1.5 mmol) and α-tetralone (1.0 mmol) in PEG400 (2 mL) and thermal
heated at 120 ◦C. Then, the mixture was allowed to cool to rt, diluted in
AcOEt, washed with brine, dried over anhydrous NaSO4, filtered and
concentrated under reduced pressure. The crude material was purified
by silica gel column with hexane:AcOEt (95:5) as solvent. Compounds
15a [38], 15b [39], 15c [40] are known.
Compounds 3a [18], 3b [17], 3c [18], 5a [37], 6a [37] are known,
new compounds follow:
5

L.G. de Souza et al.
Bioorganic Chemistry 110 (2021) 104790
Before the first step, in a microwave tube were added a suspension Pd
(dba)2 (0.0057 g, 0.01 mmol), tBu3PHBF4 (0.0058 g, 0.02 mmol), NaOH
(0.016 g, 0.4 mmol), aryl bromide (0.24 mmol) and compounds 9a-b
(0.2 mmol) in a mixture of degassed dioxane/water (4:1, v/v, 4 mL) and
heated under argon atmosphere and microwave irradiation (100 W of
initial power, 100–130 ◦C, 60 min, infrared probe). Then, the mixture
was allowed to cool to rt, diluted in AcOEt, washed with saturated
NH4Cl solution, dried over anhydrous NaSO4, filtered and concentrated
under reduced pressure. The crude material was purified by silica gel
column with hexane:AcOEt (95:5) as solvent.
35.67 (d, J = 25.0 Hz), 25.66 (d, J = 9.3 Hz).19F NMR (470 MHz, CDCl3)
δ ꢀ 144.17 (t, J = 10.9 Hz).HRMS (ESI): m/z calcd. for C20H21FO5:
360.1373; found: 360.1387.
4.3. Biological evaluation
4.3.1. Cell lines
Breast cancer cells MCF-7 were cultured in high glucose DMEM
medium (Gibco, Grand Island, NY, USA) 10 g/L supplemented with 25
mM HEPES, 0.584 g/L glutamine, 100 mg/L streptomycin and 60 mg/L
penicillin (all from Sigma-Aldrich). MCF-10A epithelial breast cells were
cultured in complete MEGM Mammary Epithelial Cell Growth Medium
(Lonza, Basel, Switzerland), containing the epidermal growth factor and
insulin needed for stimulating cell proliferation. All media was supple-
Compounds 8a [17], 8b [17], 8c [17], 10 [41], 11 [17] are known,
new compounds follow:
4.2.2.1. 3-(2-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)-4-methox-
ybenzonitrile (9a). light brown solid (28 mg, 47%). Mp: 179–182 ◦C. 1H
NMR (400 MHz, CDCl3) δ 8.07 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 1.7 Hz,
1H), 7.64 (dd, J = 8.6, 1.9 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.37 (t, J =
7.6 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 3.65 (s,
˜
mented with 10% fetal bovine serum (FBS) (Cultilab, Sao Paulo, Brazil)
inactivated at 56 ◦C for 1 h prior to use. The chronic myeloid leukemia
cell lines K562, Lucena-1 and FEPS were cultured in RPMI-1640 medium
(Gibco BRL, Paisley, UK) supplemented with 25 mM HEPES adjusted to
pH 7.4 with NaOH, 60 mg/L penicillin and 100 mg/L streptomycin (all
obtained from Sigma-Aldrich). Briefly, K562 cells were exposed to
increasing concentrations of the chemotherapeutic drugs vincristine
sulfate and daunorubicin hydrochloride (DNR) (both from Sigma-
Aldrich), as described before [20,21]. Lucena-1 (K562/VCR) and FEPS
(K562/DNR) were cultured, respectively, in the presence of either 60 nM
VCR or 500 nM DNR in order to maintain the MDR phenotypes. For
subcultures, cells were harvested every 3 days by incubation with (for
breast cells) or without (for leukemias) 2.5 mg/mL trypsin (Sigma-
Aldrich) at 37 ◦C, followed by washing with cold FBS, and maintained at
37 ◦C in 5% CO2.
3H), 3.40 – 3.30 (m, 1H), 2.95 – 2.76 (m, 2H), 2.44 – 2.34 (m, 1H); 13
C
NMR (101 MHz, CDCl3) δ 189.56 (d, J = 18.7 Hz), 158.05 (d, J = 6.2
Hz), 143.33, 134.23, 134.02, 130.93, 130.49 (d, J = 22.7 Hz), 130.18 (d,
J = 15.8 Hz), 128.65, 128.41, 127.16, 118.96, 111.80 (d, J = 1.8 Hz),
104.63 (d, J = 2.5 Hz), 94.15 (d, J = 179.0 Hz), 55.84, 33.99 (d, J =
23.5 Hz), 24.70 (d, J = 3.8 Hz); 19F NMR (470 MHz, CDCl3)δ ꢀ 165.41
(dd, J = 42.5, 13.4 Hz). .HRMS (ESI): m/z calcd. for C18H14FNO2:
295.1009; found: 295.1003.
4.2.2.2. 3-(2,7-difluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)-4-
methoxybenzonitrile (9b). light yellow solid (21.3 mg, 34%).Mp:
154–158 ◦C. 1H NMR (400 MHz, cdcl3) δ 7.89 (d, J = 1.7 Hz, 1H), 7.75
(dd, J = 8.9, 2.4 Hz, 1H), 7.67 (dd, J = 8.5, 1.9 Hz, 1H), 7.30 (dt, J = 8.1,
5.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 1H), 3.68 (s, 3H), 3.31 (dd, J = 20.1,
4.3.2. Evaluation of mitochondrial reducing activity
Mitochondrial reducing activity was determined by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Sigma-Aldrich)
colorimetric assay. MCF-7 and MCF-10A cells (2 × 104 mLꢀ 1) were
allowed to adhere to 96-well plates for 24 h. Adhered breast cells or
leukemia cells (2 × 104 mLꢀ 1) were incubated for 72 h at 37 ◦C, followed
by treatment with compounds at a range of concentrations. MTT (5 mg/
mL) was added to each well, and plates were kept at 37 ◦C in 5% CO2 for
6.1 Hz, 1H), 2.91 – 2.78 (m, 2H), 2.41 (ddd, J = 8.7, 8.0, 3.0 Hz, 1H).13
C
NMR (101 MHz, cdcl3) δ 188.63 (dd, J = 18.6, 1.7 Hz), 161.75 (d, J =
247.0 Hz), 157.90 (d, J = 6.1 Hz), 139.09 (d, J = 3.1 Hz), 134.33, 132.42
(d, J = 6.5 Hz), 130.56 (d, J = 7.1 Hz), 130.34 – 130.03 (m), 121.45 (d, J
= 22.2 Hz), 118.85, 114.17 (d, J = 22.1 Hz), 111.82 (d, J = 1.7 Hz),
104.80 (d, J = 1.4 Hz), 93.62 (dd, J = 179.2, 0.9 Hz), 55.87, 34.02 (d, J
= 23.6 Hz), 29.68 (s), 24.05 (d, J = 3.9 Hz).19F NMR (470 MHz, CDCl3) δ
ꢀ 113.93 (dd, J = 14.1, 7.8 Hz), ꢀ 165.67 (dd, J = 43.0, 13.4 Hz).HRMS
(ESI): m/z calcd. for C18H13F2NO2: 313.0914; found: 313.0911.
3 h. After centrifugation, 200 μL of DMSO was added to dissolve the
dark-blue formazan crystals formed by MTT reduction. Absorbance was
measured by enzyme-linked immunosorbent assay (ELISA) on a plate
¨
reader (Sunrise; Tecan Group Ltd., Mannedorf, Switzerland) at 570 nm,
with absorbance being directly proportional to formazan content and
indicative of living cells. The half-maximal inhibitory concentrations
(IC50) were calculated by non-linear regression using the GraphPad
Prism version 7.0 program (GraphPad Software, San Diego, CA, USA).
The selectivity index (SI) for breast cancer cells was calculated using the
formula (SI) ¼ (IC50 MCF-10A) ⁄ (IC50 MCF-7). When IC50 exceeded the
maximal tested concentration, it was expressed as being higher than this
concentration (e.g. > 320), and this value was used for calculating the SI
(e.g. SI of 8b towards MCF-7: IC50 MCF-10A / IC50 MCF-7 = 320/84.23
= 3.67). Alternatively, the relative resistance (RR) was calculated using
the formula (RR) ¼ (IC50 MDR cell line, Lucena-1 or FEPS) ⁄ (IC50
parental cell line, K562). Similarly, values expressed as being higher
than a concentration were used for calculating the RR (e.g. RR of VCR
toward FEPS: IC50 FEPS / IC50 K562 = 960/23.72 = 40.47). If RR ≤ 0.5,
the compound exerted collateral sensitivity, and cells were considered
cross-resistant when RR ≥ 2.0 [27].
4.2.2.3. 3-(2-fluoro-7-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-
yl)-4-methoxybenzonitril (9c). light brown solid (19.5 mg, 30%). Mp:
150–155 ◦C.1H NMR (400 MHz, cdcl3) δ 7.89 (d, J = 1.8 Hz, 1H), 7.67
(dd, J = 8.5, 2.0 Hz, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.23 (d, J = 8.5 Hz,
1H), 7.14 (dd, J = 8.4, 2.7 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 3.86 (s, 3H),
3.69 (s, 3H), 3.30 (td, J = 11.0, 4.7 Hz, 1H), 2.90 – 2.75 (m, 2H), 2.43 –
2.35 (m, 1H).13C NMR (101 MHz, cdcl3) δ 189.39 (d, J = 18.6 Hz),
158.67 (s), 158.09 (d, J = 6.1 Hz), 136.01 (s), 134.19 (s), 131.64 (s),
130.55 (d, J = 22.7 Hz), 130.23 (d, J = 15.9 Hz), 129.90 (s), 122.33 (s),
118.92 (s), 111.77 (s), 110.31 (s), 104.69 (s), 94.02 (d, J = 179.1 Hz),
55.86 (s), 55.54 (s), 34.17 (d, J = 23.5 Hz), 29.67 (s), 23.94 (d, J = 3.7
Hz).19F NMR (470 MHz, CDCl3) δ ꢀ 165.38 (dd, J = 42.6, 13.2 Hz).
HRMS (ESI): m/z calcd. for C19H16FNO3: 325.1114; found: 325.1111.
4.2.2.4. 2-fluoro-7-methoxy-2-(3,4,5-trimethoxyphenyl)-3,4-dihy-
dronaphthalen-1(2H)-one (12). green solid (60.3 mg, 84%). Mp:
128–129 ◦C.1H NMR (300 MHz, CDCl3) δ 7.61 (d, J = 2.5 Hz, 1H), 7.17
(d, J = 8.5 Hz, 1H), 7.12 (dd, J = 8.5, 2.6 Hz, 1H), 6.56 (d, J = 0.9 Hz,
2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.78 (s, 6H), 3.14 – 2.99 (m, 1H), 2.92 –
2.78 (m, 1H), 2.75 – 2.58 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 193.52
(d, J = 18.5 Hz), 158.73, 153.22, 138.58 (d, J = 2.5 Hz), 135.74, 132.82,
132.22, 132.07 (d, J = 23.0 Hz), 131.91, 130.13, 122.89, 109.57,
103.46 (d, J = 6.2 Hz), 95.96 (d, J = 184.8 Hz), 60.81, 56.15, 55.61,
4.3.3. ABC-mediated efflux assays
The ABCB1 and ABCC1 transport assays were performed, respec-
tively, with the use of the rhodamine 123 (Rho 123) and 5(6)-carboxy-
fluorescein diacetate (CFDA) dyes (both from Sigma-Aldrich) as
previously described [32]. Rho 123 and CFDA passively distribute into
the cell, and while the first is fluorescent and is actively extruded by
ABCB1, the latter undergoes hydrolysis by nonspecific esterases in the
6

L.G. de Souza et al.
Bioorganic Chemistry 110 (2021) 104790
cytosol, originates the fluorescent substrate carboxyfluorescein (CF) that
only then is transported out by ABCC subfamily members, notably
ABCC1 [42]. Briefly, assays were performed in two 30-minute steps,
sufficient for the accumulation and efflux of dyes. Both steps were car-
ried on at 37 ◦C in 5% CO2 in a light-protected environment. First, 2 ×
105 FEPS cells were incubated in 96-well plates with 250 nM Rho 123 or
500 nM CFDA in presence of 3c or 8a in concentrations equal to or two
times the IC50 toward FEPS, diluted in RPMI medium to allow accu-
mulation of the dyes within cells (accumulation phase). Following, cells
were centrifuged at 200 × g for 7 min and then resuspended in fresh
RPMI medium to allow efflux of the dyes (efflux phase). Inhibition of
ABCB1 efflux was performed in the presence of the calcium channel
blocker verapamil (VP) [43], and the quinoline derivative MK-571 was
employed to inhibit ABCC efflux [44] (inhibited efflux). As negative
control, cells were exposed to medium only (free efflux). Then, cells
were again centrifuged, resuspended in cold PBS and maintained on ice
until acquisition by flow cytometry.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104790.
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