Ethyl 1,8-naphthyridone-3-carboxylates downregulate human papillomavirus-16 E6 and E7 oncogene expression

Article abstract of DOI:10.1021/jm500340h

Strong epidemiological and molecular data associate cervical cancer (CC) with high-risk human papillomavirus (HPV) infections. The carcinogenic mechanism depends mainly on the expression of E6 and E7 oncoproteins encoded by the viral genome. Using a cell-based high-throughput assay, an in-house library of compounds was screened identifying the 1,8-naphthyridone 1 that efficiently inhibited the transcription driven by the long control region of the HPV genome. A series of analogues were then synthesized, obtaining more potent derivatives able to downregulate E6 and E7 transcripts in HPV-16-positive CC CaSki cells. An unusual structural insight emerged for the C-3 position of the 1,8-naphthyridone core, where the ethyl carboxylate esters, but not the carboxylic acids, are responsible for the activity. In vitro uptake studies showed that the 3-ethyl carboxylates do not act as prodrugs. The 1,8-naphthyridones emerged as valid starting points for the development of innovative agents potentially useful for the treatment of HPV-induced CC.

Full text of DOI:10.1021/jm500340h

Article
pubs.acs.org/jmc
Ethyl 1,8-Naphthyridone-3-carboxylates Downregulate Human
Papillomavirus-16 E6 and E7 Oncogene Expression
Manuela Donalisio,† Serena Massari,*,‡ Monica Argenziano,§ Giuseppe Manfroni,‡ Valeria Cagno,†
Andrea Civra,† Stefano Sabatini,‡ Violetta Cecchetti,‡ Arianna Loregian,∥ Roberta Cavalli,§
David Lembo,*,† and Oriana Tabarrini‡
†Department of Clinical and Biological Sciences, University of Torino, 10043 Orbassano, Torino, Italy
‡Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy
§Department of Drug Science and Technology, University of Torino, 10125 Torino, Italy
∥Department of Molecular Medicine, University of Padova, 35121 Padova, Italy
S
* Supporting Information
ABSTRACT: Strong epidemiological and molecular data associate cervical cancer (CC) with high-risk human papillomavirus
(HPV) infections. The carcinogenic mechanism depends mainly on the expression of E6 and E7 oncoproteins encoded by the
viral genome. Using a cell-based high-throughput assay, an in-house library of compounds was screened identifying the 1,8-
naphthyridone 1 that efficiently inhibited the transcription driven by the long control region of the HPV genome. A series of
analogues were then synthesized, obtaining more potent derivatives able to downregulate E6 and E7 transcripts in HPV-16-
positive CC CaSki cells. An unusual structural insight emerged for the C-3 position of the 1,8-naphthyridone core, where the
ethyl carboxylate esters, but not the carboxylic acids, are responsible for the activity. In vitro uptake studies showed that the 3-
ethyl carboxylates do not act as prodrugs. The 1,8-naphthyridones emerged as valid starting points for the development of
innovative agents potentially useful for the treatment of HPV-induced CC.
through the introduction of prophylactic vaccines that prevent
infections from four HPV types that cause about 70% of CCs
(types 16 and 18) and 90% of genital warts (types 6 and 11)
worldwide.6 However, these achievements are insufficient, and
high mortality from CC still persists. Furthermore, the
currently used therapy is nonspecific with many associated
toxicity problems,7,8 and no drugs directed against viral targets
actually exist.
The continuous expression of E6 and E7 proteins encoded
by high-risk HPVs plays a critical role in CC, contributing to
the maintenance of proliferation, to genomic instability, and to
the transformation of primary human keratinocytes into
malignant cells.9−11 The physical and functional interaction of
E6 and E7 oncoproteins with tumor suppressor proteins p53
INTRODUCTION
■
Human papillomaviruses (HPVs) are small double-stranded
DNA viruses that infect the epithelial cells of the skin and the
anogenital or oropharyngeal mucosa, causing benign and
malignant neoplastic lesions. Infection with high-risk HPV
types (e.g., HPV-16, HPV-18, HPV-31, and HPV-45) is a major
cause of cervical cancer (CC), one of the most common types
of gynecological malignancies among women worldwide,1,2
with more than 528 000 estimated new cases every year, and
fourth for mortality, with 266 000 deaths in 2012.3,4
Epidemiologically, HPV-16 is the most prevalent in CC,
accounting for 65.2% of all genotypes.5 Surgery is the current
treatment for HPV-associated cancers, but the virus can persist
in healthy tissues and favor tumor growth. Significant benefits
in incidence of CC and patients survival have been achieved
through screening programs that allow the detection of
epithelial alterations and lesions in early stages and, recently,
Received: March 4, 2014
Published: June 6, 2014
© 2014 American Chemical Society
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a
Table 1. Structure of Selected Quinolone-Based Derivatives
a
The synthetic procedure for the preparation of compounds 10−13, 16, 17, 19, and 20 is reported in the Supporting Information.
and pRb leads to the subversion of cellular functions such as
cell-cycle control, apoptosis, senescence, DNA repair, and
genomic stability.12−14 Since most HPV-induced tumors still
contain wild-type p53 and pRb, the inhibition of E6 and E7
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expression may rescue the tumor suppressor protein pathways,
resulting in the inhibition of cell proliferation and induction of
cellular senescence or apoptosis. Therefore, the two viral
oncoproteins and their expression are considered the “Achilles’
heel” of CC and thus the ideal molecular targets for the
development of innovative therapies. Selective silencing of E6
and E7 expression by macromolecule-based approaches such as
rybozymes, antisense RNA, and siRNA,15−20 provided the
proof of concept, but their use is limited by difficulties in
delivering therapeutically active concentrations at specific
anatomic sites. Ideally, the identification of small-molecule
inhibitors of E6 and E7 expression would facilitate the
development of a drug against the HPV-induced CC. However,
agents aimed at targeting directly or indirectly these CC-driven
viral oncogenes are in very early stages of development.21−23
The expression of HPV-16 E6 and E7 is controlled by the
long control region (LCR), a complex regulatory DNA
sequence located immediately upstream of the E6 gene in the
HPV genome.14,24 In a previous paper, we described the
development of a cell-based high-throughput assay to screen
small molecules as inhibitors of HPV-16 LCR transcriptional
activity.25
with an IC50 value of 1.12 μM (Figure 1). Compound 1 did not
show any antiproliferative activity on the same cell line at 100
Figure 1. Dose-dependent reduction of LCR-driven transcription by
compound 1. P21 cells were seeded on 24-well plates and then treated
for 24 h with increasing concentrations of the compound. The results
represent the mean SD of three indipendent experiments performed
in duplicate.
μM concentration, indicating that the inhibition of the LCR
In the present study, this assay was used to screen a set of
compounds selected within a proprietary library. Compound 1
(Table 1) emerged as a very promising inhibitor of E6 and E7
oncogene expression, prompting the design, synthesis, and
biological characterization of a series of analogues.
activity was not a consequence of a reduced cell viability.
STRUCTURAL MODIFICATIONS OF COMPOUND 1
■
According to the IC50 value, compound 1 induced potent
inhibition of the LCR activity without showing appreciable
toxicity, prompting the design and synthesis of additional
derivatives to identify the structural features responsible for this
ability. The activity of 1, the only 1,8-naphthyridone derivative
assayed within the initial set of compounds, was quite
unexpected. Indeed, it bears an unusual benzyl moiety at the
N-1 position, but most importantly, it is a C-3 ester derivative.
In fact, the vast majority of the 6-fluoroquinolones and 6-DFQs
until now reported as antibacterial,34−39 including antituber-
cular,40−42 anti-HIV,26 and anti-HCMV30,31,43 agents are
characterized by a carboxylic acid group at the C-3 position.
Of note, compound 3 (Table 1), the other ester derivative
tested, did not show any inhibitory activity.
HIT IDENTIFICATION
■
To identify small-molecule inhibitors of HPV-16 LCR
transcriptional activity, we focused our attention on quino-
lone-based derivatives thanks to their capability to inhibit the
transcription processes of different viruses. The ability of the 6-
desfluoroquinolones (6-DFQs) to inhibit human immunodefi-
ciency virus type 1 (HIV-1) gene expression by selectively
interfering with the Tat-mediated transcription step is well
documented,26−29 as well as their anti human cytomegalovirus
(HCMV) activity ascribable to the specific inhibition of the
immediate early 2-mediated transactivation of viral pro-
moters.30,31 In particular, 22 compounds (Table 1) were
selected, including mainly anti-HIV and anti-HCMV quino-
lones and a few intermediates (compounds 1−18), together
with two antibacterial quinolones (compounds 19 and 20) and
two anti hepatitis C virus acridones (compounds 21 and 22).
The ability of the compounds to inhibit the HPV-16 LCR-
driven transcription was assayed using P21 cells that are a clone
of keratinocytes stably transfected with a reporter construct
containing the HPV-16 LCR positioned upstream from the
firefly luciferase gene.25 The cells were initially treated with two
different concentrations of compounds (100 and 30 μM) and
then assayed for luciferase activity reported as the percentage of
LCR inhibition (Table S1, Supporting Information). Many of
the compounds were essentially inactive even at 100 μM, while
others showed a weak inhibitory activity at 100 μM but were
inactive at 30 μM. However, compound 1 exhibited a high
percentage of inhibition both at 100 μM (98.8%) and at the
lower concentration of 30 μM (92%). TGF-β1 and interleukin
4 (IL-4), known inhibitors of HPV-16 LCR activity used as
internal references,32,33 reduced the LCR activity by 70.5% and
69.8%, respectively, when used at 50 ng/mL (data not shown).
When assayed in P21 cells at different concentrations
(between 0.01 and 100 μM), compound 1 inhibited the LCR
transcriptional activity in a concentration-dependent manner
Starting from the hit 1, a first set of naphthyridone analogues
was designed, maintaining both the ethyl carboxylate group and
the 3-chloro-2-fluorobenzyl moiety at the C-3 and N-1
positions, respectively, while modifying the 2-pyridylpiperazine
ring at the C-7 position (Figure 2). In particular, the pyridine
moiety was replaced by some of the heterocyclic bases that
usually impart antiviral activity to the quinolone compounds,
such as 1,3-benzothiazole, quinoline, 1,3-thiazole, 1,3-benzox-
azole, and 3-(trifluoromethyl)phenyl rings, synthesizing de-
rivatives 23−27. With the aim to investigate the role played by
the C-3 ester moiety, the corresponding acid derivatives
(compounds 29−33) were also synthesized together with
compound 28, the acid counterpart of the hit 1.
The biological evaluation of this first set of naphthyridone
analogues led to the identification of the 7-benzothiazolylpiper-
azine derivative 23 as an extremely interesting new molecule,
which was 5 times more active than compound 1 while
maintaining the absence of cytotoxicity up to the maximum
concentration tested of 100 μM. Thus, a second set of
compounds was designed starting from the new hit 23, keeping
fixed the 1,3-benzothiazolylpiperazine moiety at the C-7
position while varying the N-1 substituent (Figure 2). In
particular, the role of the halogens on the benzyl group was
studied by synthesizing compounds lacking the 3-chloro
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Figure 2. From hit compound 1 to an enlarged series of analogues.
(compound 34), the 2-fluoro (compound 35), and both the
halogens (compound 36). To explore the C-4 position of the
N1-benzyl ring, the chlorine derivative 37 as well as the 4-
trifluoromethyl derivative 38 were prepared. Then the 3-chloro-
2-fluorophenyl moiety was replaced by various heteroaromatic
rings, such as 2-pyridine (compound 39), 2-furan (compound
40), 5-indole (compound 41), and 2-benzimidazole (com-
pound 42). Finally, the aromatic ring of compounds 34, 37, and
39 was spaced from the 1,8-naphthyridone scaffold by inserting
an additional methylene unit, obtaining compounds 43−45.
Few additional 3-carboxylic acid analogues (compounds 46−
49) were synthesized within this second set of compounds.
RESULTS AND DISCUSSION
■
All the newly synthesized compounds were initially assayed for
their ability to inhibit HPV-16 LCR activity at 100 and 30 μM
concentrations in P21 cells. Unfortunately, the poor solubility
of compounds 32 and 47 in DMSO hampered their biological
evaluation. In addition, compounds 27, 33, 40, and 43 caused a
noticeable alteration of P21 cultured cells, thus preventing their
evaluation in the luciferase assay.
Analyzing the data summarized in Table 2, it clearly emerged
that many of the new derivatives were active in inhibiting the
LCR activity at both the concentrations tested. By selecting the
compounds with the higher percentage of inhibition, the IC50
value was determined for 14 derivatives. These derivatives
reduced the LCR activity in a concentration-dependent fashion
(data not shown) with IC50 values ranging from 0.13 to 10.90
μM (Table 2). In parallel, the compounds were also tested for
antiproliferative activity in the same cell line. In a few cases they
exhibited some cytotoxicity but always at concentrations higher
than those producing LCR inhibition. Comparing the biological
data obtained for the 14 derivatives with those of compound 1,
some structural considerations can be highlighted. The
modifications made at the C-7 position in most cases were
very productive, improving the activity as in compounds 23, 24,
and 26. In particular, the benzothiazolylpiperazine derivative 23
was endowed with the best profile, showing IC50 = 0.26 μM,
which, coupled with the lack of cytotoxicity (CC50 > 100 μM;
CC50 is the concentration of compound that reduced cellular
viability by 50%), led to a selectivity index (SI, ratio of CC50 to
CHEMISTRY
■
All the 1,8-naphthyridones synthesized in this study were
prepared following the cycloaracylation procedure, as depicted
in Scheme 1. Thus, acrylate 5044 was reacted with variously
substituted arylalkylamines in an Et2O/EtOH mixture to give
intermediates 51−63, which were then cyclized in the presence
of K2CO3 in DMF to give synthones 64−76.
The nucleophilic reaction of N1-(3-chloro-2-fluorobenzyl)-
naphthyridone 64 with selected arylpiperazines gave ester
derivatives 1 and 23−27, which were then hydrolyzed in basic
conditions to the corresponding acids 28−33. On the other
hand, the nucleophilic reaction of 7-chloro synthones 65, 66,
67,45 and 68−76, differently functionalized at the N-1 position,
with 2-(piperazin-1-yl)benzothiazole46 furnished the ester
derivatives 34−45, of which compounds 36, 37, 39, and 45
were then hydrolyzed to the corresponding acids 46−49.
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a
Scheme 1
a
Reagents and conditions: (i) arylalkylamine, Et2O/EtOH; (ii) K2CO3, DMF, 60 °C; (iii) 4-arylpiperazine, DMF, 80−90 °C; (iv) 4% NaOH, reflux
or 4% NaOH, dioxane, microwave, 120 °C.
IC50) of >385. A similar behavior was exhibited by its 4-
benzoxazolyl bioisostere 26. A comparable potency was shown
by compound 24 (IC50 = 0.21 μM), even if coupled with some
cytotoxicity (CC50 = 59.3 μM). The thiazolylpiperazine
appeared a less suitable C-7 substituent, since compound 25
showed the same activity (IC50 = 1.21 μM) as compound 1, but
a markedly higher antiproliferative effect (CC50 = 25.3 μM)
than compound 1. Unfortunately, the high toxicity of
compound 27 hampered the evaluation of the contribution of
the [3-(trifluoromethyl)phenyl]piperazine.
Electing 23 as a new hit compound, the modifications then
involved the 3-chloro-2-fluorobenzyl group at the N-1 position.
The removal of the chlorine (compound 34) or the fluorine
(compound 35) atom proved to be unsuccessful. On the
contrary, compound 36, lacking both the halogens, exhibited
the same good activity (IC50 = 0.23 μM) as compound 23 (IC50
contrasting results. While the presence of furan (compound
40), 2-benzimidazole (compound 42), and 2-pyridine (com-
pound 39) was not fruitful, the 5-indole moiety markedly
improved the activity. Indeed, with IC50 = 0.13 μM, derivative
41 emerged as the most active and endowed with the highest SI
value (>769). Finally, the spacing of the o-fluorobenzyl, p-
chlorobenzyl, and pyridine moieties from the N-1 position by
an additional methylenic unit was not productive, derivatives
43−45 being inactive or toxic.
For some of the 3-ethyl carboxylate target compounds, the
corresponding acids were also synthesized and tested,
permitting clear SAR insights for the C-3 position to be
obtained. Analyzing the data reported in Table 2 for the ester
(1, 23−26, 36, 39, and 45)/acid (28−31, 48, and 49) pairs, the
ester derivatives always resulted in more potent activity, while
some 3-carboxylic acids were even completely inactive. This is
true also for compound 28, the acid analogue of the hit 1.
Since the end point of the primary luciferase assay used for
hit identification is measured enzymatically, a subsequent
confirmatory assay was run to eliminate potential false positives
caused by luciferase inhibition, a possibility reported in previous
literature.47 Therefore, in the next step of the study, we tested
the ability of the molecules to repress HPV-16 oncogene
transcription in the HPV-16-positive cervical carcinoma-derived
CaSki cell line. In particular, we evaluated the effect of the
= 0.26 μM) although coupled with a higher toxicity (CC50
=
61.7 μM). These data suggested that the presence of both
halogens is important to improve the selectivity. The presence
of a substituent at the para position of the benzyl group
decreased the activity as in the p-chloro derivative 37 (IC50
value of 2.37 μM) and even more in the p-trifluoromethyl
derivative 38 (IC50 = 10.90 μM).
The insertion of various heteroaromatic rings at the N-1
position instead of the 3-chloro-2-fluorophenyl moiety gave
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Table 2. Effect of Compound 1 and Its Analogues on HPV-16 LCR Activity and Cytotoxicity on P21 Reporter Cells
LCR inhibition (%) (mean values SD)
a
c
b
d
compd
100 μM
30 μM
IC50 (μM) (95% CI)
CC50 (μM)
SI
1
23
24
25
26
28
29
30
31
34
35
36
37
38
39
41
42
44
45
46
48
49
93.1 3.1
94.9 1.7
93.7 1.8
99.7 2.3
83.9 1.8
25.8 3.7
56.0 0.7
100 0.1
2.1 0.7
75.6 0.8
96 3.2
89.2 1.8
82.8 3.1
92.3 4.5
98.0 2.1
88.1 3.2
30.5 0.5
38.3 3.8
99.3 0.7
7.39 0.9
88.6 1.4
96 1.9
1.12 (0.83−1.50)
0.26 (0.16−0.42)
0.21 (0.12−0.35)
1.21 (0.95− 1.53)
0.39 (0.02−7.74)
>100
>100
59.3
25.3
>100
NT
>89.2
>385
282
20.9
>256
e
NT
NT
NT
f
2.86 (1.34−268)
17.6
NT
6.1
NT
4.18 (0.84−20.8)
2.62 (0.02−24.5)
0.23 (0.08−0.61)
2.37 (1.62−3.47)
>100
>100
61.7
>100
78.2
34.6
>100
NT
>23.9
>38.1
268
98.1 0.2
90 0.7
98.2 4.1
90 1.7
>42.1
7.16
f
89.3 1.8
73.4 1.3
90.2 1.2
45.5 0.7
50.4 2.0
100 0.2
100 1.9
61.0 2.9
0
67.5 2.3
63.1 2.4
93.6 3.0
38.3 2.2
49.6 4.1
87 2.4
10.9 (1.61−73.8)
f
5.30 (0.15−182)
6.5
0.13 (0.0−4.20)
NT
>769
NT
NT
f
1.05 (0.54−291)
17.1
69.6
NT
16.2
15.2
100 0.5
24.0 1.3
0
4.55 (3.56−5.80)
NT
NT
NT
a
b
IC50 = concentration of compound which reduced luciferase activity by 50%. CC50 = concentration of compound that reduced cellular viability by
c
d
e
50%. CI = confidence interval. SI = CC50/IC50 ratio. NT = not tested. The IC50 and CC50 values were not determined due to the low LCR
inhibition. Wide 95% CI. All data represent the mean value of three similar experiments performed in duplicate. Compounds 32 and 47 were not
f
tested due to their low solubility in DMSO. Compounds 27, 33, 40, and 43 caused a noticeable alteration of cultured cells, not permitting the
evaluation of LCR inhibition.
molecules that showed the best biological profile (compounds
1, 23−26, 35−37, and 41) together with their corresponding 3-
carboxylic acids (compounds 28−31 and 46) on HPV-16 E6
and E7 mRNA levels. To this aim, the cells were treated with a
10 μM concentration of the compounds, a concentration that
reduced the luciferase activity more than 80% for 24 h, and
then the levels of E6 and E7 mRNAs were examined by RT-
PCR. Figure 3 shows that, with the exception of compound 37,
all the 1,8-naphthyridone 3-ethyl esters downregulated, in a
statistically significant manner, the expression of E6 and E7
transcripts, though to a different extent. In particular,
quinolinylpiperazine derivative 24 and 1,3-thiazolylpiperazine
derivative 25 strongly suppressed HPV transcription, inhibiting
E6 mRNA levels by 61.5% and 71.7% and E7 mRNA levels by
57.4% and 72.7%, respectively. TGF-β1,32,33 used as a positive
control, showed 65.3% and 60.7% inhibition of the E6 and E7
transcripts, respectively. Of note, none of the compounds
Figure 3. Effect of molecules on the steady-state levels of E6 and E7
mRNAs in CaSki cells. Cells were treated with a 10 μM concentration
tested showed any antiproliferative effect on CaSki cells at the
concentration and time point chosen for the RT-PCR (data not
shown). Confirming the data measured in the luciferase assay,
all the tested 3-carboxylic acids failed to inhibit E6 and E7
mRNA levels. Overall, the secondary screening based on the
RT-PCR assay independently confirmed the hit compounds
identified by the primary assay and put forward the luciferase-
based assay as a reliable tool for the initial identification of
candidate small molecules capable of downregulating HPV-16
oncogene expression in HPV-16-positive human cancer cells.
Since the quinolone derivatives are able to inhibit the
transcription processes of different viruses, depending on the
substitution patterns, we decided to investigate the selectivity of
the derivatives herein identified. Thus, hit compound 23 was
of the compounds or 50 ng/mL TGF-β1 for 24 h. mRNA was
retrotranscribed, and the levels of HPV-16 E6 and E7 transcripts were
determined by real-time PCR. The results shown are the mean of three
independent experiments. An asterisk indicates statistical significance
for E6 or E7 (P < 0.05).
tested against the DNA virus herpes simplex type 2 (HSV-2)
and some RNA viruses, namely, HIV-1, vesicular stomatitis
virus, respiratory syncytial virus, and human rotavirus (VSV,
RSV, and HRV, respectively). The antiviral assays showed that
compound 23 is inactive against HIV-1 and HRV, barely active
against RSV, and moderately active against HSV-2 and VSV
(see Table S2, Supporting Information).
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As mentioned before, the greater activity of the 3-ethyl ester
derivatives compared to the acid analogues is quite unusual in
the quinolone field, since the majority of the biologically active
quinolone-based compounds bear a carboxylic acid group at the
C-3 position.48 Its key role in the mechanism of action of the
antibacterial quinolone drugs, where a Mg2+ bridge is formed
between the quinolone 4-keto-3-carboxyl moiety and the DNA
phosphodiester backbone, has been extensively reported.49,50
Most of the modifications of the carboxylic acid moiety
produced no active compounds51 with few exceptions, such as
certain carboxylate esters which are converted in vivo back to a
carboxylic acid.52
To investigate the role played by the C-3 substituent in the
naphthyridones targeting HPV E6 and E7 oncogene expression,
we performed an uptake study to understand whether the
higher activity of the ethyl 3-carboxylates might be due to a
better intracellular penetration and to clarify whether they are
the active forms in the cellular context. To this end, CaSki cells
were treated with three pairs of derivatives, the esters 23−25,
selected among the most active compounds in real-time PCR
assays, and the corresponding acid derivatives 29−31 at fixed
concentrations and different times of incubation up to 16 h.
After incubation with the compounds, the cells were washed
twice with phosphate-buffered saline (PBS) and lysed with a
solution containing an excess of ammonium sulfate; then the
cell lysates were subjected to quantitative chromatographic
analyses as described in the Experimental Section.
As shown in Figure 4, after cell incubation with acid
derivatives, a marked uptake (about 20%) was detected by the
first hour. The intracellular percentage of the acid derivative
increased over time for all the compounds and reached 35%,
38%, and 63% for 29, 30, and 31, respectively, after 16 h. These
results clearly demonstrated that the acids are able to penetrate
CaSki cells, and thus, their inactivity is most likely due to the
unsuitability of the 3-carboxylic group to properly interact with
the specific target. The results obtained with ester derivatives
23−25 showed a different behavior. Indeed, after 1 h of
incubation no carboxylate ester was detected in the cell lysates.
The intracellular amount of all the esters tested increased over
time, reaching 15%, 40%, and 28% for compounds 23, 24, and
25, respectively, after 16 h. Interestingly, when the cells were
incubated with ester derivatives, a remarkable intracellular
percentage of the acid counterpart was detected at each time
point in the chromatogram, reaching 30%, 52%, and 70% for
compounds 23, 24, and 25, respectively, at 16 h. These data
suggest that the cell uptake of ester derivatives seems favored,
but as soon as they are internalized, the metabolism to the
corresponding acid forms occurs. Considering that the ester
amount inside the cells increases over time while only the acid
counterpart is initially detectable, the enzymatic hydrolysis
appears as a saturable process.
In summary, the uptake studies show that both acid and ester
derivatives are able to cross the cell membranes and the ethyl
1,8-naphthyridone-3-carboxylates are not prodrugs but the
biologically active form. Actually, since the acids are inactive
although they are capable of permeating CaSki cells, the ethyl
esters seem the sole active forms. Most likely, they would be
even more potent if they were not so extensively metabolized.
The synthesis of different naphthyridone ester derivatives less
prone to enzymatic hydrolysis is required to validate this
hypothesis.
Figure 4. Cell uptake percentages of 1,8-naphthyridone acids, ester
counterparts, and acids derived from esters determined in cell lysate 1,
2, 4, and 16 h postincubation.
CONCLUSIONS
■
Although the prophylactic vaccines recently introduced prevent
infections from HPV types 16 and 18, high mortality from CC
still persists. Furthermore, the currently used therapeutic
approach is nonspecific and is associated with toxicity
problems, and no drugs directed against specific viral targets
actually exist. Thus, the medical need for new anti-HPV drugs is
still urgent with a preference for small molecules targeting viral
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aromatic CH), 7.75 (d, J = 7.9 Hz, 1H, H-4′), 8.35 (d, J = 13.8 Hz, 1H,
H-3), 11.00−11.10 (m, 1H, NH).
components. The 1,8-naphthyridone derivatives identified in
this study could meet this urgent request. Indeed, when
properly functionalized at the N-1, C-3, and C-7 positions, they
are able to inhibit the expression of HPV-16 E6 and E7
oncogenes at subcytotoxic concentrations. The best com-
pounds are characterized by a 3-chloro-2-fluorobenzyl moiety at
the N-1 position coupled with a quinolinylpiperazine
(compound 24) or a 1,3-thiazolylpiperazine (compound 25)
at the C-7 position. An interesting SAR insight emerged for the
C-3 position of the 1,8-naphthyridone scaffold, where, in
contrast to most of the biologically active quinolones, an ethyl
carboxylate ester, instead of the usual 3-carboxylic moiety, is
crucial for the activity. However, a few recent publications
reporting quinolone carboxylate esters endowed with different
biological activities are emerging.53−56
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-[(2-
fluorobenzyl)amino]acrylate (52). The title compound was
prepared starting from 5044 through method A (20 min) using (2-
fluorobenzyl)amine in 75% yield: mp 161−162 °C; 1H NMR (DMSO-
d6) 0.68 and 0.85 (each t, J = 7.1 Hz, 0.65 and 2.35 H, CH2CH3), 3.80
(q, J = 7.1 Hz, 2H, CH2CH3), 4.60 (br d, J = 6.0 Hz, 2H, CH2), 7.10−
7.45 (m, 4H, aromatic CH), 7.51 (d, J = 8.0 Hz, 1H, H-4′), 7.75 (d, J =
8.0 Hz, 1H, H-5′), 8.30 (d, J = 14.0 Hz, 1H, H-3), 9.80−9.95 and
11.00−11.20 (each m, 0.2H and 0.8H, NH).
Ethyl 3-[(3-Chlorobenzyl)amino]-2-[(2,6-dichloropyridin-3-
yl)carbonyl]acrylate (53). The title compound was prepared starting
from 5044 through method A (40 min) using (3-chlorobenzyl)amine
1
in 77% yield: mp 156−157 °C; H NMR (DMSO-d6) 0.75 and 0.90
(each t, J = 7.1 Hz, 0.7 and 2.3 H, CH2CH3), 3.80−3.90 (m, 2H,
CH2CH3), 4.70−4.75 (m, 2H, CH2), 7.32−7.50 (m, 4H, aromatic
CH), 7.55 (d, J = 8.0 Hz, 1H, H-5′), 7.80 (d, J = 8.0 Hz, 1H, H-4′),
8.40 (d, J = 14.0 Hz, 1H, H-3), 9.95−10.00 and 11.10−11.15 (each m,
0.25H and 0.75H, NH).
Uptake studies performed on 1,8-naphthyridone acid/ester
pairs clearly showed that the ethyl esters are the active forms
responsible for the anti-HPV activity and do not act as
prodrugs.
Ethyl 3-(Benzylamino)-2-[(2,6-dichloropyridin-3-yl)-
carbonyl]acrylate (54). The title compound was prepared starting
from 5044 through method A (40 min) using 1-phenylmethanamine,
Within the limited landscape of compounds that target the
oncogenic effects of HPV E6 and E7,21−23,57 the quinolone-
based derivatives herein identified are a rare example of small
molecules able to downregulate the expression of CC-driven
players E6 and E7. Although particularly promising, preliminary
studies showed that they are not potent enough to significantly
restore the cell growth control and induce the senescent
phenotype in HPV-16-positive CC cell lines (data not shown).
Thus, these hit compounds ask for further structural
optimization, which is mandatory to achieve more potent
analogues able to rescue p53- and Rb-dependent responses and
thus potentially useful for the treatment of CC as well as of
other HPV-induced anogenital and oropharyngeal cancers.
1
in 70% yield: mp 123−124 °C; H NMR (DMSO-d6) 0.65 and 0.80
(each t, J = 7.1 Hz, 0.77 and 2.23 H, CH2CH3), 3.75−3.80 (m, 2H,
CH2CH3), 4.65−4.75 (m, 2H, CH2), 7.25−7.40 (m, 5H, aromatic
CH), 7.55 (d, J = 8.0 Hz, 1H, H-5′), 7.75 (d, J = 8.0 Hz, 1H, H-4′),
8.30 (d, J = 14.6 Hz, 1H, H-3), 9.85−10.00 and 11.00−11.10 (each m,
0.22H and 0.78H, NH).
Ethyl 3-[(4-Chlorobenzyl)amino]-2-[(2,6-dichloropyridin-3-
yl)carbonyl]acrylate (55). The title compound was prepared starting
from 5044 through method A (1 h) using (4-chlorobenzyl)amine
1
hydrochloride in 66% yield: mp 135−136 °C; H NMR (DMSO-d6)
0.65 and 0.85 (each t, J = 7.1, 1H and 2H, CH2CH3), 3.75−3.90 (m,
2H, CH2CH3), 4.65 (br d, J = 6.0 Hz, 2H, CH2), 7.30−7.45 (m, 4H,
aromatic CH), 7.50 (d, J = 8.0 Hz, 1H, H-5′), 7.70 (d, J = 8.0 Hz, 1H,
H-4′), 8.30 (d, J = 14.0 Hz, 1H, H-3), 9.80−10.00 and 11.00−11.20
(each m, 0.3H and 0.7H, NH).
EXPERIMENTAL SECTION
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-{[4-
(trifluoromethyl)benzyl]amino}acrylate (56). The title compound
was prepared starting from 5044 through method A (6 h) using [4-
(trifluoromethyl)benzyl]amine in 90% yield: mp 110−112 °C; 1H
NMR (DMSO-d6) 0.75 and 0.90 (each t, J = 7.1 Hz, 0.8H and 2.2H,
CH2CH3), 3.85−3.90 (m, 2H, CH2CH3), 4.80 (br d, J = 5.8 Hz, 2H,
CH2), 7.55−7.65 (m, 3H, H-5′ and aromatic CH), 7.70−7.80 (m, 3H,
H-4′ and aromatic CH), 8.40 (d, J = 14.5 Hz, 1H, H-3), 10.00−10.05
and 11.10−11.20 (each m, 0.3H and 0.7H, NH).
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-[(pyridin-2-
ylmethyl)amino]acrylate (57). The title compound was prepared
starting from 5044 through method A (4 h) using (pyridin-2-
ylmethyl)amine in 91% yield: mp 157−158 °C; 1H NMR (DMSO-d6)
0.75 and 0.90 (each t, J = 7.0 Hz, 1H and 2H, CH2CH3), 3.80 (q, J =
7.0 Hz, 2H, CH2CH3), 4.80 (br s, 2H, CH2), 7.25−7.35 (m, 2H,
pyridine CH), 7.55 (d, J = 8.0 Hz, 1H, H-5′), 7.60−7.75 (m, 2H, H-4′
and pyridine CH), 8.40 (d, J = 14.4 Hz, 1H, H-3), 8.45−8.55 (m, 1H,
pyridine CH), 9.90−10.00 and 11.20−11.30 (each m, 0.2H and 0.8H,
NH).
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-[(2-
furylmethyl)amino]acrylate (58). The title compound was
prepared starting from 5044 through method A (30 min) using (2-
furylmethyl)amine in 87% yield: mp 132−134 °C; 1H NMR (DMSO-
d6) 0.85 (t, J = 7.0 Hz, 3H, CH2CH3), 3.80 (q, J = 7.0 Hz, 2H,
CH2CH3), 4.70 (br s, 2H, CH2), 6.30−6.45 (m, 2H, furan CH), 7.55
(d, J = 12.0 Hz, 1H, H-5′), 7.60−7.65 (m, 1H, furan CH), 7.70−7.80
(m, 1H, H-4′), 8.25 (d, J = 14.7 Hz, 1H, H-3), 10.90−11.00 (m, 1H,
NH).
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-[(1H-indol-5-
ylmethyl)amino]acrylate (59). The title compound was prepared
starting from 5044 through method A (1.2 h) using (1H-indol-5-
ylmethyl)amine in 96% yield: mp 141−144 °C; 1H NMR (DMSO-d6)
0.85 and 0.90 (each t, J = 7.0 Hz, 0.7H and 2.3H, CH2CH3), 3.80 (q, J
■
Chemistry. All reactions were routinely checked by TLC on silica
gel 60F254 (Merck) and visualized by using UV or iodine. Flash
column chromatography separations were carried out on Merck silica
gel 60 (mesh 230−400). Melting points were determined in capillary
tubes (Buchi Electrothermal model 9100) and are uncorrected. The
̈
purity of the compounds was determined by combustion analysis
employing a Fisons elemental analyzer, model EA1108CHN, and data
for C, H, and N are within 0.4% of the theoretical values (purity of
1
≥95%). H NMR and 13C NMR spectra were recorded at 200 MHz
(Bruker Avance DPX-200) and 400 MHz (Bruker Avance DRX-400)
using residual solvents such as chloroform (δ = 7.26) or dimethyl
sulfoxide (δ = 2.48) as an internal standard. Chemical shifts are given
in parts per million (δ), and the spectral data are consistent with the
assigned structures. For the microwave synthesis, a Biotage Initiator
Sixty (400 W) was used. Reagents and solvents were purchased from
common commercial suppliers and were used as such. After extraction,
organic solutions were dried over anhydrous Na2SO4, filtered, and
concentrated with a Buchi rotary evaporator at reduced pressure.
̈
Yields are of purified product and were not optimized. All starting
materials were commercially available unless otherwise indicated.
Preparation of Ethyl 3-[(3-Chloro-2-fluorobenzyl)amino]-2-
[(2,6-dichloropyridin-3-yl)carbonyl]acrylate (51; Method A). A
mixture of ethyl 2-[(2,6-dichloropyridin-3-yl)carbonyl]-3-
(dimethylamino)acrylate (50)44 (1.0 g, 3.1 mmol) and (3-chloro-2-
fluorophenyl)methanamine (0.6 g, 3.8 mmol) in Et2O/EtOH (4:1, 15
mL) was stirred at room temperature for 2 h. The reaction mixture
was then evaporated to dryness to give a residue which after treatment
with cyclohexane gave a solid which was filtered to give 51 (1.27 g,
95%): mp 155−156 °C; 1H NMR (DMSO-d6) 0.80 (t, J = 7.1 Hz, 3H,
CH2CH3), 3.75 (q, J = 7.1 Hz, 2H, CH2CH3), 4.75 (bs, 2H, CH2),
7.20−7.45 (m, 2H, aromatic CH), 7.50−7.60 (m, 2H, H-5′ and
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= 7.0 Hz, 2H, CH2CH3), 4.70 (br s, 2H, CH2), 6.35−6.45 and 7.0−
7.10 (m, each 1H, indole CH), 7.20−7.40 (m, 2H, indole CH), 7.40−
7.55 (m, 2H, H-5′ and indole CH), 7.75 (d, J = 8.0 Hz 1H, H-4′), 8.30
(d, J = 14.4 Hz, 1H, H-3), 11.10 (br s, 2H, NH and indole NH).
Ethyl 3-[(1H-Benzimidazol-2-ylmethyl)amino]-2-[(2,6-di-
chloropyridin-3-yl)carbonyl]acrylate (60). The title compound
was prepared starting from 5044 through method A (12 h) using (1H-
benzimidazol-2-ylmethyl)amine, with the exception that the com-
pound was further purified by flash chromatography eluting with
MeOH/CHCl3 (3%), in 67% yield: mp 110−113 °C; 1H NMR
(DMSO-d6) 0.85 and 1.00 (each t, J = 7.0 Hz, 3H, CH2CH3), 3.80 (q,
J = 7.0 Hz, 2H, CH2CH3), 5.0 (br s, 2H, CH2), 7.10−7.25 (m, 2H,
benzimidazole CH), 7.50−7.60 (m, 3H, H-5′ and benzimidazole CH),
7.75−7.80 (m, 1H, H-4′), 8.45 (d, J = 14.4 Hz, 1H, H-3), 11.05−11.10
(m, 1H, NH), 12.20 (br s, 1H, benzimidazole NH).
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-{[2-(2-
fluorophenyl)ethyl]amino}acrylate (61). The title compound
was prepared starting from 5044 through method A (50 min) using
[2-(2-fluorophenyl)ethyl]amine in 75% yield: mp 61−62 °C; 1H
NMR (DMSO-d6) 0.75 and 0.90 (each t, J = 7.1 Hz, 0.6H and 2.4H,
CH2CH3), 2.80−3.10 (m, 2H, CH2), 3.70−3.75 (m, 2H, NCH2),
3.80−3.90 (m, 2H, CH2CH3), 7.10−7.20 and 7.25−7.35 (m, each 2H,
aromatic CH), 7.55 (d, J = 8.0 Hz, 1H, H-5′), 7.70 (d, J = 8.0 Hz, 1H,
H-4′), 8.00 (d, J = 14.0 Hz, 1H, H-3), 9.60−9.70 and 10.80−10.90
(each m, 0.2H and 0.8H, NH).
2H, CH2CH3), 7.18−7.35 (m, 5H, aromatic CH), 7.55 (d, J =8.3 Hz,
1H, H-6), 8.45 (d, J =8.3 Hz, 1H, H-5), 8.90 (s, 1H, H-2).
Ethyl 7-Chloro-1-(4-chlorobenzyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylate (68). The title compound was
prepared from 55 through method B (1.5 h) in 55% yield: mp
1
157−158 °C; H NMR (CDCl3) 1.40 (t, J = 7.1 Hz, 3H, CH2CH3),
4.45 (q, J = 7.1 Hz, 2H, CH2CH3), 5.50 (s, 2H, CH2), 7.30−7.40 (m,
5H, aromatic CH and H-6), 8.65−8.70 (m, 2H, H-5 and H-2).
Ethyl 7-Chloro-4-oxo-1-[4-(trifluoromethyl)benzyl]-1,4-dihy-
dro-1,8-naphthyridine-3-carboxylate (69). The title compound
was prepared from 56 through method B (4.5 h), with the exception
that the solid obtained was purified by column chromatography eluting
1
with MeOH/CHCl3 (2%), in 45% yield: mp 191−192 °C; H NMR
(DMSO-d6) 1.28 (t, J = 7.1 Hz, 3H, CH2CH3), 4.24 (q, J = 7.1 Hz,
2H, CH2CH3), 5.70 (s, 2H, CH2), 7.53 (d, J = 8.1 Hz, 2H, aromatic
CH), 7.60 (d, J = 8.2 Hz, 1H, H-6), 7.70 (d, J = 8.1 Hz, 2H, aromatic
CH), 8.55 (d, J = 8.2 Hz, 1H, H-5), 9.00 (s, 1H, H-2).
Ethyl 7-Chloro-4-oxo-1-(pyridin-2-ylmethyl)-1,4-dihydro-
1,8-naphthyridine-3-carboxylate (70). The title compound was
prepared from 57 through method B (4 h) in 81% yield: mp 128−129
°C; 1H NMR (CDCl3) 1.50 (t, J = 6.8 Hz, 3H, CH2CH3), 4.45 (q, J =
6.8 Hz, 2H, CH2CH3), 5.75 (br s, 2H, CH2), 7.35−7.50 (m, 3H, H-6
and pyridine CH), 7.70−7.80 and 8.55−8.65 (m, each 1H, pyridine
CH), 8.75 (d, J = 8.1 Hz, 1H, H-5), 8.90 (s, 1H, H-2).
Ethyl 7-Chloro-1-(2-furylmethyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylate (71). The title compound was
prepared from 58 through method B (1.5 h), with the exception
that the solid obtained was purified by crystallization with cyclo-
Ethyl 3-{[2-(4-Chlorophenyl)ethyl]amino}-2-[(2,6-dichloro-
pyridin-3-yl)carbonyl]acrylate (62). The title compound was
prepared starting from 5044 through method A (1 h) using [2-(4-
1
1
hexane/EtOAc, in 57% yield: mp 137−139 °C; H NMR (CDCl3)
chlorophenyl)ethyl]amine in 62% yield: mp 128−130 °C; H NMR
1.35 (t, J = 7.0 Hz, 3H, CH2CH3), 4.35 (q, J = 7.0 Hz, 2H, CH2CH3),
5.55 (br s, 2H, CH2), 6.30−6.35 and 6.45−6.50 (m, each 1H, furan
CH), 7.35−7.40 (m, 2H, H-6 and furan CH), 8.55−8.65 (m, 2H, H-5
and H-2).
(DMSO-d6) 0.65 and 0.85 (each t, J = 7.0 Hz, 0.5H and 2.5H,
CH2CH3), 2.85 (t, J =6.5 Hz, 2H, CH2), 3.60−3.70 (m, 2H, NCH2),
3.80 (q, J = 8.0 Hz, 2H, CH2CH3), 7.20−7.25 and 7.30−7.35 (m, each
2H, aromatic CH), 7.70 (d, J = 8.0 Hz, 1H, H-5′), 7.50 (d, J = 8.0 Hz,
1H, H-4′), 8.00 (d, J = 14.0 Hz, 1H, H-3), 10.75−10.85 (m, 1H, NH).
Ethyl 2-[(2,6-Dichloropyridin-3-yl)carbonyl]-3-[(2-pyridin-2-
ylethyl)amino]acrylate (63). The title compound was prepared
starting from 5044 through method A (4 h) using (2-pyridin-2-
ylethyl)amine in 74% yield: mp 118−122 °C; 1H NMR (CDCl3)
0.80−1.00 (m, 3H, CH2CH3), 3.10−3.20 (m, 2H, CH2), 3.90−4.00
(m, 4H, CH2CH3 and NCH2), 7.10−7.25 (m, 3H, pyridine CH), 7.45
(d, J = 8.2 Hz, H-5′), 7.60 (t, J = 7.3 Hz, pyridine CH), 8.15 (d, J =
14.0 Hz, 1H, H-3), 8.60−8.70 (m, 1H, H-4′), 9.95−10.00 and 11.20−
11.25 (each m, 1H, NH).
Ethyl 7-Chloro-1-(1H-indol-5-ylmethyl)-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carboxylate (72). The title compound was
prepared from 59 through method B (2 h), with the exception that the
solid obtained was purified by flash chromatography eluting with
MeOH/CHCl3 (1%), in 40% yield: mp 222−224 °C; 1H NMR
(CDCl3) 1.35 (t, J = 7.0 Hz, 3H, CH2CH3), 4.35 (q, J = 7.0 Hz, 2H,
CH2CH3), 5.60 (br s, 2H, CH2), 6.45−6.55 (m, 1H, indole CH),
7.05−7.40 (m, 4H, indole CH), 7.55−7.65 (m, 1H, H-6), 8.3 (br s,
1H, indole NH), 8.55−8.70 (m, 2H, H-5 and H-2).
Ethyl 1-(1H-Benzimidazol-2-ylmethyl)-7-chloro-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxylate (73). The title com-
pound was prepared from 60 through method B (2 h) in 80% yield:
mp 294−297 °C; 1H NMR (CDCl3) 1.25 (t, J = 7.0 Hz, 3H,
CH2CH3), 4.25 (q, J = 7.0 Hz, 2H, CH2CH3), 5.80 (br s, 2H, CH2),
7.10−7.20 and 7.35−7.50 (m, each 2H, aromatic CH), 7.60 (d, J = 8.2
Hz, 1H, H-6), 8.60 (d, J = 8.2 Hz, 1H, H-5), 9.05 (s, 1H, H-2).
Ethyl 7-Chloro-1-[2-(2-fluorophenyl)ethyl]-4-oxo-1,4-dihy-
dro-1,8-naphthyridine-3-carboxylate (74). The title compound
was prepared from 61 through method B (1.5 h) in 90% yield: mp
123−124 °C; 1H NMR (DMSO-d6) 1.15 (t, J = 7.1 Hz, 3H, CH2CH3),
3.05 (t, J = 6.5 Hz, 2H, CH2), 4.10 (q, J = 7.1 Hz, 2H, CH2CH3), 4.55
(t, J = 6.5 Hz, 2H, NCH2), 6.95−7.05 and 7.10−7.20 (m, each 2H,
aromatic CH), 7.50 (d, J = 8.3 Hz, 1H, H-6), 8.45 (d, J = 8.3 Hz, 1H,
H-5), 8.50 (s, 1H, H-2).
Ethyl 7-Chloro-1-[2-(4-chlorophenyl)ethyl]-4-oxo-1,4-dihy-
dro-1,8-naphthyridine-3-carboxylate (75). The title compound
was prepared from 62 through method B (2 h) in 85% yield: mp 108−
109 °C; 1H NMR (CDCl3) 1.30 (t, J =7.1 Hz, 3H, CH2CH3), 3.10 (t, J
= 7.2 Hz, 2H, CH2), 4.30 (q, J = 7.1 Hz, 2H, CH2CH3), 4.50 (t, J = 7.2
Hz, 2H, NCH2), 7.00 and 7.20 (d, J = 8.3 Hz, each 2H, aromatic CH),
7.30 (d, J = 8.2 Hz, 1H, H-6), 8.20 (s, 1H, H-2), 8.65 (d, J =8.2 Hz,
1H, H-5).
Ethyl 7-Chloro-4-oxo-1-(2-pyridin-2-ylethyl)-1,4-dihydro-
1,8-naphthyridine-3-carboxylate (76). The title compound was
prepared from 63 through method B (3 h) in 70% yield: mp 158−159
°C; 1H NMR (DMSO-d6) 1.25 (t, J = 7.0 Hz, 3H, CH2CH3), 3.25 (t, J
= 6.7 Hz, 2H, NCH2CH2), 4.20 (q, J = 7.0 Hz, 2H, CH2CH3), 4.75 (t,
J = 6.7 Hz, 2H, NCH2), 7.15−7.25 (m, 2H, pyridine CH), 7.55 (d, J =
Preparation of Ethyl 7-Chloro-1-(3-chloro-2-fluorobenzyl)-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (64; Method
B). A mixture of 51 (1.2 g, 2.9 mmol) and K2CO3 (1.2 g, 8.8 mmol) in
DMF (15 mL) was maintained at 60 °C for 4 h. After cooling, the
reaction mixture was poured into ice/water, obtaining a precipitate
which was filtered, washed with water, and treated with Et2O to give
64 (1.1 g, 96%): mp 191−192 °C; 1H NMR (CDCl3) 1.45 (t, J = 6.8
Hz, 3H, CH2CH3), 4.45 (q, J = 6.8 Hz, 2H, CH2CH3), 5.60 (br s, 2H,
CH2), 7.10−7.20 (m, 1H, aromatic CH), 7.35−7.45 (m, 3H, H-6 and
aromatic CH), 8.70−8.80 (m, 2H, H-2 and H-5).
Ethyl 7-Chloro-1-(2-fluorobenzyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylate (65). The title compound was
prepared from 52 through method B (1 h) in 87% yield: mp 155−
156 °C; 1H NMR (DMSO-d6) 1.20 (t, J =7.0 Hz, 3H, CH2CH3), 4.20
(q, J =7.0 Hz, 2H, CH2CH3), 5.60 (s, 2H, CH2), 7.00−7.35 (m, 4H,
aromatic CH), 7.50 (d, J = 8.3 Hz, 1H, H-6), 8.48 (d, J = 8.3 Hz, 1H,
H-5), 8.90 (s, 1H, H-2).
Ethyl 7-Chloro-1-(3-chlorobenzyl)-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxylate (66). The title compound was
prepared from 53 through method B (1 h) in 90% yield: mp 165−
167 °C; 1H NMR (DMSO-d6) 1.28 (t, J = 7.0 Hz, 3H, CH2CH3), 4.25
(q, J = 7.0 Hz, 2H, CH2CH3), 5.60 (s, 1H, CH2), 7.25−7.40 (m, 3H,
aromatic CH), 7.50 (br s, 1H, aromatic CH), 7.60 (d, J = 8.2 Hz, 1H,
H-6), 8.55 (d, J = 8.2 Hz, 1H, H-5), 9.00 (s, 1H, H-2).
Ethyl 1-Benzyl-7-chloro-4-oxo-1,4-dihydro-1,8-naphthyri-
dine-3-carboxylate (67).45 The title compound was prepared
1
from 54 through method B (2 h) in 80% yield: mp 136−138; H
NMR (DMSO-d6) 1.23 (t, J = 7 Hz, 3H, CH2CH3), 4.20 (q, J =7 Hz,
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7.9 Hz, 1H, H-6), 7.60−7.65 and 8.35−8.40 (m, each 1H, pyridine
CH), 8.50 (d, J = 8.2 Hz, 1H, H-5), 8.55 (s, 1H, H-2).
Preparation of 1-(3-chloro-2-fluorobenzyl)-4-oxo-7-(4-pyri-
din-2-ylpiperazin-1-yl)-1,4-dihydro-1,8-naphthyridine-3-car-
boxylic Acid Hydrochloride (28; Method D). A suspension of 1
(0.10 g, 0.2 mmol) in 4% NaOH (4 mL) was refluxed for 11 h. After
cooling, the reaction mixture was filtered, and the solid was
resuspended in 8 N HCl and the suspension refluxed for 30 min.
After cooling, the reaction mixture was filtered to give a solid which
was crystallized with DMF/EtOH to give 28 (0.96 g, 94%): mp 178−
Preparation of Ethyl 1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-(4-
pyridin-2-ylpiperazin-1-yl)-1,4-dihydro-1,8-naphthyridine-3-
carboxylate (1; Method C). A mixture of 64 (0.15g, 0.4 mmol) and
1-(pyridin-2-yl)piperazine (0.17 g, 0.8 mmol) in DMF (10 mL) was
maintained at 80 °C for 6 h. After cooling, the reaction mixture was
poured into ice/water, obtaining a precipitate which was filtered and
crystallized with EtOH/DMF to give compound 1 (0.15 g, 75%): mp
200−201 °C; 1H NMR (DMSO-d6) 1.25 (t, J = 7.0 Hz, 3H, CH2CH3),
3.40−3.50 and 3.65−3.75 (m, each 4H, piperazine CH2), 4.45 (q, J =
7.0 Hz, 2H, CH2CH3), 5.65 (s, 2H, CH2), 6.65 (dd, J = 5.1 and 6.8 Hz,
1H, pyridine CH), 6.85 (d, J = 8.8 Hz, 1H, pyridine CH), 7.00 (d, J =
9.0 Hz, 1H, H-6), 7.15−7.20 (m, 2H, aromatic CH), 7.50−7.60 (m,
2H, aromatic CH and pyridine CH), 8.15 (d, J = 4.9 Hz, 1H, pyridine
CH), 8.20 (d, J = 9.0 Hz, 1H, H-5), 8.85 (s, 1H, H-2). Anal.
(C27H25ClFN5O3) C, H, N.
1
180 °C; H NMR (DMSO-d6) 3.80−4.00 (m, 8H, piperazine CH2),
5.85 (s, 2H, CH2), 6.95 (t, J = 6.4 Hz, 1H, pyridine CH), 7.15−7.35
(m, 4H, aromatic CH, H-6, and pyridine CH) 7.50−7.60 (m, 1H,
aromatic CH), 7.95−8.10 (m, 2H, pyridine CH and aromatic CH),
8.35 (d, J = 9.1 Hz, 1H, H-5), 9.20 (s, 1H, H-2). Anal.
(C25H22Cl2FN5O3) C, H, N.
7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(3-chloro-2-flu-
orobenzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic
Acid Hydrochloride (29). The title compound was prepared from 23
1
through method D (24 h) in 82% yield: mp 281−282 °C; H NMR
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(3-chloro-
2-fluorobenzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-car-
boxylate (23). The title compound was prepared through method C
(80 °C, 6 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in 78% yield:
(DMSO-d6) 3.60−3.70 and 3.85−3.95 (m, each 4H, piperazine CH2),
5.80 (s, 2H, CH2), 7.05−7.40 (m, 5H, H-6, aromatic CH, and
benzothiazole CH), 7.45−7.55 (m, 2H, aromatic CH, and
benzothiazole CH), 7.80 (d, J = 7.6 Hz, 1H, benzothiazole CH),
8.45 (d, J = 9.1 Hz, 1H, H-5), 9.20 (s, 1H, H-2). Anal.
(C27H22Cl2FN5O3S) C, H, N.
1
mp 250−251 °C; H NMR (DMSO-d6) 1.30 (t, J = 6.9 Hz, 3H,
CH2CH3), 3.55−3.65 and 3.80−3.90 (m, each 4H, piperazine CH2),
4.25 (q, J = 6.9 Hz, 2H, CH2CH3), 5.70 (s, 2H, CH2), 7.05 (d, J = 8.7
Hz, 1H, H-6), 7.10 (t, J = 7.0 Hz, 1H, benzothiazole CH), 7.15−7.25
(m, 2H, aromatic CH), 7.30 (t, J = 7.0 Hz, 1H, benzothiazole CH),
7.45−7.60 (m, 2H, aromatic CH and benzothiazole CH), 7.80 (d, J =
7.7 Hz, 1H, benzothiazole CH), 8.20 (d, J = 8.7 Hz,1H, H-5), 8.80 (s,
1H, H-2). Anal. (C29H25ClFN5O3S) C, H, N.
1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-(4-quinolin-2-ylpipera-
zin-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxylic Acid (30).
The title compound was prepared from 24 through method D (48 h)
1
in 77% yield: mp 220−221 °C; H NMR (DMSO-d6) 3.35−3.55 and
3.85−4.05 (m, each 4H, piperazine CH2), 5.80 (s, 2H, CH2), 7.20−
7.30 (m, 3H, H-6 and aromatic CH), 7.40−7.60 (m, 3H, aromatic CH
and quinoline CH), 7.70−7.95 (m, 2H, quinoline CH), 8.10−8.40 (m,
3H, H-5 and quinoline CH), 9.15 (s, 1H, H-2), 15.50 (br s, 1H,
COOH). Anal. (C29H23ClFN5O3) C, H, N.
Ethyl 1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-(4-quinolin-2-yl-
piperazin-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxylate
(24). The title compound was prepared through method C (80 °C, 5
h) using 2-piperazin-1-ylquinoline58 in 61% yield after crystallization
1
with EtOH: mp 210−212 °C; H NMR (CDCl3) 1.45 (t, J = 7.1 Hz,
1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-[4-(1,3-thiazol-2-yl)-
piperazin-1-yl]-1,4-dihydro-1,8-naphthyridine-3-carboxylic
Acid (31). The title compound was prepared from 25 through method
D (18 h) in 38% yield: mp 268−269 °C; 1H NMR (DMSO-d6) 3.40−
3.50 and 3.80−3.90 (m, each 4H, piperazine CH2), 5.77 (s, 2H, CH2),
6.90 (d, J = 4.0, 1H, thiazole CH), 7.14−7.25 (m, 4H, H-6, thiazole
CH, and aromatic CH), 7.45−7.55 (m, 1H, aromatic CH), 8.30 (d, J =
9.1, 1H, H-5), 9.15 (s, 1H, H-2), 15.45 (s, 1H, COOH). Anal.
(C23H19ClFN5O3S) C, H, N.
3H, CH2CH3), 3.80 (br s, 8H, piperazine CH2), 4.40 (q, J = 7.1, 2H,
CH2CH3) 5.50 (s, 2H, CH2), 6.75 (d, J = 9.0 Hz, 1H, H-6), 6.90−7.10
(m, 3H, aromatic CH and quinoline CH), 7.25−7.35 (m, 2H,
quinoline CH), 7.60 (t, J = 7.4, 1H, quinoline CH), 7.65 (d, J = 7.9
Hz, 1H, quinoline CH), 7.75 (br s, 1H, aromatic CH), 7.95 (d, J = 9.0
Hz, 1H, quinoline CH), 8.50 (d, J = 9.0 Hz, H-5), 8.60 (s, 1H, H-2).
Anal. (C31H27ClFN5O3) C, H, N.
1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-[4-(1,3-thiazol-2-yl)-
piperazin-1-yl]-1,4-dihydro-1,8-naphthyridine-3-carboxylic
Acid (25). The title compound was prepared through method C (80
°C, 5 h) using 1-(1,3-thiazol-2-yl)piperazine59 in 50% yield after
7-[4-(1,3-Benzoxazol-2-yl)piperazin-1-yl]-1-(3-chloro-2-fluo-
robenzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic
Acid (32). In a microwave oven tube, 26 (0.12 g, 0.213 mmol) was
suspended in 4% NaOH (6 mL) and 1,4-dioxane (3 mL). The mixture
was irradiated at 120 °C for 35 min employing the following
experimental parameters: pressure 5 bar, cooling on, FHT on,
prestirring 30 s, very high absorption. After cooling, the sodium salt
was filtered, suspended in water, and then treated with 2 N HCl to pH
2. After being stirred for 30 min, the mixture was filtered to give 32
1
crystallization with EtOH: mp 223−224 °C; H NMR (CDCl3) 1.35
(t, J = 7.1 Hz, 3H, CH2CH3), 3.45−3.55 and 3.70−3.80 (m, each 4H,
piperazine CH2), 4.45 (q, J = 7.1 Hz, 2H, CH2CH3), 5.50 (s, 2H,
CH2), 6.60 (d, J = 4.0 Hz, 1H, thiazole CH) 6.70 (d, J = 9.0 Hz, 1H,
H-6), 6.85−7.05 (m, 2H, aromatic CH) 7.20 (d, J = 4.0 Hz, 1H,
thiazole CH), 7.30−7.40 (m, 1H, aromatic CH), 8.45 (d, J = 8 Hz, 1H,
H-5), 8.50 (s, 1H, H-2). Anal. (C25H23ClFN5O3S) C, H, N.
1
(0.05 g, 48%): mp >300 °C; H NMR (DMSO-d6) 3.50−3.60 and
3.80−3.90 (m, each 4H, piperazine CH2), 5.75 (s, 2H, CH2), 7.00−
7.50 (m, 8H, H-6, benzoxazole CH, and aromatic CH), 8.27 (d, J =
9.0, 1H, H-5), 9.10 (s, 1H, H-2), 15.70 (br s, 1H, COOH). Anal.
(C27H21ClFN5O4) C, H, N.
7-[4-(1,3-Benzoxazol-2-yl)piperazin-1-yl]-1-(3-chloro-2-fluo-
robenzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic
acid (26). The title compound was prepared through method C (80
°C, 5 h) using 2-piperazin-1-yl-1,3-benzoxazole59 in 18% yield after
1
crystallization with DMF: mp 282−284 °C; H NMR (DMSO-d6)
1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-{4-[3-(trifluoromethyl)-
phenyl]piperazin-1-yl}-1,4-dihydro-1,8-naphthyridine-3-car-
boxylic Acid (33). The title compound was prepared starting from 27
by using the same procedure as used for the synthesis of 32 in 55%
1.35 (t, J = 7.1 Hz, 3H, CH2CH3), 3.50−3.60 and 3.80−3.90 (m, each
4H, piperazine CH2), 4.30 (q, J = 7.1 Hz, 2H, CH2CH3), 5.50 (s, 1H,
CH2), 7.00−7.70 (m, 8H, H-6, benzoxazole CH, and aromatic CH),
8.20 (d, J =9.0 Hz, 1H, H-5), 8.90 (s, 1H, H-2). Anal.
(C29H25ClFN5O4) C, H, N.
1
yeld: mp 210−212 °C; H NMR (DMSO-d6) 3.20−3.30 and 3.75−
3.85 (m, each 4H, piperazine CH2), 5.75 (s, 2H, CH2), 7.10 (d, J = 7.5,
1H, aromatic CH), 7.15−7.25 (m, 5H, H-6 and aromatic CH), 7.40 (t,
J = 8.0, 1H, aromatic CH), 7.45−7.55 (m, 1H, aromatic CH), 8.30 (d,
J = 9.1 Hz, 1H, H-5), 9.15 (s, 1H, H-2), 15.45 (s, 1H, COOH). Anal.
(C27H21ClF4N4O3) C, H, N.
Ethyl 1-(3-Chloro-2-fluorobenzyl)-4-oxo-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-1,4-dihydro-1,8-naph-
thyridine-3-carboxylate (27). The title compound was prepared
through method C (80 °C, 17 h) using 1-[3-(trifluoromethyl)phenyl]-
piperazine60 in 90% yield: mp 100−102 °C; H NMR (DMSO-d6)
1
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(2-fluoro-
benzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate
(34). The title compound was prepared from 65 through method C
(80 °C, 17 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in 86%
yield: mp 270−272 °C; 1H NMR (DMSO-d6) 1.20 (t, J = 7.1 Hz, 3H,
1.30 (t, J = 7.0 Hz, 3H, CH2CH3), 3.20−3.40 and 3.60−3.80 (m, each
4H, piperazine CH2), 4.15 (q, J = 7.0 Hz, 2H, CH2CH3), 5.10 (s, 2H,
CH2), 6.90−7.50 (m, 8H, H-6 and aromatic CH), 8.10 (d, J = 8.7 Hz,
1H, H-5), 8.78 (s, 1H, H-2). Anal. (C29H25ClF4N4O3) C, H, N.
5658
dx.doi.org/10.1021/jm500340h | J. Med. Chem. 2014, 57, 5649−5663

Journal of Medicinal Chemistry
Article
CH2CH3), 3.40−3.60 and 3.60−3.20 (m, each 4H, piperazine CH2),
4.15 (q, J = 7.1 Hz, 2H, CH2CH3), 5.55 (s, 2H, CH2), 6.90−7.30 (m,
7H, H-6, benzothiazole CH, and aromatic CH), 7.40 (br d, J = 7.6 Hz,
1H, benzothiazole CH), 7.70 (br d, J = 7.6 Hz, 1H, benzothiazole
CH), 8.15 (d, J = 9.0 Hz, 1H, H-5), 8.75 (s, 1H, H-2). Anal.
(C29H26FN5O3S) C, H, N.
7.25 (t, J = 7.8 Hz, 1H, benzothiazole CH), 7.45 (d, J = 7.8 Hz, 1H,
benzothiazole CH), 7.55 (br s, 1H, furan CH), 7.75 (d, J = 7.5 Hz, 1H,
benzothiazole CH), 8.15 (d, J = 9.0 Hz, 1H, H-5), 8.70 (s, 1H, H-2).
Anal. (C27H25N5O4S) C, H, N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(1H-
indol-7-ylmethyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-car-
boxylate (41). The title compound was prepared from 72 through
method C (80 °C, 15 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in
30% yield: mp 290−293 °C; 1H NMR (DMSO-d6) 1.20 (t, J = 7.0 Hz,
3H, CH2CH3), 3.55−3.65 and 3.80−3.90 (m, each 4H, piperazine
CH2), 4.15 (q, J = 7.0 Hz, 2H, CH2CH3), 5.60 (br s, 2H, CH2), 6.35−
6.40 (m, 1H, indole CH), 6.95−7.10 (m, 3H, H-6, benzothiazole CH,
and indole CH), 7.20−7.30 (m, 3H, indole CH and benzothiazole
CH), 7.40−7.50 (m, 2H, indole CH and benzothiazole CH), 7.75 (d, J
= 7.5 Hz, 1H, benzothiazole CH), 8.15 (d, J = 9.0 Hz, 1H, H-5), 8.70
(s, 1H, H-2). Anal. (C31H28N6O3S) C, H, N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(3-chloro-
benzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate
(35). The title compound was prepared from 66 through method C
(80 °C, 18 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 and adding
1
K2CO3 (2 equiv) in 60% yield: mp 251−252 °C; H NMR (DMSO-
d6) 1.20 (t, J = 7.0 Hz, 3H, CH2CH3), 3.50−3.60 and 3.75−3.95 (m,
each 4H, piperazine CH2), 4.30 (q, J = 7.0, 2H, CH2CH3), 5.50 (s, 2H,
CH2), 6.90−7.05 (m, 2H, H-6 and aromatic CH), 7.15−7.35 (m, 4H,
aromatic CH and benzothiazole CH), 7.40−7.45 (m, 2H,
benzothiazole CH), 7.72 (d, J = 7.4 Hz, 1H, benzothiazole CH),
8.15 (d, J = 9.0 Hz, 1H, H-5), 8.80 (s, 1H, H-2). Anal.
(C29H26ClN5O3S) C, H, N.
Ethyl 1-(1H-Benzimidazol-2-ylmethyl)-7-[4-(1,3-benzothia-
zol-2-yl)piperazin-1-yl]-4-oxo-1,4-dihydro-1,8-naphthyridine-
3-carboxylate (42). The title compound was prepared from 73
through method C (80 °C, 8 h) using 1-(1,3-benzothiazol-2-
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-benzyl-4-
oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (36). The
title compound was prepared from 6745 through method C (80 °C,
4 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 and adding K2CO3 (2
equiv) in 16% yield: mp 246−247 °C; 1H NMR (CDCl3) 1.35 (t, J =
7.0 Hz, 3H, CH2CH3), 3.55−3.65 and 3.75−3.85 (m, each 4H,
piperazine CH2), 4.32 (q, J = 7.0 Hz, 2H, CH2CH3), 5.40 (s, 2H,
CH2), 6.68 (d, J = 9.0 Hz, 1H, H-6), 7.00−7.40 (m, 7H, aromatic CH
and benzothiazole CH), 7.50−7.60 (m, 2H, benzothiazole CH), 8.45
(d, J = 9.0 Hz, 1H, H-5), 8.50 (s, 1H, H-2). Anal. (C29H27N5O3S) C,
H, N.
yl)piperazine46 in 50% yield: mp 295−297 °C; H NMR (DMSO-
1
d6) 1.20 (t, J = 7.0 Hz, 3H, CH2CH3), 3.30−3.40 and 3.65−3.75 (m,
each 4H, piperazine CH2), 4.20 (q, J = 7.0 Hz, 2H, CH2CH3), 5.65 (br
s, 2H, CH2), 6.90 (d, J = 9.0 Hz, benzimidazole CH), 6.95−7.10 (m,
3H, H-6, benzothiazole CH, and benzimidazole CH), 7.25 (t, J = 7.8
Hz, 1H, benzothiazole CH), 7.40−7.50 (m, 2H, benzimidazole CH
and benzothiazole CH), 7.75 (d, J = 7.5 Hz, 1H, benzothiazole CH),
8.15 (d, J = 9.0 Hz, 1H, H-5), 8.80 (s, 1H, H-2). Anal. (C30H27N7O3S)
C, H, N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-[2-(2-
fluorophenyl)ethyl]-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxylate (43). The title compound was prepared from 74 through
method C (80 °C, 16 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in
39% yield: mp 168−170 °C; 1H NMR (DMSO-d6) 1.20 (t, J = 7.1 Hz,
3H, CH2CH3), 3.10 (bt, J = 6.5 Hz, 2H, CH2), 3.60−3.70 and 3.85−
3.95 (m, each 4H, piperazine CH2), 4.15 (q, J = 7.1, 2H, CH2CH3),
4.50 (bt, J = 6.5 Hz, 2H, NCH2), 6.90−7.30 (m, 7H, H-6,
benzothiazole CH, and aromatic CH), 7.45 and 7.75 (d, J = 7.9 Hz,
each 1H, benzothiazole CH), 7.15 (d, J = 9.0 Hz, 1H, H-5), 8.35 (s,
1H, H-2). Anal. (C30H28FN5O3S) C, H, N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(4-chloro-
benzyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate
(37). The title compound was prepared from 68 through method C
(80 °C, 30 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in 23%
yield: mp 204−206 °C; 1H NMR (DMSO-d6) 1.30 (t, J = 7.0 Hz, 3H,
CH2CH3), 3.55−3.65 and 3.80−3.90 (m, each 4H, piperazine CH2),
4.25 (q, J = 7.0 Hz, 2H, CH2CH3), 5.55 (s, 2H, CH2), 7.00 (d, J = 9.0
Hz, 1H, H-6), 7.10−7.15 (m, 1H, benzothiazole CH), 7.25−7.55 (m,
6H, aromatic CH and benzothiazole CH), 7.77 (d, J = 7.8 Hz, 1H,
benzothiazole CH), 8.20 (d, J = 8.8 Hz, 1H, H-5), 8.82 (s, 1H, H-2).
Anal. (C29H26ClN5O3S) C, H, N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-[2-(4-
chlorophenyl)ethyl]-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxylate (44). The title compound was prepared from 75 through
method C (80 °C, 15 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-4-oxo-1-[4-
(trifluoromethyl)benzyl]-1,4-dihydro-1,8-naphthyridine-3-car-
boxylate (38). The title compound was prepared from 69 through
method C (80 °C, 18 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in
66% yield: mp 205−207 °C; 1H NMR (DMSO-d6) 1.25 (t, J = 7.1 Hz,
3H, CH2CH3), 3.50−3.60 and 3.75−3.85 (m, each 4H, piperazine
CH2), 4.20 (q, J = 7.1 Hz, 2H, CH2CH3), 5.60 (s, 2H, CH2) 6.95 (d, J
= 9.0 Hz, 1H, H-6), 7.05 (d, J = 7.5 Hz, 1H, benzothiazole CH), 7.20−
7.30 (m, 1H, aromatic CH), 7.40−7.50 and 7.65−7.75 (m, each 3H,
benzothiazole CH and aromatic CH), 8.15 (d, J = 9.0 Hz, 1H, H-5),
8.80 (s, 1H, H-2). Anal. (C30H29F3N6O3S) C, H, N.
1
47% yield: mp 261−263; H NMR (DMSO-d6) 1.30 (t, J = 7.1 Hz,
3H, CH2CH3), 3.05 (t, J = 6.9, 2H, CH2), 3.70−3.80 and 3.85−3.95
(m, each 4H, piperazine CH2), 4.30 (q, J = 7.1 Hz, 2H, CH2CH3), 4.45
(t, J = 6.9 Hz, 2H, NCH2), 6.72 (d, J = 9.0 Hz, 1H, H-6), 6.95−7.05
(m, 2H, aromatic CH) 7.10 (dt, J = 1.3 and 7.5 Hz, 1H, benzothiazole
CH), 7.20−7.35 (m, 3H, aromatic CH and benzothiazole CH), 7.50−
7.65 (m, 2H, benzothiazole CH), 8.10 (s, 1H, H-2), 8.50 (d, J = 9.0
Hz, 1H, H-5). Anal. (C30H28ClN5O3S) C, H, N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-4-oxo-1-
(pyridin-2-ylmethyl)-1,4-dihydro-1,8-naphthyridine-3-carbox-
ylate (39). The title compound was prepared from 70 through
method C (80 °C, 20 h) using 1-(1,3-benzothiazol-2-yl)piperazine,46
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-4-oxo-1-(2-
pyridin-2-ylethyl)-1,4-dihydro-1,8-naphthyridine-3-carboxy-
late (45). The title compound was prepared from 76 through method
C (80 °C, 4 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 and adding
1
adding K2CO3 (2 equiv), in 85% yield: mp 225−227 °C; H NMR
1
K2CO3 (2 equiv) in 96% yield: mp 138−139; H NMR (DMSO-d6)
(DMSO-d6) 1.30 (t, J = 7.0 Hz, 3H, CH2CH3), 3.55−3.65 and 3.70−
3.80 (m, each 4H, piperazine CH2), 4.25 (q, J = 7.0 Hz, 2H, CH2CH3),
5.60 (s, 2H, CH2), 6.95 (d, J = 9.0 Hz, 1H, H-6), 7.10 (t, J = 7.5 Hz,
1H, benzothiazole CH), 7.25−7.35, 7.40−7.50, and 7.75−7.85 (m,
each 2H, pyridine CH and benzothiazole CH), 8.20 (d, J = 9.0 Hz, 1H,
H-5), 8.45 (d, J = 4.6 Hz, 1H, pyridine CH), 8.80 (s, 1H, H-2). Anal.
(C28H26N6O3S) C, H, N.
1.25 (t, J = 7.0 Hz, 3H, CH2CH3), 3.25 (t, J = 6.7 Hz, 2H, CH2),
3.65−3.75 and 3.85−3.95 (m, each 4H, piperazine CH2), 4.15 (q, J =
7.0 Hz, 2H, CH2CH3), 4.70 (t, J = 6.7 Hz, 2H, NCH2), 7.00−7.15 (m,
2H, pyridine CH and benzothiazole CH), 7.20−7.30 (m, 3H, H-6,
pyridine CH and benzothiazole CH), 7.45 (d, J = 7.9 Hz, 1H,
benzothiazole CH), 7.70 (t, J = 7.7 Hz, 1H, pyridine CH), 7.75 (d, J =
7.9 Hz, 1H, benzothiazole CH), 8.20 (d, J = 8.8 Hz, H-5), 8.35 (s, 1H,
H-2), 8.45−8.50 (m, 1H, pyridine CH). Anal. (C29H28N6O3S) C, H,
N.
Ethyl 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(2-furyl-
methyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate
(40). The title compound was prepared from 70 through method C
(80 °C, 8 h) using 1-(1,3-benzothiazol-2-yl)piperazine46 in 60% yield:
7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-benzyl-4-oxo-
1,4-dihydro-1,8-naphthyridine-3-carboxylic Acid (46). The title
compound was prepared from 36 through method D (48 h) in 42%
1
mp 188−189 °C; H NMR (DMSO-d6) 1.20 (t, J = 7.0 Hz, 3H,
CH2CH3), 3.60−3.70 and 3.80−3.90 (m, each 4H, piperazine CH2),
4.15 (q, J = 7.0 Hz, 2H, CH2CH3), 5.50 (br s, 2H, CH2), 6.35−6.50
(m, 2H, furan CH), 6.95−7.10 (m, 2H, H-6 and benzothiazole CH),
1
yield: mp 287−288 °C; H NMR (DMSO-d6) 3.60−3.70 and 3.90−
4.00 (m, each 4H, piperazine CH2), 5.70 (s, 2H CH2), 7.10 (t, J = 7.5
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Journal of Medicinal Chemistry
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Hz, 1H, benzothiazole CH), 7.20 (d, J = 9.0 Hz, 1H, H-6), 7.25−7.40
(m, 6H, aromatic CH and benzothiazole CH), 7.50 and 7.80 (d, J =
7.8 Hz, each 1H, benzothiazole CH), 8.30 (d, J = 9.0 Hz, 1H, H-5),
9.15 (s, 1H, H-2), 15.50 (s, 1H, COOH). Anal. (C27H23N5O3S) C, H,
N.
cells/well in 96-well plates. The next day, the cells were either
incubated with different concentrations of the compounds in triplicate
or treated with equal volumes of DMSO. After a 24 h treatment, cell
viability was measured by the CellTiter 96 proliferation assay kit
(Promega, Madison, WI) according to the manufacturer’s instructions,
as previously described.62 Absorbances were measured at 490 nm
using the microplate reader Multiskan FC (Thermo Fisher Scientific,
Waltham, MA). The effect on cell viability of each test compound at
different concentrations was expressed as a percentage by comparing
the absorbance of the compound-treated cells with that of the DMSO-
treated cells. The 50% cytotoxic concentrations (CC50) and 95% CIs
were determined using Prism software (Graph-Pad Software). The SI
value of each compound was calculated by the ratio of CC50 to IC50.
RT-PCR Analysis. CaSki cells were seeded at a density of 1.5 × 106
cells/well in 100 mm plates. The next day, the cells were treated with a
10 μM concentration of each compound or TGF-β1 (50 ng/mL) for
24 h. Total RNA isolation was performed using a NucleoSpin II kit
7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-(4-chloroben-
zyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic Acid
(47). The title compound was prepared from 37 through method D
1
(72 h) in 75% yield: mp >300 °C; H NMR (DMSO-d6) 3.60−3.70
and 3.80−3.90 (m, each 4H, piperazine CH2), 5.65 (s, 2H, CH2), 7.05
(t, J = 7.5 Hz, 1H, benzothiazole CH), 7.15 (d, J = 8.9 Hz, 1H, H-6),
7.25−7.30 (m, 1H, benzothiazole CH), 7.35−7.40 (m, 4H, aromatic
CH), 7.50 and 7.75 (d, J = 7.5, each 1H, benzothiazole CH), 8.30 (d, J
= 8.9 Hz, 1H, H-5), 9.15 (s, 1H, H-2), 15.50 (s, 1H, COOH). Anal.
(C27H22ClN5O3S) C, H, N.
7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-4-oxo-1-(pyridin-
2-ylmethyl)-1,4-dihydro-1,8-naphthyridine-3-carboxylic Acid
Hydrochloride (48). The title compound was prepared from 39
(Macherey-Nagel, Duren, Germany), and 2 μg of RNA was
̈
1
through method D (4 h), in 96% yield: mp 257−258 °C; H NMR
retrotranscribed with a Transcriptor First Strand cDNA synthesis kit
(Roche, Mannheim, Germany). The resulting cDNAs were
quantitated by real-time PCR using the following primers: HPV-16
E6 forward primer, 5′-GCAAGCAACAGTTACTGAGACGT-3′;
HPV-16 E6 reverse primer, 5′-GCAACAAGACATACATCGAC-
CGG-3′; HPV-16 E7 forward primer, 5′-GATGGTCCAGCTGGA-
CAAGC-3′; HPV-16 E7 reverse primer, 5′-GTGCCCATTAACAGG-
TCTTC-3′; β-actin forward primer, 5′-GTTGCTATCCAGGCTG-
TG-3′; β-actin reverse primer, 5′-TGTCCACGTCACACTTCA-3′.33
DNA amplifications were carried out by the SYBR Green real-time
PCR method in a 96-well reaction plate format in a LightCycler 480
real-time machine PCR system (Roche, Mannheim, Germany). The
results were normalized to the β-actin transcript levels, and the fold
(DMSO-d6) 3.55−3.65 and 3.80−3.90 (m, each 4H, piperazine CH2),
5.95 (s, 2H, CH2), 7.10−7.20 (m, 2H, H-6 and benzothiazole CH),
7.30 (t, J = 7.1 Hz, 1H, benzothiazole CH), 7.45−7.55 (m, 2H,
benzothiazole CH and pyridine CH), 7.70 (d, J = 7.2 Hz, 1H, pyridine
CH), 7.80 (d, J = 7.5 Hz, 1H, benzothiazole CH), 8.05−8.15 (m, 1H,
pyridine CH), 8.30 (d, J = 8.5 Hz, 1H, H-5), 8.65 (s, 1H, pyridine
CH), 9.20 (s, 1H, H-2). Anal. (C26H23ClN6O3S) C, H, N.
7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-4-oxo-1-(2-pyri-
din-2-ylethyl)-1,4-dihydro-1,8-naphthyridine-3-carboxylic
Acid (49). The title compound was prepared from 45 through method
D (1 h) in 91% yield: mp >300 °C; 1H NMR (DMSO-d6) 3.60 (t, J =
6.7 Hz, 2H, CH2), 3.65−3.75 and 3.85−3.95 (m, each 4H, piperazine
CH2), 4.95 (t, J = 6.7 Hz, 2H, NCH2), 7.05−7.15 (m, 2H, pyridine
CH and benzothiazole CH), 7.30 (t, J = 7.7 Hz, 1H, benzothiazole
CH), 7.50 (d, J = 7.9 Hz, 1H, benzothiazole CH), 7.77−7.85 (m, 3H,
H-6, pyridine CH, and benzothiazole CH), 8.25 (d, J = 8.8 Hz, H-5),
8.35 (t, J = 7.5 Hz, 1H, pyridine CH), 8.75 (d, J = 5.3 Hz, 1H, pyridine
CH), 8.90 (s, 1H, H-2). Anal. (C22H27N7O3S) C, H, N.
Biology. Cells. P21 is an HaCat-derived stably transfected cell
clone with integrated reporter plasmid pALuc HPV-16-LCR, which we
previously generated.25 This cell line grows as monolayers in
Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supple-
mented with 10% fetal calf serum (Sigma), 1% antibiotic−antimycotic
solution (Zell Shield, Minerva Biolabs GmbH, Berlin, Germany), and
0.2 mg/mL G418 (Gibco/BRL). CaSki is an HPV-16-positive human
cervical carcinoma cell line purchased from the American Type
Culture Collection (ATCC, Rockville, MD) and grown in DMEM
high glucose (4.5 g/L) (Sigma) supplemented with 10% fetal calf
serum.
increase (mean
SD) was compared with the DMSO-treated
controls. The results are expressed as the mean of three independent
experiments.
Statistical Analysis. Each data point is the mean
standard
deviation of the mean values for three determinations performed in
duplicate. Statistical analysis was performed by analysis of variance
(ANOVA) followed by a Bonferroni test if the P values showed
significant differences (P value <0.05) using GraphPad Prism 5.00
(GraphPad Software).
In Vitro Uptake Studies in CaSki Cells. The quantitative
determination of 1,8-naphthyridone derivatives was achieved by the
reversed-phase HPLC method using a UV detector, developed for this
study. A preliminary evaluation of the UV spectra of the series of
ester/acid pairs (compounds 23−25 and 29−31) was carried out by
spectrophotometric analysis to identify the value of λmax for each
compound. For this purpose a weighted amount of each 1,8-
naphthyridone derivative was dissolved in a methanol−chloroform
mixture (1:1, v/v). The solutions were then diluted with methanol and
analyzed using a spectrophotometer (Beckman Coulter DU 730),
collecting the data in the range between 200 and 300 nm to identify
the absorbance maximum (λmax).
Compounds. Human TGF-β1 was obtained from Peprotech
(RockyHill, NJ), and IL-4 was purchased from Bender MedSystems
(Burlingame, CA). All quinolones were solubilized in 100% dimethyl
sulfoxide (DMSO), aliquoted, and stored at −20 °C.
Luciferase Assay. P21 clones were seeded in 24-well culture plates
at a density of 6 × 104 cells/well. After 24 h, they were incubated with
different concentrations of the compounds in triplicate or treated with
equal volumes of DMSO. The next day, the monolayers were washed
twice with PBS 1× and lysed on ice with 40 μL of the reporter lysis
buffer (Promega, Madison, WI). The lysate was centrifuged at 13 000
rpm for 10 min at 4 °C. The supernatants (soluble proteins) were
quantified for protein concentration and assayed for luciferase activity
with the Promega luciferase assay system using a VICTOR light
luminescence counter (PerkinElmer, Waltham, MA).25,61 The end
point of the assay was the inhibitory concentration of drug which
reduced luciferase activity by 50% (IC50) in comparison to the DMSO-
treated control. The IC50 and the 95% confidence interval (95% CI)
values for inhibition curves were calculated by using the program
PRISM 4 (GraphPad Software, San Diego, CA) to fit a variable-slope
sigmoidal dose−response curve.
Table 3 reports the λmax values for each compound subsequently
used in the HPLC quantitative procedure.
Table 3. UV λmax Values
compd
λ
(nm)
compd
λ
(nm)
compd
λ
(nm)
max
max
max
23
24
278
277
25
29
275
274
30
31
276
280
For the quantitative chromatographic analyses of the 1,8-
naphthyridone derivatives an HPLC system consisting of a binary
pump (PerkinElmer PUMP 250B) equipped with a spectrophotom-
eter detector (PerkinElmer Flexar UV−vis detector) was used. The
mobile phase was a mixture of TFA (0.1%), methanol, and acetonitrile
(60:20:20, v/v), degassed and pumped through a Microsorb-MV 100-
5 C18 column (250 mm × 4.6 mm, 5 μm) with a flow rate of 1.2 mL/
min. Ultraviolet detection was set to a different wavelength (λ max) for
Cell Viability Assay. To test the cytotoxic effect of the compounds
on P21 and CaSki cultures, cells were seeded at a density of 1 × 104
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Journal of Medicinal Chemistry
Article
each compound (see Table 3). The external standard method was
used to calculate the compound concentrations.
long control region; RT-PCR, real-time polymerase chain
reaction; TGF-β1, transforming growth factor β1
For this purpose, 1 mg of each 1,8-naphthyridone derivative was
weighed, placed in a volumetric flask, and dissolved in a methanol−
chloroform mixture (1:1, v/v) to obtain stock standard solutions. The
stock solutions of each compound were diluted using the mobile
phase, providing a series of calibration solutions for each ester/acid
pair subsequently injected into the HPLC system.
Linear calibration curves were obtained for each compound
reported in Table 3 over the concentration range of 0.5−20 μg/mL
with a regression coefficient of 0.998.
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ASSOCIATED CONTENT
* Supporting Information
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̈
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DNA and RNA viruses, chemistry and experimental procedures
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and 20, and Table S3 containing elemental analysis data for
target compounds 1, 10−13, 16, 17, 19, 20, and 23−49. This
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AUTHOR INFORMATION
Corresponding Authors
*Phone: + 39 075 585 5146. Fax: +39 075 585 5115. E-mail:
serena.massari0@gmail.com.
*Phone: + 39 011 670 5484. Fax: +39 011 236 5484. E-mail:
david.lembo@unito.it.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work coordinated by O.T. was carried out with financial
support by the Ministry of Health (AIDS, Grant UPR-2009-
1301355) and PRIN 2011 (Grant 2010W2KM5L_004). We
thank Christophe Pannecouque (Rega Institute for Medical
Research, Leuven, Belgium) for the anti-HIV-1 evaluation.
ABBREVIATIONS USED
6-DFQs, 6-desfluoroquinolones; CC, cervical cancer; HCMV,
human cytomegalovirus; HPV, human papillomavirus; LCR,
■
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