Indazolyl-substituted piperidin-4-yl-aminopyrimidines as HIV-1 NNRTIs: Design, synthesis and biological activities

Article abstract of DOI:10.1016/j.ejmech.2019.111864

A series of indazolyl-substituted piperidin-4-yl-aminopyrimidines (IPAPYs) were designed from two potent HIV-1 NNRTIs piperidin-4-yl-aminopyrimidine 3c and diaryl ether 4 as the lead compounds by molecular hybridization strategy. The target molecules 5a-q

Full text of DOI:10.1016/j.ejmech.2019.111864

Journal Pre-proof
Indazolyl-substituted piperidin-4-yl-aminopyrimidines as HIV-1 NNRTIs: Design,
synthesis and biological activities
Ting Xiao, Jia-Fan Tang, Ge Meng, Christophe Pannecouque, Yuan-Yuan Zhu, Gen-
Yan Liu, Zhi-Qiang Xu, Feng-Shou Wu, Shuang-Xi Gu, Fen-Er Chen
PII:
S0223-5234(19)31016-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111864
EJMECH 111864
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 October 2019
Revised Date: 25 October 2019
Accepted Date: 6 November 2019
Please cite this article as: T. Xiao, J.-F. Tang, G. Meng, C. Pannecouque, Y.-Y. Zhu, G.-Y. Liu, Z.-Q. Xu,
F.-S. Wu, S.-X. Gu, F.-E. Chen, Indazolyl-substituted piperidin-4-yl-aminopyrimidines as HIV-1 NNRTIs:
Design, synthesis and biological activities, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111864.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Masson SAS.

Graphical abstract

Indazolyl-substituted piperidin-4-yl-aminopyrimidines as
HIV-1 NNRTIs: design, synthesis and biological activities
Ting Xiaoa, 1, Jia-Fan Tanga, 1, Ge Mengb, Christophe Pannecouquec, Yuan-Yuan Zhud,
Gen-Yan Liua, Zhi-Qiang Xua, Feng-Shou Wua, Shuang-Xi Gua, *, Fen-Er Chena, b,
*
a
Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical
Engineering & Pharmacy, Wuhan Institute of Technology, Wuhan 430205, China
b Department of Chemistry, Fudan University, Shanghai 200433, China
c
KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and
Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium
d School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan
430205, China
1 Authors Ting Xiao and Jia-Fan Tang contributed equally to this work.
* Corresponding authors
E-mail addresses: shuangxigu@163.com (S.-X. Gu), rfchen@fudan.edu.cn (F.-E. Chen)
Abstract: A series of indazolyl-substituted piperidin-4-yl-aminopyrimidines (IPAPYs) were
designed from two potent HIV-1 NNRTIs piperidin-4-yl-aminopyrimidine 3c and diaryl ether 4 as
the lead compounds by molecular hybridization strategy. The target molecules 5a-q were
synthesized and evaluated for their anti-HIV activities and cytotoxicities in MT-4 cells. 5a-q
displayed moderate to excellent activities against wild-type (WT) HIV-1 with EC50 values ranging
from 1.5 to 0.0064 µM. Among them, 5q was regarded as the most excellent compound against
WT HIV-1 (EC50 = 6.4 nM, SI = 2500). And also, it displayed potent activities against K103N
(EC50 = 0.077 µM), Y181C (EC50 = 0.11 µM), E138K (EC50 = 0.057 µM), and moderate activity
against double mutants RES056 (EC50 = 8.7 µM). Moreover, the structure-activity relationships
(SARs) were summarized, and the molecular docking was performed to investigate the binding
mode of IPAPYs and HIV-1 reverse transcriptase.
Keywords: AIDS; Anti-HIV; NNRTIs; Piperidin-4-yl-aminopyrimidines; Molecular hybridization

1. Introduction
Reverse transcriptase (RT) plays a vital role in the process of the human immunodeficiency
virus type 1 (HIV-1) replication [1,2]. Nonnucleoside reverse transcriptase inhibitors (NNRTIs),
known as one of the indispensable components of highly active antiretroviral therapy (HAART)
for specifically inhibiting HIV-1, have received wide attention due to their potent antiviral activity,
high specificity and low cytotoxicity [3-5]. In the past decades, many series of NNRTIs have
sprung up to fight against HIV-1/AIDS [6], such as benzophenones [7,8],
dihydroalkoxybenzyl-oxopyrimidines (DABOs) [9,10], diaryl ethers [11,12], diaryltriazines
(DATAs) [13,14], and diarylpyrimidines (DAPYs) [15,16] and so on. Among them,
diarylpyrimidines (DAPYs, Fig. 1), represented by FDA-approved drugs etravirine (TMC125, Fig.
1) and rilpivirine (TMC278, Fig. 1), have been recognized as one of the most successful classes of
NNRTIs developed so far due to their excellent potency against wild-type (WT) HIV-1 and its
mutant strains. In recent years, a great deal of DAPYs and their analogues emerged as HIV-1
NNRTIs [17-21].
In earlier years, the 4-cyanophenylamino structure (wing I) was regarded as the essential
pharmacophore of classical DAPYs (Fig. 1). However, in 2010, Tang & Kertesz group [22,23]
combined
the
structure
of
DAPYs
and
the
benzophenones
to
design
piperidin-4-yl-aminopyrimidines 3a-c (Fig. 1), which showed potent activities against WT HIV-1
(7-34 nM) as well as HIV-1 mutants. It demonstrated that phenyl- or benzyl-substituted piperidine
was also an excellent pharmacophore which could replace the 4-cyanophenylamino (wing I) of
classical DAPYs. More importantly, it is well-known that the pharmacokinetic profiles of most
DAPYs are not satisfactory due to low water solubility, while piperidine-linked aminopyrimidine
derivatives could improve the water solubility and bioavailability [24,25]. Based on these,
medicinal chemists have discovered dozens of new series of piperidin-4-yl-aminopyrimidines as
HIV-1 NNRTIs [15,25,26].
Fig. 1 Structures of classical DAPYs and their analogues
Herein, the structures of piperidin-4-yl-aminopyrimidine 3c [22] and diaryl ether 4 [27] as
two potent HIV-1 NNRTIs, were hybridized to design a novel series of indazolyl-substituted
piperidin-4-yl-aminopyrimidines (5, IPAPYs, Fig. 2), which were synthesized and evaluated for
their anti-HIV activities as well as their cytotoxicities. Their structure-activity relationships were
summarized, and the molecular docking was performed to investigate their binding mode.

Fig. 2 Design of target molecules 5 by molecular hybridization
2. Results and discussion
2.1 Chemistry
The synthetic route of the target compounds 5a-q is shown in Scheme 1. The key
intermediate 1-Boc-3-bromomethylindazole 9 was synthesized from 1H-indazole-3-carboxylic
acid 6 as a starting material via three-step reactions including reduction, NH protection and
bromination, successively. Meanwhile, the key intermediates 13a-q were prepared from
2,4-dichloropyrimidine 10 by two nucleophilic reactions followed by removal of the protecting
group Boc. Then, 14a-q were obtained by a nucleophilic substitution reaction of 9 and 13a-q
using K2CO3 as base in the presence of 18-crown-6 in THF. Finally, the intermediates 14a-q were
subjected to deprotection of amino group to get the corresponding target compounds 5a-q.
Compd
5a
R
Compd
5g
R
Compd
5m
5n
R
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CF3
3-CF3
4-CF3
3-CH3
4-CH3
3-CN
4-CN
2,6-diMe
5b
5h
3-Cl-5-CN
3,5-diMe-4-Cl
2,6-diMe-4-CN
5c
5i
5o
5d
5j
5p
5e
5k
5q
5f
5l
Scheme 1. Reagents and conditions: (a) LiAlH4, THF; (b) Boc2O, dimethylaminopyridine,
triethylamine, THF; (c) PBr3, DMF; (d) substituted phenol, N,N-diisopropylethylamine, DMSO; (e)
4-amino-1-Boc-piperidine, N,N-diisopropylethylamine, NMP; (f) CF3COOH, DCM, rt; (g) K2CO3,

18-crown-6, THF; (h) CF3COOH, DCM, rt.
2.2 Biological activity
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method [28,29]
was used to evaluate 17 new IPAPYs (5a-q) along with five FDA-approved drugs: zidovudine,
lamivudine, nevirapine, etravirine, efavirenz as reference drugs. The compounds were assayed for
their anti-HIV activities in MT-4 cells against WT HIV-1 (strain IIIB), common HIV-1 mutants
[K103N, Y181C, E138K, K103N+Y181C (RES056)], as well as HIV-2 (strain ROD). The results
were summarized in Table 1, which were expressed as CC50 (50% cytotoxic concentration), EC50
(50% HIV-1 replication inhibitory concentration) as well as SI (selectivity index given by the
CC50/EC50 (IIIB) ratio) values.
As shown in Table 1, compounds 5a-q displayed moderate to excellent activities against WT
HIV-1 with EC50 values ranging from 1.5 to 0.0064 µM. Among them, the two most potent
compounds 5n and 5q displayed extremely potent WT HIV-1 inhibitory activities with EC50
values of 8.6 nM and 6.4 nM, respectively, which surpassed the corresponding activities of the
FDA-approved zidovudine, lamivudine and nevirapine.
The 17 target molecules are chemically and biologically diverse. Among the compounds 5a-f,
the two molecules 5a (EC50 = 0.037 µM) and 5d (EC50 = 0.031 µM) with an ortho substituent were
obviously superior to 5b-c and 5e-f (EC50 = 0.21-0.28 µM) with a meta- or para-substituent in WT
inhibitory activities. The replacement of the halogen atoms of 5a-f with a trifluoromethyl (5g-i)
led to a dramatic decrease of activities. For compounds 5a-i bearing a halogen atom or CF3, the
substituent priority is as follows, -Br ≥ -Cl > -CF3.
For compounds 5j-k bearing an electron-donating methyl group, the para-methyl (5k, EC50
=
0.14 µM) seemed to be more favorable than meta-methyl (5j, EC50 = 0.39 µM). For compounds
5l-m bearing an electron-withdrawing cyano group, the para-CN (5m, EC50 = 0.078 µM) showed
an overwhelming priority for anti-HIV-1 IIIB activity compared with meta-CN (5l, EC50 = 1.5
µM). Not surprisingly, the compound 5q, featuring a 2,6-diMe-4-CN-Ph left wing which was the
same as the left wing of etravirine (Fig. 1), displayed the most potent activities against WT HIV-1
(EC50 = 6.4 nM). While 4-CN of 5q was removed, the corresponding compound 5n also exhibited
excellent activity against WT HIV-1 (EC50 = 8.6 nM). The two compounds 5o (EC50 = 2.3 µM) and
5p (EC50 = 0.37 µM) with 3- and 5-substituents were inferior to the two compounds 5n (EC50 =
0.086 µM) and 5q (EC50 = 0.064 µM) with a 2,6-dimethyl.
All target molecules have also been evaluated for their activities against the RT mutants
(K103N, Y181C, E138K, RES056). Most of compounds 5a-q showed moderate activities against
single mutants K103N, Y181C and E138K, and were nearly devoid of activities against double
mutants RES056 as well as HIV-2 (ROD). The most excellent compound 5q displayed potent
activities against K103N (EC50 = 0.077 µM), Y181C (EC50 = 0.11 µM), E138K (EC50 = 0.057
µM), and moderate activity against RES056 (EC50 = 8.7 µM).

Table 1 Anti-HIV activities and cytotoxicities of 5a–q in MT-4 cells.a
b
d
EC50 （µM）
RODc
CC50
Compd
SIe
IIIB
0.037
0.28
K103N Y181C E138K RES056 （µM） （µM）
5a
>5.4
9.8
1.6
9.0
0.80
4.7
>5.4
>26
>30
>5.3
>22
>24
>15
>22
>29
>32
>32
>32
>31
>18
>27
>15
8.7
>5.4
>26
>30
>5.3
>22
>24
>15
>22
>29
>32
>32
>32
>31
>18
>27
>15
>16
0.021
NA
5.4
26
146
93
5b
5c
0.23
4.0
5.6
2.3
30
130
171
88
5d
0.031
0.25
1.9
1.0
0.48
3.5
5.3
22
5e
4.9
6.1
5f
0.21
5.7
7.5
2.0
24
114
50
5g
0.30
8.3
6.8
4.4
15
5h
0.55
8.9
9.7
7.0
22
40
5i
0.47
10
>29
≥11
4.3
4.8
29
62
5j
5k
0.39
9.6
4.0
32
82
0.14
3.8
0.77
>32
1.4
32
229
21
5l
1.5
>32
2.6
>32
3.13
0.40
>27
8.7
32
5m
0.078
0.0086
2.3
31
397
2093
12
5n
0.22
>27
8.2
0.084
>27
4.7
18
5o
27
5p
0.37
15
41
5q
0.0064
0.018
3.10
0.077
0.019
2.0
0.11
0.012
2.4
0.057
0.019
2.9
16
2500
>417
>28
>100
>1438
>2423
Zidovudine
Lamivudine
Nevirapine
Etravirine
Efavirenz
0.028
5.0
>7.5
>87
>15
>4.6
>6.3
0.15
5.6
10
0.16
0.0067
0.0044
>15
0.034
0.24
>15
NA
0.0032
0.0026
0.0032
0.076
0.014
0.0050
NA
a Data represent the mean of at least three independent experiments.
b Compound concentration required to protect MT-4 cells against HIV-1-induced cytopathogenicity by 50%.
c Compound concentration required to protect MT-4 cells against HIV-2-induced cytopathogenicity by 50%.
d Compound concentration that decreases the uninfected MT-4 cell viability by 50%.
e Selectivity index: CC50/EC50 (IIIB) ratio.
2.3 Molecular modeling analysis
To investigate the binding mode of our newly synthesized compounds, the molecular docking
studies of the two representative compounds 5n and 5q were performed by using the SYBYL
Surflex-Dock program and crystal structure of WT HIV-1 RT/3c (PDB code: 3NBP) [30]. As
illustrated in Fig. 3, compounds 5q (Fig. 3a) and 5n (Fig. 3b) displayed similar binding mode as
3c in NNIBS, adopting similar “U” shape binding mode. π-π stacking interaction formed between
the left wing of compounds 5n (or 5q) and the aromatic amino acid residues Tyr181, Tyr188 and
Trp229. And a key hydrogen bond formed between the hydrogen of NH liner and the carbonyl of

Lys101. Moreover, the piperidinyl-linked indazole moiety extended to the protein-solvent
interface, and another hydrogen-bonding interaction was found to form between the NH of the
indazole and the main chain of Lys103. For another, as illustrated in Fig. 3, the binding mode of
the left wing of 5n and 5q showed almost perfect coincidence. And the 2,6-dimethyl-4-cyano
fragment of 5q could build extensive interactions with sub pocket composed by hydrophobic
amino acid residue Trp229, Thr181 and Phe227.
Fig. 3. a) Binding mode of 5q (gray) and overlap with 3c (cyan) in NNIBS; b) Binding mode of
5n (gray) and overlap with 3c (cyan) in NNIBS.
3. Conclusion
In summary, as a continuing effort to develop novel NNRTIs, a series of structurally diverse
analogues of indazolyl-substituted piperidin-4-yl-aminopyrimidines (IPAPYs) were designed and
synthesized. The results of biological activities indicated that all target compounds showed good
to excellent activities against WT HIV-1 strain. Among them, 5q was regarded as the most
excellent compound against WT HIV-1 (EC50 = 6.4 nM, SI = 2500). And also, it displayed potent
activities against K103N (EC50 = 0.077 µM), Y181C (EC50 = 0.11 µM), E138K (EC50 = 0.057
µM), and moderate activity against RES056 (EC50 = 8.7 µM). The preliminary structure-activity
relationships were summarized, and the molecular docking was performed to investigate the
binding mode of representative inhibitors and HIV-1 RT, which laid a foundation for the further
development of new DAPY analogues as NNRTIs.
4. Experimental
4.1. Chemistry
Melting points were measured on a SGW X-4B microscopic melting-point apparatus.
Nuclear magnetic resonance (NMR) spectra on two Varian spectrometers (400, 600, 800 MHz for
1H NMR; 200 MHz for 13C NMR) were recorded in DMSO-d6 or CDCl3. Chemical shifts were
reported in δ (ppm) units relative to the internal standard tetramethylsilane. For most of target

molecules, high resolution mass spectra (HRMS) was obtained on an Agilent Q-TOF6300
instrument, and the deviation between the calculated values and measured values of HRMS is
less than 5‰. And for others, mass spectra was obtained on a Waters Quattro Micromass
instrument using electrospray ionization (ESI) techniques. All chemicals and solvents were of
reagent grade and purified and dried by standard methods before use. All the reactions were
monitored by thin layer chromatography (TLC) on pre-coated silica gel G plates at 254 nm under
a UV lamp. Column chromatography separations were performed on silica gel (200–300 mesh).
4.1.1 Synthesis of 3-hydroxymethylindazole (7)
LiAlH4 (1.176 g, 31.0 mmol) was added in portions to a solution of 1H-indazole-3-carboxylic
acid (2.506 g, 15.5 mmol) in anhydrous THF (70 mL) at 0~5 . The resulting solution was stirred
for 4 h at room temperature and then the reaction was quenched by the addition of 2 mL water,
HCl (1 M) at ice bath. Then, celite was added, and the resulting mixture was stirred for 5 min. The
mixture was filtered, and the filtrate was concentrated to remove most of solvent. The residue was
extracted with ethyl acetate. The combined organic layers were washed with water, dried over
anhydrous sodium sulfate, and concentrated under vacuum to get 3-hydroxymethylindazole (7) as
1
a light brown solid (1.65 g) in a yield of 72%, H NMR (DMSO-d6, 400 MHz) δ 12.79 (s, 1H,
IndNH), 7.85-7.83 (d, 1H, ArH), 7.49-7.47 (d, 1H, ArH), 7.35-7.31 (t, 1H, ArH), 7.11-7.07 (t, 1H,
ArH), 5.24-5.21 (t, 1H, OH), 4.80-4.78 (d, 2H, -CH2-).
4.1.2 Synthesis of 1-Boc-3-hydroxymethylindazole (8)
Triethylamine (1.5 mL, 11.0 mmol), DMAP (0.182 g, 1.5 mmol), and Boc2O (1.610 g, 7.4
mmol) were successively added to the solution of compound 7 (1.09 g, 7 mmol) in dry THF (70
ml), and the mixture was stirred at room temperature for 2 h. The solvent was removed under
reduced pressure. Then the residue was dissolved in dichloromethane (80 mL). The solution was
washed with water (2 × 50 mL), dried over anhydrous sodium sulfate and then concentrated in
vacuum. The crude product was purified by column chromatography using gradient elution
(EA/PE = 8:1 to 4:1) to give 8 (0.9 g, 49.3%). 1H NMR (DMSO-d6, 400 MHz) δ 8.08-8.06 (d, 1H,
ArH), 7.97-7.95 (d, 1H, ArH), 7.60-7.56 (t, 1H, ArH), 7.38-7.34 (t, 1H, ArH), 5.56 (s, 1H, OH),
4.82-4.81 (d, 2H, -CH2-), 1.63 (s, 9H, -COOC(CH3)3-).
4.1.3 Synthesis of 1-Boc-3-bromomethylindazole (9)
Compound 8 (0.9 g, 3.6 mmol) was dissolved in anhydrous DMF (35 mL). PBr3 (0.69 mL,
7.3 mmol) was added slowly and the mixture was stirred for 1 h at room temperature. 1 M sodium
hydroxide solution (50 mL) was added, and the mixture was extracted with ethyl acetate (70 mL).
The organic layer was washed with H2O (2 × 50 mL), dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The crude product was purified by column chromatography
1
(EA:PE = 1:20) to afford 9 (0.7 g, 62.1%) as a white solid. H NMR (DMSO-d6, 400 MHz) δ
8.10-8.08 (d, 1H, ArH), 7.97-7.95 (d, 1H, ArH), 7.66-7.62 (t, 1H, ArH), 7.46-7.43 (t, 1H, ArH), 5.05
(s, 2H, -CH2-), 1.65(s, 9H, -COOC(CH3)3-).

4.1.4 Synthesis of intermediates 11a-q
A reaction mixture of 2-chlorophenol (1.726 g, 13.4 mmol) and sodium hydroxide in
N,N-dimethylformamide (30 ml) was stirred at ice bath for 5 min. Then 2,4-dichloropyrimidine
(2.005 g, 13.5 mmol) was added to the solution and continued to stir for 2 h at room temperature
(monitored by TLC). After completion of the reaction, the mixture was poured slowly to the ice
brine (100 ml) under vigorous stirring, the precipitates was gathered by filtration, washed with ice
water (3 × 10 ml) and dried to get 2-chloro-4-(2-chlorophenoxy)pyrimidine 11a as a white solid
(2.787 g, yield 75.4%), which was used directly in the next step without further purification.
The compounds 11b-q were prepared using the same method.
4.1.5 Synthesis of intermediates 12a-q
A mixture of 2-chloro-4-(2-chlorophenoxy)pyrimidine 11a (2.502 g, 10.4 mmol),
4-amino-1-Boc-piperidine (2.500 g, 12.5 mmol) and DIEA (3.4 mL, 20.6 mmol) in
N-methylpyrrolidone (40 mL) was slowly heated at 100 for 4h under argon. The reaction
mixture was cooled to room temperature and poured into water and extracted with ethyl acetate
(100 ml). Then organic layer was washed with brine (3 × 80 ml), dried over anhydrous sodium
sulfate and concentrated under reduced pressure to get crude product, which was purified by
column chromatography using gradient elution (EA/PE = 8:1 to 3:1, v/v, containing 1% TEA) to
obtain 12a.
The compounds 12b-q were prepared using the same method.
1
12a: white solid, yield 37.6%; H NMR (CDCl3, 600 MHz) δ 8.14-8.13 (d, 1H, PyH6),
7.45-7.17 (m, 4H, PhH), 6.15-6.14 (d, 1H, PyH5), 5.11 (brs, 1H, NH), 3.96-3.45 (m, 3H, PipH), 2.71
(s, 2H, PipH), 1.87 (s, 2H, PipH), 1.44 (s, 9H, 3CH3), 1.28-1.25 (m, 2H, PipH).
12b: white solid, yield 37.3%; 1H NMR (CDCl3, 400 MHz) δ 8.13 (s, 1H, PyH6), 7.34-6.89 (m,
4H, PhH), 6.12-6.10 (d, 1H, PyH5), 5.30 (brs, 1H, NH), 3.97 (s, 2H, PipH), 2.81 (s, 2H, PipH),
1.92-1.90 (m, 2H, PipH), 1.44 (s, 9H, C(CH3)3), 1.35-1.30 (m, 3H, PipH).
12c: white solid, yield 44.2%; 1H NMR (CDCl3, 400 MHz) δ 8.11 (s, 1H, PyH6), 7.36-7.06 (m,
4H, PhH), 6.10-6.09 (d, 1H, PyH5), 5.65-4.85 (brd, 1H, NH), 3.98 (s, 2H, PipH), 2.83 (s, 2H, PipH),
1.91 (s, 2H, PipH), 1.44 (s, 9H, C(CH3)3), 1.36-1.30 (m, 3H, PipH).
12d: white solid, yield 45.1%; 1H NMR (CDCl3, 400 MHz) δ 8.10-8.09 (d, 1H, PyH6),
7.60-7.08 (m, 4H, PhH), 6.13-6.12 (d, 1H, PyH5), 5.41 (brs, 1H, NH), 3.93 (s, 2H, PipH), 2.69 (s,
2H, PipH), 1.84 (s, 2H, PipH), 1.42 (s, 9H, C(CH3)3), 1.26-1.23 (m, 3H, PipH).
1
12e: white solid, yield 33.0%; H NMR (CDCl3, 400 MHz) δ (ppm): 8.15-8.14 (d, 1H, PyH6),
7.38-7.08 (m, 4H, PhH), 6.15-6.14 (d, 1H, PyH5), 5.35 (brs, 1H, NH), 4.04-3.75 (m, 3H, PipH), 2.84
(s, 2H, PipH), 1.96-1.93 (m, 2H, PipH), 1.47 (s, 9H, C(CH3)3), 1.38-1.30 (m, 2H, PipH).
1
12f: white solid, yield 33.9%; H NMR (CDCl3, 400 MHz) δ 8.11-8.10 (d, 1H, PyH6),
7.50-7.48 (d, 2H, PhH), 7.02-7.00 (d, 2H, PhH), 6.09-6.08 (d, 1H, PyH5), 5.53-4.92 (brd, 1H, NH),
3.98-3.75 (m, 3H, PipH), 2.82 (s, 2H, PipH), 1.91 (s, 2H, PipH), 1.44 (s, 9H, C(CH3)3), 1.34-1.29 (m,
2H, PipH).

12g: white solid, yield 44%; 1H NMR (CDCl3, 400 MHz) δ 8.17-8.16 (d, 1H, PyH6), 7.73-7.28
(m, 4H, PhH), 6.24-6.23 (d, 1H, PyH5), 5.26 (brs, 1H, NH), 3.97 (s, 2H, PipH), 2.77 (s, 2H, PipH),
1.88 (s, 2H, PipH), 1.47 (s, 9H, C(CH3)3), 1.31-1.29 (m, 3H, PipH).
1
12h: white solid, yield 36.3%; H NMR (CDCl3, 400 MHz) δ 8.13-8.12 (d, 1H, PyH6),
7.53-7.30 (m, 4H, PhH), 6.16-6.15 (d, 1H, PyH5), 5.50 (brs, 1H, NH), 3.95 (s, 2H, PipH), 2.73 (s,
2H, PipH), 1.86 (s, 2H, PipH), 1.43 (s, 9H, C(CH3)3), 1.32-1.24 (m, 3H, PipH).
12i: white solid, yield 27.6%; 1H NMR (CDCl3, 400 MHz) δ 8.13 (s, 1H, PyH6), 7.65-7.22 (m,
4H, PhH), 6.15-6.13 (d, 1H, PyH5), 5.62-4.86 (brd, 1H, NH), 3.95 (s, 3H, PipH), 2.78 (s, 2H, PipH),
1.88 (s, 2H, PipH), 1.42 (s, 9H, C(CH3)3), 1.30-1.27 (m, 2H, PipH).
1
12j: yellow oil, yield 44.8%; H NMR (CDCl3, 400 MHz) δ 7.99-7.98 (d, 1H, PyH6),
7.18-6.82 (d, 4H, PhH), 5.94-5.92 (d, 1H, PyH5), 3.91-3.69 (m, 3H, PipH), 2.71 (s, 2H, PipH), 2.26
(s, 3H, CH3), 1.86-1.83 (d, 2H, PipH), 1.37 (s, 9H, C(CH3)3), 1.27-1.20 (m, 2H, PipH).
1
12k: white solid, yield 39.2%; H NMR (CDCl3, 400 MHz) δ 8.07-8.06 (d, 1H, PyH6),
7.18-6.98 (m, 4H, PhH), 6.01-5.99 (d, 1H, PyH5), 5.39 (brs, 1H, NH), 3.98-3.82 (m, 3H, PipH),
2.82 (s, 2H, PipH), 2.35 (s, 3H, CH3), 1.94-1.92 (d, 2H, PipH), 1.44 (s, 9H, C(CH3)3), 1.32-1.29 (m,
2H, PipH).
1
12l: white solid, yield 30.6%; H NMR (CDCl3, 400 MHz) δ 8.09-8.08 (d, 1H, PyH6),
7.46-7.33 (m, 4H, PhH), 6.12-6.10 (d, 1H, PyH5), 5.08 (brs, 1H, NH), 3.93 (s, 2H, PipH), 2.71 (s,
2H, PipH), 1.82 (s, 2H, PipH), 1.39 (s, 9H, C(CH3)3), 1.26-1.22 (m, 3H, PipH).
1
12m: white solid, yield 21.5%; H NMR (CDCl3, 400 MHz) δ 8.17-8.15 (d, 1H, PyH6),
7.70-7.24 (m, 4H, PhH), 6.19-6.17 (d, 1H, PyH5), 5.46-4.94 (brd, 1H, NH), 3.97-3.79 (m, 3H, PipH),
2.81 (s, 2H, PipH), 1.89 (s, 2H, PipH), 1.44 (s, 9H, C(CH3)3), 1.35-1.26 (m, 2H, PipH).
12n: white solid, yield 35.9%; 1H NMR (CDCl3, 400 MHz) δ 8.08-8.07 (d, 1H, PyH6), 7.06 (s,
3H, PhH), 5.99-5.98 (d, 1H, PyH5), 5.45-4.98 (brd, 1H, NH), 3.97-3.68 (m, 3H, PipH), 2.79 (s, 2H,
PipH), 2.11 (s, 6H, 2CH3), 1.89 (s, 2H, PipH), 1.44 (s, 9H, C(CH3)3), 1.32-1.25 (m, 2H, PipH).
12o: white oil, yield 23.9%; 1H NMR (CDCl3, 400 MHz) δ 8.17-8.16 (d, 1H, PyH6), 7.49-7.38
(s, 3H, PhH), 6.20-6.18 (d, 1H, PyH5), 5.70-4.86 (brd, 1H, NH), 3.99 (s, 3H, PipH), 2.78 (s, 2H,
PipH), 1.89 (s, 2H, PipH), 1.43 (s, 9H, C(CH3)3), 1.35-1.31 (m, 2H, PipH).
1
12p: white oil, yield 46.2%; H NMR (CDCl3, 400 MHz) δ 7.97(s, 1H, PyH6), 6.77 (s, 2H,
PhH), 5.92-5.91 (d, 1H, PyH5), 5.17(brs, 1H, NH), 3.91-3.62 (s, 3H, PipH), 2.68 (s, 2H, PipH), 2.55
(s, 6H, 2CH3), 1.83-1.81 (s, 2H, PipH), 1.35 (s, 9H, C(CH3)3), 1.23-1.20 (m, 2H, PipH).
1
12q: white solid, yield 63.7%; H NMR (CDCl3, 400 MHz) δ 7.96 (s, 1H, PyH6), 7.26 (s, 2H,
PhH), 6.01-6.00 (d, 1H, PyH5), 3.80 (s, 2H, PipH), 2.60 (s, 2H, PipH), 2.01 (s, 6H, 2CH3), 1.91-1.89
(d, 1H, NH), 1.66(s, 2H, PipH), 1.31 (s, 9H, C(CH3)3), 1.15-1.09 (m, 3H, PipH).
4.1.6 Synthesis of intermediates 13a-q
Trifluoroacetic acid (2.8 mL, 37.7 mmoL) was added to a solution of 12a (1.52 g, 3.4 mmoL)
in dichloromethane (30 mL) in ice bath for 0.5 h, then the ice bath was removed. The mixture was
stirred for 3 h. After completion of the reaction, the reaction mixture was alkalized with saturated

sodium bicarbonate solution until no gas bubbled. The aqueous layer was extracted with
dichloromethane (1 × 50 ml) and washed with water (2 × 50 ml). The organic layer was dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 13a (1.101
g, yield 96.2%). The crude product was used directly for the next step.
The intermediates 13b-q were prepared using the same method.
4.1.7 Synthesis of intermediates 14a-q
Compound 13a (0.707 g, 2.3 mmol), 9 (0.789 g, 2.5 mmol) and 18-Crown-6 (0.061 g, 0.23
mmol) were dissolved in tetrahydrofuran (18 mL) in the presence of anhydrous potassium
carbonate, the mixture was stirred at room temperature for 4 h. After completion of the reaction,
water (80 ml) and ethyl acetate (80 mL) was added for extraction. The organic layer was washed
with brine (2 × 80 ml), then dried over anhydrous sodium sulfate to obtain the corresponding
crude product, which was purified by column chromatography using gradient elution (EA/PE =
3:1 to 1:1, v/v, containing 1% TEA) to afford 14a (0.47g, yield 38.1%) as white grease.
The intermediates 14b-q were prepared using the same method.
4.1.8 Synthesis of intermediates 5a-q
Trifluoroacetic acid (1.2 mL, 16.1 mmol) was added to a solution of 14a (0.418 g, 0.8 mmol)
in dichloromethane (10 mL) in ice bath and stirred for 0.5 h. Then the ice bath was removed, and
the mixture was stirred for another 3 h (monitored by TLC). Then, the reaction solution was
alkalized with saturated sodium bicarbonate solution until no gas bubble. The aqueous phase was
extracted with dichloromethane (60 ml) and washed with water (2 × 50 ml). The organic phase
was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The
crude product was purified by stirring and rinsing with
(n-hexane/dichloromethane, 6:1, v/v) to obtain 5a.
a
mixed solvent
The compounds 5b-q were prepared using the same method.
1
5a: n-hexane/dichloromethane (6:1, v/v), white solid, yield 52.9%, mp 182.9-187.3 ; H
NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, IndNH), 8.16 (s, 1H, PyH6), 7.84-7.07 (m, 8H, ArH),
6.19-6.18 (d, 1H, PyH5), 3.79-3.70 (m, 3H, -CH2- and PipH), 2.84-2.79 (m, 2H, PipH), 2.07-1.34
(m, 6H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 168.55, 161.63, 160.27, 159.84, 148.25,
140.87, 130.32, 128.55, 126.99, 126.47, 125.86, 124.54, 122.15, 120.60, 119.72, 110.03, 95.82,
54.41, 52.09, 48.33, 31.04; HRMS (ESI+) calcd for C23H23ClN6O: 435.1622. Found: 435.1698
(M+H)+.
1
5b: n-hexane/dichloromethane (10:1, v/v), white solid, yield 57.6%, mp 154.3-156.8 ; H
NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H, IndNH), 8.15 (s, 1H, PyH6), 7.84-7.01 (m, 8H, ArH),
6.14 (s, 1H, PyH5), 3.78-3.69 (m, 3H, -CH2- and PipH), 2.82 (s, 2H, PipH), 2.04-1.42 (m, 6H, PipH);
13C NMR (200 MHz, DMSO-d6): δ 168.90, 161.60, 160.35, 159.88, 153.22, 142.28, 140.88, 133.53,
130.67, 125.84, 125.34 , 122.15, 120.62, 119.68, 118.04, 110.01, 96.32, 54.55, 52.24, 48.02, 31.28;
MS (ESI+) calcd for C23H23ClN6O: 435.16. Found: 435.99 (M+H)+.
1
5c: n-hexane/dichloromethane (8:1, v/v), white solid, yield 51.9%, mp 194.3-196.4 ; H

NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, IndNH), 8.14 (s, 1H, PyH6), 7.84-7.05 (m, 8H, ArH),
6.15-6.10 (d, 1H, PyH5), 3.77-3.67 (m, 3H, -CH2- and PipH), 2.81 (s, 2H, PipH), 2.03-1.40 (m, 6H,
PipH); 13C NMR (200 MHz, DMSO-d6) δ 169.04, 161.67, 159.79, 151.17, 142.34, 140.89, 129.53,
129.25, 125.82, 123.81, 122.12, 120.60, 119.66, 110.01, 96.25, 54.62, 52.42, 48.02, 31.40; HRMS
(ESI+) calcd for C23H23ClN6O: 435.1622. Found: 435.1715 (M+H)+.
1
5d: n-hexane/dichloromethane (6:1, v/v), white solid, yield 76.4%, mp 163.5-166.7 ; H
NMR (400 MHz, DMSO-d6) δ 12.81 (s, 1H, IndNH), 8.16 (s, 1H, PyH6), 7.84-7.06 (m, 8H, ArH),
6.17-6.16 (d, 1H, PyH5), 3.79-3.69 (m, 3H, -CH2- and PipH), 2.84-2.80 (m, 2H, PipH), 2.08-1.34 (m,
6H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 168.54, 161.63, 160.23, 149.49, 142.09, 140.87,
133.31, 129.13, 127.29, 125.85, 124.55, 122.15, 120.59, 119.71, 115.98, 110.03, 95.94, 54.41, 52.40,
47.92, 31.07; HRMS (ESI+) calcd for C23H23BrN6O: 479.1117, 481.1096. Found: 479.1192,
481.1171 (M+H)+.
1
5e: n-hexane/dichloromethane (8:1, v/v), white solid, yield 76.4%, mp 152.1-155.5 ; H
NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H, IndNH), 8.15 (s, 1H, PyH6), 7.85-7.07 (m, 8H, ArH),
6.15 (s, 1H, PyH5), 3.85-3.70 (m, 3H, -CH2- and PipH), 2.88 (s, 2H, PipH), 2.12-1.44 (m, 6H, PipH);
13C NMR (200 MHz, DMSO-d6): δ 168.84, 161.41, 160.33, 159.85, 153.00, 140.86, 131.38,
128.22, 125.88, 124.95, 122.19, 121.74, 121.23, 120.56, 119.78, 110.06, 96.39, 54.19, 52.21,
48.04, 31.05; HRMS (ESI+) calcd for C23H23BrN6O: 479.1117, 481.1096. Found: 479.1168,
481.1151 (M+H)+.
5f: n-hexane/dichloromethane (6:1, v/v), light yellow solid, yield 35.3%, mp 194.3-196.8 ;
1H NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H, IndNH), 8.14 (s, 1H, PyH6), 7.84-7.06 (m, 8H, ArH),
6.15-6.10 (d, 1H, PyH5), 3.77-3.67(m, 3H, -CH2- and PipH), 2.82 (s, 2H, PipH), 2.03-1.40 (m, 6H,
PipH); 13C NMR (200 MHz, DMSO-d6): δ 168.96, 161.38, 159.80, 151.66, 142.30, 140.88, 132.48,
125.83, 124.25, 122.11, 120.59, 119.67, 117.35, 110.01, 96.27, 54.59, 52.39, 47.99, 31.37; HRMS
(ESI+) calcd for C23H23BrN6O: 479.1117, 481.1096. Found: 479.1211, 481.1196 (M+H)+.
1
5g: n-hexane/dichloromethane (8:1, v/v), white solid, yield 46.4%, mp 193.5-195.8 ; H
NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H, IndNH), 8.17 (s, 1H, PyH6), 7.83-7.06 (m, 8H, ArH),
6.21-6.20 (d, 1H, PyH5), 3.77-3.69 (m, 2H, -CH2- and PipH), 2.83-2.77 (m, 2H, PipH), 2.05-1.35 (m,
6H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 169.06, 161.38, 160.37, 149.87, 142.20, 140.88,
133.46, 126.85, 125.84, 125.70, 124.80, 123.86, 122.51, 122.14, 120.61, 119.67, 110.01, 96.17,
54.51, 52.41, 48.03, 31.33; HRMS (ESI+) calcd for C24H23F3N6O: 469.1885. Found: 469.1943
(M+H)+.
5h: n-hexane/dichloromethane (6:1, v/v), white solid, yield 37.3%, mp 158.5-161.7 ; 1H
NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H, IndNH), 8.17 (s, 1H, PyH6), 7.82-7.06 (m, 8H, ArH),
6.20-6.19 (d, 1H, PyH5), 3.76 (s, 3H, -CH2- and PipH), 2.78 (s, 2H, PipH), 2.08-1.38 (m, 6H, PipH);
13C NMR (200 MHz, DMSO-d6): δ 168.73, 161.41, 160.12, 152.57, 142.11, 140.82, 130.59, 125.99,
125.66, 124.29, 122.93, 122.05, 121.65, 120.43, 119.52, 118.98, 109.88, 96.28, 54.43, 52.12, 48.03,
31.17; HRMS (ESI+) calcd for C24H23F3N6O: 469.1885. Found: 469.1887 (M+H)+.
1
5i: n-hexane/dichloromethane (6:1, v/v), white solid, yield 65.5%, mp 169.2-173.6 ; H

NMR (400 MHz, DMSO-d6) δ 13.65 (s, 1H, IndNH), 10.58-10.45 (d, 1H, -NH-); 8.24-8.23 (d, 1H,
PyH6), 8.02-7.19 (m, 8H, ArH), 6.34-6.33 (d, 1H, PyH5), 4.65(s, 2H, -CH2-), 4.06-3.90 (m, 1H,
PipH), 3.53 (s, 2H, PipH ), 3.24-2.94 (m, 2H, PipH ), 2.00-1.72 (m, 4H, PipH); 13C NMR (200 MHz,
DMSO-d6): δ 168.76, 160.93, 159.53, 158.15, 155.30, 140.73, 134.42, 126.84, 126.37, 122.55,
122.42, 120.87, 119.94, 115.62, 110.46, 97.14, 54.76, 50.75, 47.19, 28.27; HRMS (ESI+) calcd for
C24H23F3N6O: 469.1885. Found: 469.1984 (M+H)+.
1
5j: n-hexane/dichloromethane (6:1, v/v), white solid, yield 62.7%, mp 135.8-139.4 ; H
NMR (400 MHz, DMSO-d6) δ 12.81 (s, 1H, IndNH), 8.11 (s, 1H, PyH6), 7.85-6.92 (m, 8H, ArH),
6.04 (s, 1H, PyH5), 3.82-3.69(m, 3H, -CH2- and PipH), 2.84 (s, 2H, PipH), 2.28 (s, 3H, -CH3-),
2.07-1.43 (m, 6H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 169.26, 161.62, 159.62, 152.30,
141.90, 140.82, 139.08, 129.09, 125.70, 125.65, 122.11, 122.05, 120.44, 119.59, 118.48, 109.91,
96.02, 54.27, 52.10, 47.85, 31.15, 20.67; HRMS (ESI+) calcd for C24H26N6O: 415.2168. Found:
415.2267 (M+H)+.
1
5k: n-hexane/dichloromethane (10:1, v/v), white solid, yield 77.6%, mp 141.1-145.6 ; H
NMR (400 MHz, DMSO-d6) δ 13.12 (s, 1H, IndNH), 8.12-8.11 (d, 1H, PyH6), 7.91-7.01 (m, 8H,
ArH), 6.06 (s, 1H, PyH5), 4.11(s, 2H, -CH2-), 3.06 (s, 2H, PipH), 2.29 (s, 3H, -CH3-); 1.82-1.24 (m,
7H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 169.54, 161.52, 160.04, 159.57, 150.10, 140.83,
134.35, 130.05, 129.86, 126.09, 122.35, 120.41, 120.18, 110.23, 96.40, 54.93, 51.67, 47.29, 30.18,
20.38; HRMS (ESI+) calcd for C24H26N6O: 415.2168. Found: 415.2264 (M+H)+.
1
5l: n-hexane/dichloromethane (8:1, v/v), white solid, yield 64.4%, mp 170.9-173.2 ; H
NMR (400 MHz, DMSO-d6): δ 12.78 (s, 1H, IndNH), 8.17 (s, 1H, PyH6), 7.84-7.07 (m, 8H, ArH),
6.20-6.19 (d, 1H, PyH5), 3.79-3.68 (m, 3H, -CH2- and PipH), 2.82 (s, 2H, PipH), 2.05-1.40 (m, 6H,
PipH); 13C NMR (200 MHz, DMSO-d6): δ 168.67, 161.47, 160.48, 152.41, 142.19, 140.88, 131.07,
129.20, 127.39, 125.84, 122.13, 120.61, 119.69, 118.08, 112.39, 110.02, 96.36, 54.53, 52.36, 47.98,
28.53; HRMS (ESI+) calcd for C24H23N7O: 426.1964. Found: 426.2049 (M+H)+.
1
5m: n-hexane/dichloromethane (6:1, v/v), white solid, yield 54%, mp 202.2-205.1 ; H
NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H, IndNH), 8.19 (s, 1H, PyH6), 7.91-7.06 (m, 8H, ArH),
6.21 (s, 1H, PyH5), 3.79-3.71 (m, 3H, -CH2- and PipH), 2.83 (s, 2H, PipH), 2.06-1.39 (m, 6H, PipH);
13C NMR (200 MHz, DMSO-d6): δ 168.49, 161.35, 160.23, 156.17, 142.22, 140.88, 134.16, 125.85,
122.98, 122.12, 120.58, 119.70, 118.52, 110.03, 107.74, 96.57, 54.58, 52.31, 48.02, 31.33; HRMS
(ESI+) calcd for C24H23N7O: 426.1964. Found: 426.2028 (M+H)+.
1
5n: n-hexane/dichloromethane (6:1, v/v), white solid, yield 52.3%, mp 163.5-167.4 ; H
NMR (400 MHz, DMSO-d6): δ 12.80 (s, 1H, IndNH), 8.11 (s, 1H, PyH6), 7.83-7.05 (m, 7H, ArH),
6.03 (s, 1H, PyH5), 3.76-3.67 (m, 3H, -CH2- and PipH), 2.81-2.75 (m, 2H, PipH), 2.02 (s, 7H, -CH3-
and PipH), 1.75-1.33 (m, 5H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 168.49, 161.73, 159.86,
149.29, 142.31, 140.88, 130.23, 128.64, 125.82, 125.32, 122.12, 120.62, 119.64, 109.99, 94.48,
54.59 52.43, 48.06, 31.40, 16.07; MS (ESI+) calcd for C25H28N6O: 429.23. Found: 429.74 (M+H)+.
1
5o: n-hexane/dichloromethane (6:1, v/v), white solid, yield 53.8%, mp 167.1-169.8 ; H
NMR (400 MHz, DMSO-d6): δ 12.78 (s, 1H, IndNH), 8.19 (s, 1H, PyH6), 7.90-7.06 (m, 7H, ArH),

6.24-6.23 (d, 1H, PyH5), 3.78-3.70 (m, 2H, -CH2- and PipH), 2.82 (m, 2H, PipH), 2.04-1.41 (m, 6H,
PipH); 13C NMR (200 MHz, DMSO-d6): δ 168.46, 161.36, 160.15, 153.06, 142.14, 140.87, 134.27,
128.54, 127.98, 125.81, 125.05, 122.15, 120.60, 119.68, 116.91, 113.27, 110.02, 96.32, 54.51, 52.40,
48.07, 31.22; HRMS (ESI+) calcd for C24H22ClN7O: 460.1574. Found: 460.1660 (M+H)+.
1
5p: n-hexane/dichloromethane (6:1, v/v), white solid, yield 62.3%, mp 198.3-200.5 ; H
NMR (400 MHz, DMSO-d6): δ 12.80 (s, 1H, IndNH), 8.12 (s, 1H, PyH6), 7.83-7.03 (m, 6H, ArH),
6.11-6.07 (d, 1H, PyH5), 3.77-3.67 (m, 2H, -CH2- and PipH), 2.82 (s, 2H, PipH), 2.30 (s, 6H, 2CH3),
2.02-1.40 (m, 6H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 169.07, 161.50, 159.64, 150.31,
142.31, 140.85, 137.06, 129.96, 125.81, 122.14, 121.96, 120.56, 119.66, 110.00, 96.13, 54.54,
52.46, 48.02, 31.39, 20.23; HRMS (ESI+) calcd for C25H27ClN6O: 463.1935. Found: 463.2015
(M+H)+.
5q: column chromatography on silica gel (ethyl acetate), white solid, yield 42%, mp
1
160.7-164.5 ; H NMR (400 MHz, DMSO-d6): δ 12.80 (s, 1H, IndNH), 8.17 (s, 1H, PyH6),
7.83-7.03 (m, 6H, ArH), 6.20 (s, 1H, PyH5), 3.76-3.65 (m, 3H,-CH2- and PipH), 2.81-2.74 (m, 2H,
PipH), 2.06 (s, 6H, -CH3-), 1.98-1.29 (m, 6H, PipH); 13C NMR (200 MHz, DMSO-d6): δ 167.79,
161.51, 160.04, 153.23, 142.32, 140.88, 132.66, 132.13, 125.82, 122.09, 120.58, 119.66, 118.62, 110.01,
108.13, 95.22, 54.58, 52.42, 48.07, 31.36, 15.87; HRMS (ESI+) calcd for C26H27N7O: 454.2277.
Found: 454.2341 (M+H)+.
4.2. Anti-HIV activity and cytotoxicity assay in MT-4 cells
The anti-HIV activities and cytotoxicities of the target molecules were evaluated against
wild-type (WT) HIV-1 strain IIIB, common single mutants K103N, Y181C, E138K, double
mutant (K103N+Y181C) (RES056) and HIV-2 strain (ROD) in MT-4 cell cultures using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [28,29]. Briefly,
stock solutions (10 × final concentration) of test compounds were added in 25 µL volumes to two
series of triplicate wells so as to allow simultaneous evaluation of their effects on mock- and
HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds
were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot
(Beckman instruments, Fullerton, CA). Untreated control HIV- and mock-infected cell samples
were included for each sample. Virus stock (50 µL) at 100–300 CCID50 (50% cell culture
infectious dose) or culture medium was added to either the virus-infected or mock-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 the cytotoxicity of the test compounds. Exponentially growing
MT-4 cells were centrifuged for 5 min at 1000 rpm (220 g) and the supernatant was discarded. The
MT-4 cells were resuspended at 6 × 105 cells/mL and 50-µL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of mock- and HIV-infected cells was
examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) (Acros Organics) by mitochondrial dehydrogenase

activity in metabolically active cells to a blue-purple formazan that can be measured
spectrophotometrically. The absorbances were read in an eight-channel computer-controlled
photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were
calculated using the median absorbance value of three wells. The 50% cytotoxic concentration
(CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540
)
of the mock-infected control sample by 50%. The concentration achieving 50% protection against
the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration
(EC50).
4.3. Molecular docking
Molecular docking was performed with the Tripos molecular modeling packages Sybyl-X 1.2.
All the molecules for docking were built using standard bond lengths and angles from Sybyl-X
1.2/base Builder and were then optimized using the Tripos force field for 2000 generations two
times or more, until the minimized conformers of the ligand were the same. The flexible docking
method, called Surflex-Dock, docks the ligand automatically into the ligand binding site of the
receptor by using a protocol-based approach and an empirically-derived scoring function [31-33].
The protocol is a computational representation of a putative ligand that binds to the intended
binding site and is a unique and essential element of the docking algorithm. The scoring function
in Surflex-Dock, which contains hydrophobic, polar, repulsive, entropic, and solvation terms, was
trained to estimate the dissociation constant (Kd) expressed in –log (Kd)2. Prior to docking, the
protein was prepared by removing water molecules, the ligand 3c, and other unnecessary small
molecules from the crystal structure of the 3c–HIV-1 RT complex (PDB code: 3NBP) [30];
simultaneously, polar hydrogen atoms were added to the protein. Surflex-Dock default settings
were used for other parameters, such as the number of starting conformations per molecule (set to
0), the size to expand search grid (set to 8 Å), the maximum number of rotatable bonds per
molecule (set to 100), and the maximum number of poses per ligand (set to 20). During the
docking procedure, all of the single bonds in residue side-chains inside the defined RT binding
pocket were regarded as rotatable or flexible, and the ligand was allowed to rotate at all single
bonds and move flexibly within the tentative binding pocket. The atomic charges were
recalculated using the Kollman all-atom approach for the protein and the Gasteiger–Hückel
approach for the ligand. The binding interaction energy was calculated to include van der Waals,
electrostatic, and torsional energy terms defined in the Tripos force field. The structure
optimization was performed for 20,000 generations using a genetic algorithm, and the
20-best-scoring ligand–protein complexes were kept for further analyses. The –log (Kd)2 values of
the 20-best-scoring complexes, which represented the binding affinities of ligand with RT,
encompassed a wide scope of functional classes (10-2–10-9). Therefore, only the highest-scoring
3D structural model of the ligand-bound RT was chosen to define the binding interaction [34-37].
Acknowledgments

The financial support from the National Natural Science Foundation of China (Nos. 21877087,
21602164, 21807082, 21402148), Wuhan International Scientific and Technological Cooperation
Project (No. 2017030209020257), Natural Science Foundation of Hubei Province of China (No.
2017CFB121) and Hubei Provincial Department of Education of China (No. Q20171503) are
greatly appreciated.
References
[1] E. De Clercq, New developments in anti-HIV chemotherapy. Curr. Med. Chem. 8 (2001)
1543-1572.
[2] Y. Mehellou, E. De Clercq, Twenty-six years of anti-HIV drug discovery: where do we stand
and where do we go? J. Med. Chem. 53 (2010) 521-538.
[3] V. Namasivayam, M. Vanangamudi, V.G. Kramer, S. Kurup, P. Zhan, X. Liu, J. Kongsted, S.N.
Byrareddy, The journey of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs)
from lab to clinic. J. Med. Chem. 62 (2018) 4851-4883.
[4] H.-H. Lu, P. Xue, Y.-Y. Zhu, X.-L. Ju, X.-J. Zheng, X. Zhang, T. Xiao, C. Pannecouque, T.-T.
Li, S.-X. Gu, Structural modifications of diarylpyrimidines (DAPYs) as HIV-1 NNRTIs:
synthesis, anti-HIV activities and SAR. Bioorg. Med. Chem. 25 (2017) 2491-2497.
[5] P. Xue, H.-H. Lu, Y.-Y. Zhu, X.-L. Ju, C. Pannecouque, X.-J. Zheng, G.-Y. Liu, X.-L. Zhang,
S.-X. Gu, Design and synthesis of hybrids of diarylpyrimidines and diketo acids as HIV-1
inhibitors. Bioorg. Med. Chem. Lett. 27 (2017) 1640-1643.
[6] P. Shirvani, A. Fassihi, L. Saghaie, Recent advances in the design and development of
nonnucleoside reverse transcriptase inhibitor scaffolds. ChemMedChem 14 (2019) 52-77.
[7] K.R. Romines, G.A. Freeman, L.T. Schaller, J.R. Cowan, S.S. Gonzales, J.H. Tidwell, C.W.
Andrews III, D.K. Stammers, R.J. Hazen, R.G. Ferris, S.A. Short, J.H. Chan, L.R. Boone,
Structure-activity relationship studies of novel benzophenones leading to the discovery of a
potent, next generation HIV nonnucleoside reverse transcriptase inhibitor. J. Med. Chem. 49
(2006) 727-739.
[8] J.H. Chan, G.A. Freeman, J.H. Tidwell, K.R. Romines, L.T. Schaller, J.R. Cowan, S.S.
Gonzales, C.W. Lowell, C. Andrews III, D.J. Reynolds, M.St. Clair, R.J. Hazen, R.G. Ferris,
K.L. Creech, G.B. Roberts, S.A. Short, K. Weaver, G.W. Koszalka, L.R. Boone, Novel
benzophenones as non-nucleoside reverse transcriptase inhibitors of HIV-1. J. Med. Chem. 47
(2004) 1175-1182.
[9] S. Yang, F.-E. Chen, E. De Clercq, Dihydro-alkoxyl-benzyl-oxopyrimidine derivatives
(DABOs) as non-nucleoside reverse transcriptase inhibitors: an update review (2001-2011).
Curr. Med. Chem. 19 (2012) 152-162.
[10]
[11]
Y.-P. Wang, F.-E. Chen, E. De Clercq, J. Balzarini, C. Pannecouque, Synthesis and in vitro
anti-HIV evaluation of a new series of 6-arylmethyl-substituted S-DABOs as potential
non-nucleoside HIV-1 reverse transcriptase inhibitors. Eur. J. Med. Chem. 44 (2009)
1016-1023.
S.-X. Gu, X. Zhang, Q.-Q. He, L.-M. Yang, X.-D. Ma, Y.-T. Zheng, S.-Q. Yang, F.-E.
Chen, Synthesis and biological evaluation of naphthyl phenyl ethers (NPEs) as novel
nonnucleoside HIV-1 reverse transcriptase inhibitors. Bioorg. Med. Chem. 19 (2011)
4220-4226.

[12]
[13]
X. Chen, S. Ding, P. Zhan, X. Liu, Recent advances of diaryl ether family as HIV-1
non-nucleoside reverse transcriptase inhibitors. Curr. Pharm. Design 19 (2013) 2829-2838.
D.W. Ludovici, R.W. Kavash, M.J. Kukla, C.Y. Ho, H. Ye, B.L.D. Corte, K. Andries, M.-P.
de Béthune, H. Azijn, R. Pauwels, H.E.L. Moereels, J. Heeres, L.M.H. Koymans, M.R. de
Jonge, K.J.A. Van Aken, F.F.D. Daeyaert, P.J. Lewi, K. Das, E. Arnold, P.A.J. Janssen,
Evolution of anti-HIV drug candidates.part 2:diaryltriazine (DATA) analogues. Bioorg. Med.
Chem. Lett. 11 (2001) 2229-2234.
[14]
[15]
Y. Van Herrewege, G. Vanham, J. Michiels, K. Fransen, L. Kestens, K. Andries, P.
Janssen, P. Lewi, A series of diaryltriazines and diarylpyrimidines are highly potent
nonnucleoside reverse transcriptase inhibitors with possible applications as microbicides.
Antimicrob. Agents Chemother. 48 (2004) 3684-3689.
S.-X. Gu, H.-H. Lu, G.-Y. Liu, X.-L. Ju, Y.-Y. Zhu, Advances in diarylpyrimidines and
related analogues as HIV-1 nonnucleoside reverse transcriptase inhibitors. Eur. J. Med. Chem.
158 (2018) 371-392.
[16]
[17]
X. Chen, P. Zhan, D. Li, C.E. De, X. Liu, Recent advances in DAPYs and related
analogues as HIV-1 NNRTIs. Curr. Med. Chem. 18 (2011) 359-376.
S.-X. Gu, Q.-Q. He, S.-Q. Yang, X.-D. Ma, F.-E. Chen, E. De Clercq, J. Balzarini, C.
Pannecouque, Synthesis and structure-activity relationship of novel diarylpyrimidines with
hydroxymethyl linker (CH(OH)-DAPYs) as HIV-1 NNRTIs. Bioorg. Med. Chem. 19 (2011)
5117-5124.
[18]
[19]
S.-X. Gu, S.-Q. Yang, Q.-Q. He, X.-D. Ma, F.-E. Chen, H.-F. Dai, E.D. Clercq, J.
Balzarini, C. Pannecouque, Design, synthesis and biological evaluation of cycloalkyl
arylpyrimidines (CAPYs) as HIV-1 NNRTIs. Bioorg. Med. Chem. 19 (2011) 7093-7099.
S.-X. Gu, Z.-M. Li, X.-D. Ma, S.-Q. Yang, Q.-Q. He, F.-E. Chen, E. De Clercq, J.
Balzarini, C. Pannecouque, Chiral resolution, absolute configuration assignment and
biological activity of racemic diarylpyrimidine CH(OH)-DAPY as potent nonnucleoside
HIV-1 reverse transcriptase inhibitors. Eur. J. Med. Chem. 53 (2012) 229-234.
S.-X. Gu, Y.-Y. Zhu, F.-E. Chen, E. De Clercq, J. Balzarini, C. Pannecouque, Structural
modification of diarylpyrimidine derivatives as HIV-1 reverse transcriptase inhibitors. Med.
Chem. Res. 24 (2015) 220-225.
[20]
[21]
[22]
S.-X. Gu, H. Qiao, Y.-Y. Zhu, Q.-C. Shu, H. Liu, X.-L. Ju, E. De Clercq, J. Balzarini, C.
Pannecouque, A novel family of diarylpyrimidines (DAPYs) featuring a diatomic linker:
design, synthesis and anti-HIV activities. Bioorg. Med. Chem. 23 (2015) 6587-6593.
D.J. Kertesz, C. Brotherton-Pleiss, M. Yang, Z. Wang, X. Lin, Z. Qiu, D.R. Hirschfeld, S.
Gleason, T. Mirzadegan, P.W. Dunten, Discovery of piperidin-4-yl-aminopyrimidines as
HIV-1 reverse transcriptase inhibitors. N-Benzyl derivatives with broad potency against
resistant mutant viruses. Bioorg. Med. Chem. Lett. 20 (2010) 4215-4218.
[23]
[24]
D.J. Kertesz, C. Brotherton-Pleiss, M. Yang, Z. Wang, X. Lin, Z. Qiu, D.R. Hirschfeld, S.
Gleason, T. Mirzadegan, P.W. Dunten, Exploration of piperidin-4-yl-aminopyrimidines as
HIV-1 reverse transcriptase inhibitors. N-Phenyl derivatives with broad potency against
resistant mutant viruses. Bioorg. Med. Chem. Lett. 20 (2010) 6020-6023.
D. Li, P. Zhan, E. De Clercq, X. Liu, Strategies for the design of HIV-1 non-nucleoside
reverse transcriptase inhibitors: lessons from the development of seven representative
paradigms. J. Med. Chem. 55 (2012) 3595-3613.

[25]
[26]
Z.-Y. Wan, J. Yao, Y. Tao, T.-Q. Mao, X.-L. Wang, Y.-P. Lu, H.-F. Wang, H. Yin, Y. Wu,
F.-E. Chen, E. De Clercq, D. Daelemans, C. Pannecouque, Discovery of
piperidin-4-yl-aminopyrimidine derivatives as potent non-nucleoside HIV-1 reverse
transcriptase inhibitors. Eur. J. Med. Chem. 97 (2015) 1-9.
D. Kang, Z. Fang, Z. Li, B. Huang, H. Zhang, X. Lu, H. Xu, Z. Zhou, X. Ding, D.
Daelemans, E. De Clercq, C. Pannecouque, P. Zhan, X. Liu, Design, synthesis and evaluation
of thiophene[3,2-d]pyrimidine derivatives as HIV-1 non-nucleoside reverse transcriptase
inhibitors with significantly improved drug resistance profiles. J. Med. Chem. 59 (2016)
7991-8007.
[27]
T.J. Tucker, S. Saggar, J.T. Sisko, R.M. Tynebor, T.M. Williams, P.J. Felock, J.A. Flynn,
M.-T. Lai, Y. Liang, G. McGaughey, M. Liu, M. Miller, G. Moyer, V. Munsh, R.
Perlow-Poehneit, S. Prasad, R. Sanchez, M. Torrent, J.P. Vacca, B.L. Wan, Y. Yan, The design
and synthesis of diaryl ether second generation HIV-1 non-nucleoside reverse transcriptase
inhibitors (NNRTIs) with enhanced potency versus key clinical mutations. Bioorg. Med. Chem.
Lett. 18 (2008) 2959-2966.
[28]
R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, C.E.
De, Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV
compounds. J. Virol. Methods 20 (1988) 309-321.
[29]
[30]
P. Christophe, D. Dirk, D.C. Erik, Tetrazolium-based colorimetric assay for the detection
of HIV replication inhibitors: revisited 20 years later. Nat. Protoc. 3 (2008) 427-434.
G. Tang, D.J. Kertesz, M. Yang, X. Lin, Z. Wang, W. Li, Z. Qiu, J. Chen, J. Mei, L. Chen,
T. Mirzadegan, S.F. Harris, A.G. Villasenor, J. Fretland, W.L. Fitch, J.Q. Hang, G. Heilek, K.
Klumpp, Exploration of piperidine-4-yl-aminopyrimidines as HIV-1 reverse transcriptase
inhibitors. N-Phenyl derivatives with broad potency against resistant mutant viruses. Bioorg
Med Chem Lett 20 (2010) 6020-6023.
[31]
[32]
[33]
A.N. Jain, Scoring noncovalent protein-ligand interactions: a continuous differentiable
function tuned to compute binding affinities. J. Comput. Aided Mol. Des. 10 (1996) 427-440.
A.N. Jain, Surflex: fully automatic flexible molecular docking using a molecular
similarity-based search engine. J. Med. Chem. 46 (2003) 499-511.
L. Qian, M. Brian, S. Karl, S. Julian, Tagged fragment method for evolutionary
structure-based de novo lead generation and optimization. J. Med. Chem. 50 (2007)
5392-5402.
[34]
[35]
J.-Z. Chen, J. Wang, X.-Q. Xie, GPCR structure-based virtual screening approach for
CB2 antagonist search. J. Chem Inf. Model. 47 (2007) 1626-1637.
S. Raduner, A. Majewska, J.Z. Chen, X.Q. Xie, J. Hamon, B. Faller, K.H. Altmann, J.
Gertsch, Alkylamides from echinacea are a new class of cannabinomimetics. J. Biol. Chem.
281 (2006) 14192-14206.
[36]
[37]
J. Ruppert, W. Welch, A.N. Jain, Automatic identification and representation of protein
binding sites for molecular docking. Protein Sci. 6 (2010) 524-533.
W. Welch, J. Ruppert, A.N. Jain, Hammerhead: fast, fully automated docking of flexible
ligands to protein binding sites. Chem. Biol. 3 (1996) 449-462.

Highlights
1. A series of indazolyl-substituted piperidin-4-yl-aminopyrimidines (IPAPYs) as HIV-1
NNRTIs were designed and synthesized.
2. The target molecules displayed moderate to excellent activities against wild-type (WT)
HIV-1 with EC50 values ranging from 1.5 to 0.0064 µM.
3. 5q displayed the most excellent activities against WT HIV-1 (EC50 = 6.4 nM, SI = 2500).

Declaration of Interest Statement
This is to state that all of our co-authors approved the submission of the article titled
“Indazolyl-substituted piperidin-4-yl-aminopyrimidines as HIV-1 NNRTIs: design,
synthesis and biological activities” to Eur. J. Med. Chem. We declare that we have no
conflict of interest.
Ting Xiao, Jia-Fan Tang, Ge Meng, Christophe Pannecouque, Yuan-Yuan Zhu,
Gen-Yan Liu, Zhi-Qiang Xu, Feng-Shou Wu, Shuang-Xi Gu*, Fen-Er Chen*