Discovery, optimization, and target identification of novel coumarin derivatives as HIV-1 reverse transcriptase-associated ribonuclease H inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113769

Despite significant advances in antiretroviral therapy, acquired immunodeficiency syndrome remains as one of the leading causes of death worldwide. New antiretroviral drugs combined with updated treatment strategies are needed to improve convenience, tolerability, safety, and antiviral efficacy of available therapies. In this work, a focused library of coumarin derivatives was exploited by cell phenotypic screening to discover novel inhibitors of HIV-1 replication. Five compounds (DW-3, DW-4, DW-11, DW-25 and DW-31) showed moderate activity against wild-type and drug-resistant strains of HIV-1 (IIIB and RES056). Four of those molecules were identified as inhibitors of the viral RT-associated RNase H. Structural modification of the most potent DW-3 and DW-4 led to the discovery of compound 8a. This molecule showed increased potency against wild-type HIV-1 strain (EC₅₀ = 3.94 ± 0.22 μM) and retained activity against a panel of mutant strains, showing EC₅₀ values ranging from 5.62 μM to 202 μM. In enzymatic assays, 8a was found to inhibit the viral RNase H with an IC₅₀ of 12.3 μM. Molecular docking studies revealed that 8a could adopt a binding mode similar to that previously reported for other active site HIV-1 RNase H inhibitors.

Full text of DOI:10.1016/j.ejmech.2021.113769

European Journal of Medicinal Chemistry 225 (2021) 113769
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery, optimization, and target identification of novel coumarin
derivatives as HIV-1 reverse transcriptase-associated ribonuclease H
inhibitors
,
*
b
a
b
a
Dongwei Kang a , Çagil Urhan , Fenju Wei , Estrella Frutos-Beltran , Lin Sun ,
ꢀ
b
a
a
c
c
ꢀ
Mar Alvarez , Da Feng , Yucen Tao , Christophe Pannecouque , Erik De Clercq ,
Luis Menendez-Arias **, Xinyong Liu a ***, Peng Zhan a
b,
,
, ****
ꢀ
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology, Ministry of Education, School of Pharmaceutical Sciences, Cheeloo College of
Medicine, Shandong University, Ji'nan, 250012, China
b
ꢀ
Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad Autonoma de Madrid), Madrid, Spain
c Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, B-3000, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Despite significant advances in antiretroviral therapy, acquired immunodeficiency syndrome remains as
one of the leading causes of death worldwide. New antiretroviral drugs combined with updated treat-
ment strategies are needed to improve convenience, tolerability, safety, and antiviral efficacy of available
therapies. In this work, a focused library of coumarin derivatives was exploited by cell phenotypic
screening to discover novel inhibitors of HIV-1 replication. Five compounds (DW-3, DW-4, DW-11, DW-
25 and DW-31) showed moderate activity against wild-type and drug-resistant strains of HIV-1 (IIIB and
RES056). Four of those molecules were identified as inhibitors of the viral RT-associated RNase H.
Structural modification of the most potent DW-3 and DW-4 led to the discovery of compound 8a. This
Received 3 May 2021
Received in revised form
29 July 2021
Accepted 31 July 2021
Available online 11 August 2021
Keywords:
HIV-1
RNase H inhibitor
Coumarin
Phenotypic screen
Drug design
molecule showed increased potency against wild-type HIV-1 strain (EC50 ¼ 3.94 0.22
activity against a panel of mutant strains, showing EC50 values ranging from 5.62
enzymatic assays, 8a was found to inhibit the viral RNase H with an IC50 of 12.3 M. Molecular docking
m
M) and retained
m
M to 202 M. In
m
m
studies revealed that 8a could adopt a binding mode similar to that previously reported for other active
site HIV-1 RNase H inhibitors.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
the incidence of opportunistic infections, their success is still
hampered by their adverse side effects and the emergence of drug
Acquired immunodeficiency syndrome (AIDS) is caused by hu-
man immunodeficiency virus type 1 (HIV-1), and remains as a
major threat to human health worldwide. Currently, the number of
people infected with HIV-1 in the world is estimated at 38 million,
causing 690,000 deaths due to AIDS in 2019 [1]. Although treat-
ment with potent combination antiretroviral therapies has resulted
in major improvements in overall survival, immune function and
resistance [2e4]. Therefore, the development of potent, affordable
and highly effective antiretroviral drugs blocking the replication of
drug-resistant HIV strains is an important research priority.
Drug discovery through cell-based phenotypic screening has a
long track record of delivering innovative drugs and has received
renewed attention [5,6]. However, phenotypic screening has also
disadvantages, since it is time-consuming, labor intensive and often
shows a low success rate. On the other hand, drug repositioning
strategies have become increasingly popular, and increasing
numbers of approved drugs are being re-evaluated, especially for
the development of antiviral drugs [7,8]. Nevertheless, most of the
discoveries of new therapeutic uses of existing drugs are based on
clinical observations or serendipitous findings.
* Corresponding authors.
** Corresponding author.
*** Corresponding authors.
**** Corresponding author.
E-mail addresses: kangdongwei@sdu.edu.cn (D. Kang), lmenendez@cbm.csic.es
ꢀ
(L. Menendez-Arias), xinyongl@sdu.edu.cn (X. Liu), zhanpeng1982@sdu.edu.cn
(P. Zhan).
https://doi.org/10.1016/j.ejmech.2021.113769
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
Our labs have been interested in the development of novel in-
hibitors blocking HIV-1 replication, active against drug-resistant
variants, and most notably those emerging during treatment with
HIV-1 reverse transcriptase (RT) inhibitors [9e14]. Most of this
work was done through rational drug design. Now, we report on
the discovery of novel coumarin derivatives with anti-HIV activity
through a drug-repositioning strategy.
IC50 of 6.75
m
M in RNase H inhibition assays. Unfortunately, none of
those compounds was active in cell-based assays.
Therefore, following a drug repositioning strategy, the library
compounds were screened for their antiviral activity in vitro against
the wild-type (WT) HIV-1 strain IIIB, a double-mutant HIV-1 strain
RES056 (K103 N/Y181C) and HIV-2 (ROD strain), and their cellular
cytotoxicity was determined using the MTT method [32]. Their
antiviral effect in cell culture (EC50; 50% effective concentration)
and cytotoxicity (CC50; 50% cytotoxic concentration) were obtained
for the synthesized compounds, using the approved NNRTI drug
nevirapine (NVP) as reference. Results revealed that five com-
pounds (DW-3, DW-4, DW-11, DW-25 and DW-31) were active
against HIV-1 IIIB and the NVP-resistant strain RES056, with EC50
Coumarin is one of the most common structures found in
bioactive molecules. Its derivatives have attracted a lot of attention
due to their pharmacological properties as antibacterial [15], anti-
viral [16] and antitumoral drugs [17,18], as well as their effects
promoting lymph circulation and preventing renal failure [19].
Among them, warfarin has been used for over 60 years as antico-
agulant and is considered as an essential medicine by the World
Health Organization [20]. A more complex coumarin-based natural
product known as (þ)-calanolide A has been identified as an HIV-1
nonnucleoside RT inhibitor (NNRTI), and developed as antiretro-
viral agent while completing phase 1 clinical trials [21]. However,
its development was extremely slow and eventually halted. In our
study, we have identified novel coumarin derivatives targeting the
HIV-1 RT-associated RNase H. Optimization via structure-based
drug design of a series of hit compounds identified through a
repositioning strategy led to the identification of a coumarin de-
rivative (compound 8a) that showed antiviral efficacy against the
wild-type HIV-1 strain, as well as a panel of HIV-1 strains bearing
mutations associated with resistance to NNRTIs.
values ranging from 19.6 mM to 213 mM (Table 1). All five com-
pounds were particularly effective against both HIV-1 strains,
although DW-3, DW-4, and DW-25 were also active against the
wild-type HIV-2 ROD strain, with EC50 values of 116e144 mM.
Analysis of the structures of DW-3, DW-4, DW-11, DW-25 and
DW-31 revealed that these molecules contain three main compo-
nents: a coumarin scaffold (red), an aromatic (dominant) hydro-
phobic substituent (cyan) and a linker (yellow) (Fig.1). Based on the
current knowledge of the interactions at the catalytic site of RNases
H and their inhibitors [33,34], we hypothesized that the coumarin
scaffold could be a relevant pharmacophore to inhibit HIV-1 RNase
H activity. Therefore, we determined the inhibitory activity of the
selected DW compounds in enzymatic assays, using as substrate a
template-primer containing a 31-nucleotide RNA labeled at its
50end with 32P, annealed to a 21-nucleotide DNA [35]. RNA cleavage
(in the presence or absence of the candidate inhibitors) was fol-
lowed by separation of the labeled RNA products by electrophoresis
in denaturing polyacrylamide gels and phosphorimaging analysis.
2. Results and discussion
2.1. Discovery of coumarin derivatives as novel HIV-1 inhibitors via
a phenotypic screen
The results are shown in Table 1. While DW-11 (IC50 > 250 mM)
failed to inhibit the HIV-1 RNase H activity, related compounds
DW-3, DW-4, DW-25 and DW-31 were effective inhibitors. DW-3
was identified as the most potent, showing an IC50 of 9.9 mM.
In a previous project involving the development of anti-renal
failure inhibitors, we constructed a mini focused library consist-
ing of ~100 coumarin derivatives [22]. These compounds (struc-
turally related to urolithin A, daphnetin, esculetin or
wedelolactone, among others) were expected to act as inhibitors of
caspase-dependent apoptosis by regulating the balance between
Bcl-2 and Bax expression [17,23,24]. However, as described above,
coumarin is a privileged scaffold that targets many biological ac-
tivities, including antiviral effects against HIV-1, due to the inhibi-
tion of various steps of the viral replication cycle (e.g. reverse
transcription, integration and maturation) [25e29]. Among them,
natural products such as prenylated coumarins of Antocarpus het-
erophyllus or Clausena lenis showed EC50 values in the range of
The stronger RNase H inhibitory activity of DW-3 and DW-4 in
comparison with DW-25 and DW-31 was not reflected in the EC50
values obtained in cell-based assays. This phenomenon has been
previously observed with other antiretroviral drug candidates
[10,14,36] and could be attributed to a decreased cell permeability
of DW-3 and DW-4. In addition, these compounds contain multiple
hydrogen bond donor and acceptor atoms that could favor the
formation of cocrystals and salts, thereby reducing their
bioavailabilty.
Guided by the pharmacophore model of HIV-1 RNase H in-
hibitors, three novel series of coumarinamide derivatives were
designed and synthesized with DW-3, DW-4, DW-11, DW-25 and
DW-31 as leads, with the aim to discover more potent HIV-1 RNase
H inhibitors. As shown in Fig. 1, the newly designed derivatives
retained the privileged divalent metal ion chelating group and
linker, while exploring elaborated structural modifications of the
7.4e9.1 mM, although their molecular target was not identified in
those studies [30,31]. Esposito et al. [25] obtained a series of
substituted coumarin compounds with RNase H inhibitory activity
in enzymatic assays. Although most of those compounds showed
weak activity, the most potent derivative (i.e., 3-acetyl-4-hydroxy-
9-methyl-2H,5H-pyrano[3,2-c]chromene-2,5-dione) showed an
Table 1
Antiviral activity and cytotoxicity in MT-4 cells, and RNase H inhibitory activity of representative compounds of the DW series.
Compounds
EC50
IIIB
(
m
M)a
CC50
(m
M)b
HIV-1 RNase HIC50 (m
M)c
ROD
RES056
DW-3
DW-4
DW-11
DW-25
DW-31
NVP
121 10.5
101 9.12
208 56.1
28.5 7.10
82.7 9.44
0.28 0.04
121 22.4
116 9.37
>280
113 3.65
107 3.58
213 55.1
19.6 0.52
123 15.4
>15.02
>312
>310
>280
>271
>231
9.90 1.50
20.8 5.7
>250
45.1 15.0
68.5 33.3
NDd
144 37.3
>231
NDd
>15.02
a
EC50: concentration of the compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathicity, as determined using the MTT method.
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined using the MTT method.
IC50: Concentration required to inhibit by 50% the in vitro RNase H activity.
b
c
d
ND, not determined.
2

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
3

Scheme 1. Reagents and conditions: (i) HATU, K2CO3, DMF, r.t.; (ii) TFA, DCM, r.t.; (iii) K2CO3, DMF, r.t.; (iv) Et3N, DMF, r.t.

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
hydrophobic pocket by introducing structural diversity groups,
such as substituted anilines, substituted benzylamines and
substituted piperylhydrazines.
2.2. Optimization of compound 8a as a novel HIV-1 RNase H
inhibitor
The target compounds were prepared via a concise synthetic
route as outlined in Schemes 1 and 2. The starting material DW-1
was reacted with different anilines, benzylamines and tert-butyl 4-
aminopiperidine-1-carboxylate to form target compounds 1a-d,
2a-d, and intermediate 3, respectively. Then, 3 was treated with
trifluoroacetic acid to give the key intermediate 4. Subsequent
treatment of 4 with substituted benzyl bromine, substituted acyl
chloride and dimethylsulfamoyl chloride rendered target com-
pounds 5a-k, 6a-b and 7, respectively (Scheme 1). In an analogous
way, target compounds 8a-c, 9a-d, and 12a-g were prepared, only
with the difference that the starting material was DW-2 (Scheme
2).
As shown in Table 2, when R is a CH3 group, all compounds of
the 1, 2 and 6 subseries showed no antiviral activity against HIV-1
IIIB in cell culture at their highest tested concentrations. Three
compounds of the 5 subseries (5c, 5e and 5f) exhibited moderate
potency against HIV-1 IIIB (EC50 ¼ 167
mM, 234 mM, and 138 mM,
respectively), with similar efficacy in comparison with the lead
compound DW-3. In addition, 5c was also effective against HIV-2
ROD, showing an EC50 value of 148
activity was much lower than that of the approved drug NVP
M).
mM. However, their antiviral
(EC50 ¼ 0.09
m
Newly designed compounds containing an OH group at position
8 of the chromenone ring (i.e. compounds 8a, 8c, 9d, 12c and 12e)
showed moderate inhibitory activity of the replication of HIV-1 IIIB,
with EC50 values within 3.94 and 237
compound 8a turned out to be the most potent inhibitor, exhibiting
an EC50 value of 3.94 M, about 25 times more potent than the lead
DW-4 (EC50 101.15 M). For HIV-2 ROD, compound 8a
M) exhibited comparable activity with that of DW-4
M). In addition, 8a exhibited no cytotoxicity at the
M). Besides, the activity
M) was also superior to that of DW-
mM (Table 3). Among them,
m
¼
m
(EC50 ¼ 169
(EC50 ¼ 116
m
m
highest tested concentrations (CC50 > 300
m
of compound 9d (EC50 ¼ 46.1
m
4. Although it was difficult to establish a clear structure-activity
relationship for these novel compounds, it is clear that an OH
group at position 8 of the chromenone ring would be more bene-
ficial to improve the antiviral activity.
Compounds DW-4 and 8a differ at the substituent group on the
benzene ring which is COOH in DW-4 and CO2CH3 in 8a. Differences
in antiviral activity between both inhibitors could be attributed to
their cellular permeability. Although both compounds showed
relatively low permeability in Caco-2 cells, obtained values were
higher for compound 8a (Papp ¼ 9.06 ꢀ 10ꢁ6 cm/s) than for DW-4
(Papp ¼ 5.71 ꢀ 10ꢁ6 cm/s), thereby suggesting that this property is
an important contributor to the stronger antiviral effect of 8a in cell
culture..
Compound 8a was further tested against a panel of mutant HIV-
1 strains, including NNRTI-resistant variants containing amino acid
substitutions in the viral RT, such as L100I, K103 N, Y181C, Y188L,
E138K, V106A/F227L, and RES056. As shown in Table 4, the com-
pound exhibited some activity against all mutant strains, with EC50
values ranging from 5.62 mM to 202 mM. Interestingly, compound 8a
was found to be a weak but effective inhibitor of NVP-resistant
variants such as Y188L and V106A/F227L, as well as the RES056
strain. These results demonstrated that 8a could be regarded as a
promising lead compound for further structural modification to
discover novel potent inhibitors with improved drug resistance
profiles.
5

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
Table 2
Structure, antiviral activity against HIV-1 IIIB and HIV-2 ROD, and cytotoxicity of target compounds 1a-d, 2a-d, 5a-k, 6a-b and 7.
Compounds
Structure
EC50
IIIB
(m
M)a
CC50 (m
M)b
ROD
1a
>182
>182
182 90.3
1b
1c
1d
>6.50
>12.5
>10.7
>6.50
>12.5
>10.7
6.50 2.84
12.5 2.82
10.7 3.05
2a
>44.0
>44.0
44.0 8.31
2b
2c
>47.5
>45.0
>47.5
>45.0
47.5 16.4
45.0 11.6
2d
>32.5
>32.5
32.5 17.0
5a
5b
5c
>7.80
>42.6
>7.80
7.80 7.19
42.6 4.72
>264
>42.6
167 82.5
148 15.0
5d
5e
5f
>72.3
>72.3
>259
>260
>276
72.3 52.9
>259
234 18.1
138 25.9
>276
>260
5g
>276
5h
5i
>3.88
>50.5
>9.38
>3.88
>50.5
>9.38
3.88 0.69
50.5 41.2
9.37 1.26
5j
5k
6a
>14.7
>251
>14.7
>251
14.7 3.22
>251
6

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
Table 2 (continued)
Compounds
Structure
EC50
IIIB
(m
M)a
CC50 (m
M)b
ROD
6b
>19.9
>19.9
19.9 6.60
7
e
e
>50.8
1.9 0.01
121 10.5
>50.8
50.8 9.87
>15.0
>312
NVP
DW3
NDc
121 22.4
a
EC50: concentration of the compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathicity, as determined using the MTT method.
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined using the MTT method.
ND, not determined.
b
c
2.3. HIV-1 RNase H inhibition and mechanism of action of
compound 8a
the cellular level demonstrated that five compounds (DW-3, DW-4,
DW-11, DW-25 and DW-31) exhibited moderate potency (EC50
values within 19.6 and 213 mM) against HIV-1 IIIB and the NNRTI-
Representative compounds of the newly synthesized com-
pounds were tested in vitro for their inhibitory activity against the
resistant strain RES056. These compounds were then identified as
HIV-1 RNase H inhibitors after being tested in enzymatic assays
using RNA-DNA template-primers as substrates. Further optimi-
zation of DW-3 and DW-4 led to the discovery of compound 8a
which showed similar inhibitory potency than DW-3 in in vitro
RNase H activity assays, but an increased antiviral effect against
HIV-1 IIIB and a panel of mutant strains bearing amino acid sub-
stitutions associated with resistance to NNRTIs. Molecular docking
studies of 8a bound to HIV-1 RT revealed that the compound could
adopt a binding mode similar to that previously reported for other
active site HIV-1 RNase H inhibitors.
RNase H of HIV-1 RT (Table 5). The initial screening at 100
mM
showed that compounds 1c, 2c, 5c, 9a and 12b had less than 50%
inhibitory activity at that concentration. Most of the other com-
pounds were classified as weak inhibitors, with IC50s above 50 mM.
Among them, 5f, 12g, 12a and 8c were the most potent and showed
37.1, 32.4, 31.8 and 12.0% inhibition at such a concentration. In
contrast, the inhibitory effect of compounds 5h, 5k, 8b, 9d and 12f
was almost negligible at 50 mM concentration (<10% inhibition).
Interestingly, the inhibitory activity of compounds 8a and 12a
was similar to that of the lead compound DW-3, with IC50 values
around 10
mM. As discussed above, compound 8a was the most
4. Experimental section
potent antiviral molecule in antiviral assays, while 12a showed
important cellular toxicity and was not effective in cell culture.
Further insight into the mechanism of action of compound 8a
was obtained from molecular docking studies of this molecule in
4.1. Chemistry
All melting points were determined on a micro melting point
apparatus and are uncorrected. 1H NMR spectra were recorded in
DMSO‑d6 on a Bruker AV-400 spectrometer with tetramethylsilane
(TMS) as the internal standard. A G1313A Standard LC Autosampler
(Agilent) was used to collect samples for measurement of mass
spectra. All reactions were monitored by thin layer chromatog-
raphy (TLC), and spots were visualized with iodine vapor or by
irradiation with UV light. Flash column chromatography was per-
formed on columns packed with Silica Gel (200e300 mesh). Sol-
vents were of reagent grade and were purified by standard
methods when necessary.
€
the HIV-1 RNase H catalytic site, using the Schrodinger software
and the crystal structure of HIV-1 RT bound to 5-hydroxy-4-oxo-1-
[(40-sulfamoyl[1,10-biphenyl]-4-yl)methyl]-1,4-dihydropyridine-3-
carboxylic acid, an RNase H inhibitor with sub-micromolar efficacy
in enzymatic assays [37].
As depicted in Fig. 2, the predicted binding mode of 8a was
similar to that previously reported for other HIV-1 RNase H in-
hibitors. According to the obtained models, the two phenolic hy-
droxyl groups and the nitryl moiety of compound 8a could chelate
the two Mg2þ ions, which are coordinated to the conserved active
site acidic residues Asp443, Glu478, Asp498 and Asp549, and
required for RNase H activity. In addition, the hydroxyl group
located in the hydrophobic phenyl ring and in a distal location
relative to the active site of the RNase H could establish hydrogen
bond interactions with the side-chain of Lys540. Docking accuracy
was rather good, since a root mean square deviation (RMSD) value
of 1.08 Å was obtained between the docked conformation of the
inhibitor and the active ligand conformation found in the crystal
structure (PDB file 5J1E).
4.1.1. General procedure for the preparation of target compounds
1a-d and 2a-d
A solution of starting material DW-1 (0.10 g, 0.377 mmol) and 2-
(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexa-
fluorophosphate (HATU) (0.21 g, 0.566 mmol) in DMF (4 mL) was
stirred at room temperature for 10 min; then K2CO3 (0.052 g,
0.377 mmol) and different anilines or benzylamines (0.377 mmol)
were added, and the mixture was stirred for another 5e10 h
(monitored by thin layer chromatography). The solvent was
removed under reduced pressure and 30 mL water was added to
the residue. Then the mixture solution was extracted with ethyl
acetate and washed with saturated sodium chloride, purified by
flash column chromatography and recrystallized from ethyl acetate
(EA)/petroleum ether (PE) to afford the target compounds 1a-d and
2a-d.
Docking results were also consistent with the experimental
observations showing that 8a is a potent HIV-1 RNase H inhibitor,
validating the advantages of ambident scaffolds in metal ion
chelating and providing
optimization.
a basis for further rational drug
3. Conclusion
To discover new potent HIV-1 inhibitors with novel mechanisms
of action and scaffolds, a mini focused library of coumarin de-
rivatives was screened for anti-HIV-1 activity through a drug
repositioning strategy. Preliminary phenotypic screening results at
4.1.1.1. Methyl 2-hydroxy-5-(7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamido)benzoate(1a). 1a was synthesized from
DW-1 (0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU
(215 mg, 0.566 mmol), and methyl 5-amino-2-hydroxybenzoate
7

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
Table 3
Structure, antiviral activity against HIV-1 IIIB and HIV-2 ROD, and cytotoxicity of target compounds 8a-c, 9a-d, and 12a-g.
Compounds
Structure
EC50
IIIB
(
m
M)a
CC50 (m
M)b
ROD
8a
3.94 0.22
169 17.4
>300
8b
8c
>200
>200
>330
200 39.0
237 17.2
>330
9a
9b
9c
>272
>228
>259
>272
>228
>259
272 10.0
228 33.2
259 39.7
9d
46.1 14.0
>266
266 17.5
12a
12b
12c
12d
>21.7
>21.7
>112
>269
>217
21.7 4.60
112 58.2
>269
>112
160 77.3
ꢂ101
ꢂ217
12e
12f
12g
210 47.5
>143
>220
>143
>153
ꢂ220
143 45.7
153 50.6
>153
NVP
DW-4
e
e
0.09 0.01
101 9.12
NDc
116 9.37
>15.0
>310
a
EC50: concentration of the compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathicity, as determined using the MTT method.
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined using the MTT method.
ND, not determined.
b
c
Table 4
Activity against mutant HIV-1 strains of compound 8a and NVP.
Cpds
EC50
L100I
(m
M)a
K103 N
Y181C
Y188L
E138K
V106A/F227
RES056
8a
NVP
16.5 0.79
0.70 0.29
5.62 1.99
2.85 0.37
22.4 4.40
4.09 0.65
44.3 8.96
ꢂ2.97
22.5 4.40
0.12 0.04
101 48.7
ꢂ5.78
202 61.2
>9.51
a
EC50: concentration of the compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathicity, as determined using the MTT method.
8

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
Table 5
160.8,160.1,156.0,147.8,140.1, 135.1, 131.1 (JCF ¼ 9.0 Hz),125.8,117.5,
116.2 (JCF ¼ 3.0 Hz), 115.5, 111.2, 110.8, 107.3, 9.1. HRMS m/z
HIV-1 RNase H inhibitory activity of selected compounds.
17H11FN2O6: calcd 358.0601, found 357.1124 [M ꢁ H]-.
a
Compounds
IC50
(
mM)
Compounds
IC50 (m
M) a
C
1c
2c
5c
5f
5h
5i
5k
8a
8b
8c
>100 (28.6)
>100 (38.7)
>100 (23.8)
>50 (87.2)
>50 (72.1)
45.2 8.8
>50 (62.9)
12.3 2.8
>50 (72.5)
>50 (78.0)
9a
9d
>100 (48.0)
>50 (68.6)
8.4 2.7
>100 (47.4)
>50 (57.7)
>50 (72.5)
>50 (57.4)
9.9 1.5
4.1.1.4. N-(2-fluorophenyl)-7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamide(1d). 1d was synthesized from DW-1
(0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU (215 mg,
12a
12b
12e
12f
12g
DW-3
DW-4
0.566 mmol), and 2-fluoroaniline (36
m
L, 0.377 mmol). Yellow solid,
42% yield, carbonize at 280 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
10.84
(s, 1H, NH), 9.01 (s, 1H, CH), 8.66 (s, 1H, Ph-H), 8.36 (t, J ¼ 7.7 Hz, 1H,
Ph-H), 7.40e7.28 (m,1H, Ph-H), 7.28e7.12 (m, 2H, Ph-H), 2.27 (s, 3H,
20.8 5.7
CH3). 13C NMR (100 MHz, DMSO‑d6)
d 161.5, 160.1, 156.0
a
Concentration required to inhibit by 50% the in vitro RNase H activity. Numbers
(JCF ¼ 194 Hz), 149.0, 135.6, 126.6 (JCF ¼ 10 Hz), 126.2, 125.4, 125.4,
125.3, 122.1, 115.8 (JCF ¼ 19 Hz), 110.6, 9.1. HRMS m/z C17H11FN2O6:
calcd 358.0601, found 357.1584 [M ꢁ H]-.
between parentheses indicate the maximum percent inhibition observed with the
corresponding coumarinamide derivative at 100
are averages of two independent determinations).
mM concentration (reported values
4.1.1.5. N-(4-fluorobenzyl)-7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamide(2a). 2a was synthesized from DW-1
(0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU (215 mg,
(63 mg, 0.377 mmol). Yellow solid, 22% yield, carbonize at 320 ꢃC.
1H NMR (400 MHz, DMSO‑d6)
10.45 (s, 1H, NH), 8.89 (s, 1H, CH),
8.68 (s, 1H, Ph-H), 8.26 (d, J ¼ 2.6 Hz, 1H, Ph-H), 7.76 (dd, J ¼ 8.9,
2.7 Hz, 1H, Ph-H), 7.02 (d, J ¼ 8.9 Hz, 1H, Ph-H), 3.93 (s, 3H, CH3),
2.30 (s, 3H, CH3). HRMS m/z C19H14N2O9: calcd 414.0699, found
413.1054 [M ꢁ H]-.
d
0.566 mmol), and (4-fluorophenyl)methanamine (43
0.377 mmol). Yellow solid, 43% yield, mp: 234e236 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
mL,
d
9.00 (t, J ¼ 5.9 Hz, 1H, NH), 8.87 (s, 1H, CH),
8.64 (s, 1H, Ph-H), 7.39 (dd, J ¼ 8.1, 5.8 Hz, 2H, Ph-H), 7.15 (t,
J ¼ 8.8 Hz, 2H, Ph-H), 4.51 (d, J ¼ 6.0 Hz, 2H, CH2), 2.26 (s, 3H, CH3).
HRMS m/z C18H13FN2O6: calcd 372.0758, found 371.0028 [M ꢁ H]-.
4.1.1.2. N-(4-fluorophenyl)-7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamide(1b). 1b was synthesized from DW-1
(0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU (215 mg,
4.1.1.6. N-(3-fluorobenzyl)-7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamide(2b). 2b was synthesized from DW-1
(0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU (215 mg,
0.566 mmol), and 4-fluoroaniline (36
solid, 40% yield, carbonize at 280 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
10.54 (s, 1H, NH), 8.84 (s, 1H, CH), 8.56 (s, 1H, Ph-H), 7.72 (s, 2H,
Ph-H), 7.20 (s, 2H, Ph-H), 2.23 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO‑d6) 160.9, 160.5, 160.1, 158.8, 157.7, 155.8, 148.1, 135.7, 134.9,
mL, 0.377 mmol). Brick red
d
0.566 mmol), and (3-fluorophenyl)methanamine (43
0.377 mmol). Yellow solid, 32% yield, mp: 235e236 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
mL,
d
d
9.05 (t, J ¼ 5.9 Hz, 1H, NH), 8.87 (s, 1H, CH),
126.2, 122.2 (JCF ¼ 8.0 HZ), 116.1 (JCF ¼ 22.0 HZ), 115.4, 113.8, 109.5,
9.0. HRMS m/z C17H11FN2O6: calcd 358.0601, found 357.0842
[M ꢁ H]-.
8.65 (s,1H, Ph-H), 7.37 (q, J ¼ 7.7 Hz,1H, Ph-H), 7.17 (t, J ¼ 9.4 Hz, 2H,
Ph-H), 7.07 (t, J ¼ 8.6 Hz,1H, Ph-H), 4.55 (d, J ¼ 6.0 Hz, 2H, CH2), 2.27
(s, 3H, CH3). HRMS m/z C18H13FN2O6: calcd 372.0758, found
371.1425 [M ꢁ H]-.
4.1.1.3. N-(3-fluorophenyl)-7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamide(1c). 1c was synthesized from DW-1
(0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU (215 mg,
4.1.1.7. N-(2-fluorobenzyl)-7-hydroxy-8-methyl-6-nitro-2-oxo-2H-
chromene-3-carboxamide(2c). 2c was synthesized from DW-1
(0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU (215 mg,
0.566 mmol), and 3-fluoroaniline (36
m
L, 0.377 mmol). Yellow solid,
10.71
37% yield, carbonize at 300 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
0.566 mmol), and (2-fluorophenyl)methanamine (43
0.377 mmol). Yellow solid, 28% yield, carbonize at 220 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
9.00 (t, J ¼ 5.9 Hz, 1H, NH), 8.88 (s, 1H, CH),
8.66 (s, 1H, Ph-H), 7.39 (t, J ¼ 7.6 Hz, 1H, Ph-H), 7.33 (q, J ¼ 5.9 Hz,
mL,
(s, 1H, NH), 8.86 (s, 1H, CH), 8.61 (s, 1H, Ph-H), 7.74 (d, J ¼ 11.2 Hz,
1H, Ph-H), 7.51e7.34 (m, 2H, Ph-H), 7.04e6.87 (m, 1H, Ph-H), 2.28
d
(s, 3H, CH3). 13C NMR (100 MHz, DMSO‑d6)
d
163.8 (JCF ¼ 241 Hz),
Fig. 2. Predicted binding modes of 8a (green) in the HIV-1 RNase H active site (PDB code: 5J1E [37]). Protein carbon atoms are shown in cyan, hydrogen bonds between 8a and
amino acid residues are indicated with dashed lines (yellow). Figures were prepared with the PyMOL visualization program (http://www.pymol.org).
9

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
1H, Ph-H), 7.19 (q, J ¼ 9.4, 7.6 Hz, 2H, Ph-H), 4.59 (d, J ¼ 5.9 Hz, 2H,
(s, 3H, CH3), 1.88e1.65 (m, 2H, CH2). HRMS m/z C23H23N3O6: calcd
437.1587, found 436.0745 [M ꢁ H]-.
CH2), 2.27 (s, 3H, CH3). 13C NMR (100 MHz, DMSO‑d6)
d 161.7, 160.2,
159.3, 156.2, 154.9, 147.9, 134.3, 129.9, 129.5 (JCF ¼ 8.0 Hz), 126.1,
125.9 (JCF ¼ 3.0 Hz), 124.8,117.6, 115.7 (JCF ¼ 21 Hz),115.3, 111.6, 37.3,
8.9. HRMS m/z C18H13FN2O6: calcd 372.0758, found 371.1125
[M ꢁ H]-.
4.1.2.2. N-(1-(4-fluorobenzyl)piperidin-4-yl)-7-hydroxy-8-methyl-6-
nitro-2-oxo-2H-chromene-3-carboxamide(5b). 5b was synthesized
from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and 1-
(bromomethyl)-4-fluorobenzene (43
mL, 0.346 mmol). Orange
4.1.1.8. N-(2-chloro-4-fluorobenzyl)-7-hydroxy-8-methyl-6-nitro-2-
oxo-2H-chromene-3-carboxamide(2d). 2d was synthesized from
DW-1 (0.1 g, 0.377 mmol), K2CO3 (52 mg, 0.377 mmol), HATU
(215 mg, 0.566 mmol), and (2-chloro-4-fluorophenyl)methan-
solid, 24% yield, carbonize at 245 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.58 (d, J ¼ 7.1 Hz, 1H, NH), 8.54 (s, 1H, CH), 8.16 (s, 1H, Ph-H), 7.53
(dd, J ¼ 8.2, 5.7 Hz, 2H, Ph-H), 7.30 (t, J ¼ 8.7 Hz, 2H, Ph-H), 4.22 (s,
2H, CH2), 4.06e3.92 (m, 1H, CH), 3.24 (d, J ¼ 11.2 Hz, 2H, CH2),
3.07e2.95 (m, 2H, CH2), 2.05 (d, J ¼ 12.3 Hz, 2H, CH2), 1.99 (s, 3H,
amine (36
m
L, 0.377 mmol). Yellow solid, 44% yield, carbonize at
260 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
9.06 (s, 1H, NH), 8.87 (s, 1H,
CH3), 1.84e1.63 (m, 2H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d 167.4,
CH), 8.64 (s, 1H, Ph-H), 7.64e7.31 (m, 2H, Ph-H), 7.29e7.07 (m, 1H,
164.2, 162.6 (JCF ¼ 16 Hz), 161.8, 155.1, 148.9, 138.5, 133.7, 133.6,
127.2, 116.2 (JCF ¼ 21 Hz),115.3, 107.1, 104.3, 50.6, 44.3, 9.0. HRMS m/
z C23H22FN3O6: calcd 455.1493, found 454.1029 [M ꢁ H]-.
Ph-H), 4.86e4.35 (m, 2H, CH2), 2.26 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO‑d6)
d
162.7, 161.7 (JCF ¼ 185 Hz), 160.2, 156.2, 155.6, 148.0,
134.5, 133.2, 132.8, 130.9 (JCF ¼ 9.0 Hz), 125.8, 117.0 (JCF ¼ 4.0 Hz),
116.8, 115.4, 114.8 (JCF ¼ 21 Hz), 111.3, 40.7, 9.1. HRMS m/z
4.1.2.3. N-(1-(4-chlorobenzyl)piperidin-4-yl)-7-hydroxy-8-methyl-6-
nitro-2-oxo-2H-chromene-3-carboxamide(5c). 5c was synthesized
from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and 1-
C
18H12ClFN2O6: calcd 406.0368, found 405.1320 [M ꢁ H]-.
4.1.1.9. tert-butyl
4-(7-hydroxy-8-methyl-6-nitro-2-oxo-2H-chro-
(bromomethyl)-4-chlorobenzene (45
mL, 0.346 mmol). Orange
mene-3-carboxamido)piperidine-1-carboxylate(3). A solution of
DW-1 (0.10 g, 0.377 mmol) and HATU (0.21 g, 0.566 mmol) in DMF
(4 mL) was stirred at room temperature for 10 min; then K2CO3
(0.052 g, 0.377 mmol) and tert-butyl 4-aminopiperidine-1-
carboxylate (76 mg, 0.377 mmol) were added, and the mixture
was stirred for another 4 h. The solvent was removed under
reduced pressure and 30 mL water was added. Then the solution
was extracted with ethyl acetate and washed with saturated so-
dium chloride, purified by flash column chromatography and
recrystallized from EA/PE to afford the target compound 3 as a
yellow solid in 43% yield. ESI-MS: 446.32 [M ꢁ H]-. C21H25N3O8
[447.16].
solid, 25% yield, carbonize at 260 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.57 (d, J ¼ 7.3 Hz, 1H, NH), 8.55 (s, 1H, CH), 8.18 (s, 1H, Ph-H),
7.59e7.43 (m, 4H, Ph-H), 4.19 (s, 2H, CH2), 4.04e3.92 (m, 1H, CH),
3.22 (d, J ¼ 11.7 Hz, 2H, CH2), 2.99 (t, J ¼ 11.0 Hz, 2H, CH2), 2.10e2.02
(m, 2H, CH2), 1.99 (s, 3H, CH3), 1.84e1.65 (m, 2H, CH2). 13C NMR
(100 MHz, DMSO‑d6) d 167.0, 162.5, 162.3, 155.2, 148.8, 138.3, 134.4,
133.1, 130.8, 129.2, 127.1, 115.3, 107.6, 104.6, 58.6, 50.7, 44.4, 29.1,
9.07. HRMS m/z C23H22ClN3O6: calcd 471.1197, found 470.1523
[M ꢁ H]-.
4.1.2.4. N-(1-(4-bromobenzyl)piperidin-4-yl)-7-hydroxy-8-methyl-
6-nitro-2-oxo-2H-chromene-3-carboxamide(5d). 5d was synthe-
sized from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and
4.1.1.10. 7-hydroxy-8-methyl-6-nitro-2-oxo-N-(piperidin-4-yl)-2H-
chromene-3-carboxamide(4). To a solution of 3 (0.45 g, 1.0 mmol) in
dichloromethane (DCM) (4 mL) was added trifluoroacetic acid
(TFA) (0.74 mL, 10 mmol) at room temperature, and the mixture
was stirred for 5 h (monitored by TLC). Then, the reaction solution
was alkalized to pH 9 with saturated sodium bicarbonate solution
and extracted with DCM. The organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to give 4 as a yellow solid in 66% yield. ESI-MS:
346.41 [M ꢁ H]-. C16H17N3O6 [347.11].
1-bromo-4-(bromomethyl)benzene (48
mL, 0.346 mmol). Orange
solid, 39% yield, carbonize at 245 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.57 (d, J ¼ 7.4 Hz, 1H, NH), 8.55 (s, 1H, CH), 8.18 (s, 1H, Ph-H), 7.66
(d, J ¼ 8.3 Hz, 2H, Ph-H), 7.43 (d, J ¼ 8.3 Hz, 2H, Ph-H), 4.17 (s, 2H,
CH2), 4.04e3.92 (m, 1H, CH), 3.21 (d, J ¼ 11.3 Hz, 2H, CH2), 2.97 (t,
J ¼ 10.8 Hz, 2H, CH2), 2.10e2.01 (m, 2H, CH2), 1.99 (s, 3H, CH3),
1.83e1.66 (m, 2H, CH2). HRMS m/z C23H22BrN3O6: calcd 515.0692,
found 514.1020 [M ꢁ H]-.
4.1.2.5. 7-hydroxy-8-methyl-6-nitro-N-(1-(4-nitrobenzyl)piperidin-
4-yl)-2-oxo-2H-chromene-3-carboxamide(5e). 5e was synthesized
from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and 1-
4.1.2. General procedure for the preparation of target compounds
5a-k
(bromomethyl)-4-nitrobenzene (45
m
L, 0.346 mmol). Orange solid,
8.54
To a solution of 4 (0.35 g, 1.0 mmol) in anhydrous DMF (10 mL)
was added K2CO3 (0.28 g, 2.0 mmol) and substituted benzyl
bromine (1.2 mmol), and the solution was stirred for 4e7 h
(monitored by TLC) at room temperature. The solvent was removed
under reduced pressure and 30 mL water was added to the residue.
Then the mixture solution was extracted with ethyl acetate and
washed with saturated sodium chloride, purified by flash column
chromatography and recrystallized from EA/PE to afford the target
compounds 5a-k.
19% yield, carbonize at 250 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
(s, 1H, CH), 8.51 (d, J ¼ 7.2 Hz, 1H, NH), 8.23 (d, J ¼ 8.5 Hz, 2H, Ph-H),
8.20 (s, 1H, Ph-H), 7.67 (d, J ¼ 8.5 Hz, 2H, Ph-H), 4.13 (s, 2H, CH2),
3.93e3.82 (m, 1H, CH), 3.06 (d, J ¼ 12.6 Hz, 2H, CH2), 2.81 (d,
J ¼ 12.4 Hz, 2H, CH2), 1.98 (s, 3H, CH3), 1.96e1.87 (m, 2H, CH2),
1.73e1.58 (m, 2H, CH2). HRMS m/z C23H22N4O8: calcd 482.1438,
found 481.1007 [M ꢁ H]-.
4.1.2.6. N-(1-(4-carbamoylbenzyl)piperidin-4-yl)-7-hydroxy-8-
methyl-6-nitro-2-oxo-2H-chromene-3-carboxamide(5f). 5f
synthesized from (0.1 g, 0.288 mmol), K2CO3 (80 mg,
0.576 mmol), and 4-(bromomethyl)benzamide (47 L, 0.346 mmol).
Orange solid, 22% yield, carbonize at 205 ꢃC. 1H NMR (400 MHz,
DMSO‑d6)
Ph-H), 8.02 (s, 1H, NH), 7.93 (d, J ¼ 8.1 Hz, 2H, Ph-H), 7.56 (d,
J ¼ 8.1 Hz, 2H, Ph-H), 7.43 (s, 1H, NH), 4.21 (s, 2H, CH2), 4.02e3.93
(m, 1H, CH), 3.21 (d, J ¼ 11.1 Hz, 2H, CH2), 2.97 (t, J ¼ 10.8 Hz, 2H,
CH2), 2.11e2.00 (m, 2H, CH2), 1.99 (s, 3H, CH3), 1.83e1.68 (m, 2H,
was
4.1.2.1. N-(1-benzylpiperidin-4-yl)-7-hydroxy-8-methyl-6-nitro-2-
oxo-2H-chromene-3-carboxamide(5a). 5a was synthesized from 4
(0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and (bromo-
4
m
methyl)benzene (41
m
L, 0.346 mmol). Brick red solid, 20% yield,
d
8.58 (d, J ¼ 7.2 Hz, 1H, NH), 8.54 (s, 1H, CH), 8.17 (s, 1H,
carbonize at 240 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.59 (d,
J ¼ 7.2 Hz, 1H, NH), 8.53 (s, 1H, CH), 8.14 (s, 1H, Ph-H), 7.55e7.40 (m,
5H, Ph-H), 4.23 (s, 2H, CH2), 4.07e3.92 (m, 1H, CH), 3.24e3.16 (m,
2H, CH2), 3.11e3.00 (m, 2H, CH2), 2.06 (d, J ¼ 13.4 Hz, 2H, CH2), 1.98
10

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
CH2). 13C NMR (100 MHz, DMSO‑d6)
d
167.8, 162.5, 162.4, 157.2,
2H, CH2), 2.09e2.01 (m, 2H, CH2), 2.00 (s, 3H, CH3), 1.83e1.67 (m,
2H, CH2). HRMS m/z C23H22BrN3O6: calcd 515.0692, found 514.1120
[M ꢁ H]-.
155.1,148.9,138.5,135.2,130.9,128.2,127.2,115.3,107.1,104.2,102.9,
98.6, 75.6, 29.2, 9.0. HRMS m/z C24H24N4O7: calcd 480.1645, found
479.1126 [M ꢁ H]-.
4.1.3. General procedure for the preparation of target
compounds 6a-b and 7
4.1.2.7. 7-hydroxy-8-methyl-N-(1-(4-methylbenzyl)piperidin-4-yl)-
6-nitro-2-oxo-2H-chromene-3-carboxamide(5g). 5g was synthe-
sized from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and
To a solution of 4 (0.35 g, 1.0 mmol) and Et3N (0.20 g, 2.0 mmol)
in anhydrous DMF (10 mL) was added substituted acyl chloride or
dimethylsulfamoyl chloride (1.2 mmol), and the solution was stir-
red for 3e5 h (monitored by TLC) at room temperature. Then 30 mL
water was added, and the mixture was extracted with ethyl acetate
and washed with saturated sodium chloride. Purified by flash col-
umn chromatography and recrystallized from EA/PE to afford the
target compounds 6a-b and 7.
1-(bromomethyl)-4-methylbenzene (47
mL, 0.346 mmol). Orange
solid, 29% yield, carbonize at 220 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.58 (d, J ¼ 7.7 Hz, 1H, NH), 8.52 (s, 1H), 8.13 (s, 1H, CH), 7.37 (d,
J ¼ 7.9 Hz, 2H, Ph-H), 7.28 (d, J ¼ 7.9 Hz, 2H, Ph-H), 4.21 (s, 2H, CH2),
4.06e3.91 (m, 1H, CH), 3.29e3.20 (m, 2H, CH2), 3.13e2.99 (m, 2H,
CH2), 2.33 (s, 3H, CH3), 2.13e2.01 (m, 2H, CH2), 1.97 (s, 3H, CH3),
1.86e1.65 (m, 2H, CH2). HRMS m/z C24H25N3O6: calcd 451.1743,
found 450.1338 [M ꢁ H]-.
4.1.3.1. 7-hydroxy-8-methyl-6-nitro-N-(1-(4-nitrobenzoyl)piperidin-
4-yl)-2-oxo-2H-chromene-3-carboxamide(6a). 6a was synthesized
4.1.2.8. 7-hydroxy-8-methyl-N-(1-(3-methylbenzyl)piperidin-4-yl)-
6-nitro-2-oxo-2H-chromene-3-carboxamide(5h). 5h was synthe-
sized from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and
from 4 (0.1 g, 0.288 mmol), Et3N (80
nitrobenzoyl chloride (42 L, 0.346 mmol). Yellow solid, 15%
yield, carbonize at 270 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
8.87 (s,
mL, 0.576 mmol), and 4-
m
d
1-(bromomethyl)-3-methylbenzene (47
mL, 0.346 mmol). Orange
1H, CH), 8.65 (s, 1H, Ph-H), 8.53 (d, J ¼ 7.7 Hz, 1H, NH), 8.30 (d,
J ¼ 8.7 Hz, 2H, Ph-H), 7.70 (d, J ¼ 8.7 Hz, 2H, Ph-H), 4.44e4.33 (m,
1H, CH), 3.30e3.02 (m, 4H, CH2 ꢀ 2), 2.27 (s, 3H, CH3), 2.01e1.79 (m,
2H, CH2), 1.67e1.52 (m, 2H, CH2). HRMS m/z C23H20N4O9: calcd
496.1230, found 495.1007 [M ꢁ H]-.
solid, 18% yield, mp: 170e172 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.58 (d, J ¼ 6.3 Hz, 1H, NH), 8.53 (s, 1H, CH), 8.14 (s, 1H, Ph-H), 7.35
(t, J ¼ 7.4 Hz, 1H, Ph-H), 7.28 (dd, J ¼ 11.7, 6.2 Hz, 3H, Ph-H), 4.19 (s,
2H, CH2), 4.04e3.91 (m, 1H, CH), 3.29e3.23 (m, 2H, CH2), 3.10e3.00
(m, 2H, CH2), 2.34 (s, 3H, CH3), 2.10e2.02 (m, 2H, CH2), 1.97 (s, 3H,
CH3), 1.82e1.68 (m, 2H, CH2). HRMS m/z C24H25N3O6: calcd
451.1743, found 450.1097 [M ꢁ H]-.
4.1.3.2. 4-(7-hydroxy-8-methyl-6-nitro-2-oxo-2H-chromene-3-
carboxamido)-N,N-dimethylpiperidine-1-carboxamide(6b). 6b was
synthesized from 4 (0.1 g, 0.288 mmol), Et3N (80
and methyl dimethylcarbamoyl chloride (32
Brown solid, 15% yield, mp: 246e248 ꢃC. 1H NMR (400 MHz,
DMSO‑d6)
m
L, 0.576 mmol),
4.1.2.9. N-(1-(3-fluorobenzyl)piperidin-4-yl)-7-hydroxy-8-methyl-6-
nitro-2-oxo-2H-chromene-3-carboxamide(5i). 5i was synthesized
from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and 1-
m
L, 0.346 mmol).
d
8.86 (s, 1H, CH), 8.65 (s, 1H, Ph-H), 8.49 (d, J ¼ 7.7 Hz,
(bromomethyl)-3-fluorobenzene (43
mL, 0.346 mmol). Orange
1H, NH), 4.06e3.91 (m, 1H, CH), 3.48 (d, J ¼ 13.3 Hz, 2H, CH2),
2.91e2.82 (m, 2H, CH2), 2.74 (s, 6H, CH3 ꢀ 2), 2.26 (s, 3H, CH3),
1.93e1.78 (m, 2H, CH2), 1.57e1.39 (m, 2H, CH2). HRMS m/z
solid, 22% yield, carbonize at 210 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.58 (d, J ¼ 7.5 Hz, 1H, NH), 8.51 (s, 1H, CH), 8.11 (s, 1H, Ph-H),
7.56e7.51 (m, 1H, Ph-H), 7.46 (d, J ¼ 7.8 Hz, 1H, Ph-H), 7.37e7.27
(m, 2H, Ph-H), 3.87e3.73 (m, 1H, CH), 3.53 (s, 2H, CH2), 2.78e2.64
(m, 2H, CH2), 2.22 (t, J ¼ 9.6 Hz, 2H, CH2), 1.96 (s, 3H, CH3), 1.91e1.81
(m, 2H, CH2), 1.59e1.44 (m, 2H, CH2). 13C NMR (100 MHz, DMSO‑d6)
C
19H22N4O7: calcd 418.1488, found 417.1224 [M ꢁ H]-.
4.1.3.3. N-(1-(N,N-dimethylsulfamoyl)piperidin-4-yl)-7-hydroxy-8-
methyl-6-nitro-2-oxo-2H-chromene-3-carboxamide(7). 7 was syn-
d
169.5, 162.8, 162.4, 155.0, 149.1, 138.8, 136.1, 133.6, 131.8, 131.4,
thesized from 4 (0.1 g, 0.288 mmol), Et3N (80
dimethylsulfamoyl chloride (37 L, 0.346 mmol). Brown solid, 27%
yield, mp: 230e232 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
8.85 (d,
mL, 0.576 mmol), and
130.3, 128.3, 127.5, 122.0, 115.3, 106.0, 103.2, 61.4, 51.7, 45.9, 31.8, 9.1.
HRMS m/z C23H22FN3O6: calcd 455.1493, found 454.1023 [M ꢁ H]-.
m
d
J ¼ 5.6 Hz,1H, NH), 8.64 (s,1H, CH), 8.52 (s,1H, Ph-H), 4.29e4.14 (m,
1H, CH), 3.60e3.40 (m, 4H, CH2 ꢀ 2), 2.76 (s, 6H, CH3 ꢀ 2), 2.26 (s,
3H, CH3), 2.08e1.82 (m, 2H, CH2), 1.71e1.48 (m, 2H, CH2). HRMS m/z
4.1.2.10. N-(1-(3-chlorobenzyl)piperidin-4-yl)-7-hydroxy-8-methyl-
6-nitro-2-oxo-2H-chromene-3-carboxamide(5j). 5j was synthesized
from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and 1-
C
18H22N4O8S: calcd 454.1158, found 453.0885 [M ꢁ H]-.
(bromomethyl)-3-chlorobenzene (45
mL, 0.346 mmol). Orange
solid, 24% yield, carbonize at 240 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
4.1.4. General procedure for the preparation of target
compounds 8a-c and 9a-d
The synthetic route was similar to that of 1a-d, only with the
difference that the starting material was DW-2 (0.10 g, 0.377 mmol)
d
8.58 (d, J ¼ 7.8 Hz, 1H, NH), 8.57 (s, 1H, CH), 8.20 (s, 1H, Ph-H), 7.58
(s, 1H, Ph-H), 7.49 (d, J ¼ 6.8 Hz, 2H, Ph-H), 7.45 (t, J ¼ 6.2 Hz,1H, Ph-
H), 4.19 (s, 2H, CH2), 4.05e3.92 (m, 1H, CH), 3.23 (d, J ¼ 11.5 Hz, 2H,
CH2), 2.99 (t, J ¼ 10.9 Hz, 2H, CH2), 2.11e2.03 (m, 2H, CH2), 2.00 (s,
3H, CH3), 1.75 (q, J ¼ 10.1 Hz, 2H, CH2). HRMS m/z C23H22ClN3O6:
calcd 471.1197, found 470.0852 [M ꢁ H]-.
4.1.4.1. Methyl 5-(7,8-dihydroxy-6-nitro-2-oxo-2H-chromene-3-
carboxamido)-2-hydroxy benzoate(8a). 8a was synthesized from
DW-2 (0.1 g, 0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU
(215 mg, 0.563 mmol), and methyl 5-amino-2-hydroxybenzoate
(63 mg, 0.375 mmol). Brown solid, 14% yield, carbonize at 250 ꢃC.
4.1.2.11. N-(1-(3-bromobenzyl)piperidin-4-yl)-7-hydroxy-8-methyl-
6-nitro-2-oxo-2H-chromene-3-carboxamide(5k). 5k was synthe-
sized from 4 (0.1 g, 0.288 mmol), K2CO3 (80 mg, 0.576 mmol), and
1-bromo-3-(bromomethyl)benzene (55
mL, 0.346 mmol). Orange
1H NMR (400 MHz, DMSO‑d6)
d
10.58 (d, J ¼ 15.9 Hz, 1H, NH),
solid, 18% yield, carbonize at 240 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
8.91e8.66 (m, 1H, CH), 8.32e8.19 (m, 1H, Ph-H), 8.08e7.96 (m, 1H,
Ph-H), 7.83e7.69 (m,1H, Ph-H), 6.98 (d, J ¼ 7.9 Hz,1H, Ph-H), 3.91 (s,
3H, CH3). HRMS m/z C18H12N2O10: calcd 416.0492, found 415.0217
[M ꢁ H]-.
d
8.59 (d, J ¼ 7.0 Hz, 1H, NH), 8.56 (s, 1H, CH), 8.19 (s, 1H, Ph-H), 7.51
(q, J ¼ 7.8 Hz, 1H, Ph-H), 7.39e7.24 (m, 3H, Ph-H), 4.19 (s, 2H, CH2),
4.03e3.92 (m, 1H, CH), 3.21 (d, J ¼ 12.3 Hz, 2H, CH2), 3.03e2.90 (m,
11

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
4.1.4.2. N-(4-fluorophenyl)-7,8-dihydroxy-6-nitro-2-oxo-2H-chro-
mene-3-carboxamide(8b). 8b was synthesized from DW-2 (0.1 g,
0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU (215 mg,
0.563 mmol), and 4-fluoroaniline (42 mg, 0.375 mmol). Brown
solid, 31% yield, carbonize at 170 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
was similar to that of 3, only with the difference that the starting
material was DW-2 (0.10 g, 0.377 mmol). Yellow solid, 49% yield,
carbonize at 270 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d 8.80 (s, 1H, CH),
8.49 (d, J ¼ 7.7 Hz, 1H, NH), 8.19 (s, 1H, Ph-H), 4.06e3.90 (m, 2H,
CH2), 3.90e3.72 (m, 3H, CH2 þ CH), 3.03e2.91 (m, 2H, CH2),
1.93e1.79 (m, 2H, CH2), 1.41 (s, 9H, CH3 ꢀ 3). ESI-MS: 448.57
[M ꢁ H]-. C20H23N3O9 [449.14].
d
10.47 (s, 1H, NH), 8.81 (s, 1H, CH), 8.42 (s, 1H, Ph-H), 7.76e7.70 (m,
2H, Ph-H), 7.23e7.17 (m, 2H, Ph-H). HRMS m/z C16H9FN2O7: calcd
360.0394, found 359.1042 [M ꢁ H]-.
4.1.4.9. 7,8-Dihydroxy-6-nitro-2-oxo-N-(piperidin-4-yl)-2H-chro-
mene-3-carboxamide(11). The synthetic route was similar to that of
4, only with the difference that the starting material was 10 (0.45 g,
1.0 mmol). Yellow solid, 67% yield, carbonize at 300 ꢃC. ESI-MS:
348.38 [M ꢁ H]-. C15H15N3O7 [349.09].
4.1.4.3. N-(2,4-difluorophenyl)-7,8-dihydroxy-6-nitro-2-oxo-2H-
chromene-3-carboxamide(8c). 8c was synthesized from DW-2
(0.1 g, 0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU (215 mg,
0.563 mmol), and 2,4-difluoroaniline (48 mg, 0.375 mmol). Yellow
solid, 24% yield, carbonize at 160 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
10.72 (s, 1H, NH), 8.87 (s, 1H, CH), 8.45 (s, 1H, Ph-H), 7.51 (dd,
4.1.5. General procedure for the preparation of target compounds
12a-g
The synthetic route was similar to that of 5a-k, only with the
difference that the starting material was 11 (0.35 g, 1.0 mmol).
J ¼ 8.2, 4.2 Hz, 2H, Ph-H), 7.45e7.34 (m, 1H, Ph-H), 7.12 (t, J ¼ 8.3 Hz,
1H, Ph-H). HRMS m/z C16H8F2N2O7: calcd 378.0300, found 377.0745
[M ꢁ H]-.
4.1.4.4. N-(4-fluorobenzyl)-7,8-dihydroxy-6-nitro-2-oxo-2H-chro-
mene-3-carboxamide(9a). 9a was synthesized from DW-2 (0.1 g,
0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU (215 mg,
4.1.5.1. N-(1-(4-chlorobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12a). 12a was synthesized
from 11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 1-
0.563 mmol), and (4-fluorophenyl)methanamine (43
0.375 mmol). Yellow solid, 18% yield, carbonize at 180 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
mL,
(bromomethyl)-4-chlorobenzene (45
mL, 0.344 mmol). Brick red
solid, 33% yield, carbonize at 180 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.89 (t, J ¼ 6.0 Hz, 1H, NH), 8.73 (s, 1H, CH),
d
8.55 (s, 1H, CH), 8.41 (d, J ¼ 7.2 Hz, 1H, NH), 7.94 (s, 1H, Ph-H),
8.37 (s, 1H, Ph-H), 7.37 (dd, J ¼ 8.3, 5.8 Hz, 2H, Ph-H), 7.15 (t,
J ¼ 8.8 Hz, 2H, Ph-H), 4.49 (d, J ¼ 6.0 Hz, 2H, CH2). HRMS m/z
7.50e7.48 (m, 4H, Ph-H), 4.14e4.10 (m, 2H, CH2), 3.95 (s, 1H, CH),
3.10e3.04 (m, 2H, CH2), 2.93e2.86 (m, 2H, CH2), 2.01e1.97 (m, 2H,
C
17H11FN2O7: calcd 374.0550, found 373.0126 [M ꢁ H]-.
CH2), 1.72e1.67 (m, 2H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d 162.8,
162.4, 161.9, 149.7, 146.7, 139.8, 136.6, 135.5, 132.8, 130.2, 129.1,
4.1.4.5. N-(3-fluorobenzyl)-7,8-dihydroxy-6-nitro-2-oxo-2H-chro-
mene-3-carboxamide(9b). 9b was synthesized from DW-2 (0.1 g,
0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU (215 mg,
128.8, 127.1, 120.9, 108.6, 104.1, 101.9, 72.0, 50.9, 38.7, 9.1. HRMS m/z
C
22H20ClN3O7: calcd 473.0990, found 473.0115 [M ꢁ H]-.
0.563 mmol), and (3-fluorophenyl)methanamine (43
0.375 mmol). Yellow solid, 19% yield, carbonize at 180 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
8.37 (s, 1H, Ph-H), 7.37 (q, J ¼ 7.7 Hz, 1H, Ph-H), 7.15 (t, J ¼ 10.3 Hz,
2H, Ph-H), 7.11e7.02 (m, 1H, Ph-H), 4.53 (d, J ¼ 6.1 Hz, 2H, CH2).
HRMS m/z C17H11FN2O7: calcd 374.0500, found 373.2558 [M ꢁ H]-.
mL,
4.1.5.2. N-(1-(4-bromobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12b). 12b was synthesized
from 11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 1-
d
8.95 (t, J ¼ 6.0 Hz, 1H, NH), 8.73 (s, 1H, CH),
bromo-4-(bromomethyl)benzene (47
mL, 0.344 mmol). Orange
solid, 16% yield, carbonize at 190 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.70 (s, 1H, CH), 8.40 (d, J ¼ 7.5 Hz, 1H, NH), 8.36 (s, 1H, Ph-H), 7.52
(d, J ¼ 7.9 Hz, 2H, Ph-H), 7.29 (d, J ¼ 7.8 Hz, 2H, Ph-H), 3.86e3.74 (m,
1H, CH), 3.57e3.41 (m, 2H, CH2), 2.80e2.63 (m, 2H, CH2), 2.29e2.09
(m, 2H, CH2), 1.95e1.78 (m, 2H, CH2), 1.60e1.42 (m, 2H, CH2). HRMS
m/z C22H20BrN3O7: calcd 517.0485, found 516.0226 [M ꢁ H]-.
4.1.4.6. N-(2-fluorobenzyl)-7,8-dihydroxy-6-nitro-2-oxo-2H-chro-
mene-3-carboxamide(9c). 9c was synthesized from DW-2 (0.1 g,
0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU (215 mg,
0.563 mmol), and (2-fluorophenyl)methanamine (43
0.375 mmol). Yellow solid, 21% yield, carbonize at 220 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
8.88 (t, J ¼ 6.0 Hz, 1H, NH), 8.73 (s, 1H, CH),
mL,
4.1.5.3. N-(1-(4-cyanobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12c). 12c was synthesized
from 11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 4-
(bromomethyl)benzonitrile (67 mg, 0.344 mmol). Orange solid, 15%
d
8.37 (s,1H, Ph-H), 7.37 (t, J ¼ 7.7 Hz,1H, Ph-H), 7.34e7.28 (m,1H, Ph-
H), 7.18 (q, J ¼ 8.9, 7.3 Hz, 2H, Ph-H), 4.56 (d, J ¼ 5.9 Hz, 2H, CH2). 13
C
NMR (100 MHz, DMSO‑d6)
d
162.8 (JCF ¼ 155 Hz), 160.9, 159.4, 149.8,
yield, carbonize at 220 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d 8.70 (s,
146.8, 138.0, 132.5, 130.0 (JCF ¼ 5.0 Hz), 129.5 (JCF ¼ 8.0 Hz), 127.1,
126.4 (JCF ¼ 15 Hz), 124.8 (JCF ¼ 3.0 Hz), 115.7 (JCF ¼ 21 Hz), 108.6,
101.9, 37.1, 14.5. HRMS m/z C17H11FN2O7: calcd 374.0500, found
373.2014 [M ꢁ H]-.
1H, CH), 8.41 (d, J ¼ 7.6 Hz, 1H, NH), 8.36 (s, 1H, Ph-H), 7.81 (d,
J ¼ 7.8 Hz, 2H, Ph-H), 7.54 (d, J ¼ 7.9 Hz, 2H, Ph-H), 3.88e3.78 (m,
1H, CH), 3.72e3.56 (m, 2H, CH2), 2.83e2.71 (m, 2H, CH2), 2.37e2.17
(m, 2H, CH2), 1.91e1.80 (m, 2H, CH2), 1.58e1.47 (m, 2H, CH2). HRMS
m/z C23H20N4O7: calcd 464.1332, found 463.1044 [M ꢁ H]-.
4.1.4.7. N-(2-chloro-4-fluorobenzyl)-7,8-dihydroxy-6-nitro-2-oxo-
2H-chromene-3-carboxamide(9d). 9d was synthesized from DW-2
(0.1 g, 0.375 mmol), K2CO3 (52 mg, 0.375 mmol), HATU (215 mg,
4.1.5.4. N-(1-(4-cyanobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12d). 12d was synthesized
from 11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 1-
(bromomethyl)-4-nitrobenzene (74 mg, 0.344 mmol). Brick red
solid, 25% yield, carbonize at 160 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
0.563 mmol), and (2-chloro-4-fluorophenyl)methanamine (47
0.375 mmol). Orange solid, 28% yield, mp: 180e182 ꢃC. 1H NMR
(400 MHz, DMSO‑d6)
mL,
d
9.08 (t, J ¼ 5.9 Hz, 1H, NH), 8.81 (s, 1H, CH),
8.18 (s, 1H, Ph-H), 7.57e7.50 (m, 1H, Ph-H), 7.46e7.42 (m, 1H, Ph-H),
7.24e7.19 (m, 1H, Ph-H), 4.57 (d, J ¼ 6.0 Hz, 2H, CH2). HRMS m/z
d
8.71 (s, 1H, CH), 8.42 (d, J ¼ 7.2 Hz, 1H, NH), 8.37 (s, 1H, Ph-H),
C
16H8F2N2O7: calcd 408.0161, found 407.3325 [M ꢁ H]-.
8.26e8.19 (m, 2H, Ph-H), 7.70e7.61 (m, 2H, Ph-H), 3.87 (s, 1H,
CH), 3.85e3.74 (m, 2H, CH2), 2.91e2.79 (m, 2H, CH2), 2.46e2.33 (m,
2H, CH2), 1.92 (t, J ¼ 12.2 Hz, 2H, CH2), 1.61 (t, J ¼ 17.0 Hz, 2H, CH3).
HRMS m/z C22H20N4O9: calcd 484.1230, found 483.0021 [M ꢁ H]-.
4.1.4.8. tert-butyl4-(7,8-dihydroxy-6-nitro-2-oxo-2H-chromene-3-
carboxamido) piperidine-1-carboxylate(10). The synthetic route
12

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
4.1.5.5. N-(1-(3-fluorobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12e). 12e was synthesized
from 11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 1-
metabolically active cells to form a blue-purple formazan that can
be measured spectrophotometrically. The absorbances were read in
an eight-channel computer-controlled photometer at wavelengths
of 540 and 690 nm. All data were calculated using the median
optical density (OD) value of three wells. The EC50 was defined as
the concentration of the test compound allowing 50% protection
from viral cytopathogenicity. The CC50 was defined as the com-
pound concentration that reduced the absorbance of mock-infected
cells by 50%.
(bromomethyl)-3-fluorobenzene (42
mL, 0.344 mmol). Orange
solid, 23% yield, carbonize at 210 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.70 (s, 1H, CH), 8.41 (d, J ¼ 7.5 Hz, 1H, NH), 8.36 (s, 1H, Ph-H), 7.39
(q, J ¼ 7.5 Hz, 1H, Ph-H), 7.21e7.09 (m, 3H, Ph-H), 3.91e3.78 (m, 1H,
CH), 3.71e3.53 (m, 2H, CH2), 2.87e2.72 (m, 2H, CH2), 2.43e2.14 (m,
2H, CH2), 1.94e1.82 (m, 2H, CH2), 1.61e1.46 (m, 2H, CH2). HRMS m/z
C
22H20FN3O7: calcd 457.1285, found 456.1146 [M ꢁ H]-.
4.3. Cell permeability assays
4.1.5.6. N-(1-(3-chlorobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12f). 12f was synthesized from
11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 1-(bro-
The permeability of compounds DW-4 and 8a was determined
with the human colon epithelial cancer cell line, Caco-2, as previ-
ously described [38]. The rate of membrane transport for both
compounds was measured and expressed as apparent permeability
(Papp). Nadolol and metoprolol were used as controls in the assays.
momethyl)-3-chlorobenzene (45
m
L, 0.344 mmol). Orange solid,
21% yield, carbonize at 200 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.70
(s, 1H, CH), 8.40 (d, J ¼ 7.6 Hz, 1H, NH), 8.36 (s, 1H, Ph-H), 7.40e7.27
(m, 4H, Ph-H), 3.88e3.78 (m, 1H, CH), 3.63e3.48 (m, 2H, CH2),
2.79e2.70 (m, 2H, CH2), 2.32e2.12 (m, 2H, CH2), 1.86 (d, J ¼ 9.9 Hz,
2H, CH2), 1.62e1.45 (m, 2H, CH2). 13C NMR (100 MHz, DMSO‑d6)
4.4. HIV-1 RNase H inhibition assay
d
162.8, 161.6, 161.2, 161.1, 151.3, 149.5, 146.7, 138.0, 135.4, 133.4,
Wild-type HIV-1 BH10 reverse transcriptase (RT) was expressed
in E. coli and purified as previously described [39,40]. RNase H ac-
tivity was determined with template-primer 31Trna/21P [35].
Briefly, the synthetic RNA oligonucleotide 31Trna (50-
UUUUUUUUUAGGAUACAUAUGGUUAAAGUAU-30) was labeled at
132.4, 130.5, 127.6, 127.1, 115.3, 108.7, 101.9, 99.9, 51.8, 38.7, 31.6.
HRMS m/z C22H20ClN3O7: calcd 473.0990, found 472.8017 [M ꢁ H]-.
4.1.5.7. N-(1-(3-bromobenzyl)piperidin-4-yl)-7,8-dihydroxy-6-nitro-
2-oxo-2H-chromene-3-carboxamide(12g). 12g was synthesized
from 11 (0.1 g, 0.286 mmol), K2CO3 (79 mg, 0.572 mmol), and 1-
its 50 end with [ 32P]ATP (PerkinElmer) and T4 polynucleotide
g-
kinase (New England Biolabs), and purified with a mini Quick
SpinTM column (Roche) before annealing to the DNA primer 21P
(50-ATACTTTAACCATATGTATCC-30). Candidate RNase H inhibitors
were available in 50% DMSO, and reactions in the presence or
absence of inhibitor were performed as previously described [38].
Assays were carried out for 15 s at 37 ꢃC with 20e40 nM HIV-1 RT in
bromo-3-(bromomethyl)benzene (55
mL, 0.344 mmol). Orange
solid, 22% yield, carbonize at 180 ꢃC. 1H NMR (400 MHz, DMSO‑d6)
d
8.70 (s, 1H, CH), 8.40 (d, J ¼ 7.5 Hz, 1H, NH), 8.36 (s, 1H, Ph-H), 7.54
(s, 1H, Ph-H), 7.47 (d, J ¼ 7.1 Hz, 1H, Ph-H), 7.38e7.27 (m, 2H, Ph-H),
3.90e3.77 (m, 1H, CH), 3.68e3.49 (m, 2H, CH2), 2.85e2.72 (m, 2H,
CH2), 2.39e2.13 (m, 2H, CH2), 1.87 (d, J ¼ 10.3 Hz, 2H, CH2),
16 mL of 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 25 nM
1.61e1.48 (m, 2H, CH2). 13C NMR (100 MHz, DMSO‑d6)
d
162.8, 161.7,
32P-labeled template-primer, and 5% DMSO. Products of the hy-
drolytic reaction were separated by denaturing polyacrylamide gel
electrophoresis, and quantified by phosphorimaging as described
161.2, 161.1, 149.5, 146.7, 138.0, 132.5, 130.9, 128.6, 127.1, 122.1, 120.9,
114.0, 108.7, 103.9, 101.9, 51.7, 38.7, 31.2. HRMS m/z C22H20BrN3O7:
calcd 517.0485, found 516.0332 [M ꢁ H]-.
[35,38]. IC50 values (mM) were determined from dose-response
curves after calculating the amount of cleaved substrate in the
absence of inhibitor (5% DMSO) and in the presence of drug at
4.2. In vitro anti-HIV assay
concentrations in the range of 0.2e100 mM.
The anti-HIV activity and cytotoxicity of synthesized derivatives
were evaluated in MT-4 cell cultures using the MTT method, with
wild-type HIV-1 strain IIIB, and mutant strains containing single
amino acid substitutions in the viral RT (L100I, K103 N, E138K,
Y181C and Y188L), or double-mutants (V106A/F227L and RES056),
as well as the HIV-2 strain ROD [32]. Stock solutions (10 ꢀ final
4.5. Molecular modeling
The molecular structure of compound 8a was sketched using the
Molecular Builder module implemented in Maestro (Maestro,
Schrodinger, LLC, New York, NY, 2019) and then preprocessed with
LigPrep (LigPrep, Schrodinger, LLC, New York, NY, 2019). Multiple
concentration) of test compounds were added in 25 mL volumes to
two series of triplicate wells to allow simultaneous evaluation of
the effects on mock- and HIV-infected cells. Using a Biomek 3000
robot (Beckman Instruments, Fullerton, CA), serial five-fold di-
lutions of the test compounds (final 200 mL volume per well) were
made directly in flat-bottomed 96-well microtiter trays, including
protonation and tautomerization states at pH of 7.0
2.0 were
requested. Docking experiments were performed employing the
crystal structure of a hydroxypyridone carboxylic acid active-site
RNase H inhibitor in complex with HIV-1 RT (PDB ID: 5J1E). The
protein setup was carried out using the Protein Preparation Wizard
implemented in Maestro. Crystallographic water molecules were
removed; hydrogen atoms were added to the protein consistent
with the neutral physiologic pH and ensuring all other atoms po-
sitions remained fixed. The centroid of the x-ray ligand was defined
as the grid box. van der Waals radius scaling factor 1.00, partial
charge cutoff 0.25, and OPLS3e force filed were used for receptor
grid generation. Docking was carried out using the Ligand Docking
module with extra precision. Each generated conformation of 8a
was subsequently docked and scored, and the conformation with
the highest score was chosen as the final result. Docking was vali-
dated by measuring the RMSD between the predicted conformation
of 8a and that of the RNase H inhibitor found in the crystal structure
5J1E.
untreated control HIV-1 and mock-infected cell samples for each
sample. HIV-1 strains stock (50 mL at 100e300 CCID50) or culture
medium was added to either the infected or mock-infected wells of
the microtiter tray. Mock-infected cells were used to evaluate the
effect of compounds on uninfected cells to assess cytotoxicity.
Exponentially growing MT-4 cells were centrifuged for 5 min at
1000 rpm and the supernatant was discarded. The MT-4 cells were
resuspended at 6 ꢀ 105 cells/mL, and 50
mL aliquots were trans-
ferred to the microtiter tray wells. At five days after infection, the
viability of mock- and HIV-infected cells was determined spectro-
photometrically with the MTT assay.
This assay is based on the reduction of yellow-colored MTT
(Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase of
13

D. Kang, Ç. Urhan, F. Wei et al.
European Journal of Medicinal Chemistry 225 (2021) 113769
with improved potency against resistance-associated variants, J. Med. Chem.
60 (2017) 4424e4443.
Declaration of competing interest
[14] D. Kang, Z. Fang, Z. Li, B. Huang, H. Zhang, X. Lu, H. Xu, Z. Zhou, X. Ding,
D. Daelemans, E. De Clercq, C. Pannecouque, P. Zhan, X. Liu, Design, synthesis,
and evaluation of thiophene[3,2-d]pyrimidine derivatives as HIV-1 non-
nucleoside reverse transcriptase inhibitors with significantly improved drug
resistance profiles, J. Med. Chem. 59 (2016) 7991e8007.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[15] D. Feng, A. Zhang, Y. Yang, P. Yang, Coumarin-containing hybrids and their
antibacterial activities, Arch. Pharm. 353 (2020), e1900380.
[16] S. Mishra, A. Pandey, S. Manvati, Coumarin: an emerging antiviral agent,
Heliyon 6 (2020), e03217.
Acknowledgments
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (NSFC Nos. 81973181,
81903453), Shandong Provincial Key research and development
project (Nos. 2019JZZY021011), Shandong Provincial Natural Sci-
ence Foundation (ZR2019BH011, ZR2020YQ61, ZR2020JQ31),
[17] D. Pinto, A.M.S. Silva, Anticancer natural coumarins as lead compounds for the
discovery of new drugs, Curr. Top. Med. Chem. 17 (2017) 3190e3198.
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compounds for their pharmacological activity, BioMed Res. Int. 2013 (2013)
963248.
Foreign
cultural
and
educational
experts
Project
(GXL20200015001), Qilu Young Scholars Program of Shandong
University, the Taishan Scholar Program at Shandong Province, and
KU Leuven (GOA 10/014). Work in Madrid was supported by the
Spanish Ministry of Science and Innovation (grant PID2019-
104176RB-I00/AEI/10.13039/501100011033), and an institutional
[20] World Health Organization, World Health Organization Model List of Essential
Medicines: 21st List 2019, World Health Organization, Geneva, 2019.
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grant of Fundacion Ramon Areces (Madrid, Spain). The technical
assistance of Mr. Kris Uyttersprot and Mrs. Kristien Erven, for the
HIV experiments is gratefully acknowledged.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113769.
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