Modulation of cell differentiation, proliferation, and tumor growth by dihydrobenzyloxopyrimidine non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm200734j

A series of 5-alkyl-2-(alkylthio)-6-(1-(2,6-difluorophenyl)propyl)-3,4- dihydropyrimidin-4(3H)-one derivatives (3a-h) belonging to the F ₂-DABOs class of non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) are endowed with a strong a

Full text of DOI:10.1021/jm200734j

ARTICLE
pubs.acs.org/jmc
Modulation of Cell Differentiation, Proliferation, and Tumor Growth
by Dihydrobenzyloxopyrimidine Non-Nucleoside Reverse
Transcriptase Inhibitors
Gianluca Sbardella,*,† Antonello Mai,*,‡ Sara Bartolini,§ Sabrina Castellano,† Roberto Cirilli,§ Dante Rotili,‡
Ciro Milite,† Marisabella Santoriello,† Serena Orlando,|| Ilaria Sciamanna,§ Annalucia Serafino,^
Patrizia Lavia,|| and Corrado Spadafora§
†Dipartimento di Scienze Farmaceutiche e Biomediche, Epigenetic Med Chem Lab, Universitꢀa degli Studi di Salerno,
Via Ponte Don Melillo, I-84084 Fisciano (SA), Italy
‡Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universitꢀa di Roma,
P.le A. Moro 5, I-00185 Rome, Italy
§Istituto Superiore di Sanitꢀa, Viale Regina Elena 299, I-00161 Rome, Italy
Istituto di Biologia Molecolare e Patologia, CNR, c/o Sapienza Universitꢀa di Roma, Via degli Apuli 4, I-00185 Rome, Italy
^Istituto di Farmacologia Traslazionale, CNR, Via Fosso del Cavaliere 100, 00133, Rome, Italy
S Supporting Information
b
ABSTRACT: A series of 5-alkyl-2-(alkylthio)-6-(1-(2,6-di-
fluorophenyl)propyl)-3,4-dihydropyrimidin-4(3H)-one deri-
vatives (3aꢀh) belonging to the F2-DABOs class of non-
nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs)
are endowed with a strong antiproliferative effect and induce
cytodifferentiation in A375 melanoma cells. Among tested
compounds, the most potent is 3g (SPV122), which also
induces apoptosis in a cell-density-dependent manner and
antagonizes tumor growth in animal models. All these effects
are similar or even more pronounced than those previously reported for other nucleoside or non-nucleoside inhibitors of reverse
transcriptase or by functional knockout of the reverse-transcriptase-encoding long interspersed element 1 by RNA interference
(RNAi). Taken together with our previously reported results, these data further confirm our idea that cellular alterations induced by
NNRTIs are a consequence of the inhibition of the endogenous reverse transcriptase in A375 cells and support the potential of
NNRTIs as valuable agents in cancer therapy.
’ INTRODUCTION
retrotransposition machinery provided by LINE.2,5,6 The RT-
dependent mechanism plays an essential catalytic role in retro-
transposition and determines the successful progressive spread-
ing of retrotransposon copies in the genomes of a variety of
species. Interestingly, such copy number amplification is con-
sistent with the expansion of evolutionary complexity.7,8
Retrotransposons have traditionally been considered as use-
less parasitic elements.9,10 It is now well established, however,
that their expression is carefully modulated, being very low in
nonpathological differentiated tissues11,12 and significantly up-
regulated in embryonic13ꢀ15 and transformed cells and tissues.16ꢀ18
It is worth mentioning that high reverse transcriptase titers are
found in the plasma of patients with lymphoma and breast
cancer, which drop dramatically after cancer treatment.19,20
In past studies, we reported that inhibiting the endogenous RT
has striking effects on the biology of transformed and tumor cells:
A surprising finding emerging after completion of the human
genome sequence is the tiny proportion of the protein-coding
gene component, which accounts for a mere 1.2% of the entire
genome while the rest of the genome is devoid of conventional
protein-coding functions.1ꢀ3 A much larger proportion, repre-
senting about 45% of the genome, is constituted mostly by
repetitive elements of retrotransposon origin.1ꢀ3 Retrotranspo-
sons are subdivided into two groups, the long terminal repeats
(LTRs)-containing endogenous retroviruses (HERVs) and the
non-LTR elements, which include LINE-1 (long interspersed
nuclear element 1) and SINE-VNTR-Alu (SVA) families.2
HERV and LINE-1 are autonomously replicating elements2,4
that harbor genes encoding their own reverse-transcriptase
(RT): this enables them to move within the genome using a
“copy-and-paste” mechanism involving the reverse transcription
of RNA intermediates and the insertion of the resulting cDNA
copies into new chromosomal sites of the host genome. Alu and
SVA, which lack the RT-coding gene, instead exploit the
Received: June 8, 2011
Published: July 14, 2011
r
2011 American Chemical Society
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Chart 1
Figure 1
Scheme 1a
pharmacological inhibition of endogenous RT activity using non-
nucleoside inhibitors (NNRTIs), such as nevirapine21 and
efavirenz22 (Chart 1), or down-regulating RT expression using
LINE-1-specific interfering RNAs (siRNAs) causes a significant
reduction of cell proliferation, promotes differentiation in tu-
morigenic cell lines, and strongly antagonizes human tumor
progression in murine models.23ꢀ27 More recently, nucleoside
reverse transcriptase inhibitors (NRTIs) have also been reported
to suppress LINE-1 retrotransposition activity and to exert
antiproliferative effects on tumor cell lines.28,29 On the whole,
these results suggest that an RT-dependent mechanism operates
in transformed cells. Suppressing the RT-dependent mechanism
restores control of cell differentiation and proliferation.30 In this
context, NNRTIs can be viewed as useful therapeutic tools, able
to counteract the loss of differentiation in dedifferentiating pathol-
ogies and as antiproliferative drugs in tumor therapy.27,31
Inspired by this evidence, we resolved to investigate the effect
on tumor cells of F2-DABOs (Chart 1),25,26 potent non-nucleo-
side HIV-1 reverse transcriptase inhibitors developed by our
group after a decade of lead optimization studies.32ꢀ43 We found
that two compounds (MC1047 and MC1220) significantly
reduced cell proliferation and facilitated the morphological
differentiation of human melanoma A375 cells.25 We next
decided to replace the substituted C5 of the pyrimidine ring
with a nitrogen atom, as triazine-containing compounds are re-
ported to induce the down-regulation of telomerase (hTERT),
another human reverse transcriptase.44 Although the resulting
6-alkylthio-4-[1-(2,6-difluorophenyl)alkyl]-1H-[1,3,5]triazin-2-
one (ADAT, Chart 1) derivatives were generally less active than
F2-S-DABOs, the effects that they induced in the A375 melano-
ma cell line did closely resemble those previously observed with
either NNRTIs or RNA interference (RNAi)-mediated silencing
of RT-encoding LINE-1 elements. Moreover, cell growth inhibi-
tion by either established NNRTIs or ADATs was reversible and
not inherited as a permanent change through cell division. This
suggests that the phenotypic variations induced by RT inhibition
in cultured cells are of an epigenetic nature and are independent
of insertional mutagenic events.26 Surprisingly, we noticed that
compound 1 (Chart 1), characterized by a 6-thioxotriazin-2-one
a Reagents and conditions: (a) n-BuLi, EtI, THF, ꢀ10 ꢀC; (b) CDI,
CH3CN; (c) potassium ethylmalonate or potassium ethyl-2-methylmalo-
nate, MgCl2, TEA, CH3CN; (d) 12% HCl; (e) thiourea, Na, EtOH, reflux;
(f) alkyl iodide, K2CO3, DMF.
structure and by the presence of an ethyl substituent at the linker
position, though not affecting cell proliferation, showed a strong
cytodifferentiating effect and a marked up-regulation of the
E-cadherin (e-cad) gene; in contrast, the corresponding linker-
unsubstituted analogue was found to be substantially inactive in
the same assay.26 This evidence suggested a crucial role of the
ethyl substituent in this position.
Here we report on the activity of a series of pyrimidinones
derivatives designed on the basis of structureꢀactivity relation-
ships resulting from both F2-DABOs and ADATs series. Among
them we identified 3g (SPV122, Figure 1), a compound that
significantly inhibits cell proliferation and induces differentiation
in A375 melanoma cells and is able to inhibit tumor growth in
nude mice.
’ CHEMISTRY
Derivatives 2 and 3 were prepared, following a straightforward
procedure previously described by us, starting from 2-(2,
6-difluorophenyl)butanoic acid (compound 5),26,38 promptly
obtained by alkylation of (2,6-difluoro)phenylacetic acid with
ethyl iodide in the presence of n-butyl lithium in anhydrous THF
(Scheme 1). The imidazolide obtained from the acid 5 by
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Table 1. Inhibition of Proliferation by Compounds 2a,b and
3aꢀha
compd
control
R
R0
cell growth (% at 25 μM)b
100
100
13
19
13
22
76
40
12
5
2a
H
H
3a
H
Me
3b
H
i-Pr
s-Bu
c-Pen
H
3c
H
3d
H
2b
Me
Me
Me
Me
Me
3e
Me
3f
i-Pr
s-Bu
c-Pen
3g
3h
5
NEV (350 μM)
EFV (15 μM)c
35
51
a Cell growth in human A375 melanoma cultures treated with DMSO
(control), test compounds, nevirapine (NEV), and efavirenz (EFV).
b Cells were treated for 96 h, then harvested and counted. Counted cells
are expressed as the % of controls, taken as 100. Values represent pooled
data from three experiments. c In the condition of the assay, this is the
highest testable concentration of efavirenz before the occurrence of
cytoxic effects.
Figure 2. Effects of 3g (A) on the human melanoma A375 cell line. (B)
Cell growth in human A375 melanoma cultures treated with DMSO
(control), 3g at increasing concentrations (2.5, 5, 10, 25 μM), nevirapine
(NEV, 350 μM), and efavirenz (EFV, 15 μM). Cells were harvested and
counted after 96 h. Counted cells are expressed as the % of controls,
taken as 100. Values represent pooled data from three experiments. (C)
Morphological differentiation of A375 melanoma cells in the presence of
DMSO (control) and 3g (5, 7.5, and 10 μM) under phase-contrast
microscopy.
treatment with N,N0-carbonyldiimidazole (CDI) was then re-
acted with potassium ethylmalonate or potassium ethyl-2-
methylmalonate in the presence of the magnesium dichloride/
triethylamine system to yield, after decarboxylation with 12%
hydrochloric acid, the ethyl 4-(2,6-difluorophenyl)-3-oxohex-
anoates 6a,b.38,43 Condensation of these β-oxoesters with thiour-
ea in the presence of sodium ethoxide gave the intermediate
6-(1-(2,6-difluorophenyl)propyl)-2-thioxo-2,3-dihydropyrimidin-
4(1H)-one 2a and 6-(1-(2,6-difluorophenyl)propyl)-5-methyl-
2-thioxo-2,3-dihydropyrimidin-4(1H)-one 2b,45 respectively,
which were finally S-alkylated in dry DMF with the proper alkyl
halide in the presence of potassium carbonate to yield the
corresponding compounds 3aꢀh.
analogues. For example, when A375 cells were treated with
derivative 3h at 25 μM, a very low growth rate (5%) was
observed. Under the same conditions, cells treated with
MC1047, the methyl substituted analogue of 3h, displayed a
significantly higher growth rate (72%).25 This result substanti-
ates the importance of the ethyl substituent on the methylene
connecting phenyl with triazine or, in this case, pyrimidine ring
(linker position). In general, the antiproliferative capability of the
compounds is related to the size of the substituent on the sulfur
atom at the C2 position of the pyrimidine ring. This effect is
further increased by the insertion of a methyl group at the C5
position (compare, for example, the activities of compounds 3b,
3c, and 3d with those of 3f, 3g, and 3h, respectively).
The derivative 3g (SPV122) (Figure 2A) showed the most
powerful antiproliferative effect. To characterize this effect in
more depth, we carried in vitro growth assays out by exposing
A375 cultures to increasing doses of the compound. As shown in
Figure 2B, the antiproliferative effect exerted by 3g was highly
dose-dependent and markedly stronger than that induced by
efavirenz at approximately equivalent dosage. In addition, 3g,
even in low concentrations (i.e., 5, 7.5, and 10 μM), induced
remarkable morphological changes in A375 cells compared to
DMSO-treated controls (Figure 2C); dendritic-like extensions
’ RESULTS
Compounds 2a,b and 3aꢀh were tested (25 μM in DMSO
solution) on the human A375 cell line, derived from metastatic
malignant melanoma (ATCC CRL-1619), previously character-
ized in depth for being sensitive to both siRNA-mediated
silencing of RT-encoding LINE-1 elements and to NNRTI-
mediated (nevirapine and efavirenz) RT inhibition.24,46 Nevir-
apine and efavirenz were used as reference drugs (Table 1). With
the exception of the two S-unsubstituted compounds 2a and 2b,
all tested derivatives exhibited a strong antiproliferative effect,
being more effective than references at concentrations 14-fold
lower than that employed for nevirapine.38 Moreover, derivatives
3aꢀh are more active than the corresponding methyl-substituted
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Figure 3. HPLC separation of the four stereoisomers of 3g.48 (A)
Polarimetric (365 nm) (top) and UV (330 nm) (bottom) chromato-
grams of 3g: column, Chiralpak IA (250 mm ꢁ 10 mm i.d.); eluent
MTBEꢀEtOAc (75:25, v/v); flow rate of 1 mL/min; column tempera-
ture of 25 ꢀC. (B) CD spectra of the stereoisomers of 3g in ethanol at 25
ꢀC.
Figure 4. Stereoisomers 3g inhibit proliferation of A375 melanoma
cells in a dose-dependent (A) and time-dependent (B) manner. (A) The
histograms represent the extent of cell proliferation in A375 cell cultures
exposed to the indicated treatments for 96 h (mean values and SD from
four independent experiments). The ratio of proliferating cells after
treatment/seeded cells is expressed relative to that measured in DMSO-
exposed control samples, taken as 100%. For comparison, 20 μM 3g and
350 μM nevirapine were used. (B) A375 melanoma cell cultures were
exposed to 3g stereoisomers (5 or 10 μM) or to DMSO only (control),
and cells were counted at the indicated times (mean values and SD from
three experiments).
were induced and adhesion increased, strongly suggesting that a
differentiation process was activated.24ꢀ26,47
Separation of 3g Stereoisomers. Compound 3g has two
stereogenic centers; therefore, the biological effects ascribed to
this compound are in fact the resultant of a mixture of four
stereoisomers. In order to assess the biological effect of each
single stereoisomer, we decided to separate them using HPLC in
a single run on the immobilized-type Chiralpak IA chiral
stationary phase (CSP) using ethyl acetate based eluents.48
Different from closely related compounds,49 in this case the
assignment of absolute configuration by X-ray diffraction analysis
was not possible because of unfavorable physical properties of
the isolated stereoisomers and difficulty in obtaining crystalline
derivatives. By analysis of the sign of optical rotation and relative
peak areas (Figure 3), it was possible to identify the first and the
fourth eluted stereoisomer as belonging to the same enantio-
meric pair (compound 3g0) and the second and third one to the
diastereomeric pair of enantiomers (compound 3g00). Moreover,
the analogy between the circular dichroism (CD) profiles of 3g
and its dichlorophenyl analogue49 permitted assignment of the
(R) absolute configuration to the stereogenic center adjacent to
the 2,6-difluorophenyl moiety of (ꢀ)-3g0 and (ꢀ)-3g00. The
configurational assignment about the stereogenic center of sec-
butylthio group of 3g still remains an unsolved question. After
separation, the four stereoisomers were individually tested in
biological experiments, in comparison with the 3g mixture
(hereafter simply referred to as 3g) and nevirapine.
unfractionated 3g (20 μM), nevirapine (NEV, 350 μM), or
DMSO alone for control. The results in Figure 4 show that all
four stereoisomers inhibited proliferation of A375 melanoma
cells compared to solvent-treated control samples. The stereo-
isomers exhibited different individual effectiveness in terms of
their growth inhibitory properties: (ꢀ)-3g0 and (+)-3g00 were
identified as the most effective of the four isolated stereoisomers
and in comparison to 3g. The repressive effect is both dose-
dependent, with 20 μM yielding the most powerful antiproli-
ferative effect (70ꢀ80% suppression of the proliferation,
Figure 4A), and time-dependent (with a most pronounced effect
after 96 h compared to 48 h for all compounds, Figure 4B) for all
stereoisomers. It is noteworthy that repression of proliferation by
the most effective stereoisomers at 20 μM was consistently
higher than observed with 350 μM nevirapine, which leaves a
cycling subpopulation representing almost 50% of the original
culture after 96 h of treatment.
Induction of Apoptosis. In preliminary experiments we
found that 3g induces apoptotic cell death in a dose-dependent
manner, as shown by combined staining with propidium iodide
(PI) to reveal permeable necrotic cells, 40,6-diamidino-2-pheny-
lindole lactate (DAPI) to visualize apoptotic nuclei, and 3,3-
dihexyloxacarbocyanine [DiOC6(3)]24,50 to monitor the loss of
mitochondrial transmembrane potential (Supporting Informa-
tion, Figure S1), similar to previously analyzed NNRTIs.23ꢀ26
Inhibition of Cell Proliferation. In the first set of experi-
ments, stereoisomers were tested in increasing doses (5, 10, and
20 μM) for their antiproliferative efficacy in comparison to
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A fraction of necrotic cells only became apparent with the highest
tested dose (25 μM).
metric FACS analysis of the nuclear DNA content (PI) com-
bined with annexin V (which detects phosphatidylserine residues
translocated from the inner to the outer cell membrane in early
apoptotic stages) and in indirect immunofluorescence (IF)
analysis of cells expressing active caspase-3 (data not shown).
Together these assays indicate that the 3g mixture, as well as each
single stereoisomer, induces a specific apoptotic response in
A375 melanoma cells in a cell-density-dependent manner.
Morphological Differentiation. The observation (under
bright-field microscopy) of A375 cells cultured in the presence
of the stereoisomers, particularly with (ꢀ)-3g0 and (+)-3g0,
indicated clear signs of morphological differentiation, with the
appearance of dendritic-like extensions (Figure 2C and Figure
S2, Supporting Information). To refine this analysis, we used
antibodies to tubulin and vimentin intermediate filament in IF
assays. As shown in Figure 6, after 96 h of exposure a clear
reorganization of microtubules and of the vimentin intermediate
filament network was revealed, with an evident induction of
elongated extensions. Scoring of cells that underwent these
morphological changes (representative examples are shown in
Figure S3A, Supporting Information) indicated that all four
isomers induce differentiation more effectively than nevirapine
(Figure S3B), albeit to varying extent: both enantiomers (ꢀ)-3g0
and (+)-3g0 were particularly effective, whereas surprisingly (+)-
3g00, which had a strong antiproliferative effect, was a somewhat
weaker inducer of differentiation.
We next investigated the ability of the single separated
stereoisomers to induce apoptotic cell death. A375 cells were
seeded at low density (100 000 cells per 25 mm2 flask), cultured
for 96 h in the presence of compounds (20 μM), then harvested
and subjected to biparametric fluorescence-activated cell sorting
(FACS) analysis (the nuclear DNA profiles were revealed by PI
incorporation and the cell granular density by side scatter
analysis, SSC). This revealed a very low induction of apoptosis.
When cells were seeded at a higher density (i.e., 300 000 cells per
25 mm2 flask), however, a significant induction of apoptosis was
recorded, especially with (ꢀ)-3g0 and, even more evidently, with
(+)-3g0 (Figure 5). In contrast, nevirapine failed to induce
significant apoptosis in either condition. To distinguish apoptotic
cell death from necrosis, the results were confirmed in bipara-
Compound 3g Antagonizes Tumor Progression. Given
that 3g can suppress proliferation and induce differentiation in
transformed cells, we next asked whether it also affects tumor
growth in vivo. To that aim, A375 cells were inoculated sub-
cutaneously in the limb of athymic nude mice. Animals were then
subjected to treatment with either 3g or isomer (ꢀ)-3g0,
endowed with the strongest antiproliferative activity (vide
supra); efavirenz was used as the reference drug because it had
shown higher in vivo effectiveness than nevirapine in previous
assays.24
Figure 5. Density-dependent effect induction of apoptosis by 3g
stereoisomers. The histograms represent the mean and SD values
(three experiments) of the apoptotic cell population, revealed by FACS
analysis in A375 melanoma cell cultures plated at low and high density.
Figure 6. Reorganization of the cytoskeleton and appearance of dendritic-like extensions in A375 human melanoma cells after treatment with 3g
stereoisomers. A375 cell samples were cultured for 96 h as indicated, then processed for IF to tubulin (red) and vimentin intermediate filament (green).
Nuclei are stained with DAPI (blue). Arrows indicate the appearance of dendritic-like extensions, absent in control (DMSO-treated) cultures (100ꢁ
objective).
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inhibiting non-telomerase activities, as the antiproliferative effect
observed after chemical59 or genetic60 inhibition of the telomer-
ase-associated RT requires several months of continuous expo-
sure to display antiproliferative effects, consistent with a
progressive mode of action at telomeres.
Compound 3g induces apoptosis in a manner that is depen-
dent on the initial cell density, and it antagonizes tumor growth in
animal models. These effects are similar or more pronounced
than those previously reported for other nucleoside or non-
nucleoside inhibitors of reverse transcriptase (abacavir, nevira-
pine, efavirenz, F2-DABOs, ADATs). Our results also reveal a
functional specificity in the activity of the single stereoisomers
separated from the 3g mixture: (ꢀ)-3g0 is the best inducer of
differentiation, the most effective repressor of proliferation, and a
good inducer of apoptosis in dense cell cultures; (+)-3g00 is nearly
as effective as (ꢀ)-3g0 in repressing proliferation, but it is less
active as a differentiation-inducing agent; (+)-3g0 has little
activity in repressing proliferation, but nevertheless, it is a good
inducer of morphological differentiation and apoptosis. This
finding may have interesting possible implications. The stereo-
isomers may differentially inhibit the bulk of the endogenous
cellular RT, with the RT “core” activity being fully blocked by
(ꢀ)-3g0, while residual proapoptotic and differentiating activities
would be only partially affected by stereoisomers of different
structure. Not mutually exclusive, the stereoisomers may differ-
entially prevent the interaction of the cellular RT with distinct
target RNAs and thus differentially affect downstream cellular
pathways. No selective antibodies that would discriminate differ-
entially active RT subpopulations are currently available to test
these alternatives, but the identification of distinctive functional
spectra for individual 3g stereoisomers can provide promising
tools to unravel RT-dependent targets in cancer cells in
prospective work.
Figure 7. Treatment with 3g antagonizes human tumor growth in nude
mice. The growth of tumors formed by A375 melanoma cells was
monitored in nude mice untreated or treated as indicated. Curves show
the mean value of tumor size in groups of five animals.
Previous doseꢀresponse experiments with efavirenz (testing
4ꢀ40 mg drug/kg animal body weight) indicated that 20 mg/kg
was the optimal dose for inhibition of progression of xenografted
tumors in nude mice.24 All compounds were therefore used at
that dose. The growth of A375 melanoma-derived tumor xeno-
grafts was monitored, and Figure 7 shows the recorded curves in
nude mice untreated or treated with solvent (20% ethanol),
efavirenz, 3g, and isomer (ꢀ)-3g0. Tumor growth was markedly
reduced in both 3g- and (ꢀ)-3g0-treated animals compared to
untreated controls (Figure 7). Remarkably, the inhibitory effi-
cacy of the latter was comparable or higher than that of efavirenz.
A slight enhancement of tumor growth was observed in solvent-
treated compared to untreated control animals (Figure 7, green
and red curves, respectively).
Taken together, the data indicate a strong antiproliferative and
antitumor potential of SPV compounds. Furthermore, they
provide further support to the notion, emerging from previous
work with NNRTIs, that changes in the tumorigenic phenotypes
of A375 cells can arise in consequence of the inhibition of the
endogenous RT. These results have important implications for
cancer therapy and help to identify activities orchestrated by the
endogenous RT at a crucial crossroad between cell proliferation,
differentiation, and apoptosis.
’ CONCLUSIONS
A growing body of evidence now indicates that high levels of
endogenous RT activity are typically associated with trans-
formed/tumorigenic phenotypes of mammalian cells. In addi-
tion, the inhibition of LINE-1-encoded RT by either nucleosidic
or non-nucleosidic inhibitors has been proposed as a novel
promising approach in cancer therapy.18,23ꢀ29,31,51ꢀ58
In this work, we report that ethyl-substituted derivatives
3aꢀh, belonging to the F2-DABOs class of non-nucleoside
HIV-1 reverse transcriptase inhibitors, are endowed with a strong
antiproliferative effect on A375 melanoma cells. The importance
of the ethyl substituent on the methylene connecting phenyl with
the pyrimidine ring (linker position) is further substantiated by
the fact that, even when lacking any substitution on the sulfur
atom, derivatives bearing an ethyl group at linker position were
able to induce differentiation of human A375 cells (see, for
example, Supporting Information, Figure S4), associated with
up-regulation of the e-cad gene (not shown). Particularly strong
effects in suppression of cell proliferation and induction of
differentiation were induced by compound 3g (SPV122). Those
effects resembled those observed when treating A375 cells with
NNRTIs23ꢀ26 or after RNA interference (RNAi)-mediated
silencing of the RT-encoding LINE-1 elements .24,46 The effects
of these treatments, as well as those presently reported for 3g and
its stereoisomers, are induced quite rapidly (within a few days),
supporting the conclusion that all tested molecules share a
common target in A375 cells. This is distinctive of molecules
’ EXPERIMENTAL SECTION
Chemistry. All chemicals were purchased from Aldrich Chimica
(Milan, Italy) or from Alfa Aesar GmbH (Karlsruhe, Germany) and were
of the highest purity. All solvents were reagent grade and, when
necessary, were purified and dried by standard methods. All reactions
requiring anhydrous conditions were conducted under a positive atmo-
sphere of nitrogen in oven-dried glassware. Standard syringe techniques
were used for anhydrous addition of liquids. Reactions were routinely
monitored by TLC performed on aluminum-backed silica gel plates
(Merck DC, Alufolien Kieselgel 60 F254) with spots visualized by UV
light (λ = 254, 365 nm) or using a KMnO4 alkaline solution. Solvents
were removed using a rotary evaporator operating at a reduced pressure
of ∼10 Torr. Organic solutions were dried over anhydrous Na2SO4.
Chromatographic separations were performed on silica gel (silica gel 60,
0.015ꢀ0.040 mm; Merck DC) columns. Melting points were deter-
mined on a Stuart SMP30 melting point apparatus in open capillary
tubes and are uncorrected. 1H NMR spectra were recorded at 300 MHz
on a Bruker Avance 300 spectrometer. Chemical shifts are reported in δ
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(ppm) relative to the internal reference tetramethylsilane (TMS). Mass
spectra were recorded on a Finnigan LCQ DECA TermoQuest (San
Jose, CA, U.S.) mass spectrometer in electrospray positive and negative
ionization modes (ESI-MS). Purity of tested compounds was established
by combustion analysis, confirming a purity g95%. Elemental analyses
(C, H, N) were performed on a Perkin-Elmer 2400 CHN elemental
analyzer at the Laboratory of Microanalysis of the Department of
Chemistry and Biology, University of Salerno (Italy); the analytical
results were within (0.4% of the theoretical values. When the elemental
analysis is not included, compounds were used in the next step without
further purification.
0.89ꢀ0.94 (t, J = 7.4 Hz, 3H, CHCH2CH3), 1.55ꢀ1.67 (m, 6H, C3,4
-
H2 + C2,5-H cyclopentane, overlapped signals), 1.89ꢀ1.93 (m, 1H,
CHCHHACH3), 2.02ꢀ2.10 (m, 2H, C2,5-H cyclopentane), 2.24ꢀ2.28
(m, 1H, CHCHHBCH3), 3.86ꢀ3.90 (m, 1H, SCH), 4.12ꢀ4.15 (m, 1H,
CHCH2CH3), 6.14 (s, 1H, C-5 H), 6.81ꢀ6.87 (m, 2H, C3,5-H Ar),
7.15ꢀ7.21 (m, 1H, C4ꢀH Ar), 12.74 (bs, 1H, NH exchangeable with
D2O). ESI-MS m/z: 351 (M + H)+. Anal. (C18H20F2N2OS) C, H, N.
6-(1-(2,6-Difluorophenyl)propyl)-5-methyl-2-(methylthio)-
pyrimidin-4(3H)-one (3e). A mixture of 6-(1-(2,6-difluorophenyl)-
propyl)-5-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (2b, 1.00
g, 3.37 mmol), iodomethane (235 μL, 3.71 mmol), potassium carbonate
(466 mg, 3.37 mmol), and 2.00 mL of anhydrous N,N-dimethylforma-
mide was stirred at room temperature under N2 atmosphere for 3 h.
After completion (TLC: silica gel/n-hexane/EtOAc/MeOH 12:3:1),
the mixture was diluted with water (20 mL) and extracted with ethyl
acetate (3 ꢁ 40 mL). The organic layers were collected, washed with
brine (3 ꢁ 50 mL), dried, and evaporated to furnish a solid residue,
which was purified by silica gel chromatography (n-hexane/EtOAc/
MeOH 12:3:1) to yield compound 3e (60%)as a white solid. Mp
183ꢀ185 ꢀC (benzene/cyclohexane). 1H NMR (300 MHz, CDCl3) δ
0.90ꢀ0.97 (t, J = 7.1 Hz, 3H, CHCH2CH3), 2.04ꢀ2.29 (m, 2H,
CHCHHACH3 + CHCHHBCH3, overlapped signals), 2.05 (s, 3H,
C-5 CH3), 2.55 (s, 3H, SCH3), 4.37ꢀ4.48 (m, 1H, CHCH2CH3),
6.80ꢀ6.86 (m, 2H, C3,5-H Ar), 7.13ꢀ7.20 (m, 1H, C4-H Ar), 11.68 (bs,
1H, NH exchangeable with D2O). ESI-MS m/z: 311 (M + H)+. Anal.
(C15H16F2N2OS) C, H, N.
Ethyl 4-(2,6-difluoro)phenyl-3-oxohexanoates 6a26,38 and 6b43 as
well as the corresponding 6-(1-(2,6-difluorophenyl)propyl)-2-thioxo-
2,3-dihydropyrimidin-4(1H)-one 2a38 and 6-(1-(2,6-difluorophenyl)-
propyl)-5-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one 2b45 were
prepared as previously described.
6-(1-(2,6-Difluorophenyl)propyl)-2-(methylthio)pyrimidin-
4(3H)-one (3a). A mixture of 6-(1-(2,6-difluorophenyl)propyl)-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one (2a, 1.00 g, 3.54 mmol), iodo-
methane (245 μL, 3.89 mmol), potassium carbonate (490 mg, 3.54
mmol), and 2.00 mL of anhydrous N,N-dimethylformamide was stirred
at room temperature under N2 atmosphere for 3 h. After completion
(TLC: silica gel/n-hexane/EtOAc/MeOH 12:3:1), the mixture was
diluted with water (20 mL) and extracted with ethyl acetate (3 ꢁ
40 mL). The organic layers were collected, washed with brine (3 ꢁ
50 mL), dried, and evaporated to furnish a solid residue, which was
purified by silica gel chromatography (n-hexane/EtOAc/MeOH 12:3:1)
to yield compound 3a (75%) as a white solid. Mp 150ꢀ151 ꢀC
6-(1-(2,6-Difluorophenyl)propyl)-2-(isopropylthio)-5-methyl-
pyrimidin-4(3H)-one (3f). Title compound 3f was obtained as a
white solid (60%) starting from 2b (1.00 g, 3.37 mmol) and 2-iodopro-
pane (370 μL, 3.71 mmol) according to the same procedure used for 3e.
Mp 162ꢀ163 ꢀC (benzene/cyclohexane). 1H NMR (300 MHz, CDCl3)
δ 0.92ꢀ0.97 (t, J = 7.2 Hz, 3H, CHCH2CH3), 1.28ꢀ1.30 (d, J = 6.8 Hz,
3H, CHCH3), 1.41ꢀ1.43 (d, J = 6.8 Hz, 3H, CHCH3), 2.04 (s, 3H, C-5
CH3), 2.16ꢀ2.25 (m, 2H, CHCHHACH3 + CHCHHBCH3, overlapped
signals), 3.94ꢀ4.03 (s, 1H, CH), 4.33ꢀ4.44 (m, 1H, CHCH2CH3),
6.79ꢀ6.85 (m, 2H, C3,5-H Ar), 7.11ꢀ7.18 (m, 1H, C4-H Ar), 11.97 (bs,
1H, NH exchangeable with D2O). ESI-MS m/z: 339 (M + H)+. Anal.
(C17H20F2N2OS) C, H, N.
2-(sec-Butylthio)-6-(1-(2,6-difluorophenyl)propyl)-5-methyl-
pyrimidin-4(3H)-one (3g). Title compound 3g was obtained (60%)
as a white solid mixture of stereoisomers (NMR; see Supporting
Information, Figures S5 and S6) starting from 2b (1.00 g, 3.37 mmol)
and 2-iodobutane (430 μL, 3.71 mmol) according to the same
procedure used for 3e. Mp 137ꢀ138 ꢀC (benzene/cyclohexane).
1H NMR (300 MHz, CDCl3) δ 0.90ꢀ1.06 (m, 6H, ArCHCH2CH3 +
SCHCH2CH3, overlapped signals), 1.24ꢀ1.27, 1.40ꢀ1.42 (2 d, 3H,
SCHCH3 first enantiomeric pair + SCHCH3 second enantiomeric pair),
1.55ꢀ1.81 (m, 2H, SCHCH2), 2.03 (s, 3H, C-5 CH3), 2.07ꢀ2.24 (m,
2H, CHCHHACH3 + CHCHHBCH3, overlapped signals), 3.87ꢀ3.89
(m, 1H, SCH), 4.31ꢀ4.36 (m, 1H, CHCH2CH3), 6.79ꢀ6.91 (m, 2H,
C3,5-H Ar), 7.10ꢀ7.20 (m, 1H, C4-H Ar), 9.80 (bs, 1H, NH exchange-
able with D2O). ESI-MS m/z: 353 (M + H)+. Anal. (C18H22F2N2OS)
C, H, N.
1
(acetonitrile). H NMR (300 MHz, CDCl3) δ 0.88ꢀ0.93 (t, J = 7.4
Hz, 3H, CHCH2CH3), 1.97ꢀ2.07 (m, 1H, CHCHHACH3),61
2.24ꢀ2.31 (m, 1H, CHCHHBCH3), 2.46 (s, 3H, SCH3), 4.11ꢀ4.16
(m, 1H, CHCH2CH3), 6.11 (s, 1H, C-5 H), 6.81ꢀ6.87 (m, 2H, C3,5-H
Ar), 7.15ꢀ7.21 (m, 1H, C4-H Ar), 11.71 (bs, 1H, NH exchangeable with
D2O). ESI-MS m/z: 297 (M + H)+. Anal. (C14H14F2N2OS) C, H, N.
6-(1-(2,6-Difluorophenyl)propyl)-2-(isopropylthio)pyrimidin-
4(3H)-one (3b)38. Title compound 3b was obtained as a white solid
(60%) starting from 2a (1.00 g, 3.54 mmol) and 2-iodopropane (400 μL,
3.89 mmol) according to the same procedure used for 3a. Mp
1
148ꢀ150 ꢀC (acetonitrile). H NMR (300 MHz, CDCl3) δ 0.88ꢀ
0.99 (t, J = 7.4 Hz, 3H, CHCH2CH3), 1.24ꢀ1.26 (d, J = 6.8 Hz, 3H,
CHCH3), 1.32ꢀ1.34 (d, J = 6.8 Hz, 3H, CHCH3), 2.00ꢀ2.11 (m, 1H,
CHCHHACH3), 2.21ꢀ2.35 (m, 1H, CHCHHBCH3), 3.81ꢀ3.97 (s,
1H, CH), 4.14ꢀ4.19 (m, 1H, CHCH2CH3), 6.18 (s, 1H, C-5 H),
6.81ꢀ6.87 (m, 2H, C3,5-H Ar), 7.15ꢀ7.21 (m, 1H, C4-H Ar), 11.97 (bs,
1H, NH exchangeable with D2O). ESI-MS m/z: 325 (M + H)+. Anal.
(C16H18F2N2OS) C, H, N.
2-(sec-Butylthio)-6-(1-(2,6-difluorophenyl)propyl)pyrimidin-
4(3H)-one (3c). Title compound 3c was obtained as a white solid
mixture of stereoisomers (60%) starting from 2a (1.00 g, 3.54 mmol)
and 2-iodobutane (450 μL, 3.89 mmol) according to the same proce-
dure used for 3a. Mp 108ꢀ109 ꢀC (acetonitrile). 1H NMR (300 MHz,
CDCl3) δ 0.85ꢀ0.98 (m, 6H, ArCHCH2CH3 + SCHCH2CH3, over-
lapped signals), 1.21ꢀ1.23, 1.30ꢀ1.32 (2 d, 3H, SCHCH3 first en-
antiomeric pair + SCHCH3 second enantiomeric pair), 1.48ꢀ1.72 (m,
2H, SCHCH2), 2.00ꢀ2.10 (m, 1H, CHCHHACH3), 2.24ꢀ2.34 (m,
1H, CHCHHBCH3), 3.70ꢀ3.79 (m, 1H, SCH), 4.10ꢀ4.15 (m, 1H,
CHCH2CH3), 6.14 (s, 1H, C-5 H), 6.81ꢀ6.86 (m, 2H, C3,5-H Ar),
7.12ꢀ7.22 (m, 1H, C4-H Ar), 9.86 (bs, 1H, NH exchangeable with
D2O). ESI-MS m/z: 339 (M + H)+. Anal. (C17H20F2N2OS) C, H, N.
2-(Cyclopentylthio)-6-(1-(2,6-difluorophenyl)propyl)pyri-
midin-4(3H)-one (3d)38. Title compound 3d was obtained as a white
solid (60%) starting from 2a (1.00 g, 3.54 mmol) and bromocyclopen-
tane (450 μL, 3.89 mmol) according to the same procedure used for 3a.
Mp 143ꢀ144 ꢀC (acetonitrile). 1H NMR (300 MHz, CDCl3) δ
2-(Cyclopentylthio)-6-(1-(2,6-difluorophenyl)propyl)-5-
methylpyrimidin-4(3H)-one (3h). Title compound 3h was ob-
tained as a white solid (60%) starting from 2b (1.00 g, 3.37 mmol) and
bromocyclopentane (430 μL, 3.71 mmol) according to the same
1
procedure used for 3e. Mp 195ꢀ197 ꢀC (benzene/cyclohexane). H
NMR (300 MHz, CDCl3) δ 0.89ꢀ0.94 (t, J = 7.2 Hz, 3H,
CHCH2CH3), 1.63ꢀ1.75 (m, 6H, C3,4-H2 + C2,5-H cyclopentane,
overlapped signals), 1.95ꢀ2.24 (m, 4H, C2,5-H cyclopentane +
CHCHHACH3 + CHCHHBCH3, overlapped signals), 2.05 (s, 3H,
C-5 CH3), 3.90ꢀ3.94 (m, 1H, SCH), 4.32ꢀ4.35 (m, 1H, CHCH2CH3),
6.80ꢀ6.86 (m, 2H, C3,5-H Ar), 7.14ꢀ7.20 (m, 1H, C4-H Ar), 12.70 (bs,
5933
dx.doi.org/10.1021/jm200734j |J. Med. Chem. 2011, 54, 5927–5936

Journal of Medicinal Chemistry
ARTICLE
1H, NH exchangeable with D2O). ESI-MS m/z: 365 (M + H)+. Anal.
(C19H22F2N2OS) C, H, N.
Tumor Xenografts and Treatment of Animals. Five-week-old
athymic nude mice (Harlan, Italy) were inoculated subcutaneously with
A375 melanoma cells (5 ꢁ 106) in 100 μL of PBS. Mice were
intraperitoneally injected daily 5 days a week with efavirenz, 3g, or
isomer (ꢀ)-3g0 (all at 20 mg/kg) using a 4 mg/mL stock in EtOH
freshly diluted 1:5 with physiological solution. Controls were injected
with 20% EtOH. Treatment started 1 week after tumor implant and was
discontinued after 30 days. Tumor growth was monitored every other
day by caliper measurement. Tumor volumes (VT) were calculated using
the following formula:63
Biology. Cell Cultures. Human A375 melanoma (ATCC-CRL-
1619) cells were seeded in six-well plates at a density of 1 ꢁ 104 to 5 ꢁ
104 cells/well and cultured in Dulbecco's modified Eagle medium
(DMEM, Euroclone) with 10% (v/v) fetal calf serum, L-glutamine
(2 mM), penicillin (100 IU/mL), and streptomycin (100 mg/mL) at
37 ꢀC in a humidified 5% CO2 atmosphere. After 5ꢀ6 h, when cells were
adherent, RT inhibitors were dissolved in DMSO at the indicated
concentrations, or the same volume of dimethylsulfoxide (DMSO,
Aldrich; 0.005% final concentration) was added. Nevirapine and efavir-
enz were purified from commercially available Viramune (Boehringer-
Ingelheim) and Sustiva (Bristol-Myers Squibb) as described.62 Com-
pounds 2a,b and 3aꢀh, as well as the reference drugs nevirapine and
efavirenz, were solubilized in DMSO. Fresh RT inhibitor-containing
medium was changed every 48 h.
VT ¼ length ꢁ width ꢁ height ꢁ 0:52
All animals were maintained in accordance with the European Union
guidelines.
Proliferation Assays. To measure proliferation, cells were plated
at an initial density of (20ꢀ50) ꢁ 103 cells/35 mm diameter plate. After
48 or 96 h of culture, viable cells were scraped off and counted in a ZM
Coulter counter (two countings per sample). Cell viability was also
assessed using the trypan blue dye exclusion method in a Burker
chamber. The basal proliferation rate was estimated using the formula
[cell n(t)]/[cell n(t0)], where the number of cells at t0 is the number of
seeded cells and the number of cells at time t is given by cell counts after
48 or 96 h of culture.
Apoptosis Analysis. For single-cell analysis of apoptotic cell death,
A375 cells grown in culture dishes on sterile coverslips were stained with
40,6-diamidino-2-phenylindole (DAPI, Sigma) to visualize the nuclear
morphology and with 3,30-dihexyloxacarbocyanine iodide [DiOC6(3),
Molecular Probes], a fluorescent probe for mitochondrial transmem-
brane potential. As a second assay, processed caspase-3 was detected
using rabbit antiactive caspase-3 (ab13847, Abcam) followed by Cy3-
conjugated donkey anti-rabbit secondary antibody (711-165-152, Jack-
son Immunoresearch Laboratories) in fixed A375 cells samples counter-
stained with DAPI (data not shown). Cells were then analyzed under an
epifluorescence Olympus AX70 microscope with a CCD camera
(Photometrics) or Leica DMR with a CoolSnap (Photometrics).24
Apoptosis was also assessed at the whole cell population level by
biparametric FACS analysis of the nuclear DNA content (PI incorpora-
tion, linear scale) and of the cell granular density (side scatter analysis,
SSC, logarithmic scale) or after incubation with annexin V-FITC
(Immunological Sciences, IK-11120) and propidium iodide (PI, Sig-
ma-Aldrich), as recommended by the suppliers. Cell samples were
analyzed in a Coulter Epics XL cytofluorimeter (Beckman Coulter)
equipped with EXPO 32 ADC software. At least 10 000 cells per sample
were acquired.
Indirect Immunofluorescence and Confocal Microscopy.
A375 cell preparations were fixed with 4% paraformaldehyde for 10 min
and permeabilized in 0.2% Triton-X100 in phosphate buffered saline
(PBS) for 5 min. Mouse monoclonal antibovine R-tubulin (Molecular
Probes, A-11126) was revealed by Alexa Fluor 488 conjugated secondary
antibody (Molecular Probes, A-11001) or with horse anti-mouse Texas
Red (Vector, TI-2000). Primary antibodies to human vimentin inter-
mediate filaments were a generous gift from Genrich Tolstonog
(Heinrich-Pette Instit€ute fur Experimentelle Virologie und Immunolo-
gie, Hamburg University, Germany) and were detected with FITC-
conjugated secondary antibodies (Santa Cruz, sc-2090). Nuclei were
stained either with 2 μg/mL PI in the presence of 0.1 μg/mL
ribonuclease A or with 0.1 μg/mL DAPI. Coverslips were mounted in
Vectashield (Vector) and examined under an epifluorescence Olympus
AX70 microscope with a CCD camera (Photometrics). For confocal
analysis, samples were imaged under a confocal Leica TCS 4D micro-
scope equipped with an argon/krypton laser. Confocal sections were
taken at 0.5ꢀ1 μm intervals.
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis results for
b
compounds 3aꢀh and additional biological data. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For G.S.: phone, +39-089-96-9770; fax, +39-089-96-9602;
e-mail, gsbardella@unisa.it. For A.M.: phone, +39-06-4991-
3392; fax, +3906-49693268; e-mail, antonello.mai@uniroma1.it.
’ ACKNOWLEDGMENT
We acknowledge the skillful technical assistance of Enrico
Cardarelli in the in vivo experiments with the murine models.
This work was supported by grants from Universitꢀa di Salerno,
Italy (G.S.), COST Action TD09/05 Epigenetics (G.S. and A.
M.), Fondazione Roma (A.M.), Ministero dell’Universitꢀa e della
Ricerca Scientifica e Tecnologica, PRIN 2008 (S.C. and P.L.),
AIRC (P.L.), Ministero della Salute (Grants 7OCF/6 and Q5F,
C.S.). C.M. was supported by a predoctoral fellowship from
Universitꢀa di Salerno, Italy.
’ ABBREVIATIONS USED
Alu, short interspersed element recognized by the Alu endonuclease,
from Arthrobacter luteus; cDNA, complementary DNA; DAPI, 40,6-
diamidino-2-phenylindole; DiOC6(3), 3,30-dihexyloxacarbocya-
nine iodide; HERV, human endogenous retrovirus; hTERT, hu-
man telomerase reverse transcriptase; LINE-1, long interspersed
element 1; LTR, long terminal repeat; NNRTI, non-nucleoside
reverse transcriptase inhibitor; PBS, phosphate buffer saline; RT,
reverse transcriptase; RNAi, RNA interference; SINE, short in-
terspersed element; siRNA, small interfering RNA; SVA, SINE-
VNTR-Alu; VNTR, variable number tandem repeat
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