Anti-HIV diarylpyrimidine-quinolone hybrids and their mode of action

Article abstract of DOI:10.1016/j.bmc.2015.03.037

Abstract A molecular hybridization approach is a powerful tool in the design of new molecules with improved affinity and efficacy. In this context, a series of diarylpyrimidine-quinolone hybrids were synthesized and evaluated against both wt HIV-1 and mut

Full text of DOI:10.1016/j.bmc.2015.03.037

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-HIV diarylpyrimidine–quinolone hybrids and their mode of
action
Tian-Qi Mao a,b, Qiu-Qin He a, , Zheng-Yong Wan , Wen-Xue Chen , Fen-Er Chen
⇑
a
a
a,b,
⇑
, Gang-Feng Tang a,
c
c
c
Erik De Clercq , Dirk Daelemans , Christophe Pannecouque
a
Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
Institute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A molecular hybridization approach is a powerful tool in the design of new molecules with improved
affinity and efficacy. In this context, a series of diarylpyrimidine–quinolone hybrids were synthesized
and evaluated against both wt HIV-1 and mutant viral strains. The most active hybrid 5a displayed an
Received 24 February 2015
Revised 12 March 2015
Accepted 13 March 2015
Available online xxxx
B
EC50 value of 0.28 ± 0.07 lM against HIV-1 III . A couple of enzyme-based assays clearly pinpoint a RT-
targeted mechanism of action. Docking studies revealed that these hybrids could be well located in the
NNIBP of HIV-1 RT despite the bulky and polar properties of a quinolone 3-carboxylic acid moiety in
the molecules.
Keywords:
Diarylpyrimidine–quinolone hybrids
HIV-1 reverse transcriptase
Integrase
Ó 2015 Published by Elsevier Ltd.
Anti-HIV
1
. Introduction
HIV-1 reverse transcriptase (RT) is an essential enzyme for ret-
considered to be essential for the biological activity. In addition,
quinolones have been reported as anti-HIV agents with diverse
mechanisms of action. For instance, recently approved elvitegravir6
(3, EVG, Fig. 1) is the first quinolone-based anti-HIV drug, exhibit-
ing potent inhibitory activity against integrase-catalyzed DNA
strand transfer, while anti-HIV 6-aminoquinolone WP7-57 (4,
Fig. 1) effectively inhibits the Tat-mediated long terminal repeat
driven transcription, another essential step in the HIV-1 replication
cycle. As the structure of the target compounds combines the fea-
tures of reverse transcriptase (RT), integrase (IN), RT-associated
ribonuclease H (RNase H) and transcription inhibitors, we tested
their inhibitory activity on HIV-1 RT, IN, RNase H as well as in
latently infected cells to pinpoint their mode of action.
roviral replication and represents an important target for the
development of anti-HIV drugs. Non-nucleoside RT inhibitors
(
NNRTIs) bind in a noncompetitive manner to an allosteric
hydrophobic site located around 10 Å away from the polymerase
active site, which is unique to HIV-1 RT and absent in host cell
polymerases. In general, NNRTIs are minimally toxic. To date,
1
there are five structurally diverse NNRTIs approved by FDA for
clinical use: nevirapine (NVP), delavirdine (DLV), efavirenz (EFV),
etravirine (ETV, TMC125) and rilpivirine (RPV, TMC278). The effi-
cacy of the first-generation NNRTIs NVP, DLV and EFV is remark-
ably compromised by RT mutations.2 The second-generation
NNRTIs TMC125 and TMC278 belonging to diarylpyrimidine ana-
logues (DAPYs, Fig. 1) overcome the deficiency of early NNRTIs,
demonstrating high activity against wild-type (wt) and mutant
2
. Chemistry
The synthesis of the diarylpyrimidine–quinolone hybrids is
3,4
virus.
depicted in Scheme 1. The 2,4-difluorobenzoyl chloride 7 were cou-
pled with ethyl 3-(dimethylamino)acrylate to afford the acrylate 8.
Substitution with appropriate aliphatic amines and followed by
cyclization with potassium carbonate and subsequent hydrolysis
produced quinolones 11. The target compounds 5 were obtained
from 11 by replacement of 7-F with 7-OH under basic condition
and followed by condensation with 4-((4-chloropyrimidin-2-
yl)amino)benzonitrile. The target compounds 6 were synthesized
from 11 by treatment with 2, 4-dimethoxybenzylamine and
In this context, we explored ‘molecular hybridization’ strategy5
to generate a series of diarylpyrimidine–quinolone hybrids (Fig. 2).
These hybrids are characterized by a bulky and polar quinolone 3-
carboxylic acid moiety as wing I, as opposed to the above features
⇑
Corresponding authors. Tel.: +86 21 65643809; fax: +86 21 65643811.
E-mail addresses: qqhe@fudan.edu.cn (Q.-Q. He), rfchen@fudan.edu.cn
(
F.-E. Chen).
http://dx.doi.org/10.1016/j.bmc.2015.03.037
968-0896/Ó 2015 Published by Elsevier Ltd.
0
Please cite this article in press as: Mao, T.-Q.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.037

2
T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
CN
the compounds bearing a straight alkyl chain of variable length
on the N-1 position, the trend of potency is ethyl (5a) ꢀ butyl
5e) > propyl (5b). Compound 5a showed an EC50 value of
.28 ± 0.07 M, coupled with a high SI value of 880, and the less
active compounds 5b still showed potency at the submicromolar
level. The replacement of a straight chain butyl group of 5e with
branched ones (5f–g) profile was also shown by the N-1 propyl
analogue 5b. Although N-1 cyclopropyl and cyclohexyl compounds
CN
O
CN
NH
CN
NH
(
0
l
N
HN
N
N
Br
N
NH
2
(
5d, 5i) proved to be inactive, the N-1 cyclopentyl analogue 5h
1
2
exhibited an EC50 value in the submicromolar range, but this activ-
ity was coupled with high cytotoxicity.
However, when assessing anti-HIV-1 activity against a typical
NNRTI-resistant double mutant strain (RT K103N/Y181C), all
diarylpyrimidine–quinolone hybrids totally lose their inhibitory
activity, as is also the case for the first generation NNRTI NVP.
EFV and ETV have EC50 values 26-fold and 7-fold higher, respec-
tively, as compared to their activity against the wt virus. The
NRTI AZT and the INSTI EVG are equipotent against wt and
NNRTI-resistant strain.
O
O
F
O
N
O
H N
2
Cl
OH
OH
N
N
O
N
OH
N
3
4
Figure 1. The structures of the inhibitors of HIV-1 RT (1, 2), IN (3) and transcription
4).
3.2. Assays in latently infected cells
(
Due to the structural similarity of our newly synthesized
hybrids and the reported quinolone derivatives, as for example
WP7-5, as HIV transcription inhibitors, we also tested the target
molecules 5a, 5e and EVG for their activity in latently HIV-1
infected promyelocytic (OM-10.1) cells, in which the virus remains
followed by removal of 2, 4-dimethoxybenzyl group and subse-
quent condensation with 4-((4-chloropyrimidin-2-yl)amino)
benzonitrile.
7
in a latent state. Current antiviral regimens are unable to com-
3
3
. Evaluation of biological activities
.1. Assays in MT-4 Cells
pletely eradicate the virus in HIV-1 infected individuals, which is
mainly caused by the occurrence of stable latent reservoirs. In con-
trast to MT-4 cells, OM-10.1 cell lines should be stimulated to pro-
duce virus. After stimulation with phorbol myristate acetate
The newly synthesized diarylpyrimidine–quinolone hybrids
(
PMA), a dramatic increase in HIV-1 expression could be detected
5
a–i and 6a–e were evaluated for their ability to inhibit the repli-
in these cells. However, none of the tested quinolone derivatives
could inhibit virus production.
cation of wild-type (wt) HIV-1 strain III
B
in MT-4 cells. The
cytotoxicity of the compounds was determined in parallel. FDA-ap-
proved drugs including the NRTI AZT, the NNRTIs NVP, EFV, ETV
and the INSTI EVG were used as reference according to the same
procedure.
As shown in Table 1, all the compounds with NH linker between
the quinolone moiety and the central pyrimidine ring (6a–e) were
devoid of antiviral activity, regardless of the substituent on N-1
position. Nevertheless, a few of the target molecules with O-linker
displayed moderate inhibitory activity against wt HIV-1 replication
3
.3. RT polymerase, RNase H and IN inhibition assay
Based on the metal chelating properties of the quinolone car-
boxylic acid moiety of our newly synthesized compounds and
the known similarities between the arrangement of the residues
in the active site of HIV IN and RNase H, we investigated whether
our diarylpyrimidine–quinolone hybrids could inhibit these
enzyme activities in an RNase H inhibitory activity assay. All com-
pounds in the series were totally devoid of inhibitory activity at the
with EC50 values ranging from 0.28 ± 0.07 to 1.16 ± 0.26 lM. For
highest tested concentrations (P300 lM). Therefore the RNaseH
inhibitory activity can be excluded as the mode of action of the
compounds.
CN
CN
NH
None of the compounds markedly inhibited the IN activity
in vitro at the highest evaluated concentrations (P800
for compound 5e that inhibits the IN activity with an IC50 value of
32.49 ± 0.09 M, which is far above the values assessed for EVG in
the same experiments (0.0018 ± 0.0013 M).
lM), except
O
N
HO
N
O
X
N
l
Br
O
CN
NH
l
NH2
Molecular
Based on the activity spectrum of our newly synthesized com-
pounds in cell culture, active against HIV-1, inactive against HIV-
Hybridization
R1
1
(DAPY)
2
and total loss of activity against an NNRTI-resistant HIV-1 strain,
N
we decided to evaluate the in vitro activity against the recombi-
nant HIV-1 RT. As shown in Table 1, all the compounds active in
cell-based assay exhibited moderate inhibitory activity against
wt HIV-1 RT at micromolar level, which clearly pinpoint a RT-tar-
geted mechanism of action.
F
O
N
O
Cl
N
OH
DAPY-Quinolone Hybrids
O
OH
3
.4. Time-of-addition assay
3
(Quinolone)
In order to confirm RT is the biological target we also performed
Figure 2. The design of diarylpyrimidine–quinolone hybrids through a ‘merging’
strategy.
time-of-addition experiments in MT-4 cells in which the test
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T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
F
O
N
O
O
O
F
O
O
Cl
a
b
c
F
F
F
NH
R1
F
F
7
8
9a-i
O
O
O
O
O
O
O
OH
OH
d
e
N
F
N
HO
N
R1
R1
R1
1
0a-i
1
1a-i
12a-i
O
O
CN
f
HO
N
N
R1
O
N
N
H
5
a-i
9
a, 10a, 11a, 12a, 5a: R = Ethyl, R = H
9f, 10f, 11f, 12f, 5f: R1 = i-Butyl, R2 = H
1
2
9
9
9
9
b, 10b, 11b, 12b, 5b: R = n-Propyl, R = H
9g, 10g, 11g, 12g, 5g: R =s-Butyl, R2 = H
1
2
1
c, 10c, 11c, 12c, 5c: R = i-Propy, R2 = H
9h, 10h, 11h, 12h, 5h: R1 = Cyclopentyl, R2 = H
9i, 10i, 11i, 12i, 5i: R1 = Cyclohexyl, R2 = H
1
d, 10d, 11d, 12d, 5d: R = Cyclopropyl, R = H
1
2
e, 10e, 11e, 12e, 5e: R = n-Butyl, R = H
1
2
O
O
OH
O
O
O
N
O
g
HN
O
N
R1
h
i
OH
OH
H N
N
R1
F
2
R
1
O
1
1
1
1
1
1a: R = Ethyl, R2 = H
13a: R = Ethyl, R2 = H
14a: R = Ethyl, R = H
1 2
1
1
1b: R = n-Propyl, R2 = H
13b: R = n-Propyl, R = H
1
1
1
2
14b: R = n-Propyl, R = H
1 2
1c: R = i-Propy, R2 = H
13c: R = i-Propy, R = H
14c: R = i-Propy, R2 = H
1
1
2
1e: R = n-Butyl, R2 = H
13d: R = n-Butyl, R2 = H
14d: R = n-Butyl, R = H
1 2
1
1
1i: R = Cyclohexyl, R2 = H
13e: R = Cyclohexyl, R2 = H
1
1
14e: R = Cyclohexyl, R = H
1 2
O
O
6
6
6
6
a: R = Ethyl, R = H
1 2
b: R = n-Propyl, R = H
CN
1
1
2
HO
N
c: R = i-Propyl, R2 = H
d: R = n-Butyl, R = H
N
N
H
N
N
H
1
2
6e: R = Cyclohexyl, R = H
1
2
R
1
Scheme 1. Synthesis of diarylpyrimidine–quinolone hybrids 5a–i and 6a–e. Reagents and conditions: (a) ethyl 3-(dimethylamino)acrylate, Et
aliphatic amines, THF, 50 °C, 3 h; (c) K CO
yl)amino)benzonitrile, CO DMF, 60–90 °C, 8–24 h; (g) 2,4-dimethoxybenzylamine; DMSO, 85 °C, 6 h; (h) CF
3
N, toluene, 90 °C, 4 h; (b)
2
3
, DMF, 60–90 °C, 1–6 h; (d) 5 M NaOH, THF, 50 °C, 3.5 h; (e) 12.5 M KOH, DMSO, 80 °C, 3–8 h; (f) 4-((4-chloropyrimidin-2-
COOH, DCM, rt, 5 h; (i) 4-((4-chloropyrimidin-2-
K
2
3
,
3
yl)amino)benzonitrile, iPrOH, conc. HCl, reflux, 5–12 h.
compounds were added at different time points after infection of
4. Molecular modeling studies
B
the cell cultures with HIV-1(III ). Reference compounds with
known mode of action were included such as ETV that inhibits
HIV RT (3–5 h post infection), the diketo acid EVG that inhibits
the strand transfer step of the integration (7–8 h post-infection)
and WP7-5 that is an inhibitor of transcription (10–16 h post-in-
fection). Addition of the diarylpyrimidine–quinolone hybrids, 5a,
To investigate the potential binding mode of the newly synthe-
sized hybrids, molecular simulation was conducted by using Sybyl
Surflex-Dock program and the crystallographic structures of wt RT/
8
RPV (PDB ID: 3MEC) complex. The most active compound in RT
assay 5e was chosen as a representative to be docked into the
NNIBP of wt HIV-1 RT. The co-crystallized ligand was extracted
and the protein was prepared by removing water molecules and
adding hydrogens. Gasteiger–Hückel charges were assigned to
the ligand atoms. The structure of 5e was energy minimized using
the conjugate gradient method. As shown in Figure 3, 5e was
superposed into NNIBP of wt HIV-1 RT in a horseshoe con-
formational shape and established several ligand-receptor interac-
tions similar to those of ETV: (i) Two typical hydrogen bonds
5
e and 5f, all can be postponed for 4–6 h post-infection, coinciding
with the process of reverse transcription, which is also confirmed
by a similar behavior of the NNRTI ETV in the experiments. The
addition of the well-characterized INSTI EVG clearly can postpone
for additional 2 h as compared to the addition of NNRTIs. As time of
addition experiments only reveal the last step targeted by
inhibitors, the NNRTI mode of action of the newly synthesized
compounds is confirmed.
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4
T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Anti-HIV-1 activity and cytotoxicity of diarylpyrimidine–quinolone hybrids 5a–i and 6a–e in MT-4 cells
Compd
EC50a
(
lM)
IC50b
RT
(l
M)
CC50c
HIV-1(III
(
l
M)
SId
HIV-1(III
B
)
K103N/Y181C
B
)
B
HIV-1(III )
5
5
5
5
5
5
5
5
5
6
6
6
6
6
a
b
c
d
e
f
g
h
i
a
b
c
d
e
0.28 ± 0.07
P0.43
>283.16
>246.37
>244.84
>283.16
>221.43
>199.35
>274.44
>274.44
>4.96
7.15 ± 0.51
9.12 ± 0.49
Nd
246.37 ± 29.08
244.84 ± 25.92
>283.16
P221.43
P199.35
880
6569
1
e
>221.43
Nd
61
0.33 ± 0.06
1.16 ± 0.26
>274.44
0.98 ± 0.19
>3.22
>123.44
>188.67
>10.94
>13.62
3.49 ± 0.51
13.98 ± 4.16
Nd
6.08 ± 0.01
Nd
7.07 ± 0.00
Nd
Nd
P604
>237
1
>274.44
>274.44
4.96 ± 0.40
3.22 ± 0.46
123.44 ± 79.38
188.67 ± 54.85
10.94 ± 4.09
13.62 ± 6.09
2.18 ± 1.24
>94
>15
>6.5
14.24 ± 7.49
4.60 ± 0.69
5
<1
<1
<1
<1
<1
<1
16,785
>61
>1204
7495
1484
>3.22
>123.44
>188.67
>10.94
>13.62
>2.18
0.0052 ± 0.0004
P11
0.14 ± 0.00
0.014 ± 0.001
0.0022 ± 0.0013
Nd
Nd
Nd
>2.18
AZT
NVP
EFV
ETV
EVG
0.0056 ± 0.0011
0.244 ± 0.041
0.0054 ± 0.0006
0.0019 ± 0.0005
0.0031 ± 0.0020
0.62 ± 0.16
0.022 ± 0.0030
0.011 ± 0.00
Nd
a
Compound concentration required to protect MT-4 cells from HIV-1-induced cytopathogenic effect by 50% and the data represent the mean of at least three separate
experiments.
b
Compound concentration required to inhibit in vitro HIV-1 RT wt activity by 50%.
Compound concentration that decreases the uninfected MT-4 cell viability by 50%.
Selectivity index: CC50/EC50 ratio.
Not detected.
c
d
e
O-linker showed potent activity against wt HIV-1. In particular,
compounds 5a, 5e and 5h showed low EC50 value of 0.28, 0.33
and 0.98
l
M, respectively.
6
6
. Material and methods
.1. Chemistry
Melting points were measured with a SGW X-1 microscopic
1
13
melting-point apparatus and are uncorrected. H NMR and
C
NMR spectra were recorded on a Bruker AV 400 MHz spectrometer.
Chemical shifts are reported in d (ppm) units relative to the inter-
nal standard tetramethylsilane (TMS). HRMS were obtained on a
Bruker micrOTOF II instrument using electrospray ionization
(
ESI) techniques. All chemicals and solvents used were of reagent
grade and were purified and dried by standard methods before
use. All air-sensitive reactions were run under a nitrogen atmo-
sphere. All the reactions were monitored by thin layer chro-
matography (TLC) on pre-coated silica gel G plates at 254 nm
under a UV lamp using ethyl acetate/hexane as eluent. Column
chromatography separations were obtained on silica gel (300–
Figure 3. Predicted binding mode of 5e (blue) within NNIBP of wt HIV-1 RT. ETV
orange) was superposed into the same pocket for comparison. Hydrogen bonds are
indicated with dashed lines in yellow.
4
00 mesh) using dichloromethane and methanol as eluents.
Analysis of sample purity was performed on an Agilent 1200 series
HPLC system with a Phenomenex Synergi 4 Hydro-RP 80A
(
l
(
4.6 mm ꢁ 250 mm). HPLC conditions were the following: solvent
formed between the nitrogen atom of pyrimidine ring, the NH
linker and Lys101, and (ii) Favorable p–p interaction of the quino-
lone ring with a hydrophobic pocket, as defined by the side chains
of Tyr181, Tyr188, Phe227 and Tyr229.
A = water, solvent B = MeCN; flow rate = 1.0 mL/min. Condition 1:
compounds were eluted with 55% MeCN/water in 40–60 min.
Condition 2: compounds were eluted with 50% MeCN/water con-
taining 0.05% TFA in 40–60 min. Purity was determined by the
absorbance at 254 nm. All tested compounds have a purity of >95%.
5
. Conclusion
6
.1.1. Ethyl 2-(2,4-difluorobenzoyl)-3-(dimethylamino)acrylate
On the basis of ‘hybridization’ strategy, diarylpyrimidine–qui-
(8)
nolone hybrids were identified as a new class of potent NNRTIs.
As opposed to the structural features considered to be essential
for the biological activity of typical NNRTIs, these hybrids are
characterized by a bulky and polar quinolone 3-carboxylic acid
moiety as wing I in the molecules. Most of the hybrids with
A mixture of substituted 2, 4-difluorobenzoyl chloride (3.19 g,
18.1 mmol), ethyl 3-(dimethylamino)acrylate (2.59 g, 18.1 mmol,
1.0 equiv), and triethylamine (2.74 g, 27.1 mmol, 1.5 equiv) in
toluene (30 mL) was stirred at 90 °C for 4 h. After cooling, the reac-
tion mixture was filtered. The filtrate was concentrated under
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T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
reduced pressure. The residue was purified by column chro-
matography on silica gel (petroleum ether/ethyl acetate 2:1 to
1H), 4.45–4.41 (t, J = 7.2 Hz, 2H), 1.78–1.74 (m, 2H), 1.37–1.32
(m, 2H), 0.93–0.89 (t, J = 7.2 Hz, 3H).
1
:2, v/v) to give the desired product 8 as yellow oil. Yield 65%;
1
H NMR (400 MHz, CDCl
3
) d 7.79 (s, 1H), 7.62–7.68 (m, 1H),
6.1.3.6.
7-Hydroxy-1-isobutyl-4-oxo-1,4-dihydroquinoline-3-
1
6
2
.89–6.93 (m, 1H), 6.74–6.80 (m, 1H), 4.03–3.98 (q, J = 7.2 Hz,
H), 3.31 (s, 3H), 2.89 (s, 3H), 0.95–0.99 (t, J = 7.2 Hz, 3H).
carboxylic acid (12f). Yield 69% (for 4 steps from 8); H NMR
(400 MHz, MeOD) d 8.77 (s, 1H), 8.31–8.29 (d, J = 8.0 Hz, 1H),
7
.10 (s, 1H), 7.10–7.08 (d, J = 8.0 Hz, 1H), 4.21–4.20 (d, J = 6.8 Hz,
6
4
.1.2. General procedure for the preparation of 7-fluoro-1-alkyl-
-oxo-1,4-dihydroquinoline-3-carboxylic acids (11a–i)
2H), 2.29–2.27 (m, 1H), 1.01–0.99 (d, J = 7.2 Hz, 6H).
To a stirred solution of the acrylate 8 (0.85 g, 3.0 mmol) in THF
6.1.3.7. 1-(sec-Butyl)-7-hydroxy-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (12g). Yield 69% (for 4 steps from 8); 1H NMR
(400 MHz, MeOD) d 8.81 (s, 1H), 8.34–8.32 (d, J = 9.2 Hz, 1H),
7.27–7.26 (d, J = 1.2 Hz, 1H), 7.11–7.09 (dd, J = 8.8, 1.2 Hz, 1H),
4.90–4.86 (m, 1H), 2.05–1.96 (m, 2H), 1.61–1.60 (d, J = 6.4 Hz,
3H), 0.97–0.93 (t, J = 7.2 Hz, 3H).
(
1
30 mL) was added
a
selected aliphatic amine (3.6 mmol,
.2 equiv). After stirring at 50 °C for 3 h, the solution was concen-
trated under reduced pressure and then diluted with DMF
10 mL). To this solution was added K CO (1.04 g, 7.5 mmol,
.5 equiv). The resulting mixture was stirred at 60–90 °C for 1–
h. After cooling, ice-water (40 mL) was added and the precipitate
(
2
6
2
3
was filtered. The collected precipitate was dissolved in THF (5 mL)
and 5 M NaOH (5 mL) was added. The mixture was stirred at 50 °C
for 3.5 h and then concentrated under reduced pressure. The resi-
due was poured into ice-water (5 mL) and acidified with 4 M HCl to
pH ꢂ2. The resulting precipitate was filtered, washed by water, and
dried to afford 11a–i, which was used directly for the next step
without further purification.
6.1.3.8. 1-Cyclopentyl-7-hydroxy-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (12h). Yield 90% (for 4 steps from 8); H NMR
(400 MHz, MeOD) d 8.82 (s, 1H), 8.34–8.32 (d, J = 9.2 Hz, 1H),
7.31–7.30 (d, J = 1.6 Hz, 1H), 7.13–7.11 (dd, J = 8.8, 1.2 Hz, 1H),
5.17–5.11 (m, 1H), 2.41–2.36 (m, 2H), 2.07–1.91 (m, 6H).
1
6.1.3.9. 1-Cyclohexyl-7-hydroxy-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (12i). Yield 78% (for 4 steps from 8); 1H NMR
(400 MHz, MeOD) d 8.88 (s, 1H), 8.34 (d, J = 8.8 Hz, 1H), 7.26 (d,
J = 1.6 Hz, 1H), 7.12 (dd, J = 8.8, 1.6 Hz, 1H), 4.68–4.56 (m, 1H),
2.28–1.34 (m, 10H).
6
.1.3. General procedure for the preparation of 7-hydroxy-1-
alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acids (12a–i)
To a stirred solution of 7-fluoro-1-alkyl-4-oxo-1,4-dihydro-
quinoline-3-carboxylic acid 11a–i (1.0 mmol) in DMSO (5 mL)
was added 12.5 M KOH (5 mL). After stirring at 80 °C for 3 to 8 h,
the mixture was poured into ice-water (8 mL) and acidified with
6.1.4. General procedure for the preparation of diarylpyrimidine–
quinolone hybrids (5a–i)
6
M HCl to pH ꢂ2. The resulting precipitate was filtered, washed
A mixture of 7-hydroxy-1-alkyl-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid 12a–i (1.0 mmol), 4-((4-chloropyrimidin-2-
by water, and dried to afford 12a–i as a solid, which was used
directly for the next step without further purification.
yl)amino)benzonitrile (242 mg, 1.05 mmol, 1.05 equiv) and K
2 3
CO
(
345 mg, 2.5 mmol, 2.5 equiv) in DMF (10 mL) was stirred at 60–
6
.1.3.1. 1-Ethyl-7-hydroxy-4-oxo-1,4-dihydroquinoline-3-car-
90 °C for 8–24 h. After cooling, the mixture was poured into ice-
water (10 mL), acidified with HAc to pH 5–6, and filtered. The col-
lected precipitate was purified by column chromatography on sil-
ica gel (dichloromethane/methanol 100:1 to 50:1, v/v) to give the
desired product.
1
boxylic acid (12a). Yield 68% (for 4 steps from 8); H NMR
400 MHz, MeOD) d 8.86 (s, 1H), 8.33–8.31 (d, J = 8.8 Hz, 1H),
(
7
4
.13–7.14 (d, J = 1.6 Hz, 1H), 7.12–7.09 (dd, J = 8.8, 1.6 Hz, 1H),
.48–4.43 (q, J = 7.2 Hz, 2H), 1.54–1.51 (t, J = 7.2 Hz, 3H).
6
.1.3.2. 7-Hydroxy-4-oxo-1-propyl-1,4-dihydroquinoline-3-car-
6.1.4.1.
7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-1-
1
boxylic acid (12b). Yield 64% (for 4 steps from 8); H NMR
ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5a). White
solid. Yield 42%; mp 291–293 °C; H NMR (400 MHz, DMSO) d
1
(
400 MHz, DMSO) d 15.56 (s, 1H), 10.97 (br s, 1H), 8.92 (s, 1H),
8
7
1
.24–8.22 (d, J = 8.8 Hz, 1H), 7.16–7.15 (d, J = 1.6 Hz, 1H), 7.13–
.09 (dd, J = 8.8, 1.6 Hz, 1H), 4.41–4.38 (t, J = 7.2 Hz, 2H), 1.83–
.77 (m, 2H), 0.92–0.89 (t, J = 7.2 Hz, 3H).
15.19 (s, 1H), 10.15 (s, 1H), 9.10 (s, 1H), 8.55–8.53 (d, J = 5.6 Hz,
1H), 8.51–8.48 (d, J = 8.8 Hz, 1 H), 8.04 (s, 1H), 7.71–7.68 (d,
J = 8.8 Hz, 2H), 7.62–7.59 (dd, J = 8.8 Hz, 1.6 Hz, 1H), 7.49–4.47 (d,
J = 8.8 Hz, 2H), 6.77–6.75 (d, J = 5.6 Hz, 1H), 4.59–4.54 (q, J = 6.8 Hz,
2H), 1.35–1.31 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO) d
177.71, 169.29, 166.48, 161.03, 159.41, 157.34, 150.22, 144.81,
140.99, 133.13, 128.63, 123.62, 121.66, 119.81, 118.94, 111.36,
1
3
6
.1.3.3. 7-Hydroxy-1-isopropyl-4-oxo-1,4-dihydroquinoline-3-
1
carboxylic acid (12c). Yield 86% (for 4 steps from 8); H NMR
(
400 MHz, DMSO) d 15.58 (s, 1H), 11.03 (br s, 1H), 8.76 (s, 1H),
8
7
.26–8.24 (d, J = 8.8 Hz, 1H), 7.30–7.29 (d, J = 1.6 Hz, 1H), 7.14–
.12 (dd, J = 8.8, 1.6 Hz, 1H), 5.05–5.02 (m, 1H), 1.55–1.53 (d,
108.48, 103.21, 100.75, 49.66, 14.97; HRMS-ESI (ꢃ) m/z calcd for
ꢃ
C H N O
23 17 5 4
[MꢃH] : 426.1202, Found: 426.1196.
J = 6.8 Hz, 6H).
6
.1.4.2. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-4-oxo-
6
3
.1.3.4. 1-Cyclopropyl-7-hydroxy-4-oxo-1,4-dihydroquinoline-
-carboxylic acid (12d). Yield 81% (for 4 steps from 8); H NMR
1-propyl-1,4-dihydroquinoline-3-carboxylic acid (5b). White
solid. Yield 34%; mp 252–254 °C; H NMR (400 MHz, DMSO) d
1
1
(
7
1
400 MHz, MeOD) d 8.82 (s, 1H), 8.29–8.27 (d, J = 8.8 Hz, 1H),
.54 (s, 1H), 7.12–7.09 (d, J = 8.8, 1H), 3.71–3.68 (m, 1H), 1.38–
.36 (m, 2H), 1.20–1.99 (m, 2H).
15.17 (s, 1H), 10.13 (s, 1H), 9.05 (s, 1H), 8.53–8.52 (d, J = 4.2 Hz,
1H), 8.50–8.48 (d, J = 8.8 Hz, 1H), 8.02 (s, 1H), 7.67–7.65 (d,
J = 8.4 Hz, 2H), 7.60–7.58 (d, J = 8.8 Hz, 1H), 7.47–7.45 (d, J = 8.8 Hz,
2
H), 6.75–6.73 (d, J = 5.6 Hz, 1H), 4.49–4.46 (t, J = 7.2 Hz, 2H),
13
6
.1.3.5. 1-Butyl-7-hydroxy-4-oxo-1,4-dihydroquinoline-3-car-
1.75–1.69 (m, 2H), 0.84–0.80 (t, J = 7.2 Hz, 3H);
C NMR
1
boxylic acid (12e). Yield 70% (for 4 steps from 8); H NMR
(100 MHz, DMSO) d 177.71, 169.30, 166.48, 161.05, 159.42, 157.33,
150.55, 144.82, 141.21, 133.14, 128.59, 123.61, 121.68, 119.81,
118.91, 111.51, 108.24, 103.20, 100.77, 55.49, 22.45, 10.92; HRMS-
(
8
400 MHz, DMSO) d 15.54 (s, 1H), 10.99 (s, 1H), 8.91 (s, 1H),
.24–8.22 (d, J = 9.2 Hz, 1H), 7.15 (s, 1H), 7.13–7.10 (d, J = 8.8 Hz,
Please cite this article in press as: Mao, T.-Q.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.037

6
T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
ꢃ
ESI (ꢃ) m/z calcd for C24
H N O
19 5 4
[MꢃH] : 440.1358, Found:
165.99, 160.59, 158.97, 157.11, 144.99, 144.37, 141.54, 132.68,
128.27, 123.16, 121.17, 119.33, 118.46, 110.56, 108.25, 102.73,
4
40.1350.
1
00.30, 57.51, 28.23, 19.43, 9.97; HRMS-ESI (ꢃ) m/z calcd for
ꢃ
6
.1.4.3. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-1-iso-
C
24
H
19
N
5
O
4
[MꢃH] : 454.1515, Found: 454.1508.
propyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5c). White
1
solid. Yield 41%; mp 273–274 °C; H NMR (400 MHz, DMSO) d
6.1.4.8. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-1-cyclo-
1
1
7
6
5.18 (s, 1H), 10.12 (s, 1H), 8.89 (s, 1H), 8.53–8.52 (d, J = 5.6 Hz,
H), 8.52–8.51 (d, J = 8.8 Hz, 1H), 7.68–7.66 (d, J = 8.8 Hz, 2H),
.62–7.60 (dd, J = 8.8, 2.0 Hz, 1H), 7.47–7.45 (d, J = 8.4 Hz, 2H),
.75–6.73 (d, J = 5.6 Hz, 1H), 5.21–5.15 (m, 1H), 1.51–1.50 (d,
pentyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5h). White
solid. Yield 51%; mp 278–279 °C; H NMR (400 MHz, DMSO) d 15.12
1
(s, 1H), 10.12 (s, 1H), 8.79 (s, 1H), 8.53–8.52 (d, J = 5.6 Hz, 1H), 8.51–
8.49 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 7.68–7.66 (d, J = 8.8 Hz, 2H),
7.62–7.60 (dd, J = 8.8, 1.2 Hz, 1H), 7.47–7.45 (d, J = 8.8 Hz, 2H), 6.74–
13
J = 6.8 Hz, 6H);
C NMR (100 MHz, DMSO) d 177.45, 169.30,
1
1
1
66.50, 161.03, 159.43, 157.50, 145.37, 144.83, 141.50, 133.14,
28.69, 123.66, 121.64, 119.81, 118.94, 111.06, 108.54, 103.20,
00.76, 53.36, 21.84; HRMS-ESI (ꢃ) m/z calcd for C24
6.73 (d, J = 5.6 Hz, 1H), 5.25–5.18 (m, 1H), 2.21–1.73 (m, 8 H); 13
C
NMR (100 MHz, DMSO) d 177.44, 169.30, 166.54, 161.04, 159.43,
157.33, 145.40, 144.82, 142.06, 133.14, 128.54, 123.69, 121.70,
H N O
19 5 4
ꢃ
[MꢃH] : 440.1358, Found: 440.1348.
119.81, 118.96, 111.60, 108.27, 103.22, 100.73, 62.92, 32.00, 23.75;
ꢃ
HRMS-ESI (ꢃ) m/z calcd for C26
H
21
N
5
O
4
[MꢃH] : 466.1515, Found:
6
1
.1.4.4.
7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-
acid
466.1500.
-cyclopropyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
1
(
5d). White solid. Yield 42%; mp 272–273 °C; H NMR (400 MHz,
6.1.4.9. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-1-cyclo-
DMSO) d 15.02 (s, 1H), 10.14 (s, 1H), 8.78 (s, 1H), 8.55–8.53 (d,
J = 5.6 Hz, 1H), 8.47–8.45 (d, J = 8.8 Hz, 1H), 8.13–8.12 (d,
J = 2.0 Hz, 1H), 7.71–7.69 (d, J = 8.4 Hz, 2H), 7.64–7.61 (dd, J = 8.8,
hexyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5i). White
solid. Yield 37%; mp 288–290 °C; H NMR (400 MHz, DMSO) d 15.16
1
(s, 1H), 10.11 (s, 1H), 8.88 (s, 1H), 8.53–8.52 (d, J = 4.8 Hz, 1H), 8.52–
8.50 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 7.69–7.67 (d, J = 8.4 Hz, 2H),
7.62–7.60 (d, J = 8.8 Hz, 1H), 7.48–7.46 (d, J = 8.4 Hz, 2H), 6.74–6.73
(d, J = 5.6 Hz, 1H), 4.83–4.77 (m, 1H), 2.03–2.01 (m, 2H), 1.85–1.77
2
.0 Hz, 1H), 7.51–7.49 (d, J = 8.8 Hz, 2H), 6.76–6.75 (d, J = 5.6 Hz,
H), 3.79–3.76 (m, 1H), 1.27–1.25 (m, 2H), 1.16–1.15 (m, 2H);
C NMR (100 MHz, DMSO) d 177.93, 169.14, 166.28, 161.15,
59.45, 157.08, 149.76, 144.81, 143.01, 133.21, 128.34, 123.04,
1
1
3
13
1
1
8
(m, 4H), 1.64–1.49 (m, 3H), 1.33–1.22 (m, 1H); C NMR (100 MHz,
DMSO) d 177.36, 169.25, 166.51, 161.03, 159.41, 157.45, 145.60,
144.83, 141.49, 133.14, 128.62, 123.70, 121.73, 119.80, 118.94,
21.71, 119.84, 118.96, 111.27, 108.13, 103.25, 100.84, 36.51,
.08; HRMS-ESI (ꢃ) m/z calcd for C24
ꢃ
H
17
N
5
O
4
[MꢃH] : 438.1202,
Found: 438.1197.
110.91, 108.48, 103.22, 100.78, 60.17, 32.27, 25.28, 24.99; HRMS-ESI
ꢃ
(
ꢃ) m/z calcd for C27
H
23
N
5
O
4
[MꢃH] : 480.1671, Found: 480.1667.
6
.1.4.5. 1-Butyl-7-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)
oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5e). White
solid. Yield 54%; mp 267–268 °C; H NMR (400 MHz, DMSO) d
6.1.5. Generalprocedureforthepreparationof7-((2,4-dimethoxy-
benzyl)amino)-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
acids (13a–e)
To a solution of 7-fluoro-1-alkyl-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid 11a–c, e, i (1.0 mmol) in DMSO (2 mL) was added
2,4-dimethoxybenzylamine (3.0 mmol, 3.0 equiv). After stirring at
85 °C for 6 h, the solution was poured into ice-water (5 mL), acidi-
fied with 6 M HCl to pH ꢂ5, filtered, washed by ethanol and dried
to give the desired compound as a solid.
1
1
1
7
7
4
0
1
1
1
5.16 (s, 1H), 10.13 (s, 1H), 9.04 (s, 1H), 8.53–8.52 (d, J = 5.6 Hz,
H), 8.50–8.48 (d, J = 8.8 Hz, 1H), 7.98–7.97 (d, J = 1.2 Hz, 1H),
.66–7.64 (d, J = 8.4 Hz, 2H), 7.60–7.57 (dd, J = 8.8, 1.6 Hz, 1H),
.45–7.43 (d, J = 8.4 Hz, 2H), 6.75–6.74 (d, J = 5.6 Hz, 1H), 4.52–
.48 (d, J = 7.2 Hz, 2H), 1.68–1.64 (m, 2H), 1.27–1.21 (m, 2H),
.77–0.73 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO) d 177.70,
69.30, 166.47, 161.06, 159.41, 157.32, 150.46, 144.82, 141.16,
33.11, 128.63, 123.62, 121.61, 119.79, 118.87, 111.55, 108.27,
03.20, 100.80, 54.03, 31.18, 19.48, 13.83; HRMS-ESI (ꢃ) m/z calcd
6.1.5.1.
dihydroqui noline-3-carboxylic acid (13a). Yield 100%; H NMR
400 MHz, DMSO) d 16.03 (s, 1H), 8.75 (s, 1H), 8.01–7.99 (d,
7-((2,4-Dimethoxybenzyl)amino)-1-ethyl-4-oxo-1,4-
ꢃ
1
for C24
H
19
N
5
O
4
[MꢃH] : 454.1515, Found: 454.1503.
(
6
.1.4.6. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)oxy)-1-iso-
J = 8.8 Hz, 1H), 7.44–7.41 (t, J = 5.2 Hz, 1H), 7.21–7.19 (d,
J = 8.4 Hz, 1H), 6.96–6.93 (dd, J = 1.6, 8.8 Hz, 1H), 6.59–6.57 (m,
2H), 6.49–6.47 (dd, J = 8.4, 2.0 Hz, 1H), 4.37–4.30 (m, 4H), 3.83 (s,
3H), 3.72 (s, 3H), 1.30–1.27 (t, J = 7.2 Hz, 3H).
butyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5f). White
solid. Yield 36%; mp 251–253 °C; H NMR (400 MHz, DMSO) d
1
15.15 (s, 1H), 10.12 (s, 1H), 9.00 (s, 1H), 8.53–8.52 (d, J = 5.6 Hz,
1H), 8.50–8.48 (d, J = 8.8 Hz, 1H), 8.00–7.99 (d, J = 1.6 Hz, 1H),
7.66–7.64 (d, J = 8.4 Hz, 2H), 7.60–7.58 (dd, J = 8.8, 1.6 Hz, 1H),
7.46–7.44 (d, J = 8.8 Hz, 2H), 4.36–4.34 (d, J = 7.2 Hz, 2H), 2.08–
2
.03 (m, 1H), 0.81–0.79 (d, J = 6.8 Hz, 6H); 13C NMR (100 MHz,
6.1.5.2. 7-((2,4-Dimethoxybenzyl)amino)-4-oxo-1-propyl-1,4-
1
dihydroqui noline-3-carboxylic acid (13b). Yield 96%; H NMR
(400 MHz, DMSO) d 16.02 (s, 1 H), 8.74 (s, 1H), 8.02–7.99 (d,
J = 8.8 Hz, 1H), 7.44–7.42 (t, J = 4.8 Hz, 1H), 7.18–7.16
(d, J = 8.0 Hz, 1H), 6.97–6.94 (d, J = 8.8 Hz, 1H), 6.60–6.59
(d, J = 1.6 Hz, 1H), 6.52 (s, 1 H), 6.49–6.46 (dd, J = 8.4, 2.0 Hz, 1H),
4.32–4.30 (d, J = 5.2 Hz, 2H), 4.28–4.24 (t, J = 7.2 Hz, 2H),
DMSO) d 177.72, 169.27, 166.49, 161.07, 159.40, 157.30, 150.67,
1
1
44.81, 141.39, 133.13, 128.55, 123.55, 121.67, 119.79, 118.87,
11.59, 108.04, 103.19, 100.81, 60.29, 27.88, 19.54; HRMS-ESI (ꢃ)
ꢃ
m/z calcd for C24
H N O
19 5 4
[MꢃH] : 454.1515, Found: 454.1502.
3
.82 (s, 3H), 3.72 (s, 3H), 1.68–1.63 (m,
2 H), 0.84–0.81
6.1.4.7. 1-(sec-Butyl)-7-((2-((4-cyanophenyl)amino)pyrimidin-4-
(t, J = 7.2 Hz, 3 H).
yl)oxy)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5g). White
1
solid. Yield 49%; mp 273–275 °C; H NMR (400 MHz, DMSO) d
1
6.1.5.3. 7-((2,4-Dimethoxybenzyl)amino)-1-isopropyl-4-oxo-1,4-
5.18 (s, 1H), 10.15 (s, 1H), 8.81 (s, 1H), 8.54–8.53 (d, J = 5.6 Hz,
dihydro quinoline-3-carboxylic acid (13c). Yield 95%; 1H NMR
1H), 8.52–8.51 (d, J = 8.8 Hz, 1H), 8.19 (s, 1H), 7.68–7.66 (d,
(400 MHz, DMSO)
d
15.78 (s,
1
H), 8.74 (s, 1H),
J = 8.8 Hz, 2H), 7.61–7.59 (dd, J = 8.8, 1.2 Hz, 1H), 7.48–7.46 (d,
J = 8.8 Hz, 1H), 6.76–6.74 (d, J = 5.6 Hz, 1H), 5.07–4.99 (m, 1H),
8.30–8.28 (d, J = 8.8 Hz, 1H), 7.28 (s, 1H), 7.22–7.20 (d, J = 8.4 Hz,
1H), 6.86–6.84 (d, J = 8.8 Hz, 1H), 6.59 (s, 1 H), 6.53 (s, 1 H), 6.49–
6.47 (d, J = 8. 0 Hz, 1H), 5.01–4.98 (t, J = 5.2 Hz, 1H), 4.84–4.81 (m,
1H), 4.42–4.40 (d, J = 5.6 Hz, 2 H), 1.60–1.59 (d, J = 6.4 Hz, 6 H).
1
.96–1.83 (m, 2H), 1.48–1.46 (d, J = 7.2 Hz, 3H), 0.78–0.75 (t,
13
J = 7.2 Hz, 3H);
C NMR (100 MHz, DMSO) d 176.69, 168.85,
Please cite this article in press as: Mao, T.-Q.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.037

T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
6
.1.5.4.
dihydro quinoline-3-carboxylic acid (13d). Yield 95%; H NMR
400 MHz, DMSO) d 15.98 (s, 1 H), 8.74 (s, 1H), 8.02–8.00 (d,
J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.16–7.14 (d, J = 8.0 Hz, 1H), 6.98–
.96 (d, J = 8.4 Hz, 1H), 6.60 (s, 1H), 6.48 (s, 1H), 6.48–6.46 (d,
J = 8.0 Hz, 1H), 4.32–4.29 (m, 4H), 3.83 (s, 3H), 3.72 (s, 3H), 1.59–
1-Butyl-7-((2,4-dimethoxybenzyl)amino)-4-oxo-1,4-
yl)amino)benzonitrile (345 mg, 1.5 mmol, 1.5 equiv) in a solution
of isopropanol (5 mL) containing 20% conc. HCl was stirred at
reflux for 5–12 h. The mixture was cooled and filtered. The filter
1
(
3
cake was washed by isopropanol, neutralize with NaHCO to pH
6
5–6, and filtered. The crude was purified by column chro-
matography on silica gel (dichloromethane/methanol 20:1, v/v)
to give the desired product.
1
.56 (m, 2 H), 1.25–1.19 (m, 2 H), 0.84–0.81 (t, J = 7.2 Hz, 3 H).
6
1
.1.5.5. 1-Cyclohexyl-7-((2,4-dimethoxybenzyl)amino)-4-oxo-
6.1.7.1. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)amino)-1-
ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6a). White
1
,4-dihydro quinoline-3-carboxylic acid (13e). Yield 100%;
H
NMR (400 MHz, DMSO) d 16.02 (s, 1 H), 8.59 (s, 1H), 8.05–8.03
d, J = 9.2 Hz, 1H), 7.39 (s, 1H), 7.16–7.14 (d, J = 8.4 Hz, 1H), 6.98–
solid. Yield 28%; mp >300 °C; IR
(C„N), 1710 (C@O acid), 1629 (C@O ketone) cm
m 3427, 3322 (OH, NH), 2220
ꢃ1
1
(
;
H NMR
6
6
.96 (d, J = 9.2 Hz, 1H), 6.66 (s, 1H), 6.60–6.59 (d, J = 2.0 Hz, 1H),
.47–6.44 (dd, J = 2.0, 8.0 Hz, 1H), 4.44–4.38 (m, 1H), 4.35–4.33
(400 MHz, DMSO) d 10.90 (s, 1H), 10.41 (s, 1H), 9.02 (s, 1H),
8.35–8.32 (d, J = 8.8 Hz, 1H), 8.25–8.23 (d, J = 6.0 Hz, 1H), 8.12 (s,
1H), 8.09–8.07 (d, J = 8.8 Hz, 1H), 7.87–7.85 (d, J = 8.8 Hz, 2H),
7.78–7.76 (d, J = 8.8 Hz, 2H), 6.62–6.61 (d, J = 6.0 Hz, 1H), 4.45–
(
d, J = 5.2 Hz, 1H), 3.82 (s, 3H), 3.72 (s, 3H), 1.95–1.67 (m, 7 H),
1
.51–1.25 (m, 3 H).
4
.40 (q, J = 7.2 Hz, 2H), 1.36–1.33 (t, J = 7.2 Hz, 3H); HRMS-ESI (ꢃ)
ꢃ
6
.1.6. General procedure for the preparation of 7-amino-1-
m/z calcd for C23
H
18
N
6
O
3
[MꢃH] : 425.1362, Found: 425.1341.
alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (14a–e)
To a solution of 7-((2,4-dimethoxybenzyl)amino)-1-alkyl-4-
oxo- 1,4-dihydroquinoline-3-carboxylic acids 13a–e (5.0 mmol)
in dichloromethane (30 mL) was added trifluoroacetic acid
6.1.7.2. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)amino)-4-
oxo-1-propyl-1,4-dihydroquinoline-3-carboxylic
acid
(6b). Offwhite solid. Yield 32%; mp >300 °C; IR
m
3313, 3186,
(
1.71 g, 15.0 mmol, 3.0 equiv). After stirring at rt for 5 h, the solu-
tion was poured into ice-water (250 mL) and stirred violently for
0 min. The resulting precipitate was collected, dissolved in
3090 (OH, NH), 2220 (C„N), 1698 (C@O acid), 1608 (C@O ketone)
ꢃ1
1
cm ; H NMR (400 MHz, DMSO) d 15.48 (s, 1H), 10.22 (s, 1H), 9.83
(s, 1H), 8.98 (s, 1H), 8.33–8.31 (d, J = 9.2 Hz, 1H), 8.26–8.25 (d,
J = 5.6 Hz, 1H), 8.19 (s, 1H), 8.06–8.04 (d, J = 9.2 Hz, 1H), 7.94–
7.92 (d, J = 8.8 Hz, 2H), 7.72–7.70 (d, J = 8.8 Hz, 2H), 6.50–6.49 (d,
J = 5.6 Hz, 1H), 4.43–4.40 (t, J = 7.2 Hz, 2H), 1.82–1.76 (m, 2H),
2
dichloromethane (20 mL) containing 5% methanol and filtered.
The filtrate was concentrated under reduced pressure to afford
the desired compound (14a–e) as a solid.
0
C
.86–0.82 (t, J = 7.2 Hz, 3H); HRMS-ESI (ꢃ) m/z calcd for
ꢃ
6
.1.6.1. 7-Amino-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
24
20
H N
6
O
3
[MꢃH] : 439.1518, Found: 439.1511.
1
acid (14a). Yield 93%; H NMR (400 MHz, DMSO) d 16.02 (s, 1H), 8.77
s, 1H), 8.02–8.00 (d, J = 8.8 Hz, 1H), 6.87–6.85 (d, J = 9.2 Hz, 1H), 6.78
s, 1H), 6.49 (s, 2H), 4.37–4.32 (q, J = 6.8 Hz, 2H), 1.40–1.37 (t,
J = 6.8 Hz, 3 H).
(
(
6.1.7.3. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)amino)-1-
isopropyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(6c). Offwhite solid. Yield 30%; mp >300 °C; IR
m
3323, 3203,
3
105 (OH, NH), 2221 (C„N), 1699 (C@O acid), 1611 (C@O ketone)
ꢃ1
1
6
.1.6.2. 7-Amino-4-oxo-1-propyl-1,4-dihydroquinoline-3-car-
cm ; H NMR (400 MHz, DMSO) d 15.50 (s, 1H), 10.23 (s, 1H), 9.87
(s, 1H), 8.80 (s, 1H), 8.36–8.34 (d, J = 8.8 Hz, 1H), 8.26 (m, 2H),
8.11–8.09 (d, J = 8.4 Hz, 1H), 7.93–7.91 (d, J = 7.6 Hz, 2H), 7.71–
7.70 (d, J = 7.6 Hz, 2H), 6.50–6.49 (d, J = 5.6 Hz, 1H), 5.01–4.95 (m,
1
boxylic acid (14b). Yield 95%; H NMR (400 MHz, DMSO) d
6.02 (s, 1H), 8.76 (s, 1H), 8.02–8.00 (d, J = 8.8 Hz, 1H), 6.87–6.85
dd, J = 1.2, 9.2 Hz, 1H), 6.77–6.76 (d, J = 0.8 Hz, 1H), 6.49 (s, 2H),
.29–4.25 (t, J = 7.2 Hz, 2H), 1.83–1.77 (m, 2H), 0.92–0.89 (t,
J = 7.2 Hz, 3H).
1
(
4
1H), 1.52–1.51 (d, J = 5.2 Hz, 6H); HRMS-ESI (ꢃ) m/z calcd for
ꢃ
C
24
20
H N
6
O
3
[MꢃH] : 439.1518, Found: 439.1519.
6
.1.6.3.
7-Amino-1-isopropyl-4-oxo-1,4-dihydroquinoline-3-
6.1.7.4. 1-Butyl-7-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)a-
1
carboxylic acid (14c). Yield 96%; H NMR (400 MHz, DMSO) d
mino)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
1
1
6.05 (s, 1H), 8.63 (s, 1H), 8.05–8.03 (d, J = 8.8 Hz, 1H), 6.93 (s,
H), 6.89–6.86 (dd, J = 1.2, 8.8 Hz, 1H), 6.49 (br s, 2H), 4.91–4.84
(6d). Offwhite solid. Yield 35%; mp 293–295 °C; IR
m
3343, 3176,
3074 (OH, NH), 2221 (C„N), 1700 (C@O acid), 1606 (C@O ketone)
ꢃ1
1
(
m, 1H), 1.54–1.52 (d, J = 6.4 Hz, 6H).
cm ; H NMR (400 MHz, DMSO) d 15.50 (s, 1H), 10.24 (s, 1H), 9.85
s, 1H), 8.97 (s, 1H), 8.33–8.30 (d, J = 9.2 Hz, 1H), 8.26–8.25 (d,
(
6
.1.6.4.
boxylic acid (14d). Yield 97%; H NMR (400 MHz, DMSO) d
6.00 (s, 1H), 8.76 (s, 1H), 8.02–8.00 (d, J = 9.2 Hz, 1H), 6.87–6.85
d, J = 8.8 Hz, 1H), 6.76 (s, 1H), 6.50 (s, 2H), 4.29–4.31 (t,
J = 7.2 Hz, 2H), 1.79–1.72 (m, 2H), 1.37–1.31 (m, 2H), 0.94–0.90
t, J = 8.8 Hz, 3H).
7-Amino-1-butyl-4-oxo-1,4-dihydroquinoline-3-car-
J = 5.6 Hz, 1H), 8.21 (s, 1H), 8.03–8.01 (d, J = 9.2 Hz, 1H), 7.94–
7.92 (d, J = 8.8 Hz, 2H), 7.72–7.70 (d, J = 8.8 Hz, 2H), 6.51–6.49 (d,
J = 5.6 Hz, 1H), 4.47–4.43 (t, J = 6.8 Hz, 2H), 1.76–1.72 (m, 2H),
1
1
(
1.30–1.24 (m, 2H), 0.85–0.81 (t, J = 7.2 Hz, 3H); HRMS-ESI (ꢃ) m/
ꢃ
z calcd for C25
H
22
N
6
O
3
[MꢃH] : 453.1675, Found: 453.1677.
(
6
.1.7.5. 7-((2-((4-Cyanophenyl)amino)pyrimidin-4-yl)amino)-1-
6
.1.6.5. 7-Amino-1-cyclohexyl-4-oxo-1,4-dihydroquinoline-3-
cyclo
hexyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
1
carboxylic acid (14e). Yield 84%; H NMR (400 MHz, DMSO) d
(6e). White solid. Yield 38%; mp 227–230 °C; IR
m
3511, 3400,
16.00 (s, 1H), 8.63 (s, 1H), 8.05–8.03 (d, J = 8.8 Hz, 1H), 6.94 (s,
1H), 6.89–6.86 (dd, J = 2.0, 8.8 Hz, 1H), 6.48 (s, 2H), 5.74 (s, 1H),
4.45–4.39 (m, 1H), 2.07–1.70 (m, 7H), 1.57–1.31 (m, 3H).
3048 (OH, NH), 2229 (C„N), 1715 (C@O acid), 1605 (C@O ketone)
cm ; H NMR (400 MHz, DMSO) d 15.49 (s, 1H), 10.26 (s, 1H), 9.93
(s, 1H), 8.79 (s, 1H), 8.38 (s, 1H), 8.36–8.34 (d, J = 8.8 Hz, 1H), 8.28–
ꢃ1
1
8
.26 (d, J = 5.6 Hz, 1H), 8.01–7.98 (d, J = 8.8 Hz, 1H), 7.96–7.94 (d,
6
.1.7. Procedure for the preparation of diarylpyrimidine–
J = 8.8 Hz, 2H), 7.72–7.69 (d, J = 8.8 Hz, 2H), 6.51–6.50 (d,
J = 5.6 Hz, 1H), 4.61–4.60 (m, 1H), 2.09–2.06 (m, 2H), 1.83–1.81
quinolone hybrids (6a–e)
A mixture of 7-amino-1-alkyl-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid 14a–e (1.0 mmol), 4-((4-chloropyrimidin-2-
(m, 4H), 1.61–1.58 (m, 1H), 1.41–1.21 (m, 3H); HRMS-ESI (ꢃ) m/z
ꢃ
calcd for C27
H
24
N
6
O
3
[MꢃH] : 479.1831, Found: 479.1810.
Please cite this article in press as: Mao, T.-Q.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.037

8
T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
6
6
.2. Biology and biochemistry
relative fluorescence, that is, the fluorescence signal of the reaction
mix with compound divided by the signal of the same reaction mix
without compound.
.2.1. Anti-HIV activity assays
Evaluation of the antiviral activity of the compounds against
HIV-1 strain III
as previously described. Stock solutions (10ꢁ final concentration)
of test compounds were added in 25 L volumes to two series of
triplicate wells so as to allow simultaneous evaluation of their
effects on mock-and HIV-infected cells at the beginning of each
experiment. Serial 5-fold dilutions of test compounds were mad
directly in flat-bottomed 96-well microtiter trays using a Biomek
B
in MT-4 cells was performed using the MTT assay
6.2.3. Time-of-addition experiments
9
Time-of-addition experiments were adapted from Pauwels et al.
1
5
l
and Daelemans et al. Briefly, MT-4 cells were infected with HIV-
1(III ) or the RT double mutant virus (K103 N/Y181C) at an m.o.i. of
B
0.5. Following a 1 h adsorption period cells were distributed in a
96-well tray at 45,000 cells/well and incubated at 37 °C. Test com-
pounds were added at different times (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 24,
and 25 h) after infection. HIV-1 production was determined at 31 h
postinfection via a p24 enzyme-linked immunosorbent assay
(Perkin Elmer, Brussels, Belgium). Dextran sulfate was used at
3
000 robot (Beckman instruments, Fullerton, CA). Untreated con-
trol HIV- and mock-infected cell samples were included for each
samples.
HIV-1(III
B
)10 stock (50
lL) at 100–300 CCID50 (cell culture infec-
12.5
l
M, AZT at 1.9
l
M, ETV at 0.19
l
M, EVG at 0.31
l
M, the 6-
and the
tious dose) or culture medium was added to either the infected or
aminoquinolone derivative WP7-5 at 0.25
diarylpyrimidine–quinolone hybrids were added at their 100-fold
their EC50 concentration obtained in the MT-4/MTT assay.
lM
1
1
mock-infected wells of the microtiter tray. Mock-infected cells
were used to evaluate the effect of test compound on uninfected
cells in order to assess the cytotoxicity of the test compound.
12
Exponentially growing MT-4 cells were centrifuged for 5 min at
20 g and the supernatant was discarded. The MT-4 cells were
6.2.4. RNaseH assay
The RNaseH assay was developed also employing the dsDNA
quantitation reagent PicoGreen. A RNA/DNA heteroduplex is
2
5
resuspended at 6 ꢁ 10 cells/ml, and 50-
lL volumes were trans-
0
ferred to the microtiter tray wells. Five days after infection, the
viability of mock- and HIV-infected cells was examined spec-
trophotometrically by the MTT assay.
formed by annealing a 40 bases long RNA oligonucleotide (5 -CC
0
AGCAGGAAACAGCUAUGACGAUC UGAGCCUGGGAGCU-3 ) and 120
0
bases long DNA oligonucleotide (5 -AGCTC CCAGGCTCAGATCGTC
The MTT assayisbasedonthe reductionofyellowcoloured3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
ATAGCTGTTTCCTGCTGGCAGCTCCCAGGCTCAGATCGTCATAGCTGTT
TCCTGCTGGCAGCTCCCAGGCTCAGATCGTCATAGCTGTTTCCTGCTGG
C-3 ) in a 4:1 molar ratio.
0
(
Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase of
metabolically active cells to a blue-purple formazan that can be mea-
sured spectrophotometrically. The absorbances were read in an
eight-channel computer-controlled photometer (Infinite M1000,
Tecan, Mechelen, Belgium), at two wavelengths (540 and 690 nm).
All data were calculated using the median OD (optical density) value
of tree wells. The 50% cytotoxic concentration (CC50) was defined as
the concentration of the test compound that reduced the absorbance
An amount of 76 ng of the annealed complex is brought into
each well of a 96-well plate in a volume of 20
(60 mM Tris–HCl, 60 mM KCl, 8 mM MgCl , 13 mM DTT, pH 8.1)
and 5 L of suitably diluted RT solution is added. Reactions are
lL RNaseH buffer
2
l
stopped after 60 min. at 25 °C by the addition of EDTA (15 mM
fc). PicoGreen is then added to measure the amount of heterodu-
plexes and thus the decrease in signal upon RNA hydrolysis in
the presence of enzyme activity and signals are read as described
for the reverse transcriptase assay. The results are expressed
relative to the amount of heteroduplexes measured in a negative
control sample without RT enzyme, after subtraction of the back-
ground signal obtained with only ssDNA.
(
OD540) of the mock-infected control sample by 50%. The concentra-
tion achieving 50% protection from the cytopathic effect of the virus
ininfectedcells was definedasthe 50% effective concentration(EC50).
6
.2.2. Reverse transcriptase assay
Recombinant wt p66/p51 HIV-1 RT was expressed and purified
To test compounds for activity against RNaseH, 1 lL of com-
as described by Auwerx et al.13 The RT assay is performed with the
EnzCheck Reverse Transcriptase Assay kit (Molecular Probes,
Invitrogen), as described by the manufacturer. The assay is based
on the dsDNA quantitation reagent PicoGreen. This reagent shows
a pronounced increase in fluorescence signal upon binding to
dsDNA or RNA-DNA heteroduplexes. Single-stranded nucleic acids
generate only minor fluorescence signal enhancement when a suf-
ficiently high dye: base pair ratio is applied.1 This condition is met
in the assay.
pound in DMSO is added to each well before the addition of RT
enzyme solution. Control wells without compound contain the
same amount of DMSO. Positive compounds were tested for aut-
ofluorescence in a separate test.
6.2.5. Integrase assay
The inhibitory activity of the reference compound EVG and the
newly synthesized was assessed using the HIV-1 Integrase Kit
(catalog number EZ-1700) of XpressBio (Thurmont, MD, USA)
according to the manufacturer’s protocol.
4
A poly(rA) template of approximately 350 bases long, and an
oligo(dT)16 primer, are annealed in a molar ratio of 1:1.2
(
60 min at rt). Fifty-two ng of the RNA/DNA is brought into each
well of a 96-well plate in a volume of 20 L polymerization buffer
60 mM Tris–HCl, 60 mM KCl, 8 mM MgCl , 13 mM DTT, 100
L of RT enzyme solution, diluted to a suitable
6.2.6. Anti-HIV-1 and cytotoxic assays in latently infected OM-
10.1 cells
l
(
2
lM
The activity values of the compounds against latent HIV-1 infec-
tion were based on the inhibition of p24 antigen production in OM-
10.1 cells after stimulation with PMA (Sigma Chemical Co., Bornem
dTTP, pH 8.1). Five
l
concentration in enzyme dilution buffer (50 mM Tris–HCl, 20%
glycerol, 2 mM DTT, pH 7.6), is added. The reactions are incubated
at 25 °C for 40 min and then stopped by the addition of EDTA
5
Belgium). Briefly, OM-10.1 cells (5 ꢁ 10 cells/ml) were incubated
in the presence or absence of the compounds for 2 h in 48 wells
plates. After this short incubation period, the cells were stimulated
(
15 mM fc). Heteroduplexes are then detected by addition of
PicoGreen. Signals are read using an excitation wavelength of
90 nm and emission detection at 523 nm using a spectrofluo-
rometer (Safire2, Tecan). To test the activity of compounds against
RT, 1 L of compound in DMSO is added to each well before the
with 0.02 lM of PMA followed by transfer of 2 times 200 lL into a
4
96-well plate for toxicity evaluation. After a 2-day incubation at
37 °C, the cell culture supernatants were collected from the 48-
well plates and examined for their p24 antigen levels with the
HIV-1 p24 ELISA kit (Perkin Elmer, Boston, MA). Cytotoxicity of
the compounds for latently HIV-1-infected OM-10.1 cell line in
l
addition of RT enzyme solution. Control wells without compound
contain the same amount of DMSO. Results are expressed as
Please cite this article in press as: Mao, T.-Q.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.037

T.-Q. Mao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
the 96-well plates were based on the MTT cell viability staining as
described previously.
V.; Hughes, S. H.; Medaer, B.; De Knaep, F.; Bohets, H.; De Clerck, F.; Lampo, A.;
Williams, P.; Stoffels, P. J. Med. Chem. 2005, 48, 1901.
(a) Viegas-Junior, C.; Danuello, A.; Bolzani, V.; Barreiro, E. J.; Fraga, C. A. M. Curr.
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1
4
5
6
.
.
Acknowledgments
This research was financially supported by grants from the
National Natural Science Foundation of China (No. 21302021),
the Shanghai Municipal Natural Science of Foundation (No.
7. Tabarrini, O.; Massari, S.; Daelemans, D.; Meschini, F.; Manfroni, G.; Bottega, L.;
Gatto, B.; Palumbo, M.; Pannecouque, C.; Cecchetti, V. ChemMedChem 2010, 5,
1880.
1
3ZR1402200), the Specialized Research Fund for the Doctoral
8
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.
Lansdon, E. B.; Brendza, K. M.; Hung, M.; Wang, R.; Mukund, S.; Jin, D.; Birkus,
G.; Kutty, N.; Liu, X. J. Med. Chem. 2010, 53, 4295.
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Program of Higher Education (No. 20130071120034), and by the
KU Leuven (GOA/15/019/TBA). The technical assistance of Mr.
Kris Uyttersprot, Mrs. Kristien Erven, and Mrs. Cindy Heens for
the HIV experiments is gratefully acknowledged.
10. Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C. Science 1984, 224, 497.
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Please cite this article in press as: Mao, T.-Q.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.037