One drug for two targets: Biological evaluation of antiretroviral agents endowed with antiproliferative activity

Article abstract of DOI:10.1016/j.bmcl.2017.03.097

AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not been established yet, but a weakened immune system is certa

Full text of DOI:10.1016/j.bmcl.2017.03.097

Bioorganic & Medicinal Chemistry Letters 27 (2017) 2502–2505
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
One drug for two targets: Biological evaluation of antiretroviral agents
endowed with antiproliferative activity
b
b
b
b
Lorenzo Botta a, , Giorgio Maccari , Pierpaolo Calandro , Marika Tiberi , Annalaura Brai ,
⇑
Claudio Zamperini b, Filippo Canducci c, Mario Chiariello d, Rosa Martí-Centelles e, Eva Falomir e,
Miguel Carda e,
⇑
a Dipartimento di Farmacia, Università Federico II di Napoli, Via D. Montesano 49, 80131 Napoli, Italy
b Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, via Aldo Moro 2, 53100 Siena, Italy
c Department of Clinical and Experimental Medicine, Università degli Studi dell’Insubria, Varese, Italy
d Istituto Toscano Tumori (ITT), Core Research Laboratory (CRL), AOU Senese, Siena, Italy
e Departamento de Química Inorgánica y Orgánica, Universidad Jaume I, E-12071 Castellón, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly
subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not
been established yet, but a weakened immune system is certainly the main cause. We envisaged the pos-
sibility to screen a small library of compounds synthesized in our laboratory against opportunistic tumors
mainly due to HIV infection like Burkitt’s Lymphoma. From cellular assays and gene expression analysis
we identified two promising compounds. These derivatives have the dual action required inhibiting HIV
replication in human TZM-bl cells infected with HIV-1 NL4.3 and showing cytotoxic activity on human
colon HT-29 and breast adenocarcinoma MCF-7 cells. In addition, preclinical in vitro adsorption, distribu-
tion, metabolism, and excretion studies highlighted a satisfactory pharmacokinetic profile.
Ó 2017 Elsevier Ltd. All rights reserved.
Received 4 February 2017
Revised 30 March 2017
Accepted 31 March 2017
Available online 1 April 2017
Keywords:
HIV
Cancer
c-Myc
hTERT
Burkitt’s Lymphoma
Kaposi’s Sarcoma (KS), Hodgkin’s Lymphoma (HL) and Burkitt’s
Lymphoma (BL), are orphan AIDS-related cancers diseases.1 These
cancers are rare among the healthy population but have high inci-
dence in immune-compromised patients (e.g. persons infected by
HIV).2 The current therapy for KS, BL and HL still require the use
of surgery, debilitating radiotherapy, and anticancer compounds
such as TaxolÒ and Doxorubicin for which heavy side effects are
well known.3 Moreover, the treatment of these cancers is compli-
cated by the concomitant intake of antiretroviral drugs.
Fig. 1. General structures of the compounds of the library.
The development of a preclinical drug candidates active both
against rare AIDS-related cancers and against HIV replication
(one drug for two targets) will reduce the total number of drugs
that a patient should assume. Moreover the use of a drug with a
dual mode of action has also a clear benefit in terms of possible
side effects and multi-drug interactions.4
Recently, we reported the discovery and synthesis of a series of
compounds endowed with anti-retroviral activity which proved to
be strongly active against HIV virus.5 The general structure of these
compounds is shown in Fig. 1 (general structure A). The antiretro-
viral mode of action of these molecules resides in the inhibition of
the gp120-CD4 protein-protein interaction, detrimental for the
entry process of the viral infection. A block at this stage of the
infection results in the inability for the virus to infect healthy cells.
Two compounds of that series (compounds 3 and 4, Fig. 2), com-
bined with another small library synthesized in our laboratory
(general structures B and C, Fig. 1), were screened against human
MCF-7 breast cancer cells, HT-29 colon cancer cells and Ramos
Human Caucasian Burkitt’s Lymphoma cells.
To obtain compounds with general structure A we used a one
pot microwave assisted multicomponent reaction protocol
(Scheme 1).6,7 This microwave-assisted procedure allowed us to
obtain, in short reaction time and easy workup, different final
⇑
Corresponding authors.
E-mail address: lorenzo.botta@unina.it (L. Botta).
http://dx.doi.org/10.1016/j.bmcl.2017.03.097
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

L. Botta et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2502–2505
2503
colon HT-29, the breast adenocarcinoma MCF-7 and the Human
Caucasian Burkitt’s Lymphoma Ramos, as well as two non-tumoral
cell lines, the human embryonic kidney cell line HEK-293 and the
bovine aortic endothelial cells BAE.9 Table 1 shows the cytotoxicity
values for compounds 1–7, expressed as the compound concentra-
tion (mM) that causes 50% inhibition of cell growth (IC50).
Table 1further shows the selectivity indexes, named here as
a, b,
c
, d, and f obtained by dividing the IC50 values of the non-tumoral
e
cell lines HEK-293 or BAE by those of HT-29, MCF-7 or Ramos cell
lines respectively (see footnote in Table 1). The higher the value of
either coefficients, the higher the therapeutic safety margin of the
compound in the corresponding cell line.
The cytotoxicity of compounds 1, 3, 4, 6 and 7 is in the low
micromolar range. Regarding the HT-29 line, compounds 1 and 4
showed promising inhibitory activity and as a consequence high
a
and c values. For MCF-7 cells, compounds 6 and 7 showed the
lowest IC50 values, having b and d values reasonably high. More-
over, compounds 1, 4, 6, 7 showed good IC50 values on Ramos cell
line. Among all these compounds, derivatives 1 and 4 are the ones
that combine high cytotoxicity towards HT-29 cell line, acceptable
inhibitory activity against MCF-7 and Ramos and low cytotoxicity
towards non-tumoral cell lines HEK-293 and BAE.
Moreover, all these compounds were further investigated for
the inhibition of gene expressions. Most of the viruses responsi-
ble for the AIDS-related cancers aforementioned, in fact, promote
the up-regulation of proto-oncogenes like c-Myc and genes like
hTERT.10 These two genes are overexpressed as a result of the
infection from oncoviruses like the Epstein Barr virus (EBV)
and the human virus 8 (HHV-8), responsible for BL, HL and
KS.11 To study the ability of our compounds to inhibit hTERT
and c-Myc gene expression, HT-29 cells were incubated with a
non-cytotoxic concentration of each compound (concentration
lower than their IC50 value), then the RNA was extracted and
retrotranscripted to cDNA to quantify the amount of gene
expressed.
In order to determine whether the synthesized compounds
were able to downregulate the expression of hTERT and c-Myc
genes, we have performed a reverse transcription quantitative
PCR (RT-qPCR) analysis using HT-29 tumoral cells (see Experimen-
tal Section).12
For these measurements compounds 2 and 5 were not selected
because they were not cytotoxic towards HT-29 cells (IC50 values
higher than 100 mM). In these assays, concentrations lower than
the IC50 values towards the HT-29 cell line were used. Accordingly,
concentrations were always 1 mM except for compound 6, which
was used at a concentration of 5 mM, and compound 3, which
was used at a concentration of 20 mM, because of their lower cyto-
toxicity on HT-29 cells.
Fig. 2. Structure of compounds synthesized and tested.
products by changing only the amine employed. To generate com-
pound 1 morpholine was used as secondary amine whilst 2-(3,4-
dichlorophenyl)ethanamine, 2-(4-ethylphenyl)ethanamine and 3-
chloro-4-fluoroaniline were used as primary amines to generate
compounds 2–4, respectively.
The other series of substituted rhodanine derivatives with gen-
eral structures B (compound 5) and C (compounds 6 and 7) were
obtained by exploiting a practical and rapid procedure developed
by us and showed in Scheme 2.8 This methodology consists of a
sequential, one-pot, two-steps microwave-assisted process with
the formation of the desired final compounds in few minutes and
high purity (Scheme 2).
All the synthesized compounds were tested in vitro to evaluate
their ability to inhibit HIV replication in human TZM-bl cells
infected with HIV-1 NL4.3 (a CXCR4-tropic strain) and their biolog-
ical results are listed in Table 1. All these derivatives showed
antiviral activity in the low micromolar range, with compounds 3
and 4 being the more potent of the series (see Table 1).5 Further-
more, we carried out a measurement of the cytotoxic activity of
our synthetic compounds 1–7 using three tumoral cells, the human
Results for the selected compounds are depicted in Fig. 3 which
shows the percentage of hTERT gene expression after 48 h of incu-
bation in the presence of DMSO (control experiment) and in the
presence of each of the compounds investigated at a concentration
of 1 mM (lower than their IC50 values). All values were standardized
(100%) to control (DMSO) and to b-actin.
Scheme 1. Synthesis of derivatives 1–4 (a) 1 M NaOH aq., THF/MeOH, reflux, 2 h
(99%). (b) 2-thioxothiazolidin-4-one 10, amine RNHR1, EtOH, MW (300 W), 150 °C,
20 min. Yields: 1 (49%); 2 (39%); 3 (80%); 4 (54%).
It is worth noting that all selected compounds are able to signif-
icantly downregulate hTERT gene expression around 50%.
The most remarkable compound is 7, which has the strongest
inhibitory activity on the hTERT gene expression, downregulating
hTERT gene expression to 37%.
In order to determine whether the studied compounds were
able to regulate the expression of the c-Myc gene, we have per-
formed a RT-qPCR analysis using again HT-29 tumoral cells. The
cells were incubated for 48 h in the presence of DMSO (control)
and, as above, 1 mM of each of the studied compounds (for 6,
5 mM and for 3, 20 mM) were used. Results, standardized (100%)
to the control (DMSO) and to b-actin, are depicted in Fig. 4.
Scheme 2. Synthesis of derivatives 5–7 (a) DME, Et3N, MW (300 W), 90 °C, 10 min.
(b) aldehyde 9 or 13, MW (300 W), 110 °C, 5 min. Yields: 5 (29%); 6 (18%); 7 (54%).

2504
L. Botta et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2502–2505
Table 1
IC50 values (mM), LD50 values (mM) and selectivity indexes for compounds 1–7.a
Comp.
NL4.3
TZM-blb
HT-29
MCF-7
21
>100
4,5 1,6
Ramos
HEK-293
53
>100
3,4 0,4
12,0 0,8
BAE
76
>100
20
25
86 10
2,4 0,5
2,2 0,5
–
ac
bd
ce
df
eg
fh
1
2
3
4
5
6
7
RAL
RTV
13.4 6.4
1.8 0.4
>50
>50
>50
>50
>50
>50
>50
>50
–
2,3 1,0
>100
3
3,14
1
3
9
23,04
–
2,52
–
33,04
–
3,62
–
16,9
–
24,2
–
>100
1
1
0.3
0.8
26
3
37
2
4
4
0,13
10,91
<0,29
0,51
0,52
–
0,76
0,92
<0,29
3,72
2,00
–
0,77
22,72
<0,86
0,29
0,48
–
4,44
1,92
<0,86
2,12
1,83
–
0,09
3,9
<0,29
1,1
0,59
–
0,54
8,0
<0,86
0,63
0,53
–
1,1 0,6
>100
8,3 1,9
4,6 1,8
–
13
7
3,1 0,7
>100
3,8 1,1
4,1 0,5
–
4.8 0.7
6.9 1.2
17.3 3.8
0.021
>100
29
7
1,13 0,21
1,2 0,4
–
4,2 2,3
2,4 0,9
–
–
137
8
34
6
37
5
17.0 2.6
57
5
–
–
–
–
–
–
aValues are the average ( s.d.) of three different measurements performed as described in the Experimental Section. b50% lethal dose LD50
.
a
c = IC50 (HEK-293)/IC50 (HT-29).
, b, and d
b
d = IC50 (HEK-293)/IC50 (MCF-7). e = IC50 (BAE)/IC50 (HT-29). df = IC50 (BAE)/IC50 (MCF-7). g = IC50 (HEK-293)/IC50 (Ramos). fh IC50 (BAE)/IC50 (Ramos). Values of
c
e
a
c
have been rounded off to a decimal figure. With RAL and RSV are indicated the positive controls Raltegravir and Resveratrol respectively.
120
100
100
80
53
56
51
53
60
40
20
0
37
7
28
DMSO
1
3
4
6
RSV
Fig. 3. Expression percentage of the hTERT gene after 48 h of incubation of HT-29
cells determined by means of the RT-qPCR methodology. At least three measure-
ments were performed in each case. Concentration of all compounds was 1 mM (for
3, 20 mM and for 6, 5 mM). With RSV is indicated the positive control Resveratrol.
Error bars indicate standard errors of the mean. The statistical significance was
evaluated using one-sample t-tests (P < 0.001).
Fig. 5. Percentage of hTERT gene expression vs. percentage of c-Myc gene
expression.
100
120
100
80
60
40
20
0
100
69
60
60
67
tion levels of hTERT and c-Myc gene expressions suggests that these
compounds are able to downregulate hTERT gene expression
through downregulation of c-Myc transcription factor gene expres-
sion. Compound 1 and 4, at a concentration of 1 mM, and compound
3, at 20 mM, are able to decrease both gene expressions approxi-
mately at the same level. Among them, it is important to note
the activities showed by compounds 1 and 4, which are capable
to downregulate simultaneously hTERT and c-Myc gene expression
around 50–60% with a concentration of 1 mM.
47
DMSO
1
3
4
6
7
RSV
The administration of a sub-toxic concentration of compounds
sensibly reduced the expression of the protooncogene c-Myc and
of the gene hTERT (Fig. 5).13 These results clearly showed the
antiproliferative effects of this small library of compounds espe-
cially of the two derivatives 1 and 4.
We initiated early preclinical in vitro adsorption, distribution,
metabolism, and excretion (ADME) studies with hit compounds 1
and 4 to determine their aqueous solubility, parallel artificial
membrane permeability (PAMPA), and human liver microsomes
(HLM) stability in order to early assess the absorption/stability
profiles of these drug candidates. Passive membrane permeability
was evaluated with the PAMPA assay, while compound solubility
was evaluated according to the method developed by Avdeef.14
Metabolic stability was finally evaluated by incubating the
Fig. 4. Expression percentage of the c-Myc gene after 48 h of incubation of HT-29
cells determined by means of the RT-qPCR methodology. At least three measure-
ments were performed in each case. Concentration of all compounds was 1 lM (for
6, 5 mM and for 3, 20 mM). With RSV is indicated the positive control Resveratrol.
Error bars indicate standard errors of the mean. The statistical significance was
evaluated using one-sample t-tests (P < 0.001).
Compounds 1 and 4 showed the highest inhibitory effect on the
expression of the c-Myc gene, downregulating c-Myc gene expres-
sion to 60%. In addition, compounds 3 and 7 displayed a similar
ability to inhibit the expression of the c-Myc gene (reduction to
67% and 69% of the control value, respectively). In contrast, com-
pound 6 showed no significant decrease in c-Myc gene expression.
In Fig. 5, values of the percentage of hTERT gene expression are
graphically represented versus the percentage of c-Myc gene
expression. Compounds with approximately the same inhibition
levels on both biological targets (hTERT and c-Myc gene expres-
sions) are located in the diagonal. This correlation between inhibi-
above mentioned compounds with 5 lL of human pooled HLM
for 1 h at 37 °C in order to simulate phase I metabolism.
The ADME properties of the two hit compounds 1 and 4 ana-
lyzed by means of PAMPA and HLM are shown in Table 2.15 These

L. Botta et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2502–2505
2505
Table 2
In summary, compound 4 is the most promising of the series
showing dual activity against cancer and HIV-1 infected cell lines,
and being able to downregulate hTERT and c-Myc gene expressions.
It represents a good starting point for future studies aimed at the
identification of the mode of action of this series of compounds
and also at an optimization campaign in order to increase the inhi-
bition activity and the ADME properties, especially the membrane
permeability.
ADME profile of compounds 1 and 4.
Comp.
Apparent permeability
(cm/s)
Solubility
Metabolic
stability (%)
in H2O (
l
M)
1
4
1.28 Á 10À6
1.58 Á 10À6
<0.1 Á 10À6
9.68 Á 10À6
–
9.7
11.8
–
–
–
98.7
99.1
–
–
76.2
Caffeinea
Propanololb
Testosteronec
a
b
c
Tested as reference compound for low permeability.
Tested as reference compound for high permeability.
Tested as reference compound for CYP-dependent metabolism in human liver
Acknowledgments
Funding by the Ministerio de Economía y Competitividad (pro-
ject CTQ2014-52949-P) and by the Universitat Jaume I (project PI-
1B2015-75) is gratefully acknowledged. R. M.-C. thanks the Univer-
sity Jaume I for a research stay fellowship at the University of Siena
(grant E-2013-09).
microsomes.
two molecules showed good metabolic stability in HLM assay, low
water solubility and scarce permeability in PAMPA test. However,
it has been reported that salicylic acid derivatives might pass
through cellular membranes by active transport; thus, further
experiments in this direction are necessary to better characterize
the permeability profiles of these compounds.16 According to the
obtained results, compound 4 is the most interesting because it
shows higher permeability, solubility and metabolic stability than
compound 1.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.03.
097.
In conclusion, a small library of compounds synthesized in our
laboratory exhibited a dual activity against cancer and HIV infected
cell lines inhibiting TZM-bl cells infected with HIV-1 NL4.3 in vitro
and HT-29, MCF-7 cancer cell lines. These derivatives were also
active against opportunistic tumor, due to an immunocompro-
mised state and closely associated with virus infections like Bur-
kitt’s Lymphoma as shown by the data on activity against Ramos
cells in Table 1.
The origin of this antiviral and antiproliferative activity is not
well understood but from hTERT and c-Myc gene expression evalu-
ations, downregulation of the expression of these two genes
emerged. The most active compounds were derivatives 1 and 4,
because they were able to simultaneously reduce the hTERT and
c-Myc gene expression at a concentration of 1 mM. Derivative 7
could be also considered an active compound, as it was able to
reduce the expression of the hTERT, and partially of c-Myc gene,
References
1. Boshoff C, Weiss R. Nat Rev Cancer. 2002;2:373–382.
2. Cobuccia RN, Lima PH, Carvalho de Souza P, et al. Inf Pub Health. 2015;8:1–10.
3. Berretta M, Cinelli R, Martellotta F, Spina M, Vaccher E, Tirelli U. Oncogene.
2003;22:6646–6659.
4. Monzon JG, Dancey J. Combination agents versus multitargeted agents– pros
and cons. In: Morphy JR, Harris CJ, eds. Designing Multi-Target
Drugs. Cambridge: RSC Publishing; 2012:155–181.
5. Tiberi M, Tintori C, Ceresola ER, et al. Antimicrob Agents Chemother.
2014;58:3043–3052.
6. Anderluh M, Jukic M, Petric R. Tetrahedron. 2009;65:344–350.
7. Rinaldi M, Tintori C, Franchi L, et al. Chem Med Chem. 2011;6:343–352.
8. Radi M, Botta L, Casaluce G, Bernardini M, Botta M.
2010;12:200–205.
9. Arden N, Betenbaugh MJ. Trends Biotechnol. 2004;22:174–180.
J Comb Chem.
10. Nicot Marcia Bellon Christophe. J Natl Cancer Inst. 2008;100:98–108.
11. Ahuja R, Jamal A, Nosrati N, et al. Curr Sci. 2014;107:768–785.
12. Bustin SA, Benes V, Garson JA, et al. Clin Chem. 2009;55:611–622.
13. Latil A, Vidaud D, Valéri A, et al. Int J Cancer. 2000;89:172–176.
14. Avdeef A. Curr Top Med Chem. 2001;1:277–351.
15. Radi M, Dreassi E, Brullo C, et al. J Med Chem. 2011;54:2610–2626.
16. Koljonen M, Rousu K, Cierny J, Kaukonen AM, Hirvonen J. Eur J Pharm Biopharm.
2008;70:531–538.
at 1 lM concentration. The ability of reducing the c-Myc gene
expression could also explain the high cytotoxic activity of these
compounds on Ramos cells, which showed an overexpression of
this gene.17 Moreover, compounds 1 and 4 showed high metabolic
stability as revealed by human liver microsomes (HLM) assay.
17. Habel ME, Jung D. Biochem Biophys Res Commun. 2006;341:1309–1316.