1-Hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones as novel selective HIV integrase inhibitors obtained via privileged substructure-based compound libraries

Article abstract of DOI:10.1016/j.bmc.2017.09.006

A small library containing 3-hydroxyquinazoline-2,4(1H,3H)-dione and 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one scaffolds was obtained via the copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction and evaluated for their anti-HIV activity in MT-4

Full text of DOI:10.1016/j.bmc.2017.09.006

Accepted Manuscript
1-Hydr oxypyr ido[2,3-d]pyrimidin-2(1H)-ones as Novel Selective HIV In
tegraseInhibitorsObtained via Privileged Substructure-Based Compound
Libraries
Ping Gao, Lingzi Zhang, Lin Sun, Tianguang Huang, Jing Tan, Jian Zhang,
Zhongxia Zhou, Tong Zhao, Luis Menéndez-Arias, Christophe Pannecouque,
Erik De Clercq, Peng Zhan, Xinyong Liu
PII:
S0968-0896(17)31466-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.006
BMC 13966
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 July 2017
3 September 2017
6 September 2017
Please cite this article as: Gao, P., Zhang, L., Sun, L., Huang, T., Tan, J., Zhang, J., Zhou, Z., Zhao, T., Menéndez-
Arias, L., Pannecouque, C., Clercq, E.D., Zhan, P., Liu, X., 1-Hydr oxypyr ido[2,3-d]pyr imidin-2(1H)-ones as
Novel Selective HIV Integrase Inhibitors Obtained via Privileged Substructure-Based Compound Libraries,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.006
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1-Hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones as Novel Selective HIV
Integrase Inhibitors Obtained via Privileged Substructure-Based
Compound Libraries
Ping Gaoa, Lingzi Zhanga, Lin Suna, Tianguang Huanga, Jing Tana, Jian Zhanga, Zhongxia Zhoua,
Tong Zhaoa, Luis Menéndez-Ariasc, Christophe Pannecouqueb, Erik De Clercqb, Peng Zhana,*,
Xinyong Liua,*
aDepartment of Medicinal Chemistry, Key laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road,
250012, Jinan, Shandong, P. R. China
bRega Institute for Medical Research, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
cCentro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas
& Universidad Autónoma de Madrid), Madrid, Spain
ABSTRACT:
A small library containing 3-hydroxyquinazoline-2,4(1H,3H)-dione and
1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one scaffolds was obtained via the copper(I)-catalyzed
azidealkyne cycloaddition (CuAAC) reaction and evaluated for their anti-HIV activity in MT-4
cells. Among the synthesized compounds, several 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one
derivatives showed remarkable anti-HIV potency with EC50 values ranging from 0.92 to 26.85 µM.
The most active one, IIA-2, also showed remarkable and selective potency against HIV type 1
integrase (IN). To the best of our knowledge, this is the first report showing that
1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones are selective HIV IN inhibitors. Preliminary
structure-activity relationship (SAR) studies suggested that the divalent metal ion chelators and
the nature and position of substituents around the core are important for antiviral potency.
Molecular modeling has been used to predict the binding site of the
pyrido[2,3-d]pyrimidin-2(1H)-one core in HIV type 1 IN and suggestions are made for
improvement of its inhibitory activity.

Keywords: HIV-1, CuAAC, HIV-1 integrase, HIV-1 RNase H, “privileged structure”-guided
scaffold reposition.
1. Introduction
Acquired immunodeficiency syndrome (AIDS), mainly caused by the infection of human
immunodeficiency virus type 1 (HIV-1), remains as one of the most serious public health
problems throughout the world.1 Up to now, approximately 30 drugs have been approved by the
U.S. Food and Drug Administration (FDA) for the treatment of HIV/AIDS, which target different
steps in the viral life cycle such as viral entry, reverse transcription, integration, and viral
maturation.2 Although the clinical use of highly active antiretroviral therapy (HAART) has
dramatically decreased the morbidity and mortality of HIV-1 infection, long-term successful
treatments face major threats such as the emergence of drug-resistant strains,3, 4 unwanted
drug-drug interactions, and long-term toxicity (i.e., lipodystrophy, dyslipidemia, high
cardiovascular risk, etc.).2 Therefore, there is an urgent need for new anti-HIV drug candidates
with increased potency, novel mechanisms of action, improved pharmacokinetic properties, and
reduced side effects.
Over the past few decades, the identification of novel HIV-1 inhibitors was facilitated by
traditional cellular phenotypic screening approaches (such as the MT4 cytoprotection assay), using
large collections compounds, which in comparison with target-based screening, offered some
advantages towards the identification of novel chemical structures with a unique mechanism of
action, and the discovery of first-in class drugs. The high-throughput screening (HTS) campaigns
using large diverse compound collections are time-consuming.5, 6 On the other hand, the privileged
substructure-based diversity-oriented synthesis (pDOS) strategy, has proven to be a fruitful tool to
rapidly discover biologically active lead compounds by exploring the uncharted chemical space
and constructing high-quality compound libraries.7, 8, 9
For the construction of pDOS libraries, it is crucial to select a privileged substructure with the
potential for scaffold refining.10 It is recognized that hydroxy-(iso)quinazoline-2,4(1,3)-dione and
its analogues are featured in a number of molecules displaying a broad spectrum of biological
activities. Among them, 2-hydroxyisoquinoline-1,3(2H,4H)-dione and

3-hydroxypyrimidine-2,4-dione derivatives are considered the core structures in many antiviral
agents. Especially, they show potency against a wide range of metal-dependent enzymes,
including dual inhibitors of HIV-1 ribonuclease H (RNase H, RNH) and integrase (IN) (e.g. 1, 2,11
3,12 4, 5,13), HIV-1 RNase H active-site inhibitors (compounds 6 14, 15, 16), the HIV-1 IN active-site
inhibitor 7,17 and the hepatitis C virus (HCV) NS5B polymerase inhibitor 8 18 (Figure 1),
suggesting that hydroxy-(iso)quinazoline-2,4(1,3)-diones represent a vast, untapped chemical
space for further modification.
Figure 1. Representative antiviral agents containing 2-hydroxyisoquinoline-1,3(2H,4H)-dione and
3-hydroxypyrimidine-2,4-dione, and further design of a privileged substructure-based library.
Very recently, our lab reported for the first time that novel
3-hydroxy-quinazoline-2,4(1H,3H)-diones are specific drugs acting against DNA viruses such as
vaccinia and adenovirus.19 In particular, 24b11 displayed the most potent inhibitory activity
against vaccinia virus with an EC50 value of 1.7 μM, 15 times more potent than the reference drug
cidofovir (EC50 = 25 μM). On the other hand, 24b13 was the most potent compound against

adenovirus-2 with an EC50 value of 6.2 μM, lower than the EC50s obtained with all the reference
drugs tested in our assays. These findings warranted further investigations (library expansion and
compound refinement) on this novel class of antiviral agents.
Continuing with this research, we have investigated the antiretroviral potential of the
previously unexplored chemical space surrounding the 3-hydroxy-quinazoline-2,4(1H,3H)-dione
and its isosteres. Therefore, we constructed a small library of
3-hydroxyquinazoline-2,4(1H,3H)-dione and 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one
scaffolds via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction20 and the
biological significance of the novel compounds synthesized was determined after its evaluation in
cell culture-based high-throughput screening (HTS) assays against HIV.
2. Results and discussion
2.1. Chemistry
The library of 3-hydroxyquinolin-2(1H)-one compounds was constructed by following the
synthetic route outlined in Scheme 1. The commercially available material
2-amino-5-bromobenzoic acid (G-0) was treated with methanol in the presence of concentrated
H2SO4 to give the intermediate methyl 2-amino-5-bromobenzoate (G-1) via an esterification
reaction. The intermediate 3-(benzyloxy)-6-bromoquinazoline-2,4(1H,3H)-dione (G-2) was
obtained by ring closure of G-1 with carbonyldiimidazole (CDI) and O-benzylhydroxylamine in a
sodium hydroxide solution.19 Then this heterocyclic scaffold was alkylated with
3-bromo-1-propyne in dimethylformamide (DMF) to afford N1-substituted product G-3. CuAAC
reaction of the alkyne key intermediate G-4 with different azido substituent groups generated the
corresponding key 1,2,3-triazole intermediates which were deprotected under basic conditions to
render the target compounds 1,2,3-triazole-substituted 3-hydroxy-quinazoline-2,4(1H,3H)-diones
(series I).
The library of 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one compounds was synthesized as
described in Scheme 2. The commercially available material methyl 2-fluoronicotinate (V-0) was
treated with carbonyldiimidazole (CDI) and O-benzylhydroxylamine, followed by trichloroacetyl
isocyanate to obtain the intermediate 1-(benzyloxy)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione

(V-2).21 Then this heterocyclic scaffold was treated with POCl3 and the corresponding aniline to
obtain the key intermediate V-4. The alkyne V-4 reacted with different azido substituent groups
via CuAAC reaction to generate the corresponding key 1,2,3-triazole intermediates which were
then deprotected under trifluoroacetic acid to render the target
1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones (series II).
Scheme 1. Synthesis of designed 3-hydroxyquinolin-2(1H)-one compounds. Reagents and conditions: i) CH3OH ,
H2SO4 , reflux 8 h, yield 90.8%; ii) O-benzylhydroxylamine, CDI, tetrahydrofuran (THF), reflux; NaOH, EtOH,
H2O, reflux, yield 73.7%; iii) Propargyl bromide, K2CO3, DMF, room temperature, yield 67.8%; iv) Substituted
azide, Sodium ascorbate, CuSO4·5H2O, THF, H2O, room temperature, yield 55.4%-89.2%; v) Acetic acid, HBr,
reflux, yield 9.8%-31.0%.
Scheme 2. Synthesis of 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one compounds. Reagents and conditions: i)
DIPEA, O-Benzylhydroxylamine, 140 °C, microwave, 120 min, yield: 34.3%; ii) Trichloroacetyl isocyanate, DCE,

TEA, MeONa, room temperature, yield: 21.1%; iii) DIPEA, POCl3, 100 °C; iv) DMF, corresponding aniline, room
temperature, yield over two steps, 17.4%-72.1%; v) Substituted azide, sodium ascorbate, CuSO4·5H2O, THF, H2O,
room temperature, yield 26.7%-100.0%; vi) Trifluoroacetic acid, 60 °C, overnight, yield 7.1%-100.0%.
2.2. Biological activity
2.2.1. Anti-HIV assay in MT-4 cells
The newly synthesized 3-hydroxyquinazoline-2,4(1H,3H)-dione and
1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one derivatives were evaluated for their anti-HIV activity
against wild-type (WT) HIV-1 (IIIB) and HIV-2 (ROD) strains. Elvitegravir (EVG), raltegravir
(RAL), and raltegravir-K (potassium salt), were used as reference drugs. The biological results are
expressed as EC50, CC50 and SI (selectivity index, given by the CC50/EC50 ratio).
As shown in the Table 1, tested 3-hydroxyquinazoline-2,4(1H,3H)-dione derivatives were
found to be inactive against HIV but showed remarkable cytotoxicity in MT-4 cells. In contrast,
most of meta-triazole-benzene substituted 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one
derivatives inhibited WT HIV-1 replication, with EC50 values in the range of 0.92 μM to 26.8 μM,
indicating that activity is tightly associated with the structure of the scaffold. Among them,
compound IIA-2 was the most promising one and exhibited extremely potent inhibitory activity
against HIV-1 strain IIIB with an EC50 of 0.92 μM. In addition, it showed inhibitory activity
against HIV-2 strain ROD with an EC50 value of 2.82 μM.
The preliminary SAR analysis based on the results of the antiviral assays showed that: i) the
compounds with ortho-substitution at the phenyl ring all exhibited higher cytotoxicity than those
with meta-substitutions; ii) one or two carbon-containing linkers between the triazole and the
terminal aromatic group may have a favorable effect in activity (exemplified by IIA-2, IIA-1 and
IIA-7), and the introduction of a carbanyl group has little influence in anti-HIV-1 potency
(exemplified by IIA-2 and IIA-7), but the structure modification had great influence versus HIV-2
activity; and iii) interestingly, the introduction of the NH group decreases their anti-HIV-1 activity,
while losing their anti-HIV-2 activity (e.g. IIA-5, IIA-6, and IIA-8).
2.2.2. HIV-1 IN inhibitory assay

In an initial effort to identify the target of these 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one
derivatives, some representative compounds were also tested for their ability to inhibit HIV-1 IN
and RNase H activities. The results are shown in Figure 1.
IIA-1 was tested in a HIV-1 IN inhibition assay at a concentration of 10 μg/ml (23.5 μM).
Under these conditions, it produced a 93.1% inhibition of the IN strand transfer activity, a value
similar to that obtained with the clinically approved drug EVG (97.8%). Based on this promising
result, 5 μg/mL was selected as lower final concentration to test the inhibitory efficiency of several
target compounds. As shown in Figure 1, all compounds exhibited >50% inhibitory activity,
except compound IIB-1. The compounds IIA-1, IIA-2 and IIA-7 were the most potent,
showing >80% inhibition of the strand transfer activity. These results were consistent with their
potent anti-HIV activity in cell culture. Compound IIA-2 was the most efficient compound,
showing 86.2% inhibition of the HIV-1 IN activity, close to the percentages of 90.6% and 94.1%
obtained with the reference drugs RAL and EVG, respectively.
A brief investigation of SARs in HIV-1 IN inhibitory assay revealed that i) the HIV-1 IN
inhibition efficiency of compounds with meta-substitutions at the phenyl ring was superior to
those of compounds with ortho-substitutions; ii) improved potency can be achieved by introducing
one or two carbon-containing linkers between the triazole moiety and the terminal aromatic group
(IIA-2, IIA-1 and IIA-7); iii) the introduction of a carbonyl group has little influence in IN
inhibition (compare IIA-7 with IIA-2); and iv) the introduction of an NH group produces a
significant decrease in the inhibitory efficiency of the derivatives (e.g. compare IIA-5, IIA-6 and
IIA-8 with IIA-7). The above findings were generally consistent with the SAR conclusions
regarding the anti-HIV potency of the tested compounds.
Table 1. Activity against HIV-1 (IIIB) and HIV-2 (ROD) strains, cytotoxicity and selectivity index
in MT-4 cells.

EC50 (µM)a
SIc
Compds
R
IIIB
ROD
CC50 (µM)b
IIIB
ROD
I-1
-CH2-(4-F)Ph
-CH2-(3-F)Ph
-CH2-(3,4-diF)Ph
-CH2-Ph
>25.1
>25.1
25.1±1.9
24.9±1.7
24.7±1.3
23.7±2.6
24.1±1.6
21.8±1.8
22.3±3.1
22.4±1.9
17.8±4.3
24.0±0.86
>293.8
<1
<1
I-2
>24.9
>24.9
<1
<1
I-3
>24.7
>24.7
<1
<1
I-4
>23.7
>23.7
<1
<1
I-5
-CH2-(4-Cl)Ph
-CH2-(4-Br)Ph
-CH2-(2-Cl)Ph
-CH2-(4-NO2)Ph
-CH2-(2-CH3)Ph
-CH2-(3-CH3)Ph
-(CH2)2-Ph
>24.1
>24.1
<1
<1
I-6
>21.8
>21.8
<1
<1
I-7
>22.3
>22.3
<1
<1
I-8
>22.4
>22.4
<1
<1
I-9
>17.8
>17.8
<1
<1
I-10
IIA-1
IIA-2
IIA-3
IIA-4
IIA-5
>24.
>24.0
<1
<1
9.5±2.1
0.92±0.02
26.1±8.2
27.1±9.7
26.8±0.3
14.6±6.6
2.82±0.02
>239.2
69.4±0.8
>275.1
>31
>318
9
>20
>103
<1
-CH2-(4-F)Ph
-(CH2)3-Ph
>291.1
239.2
-1-naphthalene
-CH2-CONH-Ph
>279.4
>10
>10
>4
>275.1
X1

IIA-6
-CH2-CONH-(4-F)Ph
-CH2-CO-(4-F)Ph
-CH2-CONH-(4-CF3)Ph
-CH2-(4-F)Ph
231.8±7.5
3.0±1.4
>264.6
>264.6
>273.3
>239.3
>1
X1
IIA-7
>273.3
>91
X1
X1
IIA-8
>239.3
>239.3
X1
IIB-1
>2.7
>2.7
2.7±1.3
<1
<1
IIB-2
-CH2-(4-CH3)Ph
>1.1
>1.1
1.1±0.024
6.3±2.5
>55.8
<1
<1
(2.3±0.1) x 10-3
(7.8±0.2) x 10-3
(7.7±0.5) x 10-3
(3.8±0.1) x 10-3
(11.0±0.8) x 10-3
(16.0±0.4) x 10-3
Elvitegravir
Raltegravir
Raltegravir-K
2722
1661
>5123
>3214
>7146
>6812
>51.8
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced
cytotoxicity, as determined by the MTT method.
bCC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the
MTT method.
cSI: selectivity index, the ratio of CC50/EC50.
X1: approximately equal to 1.
Figure 2. Inhibition of the HIV-1 IN strand transfer activity. Selected compounds were tested at a
concentration of 5 μg/mL. Values represent an average ± standard deviation obtained from three

independent experiments.
2.2.3 HIV-1 RNase H inhibitory assay
As shown in Figure 3, the lack of activity in the HIV-1 RNase H inhibition assays indicated
that the active compounds owed their anti-HIV activity, exclusively to their ability to inhibit the
viral IN. Although HIV IN inhibitors and HIV RNase H inhibitors share similar metal-chelating
capacities, the conformation of the metal-chelating backbone and the diversity of substituents in
candidate drugs may influence target recognition. According to the latest report by
GlaxoSmithKline in 2011, 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one and its analogues were
attractive structures as potential HIV-1 RNase H inhibitors21. However, our results with
compounds of series II having modified substituents underline the importance of substituent
diversity in the specificity of target recognition.
Figure 3. RNase H cleavage of heteropolymeric template-primer 31Trna/21P. RT-catalyzed
cleavage of [32P]RNA/DNA was carried out at 37ºC in the presence of different compounds at the
indicated concentrations.22 Reaction time points were taken at 0, 0.25, 0.5, 1, 2, 3 and 4 min.
Samples on the right correspond to a control experiment carried out in the absence of RT and
inhibitors, and show aliquots withdrawn at 0, 1, 2, and 4 min. 31Trna, stands for uncleaved RNA
(31 nucleotides), and P indicates the cleavage product of 26 nucleotides.22 GFP-1 and VII-015
refer to compounds I-1 and IIA-2, respectively.
2.3. Molecular modeling analysis

Figure 4. Predicted binding modes of IIA-2 (orange) were explored using an HIV-1 IN model built on the basis of
the crystal structure of prototype foamy virus (PFV) IN (PDB code: 3S3M). The hydrogen bonds between IIA-2
and amino acids residues are indicated by dashed lines (yellow) and metal ions are shown as green spheres. DNA
strands are represented with a stick model. Aribbon representation is used to depict the HIV-1 IN backbone and
relevant amino acid residues are shown with sticks in magenta. All nonpolar hydrogens are hid. The figure was
obtained with the Pymol visualization program.
To improve our understanding of structure-affinity relationships, the best compound of the
series (i.e IIA-2) was docked into HIV-1 IN (PDB code: 3S3M) using the software SurflexeDock
SYBYL-X 2.0. The results of the docking analysis showing its theoretical binding location and
conformation are shown in Figure 4.
According to the docking analysis, IIA-2 adopts a “Z” conformation in the active site of the
IN, facilitated by the introduction of two turns in the inhibitor that coincide with NH and CH2
groups. With this conformation, IIA-2 could fit perfectly into the HIV-1 IN binding site by
chelating the two Mg2+ ions found in the active site, making π-π interactions between the
backbone of IIA-2 and a nitrogen base, and establishing additional hydrogen bonds between the
triazole and the main chain of Tyr212. Moreover, the terminal hydrophobic substituent could

interact with the benzene group of Tyr212. These findings could explain why IIA-2 is a potent
inhibitor of HIV-1 IN, and predictions based on the docking analysis will be taken into account in
the next round of rational structural optimization.
3. Conclusion
The exploration of chemical space around privileged substructures remains one of the most
challenging steps towards the identification of novel chemical entities valuable for drug discovery.
The divalent metal ion chelators represent privileged, biologically relevant starting points for such
explorations. We sought to approximate their structural diversity by focusing on two divalent
metal ion chelators, 3-hydroxyquinazoline-2,4(1H,3H)-dione and
1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one, as frameworks. The focused compound collections
based on these scaffolds were synthesized using CuAAC reaction which is widely applied in drug
discovery23, 24, and tested in anti-HIV bioassays to generate new promising leads for further
development. In particular, some 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one derivatives
(exemplified by IIA-2) exhibited remarkable anti-HIV activity due to their ability to inhibit the
viral IN, but their structures were different from those of other available IN inhibitors. Therefore,
they could serve as good lead compounds for the development of a new generation of anti-HIV
agents. All 3-hydroxyquinazoline-2,4(1H,3H)-dione derivatives didn’t show anti-HIV potency,
possibly because the triazole lack H-donor as other derivatives of
3-hydroxyquinazoline-2,4(1H,3H)-dione scaffold reported in the literature17.
1-Hydroxypyrido[2,3-d]pyrimidin-2(1H)-one and its analogues were attractive structures as
potential HIV-1 RNase H inhibitors according to the latest report by GlaxoSmithKline in 2011,
however, our results with compounds of series II having modified substituents didn’t show activity
in the HIV-1 RNase H inhibition assays but could selectively inhibit HIV-1 IN, which indicates the
conformation of the metal-chelating backbone and the diversity of substituents in candidate drugs
may influence target recognition. Preliminary SAR studies and molecular modeling analysis are
expected to provide helpful guidance for further optimization. Furthermore, the application of
CuAAC reaction in the construction of new pDOS libraries is being actively pursued in our
laboratories.
4. Experimental section

4.1. Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer using solvents as
indicated (d6-DMSO). 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. TLC was performed on Silica Gel GF254 for TLC
(Merck) and spots were visualized by iodine vapours or by irradiation with UV light (l ¼ 254 nm).
Flash column chromatography was performed on column packed with Silica Gel60 (200-400
mesh). HPLC was performed on Agilent Infinity 1260/ G2260A.
Thin layer chromatography was performed on pre-coated HUANGHAI_HSGF254, 0.15-0.2 mm
TLC-plates. Solvents were of reagent grade and were purified and dried by standard methods
when necessary. Concentration of the reaction solutions involved the use of rotary evaporator at
reduced pressure. The key reactants including 2-amino-5-bromobenzoic acid, carbonyldiimidazole,
methyl 2-fluoronicotinate, O-benzylhydroxylamine etc. were purchased from Adamas-beta Co.
Ltd.
4.1.1. General procedure for synthesis of methyl 2-amino-5-bromobenzoate (G-1)
The commercially available material 2-amino-5-bromobenzoic acid (G-0) (0.2 g, 0.93 mmol) was
dissolved in methanol (10 mL) and concentrated H2SO4 (0.2 ml, 3.72 mmol) was slowly added.
The reaction mixture was refluxed for 6 h. After cooling, the solvent was evaporated mostly under
reduced pressure and then aqueous NaHCO3 was used to adjust the pH to 9-10. The residue was
extracted with ethyl acetate (3×20 mL) and then the combined organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was recrystallized from ethyl acetate and
petroleum ether or purified by column chromatography using ethyl acetate and petroleum ether
(1:8) as an eluent to give G-1 as white solid. yield 90.8%, mp:64-65℃.
1H NMR (400 MHz, d6-DMSO) δ 7.77 (s, 1H，PH-H), 7.38 (d, J = 8.6 Hz, 1H, Ph-H), 6.82 (s, 2H,
NH2-H), 6.77 (d, J = 8.9 Hz, 1H, Ph-H), 3.80 (s, 3H, CH3-H). 13C NMR (100 MHz, d6-DMSO) δ
167.14, 150.86, 136.90, 132.76, 119.37, 110.65, 105.22, 52.15.
4.1.2 General procedure for synthesis of 3-(benzyloxy)-6-bromoquinazoline-2,4(1H,3H)-dione
(G-2)

G-1 (0.4g, 1.74mmol) was refluxed with carbonyldiimidazole (CDI) (0.35 g, 3.17 mmol) in THF
and the O-benzylhydroxylamine (0.32 g, 2.61 mmol) was added. The mixture was continued to
reflux for 3 h. The solvent was evaporated mostly under reduced pressure and then ethyl alcohol
(8 ml) was added to dissolve the residue. Sodium hydroxide (0.10 g, 2.61 mmol) in 5 ml H2O was
added then the mixture was reflux overnight. After cooling to room temperature, acetic acid was
added until no more precipitate formed. The precipitate was filtrated and washed with MeOH to
afford G-2 as white solid. yield 73.7%, mp:238-240ºC.
1H NMR (400 MHz, d6-DMSO) δ 11.81 (s, 1H, N-H), 8.03 (d, J = 2.3 Hz, 1H, Ph-H), 7.85 (dd, J
= 8.7, 2.3 Hz, 1H, Ph-H), 7.57 (dd, J = 7.5, 1.8 Hz, 2H, Ph-H), 7.42 (m, 3H, Ph-H), 7.17 (d, J =
8.7 Hz, 1H, Ph-H), 5.09 (s, 2H, CH2).
4.1.3 General procedure for synthesis of
3-(benzyloxy)-6-bromo-1-(prop-2-yn-1-yl)quinazoline-2,4(1H,3H)-dione (G-3)
G-2 (0.1 g, 0.29 mmol), K2CO3 (0.05 g, 0.34 mmol) and 3-bromopropyne (0.04 g, 0.34 mmol)
were added in 5 mL dimethyl formamide (DMF) and stirred under room temperature overnight.
The solvent was evaporated mostly under reduced pressure and then water was added to wash the
remaining DMF. The residue was extracted with ethyl acetate (3 ×20 mL) and then the organic
layer was dried over Na2SO4 and concentrated in vacuo. The residue was recrystallized from ethyl
acetate and petroleum ether to give G-3 as white solid. yield 67.8%, mp:170-171℃.
1H NMR (400 MHz, d6-DMSO) δ 8.15 (d, J = 2.3 Hz, 1H, Ph-H), 8.02 (dd, J = 8.9, 2.3 Hz, 1H,
Ph-H), 7.61 – 7.55 (m, 2H, Ph-H), 7.52 (d, J = 9.0 Hz, 1H, Ph-H), 7.44 (dd, J = 9.9, 4.8 Hz, 3H,
Ph-H), 5.12 (s, 2H, CH2), 4.98 (d, J = 1.8 Hz, 2H, CH2), 3.43 (s, 1H, CH).
4.1.4 General procedure for synthesis of substituted
1-((1H-1,2,3-triazol-4-yl)methyl)-3-(benzyloxy)-6-bromoquinazoline-2,4(1H,3H)-dione (G-4)
G-3 (0.2 g, 0.52 mmol), corresponding azide (1.04 mmol), sodium ascorbate(0.03 g, 0.16 mmol)
and copper sulfate pentahydrate (0.01 g, 0.05 mmol) were stirred together in THF:H2O=1:1=6 mL
overnight. The mixture was extracted with ethyl acetate (3×20 mL) and then the organic layer
was dried over with Na2SO4 and concentrated in vacuo. The residue was recrystallized from ethyl
acetate and petroleum ether to give corresponding G-4.

3-(Benzyloxy)-6-bromo-1-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,
3H)-dione (G-4-1): white solid, yield 73.7%, mp:183-185ºC.
1H NMR (400 MHz, d6-DMSO) δ 8.17 (s, 1H, Ph-H), 8.13 (d, J = 2.0 Hz, 1H, Ph-H), 7.95 (dd, J = 8.9,
2.0 Hz, 1H, Ph-H), 7.59 (d, J = 8.9 Hz, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.44 – 7.32 (m, 5H, Ph-H),
7.19 (t, J = 8.8 Hz, 2H, Ph-H), 5.55 (s, 2H, CH2), 5.38 (s, 2H, CH2), 5.12 (s, 2H, CH2). C25H19BrFN5O3
3-(Benzyloxy)-6-bromo-1-((1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-d
ione (G-4-2): white solid, yield 76.9%, mp:181-183℃.
1H NMR (400 MHz, d6-DMSO) δ 8.20 (s, 1H, Ph-H), 8.13 (d, J = 2.3 Hz, 1H, Ph-H), 7.94 (dd, J = 9.0,
2.3 Hz, 1H, Ph-H), 7.59 (d, J = 9.0 Hz, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.40 (m, 4H, Ph-H), 7.20 –
7.14 (m, 1H, Ph-H), 7.14 – 7.08 (m, 2H, Ph-H), 5.59 (s, 2H, CH2), 5.40 (s, 2H, CH2), 5.13 (s, 2H,
CH2).
3-(Benzyloxy)-6-bromo-1-((1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3
H)-dione (G-4-3): white solid, yield 70.7%, mp:169-170℃.
1H NMR (400 MHz, d6-DMSO) δ 8.21 (s, 1H, Ph-H), 8.14 (d, J = 1.7 Hz, 1H, Ph-H), 7.99 – 7.91 (m,
1H, Ph-H), 7.59 (d, J = 9.1 Hz, 2H, Ph-H), 7.57 (s, 1H, triazole-H), 7.42 (d, J = 4.8 Hz, 5H, Ph-H),
7.16 (s, 1H, Ph-H), 5.57 (s, 2H, CH2), 5.40 (s, 2H, CH2), 5.12 (s, 2H, CH2).
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(benzyloxy)-6-bromoquinazoline-2,4(1H,3H)-dione
(G-4-4): white solid, yield 87.9%, mp:192-194℃.
1H NMR (400 MHz, d6-DMSO) δ 8.17 (s, 1H, Ph-H), 8.13 (d, J = 2.3 Hz, 1H, Ph-H), 7.94 (dd, J = 9.0,
2.4 Hz, 1H, Ph-H), 7.60 – 7.57 (m, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.44 – 7.38 (m, 3H, Ph-H), 7.37
– 7.25 (m, 5H, Ph-H), 5.56 (s, 2H, CH2), 5.39 (s, 2H, CH2), 5.12 (s, 2H, CH2).
3-(Benzyloxy)-6-bromo-1-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-
dione (G-4-5): white solid, yield 65.5%, mp:176-178℃.
1H NMR (400 MHz, d6-DMSO) δ 8.18 (s, 1H, Ph-H), 8.13 (d, J = 2.2 Hz, 1H, Ph-H), 7.95 (dd, J = 8.9,
2.3 Hz, 1H, Ph-H), 7.60 – 7.57 (m, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.45 – 7.37 (m, 5H, Ph-H), 7.30
(d, J = 8.3 Hz, 2H, Ph-H), 5.57 (s, 2H, CH2), 5.39 (s, 2H, CH2), 5.12 (s, 2H, CH2).
3-(Benzyloxy)-6-bromo-1-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-
dione (G-4-6): white solid, yield 62.7%, mp:197-198℃.

1H NMR (400 MHz, d6-DMSO) δ 8.18 (s, 1H, Ph-H), 8.13 (d, J = 2.4 Hz, 1H, Ph-H), 7.95 (dd, J = 9.0,
2.4 Hz, 1H, Ph-H), 7.58 (m, 4H, Ph-H), 7.55 (s, 1H, triazole-H), 7.41 (m, 3H, Ph-H), 7.24 (d, J = 8.4
Hz, 2H, Ph-H), 5.55 (s, 2H, CH2), 5.39 (s, 2H, CH2), 5.12 (s, 2H, CH2).
3-(Benzyloxy)-6-bromo-1-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-
dione (G-4-7): white solid, yield 55.4%, mp:155-157℃.
1H NMR (400 MHz, d6-DMSO) δ 8.16 (s, 1H, Ph-H), 8.13 (d, J = 2.4 Hz, 1H, Ph-H), 7.94 (dd, J = 9.0,
2.4 Hz, 1H, Ph-H), 7.59 – 7.57 (m, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.50 (dd, J = 7.8, 1.2 Hz, 1H,
Ph-H), 7.43 – 7.33 (m, 5H, Ph-H), 7.17 (dd, J = 7.4, 1.6 Hz, 1H, Ph-H), 5.67 (s, 2H, CH2), 5.41 (s, 2H,
CH2), 5.12 (s, 2H, CH2).
3-(Benzyloxy)-6-bromo-1-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3
H)-dione (G-4-8): yellow solid, yield 89.2%, mp:167-169ºC.
1H NMR (400 MHz, d6-DMSO) δ 8.25 (s, 1H, Ph-H), 8.22 (d, J = 8.7 Hz, 2H, Ph-H), 8.14 (d, J = 2.4
Hz, 1H, Ph-H), 7.96 (dd, J = 9.0, 2.4 Hz, 1H, Ph-H), 7.59 (d, J = 9.0 Hz, 2H, Ph-H), 7.56 (s, 1H,
triazole-H), 7.50 (d, J = 8.7 Hz, 2H, Ph-H), 7.44 – 7.37 (m, 3H, Ph-H), 5.75 (s, 2H, CH2), 5.41 (s, 2H,
CH2), 5.12 (s, 2H, CH2).
3-(Benzyloxy)-6-bromo-1-((1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,
3H)-dione (G-4-9): white solid, yield 72.9%, mp:157-159℃.
1H NMR (400 MHz, d6-DMSO) δ 8.14 (d, J = 2.4 Hz, 1H, Ph-H), 8.08 (s, 1H, Ph-H), 7.95 (dd, J = 9.0,
2.4 Hz, 1H, Ph-H), 7.58 (m, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.45 – 7.37 (m, 3H, Ph-H), 7.25 – 7.13
(m, 3H, Ph-H), 7.03 (d, J = 7.6 Hz, 1H, Ph-H), 5.57 (s, 2H, CH2), 5.40 (s, 2H, CH2), 5.12 (s, 2H, CH2),
2.28 (s, 3H, CH3).
3-(Benzyloxy)-6-bromo-1-((1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-
dione (G-4-10): white solid, yield 62.4%, mp:164-166℃.
1H NMR (400 MHz, d6-DMSO) δ 8.16 (s, 1H, Ph-H), 8.13 (d, J = 2.4 Hz, 1H, Ph-H), 7.94 (dd, J = 9.0,
2.4 Hz, 1H, Ph-H), 7.58 (s, 2H, Ph-H), 7.56 (s, 1H, triazole-H), 7.41 (m, 3H, Ph-H), 7.23 (t, J = 7.8 Hz,
1H, Ph-H), 7.12 (d, J = 7.6 Hz, 1H, Ph-H), 7.07 (m, 2H, Ph-H), 5.51 (s, 2H, CH2), 5.39 (s, 2H, CH2),
5.12 (s, 2H, CH2), 2.25 (s, 3H, CH3).
4.1.5. General procedure for synthesis of substituted
1-((1H-1,2,3-triazol-4-yl)methyl)-6-bromo-3-hydroxyquinazoline-2,4(1H,3H)-dione (series I)

Corresponding G-4 (0.2 g) was refluxed in a mixed solution of hydrogen bromide (2 mL) and
acetic acid (2 mL) for 6-8 h. After cooling to room temperature, H2O was added and the
precipitate was recrystallized from MeOH to give corresponding series I.
6-Bromo-1-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyquinazoline-2,4(1H,3H)-
dione (I-1): white solid, yield 13.6%, mp:270-272℃.
1H NMR (400 MHz, d6-DMSO) δ 10.98 (s, 1H, N-OH), 8.16 (s, 1H, Ph-H), 8.11 (d, J = 2.3 Hz, 1H,
Ph-H), 7.91 (dd, J = 9.0, 2.3 Hz, 1H, Ph-H), 7.56 (d, J = 9.0 Hz, 1H, Ph-H), 7.36 (dd, J = 8.4, 5.6 Hz,
2H), 7.19 (t, J = 8.8 Hz, 2H), 5.54 (s, 2H), 5.37 (s, 2H). 13C NMR (100 MHz, d6-DMSO) δ 163.57,
161.14, 157.91, 149.50, 142.68, 138.34, 137.79, 132.61 (d, J = 12 Hz), 130.82, 130.73, 129.88, 124.12,
118.25, 117.47, 116.16, 115.95, 115.36, 52.50.
6-Bromo-1-((1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyquinazoline-2,4(1H,3H)-dion
e (I-2): white solid, yield 24.8%, mp:268-270ºC.
1H NMR (400 MHz, d6-DMSO) δ 10.99 (s, 1H), 8.20 (s, 1H, triazole-H), 8.11 (d, J = 2.2 Hz, 1H), 7.91
(dd, J = 9.0, 2.2 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 14.2, 7.9 Hz, 1H), 7.15 (m, 3H), 5.58
(s, 2H), 5.39 (s, 2H). 13C NMR (100 MHz, d6-DMSO) δ 163.79 (s), 161.36 (s), 157.92 (s), 149.51 (s),
142.73 (s), 139.02 (d, J = 29.7 Hz), 138.33 (s), 137.77 (s), 131.31 (d, J = 32.8 Hz), 129.88 (s), 124.50
(d, J = 12.0 Hz), 124.38 (s), 118.25 (s), 117.48 (s), 115.60 (s), 115.37 (d, J = 1.9 Hz), 115.16 (s), 52.61
(s).
6-Bromo-1-((1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyquinazoline-2,4(1H,3H)-d
ione (I-3): white solid, yield 12.5%, mp:269-271℃.
1H NMR (400 MHz, d6-DMSO) δ 10.98 (s, 1H), 8.19 (s, 1H, triazole-H), 8.11 (d, J = 2.4 Hz, 1H), 7.90
(dd, J = 9.0, 2.4 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.47 – 7.35 (m, 2H), 7.19 – 7.12 (m, 1H), 5.55 (s,
2H), 5.38 (s, 2H). 13C NMR (100 MHz, d6-DMSO) δ 157.91, 150.95 (d, J = 49.5 Hz), 149.50, 148.50
(d, J = 51.9 Hz), 142.74, 138.33, 137.78, 133.92 (dd, J = 23.4, 15.8 Hz), 129.89, 125.67 (dd, J = 26.5,
13.9 Hz), 124.29, 118.44, 118.25 (d, J = 12 Hz), 117.95, 117.48, 117.46, 115.37, 52.12.
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-bromo-3-hydroxyquinazoline-2,4(1H,3H)-dione (I-4):
white solid, yield 11.4%, mp:269-271℃.

1H NMR (400 MHz, d6-DMSO) δ 10.97 (s, 1H), 8.16 (s, 1H), 8.11 (d, J = 2.3 Hz, 1H), 7.90 (dd, J = 9.0,
2.4 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.39 – 7.22 (m, 5H), 5.55 (s, 2H), 5.38 (s, 2H). 13C NMR (100
MHz, d6-DMSO) δ 157.91, 149.51, 142.65, 138.33, 137.77, 136.35, 129.88, 129.21(2×C), 128.62,
128.42(2×C), 124.19, 118.25, 117.48, 115.35, 53.32.
6-Bromo-1-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyquinazoline-2,4(1H,3H)-dion
e(I-5)：white solid, yield 19.3%, mp:272-273℃.
1H NMR (400 MHz, d6-DMSO) δ 10.97 (s, 1H), 8.17 (s, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.90 (dd, J = 9.0,
2.4 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 5.56 (s, 2H),
5.38 (s, 2H).13C NMR (100 MHz, d6-DMSO) δ 157.91, 149.50, 142.70, 138.32, 137.78, 135.33, 133.37,
130.38(2×C), 129.89, 129.21(2×C), 124.27, 118.23, 117.47, 115.36, 52.51.
6-Bromo-1-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyquinazoline-2,4(1H,3H)-dion
e(I-6)：white solid, yield 17.5%, mp:277-279℃.
1H NMR (400 MHz, d6-DMSO) δ 10.97 (s, 1H), 8.16 (s, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.90 (dd, J = 9.0,
2.4 Hz, 1H), 7.56 (d, J = 8.5 Hz, 3H), 7.23 (d, J = 8.4 Hz, 2H), 5.54 (s, 2H), 5.38 (s, 2H).
13C NMR (100 MHz, d6-DMSO) δ 157.91, 149.50, 142.70, 138.32, 137.79, 135.74, 132.14(2×C),
130.68(2×C), 129.89, 124.28, 121.92, 118.23, 117.47, 115.36, 52.57.
6-Bromo-1-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-hydroxyquinazoline-2,4(1H,3H)-dion
e (I-7): white solid, yield 31.0%, mp:274-276℃.
1H NMR (400 MHz, d6-DMSO) δ 10.97 (s, 1H), 8.13 (s, 1H), 8.11 (d, J = 2.3 Hz, 1H), 7.90 (dd, J = 9.0,
2.3 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.53 – 7.46 (m, 1H), 7.42 – 7.31 (m, 2H), 7.17 (d, J = 7.3 Hz,
1H), 5.66 (s, 2H), 5.40 (s, 2H). 13C NMR (100 MHz, d6-DMSO) δ 157.91, 149.53, 142.51, 138.32,
137.75, 133.60, 133.08, 130.93, 130.73, 130.10, 129.88, 128.17, 124.60, 118.26, 117.48, 115.35, 51.10.
6-Bromo-3-hydroxy-1-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-dione
(I-8): yellow solid, yield 9.8%, mp:238-240℃.
1H NMR (400 MHz, d6-DMSO) δ 10.98 (s, 1H), 8.24 – 8.21 (m, 3H), 8.11 (s, 1H), 7.91 (d, J = 5.6 Hz,
1H), 7.57 (d, J = 6.3 Hz, 1H), 7.50 (d, J = 6.8 Hz, 2H), 5.74 (s, 2H), 5.41 (s, 2H).13C NMR (100 MHz,
d6-DMSO) δ 157.90, 149.50, 147.72, 143.71, 142.81, 138.31, 137.80, 129.90, 129.52(2×C), 124.69,
124.35(2×C), 118.23, 117.46, 115.38, 52.42.

6-Bromo-3-hydroxy-1-((1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-dio
ne (I-9): white solid, yield 18.6%, mp:282-284℃.
1H NMR (400 MHz, d6-DMSO) δ 10.96 (s, 1H), 8.11 (d, J = 2.3 Hz, 1H), 8.05 (s, 1H), 7.89 (dd, J = 9.0,
2.4 Hz, 1H), 7.55 (d, J = 9.0 Hz, 1H), 7.26 – 7.13 (m, 3H), 7.03 (d, J = 7.5 Hz, 1H), 5.55 (s, 2H), 5.38
(s, 2H), 2.26 (s, 3H). 13C NMR (100 MHz, d6-DMSO) δ 157.91, 149.52, 142.52, 138.33, 137.74,
136.79, 134.45, 130.89, 129.87, 129.13, 128.81, 126.70, 124.20, 118.25, 117.48, 115.34, 51.48, 19.08.
6-Bromo-3-hydroxy-1-((1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)quinazoline-2,4(1H,3H)-dio
ne (I-10): white solid, yield 22.4%, mp:264-266℃.
1H NMR (400 MHz, d6-DMSO) δ 10.97 (s, 1H), 8.14 (s, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.89 (dd, J = 9.0,
2.4 Hz, 1H), 7.55 (d, J = 9.0 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 6.0
Hz, 2H), 5.50 (s, 2H), 5.38 (s, 2H), 2.26 (s, 3H). 13C NMR (100 MHz, d6-DMSO) δ 157.90, 149.51,
142.62, 138.44, 138.31, 137.75, 136.24, 129.88, 129.24, 129.11, 128.94, 125.51, 124.17, 118.25,
117.49, 115.34 , 53.33, 21.36.
4.1.6. General procedure for synthesis of methyl 2-((benzyloxy)amino)nicotinate
(V-1)
A mixture of V-0 (1.0g, 6.45mmol) and O-benzylhydroxylamine (0.94g, 7.63mmol) in
diisopropylethylamine (DIPEA, 12mL, 68.70mmol) was irradiated in microwave at 120℃ for 2h.
The reaction mixture was concentrated in vacuo and the residue was purified by silica gel
chromatography using ethyl acetate and petroleum ether (1:10) as an eluent to provide V-1. Yellow
oil. Yield: 34.3%. ESI-MS: m/z 259.0(M+1), 281.3(M+23). C14H14N2O3 [258.27].
4.1.7. General procedure for synthesis of 1-(benzyloxy)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
(V-2)
A solution of V-1 (0.18 g, 0.697 mmol) in 1,2-dichloroethane (DCE) (1.89 mL) was treated with
Trichloroacetyl isocyanate (0.77 M in DCE) (1.93 mL, 1.49 mmol) and stirred t ambient
temperature. After 30 minutes, triethylamine (TEA) (0.20 mL, 1.47 mmol) was added and the
mixture was stirred at r.t. for 30 min. Then the solvent was removed under reduced pressure, and
the residue was dissolved in 5ml MeOH and MeONa solution (25% wt. in MeOH) (0.76 mL, 3.5
mmol) was added. After 3 hours, the solvent was removed under under reduced pressure and 1 N

HCl was added to adjust the pH value to 3. Twenty milliliters of EA were then added and the
aqueous phase was extracted with EA 3 times. The combined organic layer was washed with brine,
dried over with Na2SO4 and purified by silica gel chromatography using ethyl acetate and
petroleum ether (2:3) as an eluent to give V-2. White solid. Yield: 21.1%. mp:197-199℃。
1H NMR (400 MHz, d6-DMSO) δ 11.93 (s, 1H, NH), 8.79 (dd, J = 4.8, 1.7 Hz, 1H, C2-Py-H),
8.36 (dd, J = 7.7, 1.7 Hz, 1H, C4-Py-H), 7.70-7.56 (m, 2H, C5-Py-H, Ph-H), 7.49-7.33 (m, 4H,
Ph-H), 5.18 (s, 2H, CH2). ESI-MS: m/z 270.5(M+1), 292.5(M+23). C14H11N3O3 [269.26].
4.1.8. General procedure for synthesis of V-4
A solution of V-2 (0.6 g, 2.23 mmol) in DIPEA was treated with POCl3 (1.04 mL, 11.15 mmol)
and heated to 100℃. After 45 min, the excess solvent was removed in vacuo and the black residue
was dissolved in 10 mL DMF immediately. The corresponding amine (3.5 mmol) was added and
then stirred at room temperature for 1 h. The mixture was poured into 200 mL H2O and extracted
with EA three times. The combined organic layers were dried over Na2SO4 and purified by silica
gel chromatography to give corresponding compound V-4.
1-(benzyloxy)-4-((3-ethynylphenyl)amino)pyrido[2,3-d]pyrimidin-2(1H)-one (V-4a): white solid,
yield: 72.1%, mp:205-207°C
1H NMR (400 MHz, d6-DMSO) δ 10.08 (s, 1H, NH), 8.82 (d, J = 5.4 Hz, 2H, Ar-H), 8.03 (s, 1H,
Ar-H), 7.86 (d, J = 8.0 Hz, 1H, Ar-H), 7.65 (d, J = 6.5 Hz, 2H, Ar-H), 7.51-7.39 (m, 5H, Ar-H),
7.32 (d, J = 7.6 Hz, 1H, Ar-H), 5.15 (s, 2H, CH2), 4.27 (s, 1H, C≡CH). ESI-MS: m/z 369.3(M+1),
391.4(M+23). C22H16N4O2 [368.39].
1-(benzyloxy)-4-((2-ethynylphenyl)amino)pyrido[2,3-d]pyrimidin-2(1H)-one (V-4b): brown
needles, yield: 17.4%，mp: 187-189°C.
1 H NMR (400 MHz, d6-DMSO) δ 10.22 (s, 1H, NH), 8.83 (d, J = 3.9 Hz, 1H, C2-Py-H), 8.76 (d,
J = 7.9 Hz, 1H, C4-Py-H), 7.62 (d, J = 7.3 Hz, 3H, Ar-H), 7.57-7.50 (m, 2H, Ar-H), 7.46-7.37 (m,
5H, Ar-H), 5.12 (s, 2H, CH2), 4.33 (s, 1H, C≡CH). ESI-MS: m/z 369.2(M+1), 391.4(M+23).
C22H16N4O2 [368.39].
4.1.9. General procedure for synthesis of series II

Corresponding V-4 (0.52 mmol), corresponding azide (1.04 mmol), sodium ascorbate (0.03 g, 0.16
mmol) and copper sulfate pentahydrate (0.01 g, 0.05 mmol) were stirred together in THF:H2O =
1:1 = 6 mL overnight. The residue was extracted with ethyl acetate (3×20 mL) and then the
organic layer was dried over with Na2SO4 and concentrated in vacuo. The residue was
recrystallized from ethyl acetate and petroleum ether to give corresponding compound V.
1-(benzyloxy)-4-((3-(1-phenethyl-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimidin-2(1
H)-one (VA -1): white solid, yield 100%.
1 H NMR (400 MHz, d6-DMSO) δ 10.16 (s, 1H), 8.87 (d, J = 7.8 Hz, 1H), 8.82 (d, J = 4.4 Hz,
1H), 8.53 (s, 1H), 8.21 (s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 6.5 Hz, 2H), 7.59 (d, J = 7.7
Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.47-7.39 (m, 4H), 7.32-7.26 (m, 2H), 7.23 (dd, J = 7.6, 3.1 Hz,
3H), 5.16 (s, 2H), 4.68 (t, J = 7.3 Hz, 2H), 3.24 (t, J = 7.3 Hz, 2H). ESI-MS: m/z 516.7(M+1),
533.6(M+18), 538.6(M+23). C30H25N7O2 [515.57].
1-(benzyloxy)-4-((3-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimi
din-2(1H)-one (VA -2): light yellow solid，yield 21.4%. ESI-MS: m/z 520.5(M+1), 537.6(M+18),
542.5(M+23). C29H22FN7O2 [519.53].
1-(benzyloxy)-4-((3-(1-(3-phenylpropyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimi
din-2(1H)-one (VA -3): white solid, yield 85.7%.
1H NMR (400 MHz, d6-DMSO) δ 10.17 (s, 1H), 8.88 (d, J = 7.0 Hz, 1H), 8.85-8.80 (m, 1H), 8.63
(s, 1H), 8.26 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 6.8 Hz, 3H), 7.52 (d, J = 7.9 Hz, 1H),
7.48-7.39 (m, 4H), 7.30 (d, J = 7.3 Hz, 2H), 7.28-7.17 (m, 3H), 5.16 (s, 2H), 4.44 (t, J = 7.0 Hz,
2H), 2.70-2.55 (m, 2H), 2.31-2.15 (m, 2H). ESI-MS: m/z 530.4(M+1), 552.6(M+23). C31H27N7O2
[529.59].
1-(benzyloxy)-4-((3-(1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimi
din-2(1H)-one (VA -4): yellow solid, yield 53.3%.
1H NMR (400 MHz, d6-DMSO) δ 10.24 (s, 1H), 9.17 (s, 1H), 8.90 (d, J = 7.0 Hz, 1H), 8.83 (d, J =
4.7 Hz, 1H), 8.42 (s, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 7.3 Hz, 1H), 7.96 (d, J = 8.1 Hz,
1H), 7.85 (d, J = 7.2 Hz, 1H), 7.82-7.72 (m, 2H), 7.72-7.62 (m, 5H), 7.59 (dd, J = 16.3, 8.3 Hz,

1H), 7.49-7.39 (m, 4H), 5.17 (s, 2H). ESI-MS: m/z 538.6(M+1), 555.5(M+18), 560.5(M+23).
C32H23N7O2 [537.57].
2-(4-(3-((1-(benzyloxy)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)amino)phenyl)-1H-1,2,3-tr
iazol-1-yl)-N-phenylacetamide (VA -5): white solid, yield 26.7% .ESI-MS: m/z 545.5(M+1),
562.6(M+18), 567.6(M+23). C30H24N8O3 [544.56].
2-(4-(3-((1-(benzyloxy)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)amino)phenyl)-1H-1,2,3-tr
iazol-1-yl)-N-(4-fluorophenyl)acetamide (VA -6): white solid, yield 66.7%. ESI-MS: m/z
563.5(M+1), 580.5(M+18), 585.5(M+23). C30H23FN8O3 [562.55].
1-(benzyloxy)-4-((3-(1-(2-(4-fluorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrid
o[2,3-d]pyrimidin-2(1H)-one (VA -7): white solid, yield 33.3%. ESI-MS: m/z 548.6(M+1),
565.6(M+18), 570.6(M+23), 586.5(M+39). C30H22FN7O3 [547.54].
1-(benzyloxy)-4-((3-(1-(2-oxo-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)a
mino)pyrido[2,3-d]pyrimidin-2(1H)-one (VA -8): white solid, yield 47.1%。ESI-MS: m/z
613.5(M+1), 630.6(M+18), 635.4(M+23). C31H23F3N8O3 [612.56].
1-(benzyloxy)-4-((2-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimi
din-2(1H)-one (VB-1): white solid, yield 21.4%. ESI-MS: m/z 520.7(M+1), 542.5(M+23).
C29H22FN7O2[519.53].
1-(benzyloxy)-4-((2-(1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimi
din-2(1H)-one (VB-2): light yellow solid, yield 100%. ESI-MS: m/z 516.6(M+1), 538.5(M+23).
C30H25N7O2 [515.57].
4.1.10. General procedure for synthesis of final product of series II.
Trifluoroacetic acid (TFA, 1 mL) was added to a solution of V in DCM and stirred at 60ºC
overnight. The excess solvent was removed in vacuo and the residue was washed with methanol or
separated via HPLC to obtain the final product.
1-hydroxy-4-((3-(1-phenethyl-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimidin-2(1H)-
one (IIA-1): Grey solid, yield 37.5%. 1H NMR (400 MHz, d6-DMSO) δ10.56 (s, 1H, OH), 10.02

(s, 1H, NH), 8.83 (d, J = 6.7 Hz, 1H, C2-Py-H), 8.75 (d, J =3.9 Hz, 1H, C4-Py-H), 8.51 (s, 1H,
Ar-H), 8.20 (s, 1H, Ar-H), 7.91 (d, J = 7.5 Hz, 1H, Ar-H), 7.56 (d, J = 7.3 Hz, 1H, Ar-H), 7.49 (d,
J = 7.9 Hz, 1H, Ar-H), 7.37 (s, 1H, CH), 7.26 (m, 5H, Ar-H), 4.68 (t, J = 7.0 Hz, 2H, N-CH2-C),
3.24 (t, J = 6.9 Hz, 2H, C-CH2-C). 13C NMR (100 MHz, d6-DMSO) δ 156.99, 154.69, 153.22,
151.76, 146.37, 139.39, 138.10, 134.13, 131.65, 129.57, 129.16(2×C), 128.92(2×C), 127.08,
122.95, 121.94, 119.93, 118.09, 106.40, 51.15, 36.06. ESI-MS: m/z 426.4 (M+1), 443.7 (M+18),
448.5 (M+23). C23H19N7O2 [425.44].
4-((3-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)-1-hydroxypyrido[2,3-d]pyrimidin-
2(1H)-one (IIA-2): Grey solid, yield 100.0%, HPLC purity: 97.5%. 1H NMR (400 MHz,
d6-DMSO) δ 10.58 (s, 1H, OH), 10.03 (s, 1H, NH), 8.83 (d, J = 7.6 Hz, 1H, C2-Py-H), 8.75 (d,
J = 4.0 Hz, 1H, C4-Py-H), 8.64 (s, 1H), 8.24 (s, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.6 Hz,
1H), 7.48 – 7.41 (m, 3H), 7.37 (dd, J = 7.6, 4.8 Hz, 1H), 7.24 (t, J = 8.8 Hz, 2H), 5.67 (s, 2H,
CH2). 13C NMR (100 MHz, d6-DMSO) δ 156.97, 154.69, 153.22, 151.75, 146.92, 139.39, 134.14,
130.82, 130.74, 129.58(2×C), 123.03, 122.08(2×C), 119.97, 118.11, 116.24(2×C), 116.03,
106.40, 52.74. ESI-MS: m/z 430.5(M+1), 447.5(M+18), 452.4(M+23). C22H16FN7O2 [429.41].
1-hydroxy-4-((3-(1-(3-phenylpropyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimidin-
2(1H)-one (IIA-3): Yellow solid, yield 16.7%.mp: 215-217°C. 1H NMR (400 MHz, d6-DMSO) δ
10.18 (s, 1H), 10.05 (s, 1H), 8.84 (d, J = 7.7 Hz, 1H), 8.75 (d, J = 3.5 Hz, 1H), 8.62 (d, J = 7.5 Hz,
1H), 8.25 (s, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.37
(dd, J = 7.3, 4.8 Hz, 1H), 7.33 – 7.27 (m, 2H), 7.26 – 7.20 (m, 3H), 4.44 (t, J = 7.1 Hz, 2H), 2.62
(t, J = 7.6 Hz, 2H), 2.21 (dt, J = 14.4, 7.1 Hz, 2H). 13C NMR (100 MHz, d6-DMSO) δ 157.00,
154.69, 153.26, 151.76, 146.62, 141.23, 139.38, 134.15, 131.70, 129.55, 128.88(2×C), 128.83,
126.49, 122.98, 122.10, 121.92, 120.02, 118.11, 106.41, 49.60, 32.39, 31.72. ESI-MS: m/z
440.5(M+1), 457.6(M+18). C24H21N7O2 [439.47].
1-hydroxy-4-((3-(1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimidin
-2(1H)-one (IIA-4): Light yellow solid, yield 12.5%. mp: 263-265°C. 1H NMR (400 MHz,
d6-DMSO) δ 10.60 (s, 1H), 10.11 (s, 1H), 9.17 (s, 1H), 8.87 (s, 1H), 8.76 (s, 1H), 8.42 (s, 1H),
8.24 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 7.4 Hz, 1H), 7.85 (d, J = 7.1 Hz,
1H), 7.76 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 5.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.43 (dd, J = 15.1,

7.6 Hz, 2H). 13C NMR (100 MHz, d6-DMSO) δ 157.07, 154.72, 153.28, 151.77, 146.93, 139.51,
134.18, 133.76, 131.19, 130.86, 129.68, 128.85, 128.59, 128.39, 127.66, 125.95, 124.85, 124.43,
123.41, 122.58, 122.46, 120.32, 118.13, 106.43. ESI-MS: m/z 448.6(M+1), 465.5(M+18),
470.5(M+23). C25H17N7O2 [447.45]
2-(4-(3-((1-hydroxy-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)amino)phenyl)-1H-1,2,3-triaz
ol-1-yl)-N-phenylacetamide (IIA-5): Yellow solid, yield 100.0%. mp: 285-287°C. 1H NMR (400
MHz, d6-DMSO) δ 10.53 (s, 1H), 10.11 (d, J = 53.4 Hz, 1H), 8.93 – 8.73 (m, 2H), 8.61 (d, J = 4.5
Hz, 1H), 8.28 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 17.2, 7.6 Hz, 3H), 7.52 (dd, J = 15.9,
8.0 Hz, 1H), 7.47 – 7.40 (m, 1H), 7.36 (dd, J = 15.2, 7.6 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 5.42 (s,
2H), 5.16 (s, 1H). 13C NMR (100 MHz, d6-DMSO) δ 164.64, 156.98, 154.68, 153.25, 151.76,
146.46, 139.46, 138.90, 134.18, 131.55, 129.95, 129.62(2×C), 129.39, 124.29, 123.67, 122.91,
122.03, 119.89, 119.76, 118.10, 106.44, 52.90. ESI-MS: m/z 455.5(M+1), 472.5(M+18),
477.4(M+23). C23H18N8O3 [454.44].
N-(4-fluorophenyl)-2-(4-(3-((1-hydroxy-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)amino)ph
enyl)-1H-1,2,3-triazol-1-yl)acetamide (IIA-6): Yellow solid, yield 10.0%. mp: 181-183°C. 1H
NMR (400 MHz, d6-DMSO) δ 10.60 (s, 1H), 10.12 (d, J = 50.9 Hz, 1H), 8.85 (dd, J = 23.6, 6.3
Hz, 1H), 8.61 (d, J = 4.4 Hz, 1H), 8.28 (s, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.66-7.60 (m, 3H), 7.52
(dd, J = 15.9, 8.0 Hz, 1H), 7.48-7.40 (m, 2H), 7.20 (t, J = 8.8 Hz, 2H), 5.42 (s, 2H), 5.16 (s, 1H).
13C NMR (100 MHz, d6-DMSO) δ 164.60, 159.95, 157.56, 154.76, 146.47, 135.29, 131.55,
129.88(2×C), 129.65, 129.17, 128.78(2×C), 123.66, 122.30, 121.62, 121.54, 120.18, 119.87,
118.83, 116.12, 115.90, 52.82. ESI-MS: m/z 473.4(M+1), 490.6(M+18), 495.4(M+23).
C23H17FN8O3 [472.43].
4-((3-(1-(2-(4-fluorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)-1-hydroxypyrido[2,
3-d]pyrimidin-2(1H)-one (IIA-7): Light yellow solid, yield 40.0%. mp: 261-263°C. 1H NMR (400
MHz, d6-DMSO) δ 10.60 (s, 1H), 10.06 (s, 1H), 8.85 (d, J = 7.5 Hz, 1H), 8.76 (d, J = 4.1 Hz, 1H),
8.53 (s, 1H), 8.30 (s, 1H), 8.26-8.16 (m, 2H), 7.96 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 7.4 Hz, 1H),
7.54-7.46 (m, 3H), 7.38 (dd, J = 7.6, 4.7 Hz, 1H), 6.29 (s, 2H). 13C NMR (100 MHz, d6-DMSO) δ
191.34, 156.96, 154.69, 153.23, 152.38, 151.77, 139.48, 131.89, 131.79(2×C), 131.54, 129.65,
123.63, 122.89, 122.01, 119.88, 118.10, 116.73(2×C), 116.51, 106.43, 56.45. ESI-MS: m/z

458.6(M+1), 475.4(M+18), 480.4(M+23). C23H16FN7O3 [457.42].
2-(4-(3-((1-hydroxy-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)amino)phenyl)-1H-1,2,3-triaz
ol-1-yl)-N-(4-(trifluoromethyl)phenyl)acetamide (IIA-8): Light yellow solid, yield 37.5%. mp>
300°C. 1H NMR (400 MHz, d6-DMSO) δ 10.92 (s, 1H), 10.06 (s, 1H), 8.85 (d, J = 12.8 Hz, 1H),
8.76 (s, 1H), 8.62 (s, 1H), 8.29 (s, 1H), 7.94 (s, 1H), 7.83 (d, J = 7.9 Hz, 2H), 7.73 (d, J = 8.0 Hz,
2H), 7.66 (d, J = 7.0 Hz, 2H), 7.59 – 7.47 (m, 1H), 7.43 (d, J = 7.2 Hz, 1H), 5.48 (s, 2H). ESI-MS:
m/z 523.6(M+1), 540.6(M+18), 545.5(M+23). C24H17F3N8O3 [522.44]
4-((2-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)-1-hydroxypyrido[2,3-d]pyrimidin-
2(1H)-one (IIB-1): Yellow solid, yield 33.3%. mp: 233-235°C. 1H NMR (400 MHz, d6-DMSO) δ
10.94 (s, 1H, OH), 10.56 (s, 1H), 8.76 (s, 1H), 8.58 (s, 1H), 8.52 (s, 1H), 8.13 (s, 1H), 7.94 (d, J =
6.4 Hz, 1H), 7.64 (d, J = 6.7 Hz, 1H), 7.44 (d, J = 5.6 Hz, 1H), 7.42 (s, 1H, CH), 7.33 (s, 2H), 7.16
(t, J = 8.3 Hz, 2H), 5.63 (s, 2H). 13C NMR (100 MHz, d6-DMSO) δ 163.54, 161.12, 154.69,
151.76, 133.50, 132.41, 130.69, 130.61, 128.92(2×C), 128.35(2×C), 126.81, 126.36, 123.67,
118.32(2×C), 116.15(2×C), 115.94, 52.68. ESI-MS: m/z 430.5(M+1). C22H16FN7O2 [429.41].
1-hydroxy-4-((2-(1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)phenyl)amino)pyrido[2,3-d]pyrimidin-
2(1H)-one (IIB-2): Light yellow solid, yield 7.1%. mp: 225-227°C. 1H NMR (400 MHz,
d6-DMSO) δ 10.97 (s, 1H, OH), 10.58 (s, 1H), 8.77 (d, J = 4.1 Hz, 1H), 8.60 (d, J = 7.8 Hz, 1H),
8.50 (d, J = 11.3 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.49 – 7.35 (m, 3H),
7.14 (dd, J = 16.4, 7.9 Hz, 4H), 5.58 (s, 2H), 2.26 (d, J = 7.7 Hz, 3H). 13C NMR (100 MHz,
DMSO) δ 157.50, 154.68, 153.26, 151.77, 145.50, 138.03, 135.48, 133.42, 133.08, 129.73, 128.87,
128.39(2×C), 128.32, 126.56, 126.26, 124.25, 123.58, 118.32, 106.57, 53.36, 21.18.. ESI-MS:
m/z 426.4(M+1). C23H19N7O2 [425.44].
4.2. In vitro anti-HIV assay
By using the MTT method described previously,25, 26 the synthesized compounds were evaluated
for their activity against WT HIV-1 27 (strain HIV-IIIB), and HIV-2 28 (strain ROD) in MT-4 cells.
At the beginning, stock solutions (10×final concentration) of test compounds were added at 25
μM 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 CCID50) (50% cell culture infectious dose) 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. The MT-4 cells were resuspended at 6 ×105
cells/mL, and transferred 50 mL volumes 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.
The 50% effective antiviral concentration (EC50) was defined as the concentration of the tested
compound achieving 50% protection from viral cytopathicity. The 50% cytotoxic concentration
(CC50) was defined as the compound concentration that reduced the inability of mock-infected
cells by 50%. The symbol ‘>’ indicated the highest concentration at which the compound was
tested and still found to be non-cytotoxic.
4.3. HIV-1 RNase H inhibition assay
Wild-type (WT) HIV-1 BH10 reverse transcriptase (RT) was expressed in E. coli and purified as
previously described.29, 30 Synthetic oligonucleotides 31Trna
(5´-UUUUUUUUUAGGAUACAUAUGGUUAAAGUAU-3´) and 21P
(5´-ATACTTTAACCATATGTATCC-3´) were obtained from Sigma. The RNase H inhibitor
RD1759 31, 32 was kindly provided by Drs. Roberto Di Santo (Sapienza University of Rome, Italy)
and Enzo Tramontano (University of Cagliari, Italy).

The RNase H activity was determined using the template-primer 31Trna/21P.22 The template RNA
was labeled at its 5´ end with [γ-32P]ATP (Perkin Elmer) and T4 polynucleotide kinase (New
England Biolabs), and then purified with a mini Quick SpinTM column (Roche). The labeled
template was annealed to the 21-nt primer (21P) in 50 mM Tris-HCl (pH 8.0) containing 50 mM
NaCl, as previously described.22 RNase H cleavage assays were carried out for 0 to 4 min 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). The potential
RNase H inhibitors were available in 50% DMSO. Appropriate dilutions of those drugs were
preincubated with the RT for 5 min at 37ºC in the assay buffer, and reactions were initiated by
adding the labeled template-primer. Aliquots were removed at appropriate times and quenched
with an equal volume of sample loading buffer [10 mM EDTA in 90% formamide containing
xylene cyanol FF (3 mg/mL) and bromophenol blue (3 mg/mL)] and analyzed by denaturing
polyacrylamide gel electrophoresis.
4.4 HIV-1 IN inhibition assay
HIV-1 IN inhibition assay was carried out using an ELISA method 33 and the XpressBio HIV-1
Integrase Assay Kit EZ-1700 (purchased from Suzhou Xishan Biological technology co., LTD,
China). Briefly, streptavidin-coated 96-well plates were coated with a double-stranded HIV-1 LTR
U5 donor substrate (DS) DNA containing an end-labeled biotin for 30 min at 37ºC. Full-length
recombinant HIV-1 IN protein was then added, and the mixture incubated for another 30 min at
37ºC. Test compounds were added to the enzyme reaction and incubated for 5 min at room
temperature. Then a different double stranded target substrate (TS) DNA containing a 3’-end
modification was added to the reaction mixture which was then incubated for 30 min at 37ºC. The
HIV-1 IN cleaves the terminal two bases from the exposed 3’-end of the HIV-1 LTR DS DNA and
then catalyzes a strand-transfer recombination reaction to integrate the DS DNA into the TS DNA.
The products of the reaction are detected spectrophotometrically using a horse radish peroxidase
(HRP)-labeled antibody directed against the TS 3’-end modification. Therefore, HRP antibody
solutions were added to the reaction and incubated during 30 min at 37ºC. Following the
incubation with the antibody, 100 µl of a 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase
substrate solution is added to each well and incubated for 10 minutes at room temperature. The

reaction is stopped by adding 100 μL of the commercial TMB stop solution and the absorbance of
the product is determined at 450 nm using a microtiter plate ELISA reader. The percentage
inhibition obtained with the tested compounds was calculated by using the formula:
% Inhibition= [OD value with IN but without inhibitors – OD value with IN and inhibitors]/[OD
value with IN and inhibitors – OD value without IN and inhibitors]
4.5. Molecular docking
Molecular modeling of molecule IIA-2 was performed using the Tripos molecular modeling
packages Sybyl-X 2.0. IIA-2 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 IN complex was retrieved from the Protein Data Bank (PDB code: 3S3M).
The protein was prepared by using the Biopolymer application accompanying Sybyl. The
dolutegravir (DTG) ligand was extracted from the complexes, water molecules were removed,
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 IIA-2 was surflex-docked into the binding pocket of DTG.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We thank K. Erven, K. Uyttersprot and C. Heens for technical assistance with the HIV assays.
The financial support from the National Natural Science Foundation of China (NSFC No.
81573347), Key Project of NSFC for International Cooperation (No. 81420108027), Young
Scholars Program of Shandong University (YSPSDU, No. 2016WLJH32), the Science and
Technology Development Project of Shandong Province (No. 2014GSF118012), Key Research
and Development Project of Shandong Province (No. 2017CXGC1401) and the Major Project of
Science and Technology of Shandong Province (2015ZDJS04001) is gratefully acknowledged.
Work in Madrid was supported by grant BIO2016-76716-R (AEI/FEDER, UE) (Spanish
Ministery of Economy, Industry and Competitiveness) and an institutional grant of Fundación

Ramón Areces.
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