Synthesis and evaluation of the tumor cell growth inhibitory potential of new putative HSP90 inhibitors

Article abstract of DOI:10.3390/molecules23020407

Background: Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones as inhibitors of HSP90. Methods: In the present work, various compounds

Full text of DOI:10.3390/molecules23020407

molecules
Article
Synthesis and Evaluation of the Tumor Cell Growth
Inhibitory Potential of New Putative HSP90 Inhibitors
Ana Bizarro 1,2, Diana Sousa 1,3,4, Raquel T. Lima 3,4,5, Loana Musso 6, Raffaella Cincinelli 6,
Vantina Zuco 7, Michelandrea De Cesare 7, Sabrina Dallavalle 6,* and
M. Helena Vasconcelos 1,3,4,
*
1
Department of Biological Sciences, Faculty of Pharmacy of the University of Porto (FFUP), Rua de Jorge
Viterbo Ferreira 228, 4050-313 Porto, Portugal; sofiabizarro@gmail.com (A.B.); dsousa@ipatimup.pt (D.S.)
Department of Biology, School of Sciences, University of Minho, 4710-057 Braga, Portugal
i3S—Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Rua Alfredo Allen 208,
4200-135 Porto, Portugal; rlima@ipatimup.pt
2
3
4
5
6
Cancer Drug Resistance Group, Institute of Molecular Pathology and Immunology of the University of
Porto (IPATIMUP), 4200-135 Porto, Portugal
Department of Pathology, Faculty of Medicine of the University of Porto (FMUP), Alameda Prof. Hernâni
Monteiro, 4200-319 Porto, Portugal
Department of Food, Environmental and Nutritional Sciences Division of Chemistry and Molecular Biology,
Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy; loana.musso@unimi.it (L.M.);
raffaella.cincinelli@unimi.it (R.C.)
Department of Experimental Oncology and Molecular Medicine, Fondazione, IRCCS—Istituto Nazionale
dei Tumori, Via Amadeo 42, 20133 Milano, Italy; valentina.zuco@istitutotumori.mi.it (V.Z.);
Andrea.DeCesare@istitutotumori.mi.it (M.D.C.)
7
*
Correspondence: sabrina.dallavalle@unimi.it (S.D.); hvasconcelos@ipatimup.pt (M.H.V.);
Tel.: +39-2-50316818 (S.D.); +351 22 5570772 (M.H.V.)
Received: 30 December 2017; Accepted: 11 February 2018; Published: 13 February 2018
Abstract: Background: Heat shock protein 90 (HSP90) is a well-known target for cancer therapy.
In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones
as inhibitors of HSP90. Methods: In the present work, various compounds with new
chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90
inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds (5 and 8) were
further studied in various tumor cell lines regarding their potential to cause cell growth inhibition,
alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client
protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound
studied in A431 squamous cell carcinoma xenografts in nude mice. Results: Our results indicated
that treatment with compounds and decreased the proliferation of tumor cell lines and compound
induced apoptosis. In addition, these two compounds were able to downregulate selected proteins
known as “clients” of HSP90. Finally, treatment of xenografted mice with compound resulted in a
8 was
5
8
8
5
considerable dose-dependent inhibition of tumor growth. Conclusions: Our results show that two
new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising
putative HSP90 inhibitors causing tumor cell growth inhibition.
Keywords: HSP90; naphthoquinones; (thio)chromenopyridinones; antitumor; cell proliferation; cell
cycle; apoptosis; tumor xenographs
1. Introduction
Heat shock proteins (HSPs) are key elements in protein synthesis, acting in protein folding
and in the stabilization of newly synthesized proteins [1]. The expression of HSPs is induced as
Molecules 2018, 23, 407; doi:10.3390/molecules23020407
www.mdpi.com/journal/molecules

Molecules 2018, 23, 407
2 of 15
a response to environmental stress conditions [
2
] and they have been associated with tumor cell
proliferation, differentiation, metastatic spread, invasion, and drug resistance [3,4]. Since tumor cells
must extensively renew their metabolic and signal transduction pathways, they are more dependent
on “stress-related proteins” (such as HSPs) than non-tumor cells [4]. Indeed, overexpression of HSPs
was described in several types of cancer [5].
Amongst the heat-shock protein family, the heat-shock protein 90 (HSP90) is the most widely
studied target for cancer therapy, being important to the maturation and stabilization of different client
oncoproteins such as Akt, Bcr-Abl, HER-2/ErbB2, Raf-1, CDK, survivin, and mutated p53 [
6]. HSP90
overexpression was related to medulloblastoma, breast cancer and colorectal cancer [ ]. Thus, HSP90
3
has attracted a great deal of interest as a potential anticancer target. The HSP90 chaperone cycle is a
dynamic process mediated by interactions with co-chaperones and other binding partners to form a
multi-chaperone complex machinery. HSP90 exists in diverse conformational states and co-chaperones
may function by favoring distinct conformations that help to drive the cycle [7]. The dynamic nature
of the cycle allows modulation by small molecule ligands through a number of distinct mechanisms
that include targeting the N-terminal domain, the C-terminal domain, co-chaperone binding and client
protein binding. HSP90 inhibitors based on the benzoquinone ansamycin scaffold of geldanamycin, the
resorcinol scaffold of radicicol, the purine scaffold of ATP, and other unique chemical scaffolds which
were discovered to inhibit HSP90, are currently being evaluated in numerous ongoing or completed
clinical trials [8,9]. While some of the results from such trials have been encouraging, reports of hepato-,
cardio-, ocular-toxicity, peripheral neuropathy and in some cases poor solubility, limited bioavailability
and difficult dosing schedules have discontinued clinical development, and to date, no HSP90 inhibitor
has obtained regulatory approval [7].
Therefore, due to HSP90’s role in maintaining the viability of cancer cells, the search for novel and
more efficient HSP90 inhibitors represents an interesting challenge from a drug discovery perspective.
Quinones are a class of organic compounds endowed with a variety of biological activities, mostly
connected with their redox properties. A number of natural and synthetic quinones showed remarkable
anticancer activity, and 1,4-naphthoquinones have been identified as HSP90 inhibitors [10–12]. In a
previous work, some of us have reported a series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones as
inhibitors of HSP90 [13]. As a continuation of our previous investigation, in the present work a series
of new pyridine-fused naphthoquinones, chromenopyridinones and thiochromenopyridinones, were
synthesized aiming to enhance their ability to inhibit HSP90 and to reduce tumor cell growth both
in vitro and in vivo.
2. Results and Discussion
2.1. Chemistry
Initially, we had planned to replace the isoxazole group fused to the 1,4-naphthoquinone ring [
3]
in previously reported isoxazolonaphtoquinones with a pyridine moiety ( )). The advantage of the
1
replacement could be the possibility of N-alkylation to give the corresponding ammonium salts,
possibly more water soluble.
Thus, we prepared compounds 2a–c by treatment of commercially available benzo[g]isoquinoline
-5,10-dione (1) with benzyl bromides in DMSO at room temperature (Scheme 1).
Successively, we planned to investigate the role of the quinone nucleus by replacing it with
a heterocyclic ring. Changing the quinone structure into a xanthone or thioxanthone could
reduce the risk of idiosyncratic drug reactions [14]. Compound
N,N-diethyl-2-hydroxybenzamide, which was converted into compound amide
3-bromopyridine in the presence of KOtBu at 210 ◦C. An LDA-induced regiospecific acylation of
gave the corresponding 5H-chromeno[2,3-c]pyridin-5-one . Alkylation with benzyl bromide afforded
chromenopyridinone . To prepare thiochromenopyridinone diazotised 3-aminoisonicotinic acid
was condensed with thiophenol in alkaline solution to form 3-(phenylthio)isonicotinic acid . This was
4
was obtained starting from
3
by reaction with
3
4
5
7
6

Molecules 2018, 23, 407
3 of 15
converted into the corresponding acyl chloride by treatment with thionyl chloride. Heating in
nitrobenzene with AlCl3 afforded compound
7
, which was then alkylated with benzyk bromide
to obtain 8 in 55% yield (Scheme 2).
O
O
2a
R =
2b R =
2c
a
F
F
N
N
R
-
O
R =
Br
O
1
Scheme 1. Synthesis of N-substituted benzo[g]isoquinoline-5,10-diones. Reagents and conditions: (a) for
2a: benzyl bromide, DMSO, r.t., 48 h, 86%; for 2b: 2-bromomethyl-1,4-difluorobenzene, DMSO, r.t., 24
h, 60%; for 2c: 1-bromomethyl-4-tert-butylbenzene, DMSO, 24 h, r.t., 65%.
Scheme 2. Synthesis of (thio)chromenopyridinones. Reagents and conditions: (a) 3-bromopyridine,
◦
◦
KOtBu, 210 C, 3 h, 74%; (b) LDA, THF, 0 C, then 1 h at r.t., 90%; (c) benzyl bromide, DMSO, r.t., 48 h,
86%; (d) i: NaNO2, H2O, HCl conc., 5 ◦C; ii: thiophenol, NaOH, H2O, reflux, 3 h; (e) i: SOCl2, 1 h,
reflux; ii: AlCl3, PhNO2, 100 C, 4 h, 72%; (f) benzyl bromide, DMSO, overnight at r.t., 55%; (g) benzyl
◦
bromide, DMSO, overnight at r.t., 34%.
2.2. Binding Affinity to HSP90, Cell Growth Inhibitory Activity and Structure–Activity Relationship Studies
of the Newly Synthesized Compounds
The prepared compounds were tested for their affinity to HSP90 (with a binding assay) and for
their cell growth inhibitory activity (with the cell counting assay) on a number of human tumor cell lines
(non-small cell lung cancer NCI-H460, squamous-cell carcinoma A431 and peritoneal mesothelioma
STO). Results are presented in Table 1.
Unfortunately, for some of the compounds (e.g., compounds 2a
not give valuable results since the compounds themselves were autofluorescent. However, compounds
2a showed significant cell growth inhibitory activity. As these results suggested that other off-target
effects are likely involved in the mechanism of action of the tested compounds, we planned to
investigate the role of the quinone nucleus by replacing it with a xanthone moiety (compound ).
This replacement led to a complete loss of cell growth inhibitory activity and binding affinity to HSP90.
However, the introduction of the benzyl group (compound ) restored the cell growth inhibitory
activity, in spite of lack of evidence of HSP90 binding. The position of the nitrogen was crucial; in fact,
–c) the binding affinity assay did
–
c
4
5

Molecules 2018, 23, 407
4 of 15
shifting the nitrogen atom from position 2 (compound
the activity.
5) to 1 (compound 10) completely abolished
Table 1. Binding affinity (µM) to heat shock protein 90 (HSP90) protein and cell growth inhibitory
activity (µM) of the compounds.
Binding Affinity (µM)
Cell Growth Inhibitory Activity (µM)
Compound
Binding HSP90 (FP) IC50
NCI-H460 IC50 72 h
STO IC50 72 h
A431 IC50 72 h
2a
2b
2c
4
n.a.
n.a.
n.a.
0.7 ± 0.01
3.2 ± 0.5
n.d.
n.d.
n.d.
n.d.
0.2 ± 0.01
3.1 ± 0.5
0.46 ± 0.8
>50
>100
>50
>50
5
7
8
10
n.a.
4.8 ± 0.1
2.7 ± 0.8
1.36 ± 0.05
2.47 ± 0.09
3.7 ± 0.03
>100
>50
46.9
46.9
n.d.
>50
0.26 ± 0.08
>50
0.26 ± 0.40
>50
17-AAG
1.095 ±0.05
0.010 ± 0.002
-
0.069 ± 0.005
n.a. = non-available results since the compounds were autofluorescent; n.d. = not determined; 17-AAG =
tanespimycin, 17-N-allylamino-17-demethoxygeldanamycin.
Interestingly, compound 7, with a sulphur atom in place of the oxygen in the xanthone ring
showed a high affinity to HSP90, in spite of the low cell growth inhibitory activity. In this case, the
chemico-physical features of the compound may influence the cellular pharmacokinetic behavior and
therefore cellular response. Therefore, the corresponding alkylated compound
This compound showed appreciable cell growth inhibition associated with a strong HSP90 interaction.
Compounds and appeared to be some of the most promising ones regarding tumor cell growth
8 was synthesized.
5
8
inhibition, thus we further investigated their cellular/biochemical profile of activity.
2.3. Compounds 5 and 8 Induced Cytotoxicity in Different Tumor Cell Lines
Cytotoxic effects of the compounds against MCF7 (breast adenocarcinoma), AGS (gastric
adenocarcinoma) and NCI-H460 (non-small cell lung cancer) cells were evaluated after 48 h of
treatment, using the SRB assay. Cytotoxicity of each compound was evaluated by its GI50 (concentration
which inhibits cell growth by 50%). The compounds presented GI50 bellow 7
tested (Table 2).
µM in all the cell lines
Moreover, in order to verify the possible toxicity of the compounds for normal cells, their growth
inhibitory effect was also analyzed in the non-tumor human fibroblast cell line HFF-1. Both compounds
presented no cytotoxicity towards this cell line after seven days of treatment (data not shown).
Table 2. GI50 values of compounds 5 and 8 following 48 h treatment of three human tumor cell lines.
GI50 (µM)
Compound
MCF7
NCI-H460
AGS
5
8
5.4 ± 0.6
5.2 ± 1.2
6.9 ± 1.2
3.2 ± 0.1
3.2 ± 0.1
3.1 ± 0.3
Data refers to mean
±
SEM of at least three independent experiments. The maximum vehicle concentration used
(H2O or DMSO; 0.25%) was also analyzed as control treatments and presented no effect on cell growth (data
not shown).
The cell growth inhibitory effect of compounds
5 and 8 was further investigated in the NCI-H460
cell line. This study confirmed that both these compounds decreased NCI-H460 viable cell number,
as previously indirectly indicated by the SRB assay. By counting the number of viable cells with
trypan blue 48 h following treatment, it was possible to observe that compound
7.8 M (corresponding to the previously determined GI50 and to 1.5 GI50 concentration, respectively)
significantly reduced the number of viable cells to 71% and 49% (in relation to Blank cells), respectively
5 at 5.2 µM and
µ
×

Molecules 2018, 23, 407
5 of 15
(Figure 1A). Likewise, this decrease in the number of viable cells was also observed following treatment
with compound at 3.2 M and 4.8 M (corresponding to the previously determined GI50 and to 1.5
GI50 concentration, respectively) to 57% and 29% (in relation to Blank cells), respectively (Figure 1B).
8
µ
µ
×
Figure 1. NCI-H460 viable cell number 48 h following treatment with compounds
analyzed with trypan blue exclusion assay. Cells were treated with the GI50 (5.2 M) and 1.5
(7.8 M) of compound and with the GI50 (3.2 M) and 1.5 GI50 (4.8 M) of compound . Cells were
5 (A) and 8 (B)
µ
×
GI50
µ
5
µ
×
µ
8
also treated with the corresponding highest concentration of vehicle (solvent) of the compounds (H2O).
Results are presented as % of viable cells in relation to blank cells (treated with medium) and are the
mean ± SEM of six independent experiments. * p ≤ 0.001 Blank vs. treatment.
2.4. Apoptotic Induction in NCI-H460 Cells after Treatment with Compound 8
Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. To assess if
the growth inhibition observed after compounds treatment was due to apoptosis, cells were treated
with two concentrations of compounds
5
and
8
(corresponding to GI50 and 1.5
×
GI50) for 48 h and
apoptosis was detected with Annexin V and propidium iodide double staining by flow cytometry.
Annexin (+) and PI (+) cells were not found in compound 5 treated cells (Table 3(A)), suggesting that
this compound did not induce apoptosis at cytotoxic concentrations. However, Annexin (+) and PI (+)
cells were detected in compound
8 treated cells, indicating that this compound induced apoptosis at
cytotoxic concentrations (Table 3(B)).
Table 3. Levels of apoptosis in non-small cell lung cancer (NCI-H460) cells following 48 h treatment
with compounds 5 (A) and 8 (B).
(A)
Treatments
% Apoptosis
(B)
Treatments
% Apoptosis
Blank
H2O
7.0 ± 0.6
Blank
H2O
5.2 ± 0.9
7.6 ± 1.2
6.4 ± 1.8
5.2 µM
7.8 µM
7.2 ± 1.4
7.3 ± 0.9
3.2 µM
4.8 µM
6.7 ± 1.4
Compound 5
Compound 8
12.5 ± 1.7 **
Results are the mean
±
SEM of at least 3 independent experiments; ** p
≤
0.05 between Blank vs. treatment;
% Apoptosis refers to the sum of early and late apoptosis.

Molecules 2018, 23, 407
6 of 15
The reason why compound
8
is a stronger inducer of apoptosis than compound
5
might be related
to differences in the affinity for HSP90. This cannot be confirmed since it was not possible to determine
the capacity of compound 5 to bind HSP90 due to its autofluorescence (Table 1).
2.5. Effect of the Compounds 5 and 8 on NCI-H460 Cell Cycle Profile and Cellular Proliferation
To determine whether the effect of compounds on cell proliferation was related to cell cycle
control, we analyzed the effects on cell cycle in NCI-H460 cells at 48 h after drug treatment by flow
cytometry. As shown in Figure 2, the percentages of cells in each cell cycle phase were similar to
untreated cells, indicating that the compounds did not affect cell cycle profile.
Figure 2. NCI-H460 cell cycle profile 48 h following treatment with compounds
analyzed by flow cytometry. Cells were treated with the GI50 (5.2 M) and 1.5
compound and with the GI50 (3.2 M) and 1.5 GI50 (4.8 M) of compound . Cells were also treated
5
(
A
) and
8
(
B),
µ
×
GI50 (7.8
µM) of
5
µ
×
µ
8
with the corresponding highest concentration of the vehicle (solvent) of the compounds (H2O). Results
represent the mean ± SEM of at least three independent experiments.
Detection and quantification of cells actively synthesizing DNA in the S-phase of cell cycle
progression is important in defining the cellular responses to drug treatments, assessing cell health,
and determining genotoxicity. Thus, we have performed the BrdU incorporation assay [15
NCI-H460 treated cells. A statistically significant decrease in cellular proliferation was observed
after treatment with both compounds (Figure 3). Particularly, for compound the percentage of
BrdU-incorporating cells decreased from 32% (in untreated cells) to 25% and 22% (with the GI50 and
1.5 GI50 treatments, respectively), and for compound the percentage of BrdU-incorporating cells
decreased from 31% (in untreated cells) to 26% and 21% (with the GI50 and 1.5 GI50 treatments,
,16] in
5
×
8
×
respectively), indicating a dose-dependent decrease of cell proliferation after compounds exposure.
2.6. Effect of Compounds 5 and 8 on HSP90 Client Proteins
The effect of compounds in cellular apoptosis/proliferation led us to the analysis of HSP90 client
proteins involved in those mechanisms.

Molecules 2018, 23, 407
7 of 15
The most effective anti-proliferative agents, i.e., compounds
5
and
8
, were investigated for their
ability to downregulate selected proteins known as “clients” of HSP90. As expected on the basis of the
putative mechanism of action, the tested compounds induced a partial downregulation with a different
pattern of inhibition. Specifically, compound
and a partial downregulation of survivin in STO and A431 cells (Figure 4). Compound
5
induced an almost complete downregulation of CDK4
still caused
8
degradation of survivin in STO cells, but the effect was less marked in A431 cells. In the latter cell line,
the most evident effects were a partial downregulation of Akt and EGFR and a strong downregulation
of RAF (Figure 5). The different pattern of HSP90 client protein downregulation after treatment with
compounds
5 or 8 (in both cell lines) is most likely due to their different physico-chemical features,
which may most likely influence the interactions at a cellular level and, as a consequence, the activity
of the compounds and the effect on client protein levels. In addition, the differences observed in the
effect of the compounds between the two cell lines may be due to different basal levels of expression of
these proteins between the two cell lines. Nevertheless, the observed modulations are consistent with
an effect mediated by interaction of the selected compounds with HSP90.
Figure 3. NCI-H460 cellular proliferation following 48 h treatment with compounds
analyzed with the BrdU incorporation assay. Cells were treated with the GI50 (5.2 M) and 1.5
(7.8 M) of compound and with the GI50 (3.2 M) and 1.5 GI50 (4.8 M) of compound . Cells were
also treated with the corresponding highest concentration of vehicle (solvent) of the compounds (H2O).
5
(
A) and
8
(
B),
µ
×
GI50
µ
5
µ
×
µ
8
Results represent the mean
Blank vs. treatment.
±
SEM of three independent experiments. * p
≤
0.001, ** p
≤
0.05 between
Figure 4. Analysis of HSP90 client protein levels in squamous-cell carcinoma (A431) and peritoneal
mesothelioma (STO) cells after 24 h of treatment with compound (5.4 M in A431 cells; 2.7 M in
5
µ
µ
STO cells). Total cellular extracts were obtained 24 h after cell treatment. Actin is shown as a control for
protein loading.

Molecules 2018, 23, 407
8 of 15
Figure 5. Analysis of HSP90 client protein levels in A431 and STO cells after 24 h of treatment with
compound (2.6 M in A431 cells; 2.6 M in STO cells). Total cellular extracts were obtained 24 h after
8
µ
µ
cell treatment Actin is shown as a control for protein loading.
2.7. Effect of Compound 5 in the Growth of a Tumor Xenograft Mouse Model
On the basis of the cellular/biochemical profile described above (Table 1), the therapeutic effect
of compound was determined in a human epidermoid carcinoma xenografted mouse model. Two
5
experimental groups were used: (i) animals administrated with saline solution and (ii) animals
intraperitoneally administrated with newly synthesized compound. The daily administration of
compound
compared with the control group (Figure 6). The antitumor effect was dose-dependent, as the increase
of the dose of compound (to 10 mg/kg), accentuated this difference to statistically significant values.
5 (5 mg/kg) for three weeks resulted in a considerable inhibition of tumor growth when
5
Interestingly, at both doses used in this study no evident manifestations of toxicity were detected
(specifically, death and body weight loss).
Figure 6. Antitumor activity of compound
5 in A431 squamous cell carcinoma xenografts in nude mice.
Compound dissolved in physiologic saline was administered i.p. (5 mg/kg and 10 mg/kg) for five
5
consecutive days/week. The treatment started when the tumors were just palpable and was repeated
for three weeks.

Molecules 2018, 23, 407
9 of 15
3. Material and Methods
3.1. Chemistry
All reagents and solvents were reagent grade or were purified by standard methods before use.
Melting points were determined in open capillaries. NMR spectra were recorded at 300 MHz. NMR
spectra were recorded on Varian Mercury 300 MHz and Bruker AV600 spectrometers The accurate
mass spectra were recorded using a Bruker Daltonic model ICR-FTMS APEX II. Solvents were routinely
distilled prior to use; dry methylene chloride was obtained by distillation from phosphorus pentoxide
and toluene from CaCl2. All reactions requiring anhydrous conditions were performed under a
positive nitrogen flow, and all glassware was oven dried. Isolation and purification of the compounds
were performed by flash column chromatography on silica gel 60 (230–400 mesh). Analytical thin-layer
chromatography (TLC) was conducted on TLC plates (silica gel 60 F254, aluminium foil) and spots
were visualized by UV light and
to literature procedure [17].
⁄or by means of dyeing reagents. Compound 4 was prepared according
Compound 7 was prepared according to literature procedure [18].
Compound 9 (1-azaxanthone) was purchased from Alfa Aesar (76057, Karlsruhe, Germany).
2-Benzyl-5,10-dioxo-5,10-dihydro-benzo[g]isoquinolinium bromide (2a): To a solution of benzo[g]
isoquinoline-5,10-dione
1 (70 mg, 0.33 mmol) in anhydrous DMSO (4 mL), benzyl bromide (56 µL,
0.47 mmol) was added under nitrogen. The solution was stirred 48 h at room temperature. The mixture
was cooled at 0 ◦C and washed with ethyl ether to remove benzyl bromide and DMSO. Compound
◦
2a (110 mg, 86% yield) was obtained after crystallization in ethyl ether; m.p. 227 C, Rf = 0.35
(CH2Cl2:CH3OH 19.5:5), 1H-NMR (DMSO-d6, 300 MHz):
δ
: 10.01 (1H, s); 9.57 (1H, d, J = 6 Hz); 8.72
(1H, d, J = 6 Hz); 8.41–8.22 (2H, m); 8.12–7.98 (2H, m); 7.69–7.39 (5H, m); 6.11 (2H, s). 13C-NMR
(DMSO-d6, 150 MHz): : 179.3, 179.2, 149.0, 145.0, 143.7, 135.6 ( 2C), 134.0, 133.0, 132.7, 131.3, 129.6,
129.3 ( 2C), 129.0 (
δ
×
×
×
2C), 127.3, 127.1, 124.5, 64.0. Anal. Calcd. for: C20H14BrNO2: C, 63.18; H, 3.71; N,
3.68. Found: C, 63.25; H, 3.70; N, 3.67. HRMS (ES+) m/z: Calcd. for C20H14NO2 [M]+ 300.1025. Found:
300.1024.
2-(2,4-Difluoro-benzyl)-5,10-dioxo-5,10-dihydro-benzo[g]isoquinolinium bromide (2b): To a solution
of benzo[g]isoquinoline-5,10-dione
1 (15 mg, 0.072 mmol) in anhydrous DMSO (1 mL),
2-bromomethyl-1,4-difluorobenzene (13
was stirred 24 h at room temperature. The mixture was cooled at 0 C and washed with ethyl
µ
L, 0.107 mmol) was added under nitrogen. The solution
◦
ether to remove benzyl bromide and DMSO. Compound 2b (60%) was obtained after cristallization in
◦
CH3OH/ethyl ether m.p. 210 C dec, Rf = 0.32 (CH2Cl2:CH3OH 19.5:5) 1H-NMR (DMSO-d6, 300 MHz):
δ
: 9.99 (1H, s); 9.49 (1H, d, J = 6.0 Hz); 8.70 (1H, d, J = 6.0 Hz); 8.38–8.221 (2H, m); 8.12–8.00 (2H, m);
7.89–7.25 (1H, m); 7.51–7.38 (1H, m); 7.33–7.19 (1H, m); 6.20 (2H, s). Anal. Calcd. for: C20H12BrF2NO2:
C, 57.71; H, 2.91; N, 3.37. Found: C, 57.65; H, 2.92; N, 3.37. HRMS (ES+) m/z: Calcd. for C20H12F2NO2
[M]+ 336.0836. Found: 336.0834.
2-(4-tert-Butyl-benzyl)-5,10-dioxo-5,10-dihydro-benzo[g]isoquinolinium bromide (2c): To a solution
of benzo[g]isoquinoline-5,10-dione
1 (22 mg, 0.105 mmol) in anhydrous DMSO (1.5 mL),
1-bromomethyl-4-tert-butyl-benzene (23
µ
L, 0.125 mmol) was added under nitrogen. The solution
◦
was stirred 24 h at room temperature. The mixture was cooled at 0 C and washed with ethyl
ether to remove benzyl bromide and DMSO. Compound 2c (30 mg, 65% yield) was obtained after
crystallization in CH3OH/ethyl ether. m.p. 238 ◦C dec, Rf = 0.29 (CH2Cl2:CH3OH 19.5:5) 1H-NMR
(DMSO-d6, 300 MHz):
8.12–7.98 (2H, m); 7.69–7.39 (4H, m); 6.08 (2H, s); 1.22 (9H, s). 13C-NMR (DMSO-d6, 75 MHz):
179.2, 149.3, 149.1, 145.2, 143.5, 135.6 ( 2C), 134.1, 132.9, 132.5, 129.6, 129.2, 129.0 ( 2C), 128.1 (
127.3, 127.0, 63.8, 34.3, 31.6 (
3C). HRMS (ES+) m/z: Calcd. for C24H22NO2 [M]+ 356.1651. Found:
356.1645. Anal. Calcd. for: C24H22BrNO2: C, 66.06; H, 5.08; N, 3.21. Found: C, 66.16; H, 5.09; N, 3.20.
δ
: 10.11 (1H, s); 9.60 (1H, d, J = 6.1 Hz); 8.70 (1H, d, J = 6.1 Hz); 8.41–8.22 (2H, m);
: 179.4,
2C),
δ
×
×
×
×

Molecules 2018, 23, 407
10 of 15
2-Benzyl-10-oxo-10H-9-oxa-2-azonia-anthracene bromide (
5
): To a solution of compound
4
(100 mg,
0.51 mmol) in anhydrous DMSO (2 mL), benzylbromide (90
µL, 0.75 mmol) was added under nitrogen.
The solution was stirred overnight at room temperature. The mixture was cooled at 0 ◦C and washed
with hexane and ethyl ether to remove benzyl bromide and DMSO. Compound
5 (161 mg, 86% yield)
was obtained after crystallization in CH3OH/ethyl ether; m.p. = 231 ◦C, Rf = 0.43 (CH2Cl2:CH3OH
1
9:1). H-NMR (DMSO-d6, 300 MHz):
δ
: 10.22 (1H, s); 9.13 (1H, d, J = 5.5 Hz); 8.70 (1H, d, J = 5.5 Hz);
8.25 (1H, dd, J = 8.0 Hz); 8.05 (1H, dd, J = 8, 8.3 Hz); 7.85 (1H, d, J = 8.3 Hz); 7.71–7.58 (3H, m); 7.51–7.41
(3H, m); 6.06 (2H, s). 13C-NMR (DMSO-d6, 150 MHz):
: 174.2, 156.1, 153.2, 140.2, 139.0, 138.0, 134.3,
131.6, 130.0, 129.6 ( 2C), 129.4 ( 2C), 126.7 (
2C), 124.7, 122.3, 119.1, 64.5. HRMS (ES+) m/z: Calcd.
δ
×
×
×
for C19H14NO2 [M]+ 288.1025. Found: 288.1024. Anal. Calcd. for: C19H14BrNO2: C, 61.97; H, 3.83; N,
3.80. Found: C, 62.03; H, 3.83; N, 3.79.
2-Benzyl-5-oxo-5H-thiochromeno[2,3-c]pyridin-2-ium bromide (
0.23 mmol) in anhydrous DMSO (1.5 mL), benzylbromide (40
nitrogen. The solution was stirred overnight at room temperature. The mixture was cooled at 0 ◦C
8): To a solution of compound
7 (50 mg,
µL, 0.34 mmol) was added under
and washed with hexane and ethyl ether to remove benzyl bromide and DMSO. Compound
55% yield) was obtained after crystallization in ethyl ether; m.p. 202 C, Rf = 0.43(CH2Cl2:CH3OH 9:1).
8 (50 mg,
◦
1H-NMR (DMSO-d6, 300 MHz):
(1H, dd, J = 8.0 Hz); 8.05 (1H, dd, J = 8, 8.3 Hz); 7.85 (1H, d, J = 8.3 Hz); 7.71–7.58 (3H, m); 7.51–7.41
(3H, m); 6.06 (2H, s).13C-NMR (DMSO-d6, 150 MHz):
: 176.4, 146.1, 139.9, 137.9, 137.0, 135.6, 134.5,
133.7, 129.6, 129.3 ( 2C), 129.2 (
2C), 129.1, 128.6, 128.3, 127.5, 126.4, 63.9. HRMS (ES+) m/z: Calcd.
δ: 10.21 (1H, s); 9.10 (1H, d, J = 5.3 Hz); 8.81 (1H, d, J = 5.5 Hz); 8.25
δ
×
×
for C19H14NOS [M]+ 304.0786. Found: 304.0799. Anal. Calcd. for: C19H14BrNOS: C, 59.38; H, 3.67; N,
3.64. Found: C, 59.31; H, 3.66; N, 3.65.
1-Benzyl-10-oxo-10H-9-oxa-1-azonia-anthracene bromide (10): To a solution of compound
9 (40 mg,
0.20 mmol) in anhydrous DMSO (1.5 mL), benzylbromide (30
nitrogen. The solution was stirred overnight at room temperature. The mixture was cooled at 0 C and
µ
L, 0.25. mmol) was added◦under
washed with hexane and ethyl ether to remove benzyl bromide and DMSO. Compound 10 (25 mg,
1
34% yield) was obtained after crystallization in ethyl ether. H-NMR (DMSO-d6, 300 MHz):
δ: 9.53
(1H, d, J = 4.5 Hz); 9.26 (1H, d, J = 6.5 Hz); 8.2◦7 (1H, d, J = 7.1 Hz); 8.21–7.99 (3H, m); 7.82–7.60 (3H,
m); 7.11–7.38 (3H, m); 6.12 (2H, s). m.p. 240 C, Rf = 0.47 (CH2Cl2:CH3OH 9:1). Anal. Calcd. for:
C19H14BrNO2: C, 61.97; H, 3.83; N, 3.80. Found: C, 61.92; H, 3.82; N, 3.81. HRMS (ES+) m/z: Calcd. for
C19H14NO2 [M]+ 288.1025. Found: 288.1018.
3.2. Cell Lines and Culture Conditions
For the cytotoxic activity studies and determination of GI50 concentrations in three different
tumor cell lines, the cell culture conditions are described in Section 3.5. For the studies on the effect
on % viable cells, cell cycle, cellular proliferation, and apoptosis, culture conditions are described in
Section 3.6.1. For the remaining studies, the epithelial carcinoma cell line A431 and the non-small cell
lung carcinoma cell line NCI-H460 were routinely grown in RPMI 1640 (Lonza, Vierviers, Belgium),
supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, GIBCO, Invitrogen, Paisley,
UK). The human peritoneal mesothelioma cell line STO was kindly provided by Dr. N. Zaffaroni
(IRCCS Istituto Nazionale Tumori, Milan, Italy) and grown in 50/50 DMEM-Ham’s F12 medium
(Lonza, Vierviers, Belgium), supplemented with 10% (v/v) heat-inactivated foetal bovine serum and
2 mM L-glutamine (L-Gln). All cell lines were maintained at 37 ◦C in a 5%/95% CO2/air atmosphere.
All the compounds tested were freshly prepared by dissolving powder in dimethylsulfoxide (DMSO)
(BDH Prolabo, Milan, Italy) and following dilution in culture medium. Cells were incubated with
drugs for 48 h or 72 h (unless otherwise stated) at 37 ◦C in culture medium supplemented with 10%
(v/v) heat-inactivated FBS; drug concentrations used for each assay and duration of treatments are
reported in Legends to Figures.

Molecules 2018, 23, 407
11 of 15
3.3. Cell-Counting Assay and Determination of IC50 Concentrations
Cellular sensitivity to drugs was evaluated by a cell-counting assay after 72-h drug exposure.
Cells in the logarithmic phase of growth were seeded in duplicate into 6-well plates. Twenty-four
hours after seeding, the drug was added to the medium. Cells were harvested 72 h after drug exposure
and counted with a cell counter. IC50 is defined as the drug concentration causing a 50% reduction of
cell number compared with that of untreated control.
3.4. HSP90 Binding Activity
GM-FITC Geldanamycin-fluorescein-5-isothiocyanate (working concentration: 1–400 nM),
supplied by Invivogen (06C23-MT, San Diego, CA, USA), was previously dissolved in DMSO to
obtain 10 mM stock solutions and kept at
No. SPP-776, Victoria, BC, Canada), was previously dissolved in assay buffer (HFB) to form 2.2 mM
stock solutions and kept at
80 ◦C until use. The compounds were previously dissolved in DMSO
to obtain stock solutions and kept at
20 ◦C. The day of experiment, the compounds were prepared
−
20 ◦C until use. HSP90, purchased from (Stressgen cat.
−
−
by serial dilutions in assay buffer (HFB) containing 20 mM HEPES (K) pH 7.3, 50 mM KCl, 5 mM
MgCl2, 20 mM Na2MoO4 and 0.01% NP40. Before each use, 0.1 mg/mL Bovine Gamma globulin and
2 mM DTT were freshly added. Fluorescence Polarization (FP) was performed in Opti-Plate™-96F well
plates (Perkin Elmer, Zaventem, Belgium) using a plate reader (Wallac Envision 2101 multilabel reader,
Perkin Elmer, Zaventem, Belgium). To evaluate the binding affinity of the molecules, 50 mL of the
GM-FITC solution (5 nM) were added to 30 nM of HSP90 in the presence of 5
µ
L of the test compounds
◦
at increasing concentrations. The plate was mixed on a shaker at 4 C for 4 h, and the FP values in mP
(millipolarization units) were recorded. The IC50 values were calculated as the inhibitor concentration
where 50% of the tracer is displaced; each data point is the result of the average of triplicate wells, and
was determined from a plot using nonlinear least-squares analysis. Curve fitting was performed using
Prism GraphPad software program (GraphPad software, Inc., San Diego, CA, USA).
3.5. Analysis of Cell Growth Inhibition with the Sulforhodamine B Assay and Determination of
GI50 Concentrations
The following human tumor cell lines were used and maintained in RPMI-1640 medium with
UltraGlutamine I and 25 mM Hepes (Lonza, Basel, Switzerland), supplemented with 5% FBS (Biowest):
NCI-H460 (non-small cell lung adenocarcinoma), AGS (gastric adenocarcinoma) and MCF-7 (breast
adenocarcinoma). The HFF-1 cell line (human foreskin fibroblasts) was cultured in DMEM High
Glucose with L-Glutamine supplemented with 15% FBS. All cell lines were routinely maintained at
37 ◦C in a humidified incubator with 5% CO2. Cell number and viability were determined with the
trypan blue exclusion assay. All the experiments were performed with exponentially growing cells
presenting more than 90% cell viability.
The growth inhibitory effect of the compounds was assessed using the Sulforhodamine B (SRB)
assay as previously described [19]. Briefly, cells were plated into 96-well plates (5000 cells/well for
MCF-7 and NCI-H460 cells and 7500 cells/well for AGS cells) and incubated for 24 h to adhere. Serial
dilutions of the different compounds (ranging from 0 to 150 µM or from 0 to 50 µM) were then added
to the cells. Cells were further incubated for 48 h. Following this period (or at the time the compound
is added, T0), plates were fixed with 10% ice-cold trichloroacetic acid. Following staining with 0.4%
SRB, plates were washed with 1% acetic acid and the bound dye solubilized with 10 mM Tris-Base.
The corresponding absorbance was measured at 510 nm in a microplate reader (Biotek Instruments Inc.,
Synergy XS, Winooski, VT, USA), allowing to determine the GI50 concentrations (corresponding to the
concentration which results in 50% cell growth inhibition) for each of the studied compounds. For the
HFF-1 cell line, cells (15,000 cells/well) were plated into 96-well plates, allowed to adhere for 24 h
and treated with the GI50 concentration of each compound for seven days. After this period, the SRB
assay was carried out as described above. The effect of the maximum concentration of vehicle (solvent)

Molecules 2018, 23, 407
12 of 15
of the compounds (H2O: 0.31% for compound
each assay.
8
and 0.19% for compound 13) was also analyzed in
3.6. Effect of Compounds
5 and 8 on Cell Cycle Profile, Cellular Proliferation and Apoptosis in NCI-H460 Cells
3.6.1. Drug Treatments
NCI-H460 cells were plated in 6-well plates (1
UltraGlutamine I and 25 mM Hepes (Lonza) supplemented with 10% FBS (PAA) at 37 C in a
humidified incubator with 5% CO2 for 24 h. Cells were then treated for 48 h with the GI50 and
×
105 cells/well) in RPMI-1640 mediu◦m with
1.5
to 5.2
×
GI50 concentrations of compounds
5
and
8
. These concentrations corresponded respectively
M and 4.8 M for compound . Cells were also
µ
M and 7.8 M for compound and to 3.2
µ
5
µ
µ
8
incubated with medium only (Blank) or with the vehicle (solvent, H2O), corresponding to the highest
concentration used when testing the compounds. Following treatment with the compounds, cells were
further processed as follows.
3.6.2. Determination of % Viable Cells
Cell number and % viable cells was determined with the Trypan blue exclusion assay.
3.6.3. Analysis of Cell Cycle Profile
◦
Following 48 h treatment, cells were fixed in ice-cold 70% ethanol and stored at 4 C at least
overnight. Cell pellets were incubated in 5 µg/mL propidium iodide and 100 µg/mL RNase A (in
PBS) for 30 min on ice. Analysis of cellular DNA content was carried out using a FACSCalibur
flow cytometer (BD Biosciences, Erembodegem, Belgium). The FlowJo 7.6.5 software (Tree Star, Inc.,
Ashland, OR, USA) was used to determine the % of cells in the different phases of the cell cycle (G1, S
and G2/M phases of the cell cycle) after exclusion of cell debris and aggregates and analyzing at least
15,000 events [20].
3.6.4. Analysis of Cellular Proliferation
Following 47 h treatment, cells were further incubated with 10 µM bromodeoxyuridine (BrdU;
Sigma) for 1 h. Cells were then trypsinized and fixed in 4% paraformaldehyde (in PBS) for 30 min.
Cytopsins were prepared and a 20 min denaturation step was carried out by incubating slides in
2 M HCl. Following incubation with mouse anti-BrdU primary antibody (1/10, Dako) for 1 h at
room temperature (RT), cells were further incubated with anti-mouse-Ig-FITC (1/100, Dako) for
30 min, at RT [21]. Slides were then mounted with Vectashield mounting medium with DAPI (Vector
Laboratories). The percentage of BrdU incorporating cells was determined by counting a minimum
of 500 cells per slide (except for one experiment in which due to technical difficulties less cells were
counted when testing the compound
Germany ) [22].
8) using a fluorescence microscope (DM2000; Leica, Wetzlar,
3.6.5. Analysis of Apoptosis
Following 48 h treatment, the levels of apoptosis were assessed using the “Human
AnnexinV-FITC/PI apoptosis” kit (Bender MedSystems, Wien, Austria) as previously described [23].
Briefly, cell pellets were resuspended in binding buffer and further incubated with Annexin V-FITC
and with propidium iodide. Samples were analyzed in a FACSCalibur flow cytometer (BD Biosciences,
Erembodegem, Belgium), plotting at least 15,000 events and using the FlowJo 7.6.5 software (Tree Star,
Inc., Ashland, OR, USA) [24].
3.7. Effect of Compounds 5 and 8 on Protein Expression Analyzed by Western Blot
Total cell lysates were prepared as described previously [25]. Primary antibodies used in this study
are: anti-Raf-1 and anti-CDK4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Survivin

Molecules 2018, 23, 407
13 of 15
(Abcam, Cambridge, UK), anti-Akt (Transduction Laboratories, Lexington, KY, USA), and anti-Actin
Sigma Chemical Co., St. Louis, MO, USA).
3.8. Analysis of the Effect of Compound 5 on the Tumor Growth of Xenografted Mouse Models
Experiments were carried out using female athymic Swiss nude mice, 8–10 weeks old (Charles
River, Calco, Italy). Mice were maintained in laminar flow rooms keeping temperature and humidity
constant. Mice had free access to food and water. Experiments were approved by the Ethics Committee
for Animal Experimentation of the Istituto Nazionale Tumori of Milan according to institutional
guidelines. Epidermoid carcinoma cells, A-431 suspended in culture media were subcutaneously
injected on the back of mice. When tumors were just palpable, the mice were randomly divided into
two groups: control and compound-treated. Each group was constituted by 8 mice. Saline (0.9% NaCl)
or compound solution (5 or 10 mg/kg, respectively) was i.p. administered daily during 3 weeks, to
both groups. Tumor volume was assessed using calipers and determined according to the formula
(d)2xD/2.
3.9. Statistical Analysis
For the results regarding determination of GI50 concentrations, % viable cells, cellular proliferation
and apoptosis, data is presented as mean
experiments. For those experiments, statistical analysis was carried out using an unpaired student’s
±
standard error (SEM) from at least three independent
t-test.
4. Conclusions
The present study describes the screening of a series of compounds with new chromenopyridinone
and thiochromenopyridinone scaffolds as potential HSP90 inhibitors. Our results indicated that
compounds
are promising regarding in vitro tumor cell growth inhibitory activity. Treatment with these
compounds decreased the proliferation of tumor cells and compound induced apoptosis. In addition,
the compounds were able to downregulate selected proteins known as “clients” of HSP90. Specifically,
compound induced an almost complete downregulation of CDK4 and a partial downregulation of
survivin in STO and A431 cells, whereas compound produced degradation of survivin in STO cells
5 and 8 with a chromenopyridinone and thiochromenopyridinone skeleton, respectively,
8
5
8
and a significant downregulation of RAF in A431 cells. The observed modulations are consistent with
an effect mediated by interaction of the selected compounds with HSP90. In addition, treatment of
xenografted mice with compound
5 resulted in a considerable dose-dependent inhibition of tumor
growth. Work is in progress to investigate structural changes of the heterocyclic scaffold, as well as of
the N-substituent, and to improve the activity of the compounds. In addition, future work will confirm
if these compounds inhibit the ATPase activity of purified HSP90 in vitro.
Acknowledgments: This work was funded by Fundo Europeu de Desenvolvimento Regional (FEDER) através
do COMPETE 2020—Programa Operacional para a Competitividade e Internacionalização (POCI), Portugal
2020, and by portuguese fundings through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência,
Tecnologia e Inovação, no âmbito do projeto “Instituto de Investigação e Inovação em Ciências da Saúde”
(POCI-01-0145-FEDER-007274)”.
Author Contributions: S.D. and M.H.V. conceived the study and designed experiments. A.B. and D.S. performed
experiments and data organization/analysis regarding the SRB assay, cell cycle and apoptosis assays, under the
supervision of R.T.L. L.M. and R.C. synthesized the compounds, V.Z. and M.D.C. carried out the HSP90 assay,
Western Blots and xenograft studies. S.D., M.H.V., R.T.L. and S.D. wrote the manuscript. All authors read and
agreed to the final version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 407
14 of 15
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Morrow, G.; Hightower, L.E.; Tanguay, R.M. Small heat shock proteins: Big folding machines. Cell Stress
Chaperones 2015, 20, 207–212. [CrossRef] [PubMed]
Vidyasagar, A.; Wilson, N.A.; Djamali, A. Heat shock protein 27 (HSP27): Biomarker of disease and
therapeutic target. Fibrogenesis Tissue Repair 2012, 5, 7. [CrossRef] [PubMed]
Lianos, G.D.; Alexiou, G.A.; Mangano, A.; Mangano, A.; Rausei, S.; Boni, L.; Dionigi, G.; Roukos, D.H.
The role of heat shock proteins in cancer. Cancer Lett. 2015, 360, 114–118. [CrossRef] [PubMed]
Jego, G.; Hazoume, A.; Seigneuric, R.; Garrido, C. Targeting heat shock proteins in cancer. Cancer Lett. 2013
332, 275–285. [CrossRef] [PubMed]
,
Khalil, A.A.; Kabapy, N.F.; Deraz, S.F.; Smith, C. Heat shock proteins in oncology: Diagnostic biomarkers or
therapeutic targets? Biochim. Biophys. Acta 2011, 1816, 89–104. [CrossRef] [PubMed]
Neckers, L. Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol. Med. 2002, 8 (Suppl. 4),
S55–S61. [CrossRef]
Shrestha, L.; Bolaender, A.; Patel, H.J.; Taldone, T. Heat Shock Protein (HSP) Drug Discovery and
Development: Targeting Heat Shock Proteins in Disease. Curr. Top Med. Chem. 2016, 16, 16–2753. [CrossRef]
Garcia-Carbonero, R.; Carnero, A.; Paz-Ares, L. Inhibition of HSP90 molecular chaperones: Moving into the
clinic. Lancet Oncol. 2013, 14, e358–e369. [CrossRef]
Neckers, L.; Workman, P. Hsp90 molecular chaperone inhibitors: Are we there yet? Clin. Cancer Res. 2012
,
18, 64–76. [CrossRef] [PubMed]
10. Hadden, M.K.; Hill, S.A.; Davenport, J.; Matts, R.L.; Blagg, B.S. Synthesis and evaluation of Hsp90 inhibitors
that contain the 1,4-naphthoquinone scaffold. Bioorg. Med. Chem. 2009, 17, 634–640. [CrossRef] [PubMed]
11. Rios, D.; Benites, J.; Valderrama, J.A.; Farias, M.; Pedrosa, R.C.; Verrax, J.; Buc Calderon, P. Biological
evaluation of 3-acyl-2-arylamino-1,4-naphthoquinones as inhibitors of Hsp90 chaperoning function. Curr. Top.
Med. Chem. 2012, 12, 2094–2102. [CrossRef] [PubMed]
12. Davenport, J.; Balch, M.; Galam, L.; Girgis, A.; Hall, J.; Blagg, B.S.; Matts, R.L. High-throughput screen of
natural product libraries for hsp90 inhibitors. Biology 2014, 3, 101–138. [CrossRef] [PubMed]
13. Bargiotti, A.; Musso, L.; Dallavalle, S.; Merlini, L.; Gallo, G.; Ciacci, A.; Giannini, G.; Cabri, W.; Penco, S.;
Vesci, L.; et al. Isoxazolo(aza)naphthoquinones: A new class of cytotoxic Hsp90 inhibitors. Eur. J. Med. Chem.
2012, 53, 64–75. [CrossRef] [PubMed]
14. Uetrecht, J. Screening for the potential of a drug candidate to cause idiosyncratic drug reactions.
Drug Discov. Today 2003, 8, 832–837. [CrossRef]
15. Jayat, C.; Ratinaud, M.H. Cell cycle analysis by flow cytometry: Principles and applications. Biology of the
cell/under the auspices of the. Eur. Cell Biol. Organ. 1993, 78, 15–25. [CrossRef]
16. Nagashima, T.; DeArmond, S.J.; Murovic, J.; Hoshino, T. Immunocytochemical demonstration of S-phase
cells by anti-bromodeoxyuridine monoclonal antibody in human brain tumor tissues. Acta Neuropathol. 1985
,
67, 155–159. [CrossRef] [PubMed]
17. Familoni, O.B.; Ionica, I.; Bower, J.F.; Snieckus, V. Intramolecular Anionic Friedel-Crafts Equivalents.
A General Regiospecific Route to Substituted and Naturally Occurring Xanthen-9-ones. Synlett 1997, 1997,
1081–1083. [CrossRef]
18. Kruger, S.; Mann, F.G. Xanthones and thioxanthones. Part V. The preparation and properties of
9-thia-2-aza-anthrone and 9-thia-4-aza-anthrone. J. Chem. Soc. 1954, 3905–3910. [CrossRef]
19. Vaz, J.A.; Ferreira, I.C.; Tavares, C.; Almeida, G.M.; Martins, A.; Helena Vasconcelos, M. Suillus collinitus
methanolic extract increases p53 expression and causes cell cycle arrest and apoptosis in a breast cancer cell
line. Food Chem. 2012, 135, 596–602. [CrossRef] [PubMed]
20. Reis, F.S.; Sousa, D.; Barros, L.; Martins, A.; Morales, P.; Ferreira, I.C.; Vasconcelos, M.H. Leccinum vulpinum
Watling induces DNA damage, decreases cell proliferation and induces apoptosis on the human MCF-7
breast cancer cell line. Food Chem. Toxicol. 2016, 90, 45–54. [CrossRef] [PubMed]
21. Preto, A.; Goncalves, J.; Rebocho, A.P.; Figueiredo, J.; Meireles, A.M.; Rocha, A.S.; Vasconcelos, H.M.; Seca, H.;
Seruca, R.; Soares, P.; et al. Proliferation and survival molecules implicated in the inhibition of BRAF pathway
in thyroid cancer cells harbouring different genetic mutations. BMC Cancer 2009, 9. [CrossRef] [PubMed]

Molecules 2018, 23, 407
15 of 15
22. Dos Santos, T.; Oliveira, M.; Sousa, D.; Lima, R.T.; Martins, A.; Ferreira, I.C.F.R.; Vasconcelos, M.H. Suillus
luteus methanolic extract inhibits proliferation and increases expression of p-H2A.X in a non-small cell lung
cancer cell line. J. Funct. Foods 2014, 6, 100–106. [CrossRef]
23. Belaz, K.R.; Denadai, M.; Almeida, A.P.; Lima, R.T.; Vasconcelos, M.H.; Pinto, M.M.; Cass, Q.B.; Oliveira, R.V.
Enantiomeric resolution of albendazole sulfoxide by semipreparative HPLC and in vitro study of growth
inhibitory effects on human cancer cell lines. J. Pharm. Biomed. Anal. 2012, 66, 100–108. [CrossRef] [PubMed]
24. Bizarro, A.; Ferreira, I.C.; Sokovic, M.; van Griensven, L.J.; Sousa, D.; Vasconcelos, M.H.; Lima, R.T.
Cordyceps militaris (L.) Link Fruiting Body Reduces the Growth of a Non-Small Cell Lung Cancer Cell
Line by Increasing Cellular Levels of p53 and p21. Molecules 2015, 20, 13927–13940. [CrossRef] [PubMed]
25. Giommarelli, C.; Zuco, V.; Favini, E.; Pisano, C.; Dal Piaz, F.; De Tommasi, N.; Zunino, F. The enhancement of
antiproliferative and proapoptotic activity of HDAC inhibitors by curcumin is mediated by Hsp90 inhibition.
Cell. Mol. Life Sci. 2010, 67, 995–1004. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 5 and 8 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).