Design, synthesis and biological evaluation of 3-hydroxyquinazoline-2,4(1H,3H)-diones as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and integrase

Article abstract of DOI:10.1016/j.bmc.2019.07.011

A novel series of 3-hydroxyquinazoline-2,4(1H,3H)-diones derivatives has been designed and synthesized. Their biochemical characterization revealed that most of the compounds were effective inhibitors of HIV-1 RNase H activity at sub to low micromolar concentrations. Among them, II-4 was the most potent in enzymatic assays, showing an IC₅₀ value of 0.41 ± 0.13 μM, almost five times lower than the IC₅₀ obtained with β-thujaplicinol. In addition, II-4 was also effective in inhibiting HIV-1 IN strand transfer activity (IC₅₀ = 0.85 ± 0.18 μM) but less potent than raltegravir (IC₅₀ = 71 ± 14 nM). Despite its relatively low cytotoxicity, the efficiency of II-4 in cell culture was limited by its poor membrane permeability. Nevertheless, structure-activity relationships and molecular modeling studies confirmed the importance of tested 3-hydroxyquinazoline-2,4(1H,3H)-diones as useful leads for further optimization.

Full text of DOI:10.1016/j.bmc.2019.07.011

Accepted Manuscript
Design, synthesis and biological evaluation of 3-hydroxyquinazoline-2,4(1H,
3H)-diones as dual inhibitors of HIV-1 reverse transcriptase-associated RNase
H and integrase
Ping Gao, Xiqiang Cheng, Lin Sun, Shu Song, Mar Álvarez, Joanna
Luczkowiak, Christophe Pannecouque, Erik De Clercq, Luis Menéndez-Arias,
Peng Zhan, Xinyong Liu
PII:
S0968-0896(19)31003-X
https://doi.org/10.1016/j.bmc.2019.07.011
BMC 14995
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 June 2019
2 July 2019
5 July 2019
Please cite this article as: Gao, P., Cheng, X., Sun, L., Song, S., Álvarez, M., Luczkowiak, J., Pannecouque, C., De
Clercq, E., Menéndez-Arias, L., Zhan, P., Liu, X., Design, synthesis and biological evaluation of 3-
hydroxyquinazoline-2,4(1H,3H)-diones as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and
integrase, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.011
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Design, synthesis and biological evaluation of 3-hydroxyquinazoline-2,4(1H,3H)-diones
as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and integrase
Ping Gaoa, Xiqiang Chenga, Lin Suna, Shu Songa, Mar Álvarezb, Joanna Luczkowiakb,
Christophe Pannecouquec, Erik De Clercqc, Luis Menéndez-Ariasb,*, Peng Zhana,*, Xinyong Liua,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology, Ministry of
Education, School of Pharmaceutical Sciences, Shandong University, Ji’nan, 250012
bCentro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones
Científicas & Universidad Autónoma de Madrid), Madrid, Spain
cRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
*E-mail: lmenendez@cbm.csic.es (Menéndez-Arias L.); zhanpeng1982@sdu.edu.cn (Zhan
P.); xinyongl@sdu.edu.cn (Liu X.Y.).
Abstract
A novel series of 3-hydroxyquinazoline-2,4(1H,3H)-diones derivatives has been designed
and synthesized. Their biochemical characterization revealed that most of the compounds were
effective inhibitors of HIV-1 RNase H activity at sub to low micromolar concentrations. Among
them, II-4 was the most potent in enzymatic assays, showing an IC50 value of 0.41 ± 0.13 μM,
almost five times lower than the IC50 obtained with β-thujaplicinol. In addition, II-4 was also
effective in inhibiting HIV-1 IN strand transfer activity (IC50 = 0.85 ± 0.18 μM) but less potent
than raltegravir (IC50 = 71 ± 14 nM). Despite its relatively low cytotoxicity, the efficiency of II-4
in cell culture was limited by its poor membrane permeability. Nevertheless, structure-activity
relationships and molecular modeling studies confirmed the importance of tested 3-
hydroxyquinazoline-2,4(1H,3H)-diones as useful leads for further optimization.
Key words: HIV-1, RNase H, integrase, dual inhibitors
1. Introduction
HIV/AIDS continues to be a major global health epidemic. In 2017, there were 36.9 million
people living with HIV worldwide and an additional 1.8 million people became newly infected [1].
Although combinations of anti-HIV drugs targeting different steps of the viral replication cycle
are effective in maintaining a very low or undetectable viral load in infected patients,
shortcomings of antiretroviral therapies include their adverse side effects, the emergence of
extensively cross-resistant strains of HIV-1, high cost of most effective therapies, and limited
availability in many countries around the world. In this scenario, novel therapeutics that act on
1

previously untargeted steps of the viral life cycle are urgently needed to circumvent the onset of
drug resistance and to improve current treatments [2].
The HIV-1 reverse transcriptase (RT) is a validated target for AIDS therapy. RT plays an
essential role in virus replication. It converts the viral single-stranded genomic RNA into double-
stranded proviral DNA that integrates into the host cell’s genome. During this process, the DNA
polymerase activity of the RT synthesizes the proviral DNA, while the RT’s RNase H activity
degrades the viral RNA genome used as template during DNA synthesis [3, 4]. Interestingly, RT
DNA polymerase nucleoside RT inhibitors (NRTIs) constitute the backbone of current therapies,
and several non-nucleoside RT inhibitors (NNRTIs) are widely used in the clinical setting.
However, drug discovery efforts to obtain RT RNase H inhibitors have not provided any
promising candidate currently in development. Therefore, RNase H represents a novel and
attractive target for antiretroviral drug development, potentially useful to treat HIV variants
resistant to current drugs.
The RNase H active site contains a highly conserved DEDD motif consisting of four
carboxylated amino acid residues in close proximity (Asp443, Glu478, Asp498 and Asp549) that
interact with two metal ions (Mg2+) [5]. A similar arrangement is observed in the active site of
HIV-1 IN, another metalloenzyme that plays critical roles in viral infection and the establishment
of proviral latency. Three highly conserved residues in the catalytic core domain of the enzyme,
namely, Asp64, Asp116, and Glu152 (DDE motif) coordinate the two divalent cations necessary
for its strand transfer activity [6]. The geometries of the catalytic sites of HIV-1 RNase H and IN
are similar with their active site residues and magnesium ions located at structurally equivalent
positions in both enzymes. In addition, dual inhibitors of both enzymes have been previously
described [7, 8].
Continued efforts in medicinal chemistry and clinical testing have led to the approval of
four HIV-1 IN inhibitors: raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and
bictegravir [3, 9-13], while several chemotypes targeting the RNase H active site, and inhibiting
its endonuclease activity at sub to low micromolar concentration have been described [14-17]
(Fig.1).
2

O
N
HO
F
O
N
O
OH
O
N
OH
O
Cl
F
F
O
O
F
O
F
F
O
O
OH
N
H
N
H
H
H
N
N
NH
HN
O
N
N
N
O
O
O
OH
F
O
N
DTG
HIV IN IC50 = 0.5 nM (WT)
Bictegravir
RAL
HIV IN IC50 = 2 nM (WT)
EVG
HIV IN IC50 = 3.8 nM (WT)
HIV IN IC50 = 7.5 nM (WT)
Approved IN inhibitors
O
O
O
OH
HO
OH
O
O
N
O
S
OH
HN
O
O
O
N
OH
O
Cl
HIV RNase H: IC50 = 0.80 M (WT)
HIV RNase H: IC50 = 3.8 M (WT)
2. -Thujaplicinol
HIV RNase H: IC50 = 1.9 M (WT)
HIV RNase H: IC50 = 7.3 M (WT)
1. Hydroxylisoquinoline
3. Diketoacid
4. Diketoacid ester
OH
OH
O
N
N
O
O
H2N
HO
N
N
O
N
HO
S
HO
N
O
OH
O
N
N
O
Br
HN
OH
O
NH2
HIV RNase H: IC50 = 0.032 M (WT)
HIV RNase H: IC50 = 0.65 M (WT)
8. Hydroxypyridonecarboxylic acid
HIV RNase H: IC50 = 0.045 M (WT)
HIV RNase H: IC50 = 1.4 M (WT)
5. Pyrimidinol carboxylic acid
7. Naphthyridinone
6. Pyridopyrimidinone
Representative RNase H inhibitors
Fig.1 Approved IN inhibitors and representative RNase H inhibitors. IC50 values for HIV-1 IN
strand transfer and RNase H inhibition assays using purified enzymes are shown below.
Hybridization of hydroxylisoquinoline (HID) and hydroxypyrimidine-2,4-dione (HPD)
scaffolds led to the design of 3-hydroxyquinazoline-2,4(1H,3H)-diones. This chemotype is similar
to that of N-hydroxyureas, initially described as inhibitors of flap endonuclease-1 [18]. The HIV-1
RNase H inhibitory activity of 3-hydroxyquinazoline-2,4(1H,3H)-diones has been recently
reported by Kankanala et al., although their assessment was limited to compounds 6-(4-
chlorophenyl)-3-hydroxyquinazoline-2,4(1H,3H)-dione and 3-hydroxy-6-phenylquinazoline-
2,4(1H,3H)-dione [19]. In the current study, we obtained a new series of 3-hydroxyquinazoline-
2,4(1H,3H)-diones, designed by introducing different benzamide or phenylsulfonamide
substituents at positions 6 and 7 of the quinazoline ring. The new compounds were synthesized
through simple and convenient procedures in order to explore their structure-activity relationships
(SAR), and improve their inhibitory properties.
3

2. Results and Discussion
2.1. Chemistry
H
N
H
N
NH2
NH2
iv
O
O
O
O
iii
ii
i
O2N
O2N
H
N
H2N
OH
O2N
N
N
O
O
O
O
O
2
1
3
4
H
N
H
H
H
R
R
v
O
O
N
N
N
X
X
X = CO or SO2
N
N
O
OH
O
O
5 (5A1-12; 5B1-5)
6 (Series I 1-12; Series II 1-5)
Scheme 1. Reaction reagents and conditions: i) O-benzylhydroxylamine, N,N-
carbonyldiimidazole, tetrahydrofuran, 60 °C, 5 h; ii) triphosgene, triethylamine, tetrahydrofuran,
room temperature, 1 h; iii) iron powder, chlorination ammonium, ethanol, reflux, 2 h; iv)
corresponding benzoyl chloride or benzenesulfonyl chloride, pyridine, 0 °C, 3 h; v) H2, Pd/C,
room temperature, 1 h.
The target compounds were prepared by following a relatively simple procedure outlined in
Scheme 1. By treating commercially available methyl 4-nitroanthranilic acid or 5-nitroanthranilic
acid (1) with O-benzylhydroxylamine and N,N-carbonyldiimidazole in tetrahydrofuran (THF),
compound 2 was obtained. Then, a cyclization step using triphosgene yielded the intermediate 3.
After nitro reduction to obtain compound 5, followed by acylation to get compound 6, the desired
compounds were obtained by hydrogenolytic deprotection of their N-hydroxyl group.
2.2 Biological activity
2.2.1. Inhibition of HIV-1 RNase H activity
All newly synthesized compounds were tested in vitro for their inhibitory activity against the
RNase H of HIV-1 RT, and products of the endonucleolytic reaction were analyzed by denaturing
polyacrylamide gel electrophoresis [20]. Overall, series I-II were all potent RNase H inhibitors
with IC50 values ranging from 0.41 to 20.1 μM (Table 1). Compound II-4 was the most active
derivative of this series, with IC50 value of 0.41 ± 0.13 μM. It was almost 5 times more potent
than the reference compound β-thujaplicinol (IC50 = 1.98 ± 0.22 μM) in these assays. I-7 was
found to be the worst inhibitor of the series with an IC50 value of 20.1 ± 10.4 μM. The naphthyl
4

group of compounds II-4 and I-7 was located at positions 6 and 7, respectively. These differences
were responsible for the 50-fold reduction in RNase H inhibition found for I-7 as compared to II-4.
The comparison of RNase H inhibitory activities of compounds having substituents at
position 7 (series 1: I-1 to I-12) revealed that the activities of derivatives with benzoyl
substitutions were slightly higher than those obtained with molecules having a benzenesulfonyl
substitution. Among the compounds having benzoyl substituents (I-1 to I-5), the least substituted
compound (I-1) was also the best of this small series, with an IC50 value of 2.52 ± 0.78 μM. The
effects of substitutions in the benzyl group in these compounds followed the order: none (I-1) > 4-
methyl (I-3) > 4-F (I-2) ≈ 3-F (I-5) > 4-CF3 (I-4), although differences were not relevant in most
cases. I-4 was the least potent compound of the group. Regarding the benzenesulfonyl substitution
derivatives (I-6 to I-12), the naphthyl derivative (I-7, IC50 value of 20.1 ± 10.4 μM) was the
least active compound. The order of potency of these compounds was: 2,4,6-triCH3 phenyl (I-8) >
3-CH3 phenyl (I-12) > 3-CF3 phenyl (I-6) > 4-F phenyl (I-10) > 4-tert-butyl phenyl (I-10) >
biphenyl (I-11) > naphthyl (I-7).
The activity of the 6-substituted compounds (compounds II-1 to II-5) was significantly
higher than that of the 7-substituted compounds. Series II compounds with benzenesulfonyl
substitutions were more potent inhibitors than the one with the benzoyl substitution (II-1). The
naphthyl derivative (II-4, IC50 value of 0.41 ± 0.13 μM) was the most active compound. The order
of potency of the benzenesulfonyl derivatives of series II was: naphthyl (II-4) > phenyl (II-2) > 3-
CF3 phenyl (II-5) > 4-F phenyl (II-3).
5

Table 1. RNase H inhibitory activity, anti-HIV activity and cytotoxicity in MT-4 cells.
H
H
N
H
N
N
O
O
R
N
R
N
OH
N
OH
H
O
O
Series I
Series II
EC50 (µM)b
IIIB
IC50 (µM)
RNase Ha
Compounds
R
CC50 (µM)c
36.6 ± 11.4
ROD
O
I-1
2.52 ± 0.78
3.73 ± 0.94
2.74 ± 0.98
>36.6
>43.0
>10.9
>36.6
>43.0
>10.9
O
I-2
I-3
43.0 ± 4.9
10.9 ± 3.1
F
O
O
H3C
I-4
6.09 ± 2.53
>315.9
>315.9
315.9 ± 18.6
F
F
F
O
F
I-5
I-6
I-7
I-8
3.73 ± 1.04
5.33 ± 1.76
20.1 ± 10.4
>39.8
>39.8
39.8 ± 3.4
F
F
O2
S
F
>80.2
>80.2
80.2 ± 28.4
219.6 ± 3.1
153.5 ± 47.4
O2
S
>219.6
>153.5
>219.6
>153.5
O2
S
3.24 ± 1.26
6

O2
S
I-9
5.88 ± 3.04
5.68 ± 2.71
>307.0
>355.8
>307.0
>355.8
307.0 ± 9.4
>355.8
O2
S
I-10
F
O2
S
I-11
11.7 ± 5.5
>305.3
>305.3
>305.3
O2
S
H3C
F
I-12
II-1
II-2
II-3
II-4
II-5
4.10 ± 1.61
4.29 ± 0.96
1.19 ± 0.31
2.25 ± 0.85
0.41 ± 0.13
2.00 ± 0.81
>309.5
>167.7
>213.4
>199.4
>208.4
>176.1
>309.5
>167.7
>213.4
>199.4
>208.4
>176.1
309.5 ± 18.9
167.7 ± 52.9
213.4 ± 9.0
199.4 ± 7.7
208.4 ± 5.2
176.1 ± 7.1
O
O2
S
O2
S
F
O2
S
F
F
O2
S
F
d
Raltegravir
-
0.015±0.0024 0.020±0.0064 6.51 ± 0.15
-
β-thujaplicinol
-
1.98 ± 0.22
>2.83
>2.83
2.83 ± 0.46
aConcentration required to inhibit by 50% the in vitro RNase H activity.
bThe 50% effective antiviral concentration (EC50) was defined as the concentration of the tested
7

compound achieving 50% protection from viral cytopathicity.
cThe 50% cytotoxic concentration (CC50) was defined as the compound concentration that reduced
the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
dNot determined.
2.2.2 HIV-1 IN strand transfer inhibition
Based on the similarity of the HIV-1 RNase H and IN active sites, and the expected chelating
activity of designed compounds, we determined the inhibitory activity of selected compounds (i.e.
I-7, I-9 and II-4) in IN strand transfer assays [21]. As shown in Fig. 2, the three compounds were
able to inhibit the viral IN. Despite being ten times less potent than raltegravir (IC50 = 71 ± 14 nM),
II-4 was found to be more potent than I-7 and I-9 in IN strand transfer assays, with an IC50 value
of 0.85 ± 0.18 μM. All three tested 3-hydroxyquinazoline-2,4(1H,3H)-diones were found to be
effective inhibitors of both HIV-1 IN and RNase H activities. I-7 and I-9 were around ten times
less potent than II-4, showing 50-60% inhibitory activity at 10 µM in HIV-1 IN strand transfer
reactions. Interestingly, RNase H IC50 values for I-7 and I-9 shown in Table 1 (20.1 ± 10.4 µM for
I-7 and 5.88 ± 3.04 µM for I-9) were in the same order of magnitude and 14-49 times higher than
those obtained for II-4.
Fig. 2. HIV-1 IN strand transfer inhibition assays. Histograms represent percent inhibition of the
strand transfer reaction in assays carried out with heteropolymeric hybrids in the presence of
compounds I-7, I-9 and II-4 and raltegravir, at 10 µM, 1 µM and 0.1 µM concentrations (black,
grey and white bars, respectively). RAL, raltegravir.
8

2.2.3 In vitro anti-HIV assay
All compounds were evaluated in cell culture for their antiviral activity against wild-type
HIV-1 and HIV-2 strains (IIIB and ROD, respectively). Raltegravir was used as the reference drug
for IN inhibition. EC50 values (anti-HIV activity) and CC50 values (cytotoxicity) are given in
Table 1. At the highest concentration tested, series I and II compounds showed low toxicity,
although none of them revealed significant HIV-1 replication inhibitory activity. The lack of
inhibitory effect in cell culture curbed their potential development into antiviral drugs.
Table 2. Predicted physicochemical properties of compound II-4.a
Physicochemical property
Tolerance limit
II-4
Number of atoms excluding hydrogens
Molecular weight
Number of hydrogen bond acceptors
Number of hydrogen bond donors
Number of rotable bonds
Total polar surface area
Molecular volume
Octanol-water partition coefficient (log P)
-
27
383.38
8
3
3
121.26
303.61
2.30
< 500 Da
≤ 10
≤ 5
≤10
< 140 A2
-
< 5
a
Values
determined
using
on-line
cheminformatics
software:
http://www.molinspiration.com/
The reasons that explained the lack of activity of the tested compounds in cell culture was
investigated. An overall assessment of the physicochemical properties of compound II-4 using a
free online software revealed that the compound could theoretically meet the general requirements
accepted for useful drugs (Table 2), since size, polarity, volume and potentially reactive groups of
II-4 were below the tolerance limit. However, cell permeability assays were carried out and
revealed that II-4 had poor membrane permeability. Thus, in experiments carried out with Caco-2
cells, the apparent permeability (Papp) of II-4 was below 1.9 x 10-7 cm/s, while reference
compounds for low and high permeability, such as nadolol and metoprolol, showed Papp values of
9 x 10-8 cm/s and 1.47 x 10-5 cm/s, respectively. However, for II-4, the signal responses in
receiver samples were lower than the limit of detection of the apparatus, and a precise calculation
9

of Papp was not possible. The reported Papp (< 1.9 x 10-7 cm/s) was obtained by assuming the
lowest detectable value in the receiver chamber of our system (i.e. 50). In our assay conditions,
recovery of II-4 was sufficient and well above the 50% cut-off value, considered as sufficient for
reliable estimates of Papp
.
2.3. Molecular modeling analysis
To improve our understanding of structure-affinity relationships, the best compound of the
series (i.e II-4) was docked in the structures of the HIV-1 RT RNase H domain (PDB code: 5J1E)
and the prototype foamy virus (PFV) IN (PDB code: 3OYA), using the Surflex-Dock SYBYL-X
2.0 software. We used PFV IN as a surrogate model for HIV-1 IN, based on their high homology
[22, 23]. The results of the docking analysis showing the theoretical binding location of II-4 and
its conformation are shown in Fig. 3.
According to these predictions, compound II-4 binding within the RNase H active site
suggests a potential interaction between the chelating triad of the 3-hydroxyquinazoline-
2,4(1H,3H)-dione derivative and the two Mg2+ ions, which are coordinated to the conserved active
site acidic residues Asp443, Glu478, Asp498, and Asp549 (Fig. 3A). The 3-hydroxyl group and
the keto substituent at position 4 of II-4 could establish hydrogen bonds with His539, which
would stabilize the inhibitor near the active site metal-chelating residues forming the DEDD motif,
all of them required for RNase H activity.
(A)
(B)
Fig. 3. Predicted binding modes of II-4 (cyan) in the HIV-1 RNase H active site (A, PDB code:
5J1E) and the PFV IN active site (B, PDB code: 3OYA). Amino acid residues important for ligand
10

binding are represented as sticks in orange. Magnesium ions are depicted as green spheres.
Chelating and H-bond interactions are indicated by purple dashed lines. Hydrogens (nonpolar) are
not shown. Figures were prepared with the PyMOL visualization program (http://www.pymol.org).
Interatomic distances for key interactions are indicated in Å.
The predicted binding mode of compound II-4 within the IN active site (Fig. 3B) shows
chelating interactions between the chemotype core of the 3-hydroxyquinazoline-2,4(1H,3H)-dione
and the two Mg2+ ions, coordinated by the highly conserved residues (Asp128, Asp185 and
Glu221) in the active site of the enzyme. Besides, the rings containing the chelating triad of II-4
form stacking interactions with the conserved terminal deoxyadenosine, located in the DNA.
These interactions have been also observed in IN bound to approved strand transfer inhibitors such
as raltegravir [22, 23]. In addition, the naphthyl moiety of II-4 could participate in π-stacking
interactions with the side-chain of Tyr212. All mentioned interactions seem to be important to
justify the IN strand transfer inhibitory activity of II-4.
The binding models predicted for both RNase H and IN structures are consistent with our
results showing that II-4 is a potent HIV-1 RNase H-IN dual inhibitor, and provide support for
further rational drug optimization.
3. Conclusion
Most of the 3-hydroxyquinazoline-2,4(1H,3H)-dione derivatives described in this study were
highly effective in inhibiting HIV-1 RNase H activity at sub to low micromolar concentrations.
Among them, compound II-4 was the most promising one with potent inhibitory activity against
HIV-1 RNase H (IC50 = 0.41 ± 0.13 μM), about five times more potent than β-thujaplicinol in
enzymatic assays. Although less potent than the approved drug raltegravir, II-4 turned out to be
also a potent HIV-1 IN inhibitor with IC50 value of 0.85 ± 0.18 μM, providing further evidence of
the feasibility of identifying dual HIV-1 RNase H-IN inhibitors through further modification of
the 3-hydroxyquinazoline-2,4(1H,3H)-dione chemotype. Molecular modeling-based predictions
revealed expected binding conformations for dual RNase H-IN inhibitors with the ambident
chelating scaffold. Unfortunately, and despite their low cytotoxicity, herein described compounds
failed to exhibit significant inhibitory activity in infected cells, probably in part due to their poor
cell membrane permeability.
11

4. Experimental section
4.1. Synthetic procedures and analytical data
Mass spectrometry was performed on an API 4000 triple quadrupole mass spectrometer
1
(Applied Biosystems/MDS Sciex, Concord, ON, Canada). H NMR and 13C NMR spectra were
recorded on a Bruker AV-400 spectrometer (Bruker BioSpin, Switzerland), using solvents as
indicated (DMSO-d6). Chemical shifts were reported in δ values (ppm) with tetramethylsilane as
the internal reference, and J values were reported in hertz (Hz). Melting points (mp) were
determined on a micromelting point apparatus (Tian Jin Analytical Instrument Factory, Nankai,
Tianjin, China). Flash column chromatography was performed on columns packed with silica gel
60 (200-300 mesh) (Qingdao waves silica gel desiccant co., Ltd, Qingdao, China). Thin layer
chromatography was performed on pre-coated HUANGHAI® HSGF254, 0.15-0.2 mm TLC-
plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, Shandong, China). The key
reactants including 4-nitroanthranilic acid, 5-nitroanthranilic acid, O-benzylhydroxylamine, N,N-
carbonyldiimidazole etc. were purchased from Bide pharmatech Co. Ltd.
4.1.1. General procedure for the synthesis of 2-amino-N-(benzyloxy)-4-nitrobenzamide or 2-
amino-N-(benzyloxy)-5-nitrobenzamide
A mixture of 2-amino-4-nitrobenzoic acid or 2-amino-5-nitrobenzoic acid (0.50 g, 2.7 mmol)
with N,N-carbonyldiimidazole (0.55 g, 3.4 mmol, 1.25 eq), O-benzylhydroxylamine (470 μL,
4.1 mmol, 1.5 eq) in tetrahydrofuran was heated at 60 °C under agitation for 5 h. The reaction
mixture was concentrated in vacuo and the residue was purified by silica gel chromatography
using ethyl acetate and petroleum ether (eluent: petroleum ether: ethyl acetate = 12:1) to provide 2
as a yellow solid.
2-Amino-N-(benzyloxy)-4-nitrobenzamide: Yellow solid, 66% yield, mp:163-165 ºC.
2-Amino-N-(benzyloxy)-5-nitrobenzamide: Yellow solid, 78% yield, mp:150-152 ºC.
4.1.2. General procedure for the synthesis of 3-(benzyloxy)-6-nitroquinazoline-2,4(1H,3H)-
dione or 3-(benzyloxy)-7-nitroquinazoline-2,4(1H,3H)-dione (3)
Triethylamine (603 μL, 4.35 mmol) was added dropwise to a solution of 2-amino-N-
(benzyloxy)-4-nitrobenzamide or 2-amino-N-(benzyloxy)-5-nitrobenzamide (0.50 g, 1.74 mmol)
and triphosgene (206 mg, 0.70 mmol) in anhydrous tetrahydrofuran (20 mL). The mixture was
12

stirred at room temperature for 1−4 h, and then it was diluted with water (about 50 mL) to give a
solid which was collected, washed with water, and recrystallized.
3-(Benzyloxy)-7-nitroquinazoline-2,4(1H,3H)-dione. Light yellow solid, 50% yield, mp:
271-272 ℃. 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.59 – 7.55
(m, 2H), 7.46 – 7.40 (m, 4H), 7.38 (d, J = 1.8 Hz, 1H), 5.10 (s, 2H).
3-(Benzyloxy)-6-nitroquinazoline-2,4(1H,3H)-dione. Light yellow solid, 69% yield, mp:
269-271 ℃.1H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 8.67 (d, J = 2.6 Hz, 1H), 8.50 (dd, J
= 8.8, 2.7 Hz, 1H), 7.61 – 7.54 (m, 2H), 7.47 – 7.40 (m, 3H), 7.37 (d, J = 9.0 Hz, 1H), 5.13 (s, 2H).
4.1.3. General procedure for the synthesis of 6-amino-3-(benzyloxy)quinazoline- 2,4(1H,3H)-
dione or 7-amino-3-(benzyloxy)quinazoline-2,4(1H,3H)-dione (4)
A suspension of the nitro compound (1 mmol) and tin chloride dihydrate (4.8 eq) in ethanol
(10 mL) was stirred at 80 ºC for 1.5 h. Celite and solid NaHCO3 (10 equiv) were added to the
cooled solution followed by the dropwise addition of water until all acid was neutralized. The
suspension was filtered, diluted with EtOAc, washed with brine, dried, filtered, evaporated, and
flash chromatographed (EtOAc–petroleum ether) to provide the aniline.
1
7-Amino-3-(benzyloxy)quinazoline-2,4(1H,3H)-dione. Light yellow solid, 74% yield, H
NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.56 (dd, J = 7.7, 1.8 Hz,
2H), 7.45 – 7.36 (m, 3H), 6.42 (dd, J = 8.7, 2.0 Hz, 1H), 6.30 (s, 2H), 6.20 (d, J = 2.0 Hz, 1H),
5.03 (s, 2H).
1
6-Amino-3-(benzyloxy)quinazoline-2,4(1H,3H)-dione. Light yellow solid, 37% yield, H
NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 7.61 – 7.50 (m, 2H), 7.45 – 7.32 (m, 3H), 7.12 (d, J
= 2.2 Hz, 1H), 6.98 – 6.87 (m, 2H), 5.28 (s, 2H), 5.07 (s, 2H).
4.1.4. General procedure for the synthesis of 5
To a solution of 7a (1 mmol) in pyridine (8 mL) were added corresponding substituted benzyl
chloride or benzyl sulfonyl chloride (1.5 mmol). After the mixture was stirred for 3 h (monitored
by TLC), it was acidified to pH 7 with 1N hydrochloric acid solution. The resulting precipitate
was filtered and recrystallized.
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)benzamide (5A-1). White
solid, 49% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 10.68 (s, 1H), 7.96 (dd, J = 16.1,
13

8.4 Hz, 4H), 7.63 (t, J = 7.3 Hz, 1H), 7.57 (dd, J = 8.2, 4.7 Hz, 5H), 7.46 – 7.38 (m, 3H), 5.10 (s,
2H). ESI-MS: m/z 386.17 [M-H]-. C22H17N3O4 (387.40)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-4-fluorobenzamide (5A-2).
White solid, 53% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 10.69 (s, 1H), 8.07 (dd, J
= 8.6, 5.6 Hz, 2H), 7.97 – 7.90 (m, 2H), 7.62 – 7.53 (m, 3H), 7.47 – 7.38 (m, 5H), 5.10 (s, 2H).
C22H16FN3O4 (405.39)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-4-methylbenzamide (5A-3).
1
White solid, 69% yield; H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 10.59 (s, 1H), 7.98 –
7.85 (m, 4H), 7.58 (td, J = 9.5, 8.8, 5.1 Hz, 3H), 7.46 – 7.35 (m, 5H), 5.10 (s, 2H), 2.41 (s, 3H).
ESI-MS: m/z 400.17 [M-H]-. C22H19N3O4 (401.42)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-4-
1
(trifluoromethyl)benzamide (5A-4). White solid, 51% yield; H NMR (400 MHz, DMSO-d6) δ
11.74 (s, 1H), 10.88 (s, 1H), 8.31 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.04 – 7.92 (m, 3H), 7.82 (t, J
= 7.8 Hz, 1H), 7.58 (ddd, J = 8.7, 4.5, 2.0 Hz, 3H), 7.46 – 7.39 (m, 3H), 5.11 (s, 2H). ESI-MS:
m/z 454.20 [M-H]-. C23H16F3N3O4 (455.39)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-3-fluorobenzamide (5A-5).
White solid, 44% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H), 10.73 (s, 1H), 7.95 (d, J
= 8.8 Hz, 2H), 7.82 (t, J = 10.7 Hz, 2H), 7.60 (ddt, J = 14.1, 8.3, 4.8 Hz, 4H), 7.45 (qd, J = 15.2,
13.3, 8.2 Hz, 4H), 5.11 (s, 2H). ESI-MS: m/z 404.13 [M-H]-. C22H16FN3O4 (405.39)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-3-
(trifluoromethyl)benzenesulfonamide (5A-6). White solid, 61% yield; 1H NMR (400 MHz,
DMSO-d6) δ 11.60 (s, 1H), 11.25 (s, 1H), 8.15 (d, J = 6.8 Hz, 2H), 8.09 (d, J = 7.8 Hz, 1H), 7.86
(dd, J = 19.3, 8.4 Hz, 2H), 7.54 (dd, J = 7.3, 1.8 Hz, 2H), 7.45 – 7.36 (m, 3H), 7.08 (d, J = 1.8 Hz,
1H), 6.95 (dd, J = 8.7, 1.9 Hz, 1H), 5.04 (s, 2H). ESI-MS: m/z 490.09 [M-H]-. C22H16F3N3O5S
(491.44)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)naphthalene-2-sulfonamide
1
(5A-7). White solid, 77% yield; H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 11.21 (s, 1H),
8.60 (d, J = 1.9 Hz, 1H), 8.15 (dd, J = 14.7, 8.0 Hz, 2H), 8.06 – 7.99 (m, 1H), 7.83 (dd, J = 8.7,
1.9 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.71 (tt, J = 7.0, 5.4 Hz, 2H), 7.55 – 7.48 (m, 2H), 7.43 –
14

7.34 (m, 3H), 7.10 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.7, 2.1 Hz, 1H), 4.99 (s, 2H). C22H19N3O5S
(473.50)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-2,4,6-
1
trimethylbenzenesulfonamide (5A-8). White solid, 51% yield; H NMR (400 MHz, DMSO-d6) δ
11.66 (s, 1H), 11.00 (s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.58 – 7.48 (m, 2H), 7.43 – 7.34 (m, 3H),
7.05 (s, 2H), 6.84 (dd, J = 8.7, 2.0 Hz, 1H), 6.79 (d, J = 1.9 Hz, 1H), 5.02 (s, 2H), 2.63 (s, 6H),
2.23 (s, 3H). ESI-MS: m/z 464.15 [M-H]-. C24H23N3O5S (465.52)
4-(Tert-butyl)-N-(3-(benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-
yl)benzenesulfonamide (5A-9). White solid, 49% yield; ESI-MS: m/z 478.11[M-H]-.
C25H25N3O5S (479.55)
4-Fluoro-N-(3-(benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)benzenesulfonamide
(5A-10). White solid, 33% yield; ESI-MS: m/z 440.11[M-H]-. C21H16FN3O5S (441.43)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-[1,1'-biphenyl]-4-
1
sulfonamide (5A-11). White solid, 81% yield; H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H),
11.15 (s, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.7 Hz, 1H), 7.74 –
7.68 (m, 2H), 7.56 – 7.46 (m, 4H), 7.46 – 7.37 (m, 4H), 7.12 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.7,
2.0 Hz, 1H), 5.03 (s, 2H). ESI-MS: m/z 498.15 [M-H]-. C2 H21N3O5S (499.54)
7
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-3-methylbenzenesulfonamide
1
(5A-12). White solid, 76% yield; H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 11.06 (s, 1H),
7.81 (d, J = 8.7 Hz, 1H), 7.72 (s, 1H), 7.67 (dt, J = 5.9, 2.8 Hz, 1H), 7.57 – 7.52 (m, 2H), 7.50 –
7.45 (m, 2H), 7.43 – 7.36 (m, 3H), 7.06 (d, J = 2.0 Hz, 1H), 6.93 (dd, J = 8.7, 2.1 Hz, 1H), 5.03 (s,
2H), 2.38 (s, 3H). ESI-MS: m/z 436.13 [M-H]-. C22H19N3O5S (437.47)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)-2-fluorobenzamide (5B-1).
White solid, 55% yield; ESI-MS: m/z 404.14 [M-H]-. C22H16FN3O4 (405.39)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)benzenesulfonamide (5B-2).
White solid, 63% yield; ESI-MS: m/z 422.08 [M-H]-. C21H17N3O5S (423.44)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)-4-fluorobenzenesulfonamide
(5B-3). White solid, 59% yield; ESI-MS: m/z 440.12 [M-H]-. C21H16FN3O5S (441.43)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)naphthalene-2-sulfonamide
1
(5B-4). White solid, 44% yield; H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 10.55 (s, 1H),
15

8.41 (s, 1H), 8.12 (t, J = 8.2 Hz, 2H), 8.01 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.7, 1.7 Hz, 1H), 7.75
– 7.61 (m, 3H), 7.52 (dd, J = 7.2, 2.0 Hz, 2H), 7.48 (dd, J = 8.8, 2.5 Hz, 1H), 7.39 (dq, J = 7.0, 2.9,
2.2 Hz, 3H), 7.08 (d, J = 8.8 Hz, 1H), 5.02 (s, 2H). C25H19FN3O5S (473.50)
N-(3-(Benzyloxy)-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)-3-
(trifluoromethyl)benzenesulfonamide (5B-5). White solid, 62% yield; 1H NMR (400 MHz,
DMSO-d6) δ 8.05 (d, J = 7.9 Hz, 1H), 8.02 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.8 Hz,
1H), 7.60 (d, J = 2.5 Hz, 1H), 7.57 – 7.52 (m, 2H), 7.46 – 7.37 (m, 4H), 7.13 (d, J = 8.8 Hz, 1H),
5.05 (s, 2H). C22H16F3N3O5S (491.44)
4.1.5. General procedure for the synthesis of 6
Intermediate 5 was dissolved in MeOH (5 mL) and CH2Cl2 (5 mL) and the solution was
degassed and stirred at room temperature under H2 over 10% Pd/C (10% w/w, 2 h-12h). The
mixture was filtered. The filtrate was concentrated and the resulting residue was recrystallized in
MeOH and CH2Cl2.
N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)benzamide (I-1). Light yellow
solid, 34% yield, mp: 155-157 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H), 10.67 (s, 1H),
10.51 (s, 1H), 8.01 – 7.94 (m, 3H), 7.91 (d, J = 8.7 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.59 – 7.51
(m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 166.68 , 159.47, 149.46, 145.18, 139.70, 134.91,
132.46, 128.91, 128.35, 128.28, 115.41, 109.96, 105.35. ESI-MS: m/z 296.20 [M-H]-. C15H11N3O4
(297.27)
4-Fluoro-N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)benzamide (I-2). White
1
solid, 19% yield, mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.67 (s, 1H),
8.14 – 8.01 (m, 2H), 7.97 – 7.79 (m, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.39 (t, J = 8.6 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6) δ 165.54, 164.77 (J = 247 Hz), 159.48, 149.46, 145.11, 139.68,
131.31, 131.23 (2×C, J = 9 Hz), 128.25, 115.87 (2×C, J = 22 Hz), 115.46, 109.99, 105.42. ESI-
MS: m/z 314.18 [M-H]-. C15H10FN3O4 (315.26)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-4-methylbenzamide (I-3). White
solid, 55% yield, mp: 227-229 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 10.55 (s, 1H),
10.49 (s, 1H), 7.97 – 7.86 (m, 4H), 7.54 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H), 2.40 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 166.44, 159.48, 149.48, 145.25, 142.60, 139.71, 131.99,
16

129.43, 128.39, 128.24, 115.39, 109.86, 105.31, 21.52. ESI-MS: m/z 310.27 [M-H]-; m/z 334.12
[M+Na+]+. C16H13N3O4 (311.29)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-4-(trifluoromethyl)benzamide (I-
4). White solid, 25% yield, mp: 275-277 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.35
– 8.21 (m, 2H), 8.01 (d, J = 7.8 Hz, 1H), 7.93 (m, 2H), 7.81 (t, J = 7.8 Hz, 1H), 7.54 (dd, J = 8.7,
1.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 165.21, 159.45, 149.46, 144.79, 139.69, 135.80,
129.65 (2×C, J = 227 Hz), 129.24 (J = 227 Hz), 128.99 (J = 4 Hz), 124.93 (2×C, J = 4 Hz),
123.04, 115.48, 110.29, 105.59. ESI-MS: m/z 364.30 [M-H]-. C16H10F3N3O4 (365.26)
3-Fluoro-N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)benzamide (I-5). Gray
1
solid, 28% yield, mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 10.70 (s, 1H),
10.52 (s, 1H), 7.92 (d, J = 7.4 Hz, 2H), 7.82 (t, J = 10.8 Hz, 2H), 7.62 (dd, J = 13.9, 7.8 Hz, 1H),
7.57 – 7.46 (m, 2H). ESI-MS: m/z 314.27 [M-H]-; m/z 316.17 [M+H]+. C15H10FN3O4 (315.26)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-3-
1
(trifluoromethyl)benzenesulfonamide (I-6). White solid, 33% yield, mp: 198-200 °C; H NMR
(400 MHz, DMSO-d6) δ 11.46 (s, 1H), 11.19 (s, 1H), 10.46 (s, 1H), 8.13 (d, J = 5.9 Hz, 2H), 8.06
(d, J = 7.9 Hz, 1H), 7.85 (t, J = 8.1 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H),
6.90 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ159.29 , 149.22 , 140.97 ,
139.91 , 131.66 , 131.22 , 130.58 , 130.26 , 129.20 , 123.71 , 122.37 , 114.13 , 110.01 ,
103.70 .ESI-MS: m/z 400.20 [M-H]-. C15H10F3N3O5S (401.32)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)naphthalene-2-sulfonamide (I-7).
White solid, 36% yield, mp: 245-247 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1H), 11.16 (s,
1H), 10.41 (s, 1H), 8.57 (d, J = 1.9 Hz, 1H), 8.20 – 8.07 (m, 2H), 8.06 – 7.99 (m, 1H), 7.82 (dd, J
= 8.7, 1.9 Hz, 1H), 7.77 – 7.64 (m, 3H), 7.05 (d, J = 2.0 Hz, 1H), 6.93 (dd, J = 8.7, 2.0 Hz, 1H).
13C NMR (100 MHz, DMSO-d6) δ 159.31 , 149.23 , 139.85 , 136.87 , 134.82 , 131.95 , 130.14 ,
129.75 , 129.61 , 128.95 , 128.73 , 128.34 , 128.28 , 122.40 , 113.86 , 109.39 , 103.12, 99.99 . ESI-
MS: m/z 382.20 [M-H]-. C18H13N3O5S (383.38)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-2,4,6-
trimethylbenzenesulfonamide (I-8). White solid, 29% yield, mp: 278-280 °C; 1H NMR (400 MHz,
DMSO-d6) δ 11.54 (s, 1H), 10.97 (s, 1H), 10.44 (s, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.03 (s, 2H),
6.81 (dd, J = 8.7, 2.1 Hz, 1H), 6.77 (d, J = 2.0 Hz, 1H), 2.62 (s, 6H), 2.23 (s, 3H); 13C NMR (100
17

MHz, DMSO-d6) δ 159.32, 149.33, 144.47, 142.87, 139.89, 139.28 (2×C), 133.94, 132.37 (2×
C), 129.10, 112.98, 109.14, 101.83, 22.90 (2× C), 20.86. ESI-MS: m/z 374.29 [M-H]-.
C17H17N3O5S (375.40)
4-(Tert-butyl)-N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-
1
yl)benzenesulfonamide (I-9). White solid, 46% yield, mp: 153-155 °C; H NMR (400 MHz,
DMSO-d6) δ 11.45 (s, 1H), 11.02 (s, 1H), 10.46 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8.8
Hz, 1H), 7.61 (d, J = 8.5 Hz, 2H), 7.08 – 7.05 (d, J = 1.7 Hz, 1H), 6.91 (dd, J = 8.7, 1.7 Hz, 1H),
1.26 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 159.34, 156.72, 149.25, 144.53, 139.92, 137.14,
129.06, 127.11 (2×C), 126.77 (2×C), 113.54, 109.45, 102.85, 35.38 (3×C), 31.16. ESI-MS:
m/z 388.30 [M-H]-. C18H19N3O5S (389.43)
4-Fluoro-N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)benzenesulfonamide (I-
1
10). White solid, 29% yield, mp: 267-269 °C; H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H),
11.06 (s, 1H), 10.48 (s, 1H), 7.98 – 7.88 (m, 2H), 7.80 (d, J = 8.7 Hz, 1H), 7.46 (t, J = 8.8 Hz, 2H),
7.04 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.6, 2.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 166.28,
163.77, 159.31, 149.21, 143.79, 139.89, 135.90 (J = 3 Hz), 130.37 (2×C, J = 10 Hz), 129.16,
117.22 (2× C, J = 22 Hz), 111.85 (J = 378 Hz), 103.38. ESI-MS: m/z 350.21 [M-H]-.
C14H10FN3O5S (351.31)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-[1,1'-biphenyl]-4-sulfonamide
1
(I-11). White solid, 31% yield, mp > 300 ºC; H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H),
11.23 – 10.98 (m, 1H), 10.45 (s, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.78 (d, J
= 8.7 Hz, 1H), 7.71 (d, J = 7.3 Hz, 2H), 7.49 (dd, J = 8.2, 6.6 Hz, 2H), 7.43 (t, J = 7.2 Hz, 1H),
7.08 (d, J = 1.9 Hz, 1H), 6.93 (dd, J = 8.8, 2.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 159.35,
149.25, 145.13, 144.47, 139.93, 138.66, 138.60, 129.58 (2×C), 129.12, 129.09, 128.06 (2×C),
127.92 (2× C), 127.57 (2× C), 113.75, 109.59, 103.14. ESI-MS: m/z 408.20 [M-H]-.
C20H15N3O5S (409.42)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-7-yl)-3-methylbenzenesulfonamide (I-
12). White solid, 41% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H), 11.00 (s, 1H), 10.45
(s, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.70 (s, 1H), 7.67 – 7.61 (m, 1H), 7.53 – 7.42 (m, 2H), 7.02 (d, J
= 1.7 Hz, 1H), 6.89 (dd, J = 8.7, 1.8 Hz, 1H), 2.37 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
18

159.33, 149.25, 144.20, 139.87, 139.67, 139.61, 134.45, 129.77, 129.06, 127.47, 124.42, 113.61,
109.61, 102.99, 21.31. ESI-MS: m/z 346.19 [M-H]-. C15H13N3O5S (347.35)
2-Fluoro-N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)benzamide (II-1). White
solid, 18% yield, mp: 291-293 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 10.61 (s, 2H),
8.43 (d, J = 2.2 Hz, 1H), 7.92 (dd, J = 8.8, 2.3 Hz, 1H), 7.69 (t, J = 7.4 Hz, 1H), 7.60 (q, J = 6.0
Hz, 1H), 7.36 (q, J = 9.3, 7.5 Hz, 2H), 7.20 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ
163.20, 159.70, 159.35 (J = 248 Hz), 158.11, 149.02, 135.04, 134.27, 133.14 (J = 8 Hz), 130.41,
130.38, 127.44, 125.09 (J = 2 Hz), 125.05, 117.53, 116.67 (J = 22 Hz), 116.31, 114.67. ESI-MS:
m/z 314.17 [M-H]-. C15H10FN3O4 (315.26)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)benzenesulfonamide
(II-2).
White solid, 26% yield, mp: 212-214 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 10.56 (s,
1H), 10.38 (s, 1H), 7.71 (d, J = 7.4 Hz, 2H), 7.63 – 7.52 (m, 4H), 7.41 (dd, J = 8.8, 2.4 Hz, 1H),
7.09 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 159.36, 148.87, 139.50, 135.70,
133.49, 132.80, 129.78 (2×C), 128.87, 127.10 (2×C), 118.88, 116.94, 114.96. ESI-MS: m/z
332.08 [M-H]-. C14H11N3O5S (333.32)
4-Fluoro-N-(3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)benzenesulfonamide (II-
1
3). White solid, 28% yield, mp: 141-144°C; H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H),
10.57 (s, 2H), 7.77 (dd, J = 8.6, 5.1 Hz, 2H), 7.58 (d, J = 2.4 Hz, 1H), 7.45 – 7.34 (m, 3H), 7.11 (d,
J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 166.02, 163.51，159.36, 148.88, 135.92 (J =
3 Hz), 135.81, 132.73, 130.20 (2×C, J = 9 Hz), 129.13, 119.18, 116.99 (2×C, J = 23 Hz), 116.00
(J = 199 Hz). ESI-MS: m/z 350.13 [M-H]-. C14H10FN3O5S (351.31)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)naphthalene-2-sulfonamide (II-4).
White solid, 24% yield, mp: 194-195 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 10.51 (s,
2H), 8.38 (d, J = 1.8 Hz, 1H), 8.09 (dd, J = 11.0, 8.4 Hz, 2H), 7.99 (d, J = 8.0 Hz, 1H), 7.75 (dd, J
= 8.7, 1.9 Hz, 1H), 7.67 (dt, J = 11.9, 7.3 Hz, 2H), 7.61 (d, J = 2.4 Hz, 1H), 7.41 (dd, J = 8.7, 2.6
Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 159.36, 148.85, 136.92,
135.46, 134.66, 133.29, 131.99, 129.90, 129.67, 129.41, 128.82, 128.33, 128.28, 128.15, 122.51,
118.75, 116.88, 114.96. ESI-MS: m/z 382.13 [M-H]-. C18H13N3O5S (383.38)
N-(3-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)-3-
1
(trifluoromethyl)benzenesulfonamide (II-5). White solid, 33% yield, mp: 207-209 °C; H NMR
19

(400 MHz, DMSO-d6) δ 11.53 (s, 1H), 10.57 (s, 1H), 10.52 (s, 1H), 8.09 – 7.93 (m, 3H), 7.80 (t, J
= 7.8 Hz, 1H), 7.56 (d, J = 2.5 Hz, 1H), 7.41 (dd, J = 8.7, 2.5 Hz, 1H), 7.12 (d, J = 8.8 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ 159.28, 148.87, 140.60, 136.16, 132.15, 131.51, 131.19,
130.47, 130.28, 130.24, 130.14, 129.46, 123.64 (J = 4 Hz), 121.06 (J = 266 Hz), 115.03. ESI-MS:
m/z 400.14 [M-H]-. C15H10F3N3O5S (401.32)
4.2. In vitro anti-HIV assay
By using the MTT method described previously [24, 25], the synthesized compounds were
evaluated for their activity against WT HIV-1 [26] (strain IIIB), and HIV-2 [27] (strain ROD) in
MT-4 cells. First, stock solutions (10× final concentration) of test compounds were added at 25
μL volumes to two series of triplicate wells to allow simultaneous evaluation of their effects on
mock and HIV-infected cells. Serial five-fold dilutions of the test compounds were made directly
in flat-bottomed 96-well microtiter trays, including untreated control HIV-1 and mock-infected
cells for each sample, using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). HIV-1
(IIIB), or HIV-2 (ROD) stocks (50 mL at 100-300 50% cell culture infectious doses (CCID50)) or
culture medium were added to either the mock or HIV-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test compounds on uninfected cells in
order to assess their cytotoxicity. Exponentially growing MT-4 cells were centrifuged for 5 min at
1000 rpm and the supernatant was discarded. MT-4 cells were then resuspended at 6×105 cells/mL,
and 50 μL aliquots were transferred to the microtiter tray wells. Five days after infection the
viability of mock-and HIV-infected cells was determined spectrophotometrically.
The MTT assay was based on the reduction of yellow colored 3(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) (Acros Organics, Geel, Belgium) by mitochondrial
dehydrogenase of metabolically active cells to a blue-purple formazan that can be measured
spectrophotometrically. The absorbances were read in an eight-channel computer-controlled
photometer (Multiscan Ascent Reader, Labsystems, Helsinki, Finland) at the wavelengths of 540
and 690 nm. All data were calculated using the median OD (optical density) value of two or three
wells.
4.3. HIV-1 RNase H inhibition assay
Wild-type (WT) HIV-1 BH10 RT was expressed in E. coli and purified as previously
described [28, 29]. The RT RNase H activity was determined with an RNA/DNA hybrid
20

containing
32P-labeled
template
RNA
(31Trna,
5´-
UUUUUUUUUAGGAUACAUAUGGUUAAAGUAU-3´) and a DNA oligonucleotide 21P (5´-
ATACTTTAACCATATGTATCC-3´) [20]. RNase H cleavage assays were carried out at 37ºC
with 20-40 nM HIV-1 RT in 30 µl of 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 25
nM 32P-labeled template-primer (31Trna/21P), and 5% dimethyl sulfoxide (DMSO). IC50 values
were determined from dose-response curves after calculating the amount of cleaved substrate in
the absence of inhibitor (5% DMSO) or in the presence of the tested compounds at concentrations
in the range of 0.2 to 100 µM [21].
4.4. HIV-1 IN strand transfer inhibition assays
WT HIV-1 IN was expressed in E. coli and purified as previously described [30], and the
strand transfer inhibitory activity of tested compounds was determined as previously described [21,
31] using DNA complexes containing 32P-labeled INT1ST (5’-ATGTGGAAAATCTCTAGCA-3´)
and non-labeled INT2 (5’-ACTGCTAGAGATTTTCCACAT-3´). The percentage inhibitory
activity of tested compounds was determined at 1% DMSO in the absence of inhibitor and in the
presence of raltegravir (MedChemExpress) or selected compounds at 1, 10 and 50 µM
concentrations. For the determination of IC50 values of raltegravir and compound II-4, strand
transfer inhibitions were determined at additional concentrations in the range of 0.01 to 10 µM (i.e.
0.01, 0.05, 0.5 and 5 µM).
4.5. Cell permeability assays
Drug permeability was determined with the human colon epithelial cancer cell line, Caco-2,
as previously described [21]. Caco-2 cell monolayers were used for measuring the rate of
membrane transport for compound II-4, and expressed as apparent (Papp) permeability. Nadolol
and metoprolol were used as controls in the assays.
The apparent permeability coefficient Papp (cm/s) was calculated using the equation: Papp
=
(dCr/dt) x Vr / (A x C0), where dCr/dt is the cumulative concentration of compound in the receiver
chamber as a function of time (µM/s); Vr is the solution volume in the receiver chamber (0.075
mL on the apical side, 0.25 mL on the basolateral side); A is the surface area for the transport, i.e.
0.0804 cm2 for the area of the monolayer; C0 is the initial concentration in the donor chamber
(µM). Analysis was carried out using Shimadzu LC 20-AD (Shimadzu, Japan), API 4000™
System (SCIEX, USA) and Apricot Designs Dual Arm (ADDA) (Apricot Designs, USA).
21

4.6. Computational methods and materials
Molecular modeling of molecule II-4 was performed using the Tripos molecular modeling
packages Sybyl-X 2.0. II-4 was optimized for 2000-generations till the maximum derivative of
energy became 0.005 kcal/(mol*A) using the Tripos force field. The published three dimensional
crystal structure of the PFV IN complex was obtained from the Protein Data Bank (PDB code:
3OYA). The published three dimensional crystal structure of the RNase H complex was
downloaded from the Protein Data Bank (PDB code: 5J1E). The protein structure was prepared by
using the Biopolymer application accompanying Sybyl. The ligand was extracted from the
complexes, hydrogen atoms were added, side chain amides and side chains bumps were fixed, and
charges and atom types were assigned according to AMBER 99. After the protomol was generated,
the optimized II-4 was docked into the binding pocket using the Surflex-Dock workflow of the
SYBYL-X 2.0 software.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
Financial support from the National Natural Science Foundation of China (NSFC No.
81273354), the Key Project of NSFC for International Cooperation (No. 81420108027), the Key
Research and Development Project of Shandong Province (No. 2017CXGC1401), the Young
Scholars Program of Shandong University (YSPSDU No. 2016WLJH32, to P. Z.), the Major
Project of Science and Technology of Shandong Province (No. 2015ZDJS04001) is gratefully
acknowledged. Work in Madrid was supported by grant BIO2016-76716-R (AEI/FEDER, UE)
(Spanish Ministry of Economy, Industry and Competitiveness) and an institutional grant of
Fundación Ramón Areces. The technical assistance of Mr. Kris Uyttersprot, and Mrs. Kristien
Erven, for the HIV experiments is gratefully acknowledged.
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Design, synthesis and biological evaluation of 3-hydroxyquinazoline-
2,4(1H,3H)-diones as dual inhibitors of
24

HIV-1 reverse transcriptase-associated RNase H and integrase
Ping Gaoa, Xiqiang Chenga, Lin Suna, Shu Songa, Mar Álvarezb, Joanna Luczkowiakb,
Christophe Pannecouquec, Erik De Clercqc, Luis Menéndez-Ariasb,*, Peng Zhana,*, Xinyong
Liua,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology, Ministry of
Education, School of Pharmaceutical Sciences, Shandong University, Ji’nan, 250012
bCentro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones
Científicas & Universidad Autónoma de Madrid), Madrid, Spain
cRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000
Leuven, Belgium
*E-mail: lmenendez@cbm.csic.es (Menéndez-Arias L.); zhanpeng1982@sdu.edu.cn
(Zhan P.); xinyongl@sdu.edu.cn (Liu X.Y.).
Graphical Abstract
25