A combined ligand- and structure-based approach for the identification of rilmenidine-derived compounds which synergize the antitumor effects of doxorubicin

Article abstract of DOI:10.1016/j.bmc.2016.05.043

The clonidine-like central antihypertensive agent rilmenidine, which has high affinity for I₁-type imidazoline receptors (I₁-IR) was recently found to have cytotoxic effects on cultured cancer cell lines. However, due to its pharmacological effects resulting also from α₂-adrenoceptor activation, rilmenidine cannot be considered a suitable anticancer drug candidate. Here, we report the identification of novel rilmenidine-derived compounds with anticancer potential and devoid of α₂-adrenoceptor effects by means of ligand- and structure-based drug design approaches. Starting from a large virtual library, eleven compounds were selected, synthesized and submitted to biological evaluation. The most active compound 5 exhibited a cytotoxic profile similar to that of rilmenidine, but without appreciable affinity to α₂-adrenoceptors. In addition, compound 5 significantly enhanced the apoptotic response to doxorubicin, and may thus represent an important tool for the development of better adjuvant chemotherapeutic strategies for doxorubicin-insensitive cancers.

Full text of DOI:10.1016/j.bmc.2016.05.043

Accepted Manuscript
A Combined Ligand- and Structure-Based approach for the identification of
rilmenidine-derived compounds which synergize the antitumor effects of dox-
orubicin
Jelica Vucicevic, Tatjana Srdic-Rajic, Marco Pieroni, Jonne MM Laurila,
Vladimir Perovic, Sabrina Tassini, Elisa Azzali, Gabriele Costantino, Sanja
Glisic, Danica Agbaba, Mika Scheinin, Katarina Nikolic, Marco Radi, Nevena
Veljkovic
PII:
S0968-0896(16)30373-X
http://dx.doi.org/10.1016/j.bmc.2016.05.043
BMC 13027
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2016
17 May 2016
20 May 2016
Please cite this article as: Vucicevic, J., Srdic-Rajic, T., Pieroni, M., Laurila, J.M., Perovic, V., Tassini, S., Azzali,
E., Costantino, G., Glisic, S., Agbaba, D., Scheinin, M., Nikolic, K., Radi, M., Veljkovic, N., A Combined Ligand-
and Structure-Based approach for the identification of rilmenidine-derived compounds which synergize the
antitumor effects of doxorubicin, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.05.043
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A Combined Ligand- and Structure-Based
approach for the identification of
rilmenidine-derived compounds which
synergize the antitumor effects of
doxorubicin
Leave this area blank for abstract info.
Jelica Vucicevica, Tatjana Srdic-Rajicb, Marco Pieronic, Jonne MM Laurilad, Vladimir Perovice, Sabrina
Tassinic, Elisa Azzalic, Gabriele Costantinoc, Sanja Glisice, Danica Agbabaa, Mika Scheinind,f, Katarina
Nikolica*, Marco Radic* and Nevena Veljkovice*

Bioorganic & Medicinal Chemistry
A Combined Ligand- and Structure-Based approach for the identification of
rilmenidine-derived compounds which synergize the antitumor effects of doxorubicin
Jelica Vucicevic,a Tatjana Srdic-Rajic,b Marco Pieroni,c Jonne MM Laurila,d Vladimir Perovic,e Sabrina
Tassini,c Elisa Azzali,c Gabriele Costantino,c Sanja Glisic,e Danica Agbaba,a Mika Scheinin,d,f Katarina
Nikolic,a,* Marco Radic,* and Nevena Veljkovice,*
a Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000, Belgrade, Serbia
bDepartment of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia
c
, Viale delle Scienze, 27/A, 43124 Parma, Italy
dDepartment of Pharmacology, Drug Development and Therapeutics, University of Turku, Kiinamyllynkatu 10, FI-20520 Turku, Finland
eCenter for Multidisciplinary Research, Institute of Nuclear Sciences Vinca, University of Belgrade, Mihaila Petrovica Alasa 14, 11001, Belgrade, Serbia
fUnit of Clinical Pharmacology, Turku University Hospital, Turku, Finland
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The clonidine-like central antihypertensive agent rilmenidine, which has high affinity for I1-type
imidazoline receptors (I1-IR) was recently found to have cytotoxic effects on cultured cancer
cell lines. However, due to its pharmacological effects resulting also from α2-adrenoceptor
activation, rilmenidine cannot be considered a suitable anticancer drug candidate. Here, we
report the identification of novel rilmenidine-derived compounds with anticancer potential and
devoid of α2-adrenoceptor effects by means of ligand- and structure-based drug design
approaches. Starting from a large virtual library, eleven compounds were selected, synthesized
and submitted to biological evaluation. The most active compound 5 exhibited a cytotoxic
profile similar to that of rilmenidine, but without appreciable affinity to α2-adrenoceptors. In
addition, compound 5 significantly enhanced the apoptotic response to doxorubicin, and may
thus represent an important tool for the development of better adjuvant chemotherapeutic
strategies for doxorubicin-insensitive cancers.
Available online
Keywords:
Drug design
Synthesis
Rilmenidine
Doxorubicin synergism
α2-Adrenoceptors
Cytotoxic activity
Apoptosis
2016 Elsevier Ltd. All rights reserved.
1. Introduction
moderately efficacious I1-IR agonist, exerts proapoptotic
effects in fibroblasts and anti-apoptotic effects in
Centrally acting hypotensive imidazoline derivatives, such as
clonidine, rilmenidine and moxonidine, inhibit the activity of the
sympathetic nervous system by activating α2-adrenoceptors in the
brain and possibly also via actions mediated by non-adrenergic
I1-imidazoline receptors (I1-IR).1-4 The prototypical agent,
clonidine, has almost similar affinity for both types of receptors,
whereas the newer antihypertensive agents moxonidine and
rilmenidine bind more avidly I1-IR than α2-adrenoceptors.
Possibly for this reason, they are less prone to elicit the typical
side effects of clonidine, i.e. sedation, dry mouth and
bradycardia. Recently, imidazoline derivatives have been also
found to have other important biological effects not related to
cardiovascular regulation, such as control of apoptosis and cell
proliferation, both observed in vitro at micro- to millimolar
concentrations (Fig. 1). Aceros et al.5 reported that moxonidine, a
cardiomyocytes. The imidazoline compound RX871024 induces
cell death in insulin-secreting MIN6 cells.6 S43126, an I1-IR
selective inhibitor of PC12 cell growth, caused considerable
dose-dependent cell death, and apoptotic body formation after 72
h of treatment.7 Our previous study demonstrated that rilmenidine
induces significant membrane dissipation and deactivation of the
Ras/MAP kinases ERK, p38 and JNK in cultured human
leukemic K562 cells, thus exhibiting proapoptotic and
antiproliferative effects8. However, our incomplete knowledge of
I1-IR signaling pathways and the lack of an I1-IR crystallographic
structure did not allow us to claim unequivocally which is the
actual target through which rilmenidine exerts these effects. We
have assumed that the action of rilmenidine is connected with its
binding to the I1-IR candidate nischarin and its interaction partner
RAC1, a member of the Rac subfamily of Rho guanosine
———
*
Corresponding authors: Nevena Veljkovic, (POB 522, Mihaila Petrovica Alasa 14, 11001, Belgrade, Serbia, E-mail: nevenav@vinca.rs);
Marco Radi (Viale delle Scienze, 27/A, 43124 Parma, Italy, E-mail: marco.radi@unipr.it); Katarina Nikolic (Vojvode Stepe 450, 11000,
Belgrade, Serbia, E-mail: knikolic@pharmacy.bg.ac.rs.)

triphosphatases (GTPases). Nischarin is a cytoplasmic protein9
which besides rilmenidine binds numerous other imidazoline
ligands and affects cellular signaling cascades controlling cell
survival, growth and migration.10,11 Along with the proapoptotic
and antiproliferative effects, we discovered that the I1-IR agonist
rilmenidine rendered leukemic K562 cells, which are particularly
resistant to many DNA-damaging chemotherapeutic agents,
susceptible to the effects of doxorubicin.8
2. Results and discussion
Here we report the development of a novel virtual screening
protocol for the identification of structurally diverse I1-IR
agonists with proapoptotic and antiproliferative activity. To limit
the number of hits to be tested, the method included the use of an
average quasi-valence number (AQVN)-similarity search, and
ligand- and structure-based virtual screening approaches. This
combination of techniques allowed us to select eleven different
compounds for biological evaluation.
2.1. Chemoinformatic screening
Strong correlations between biological activities of organic
molecules and the values of AQVN and electron ion interaction
potential (EIIP) chemical descriptors related to long-range
interaction properties have been observed.17 In line with this
principle, we proposed a simple criterion to discriminate the
biologically relevant chemical space based on AQVN and EIIP.
Previously, we demonstrated that 92.5% of about 45 million
compounds from the PubChem database are homogeneously
distributed within the AQVN interval (2.4 – 3.3). Recently, we
reported the selection of HIV and Ebola inhibitors by means of
AQVN- and EIIP-similarity searches together with other
complementary VS approaches.18,19 Here, we define an AQVN-
based filter for the rapid in silico prescreening of large chemical
libraries in order to identify novel rilmenidine analogues. For this
purpose, we established the filter by expanding the area centered
at rilmenidine’s AQVN value (2.4828) to capture 20% of the
AQVN space occupied by the I1-IR agonists reported in the
literature (Supplementary material). In this way we defined the
rilmenidine AQVN space between 2.4296 and 2.5025. Filtering
of compound sets described in the Methods section led to the pre-
selection of 3005 compounds that were further investigated in a
Figure 1. Structures of imidazoline compounds that regulate cellular
apoptosis and proliferation in vitro
Doxorubicin, an anthracycline drug, is one of the most
effective anticancer drugs ever developed, and although in
clinical use for more than 30 years, it still plays an important role
in the treatment of many hematological cancers (leukemias and
lymphomas) and several types of solid tumors (carcinomas and
sarcomas).12 The therapeutic potential of doxorubicin is
significantly limited by the risk of cardiotoxicity, which is
dependent on the cumulative dose/treatment schedule and
unpredictably evolves towards congestive heart failure. To avoid
this serious complication, the maximum recommended
cumulative dosage of doxorubicin has been set at 500 mg/m2.13
Regardless, doxorubicin cardiotoxicity may occur at lower
cumulative doses if co-administered with other antitumor agents
(e.g. paclitaxel, trastuzumab). These combined treatment
modalities clearly offer improved response rates, but important
cardiotoxicity may surface with cumulative doses of doxorubicin
as low as 360-400 mg/m2.14,15 In the current study, we aimed at
extending our earlier investigation on the rilmenidine binding site
and I1-IR candidate nischarin.16 We used a combined ligand- and
structure-based virtual screening approach to identify ligands
that, similarly to rilmenidine, could establish effective
interactions with nischarin and affect cell viability. In addition,
by using nischarin as a target for structure-based virtual
screening, we aimed at diminishing ligand binding to α2-
adrenoceptors. Exploring a large in-house virtual library, eleven
hit compounds emerged as best candidates. Biological studies
confirmed that the most active compound 5 indeed induces
apoptosis and sustains the proliferation of K562 cells in a similar
fashion as rilmenidine but with limited effects on α2A-
adrenoceptors. More importantly, we demonstrated that co-
ligand- and structure-based virtual screening (Fig. 2).
Figure 2. Compound distribution according to their average quasi-valence
number (AQVN). 10.47% of the elements from the compound set described
in the Methods section are within the I1-IR agonist domain. The compound
sets contain 3005 elements within the rilmenidine-like domain.
2.2. Ligand- and Structure-Based Virtual Screening
A number of successful applications of FLAP (fingerprint for
ligands and proteins) for the identification of G-protein-coupled
receptor ligands have been recently reported.20-23 However, to our
knowledge, this is the first work reporting the application of VS
approaches in order to disclose selective I1-IR ligands with
potential proapoptotic and antiproliferative activity. In this work
we applied combined ligand- and structure-based virtual
screening in order to take into account the variety of available
chemical and biological information.24 After AQVN-based
filtering of the in-house virtual library (more than 9 × 106
treatment with compound
5 and doxorubicine promotes
substantial enhancement of the apoptotic responses of K562 cells
compared to the single agents. This integrated medicinal
chemistry study provides biologists and pharmacologists with a
tool that may represent a promising starting point for the
exploration of the I1-IR pathway and for the development of
improved adjuvant chemotherapeutic strategies for cancers with
limited susceptibility to doxorubicin.

compounds), the remaining library contained 3005 structurally
diverse compounds. We then performed FLAP prefiltering to
select only a subset of structures that are enriched in
pharmacophore similarity to the template. FLAP fingerprint
similarity was used to rank compounds with regard to similarity
with rilmenidine. 539 top-ranked substances were selected
according to their Glob-Sum values (> 0.01). An enriched dataset
constituted by 21 I1-IR agonists and 539 prefiltered rilmenidine-
derived compounds was built and subjected to further ligand- and
structure-based virtual screening. The results were estimated in
terms of enrichment factors and AUC. As a first step, ligand-
based VS was applied to evaluate the similarity between active
and untested compounds from the FLAP database and
rilmenidine taking into account GRID MIF interaction probes.
Various similarity scores corresponding to overlapping MIFs, as
well as their combinations, were generated for each molecule.
The second screening was performed using a structure-based
approach established on the structural homology model of
nischarin.16 The predicted active center of this modelled protein
was used to screen the FLAP database considering the similarity
between the MIFs of the binding pocket and the ligands.
According to the obtained AUC and enrichment factor values,
H*DRY*H and H similarity scores were able to efficiently
discriminate 21 known active I1-IR ligands from untested FLAP
database compounds in ligand- and structure-based virtual
screening studies, respectively, indicated by highest virtual
accuracy (AUCLBVS = 1.00; EF1%LBVS = 100 %; AUCSBVS
= 0.991; EF1%SBVS = 100 %). Based on the obtained results it
can be concluded that shape and hydrophobic interactions are
important for activity of compounds toward nischarin. Eleven
potential hits (see section 2.3) that had both H scores higher than
0.65 according to the structure-based VS and H*DRY*H scores
higher than 0.1 according to the ligand-based VS were selected,
synthesized and then tested in vitro against K562 cells (Table 1).
Scheme 2. Reaction conditions: i. NaH, DMF, reflux, 48 h
2.4. Characterization of ligands at human α2A-adrenoceptors
The apparent affinities of the eleven hit compounds and those
of the reference compounds rilmenidine and efaroxan to human
α2A-adrenoceptors were determined using competition binding
assays (Table 2). Adrenaline, dexmedetomidine, clonidine and
atipamezole, ligands known to bind to α2-adrenoceptors with
high affinity, were used as positive controls in the assays. Among
the imidazoline drugs, efaroxan was found to bind α2A-
adrenoceptors with relatively high affinity in comparison to
clonidine and rilmenidine. None of the eleven rilmenidine-
derived hit compounds exhibited appreciable affinity toward
human α2A-adrenoceptors, with the possible exception of
compounds 5 and 7 (Ki 1.2 and 11 µM). In terms of functional
activity, [35S]GTPγS binding experiments were conducted to
compare agonist potency and intrinsic activity at recombinant
human α2A-adrenoceptors. The results indicated that, similarly to
clonidine, rilmenidine is a partial agonist of α2A-adrenoceptors,
but with lower potency compared with clonidine. None of the hit
compounds nor the α2-adrenoceptor antagonist efaroxan
exhibited agonist-like properties towards the human α2A-
adrenoceptor. Some of the rilmenidine-derived compounds
produced small negative intrinsic activity values in the functional
assay, possibly acting like weak inverse agonists at α2A-
adrenoceptors, albeit at very high concentrations.
2.3. Synthesis of the selected hit compounds
Briefly, for the synthesis of compounds 3a-i, the
commercially available precursor 4-hydroxy-6-methyl-2-pyrone
1 was reacted with the suitable amine at reflux to give the N-
substituted pyridinones 2a-d in good overall yields. Pyridinones
2a-d were further substituted at C4 with the suitable alkyl- or
arylamines under microwave-assisted conditions. Compounds
3a-i were obtained in acceptable overall yields after purification
by flash column chromatography (Scheme 1). Compounds 5 and
7 were prepared according to a protocol already reported
elsewhere (Scheme 2).25
2.5. Evaluation of Cytotoxicity
K562 cells were exposed to various concentrations of
investigated compounds for 48 hours and the cytotoxicity was
determined using the MTT assay. It was observed that six
compounds exhibited cytotoxic activity at mid- to high
micromolar concentrations (Fig. 3). By comparing the effects of
compound 5 and the imidazoline compounds (rilmenidine,
5,8,26
moxonidine and efaroxan) on cell viability
and apoptosis in
K562 cells (Table 3) it was possible to propose compound 5 as a
novel hit compound. In the apoptosis assay, rilmenidine induced
apoptosis at IC50 concentration, as we previously reported,
whereas efaroxan and moxonidin were incapable of inducing
apoptosis at concentrations as high as 300 µM (Supplementary
material).
2.6. Evaluation of the capability of compound 5 to induce
apoptosis alone and in combination with doxorubicin
The proapoptotic effect of compound 5 is reflected by the
increase of early apoptotic cells in comparison to the vehicle
control. We also observed that compound 5 is two-fold more
efficient than rilmenidine in inducing cell death (Fig. 4A). FACS
was
used
to
evaluate
K562
cell
cycle
Scheme 1. Reaction conditions: i. R1-NH2, H2O, reflux, 12 h; ii. R2-NH2,
DME, MW, 120 °C, 40 min

Table 1. Structure and antiproliferative activity (IC50) on K562cells of
compounds selected by the VS protocol
Table 2. Competition binding affinities (Ki) of tested ligands obtained with
[3H]RS79948-197 at human α2A-adrenoceptors expressed in CHO cells and
estimates of their agonist potency (pEC50) and intrinsic activity obtained with
the functional [35S]GTPγS binding assay.
Compound
rilmenidine
Structure
IC50 (μM)
Intrinsic
Compound
Ki (nM)a
pEC50
activityb
(% of adrenaline)
65
3a
3b
3c
3d
3e
3f
58 000 (20 000-170 000)
62 000 (27 000-150 000)
41 000 (22 000-80 000)
80 000 (24 000-270 000)
52 000 (15 000-430 000)
27 000 (8 100-130 000)
99 000 (40 000-250 000)
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
-6 ± 2
-18 ± 3
-12 ± 4
0 ± 4
efaroxan
>300
222
-17 ± 4
-10 ± 2
-1 ± 4
3a
3g
3h
110 000 (44 000-300 000)
n.d.
-8 ± 5
3i
110 000 (67 000-1100 000)
1 200 (540-3000)
n.d.
n.d.
n.d.
-2 ± 1
-3 ± 2
-6 ± 1
5
3b
3c
115
7
11 000 (3 900-36 000)
Adrenaline
7.52 ±
0.07
6.75 ±
0.07
160 (67-430)
300 (170-520)
100
Rilmenidine
>300
54 ± 4
Efaroxan
Clonidine
4.4 (2.7-6.9)
28 (16-48)
n.d.
-13 ± 2
41 ± 6
7.52 ±
Dexmedetomidine
8.67 ±
0.04
2.5 (1.6-3.9)
63 ± 3
3d
>300
Atipamezole
1.2 (0.77-2.0)
n.d.
n.d.
aThe apparent Ki (nM) and its 95% confidence intervals are from 3-6
independent experiments, analyzed using a one-site competition mode;
bIntrinsic activity values are relative to the natural full agonist adrenaline.
Values shown for pEC50 (negative logarithm of the ligand concentration (nM)
causing 50% of the maximal observed agonist effect) and intrinsic activity are
means ± s.e.m. from 3-5 independent experiments. n.d. indicates “not
determined” for compounds which did not show any agonist activity in the
functional assay.
3e
3f
128
>300
3g
224
3h
3i
140
>300
5
7
70
Figure 3. Dose-response cytotoxicity curves of rilmenidine and six novel
compounds (3a, 3b, 3e, 3g, 3h, 5) obtained with the MTT assay in K562 cells
after 48 hours.
>300
progression after 48 hours of continual incubation with
compound 5. The accumulation of cells in sub-G1 phase was in
concordance with the observed apoptosis (Fig. 4B). To assess the
antiproliferative effect of compound 5, we performed
a
clonogenic assay of K562 cells in methylcellulose. Compound 5

inhibited in a dose-dependent manner colony formation of K562
cells after 24h treatment. We observed a significant growth
inhibition of 88±6 % at compound 5 IC50 concentration (Fig. 4C).
Then we counter-screened compound 5 against two non-related
oncogenic targets, Abl and Src kinases, in order to asses that
these cellular effects were not related to some non-specific
activity or compound 5 aggregation. Indeed, no inhibition of
selected kinases was observed when compound 5 was tested both
in the presence and absence of detergent in the reaction mixture
(Supplementary material). Furthermore, in silico analysis by the
Aggregator Advisor server27 did not find any similarity between
compound 5 and any known aggregator listed in their database.
These results tend to exclude aggregation effects or non-specific
activities of compound 5, suggesting that the dose-dependent
cytotoxic effect observed is strictly linked to the specific action
on the I1-IR pathway.
rilmenidine and doxorubicin synergistically sensitized K562 cells
to apoptosis in comparison with doxorubicin alone (44% vs.
10%).8 Here, we examined the combined application of
compound
5
and doxorubicin and observed enhanced
cytotoxicity and apoptotic responses of K562 cells compared to
doxorubicin alone (Fig. 5). The IC50 of doxorubicin in K562 cells
was 1.0 μM. To assess the cytotoxic effects of the drug
combination, we used a fixed molar ratio of compound
5/doxorubicin of 70:1, as required by the Chou-Talalay method
(Table 4). CI values significantly <1 observed over the range of
tested concentrations indicated strong synergistic effects. Similar,
albeit weaker, synergism has been previously demonstrated with
rilmenidine.8 Compound 5 is a carbazole derivative. Many
natural and semi-synthetic carbazoles show promising antitumor
activity against different cancer cell lines30-32, often by inhibiting
DNA-dependent enzymes and by causing DNA damage.
Recently, it was shown that the carbazole derivative MHY407
sensitizes breast cancer cells to doxorubicin and etoposide
toxicity by increasing DNA damage-related proteins and
inducing S phase arrest.33 Here, cell cycle experiments showed
K562 is a model cell line of blast-like chronic myeloid
leukemia which is relatively insensitive to apoptosis induced by
DNA damaging drugs, including doxorubicin.28 We have
previously demonstrated that combined application of
Figure 4. Induction of apoptotic cell death and proliferation inhibition in K562 cells treated with compound 5. (A) Early apoptosis in K562 cells
treated with vehicle, 50μM rilmenidine and 70 μM compound 5 after 48 hours. (B) Effect of compounds on cell cycle phase distribution. Cells were treated with
70 μM compound 5 for 48 hours. After the indicated time, the cells were stained with PI and analyzed for alternations in cell cycle phase distribution by flow
cytometry. The results are representative of three independent experiments. M1 - Cells with DNA content corresponding to sub-G1 peak; M2- Cells with DNA
content corresponding to G0/G1 phases. M3 - Cells with DNA content corresponding to the S phase. M4 - Cells with DNA content corresponding to G2/M
phases. (C) Compound 5 dose-dependently inhibited K562 colony formation after 24 hour treatment (upper panel). Images of representative plates of the K562
cell line incubated with 50 μM and 70 μM compound 5 (lower panel).
Table 3. Concentrations of imidazoline and imidazoline-like compounds
Table 4. Combination index (CI) value of the combination treatment with
compound 5 and doxorubicin at a molar ratio of 70:129
that induce cell death in the K562 cell line
Compound
Compound 5 (μM) doxorubicin (μM) Inhibition rate (%)
CI
Antiproliferative/proapoptotic
concentration (μM)
25
50
70
0.36
0.7
1.0
45.75
68.62
72.21
0.877
0.511
0.617
Rilmenidine
Moxonidine
Efaroxan
50
>300
-
Compound 5
70
100
1.4
78.21
0.688

that the synergistic effect obtained by combining the carbazole
derivative 5 and doxorubicin is connected to the recovery of
doxorubicin induced G2/M arrest. As depicted in Fig. 6, cells
treated with doxorubicin accumulate in G2/M phase, whereas co-
treatment significantly decreased G2/M phase accumulation on
account of an increased sub-G1 cell population.
activity at recombinant human α2A-adrenoceptors, suggesting that
unwanted side effects mediated by the activation of this receptor
may be avoided. Consistently with the observed apoptosis, the
flow cytometric analysis of cell cycle progression showed an
increase in the proportion of K562 cells in the sub-G1 phase,
complemented with decreased proportions of cells in G1, S and
G2/M. Previously, we have also shown that rilmenidine rendered
K562 cells, which are particularly resistant to chemotherapeutic
agents, susceptible to the DNA damaging drug doxorubicin.
Similarly, combined treatment with compound 5 and doxorubicin
showed synergistic increases in apoptosis compared to
doxorubicin alone (64% vs. 20%), which highlighted the
potential of compound 5 or its derivatives to complement
conventional chemotherapeutic regimens through improved
antitumor efficacy and possibly reduced side effects. In
conclusion, the integrated medicinal chemistry study herein
reported provides biologists and pharmacologists with a novel
tool (compound 5) that may represent a promising starting point
for the exploration of the I1-IR pathway and for the development
of better adjuvant chemotherapeutic strategies for cancers with
limited susceptibility to doxorubicin-like agents.
Experimental
4.1. Compound sets
The virtual library used for in silico screening was composed
of the P4T-group’s internal collection plus a focused virtual
library generated on the basis of a versatile multicomponent
synthetic protocol.34 This synthetic sequence was used as input
for the Smi-Lab software35 to combine a series of commercial
building blocks. The complete virtual library contained more
than 9 × 106 compounds.
Figure 5. Induction of apoptotic cell death in K562 cells. Proportion of
early apoptotic (FITC+/PI-) cells in a population was measured by bivariate
Annexin V/PI flow cytometry in untreated K562 cells (A) and in cells treated
with 70 μM compound 5 (B), 1 μM doxorubicin (C), and compound 5 plus
doxorubicin (D) after 48 hours.
4.2. Virtual screening protocol
The virtual screening (VS) protocol used in this study was
based on the combination of different methods. In the beginning
of the multi-step screening process we used average quasi-
valence number (AQVN) similarity searches to reduce the
number of hits. Further parallel ligand- and structure-based
screening protocols were run independently and the hits that
showed good ranking in both approaches were selected for
biological testing.
4.2.1. Chemoinformatics screen
17
Molecular descriptor AQVN was calculated as described in
using the FormCal computational tool.36 Values of AQVN of
rilmenidine and 28 I1-IR agonists (Supplementary material)
selected from the literature were used to define filtering domains
for hit-like compounds.
Figure 6. Effect of DOX and DOX/5 combination on cell cycle phase
distribution of K562 cells. The numbers indicate the fraction of cells in a
given phase of the cell cycle.
3. Conclusions
4.2.2. Ligand and Structure Based Virtual Screening
Protocols
In this work, which extends our previous investigation on the
nischarin-rilmenidine binding site, we aimed at understanding the
structure activity relationship underlying the observed
proapoptotic activity of rilmenidine. We sought to identify novel
compounds with rilmenidine-like activity and mechanism of
action but with different chemical structure. We used a VS
protocol that includes AQVN calculations accounting for the
long-range properties of organic molecules and combined ligand-
and structure-based virtual screening protocols based on GRID
MIF similarities between tested compounds and the template
rilmenidine. Among eleven prospective candidates that stemmed
from the in silico screening experiments, compound 5 was found
to be the most active derivative with cytotoxic activity against
K562 cells comparable to that of rilmenidine. In addition, none of
the identified eleven compounds showed any detectable agonist
The software FLAP (Fingerprints for Ligand and Proteins)
20,37
1.0.0
was used to perform both ligand- and structure-based
virtual screening in order to find novel I1-IR ligands with
antiproliferative/proapoptotic activity. The parallel application of
these two VS methods was expected to produce structurally
diverse hits with the desired biological activity.
4.2.2.1. FLAP database generation
During the FLAP database creation, the 3D structures of
compounds from the virtual library were generated using
molecular mechanistic MM3 force fields. The database was
enriched with rilmenidine and 20 I1-IR agonists with a reported
pKi > 6 as an affinity cutoff (Supplementary material). For each
ligand, up to 25 conformers were generated with root-mean-

square deviation (RMSD) lower than 0.3 Å. GRID molecular
interaction fields (MIFs) were calculated for each conformer and
translated into the fingerprints on which all FLAP screening
calculations were based. The GRID probes H, DRY, O, and N1
are used to describe shape, hydrophobic interaction, H-bond
acceptors and H-bond donor interactions of molecules,
respectively. Charge is implicitly described by the magnitude of
the interaction using the N1 and O probes.
150EX ion source. HRMS experiments were performed with
LTQ ORBITRAP XL THERMO.
3.3.1. General procedure for the synthesis of compounds
2a-d:
A mixture of 4-hydroxy-6-methyl-2-pyrone 1 (500 mg, 3.96
mmol) and isobutylamine (394.0 μL, 3.96 mmol, for 2a) or
pentylamine (459.6 μL, 3.96 mmol, for 2b) or isopentylamine
(459.6 μL, 3.96 mmol, for 2c) or benzylamine (432.6 μL, 3.96
mmol, for 2d) in water (16 mL) was stirred under reflux for 12
hours. At the end of the reaction the mixture was cooled to room
temperature. The solid mass was filtrated under vacuum, washed
with diethyl ether and purified by silica gel flash chromatography
(dichloromethane/methanol from 99/1 to 95/5) to give the desired
compounds as white solids.
4.2.2.2. FLAP Virtual Screening Analysis
In LBVS, compounds from the virtual library were ranked
according to their similarity with rilmenidine (ChEMBL289480),
while in SBVS they were ranked according to the active site of
nischarin, taking into account GRID MIFs. Probe scores
(representing the degree of overlap of the MIFs for each probe
individually as well as for their combinations), distance scores
(representing overall difference of probe scores between the
ligand and the template) and two global scores (Glob-Prod and
Glob-Sum) were applied to quantify similarity. All similarities
ranged from 0.0 (bad) to 1.0 (good), except for the distance score,
where 0.0 represented good.38 Since an X-ray crystallography
structure of nischarin was not available, a recently published
homology model was used in this study.16 Automatic
identification of binding pockets of proteins was performed using
the FLAPsite pocket detection algorithm.21
4-hydroxy-1-isobutyl-6-methylpyridin-2(1H)-one
(2a).
1
(90% yield). H NMR (300 MHz, DMSO-d6) δ 10.30 (s, 1H),
6.05 (s, 1H), 5.95 (s, 1H), 3.83-3.82 (m, 2H), 2.34 (s, 3H), 2.21-
2.14 (m, 1H), 0.94 (d, J = 6.5 Hz, 6H). MS (ESI) m/z 182.2 [M +
H]+, 204.3 [M + Na]+.
4-hydroxy-6-methyl-1-pentylpyridin-2(1H)-one
(2b).
(79%yield). 1H NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H), 5.98
(s, 1H), 5.91 (s, 1H), 3.96-3.91 (m, 2H), 2.34 (s, 3H), 1.67-1.62
(m, 2H), 1.37-1.32 (m, 4H), 0.93-0.89 (m, 3H). MS (ESI) m/z
196.4 [M + H]+, 218.4 [M + Na]+.
4.2.2.3. Virtual Screening Evaluation
4-hydroxy-1-isopentyl-6-methylpyridin-2(1H)-one
(2c).
The results of the VS protocol were examined in terms of
enrichment factor (EF) and the area under curve (AUC) of the
receiver operator characteristic (ROC) curve. A ROC curve is a
plot of the true positive rate (y-axis) against the false positive rate
(x-axis) for a ranked series of molecules. AUC of 0.5
corresponds to random discrimination between actives and
untested compounds (decoys), whereas an AUC of 1.0
corresponds to an ideal case, in which all known true actives are
ranked before untested compounds (decoys). EF is a cumulative
plot of the percentage of true positives (y-axis) vs. percentage of
the whole dataset (x-axis) for a ranked series of molecules. The
EFX% indicates the EF values retaining the X% of the whole
database.39
1
(83% yield). H NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H),
5.73 (s, 1H), 5.47 (s, 1H), 3.86-3.80 (m, 2H), 2.29 (s, 3H), 1.65-
1.56 (m, 1H), 1.41-1.34 (m, 2H), 0.91 (d, J = 6.6 Hz, 6H). MS
(ESI) m/z 196.2 [M + H]+, 218.3 [M + Na]+.
1-benzyl-4-hydroxy-6-methylpyridin-2(1H)-one (2d). (89%
yield). 1H NMR (300 MHz, DMSO-d6) δ 10.31 (s, 1H), 7.36-7.26
(m, 5H), 5.54 (s, 1H), 5.47 (s, 1H), 4.25 (d, J= 5.3 Hz, 2H), 2.27
(s, 3H). MS (ESI) m/z 216.2 [M + H]+, 238.3 [M + Na]+.
3.3.2. General procedure for the synthesis of compounds
3a-i:
In a 10 mL microwave tube, equipped with magnetic stir bar and
septum, a mixture of intermediate 2a-d (0.51 mmol) and the
opportune amine (0.77 mmol) in dimethoxyethane (1 mL) was
heated at 120 °C for 40 minutes in the microwave apparatus
(maximum power input: 300 W; maximum pressure: 250 PSI;
power max: OFF; stirring: ON). After cooling to room
temperature the solvent was evaporated under reduced pressure
and the residue was purified by silica gel flash chromatography
(dichloromethane/methanol from 99/1 to 98/2), affording the
desired compounds as white solids.
3.3. Chemistry
All reagents were purchased from Sigma-Aldrich, Alfa-Aesar
and Enamine at reagent purity and, unless otherwise noted, were
used without any further purification. Dry solvents used in the
reactions were obtained by distillation of technical grade
materials over appropriate dehydrating agents. MCRs were
performed using CEM Microwave Synthesizer-Discover model.
Reactions were monitored by thin layer chromatography on silica
gel-coated aluminium foils (silica gel on Al foils, SUPELCO
Analytical, Sigma-Aldrich) at both 254 and 365 nm wavelengths.
Where indicated, intermediates and final products were purified
through silica gel flash chromatography (silica gel, 0.040-0.063
mm), using appropriate solvent mixtures. 1H-NMR were recorded
on a BRUKER AVANCE spectrometer at 400 and 300 MHz,
whereas 13C-NMR spectra were recorded in the same instrument
1-isobutyl-6-methyl-4-((4-
(trifluoromethyl)benzyl)amino)pyridin-2(1H)-one (3a). (29%
1
yield). H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 7.8 Hz, 2H),
7.42 (d, J = 7.8 Hz, 2H), 5.53 (s, 1H), 5.46 (s, 1H), 4.59 (s, 1H),
4.36 (d, J= 5.2 Hz, 2H), 3.76-3.74 (m, 2H), 2.27 (s, 3H), 2.20-
2.13 (m, 1H), 0.94 (d, J = 6.6 Hz, 6H). 13C NMR (100.6 MHz,
CDCl3) δ 164.9, 154.2, 145.6, 142.3, 128.0, 127.5 (2C), 125.7
(2C), 125.6, 99.3, 91.8, 50.2, 46.4, 28.1, 20.9, 20.1 (2C). MS
(ESI) m/z 339.2 [M + H]+, 361.3 [M + Na]+. HRMS (ESI)
calculated for C18H21F3N2O [M + H]+ 339.1606, found
339.17022.
1
at 100.6 MHz. TMS was used as the internal standard. H-NMR
spectra are reported in this order: multiplicity and number of
protons. Standard abbreviations indicating the multiplicity were
used as follows: s = singlet, d = doublet, dd = doublet of
doublets, t = triplet, q = quadruplet, m = multiplet and br = broad
signal. HPLC/MS experiments were performed with an Agilent
1100 series HPLC system, equipped with a Waters Symmetry
C18, 3.5 µm, 4.6 mm x 75 mm column and an Applied
Biosystem/MDS SCIEX MS detector equipped with an API
6-methyl-1-pentyl-4-(pentylamino)pyridin-2(1H)-one (3b).
1
(35%yield). H NMR (300 MHz, CDCl3) δ 5.46 (s, 1H), 5.40 (s,
1H), 4.33-4.32 (m, 1H), 3.88-3.82 (m, 2H), 3.05-2.98 (m, 2H),
2.23 (s, 3H), 1.64-1.51 (m, 4H), 1.34-1.28 (m, 8H), 0.90-0.86 (m,
6H). 13C NMR (75 MHz, CDCl3) δ 164.7, 154.8, 144.5, 99.5,

90.4, 43.4, 42.6, 29.3, 29.1, 28.9, 28.6, 22.4, 22.3, 20.3, 14.0,
13.9. MS (ESI) m/z 265.4 [M + H]+, 287.3 [M + Na]+. HRMS
(ESI) calculated for C16H28N2O [M + H]+ 265.2202, found
265.22899.
5H), 5.54 (s, 1H), 5.51 (s, 1H), 5.24 (d, J= 5.4 Hz, 2H), 4.30 (s,
1H), 3.19-3.07 (m, 2H), 2.12 (s, 3H), 1.67-1.66 (m, 1H), 1.52-
1.46 (m, 2H), 0.94 (d, J = 6.4 Hz, 6H). 13C NMR (100.6 MHz,
CDCl3) δ 165.0, 154.9, 145.4, 137.7, 128.6 (2C), 126.9, 126.3
(2C), 99.8, 90.0, 46.0, 40.9, 37.8, 25.9, 22.5 (2C), 20.5. MS (ESI)
m/z 285.3 [M + H]+, 307.3 [M + Na]+. HRMS (ESI) calculated
for C18H24N2O [M + H]+ 285.1889, found 285.19088.
4-(benzylamino)-6-methyl-1-pentylpyridin-2(1H)-one (3c).
1
(31% yield). H NMR (400 MHz, CDCl3) δ 7.36-7.26 (m, 5H),
5.52 (s, 1H), 5.51 (s, 1H), 4.53 (s, 1H), 4.25 (d, J= 5.4 Hz, 2H),
3.90-3.86 (m, 2H), 2.27 (s, 3H), 1.65-1.63 (m, 2H), 1.36-1.34 (m,
4H), 0.93-0.90 (m, 3H). 13C NMR (100.6 MHz, CDCl3) δ 164.6,
154.5, 144.9, 137.9, 128.8 (2C), 127.6, 127.5 (2C), 99.3, 91.4,
47.0, 43.6, 29.2, 28.8, 22.5, 20.4, 14.0. MS (ESI) m/z 285.2 [M +
H]+, 307.2 [M + Na]+. HRMS (ESI) calculated for C18H24N2O
[M + H]+ 285.1889, found 285.19052.
3.3.3. General procedure for the synthesis of compounds 5
and 7.
Carbazole 4 or benzimidazole 6 (3 mmol) was added at 0 °C to a
suspension of sodium hydride (60% dispersion in mineral oil;
8.97 mmol) in dry DMF (15 mL). Reaction mixture was stirred
for 15 minutes at the same temperature and then 2-(2-
chloroethyl)-piperidine hydrochloride (3.88 mmol) was added
portion wise. The mixture was allowed to react at room
temperature for 48 hours until consumption of the starting
material. After quenching with water (20 ml), the mixture was
extracted with ethyl acetate (3 × 10 mL), and the organic layers
were washed with water and brine (3 × 10 mL), dried over
anhydrous Na2SO4 and concentrated under reduced pressure.
4-((4-chlorobenzyl)amino)-6-methyl-1-pentylpyridin-
2(1H)-one (3d). (32% yield). 1H NMR (400 MHz, CDCl3) δ 7.30
(d, J = 7.8 Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H), 5.51 (s, 1H), 5.43
(s, 1H), 4.57 (s, 1H), 4.21 (d, J= 4.4 Hz, 2H), 3.88-3.84 (m, 2H),
2.27 (s, 3H), 1.65-1.62 (m, 2H), 1.36-1.34 (m, 4H), 0.92-0.89 (m,
3H). 13C NMR (100.6 MHz, CDCl3) δ 164.5, 154.3, 145.0, 136.4,
133.3, 128.9 (2C), 128.7 (2C), 99.3, 91.5, 46.2, 43.6, 29.1, 28.8,
22.5, 20.4, 14.0. MS (ESI) m/z 319.3 [M + H]+, 341.4 [M + Na]+.
HRMS (ESI) calculated for C18H23ClN2O [M + H]+ 319.1499,
found 319.15063.
9-(2-(1-methylpiperidin-2-yl)ethyl)-9H-carbazole (5). The
crude material was purified through silica gel flash
chromatography eluting with dichloromethane/methanol 9/1.
(Yield: 53%). White powder. 1H NMR (CDCl3-400 MHz) δ 1.36-
1.40 (m, 1H), 1.65-1.68 (m, 3H), 1.82-1.85 (m, 2H), 2.07-2.3 (m,
4H), 2.37 (s, 3H), 2.92-2.97 (m, 1H), 4.36-4.48 (m, 2H),
7.24,7.52 (m, 6H), 8.12 (d, J = 8 Hz, 2H). 13C NMR (CDCl3-
100.6 MHz) δ 24.2, 25.3, 30.5, 31.4, 35.5, 39.2, 42.7, 56.9, 61.7,
108.5, 118.8, 120.4, 123.0, 125.7, 140.2. MS (ESI) m/z 293.3 [M
+ H]+, 315.4 [M + Na]+. HRMS (ESI) calculated for C20H24N2
[M + H]+ 293.1939, found 293.20123.
1-isopentyl-4-(isopentylamino)-6-methylpyridin-2(1H)-one
1
(3e). (33% yield). H NMR (400 MHz, CDCl3) δ 5.45 (s, 1H),
5.44 (s, 1H), 3.98 (s, 1H), 3.94-3.90 (m, 2H), 3.09-3.05 (m, 2H),
2.27 (s, 3H), 1.74-1.64 (m, 2H), 1.55-1.45 (m, 4H), 0.97 (d, J =
6.6 Hz, 6H), 0.94 (d, J = 6.6 Hz, 6H). 13C NMR (100.6 MHz,
CDCl3) δ 164.6, 154.6, 144.6, 99.4, 90.7, 42.1, 40.9, 37.9, 37.8,
26.5, 25.9, 22.5 (2C), 22.4 (2C), 20.2. MS (ESI) m/z 265.3 [M +
H]+, 287.2 [M + Na]+. HRMS (ESI) calculated for C16H28N2O
[M + H]+ 265.2202, found 265.22951.
1-(2-(1-methylpiperidin-2-yl)ethyl)-1H-benzo[d]imidazole
(7). The crude material was purified by flash column
chromatography petroleum ether/ethyl acetate 9/1. (Yield: 72%)
Yellow oil. 1H-NMR (CDCl3-300 MHz) δ 1.27-1.77 (m, 6H),
2.02-2.20 (m, 4H), 2.30 (s, 3H), 2.86-2.94 (m, 1H), 4.15- 4.36
(m, 2H), 7.20-7.34 (m, 2H), 7.39-7.44 (m, 1H), 7.80- 7.85 (m,
1H), 7.93 (s, 1H). 13C-NMR (CDCl3-100.6 MHz) δ 24.2, 25.1,
30.2, 32.9, 41.3, 42.5, 56.7, 61.1, 64.4, 88.8, 109.6, 120.4, 122.1,
122.9, 142.8. MS (ESI) m/z 244.2 [M + H]+, 266.3 [M + Na]+.
HRMS (ESI) calculated for C15H21N3 [M + H]+ 244.1735,
found 244.18082.
4-(benzylamino)-1-isopentyl-6-methylpyridin-2(1H)-one
(3f). (28% yield). H NMR (400 MHz, CDCl3) δ 7.35-7.26 (m,
1
5H), 5.52 (s, 1H), 5.49 (s, 1H), 4.61 (s, 1H), 4.24 (d, J= 5.4 Hz,
2H), 3.92-3.88 (m, 2H), 2.26 (s, 3H), 1.73-1.64 (m, 1H), 1.54-
1.48 (m, 2H), 0.97 (d, J = 6.6 Hz, 6H). 13C NMR (100.6 MHz,
CDCl3) δ 164.5, 154.6, 144.8, 137.9, 128.7 (2C), 128.5, 127.5
(2C), 99.4, 91.3, 46.9, 42.1, 37.8, 26.5, 22.5 (2C), 20.3. MS (ESI)
m/z 285.3 [M + H]+, 307.3 [M + Na]+. HRMS (ESI) calculated
for C18H24N2O [M + H]+ 285.1889, found 285.19034.
4-((4-chlorobenzyl)amino)-1-isopentyl-6-methylpyridin-
1
2(1H)-one (3g). (33% yield). H NMR (400 MHz, CDCl3) δ
3.4. α2-Adrenoceptor assays
7.32-7.22 (m, 4H), 5.52 (s, 1H), 5.48 (s, 1H), 4.55 (s, 1H), 4.24
(d, J= 5.4 Hz, 2H), 3.93-3.89 (m, 2H), 2.28 (s, 3H), 1.73-1.67 (m,
1H), 1.55-1.49 (m, 2H), 0.97 (d, J = 6.6 Hz, 6H). 13C NMR
(100.6 MHz, CDCl3) δ 164.4, 154.3, 144.9, 136.4, 133.3, 128.9
(2C), 128.7 (2C), 99.3, 91.5, 46.2, 42.2, 37.8, 26.5, 22.5 (2C),
20.3. MS (ESI) m/z 319.3 [M + H]+, 341.3 [M + Na]+. HRMS
(ESI) calculated for C18H23ClN2O [M + H]+ 319.1499, found
319.15082.
3.4.1. Cell culture
Recombinant Chinese hamster ovary (CHO) cells (K1 strain)
(American Type Culture Collection (ATCC), Manassas, VA,
USA), stably expressing the cDNA encoding the human α2A-
adrenoceptor subtype, were produced as previously described by
Pohjanoksa et al.40 Cells were cultured in α-minimum essential
medium (GIBCOTM
,
Invitrogen, Carlsbad, CA, USA)
1-benzyl-6-methyl-4-(pentylamino)pyridin-2(1H)-one (3h).
(31% yield). H NMR (400 MHz, CDCl3) δ 7.27-7.14 (m, 5H),
supplemented with 26 mM NaHCO3, 5% heat-inactivated fetal
bovine serum (FBS), penicillin (50 IU/ml), streptomycin (50
µg/ml) and 200 µg/ml G418. The cultured cells were tested for
their capacity to bind the α2-adrenoceptor antagonist radioligand
[3H]RS-79948-197 (GE Healthcare, London, U.K.). Confluent
cells were harvested into chilled phosphate-buffered saline,
pelleted and frozen at –70 °C.
1
5.54 (s, 1H), 5.49 (s, 1H), 5.25 (d, J= 5.4 Hz, 2H), 4.30 (s, 1H),
3.10-3.05 (m, 2H), 2.12 (s, 3H), 1.61-1.59 (m, 2H), 1.34-1.27 (m,
4H), 0.93-0.89 (m, 3H). 13C NMR (100.6 MHz, CDCl3) δ 165.0,
155.0, 145.3, 137.7, 128.6 (2C), 126.9, 126.3 (2C), 99.8, 90.0,
46.0, 42.7, 29.2, 28.6, 22.4, 20.5, 14.0. MS (ESI) m/z 285.3 [M +
H]+, 307.2 [M + Na]+. HRMS (ESI) calculated for C18H24N2O
[M + H]+ 285.1889, found 285.19074.
3.4.2. Membrane preparation
1-benzyl-4-(isopentylamino)-6-methylpyridin-2(1H)-one
(3i). (36% yield). H NMR (400 MHz, CDCl3) δ 7.30-7.13 (m,
All procedures were performed on ice. CHO cell pellets were
thawed and suspended in hypotonic lysis buffer (10 mM Tris–
1

HCl, 0.1 mM EDTA, 0.32 mM sucrose, pH 7.4) and
homogenised using an Ultra-Turrax homogeniser (3 × 10 s at
8000 rpm). The homogenate was centrifuged at 180 g for 15 min
to remove cell nuclei, unbroken cells and aggregates. The
supernatants were pooled and centrifuged at 50,200 g for 30 min.
The pellet was washed with TE buffer (10 mM Tris, 0.1 mM
EDTA) and re-centrifuged as above. The membranes were then
suspended in TE buffer, aliquoted and stored at –70 °C until
used. Protein concentrations were determined with the method of
Bradford41 using bovine serum albumin as reference.
3.5.2. Cytotoxicity assay and combination index (CI)
The cytotoxicity of compounds on K562 cells were measured
using
the
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay developed by Mosmann44
and modified by Ohno and Abe.45 After treatment in 96-well
plates, 20 µL MTT solution was added to each well. Samples
were incubated for a further 4 h, followed by the addition of 100
μl of 10% SDS. Absorbance at 570 nm was measured on the
following day. To quantify cell survival (S%), the absorbance of
a sample with cells grown in the presence of different
concentrations of the investigated agents was divided by the
absorbance of the control cells grown only in the nutrient
medium, and multiplied by 100. The absorbance of the blank was
subtracted from the corresponding absorbance of samples with
target cells. The IC50 value is the concentration of the agent that
inhibited cell survival by 50%, compared to the vehicle-treated
control. IC50 was calculated as described in 46. As a positive
control we used rilmenidine hemifumarate salt (prod. no. R134)
and as a negative control, efaroxan hydrochloride (prod. no.
E3263) (Sigma-Aldrich). To assess the cytotoxic effects of drug
combinations, we calculated CI according to the Chou-Talalay
method29 using CalcuSyn software (Biosoft, Cambridge, UK). CI
allows for quantitative definition of drug interactions;
antagonism (CI>1), additive effect (CI = 1) and synergism
(CI<1).
3.4.3.
[35S]GTPγS binding assay
Agonist-induced stimulation of [35S]GTPγS binding was
measured essentially as described previously.42 Briefly,
membranes were thawed and diluted with binding buffer (25 mM
Tris, 1 mM EDTA, 5 mM MgCl2, 20 mM NaCl, 1 μM GDP, 1
mM DTT, 30 μM ascorbic acid, pH 7.4). Incubations were
performed on 96-well Millipore MultiScreen MSFBN glass-fibre
filter plates. Samples containing 5 μg of membrane protein were
incubated with 7 or 8 serial dilutions of the test compounds and
0.1 nM [35S]GTPγS (PerkinElmer, Boston, MA, USA). Reactions
were terminated after 30 min incubation at RT by rapid vacuum
filtration using a Millipore MultiScreen Vacuum Manifold. The
filter plates were washed three times with ice-cold wash buffer
(20 mM Tris, 1 mM EDTA, 5 mM MgCl2, pH 7.4). Filters were
dried and 50 μl SuperMix scintillation cocktail was added into
each well. The incorporated radioactivity was measured using a
Wallac 1450 Betaplate scintillation counter. All experiments
were performed in duplicate and repeated at least three times.
Analysis of the results with GraphPad Prism software yielded
estimates of agonist potency (EC50) and efficacy (intrinsic
activity in comparison to the natural full agonist adrenaline).
3.5.3. Apoptotic assay
Apoptotic rates were assessed by flow cytometry using the
annexin V–fluorescein isothiocyanate (annexin
V
-
FITC)/propidium iodide (PI) kit (BD Pharmingen), in which
annexin V binds to exposed phosphatidylserines of early
apoptotic cells, whereas PI stains cells that have increased
membrane permeability, i.e., late apoptotic or necrotic cells.
Samples were prepared according to the manufacturer’s
instructions. Flow cytometry analysis was performed using a
FACS-Calibur cytometer (Becton Dickinson). The annexin
V+/PI- cells were defined as early apoptotic cells.
3.4.4.
Competition binding assays
Competition binding assays were implemented using a
MultiScreen Vacuum Manifold system (Millipore) with Millipore
MultiScreen MSFBN 96-well glass fibre filtration plates. The
experiments were performed in a total assay volume of 180 µl (in
50 mM potassium phosphate buffer, pH 7.4) using 0.2 nM
3.5.4. Flow-cytometric analysis of cell phase distribution
[3H]RS-79948-197
((8aR,12aS,13aS)-
Quantitative analysis of cell cycle phase distribution was
performed by flow-cytometric analysis of the DNA content in
fixed of K562 cells, after staining with PI (Sigma-Aldrich).47
Cells in an exponential growth phase, at a density of 2 x 105
cells/Petri dish (dimensions 60 x 15 mm, NUNC) were
continually exposed to investigated hit compounds at
concentrations of 70 µM. After indicated times of continual
treatment, cells were collected by trypsinization, washed twice
with ice-cold PBS, and fixed for 30 min in 70% EtOH. Fixed
cells were washed again with PBS, and incubated with RNase A
(1 mg/mL) for 30 min at 37°C. Just before flow-cytometric
analysis, the cells were stained with PI at a concentration of 400
µg/mL. Cell cycle phase distributions were analyzed using a
FACS Calibur Becton Dickinson flow cytometer and Cell Quest
computer software.
5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12-
(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine;
GE
Healthcare, London, U.K.), 8 serial dilutions of the competitor
ligands and crude cell membrane preparations containing 10 µg
of protein per sample. Non-specific binding was determined in
parallel wells in the presence of 100 µM oxymetazoline. Bound
radioactivity was measured with a Wallac 1450 MicroBeta
scintillation counter (PerkinElmer Wallac). All experiments were
performed in duplicate and repeated at least three times. The
apparent affinity (apparent Ki) of each ligand was determined
using nonlinear regression analysis (GraphPad Prism), assuming
one-site binding. For conversion of IC50 estimates to Ki values,
the Cheng-Prusoff equation was applied.43
3.5. Evaluation of antiproliferative and proapoptotic activity
3.5.1.
Cell culture
3.5.5. Clonogenic Assay for Proliferation Ability
Human chronic myelogenous leukemia K562 cells were
obtained from ATCC. Cells were maintained in RPMI-1640
medium (Gibco, Grand Island, NY, USA) with 10% heat-
inactivated fetal calf serum (FCS, Gibco) at 37°C in 5% CO2.
Nutrient medium RPMI 1640 was prepared in sterile deionized
water, supplemented with penicillin (192 IU/mL), streptomycin
(200 µg/mL), 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic
acid (HEPES) (25 mM), L-glutamine (3 mM) and 10% of heat-
inactivated FCS (pH 7.2).
After 24 h treatment of K562 cells with compound 5 the cells
were washed with PBS, and resuspended in Cell Resuspension
Solution (R&D Systems). Briefly, 0.5 × 104/mL treated K562
cells were mixed with 1 mL of Human methylcellulose complete
medium (R&D Systems). Cells were then plated in a 35 mm
plastic dish (Falcon). The blast colonies (> 50 cells) of K562
cells were scored on day 7. All assays were performed in
duplicate.

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Acknowledgments
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This project was supported by the Ministry of Education,
Science and Technological Development of the Republic of
Serbia, Contract #173001 and # 172033. This work was also
partially supported by the University of Parma (to MR) and by
Turku University Hospital (to MS). ST is supported by a
fellowship from the Chiesi Foundation. The COST Actions
CM1207 is also kindly acknowledged for supporting the short
term scientific mission of JV to MR laboratories. Dr. Emmanuele
Crespan and Dr. Jorge I. Armijos Rivera are kindly
acknowledged for the enzymatic screening on two unrelated
targets: the oncogenic Src and Abl kinases.
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Supplementary Material
Supplemental information entails: (1) I1-IR ligands used in VS
study and corresponding binding affinities (pKi = log(1/Ki)),
(2) 1H-NMR spectra, (3) Evaluation of proapoptotic activities of
moxonidine and efaroxan and (4) Enzymatic assays on isolated
Src and Abl kinases.
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HIGHLIGHTS
Identification of rilmenidine-derived compounds with antitumor activity
Combined VS singled out 11 candidates that were synthesized and in vitro tested
Examined compounds haven’t shown any significant activity on α2A-
adrenoceptors
The most active compound 5 exhibited a cytotoxic profile similar to rilmenidine
Compound 5 combined with doxorubicin demonstrated synergism in evoking
apoptosis