6-[1-(2,6-Difluorophenyl)ethyl]pyrimidinones antagonize cell proliferation and induce cell differentiation by inhibiting (a nontelomeric) endogenous reverse transcriptase

Article abstract of DOI:10.1021/jm0507330

Two 2,6-difluoro-DABO derivatives (MC 1047, 1, and MC 1220, 2, respectively) were tested against endogenous, nontelomeric reverse transcriptase (endo-RT) in human differentiating cell systems to investigate their antiproliferative and cytodifferentiating

Full text of DOI:10.1021/jm0507330

6776
J. Med. Chem. 2005, 48, 6776-6778
6-[1-(2,6-Difluorophenyl)ethyl]pyrim-
idinones Antagonize Cell Proliferation
and Induce Cell Differentiation by
Inhibiting (a Nontelomeric) Endogenous
Reverse Transcriptase
Sara Bartolini,† Antonello Mai,*,‡ Marino Artico,‡
Nicola Paesano,§ Dante Rotili,‡
Corrado Spadafora,† and Gianluca Sbardella§
Figure 1. F2-DABOs.
Istituto Superiore di Sanita`, Viale Regina Elena 299,
Via del Castro Laurenziano 25, 00161, Rome, Italy,
Istituto PasteursFondazione Cenci Bolognetti, Dipartimento
di Studi Farmaceutici, Universita` di Roma “La Sapienza”,
P.le A. Moro 5, 00185 Rome, Italy, and Dipartimento di
Scienze Farmaceutiche, Universita` degli Studi di Salerno,
Via Ponte Don Melillo, 84084, Fisciano (SA), Italy
Scheme 1a
Received July 28, 2005
Abstract: Two 2,6-difluoro-DABO derivatives (MC 1047, 1,
and MC 1220, 2, respectively) were tested against endogenous,
nontelomeric reverse transcriptase (endo-RT) in human dif-
ferentiating cell systems to investigate their antiproliferative
and cytodifferentiating activity. The two compounds signifi-
cantly reduced cell proliferation and facilitated the morpho-
logical differentiation of cells. These results propose F2-DABOs
as useful tools in preventive and/or curative therapy to
counteract the loss of differentiation in dedifferentiating
pathologies and as antiproliferative drugs in tumor therapy.
The recent realization that retrotransposons can
reshape the genome and contribute to modulation of
gene expression1,2 led to reconsideration of the impor-
tance of these elements. As a matter of fact, human
DNA is significantly (45%) composed of retrotranspos-
able elements, all (but the Alu family) endowed with a
reverse transcriptase (RT)-coding gene.2,3 Growing evi-
dence indicates that RT-coding genes are expressed at
low levels, if at all, in differentiated nonpathological
tissues; in contrast, high expression is distinctive of
germ cells, embryos, embryonic tissues, and undif-
ferentiated and transformed cells, suggesting that levels
of RT expression are linked to the proliferative potential
of the cell.2 Unscheduled activity of retrotransposons
and endogenous retroviruses (ERVs) is implicated in a
variety of diseases, including cancer, while inactivation
of specific RT-encoding elements using antisense oligo-
nucleotides or ribozymes inhibited proliferation of hu-
man and murine cell lines.2
Nevirapine and efavirenz, two well-known non-
nucleoside reverse transcriptase inhibitors (NNRTIs),
are able to modulate cell growth and differentiation in
several cell lines.2,4-6 They reduce proliferation, induce
morphological differentiation, and reprogram gene ex-
pression.2 These features are reversible upon discon-
tinuation of the anti-RT treatment, suggesting that RT
contributes to an epigenetic level of control. Most
importantly, inhibition of RT activity in vivo antago-
nizes tumor growth in animal experiments. Moreover,
a Reagents and conditions: (a) n-BuLi, MeI, THF, -10 °C; (b)
SOCl2, reflux; (c) potassium ethyl 2-methylmalonate, MgCl2, TEA,
CH3CN; (d) HCl 12%; (e) thiourea, Na, EtOH, reflux; (f) c-PentBr,
K2CO3, DMF; (g) N,N-dimethylguanidine sulfate, Na, EtOH,
reflux.
pretreatment with these RT inhibitors attenuated the
tumorigenic phenotype of prostate carcinoma cells
inoculated in nude mice.2 On the basis of these data,
the endogenous RT can be regarded as an epigenetic
regulator of cell differentiation and proliferation and
may represent a novel target in cancer therapy.2
2,6-Difluoro-S-DABOs and -N-DABOs (F2-S-DABOs
and F2-N-DABOs, Figure 1) are the latest generations
of a class of NNRTIs developed by our group after a
decade of rationale lead optimization studies.7-17 In both
cell-based and enzymatic assays they are active at low
nanomolar concentrations against HIV-1 RT, thus being
more potent than nevirapine and comparable to
efavirenz. With the aim to investigate the antiprolif-
erative and cytodifferentiating activity of other NNRTIs,
we chose18 MC 1047 (1) and MC 1220 (2), respectively,
as representatives of the F2-S-DABO and F2-N-DABO
classes and tested them against endo-RT.
Derivatives 113 and 2 (Scheme 1) were prepared, both
as racemic mixtures,19 starting from the same interme-
diate: in fact, ethyl 4-(2,6-difluoro)phenyl-2-methyl-3-
oxopentanoate (5) was condensed, respectively, with
* To whom correspondence should be addressed. Phone: +396-
49913392. Fax: +396491491. E-mail: antonello.mai@uniroma1.it.
† Istituto Superiore di Sanita`.
‡ Universita` di Roma “La Sapienza”.
§ Universita` degli Studi di Salerno.
10.1021/jm0507330 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/30/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6777
Table 1. Inhibition of Proliferation by F2-DABOsa
compound (µM)
alive cells (%)b
CTRL
100
77
72
65
14
91
86
42
30
35
51
1 (10 µM)
1 (25 µM)
1 (40 µM)
1 (50 µM)
2 (10 µM)
2 (25 µM)
2 (40 µM)
2 (50 µM)
NEV (350 µM)
EFV (15 µM)c
a Cell growth in human A-375 melanoma cultures treated with
DMSO (control), 1, 2, nevirapine (NEV), and efavirenz (EFV).
b 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 Maximum testable concen-
tration.
Table 2. Effect of F2-DABOs on Cell Deatha
necrotic
apoptotic
cells (%)b
compound (µM)
alive cells (%)b
cells (%)b
control
97.6
96.7
96.4
98.7
80
0.2
1
2.2
2.3
2.6
1
12.8
1.8
2.3
3.1
8.4
1 (10 µM)
1 (25 µM)
1 (40 µM)
1 (50 µM)
2 (10 µM)
2 (25 µM)
2 (40 µM)
2 (50 µM)
1
0.3
7.2
0.8
1
0.9
2.6
97.4
96.3
96
89
a Cell death in human A-375 melanoma cultures treated with
DMSO (control), 1, and 2 for 96 h. b Counted cells are expressed
as the % of controls, taken as 100. Values represent pooled data
from three experiments.
thiourea in the presence of sodium ethoxide to yield,
after S-alkylation in dry DMF with bromocyclopentane
in the presence of potassium carbonate, compound 1 or
with N,N-dimethylguanidine sulfate in the same alka-
line medium to afford compound 2. Preparation of the
above-mentioned â-oxoester 5 was accomplished start-
ing from the methylation of (2,6-difluoro)phenylacetic
acid 3 with methyl iodide in the presence of n-butyl-
lithium in anhydrous THF to afford 2-(2,6-difluoro)-
phenylpropionic acid 4. The latter acid was treated with
thionyl chloride and then reacted with potassium mono-
ethyl-2-methylmalonate in the presence of the magne-
sium dichloride/triethylamine system13 to yield, after
decarboxylation with hydrochloric acid 12%, the re-
quired intermediate 5.
Compounds 1 and 2 (DMSO solutions) were tested in
human A-375 melanoma (ATCC-CRL-1619) cell lines in
comparison with nevirapine and efavirenz as reference
drugs.
Figure 2. Morphological differentiation of A-375 melanoma
cells in the presence of F2-DABOs. Cultures were observed by
phase-contrast microscopy.
As shown in Table 1, both of them resulted more
effective than references in reducing cell proliferation.
It is particularly noteworthy that test compounds were
more active than nevirapine at concentrations 7-fold
lower20 (compare inhibition at 50 µM of tested com-
pounds with inhibition at 350 µM of reference drug).
We next asked whether this antiproliferative effect
of the two F2-DABO could be due to A-375 cell death.
Combined staining with propidium iodide (PI) to reveal
permeable necrotic cells, 4′,6-diamidino-2-phenylindole
(DAPI) to visualize apoptotic nuclei, and 3,3 dihexyl-
oxacarbocyanine [DiOC6(3)]2,21 to monitor the loss of
mitochondrial transmembrane potential revealed (Table
2) only a low induction of cell death by either compound
even at 50 µM, the highest tested concentration (20%
after exposure to 1, 11% after exposure to 2), largely
accounted for by apoptosis (12.8% for 1, 8.4% for 2).
Neither drug exerts a significant nonspecific toxicity on
A-375 cells, thus suggesting that, as in the case of
nevirapine,5 the observed reduced cell proliferation is
mainly due to the arrest or retardation of the cell cycle.
Moreover, the modified cell shape, dendritic-like
extensions, and increased adhesion in transformed cell
lines (Figure 2) may suggest the occurrence of a dif-

6778 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Letters
ferentiation process, thus hinting2 that critical regula-
tory genes are modulated in response to the RT-
inhibitory treatment.
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Tramontano, E.; La Colla, P. 3,4-Dihydro-2-alkoxy-6-benzyl-4-
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The effects induced by compounds 1 and 2 in the
A-375 melanoma cell line closely resemble those previ-
ously observed in this cell line after exposure to chemical
inhibitors of RT (nevirapine and efavirenz)2,5 or to
genetic inactivation of the RT-encoding LINE-1 ele-
ments by RNA interference (RNAi).2 The similarity
between the cellular phenotypes induced under all of
these conditions with those presently reported for
derivatives 1 and 2 are highly significant, indicating
that a common activity is targeted in A-375 cells. Of
note, these effects are induced relatively rapidly (in the
order of days), which is a distinctive feature of RT
inhibitors (nevirapine, efavirenz, and RNAi to LINE-1)
and distinguishes them from chemical22 or genetic23
inhibition of the telomerase-associated RT. Taken to-
gether, these data support the idea that cellular alter-
ations induced by 1 and 2 truly reflect the inhibition of
the endogenous RT in A-375 cells.
In conclusion, the present work describes new data
about the antiproliferative and cytodifferentiating activ-
ity of two compounds of the F2-DABO class. In experi-
ments with human differentiating cell systems, deriva-
tives 1 and 2 significantly reduced cell proliferation and
facilitated the morphological differentiation of cells.
These results support the suggestion that NNRTIs could
be useful tools in preventive and/or curative therapy to
counteract the loss of differentiation in dedifferentiating
pathologies and as antiproliferative drugs in tumor
therapy. For the above reasons, F2-DABOs are proposed
as lead compounds for further research.
(13) Mai, A.; Sbardella, G.; Artico, M.; Ragno, R.; Massa, S.; Novellino,
E.; Greco, G.; Lavecchia, A.; Musiu, C.; La Colla, M.; Murgioni,
C.; La Colla, P.; Loddo, R. Structure-based design, synthesis,
and biological evaluation of conformationally restricted novel
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pyrimidin-4(3H)-ones as non-nucleoside inhibitors of HIV-1
reverse transcriptase. J. Med. Chem. 2001, 44, 2544-2554.
(14) Sbardella, G.; Mai, A.; Artico, M.; Chimenti, P.; Massa, S.; Loddo,
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relationship studies on new DABOs: effect of substitutions at
pyrimidine C-5 and C-6 positions on anti-HIV-1 activity. Anti-
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(15) Pani, A.; Musiu, C.; Loi, A. G.; Mai, A.; Loddo, R.; La Colla, P.;
Marongiu, M. E. DABOs as candidates to prevent mucosal HIV
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(16) Ragno, R.; Mai, A.; Sbardella, G.; Artico, M.; Massa, S.; Musiu,
C.; Mura, M.; Marceddu, T.; Cadeddu, A.; La Colla, P. Computer-
aided design, synthesis, and anti-HIV-1 activity in vitro of
2-alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkyl-
pyrimidin-4-(3H)-ones as novel potent non-nucleoside reverse
transcriptase inhibitors, also active against the Y181C variant.
J. Med. Chem. 2004, 47, 928-934.
(17) Mai, A.; Artico, M.; Ragno, R.; Sbardella, G.; Massa, S.; Musiu,
C.; Mura, M.; Marturana, F.; Cadeddu, A.; Maga, G.; La Colla,
P. 5-Alkyl-2-alkyl amino-6-(2,6-difluorophenylalkyl)-3,4-dihy-
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non-nucleoside reverse transcriptase inhibitors belonging to the
DABO family. Bioorg. Med. Chem. 2005, 13, 2065-2077.
(18) We decided to select for this screening the derivatives among
the latest F2-DABOs endowed with the best selectivity index
(cytotoxic concentration/effective concentration ratio). Com-
pounds 1 and 2 meet the requirements (references 13 and 15).
(19) As a first approach we decided to test only the two racemic forms.
Biological assays for both the enantiomers of compound 1 as well
as chiral resolution of racemic 2 are in progress.
(20) Maximum testable concentration for efavirenz in this condition
was 15 µM.
(21) A very comprehensive review of experimental methods in cell
death analysis can be found in Robinson, J. P. The Purdue
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http://flowcyt.cyto.purdue.edu/flowcyt/cdseries.htm.
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P. M.; DePinho, R. A.; Greider, C. W. Telomere shortening and
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Acknowledgment. This work was supported by
grants from PRIN 2004 and Istituto Superiore di Sanita`
(Italian National Institute of Health) (501/1 and 501/
2).
Supporting Information Available: Experimental chemi-
cal and biological procedures and characterization data for
compounds 1 and 2 and for intermediates 4 and 5. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM0507330