In situ click chemistry-based rapid discovery of novel HIV-1 NNRTIs by exploiting the hydrophobic channel and tolerant regions of NNIBP

Article abstract of DOI:10.1016/j.ejmech.2020.112237

HIV-1 RT has been considered as one of the most important targets for the development of anti-HIV-1 drugs for their well-solved three-dimensional structure and well-known mechanism of action. In this study, with HIV-1 RT as target, we used miniaturized pa

Full text of DOI:10.1016/j.ejmech.2020.112237

Journal Pre-proof
In situ click chemistry-based rapid discovery of novel HIV-1 NNRTIs by exploiting the
hydrophobic channel and tolerant regions of NNIBP
Dongwei Kang, Da Feng, Lanlan Jing, Yanying Sun, Fenju Wei, Xiangyi Jiang,
Gaochan Wu, Erik De Clercq, Christophe Pannecouque, Peng Zhan, Xinyong Liu
PII:
S0223-5234(20)30204-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112237
EJMECH 112237
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 February 2020
Revised Date: 11 March 2020
Accepted Date: 11 March 2020
Please cite this article as: D. Kang, D. Feng, L. Jing, Y. Sun, F. Wei, X. Jiang, G. Wu, E. De Clercq, C.
Pannecouque, P. Zhan, X. Liu, In situ click chemistry-based rapid discovery of novel HIV-1 NNRTIs
by exploiting the hydrophobic channel and tolerant regions of NNIBP, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112237.
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Graphical abstract

In Situ Click Chemistry-Based Rapid Discovery of Novel HIV-1 NNRTIs By
Exploiting the Hydrophobic Channel and Tolerant Regions of NNIBP
Dongwei Kang,†, ,ω Da Feng, † Lanlan Jing,† Yanying Sun,† Fenju Wei,† Xiangyi
&
Jiang,† Gaochan Wu,† Erik De Clercq, § Christophe Pannecouque,§ Peng Zhan†,&,* and
Xinyong Liu,†,
*
&,
†
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
250012 Jinan, Shandong, PR China
§
Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U.
Leuven, Herestraat 49 Postbus 1043 (09.A097), B-3000 Leuven, Belgium.
&China-Belgium Collaborative Research Center for Innovative Antiviral Drugs of Shandong
Province, 44 West Culture Road, 250012 Jinan, Shandong, PR China
ω
Suzhou Research Institute, Shandong University, Room522, Building H of NUSP, NO.388
Ruoshui Road, SIP, Suzhou, 215123 Jiangsu, P.R. China
ABSTRACT:
HIV-1 RT has been considered as one of the most important targets for the
development of anti-HIV drugs for their well-solved three-dimensional structure and
well-known mechanism of action. In this study, with HIV-1 RT as target, we used
miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in
situ biological screening to discover novel potent HIV-1 NNRTIs. A 156
triazole-containing inhibitor library was assembled in microtiter plates and in
millimolar scale. The enzyme inhibition screening results showed that 22 compounds
exhibited improved inhibitory activity. Anti-HIV-1 activity results demonstrated that
A3N19 effected the most potent activity against HIV-1 IIIB (EC50 = 3.28 nM) and

mutant strain RES056 (EC50 = 481 nM). The molecular simulation analysis suggested
that the hydrogen bonding interactions of A3N19 with the main chain of Lys101 and
Lys104 was responsible for its potency. Overall, the results indicated the in situ click
chemistry-based strategy was rational and might be amenable for the future discovery
of more potent HIV-1 NNRTIs.
Keywords: HIV-1, DAPY, NNRTIs, In situ screening, CuAAC, Click Chemistry
1. INTRODUCTION
According to the Joint United Nations Program on HIV/AIDS (UNAIDS) report
2018, more than 37.9 million people are today living with human immunodeficiency
virus (HIV), including 1.7 million people newly infected with HIV in 2018.[1] HIV
infection is still pandemic around the world. Among all the targets for treatment
HIV-1 disease, reverse transcriptase (RT) has been considered as one of the most
successful. Especially, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have
gained a definitive place due to their unique antiviral potency, high specificity, and
low toxicity in highly active antiretroviral therapy (HAART) used to treat HIV.[2, 3]
According to UNAIDS, 62% of people living with HIV were receiving antiretroviral
treatment in 2018.[1] So far, more than 50 structurally diverse classes of inhibitors
have been identified as HIV-1 NNRTIs. They could non-competitive interact with the
allosteric site (known as the non-nucleoside inhibitory binding pocket, NNIBP) of
HIV-1 RT, and then induce conformational changes of the catalytic domain to inhibit
their DNA polymerase activity.[4, 5] Among them, six NNRTIs were approved for
AIDS therapy. Nevirapine (NVP), delavirdine (DLV) and efavirenz (EFV) are

first-generation NNRTIs, etravirine (ETR), rilpivirine (RPV), and doravirine (DOR)
are second-generation NNRTIs.[6, 7] However, the first-generation NNRTIs
encountered a sharp loss of activity with mutants such as Y181C, K103N and RES056
(Y181C+K103N), and the second-generation NNRTIs show loss of activity against
single-point mutations such as K101E and E138K.[8] In addition, hypersensitivity
reactions or other adverse effects have been reported with second-generation
NNRTIs.[9] Therefore, the development of novel NNRTIs with more active potency,
increased resistance profiles and less toxicity is still highly desired.
High-throughput screening (HTS) of compounds libraries has always been a
widely used approach for drug discovery, based on the construction of a high-quality
compounds library.[10] Various new synthetic methodologies and concepts have been
proposed for the assembly of compound collections.[11] Among them, click chemistry,
especially copper(I)-catalyzed azide–alkyne (3 + 2) dipolar cycloaddition (CuAAC),
is generally regarded as a powerful fragment-based assembly reaction for rapid
synthesis of small molecules libraries with its unique advantages, such as mild
reaction conditions, almost complete chemoselectivity with broad functional group
tolerance, excellent yield and excellent biorthogonality, which means the CuAAC
could rapid generate a triazole-containing compounds library for activity screening
without purification.[12-14] In recent years, the rapid assembly and in situ screening
of focused combinatorial fragment libraries using CuAAC click chemistry has
become more robust, and an efficient strategy for bioactive molecules generation.
[15-17] Although various strategies have been made to seek novel anti-HIV-1

inhibitors,[18, 19] there has been no previous report on the discovery of novel
NNRTIs based on in situ click chemistry. In this work, we report for the first time the
rapid identification of highly potent HIV-1 NNRTIs with this technique.
Figure 1. Chemical structures of U.S. FDA-approved second-generation NNRTI
drugs (1 and 2) and our previously reported potent HIV-1 NNRTI K-5a2.
Our previous efforts have led to the development of a novel potent
thiophene[3,2-d]pyrimidine NNRTIs K-5a2.[20] The co-crystal structure of HIV-1
wild-type (WT) RT in complex with K-5a2 revealed that K-5a2 adopted a horseshoe
conformation in the NNIBP, and displayed remarkable structural complementarity to
the NNIBP with substantial extensions into the three channels, namely hydrophobic
channel, tolerant region I and tolerant region II, which is similar to that observed
with ETR.[21] Specifically, the left wing of K-5a2 arches into the tunnel lined by
Tyr181, Tyr188, Phe227, and Trp229, forming π-π interactions with these residues.
The piperidine-linked aryl structure of the right wing projects into the groove
surrounded by Lys103, Val106, Pro225, Pro236, and Tyr318, establishing van der
Waals contacts with their lipophilic side chains. The terminal sulfonamide group
extends to the solvent-exposed surface and forms hydrogen-bonding with the
backbone of Lys104 and Val106. The thiophene[3,2-d]pyrimidine heterocyclic

structure of K-5a2 located at the tolerant region II formed by Glu138 and Lys101,
developing novel nonpolar interactions with the alkyl chain of Glu138. Furthermore,
the nitrogen atoms in the central thiophene pyrimidine and the amine group linking
the central thiophene pyrimidine and the piperidine ring develops double
hydrogen-bonding network with the carbonyl oxygen of Lys101, which plays an
important role in maintaining its high anti-HIV activity.
In this article, with K-5a2 and ETR as lead compounds, six alkyne building
block (A1-A6) were designed to further exploit the hydrophobic channel and tolerant
regions of NNIBP and discover novel NNRTIs with greater potency.[22-25] Moreover,
according to the chemical space of the three-dimensional structure of NNIBP, azide
substituents N1-36 and N34-39 with different aryl substituents varying in size, polar
group substitution, and electronic nature were designed to match the tolerant regions
and hydrophobic channel, respectively. Then CuAAC click chemistry in 96-wall
plates provided triazoles-containing derivatives, which were tested for HIV- RT
inhibitory activity directly without purification. Further in vitro anti-HIV activity
evaluation and molecular docking led to the identification of compound A3N19 as a
promising lead compound.

Figure 2. Design of alkynes based on the co-crystal structure of HIV-1 RT/K-5a2 and
RT/ETR
Figure 3. Structures of the designed diverse azide substituents
2. RESULTS AND DISCUSSION
2.1 Building and characterizing the libraries by click chemistry
The general synthetic steps adopted to obtain the alkynes A1–6 are outlined in

Scheme 1-5. As depicted in Scheme 1, with our previously reported compound 4 as
starting material,[26] the alkyne building block A1 was obtained underwent a SNAr
reaction with but-3-yn-1-amine. Treatment of compounds 5 with propargyl bromide in
DMF at room temperature afforded alkynes A2, while A3 was obtained by treatment 5
with propiolic acid using HATU as condensation agent.
Scheme 1. Reagents and conditions (i) K2CO3, DMF, but-3-yn-1-amine, 80°C, 59%.
Scheme 2. Reagents and conditions (i) HATU, propiolic acid, DCM, r.t., 62%; (ii) K2CO3, DMF,
propargyl bromide, r.t., 71%.
As shown in Scheme 3, the starting material 2,4,6-trichloropyrimidine 6 was
treated with 4-hydroxy-3,5-dimethylbenzonitrile to give the compound 7, which
following reaction with 4-aminobenzonitrile provided the intermediate 8. Subsequent
ammonolysis reaction using NH3.H2O in NMP via a microwave reaction yielded
compound 9, which was converted to the key intermediate 11 using PbO2 and I2 in
HOAc. Then, the target alkyne building block A4 was prepared from 11 via the
Sonogashira cross-coupling and trimethylsilyl-removal reaction.

Scheme 3. Reagents and conditions (i) 3,5-dimethyl-4-hydroxybenzonitrile, 1,4-dioxane, 70°C,
2 h, 92%; (ii) 4-aminobenzonitrile, NMP, t-BuOK, 0-5°C, 60%; (iii) NH3.H2O, NMP, 130°C, 15
min, mw, 85%; (iv) PbO2, I2, HOAc, r.t., 64%; (v) ethynyltrimethylsilane, PdCl2(PPh3)2, CuI, Et3N,
THF, r.t.; (vi) K2CO3, MeOH, r.t., 72%.
The approved drug ETR reacted with 3-bromopropyne in the presence of K2CO3
obtained A5 (Scheme 4). Synthesis of alkyne building block A6 is shown in Scheme
5. Firstly, nucleophilic substitution of 2,4-dichlorothiophene[3,2-d]pyrimidine (12)
with 4-iodo-2,6-dimethylphenol and 4-Boc-aminopiperidine in the presence of K2CO3
and DMF afforded the key intermediate 14. Deprotection with trifluoroacetic acid in
dichloromethane
gave
compound
15,
which
was
reacted
with
4-(bromomethyl)benzenesulfonamide to obtain intermediate 16. Then, the target
alkyne building block A6 was prepared from 16 via the Sonogashira cross-coupling
and trimethylsilyl-removal reaction. The synthesis of azide is detailed in our
previously published paper.[17]

Scheme 4. Reagents and conditions (i): K2CO3, DMF, propargyl bromide, r.t., 63%.
Scheme 5. Reagents and conditions (i): 4-iodo-2,6-dimethylphenol, K2CO3, DMF, r.t., 89%; (ii)
tert-butyl 4-aminopiperidine-1-carboxylate, K2CO3, DMF, 120°C; (iii) TFA, DCM, r.t., 68%; (iv)
K2CO3, DMF, r.t., 75%; (v) ethynyltrimethylsilane, PdCl2(PPh3)2, CuI, Et3N, THF, 50°C; (vi)
K2CO3, MeOH, r.t., 54%.
2.2. Generation of the combinatorial library
Using the CuAAC reaction, a 156 triazole-containing inhibitor library was
assembled in microtiter plates and at millimolar scale. Each of the A1-A4 alkynes was
mixed with each of the N1-N36 azides and A5, A6 alkynes were mixed with each of
the N34-N39 azides in DMSO solution, followed by the addition of catalytic amounts
of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), CuSO4·5H2O and
sodium ascorbate (VcNa). The detailed reaction conditions and dosage of the reactants
are shown in Table 1. The completion of the reaction was detected by TLC (about 24

hours). The compounds are shown in the supporting information Table S1.
Table 1. Detailed reaction conditions.
Reagents
Alkyne-unit (Ax)
Azide-unit (Nx)
TBTA
Concentration
25 mM DMSO
35 mM DMSO
10 mM DMSO
4 mM MilliQ
Volume
20 µL
Final Concentration
5 mM
20 µL
7 mM
10 µL
20 mol%
CuSO4.5H2O
VcNa
25 µL
20 mol%
20 mM MilliQ
25 µL
100 mol%
Total Volume
100 µL/well
2.3. In situ screening against HIV-1 RT
With the aim to rapidly discover novel potent HIV-1 NNRTIs, the generated
triazole-containing library was screened for their HIV-1 RT inhibitory activity at
0.267 µM (final concentration) without further purification. The lead compounds ETR
and K-5a2 were selected as reference drugs, a mixture solution of TBTA,
CuSO4·5H2O and VcNa was used as blank control. As shown in Figures 4-7, a total
of 22 hits emerged from the screens. For the A1 alkyne building block, the inhibition
percentages of A1N4 (89.1%), A1N7 (75.6%), A1N11 (91.8%), A1N18 (72.4%),
A1N19 (71.1%) and A1N34 (86.7%) were higher than ETR (62.3%) and K-5a2
(56.2%) against HIV-1 RT. In the case of the A2 alkyne building block, only three
compounds showed higher enzyme activity, including A2N10 (75.8%), A2N23
(80.2%), and A2N34 (71.8%). For the A3 alkyne building block, A3N1 (50.3%),

A3N4 (51.9%), A3N10(44.8%), A3N12(46.1%), A3N18(42.0%), A3N19 (66.6%),
A3N24 (60.1%), A3N33 (57.1%), A3N34 (69.7%) and A3N35 (73.0 %) exhibited
greater enzyme inhibitory activity than ETR (32.7%) and K-5a2 (44.0%). A6NX,
which was designed for exploiting the hydrophobic channel of the NNIBP, A6N9
(70.8%), A6N17 (72.5%) and A6N35 (74.0 %) showed improved inhibitory activity
compared to ETR (64.0%) and K-5a2 (58.7%). However, compounds A4NX and
A5NX showed decreased enzyme activity (Supporting Information Table S2).
100
80
60
40
20
0
-20
-40
Compounds (0.267µM)
Figure 4. HIV-1 RT inhibitory activity in the presence of 0.267 µM A1NX
90
80
70
60
50
40
30
20
10
0
-10
-20
Compounds (0.276 µM)
Figure 5. HIV-1 RT inhibitory activity in the presence of 0.267 µM A2NX

80
70
60
50
40
30
20
10
0
-10
Compounds (0.267 µM)
Figure 6. HIV-1 RT inhibitory activity in the presence of 0.267 µM A3NX
80
70
60
50
40
30
20
10
0
Compounds (0.267 µM)
Figure 7. HIV-1 RT inhibitory activity in the presence of 0.267 µM A6NX
2.4. Biological evaluation
Then the selected 22 hits were individually re-synthesized at a milligram scale
with the aim to perform further biological studies at the cellular level. In addition, we
also synthesized compound A3N3 with the aim to verify the correlation between
enzyme activity and cell activity. Their in vitro antiviral potency was evaluated in
MT-4 cell cultures infected with WT HIV-1 strain (IIIB) as well as cells infected with
double-mutant strains K103N+Y181C (RES056). NVP, EFV, and ETR were selected
as control drugs, and K-5a2 was selected as the reference compound. The values of
EC50 (anti-HIV potency), CC50 (cytotoxicity) as well as SI (selectivity index,

CC50/EC50 ratio) of the synthesized compounds are summarized.
The anti-HIV-1 activity results demonstrated that A3 was the most preferred
alkyne building block. As depicted in Table 2, most compounds of the A3NX series
exhibited potent activity against HIV-1 IIIB with low nanomolar EC50 values ranging
from 3.28 to 10.4 nM, being far more potent than the reference drug NVP (EC50 = 163
nM). A3N19 (EC50 = 3.28 nM) and A3N34 (EC50 = 4.38 nM) turned out to be the
most potent HIV-1 inhibitors, being comparable to the drug EFV (EC50 = 4.7 nM),
ETR (EC50 = 5.1 nM) and the lead K-5a2 (EC50 = 1.63 nM). Moreover, A3N19 and
A3N34 exhibited no cytotoxicity (CC50) up to the maximum tested concentration of
210 and 212 µM, which contribute to their higher SI values (SI > 64103 and 48544,
respectively). It is noteworthy that compound A3N3 displayed much decreased
potency again HIV-1 IIIB with an EC50 value of 209 nM. Compounds A6N9, A6N17
and A6N35 exhibited modest antiviral activity with EC50 values of 31.3, 21.1 and
25.8 nM, respectively.
In the case of mutant HIV-1 strain RES056, A3N18 and A3N19 exhibited modest
potency of 670 nM and 481 nM, being comparable to that of EFV (EC50 = 264 nM)
but inferior to that of ETR (EC50 = 45.4 nM). The other compounds are almost
inactive to RES056. In addition, the antiviral results also demonstrated that
compounds A1NX and A2NX showed sharply reduced activity against HIV-1 IIIB
and RES056 (See the Supporting information, Table S3).
Table 2. Activity and cytotoxicity against WT HIV-1 (IIIB) and mutant strain RES056

EC50 (nM)a
RES056
SIc
Inhibition
Ration
Compds
CC50 (µM)b
IIIB
IIIB
2444
525
RES056
6
6.64±1.15
209±65.5
6.12±0.87
10.4±1.3
7.26±1.61
5.35±0.35
3.28±0.83
6.66±1.34
82.9±23.9
4.38±0.65
5.02±2.08
31.3±11.3
21.1±7.03
25.8±7.32
1.4±0.4
2911±431
18053±1512
1173±147
>4760
16.2±7.01
110±3.92
7.66±2.79
4.76±0.19
4.72±0.22
20.7±0.66
>210
0.503
0.370
0.519
0.448
0.461
0.420
0.666
0.601
0.571
0.697
0.730
0.708
0.725
0.740
0.442
-
A3N1
A3N3
A3N4
A3N10
A3N12
A3N18
A3N19
A3N24
A3N33
A3N34
A3N35
A6N9
A6N17
A6N35
K-5a2
NVP
6
1252
454
7
<1
>4720
650
<1
670±0.33
481±16.2
1994±240
10555±385
4464±1258
7032±1174
>3395
3881
>64103
2974
1800
>48544
23325
108
31
>437
10
19.8±1.09
149±6.90
>212
14
>48
17
117±3.68
3.39±0.31
11.7±5.35
17.6±4.23
>227
<1
6186±670
6046±840
30.6±12
552
1.88
3
682
>159101
>58
>53533
NA d
>101
>24
163±41.2
5.1±0.8
>9513
>9.51
45.4±15.5
264±27.3
>4.59
>889
>1333
0.327
-
ETR
4.7±1.7
>6.38
EFV
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against
HIV-1-induced cytopathic effect, as determined by the MTT method.
b
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
determined by the MTT method.
c SI: selectivity index, the ratio of CC50/EC50.
d NA: not available.
2.5 Molecular modeling analysis
With the aim to obtain insight into the allosteric binding of the novel designed
derivatives to the NNIBP, the molecular docking studies for representative compounds
A3N19 and A3N34 was performed by using the software SurflexeDock SYBYL-X

2.0. PyMOL was used to visualize the docking results. Co-crystal structure of HIV-1
WT RT/K-5a2 (PDB code: 6c0n) and RES056 RT/K-5a2 (PDB code: 6c0r) were used
as the input structures for docking calculations.[21] The docking protocol is described
in the computational section.
Figure 8. The binding mode of lead K-5a2 (A), the predicted binding modes of
A3N19 (B) and A3N34 (C) with the HIV-1 WT RT (PDB code: 6c0n), and the
predicted binding mode of A3N19 with the HIV-1 RES056 RT (D, PDB code: 6c0r).
The hydrogen bonds are indicated with yellow dashed lines.
The docking simulations of A3N19 and A3N34 with HIV-1 WT RT (PDB code:

6c0n) demonstrated that their binding modes were similar with the lead K-5a2
(Figure 8). Firstly, the left 4-cyano-2,6-dimethylphenyl group of A3N19 and A3N34
occupies the hydrophobic sub-pocket composed by aromatic amino acid residues
Tyr181, Tyr188, and Phe227, exhibiting aryl-aryl interaction with these residues.
Secondly, the central thiophene[3,2-d]pyrimidine heterocycle effectively occupies the
NNIBP tolerant regions II, establishes nonpolar interactions with the alkyl chain of
Glu138 and favorable hydrophobic contacts with Val179. Thirdly, the NH linker
connecting the central pyrimidine ring and the right wing and pyrimidine nitrogen
develops a dual hydrogen bond with the main chain backbone of Lys101. Notably, the
sulfonamide group of the lead K-5a2 forms double-hydrogen bonding with the
carbonyl oxygen of Lys104 and the backbone nitrogen of Val106, while the amide
group of A3N19 yields hydrogen-bonding interactions with the main chain of Lys104
and the morpholine ring of A3N34 does not establish any hydrogen bond interactions,
which may responsible for their decreased activity for WT HIV-1 strain. In regard to
RT carrying the more disruptive RES056 double-mutation (PDB code: 6c0r), the
Y181C mutation abolishes the most favorable π-π interaction between the Tyr181 side
chain and the dimethyl phenyl ring of A3N19. In addition, the terminal substituent of
A3N19 is twisted and lost the hydrogen bonding with Lys104. Although the triazole
group developed novel hydrogen bonding through a water molecular with the mutant
ASN103, it was not enough to compensate for the loss of the foremost π-π interaction
and the hydrogen bonding with the minichain of Lys104. All these may be account for
the decreased anti-HIV-1 activities against HIV-1 RES056.

Furthermore, by comparing the binding mode of A3N19 and A3N34 with WT
and RES056 RT, we conclude that the newly introduced hydrogen bond receptor
triazole group plays pivotal role of keeping their terminal substituent in a reasonable
conformation in the NNIBP. In turn, the terminal substituent of the compounds also
influence the conformation of triazole, which means that it could be regarded as a
dominant site for structural modification to establish stronger hydrogen bonding
interactions between the triazole and NNIBP. The docking simulations contribute to
understand the binding mode of the newly designed compounds with NNIBP, which
helps further structure-based design of novel potent NNRTIs.
3. CONCLUSION
In conclusion, we reported the identification of highly potent HIV-1 NNRTIs
from miniaturization CuAAC click-chemistry-based combinatorial libraries. In this
exploratory project, 22 hits emerged from the enzyme-inhibitory screening, and were
then synthesized and evaluated for their anti-HIV-1 activity in vitro. All these efforts
led to the successful discovery of a potent HIV-1 NNRTI, A3N19, which exhibited
active potency against WT HIV-1 IIIB (EC50 = 3.28 nM) and mutant strain RES056
(EC50 = 481 nM). Moreover, A3N19 exhibited lower cytotoxicity (CC50 > 210 µM)
and higher SI values (SI > 64103, HIV-1 IIIB). The project indicates this in situ click
chemistry-based strategy was rational and might be amenable for the future discovery
of more potent HIV-1 NNRTIs. In addition, the molecular simulation analysis
suggested that the strong activity profile of A3N19 against WT and mutant HIV-1
strains can be attributed to its hydrogen bonding interactions with the main chain of

Lys101 and Lys104.
4. EXPERIMENTAL SECTION
4.1. Chemistry
4-((2-(but-3-yn-1-ylamino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylben
zonitrile (A1)
The starting material 4 (1.0 mmol, 0.32 g) was treated with but-3-yn-1-amine
(1.2 mmol, 0.08 g) in the presence of anhydrous K2CO3 (2.0 mmol, 0.28 g) in 10 mL
DMF. The mixture was stirred at 80°C for 8 h (monitored by TLC). The solution
cooled to room temperature and 40 mL water was added. Then the mixture was
extracted with ethyl acetate (3 × 15 mL) and the organic phase was dried over
anhydrous Na2SO4. The solvent concentrated in vacuo and the crude product was
purified by flash column chromatography to yield the target compound A1 with a
yield of 59%, mp: 203-205 . 1H NMR (400 MHz, DMSO-d6): δ 8.11 (d, J = 5.3 Hz,
1H, C6-thienopyrimidine-H), 7.72 (s, 2H, C3,C5-Ph-H), 7.29 (d, J = 5.4 Hz, 1H,
C7-thienopyrimidine-H), 6.04 (s, 1H, NH), 4.34-4.32 (m, 2H, N-CH2), 2.87(s, 1H),
13
2.32-2.28 (m, 2H), 2.10 (s, 6H). C NMR (100 MHz, DMSO-d6): δ 168.0, 162.1,
160.8, 153.6, 133.6, 132.2, 129.3, 126.7, 125.2, 119.1, 108.6, 82.2, 71.8, 50.3, 30.1,
21.5, 16.2. ESI-MS: m/z 349.2 [M + 1]+. C19H16N4OS (348.10).
3,5-dimethyl-4-((2-((1-propioloylpiperidin-4-yl)amino)thieno[3,2-d]
pyrimidin-4-yl)oxy)benzonitrile (A2)
Our previously reported compound 5 (0.5 mmol, 0.19 g) was selected as starting
material,[26] which was treated with HATU (0.6 mmol, 0.22 g), propiolic acid (0.6

mmol, 0.04 g) in 5 mL of dry DCM and stirred at room temperature. After the reaction
was completed, the solvent was removed under reduced pressure. Then the residue
was washed with 30 mL saturated aqueous sodium chloride and DCM (3 × 10 mL),
the organic layer was separated, dried over Na2SO4 and filtered. The concentrated
product was purified by flash column chromatography to give the target compound
A2 with a yield of 62%, mp: 254-256 . 1H NMR (400 MHz, DMSO-d6, ppm): δ 8.11
(d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.76 (d, J = 8.3 Hz, 2H, C3,C5-Ph-H),
7.72 (s, 2H, C3,C5-Ph’-H), 7.41 (d, J = 8.4 Hz, 2H, C2,C6-Ph-H), 7.31 (s, 2H,
SO2NH2), 7.25 (d, J = 5.4 Hz, 1H, C7-thienopyrimidine-H), 6.04 (s, 1H, NH),
3.75-3.74 (m, 1H), 2.72-2.70 (m, 2H), 2.12 (s, 6H), 1.95-1.45 (m, 6H). 13C NMR (100
MHz, DMSO-d6): δ 165.0, 163.1, 160.9, 153.7, 153.1, 143.3, 136.7, 133.6, 133.0,
129.3, 126.8, 123.8, 123.1, 119.0, 109.6, 61.9, 52.8, 31.8, 16.2. ESI-MS: m/z 432.3
[M + 1]+. C23H21N5O2S (431.14).
3,5-dimethyl-4-((2-((1-(prop-2-yn-1-yl)piperidin-4-yl)amino)thieno[3,2-d]
pyrimidin-4-yl)oxy)benzonitrile (A3)
A mixture of 5 (0.5 mmol, 0.19 g) anhydrous K2CO3 (1.0 mmol, 0.14 g) and
3-bromoprop-1-yne (0.6 mmol, 0.07 g) in 5 mL DMF was stirred at room temperature
for 8 h (monitored by TLC), and then saturated sodium chloride solution (20 mL) was
added. The mixture was extracted with ethyl acetate (3 × 15 mL) and the organic
phase was dried over anhydrous Na2SO4. The solvent concentrated in vacuo and the
crude product was purified by flash column chromatography. The product was
recrystallized from ethyl acetate/petroleum ether to afford the target compound A3

1
with a yield of 71%, mp: 272-274 . H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J =
5.3 Hz, 1H, C6-thienopyrimidine-H), 7.72 (s, 2H, C3,C5-Ph-H), 7.27 (s, 1H,
C7-thienopyrimidine-H), 6.88 (s, 1H, NH), 3.69 (s, 1H), 3.22 (s, 2H, N-CH2), 3.13 (s,
1H, CH≡), 2.74 (s, 2H), 2.12 (s, 6H), 1.90-1.30 (m, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 162.4, 160.5, 153.4, 133.2, 132.9, 123.7, 119.0, 109.0, 75.9, 51.3, 46.6,
31.5, 16.2. ESI-MS: m/z 418.5 [M + 1]+. C23H23N5OS (417.16).
4-((2,6-dichloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (7)
2,4,6-trichloropyrimidine (6, 11 mmol, 2.0 g), DIEPA (13 mmol, 1.7 g) and
4-hydroxy-3,5-dimethylbenzonitrile (11 mmol, 1.6 g) were dissolved in 10 mL
1,4-dioxane and the mixed solution were heated at 70°C for 2 h. After the reaction
mixture was brought to room temperature, 50 mL cold water was poured into the
mixture and stirred for another 30 min, filtrated. The wet cake was dried at 55-60°C
under vacuum to give the intermediate 7 as white solid with a yield of 92%, mp:
207-209℃. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 7.76 (s, 2H, C3,C5-Ph-H), 7.64 (s,
1H, pyrimidine-H), 2.12 (s, 6H). ESI-MS: m/z 294.2 [M + 1]+. C13H9Cl2N3O
(293.01).
4-((6-chloro-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenz
onitrile (8)
The obtained intermediate 7 (6.8 mmol, 2.0 g) and 4-aminobenzonitrile (6.8
mmol, 0.8 g) were dissolved in NMP (10 mL) at 0-5°C, then the solution was added
potassium tert-butoxide (13.6 mmol, 1.53 g) over a period of 30 min and stirred for
another 2 h at 0-5°C. Then the mixture was added to 50 mL cold water slowly and

filtered to get white precipitate, which was suspended in cold water (30 mL) and
acidified to pH 6-7 with 3M HCl. The formed solid was filtered, dried at 55-60°C
under vacuum to give the crude product, which was subsequently washed with 20 mL
ethyl acetate. Then the wet cake was finally dried at 50°C under vacuum to give the
1
intermediate 8 as white solid with a yield of 60%, mp: 277-279℃. H NMR (400
MHz, DMSO-d6, ppm) δ: 10.56 (s, 1H, NH), 7.79 (s, 2H, C3,C5-Ph’-H), 7.55-7.45 (m,
4H), 6.93 (s, 1H, pyrimidine-H), 2.13 (s, 6H). ESI-MS: m/z: 376.5 [M + 1]+, 393.3 [M
+ NH4]+. C20H14ClN5O (375.09).
4-((6-amino-2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenz
onitrile (9)
Intermediate 8 (0.53 mmol, 0.20 g) and 25% aq ammonia (15 mL) were
dissolved in 20 mL NMP and the mixture solution was put into a microwave reactor
in 130°C for 15 min. In the reaction process, the pressure of this reaction system can
up to 135 psi. After the reaction mixture was brought to 5-10°C, 15 mL cold water
was added and stirred for another 30 min. The yielded solid was filtered, washed with
15 mL water and dried at 45-50°C to give the intermediate 9 as white solid with a
1
yield of 85%, mp: 283-286℃. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.57 (s, 1H,
NH), 7.73 (s, 2H, C3,C5-Ph’-H), 7.65 (d, J = 8.0 Hz, 2H, C3,C5-Ph-H), 7.46 (d, J = 8.0
Hz, 2H, C2,C6-Ph-H), 6.80 (s, 2H, NH2), 5.47 (s, 1H, pyrimidine-H), 2.12 (s, 6H).
ESI-MS: m/z 357.4 [M + 1]+, 379.5 [M + Na]+. C20H16N6O (356.14).
4-((6-amino-2-((4-cyanophenyl)amino)-5-iodopyrimidin-4-yl)oxy)-3,5-dimet
hylbenzonitrile (10)

Intermediate 9 (0.84 mmol, 0.3 g) was dissolved in 5 mL glacial acetic acid, then
PbO2 (0.30 mmol, 0.07 g) and I2 (0.30 mmol, 0.08 g) were added and the mixed
solution was stirred at room temperature for 4 h. Then the reaction mixture was
diluted with H2O and stirred for another 30 min, the obtained precipitate was filtered,
washed with H2O. Then the crude product was purified by flash column
chromatography to obtain 10 with a yield of 64%, mp: 302-304 . 1H NMR (400 MHz,
DMSO-d6) δ 9.58 (s, 1H, NH), 7.74 (s, 2H, C3,C5-Ph’-H), 7.55 (d, J = 8.5 Hz, 2H,
C3,C5-Ph-H), 7.43 (d, J = 8.5 Hz, 2H, C2,C6-Ph-H), 6.98 (s, 2H, NH2), 2.11 (s, 6H).
13C NMR (100 MHz, DMSO-d6) δ 166.8, 165.5, 158.5, 154.9, 145.4, 133.1, 132.9,
132.8, 120.0, 119.1, 118.6, 108.6, 102.4, 46.8, 16.2. ESI-MS: m/z 483.4 [M + 1]+.
C20H15IN6O (482.04).
4-((6-amino-2-((4-cyanophenyl)amino)-5-ethynylpyrimidin-4-yl)oxy)-3,5-di
methylbenzonitrile (A4)
Compound 10 (1.0 mmol, 0.48 g), PdCl2(PPh3)2 (0.05 mmol, 0.04 g), CuI (0.2
mmol, 0.04 g) and triethylamine (4.0 mmol, 0.5 mL) were added to 10 mL THF. Then
trimethylsilylacetylene (2.2 mmol, 0.2 g) was added to the mixed solution under N2
and stirred at room temperature for 24 h. Then the mixture was filtered through a
celite pad. The filtrate was concentrated and purified by flash column chromatography
to afford the key intermediate 11. Then 11 was added 5 mL MeOH and potassium
carbonate (7 mmol, 1.1g), and the mixture was stirred at room temperature for 1h. To
the reaction mixture was added 10 mL saturated sodium bicarbonate solution, filtrated
and the solid was washed with water. The crude product was recrystallized in MeOH

1
to get the target product A4 as white solid with a yield of 72%, mp: 295-297 . H
NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H, NH), 7.74 (s, 2H, C3,C5-Ph’-H), 7.55 (d, J
= 8.5 Hz, 2H, C3,C5-Ph-H), 7.43 (d, J = 8.5 Hz, 2H, C2,C6-Ph-H), 6.98 (s, 2H, NH2),
3.24 (s, 1H, CH≡), 2.11 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 166.8, 165.5,
158.5, 154.9, 145.6, 133.1, 132.9, 132.7, 120.0, 119.1, 118.6, 108.7, 102.4, 81.6, 73.4,
46.8, 16.2. ESI-MS: m/z 381.5 [M + 1]+. C20H16N6O (380.14).
4-((5-bromo-2-((4-cyanophenyl)amino)-6-(prop-2-yn-1-ylamino)pyrimidin-4
-yl)oxy)-3,5-dimethylbenzonitrile (A5)
The approved drug ETR (0.5 mmol, 0.22 g) was selected as starting material and
dissolved in 10 mL anhydrous DMF in the presence of anhydrous K2CO3 (1.0 mmol,
0.14 g) and 3-bromoprop-1-yne (0.6 mmol, 0.07 g). The mixture was stirred at room
temperature for 5 h (monitored by TLC), and then the solvent was removed under
reduced pressure. The residue was taken up in water (20 mL) and extracted with ethyl
acetate (3 × 10 mL). The organic phase was dried over anhydrous Na2SO4, filtered,
and purified by flash column chromatography. The product was recrystallized from
ethyl acetate/petroleum ether to afford the target compound A5 with a yield of 63%,
1
mp: 264-267 . H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H, NH), 7.74 (s, 2H,
C3,C5-Ph’-H), 7.55 (d, J = 8.5 Hz, 2H, C3,C5-Ph-H), 7.43 (d, J = 8.5 Hz, 2H,
C2,C6-Ph-H), 7.36 (s, 1H), 3.93-3.66 (m, 2H, N-CH2), 2.94 (s, 1H, CH≡), 2.10 (s, 6H).
13C NMR (100 MHz, DMSO-d6) δ 166.8, 165.5, 158.5, 154.9, 145.5, 133.1, 132.9,
132.8, 120.0, 119.2, 118.6, 108.8, 102.4, 82.1, 72.4, 30.6, 16.2. ESI-MS: m/z 473.4
[M + 1]+. C23H17BrN6O (472.06).

2-chloro-4-(4-iodo-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidine (13)
A mixture of 2,4-dichlorothiopheno[3,2-d]pyrimidine (12, 1.0 mmol, 0.21 g),
4-iodo-2,6-dimethylphenol (1.0 mmol, 0.25 g), and anhydrous K2CO3 (1.2 mmol, 0.17
g) in 5 mL DMF was stirred at 25°C for 2 h (monitored by TLC). The mixture was
poured into ice water and then the obtained precipitate was collected by filtration,
recrystallized in DMF-H2O to provide the product 13 as white solid in 89 % yield, mp:
243-245°C. ESI-MS: m/z 417.1 [M + 1]+. C14H10ClIN2OS (415.92).
4-(4-iodo-2,6-dimethylphenoxy)-N-(piperidin-4-yl)thieno[3,2-d]pyrimidin-2-amin
e (15)
A solution of 13 (1.0mmol, 0.42 g), N-Boc-4-aminopiperidine (1.2 mmol, 0.24 g),
and anhydrous K2CO3 (2.0 mmol, 0.28 g) in DMF (5 mL) was heated at 120°C for 12
h (monitored by TLC). Then the solution was cooled to room temperature, and 30 mL
ice water was added. The resulting precipitate was collected by filtration, and dried to
give crude 14, which was used directly in the next step without further purification.
To a solution of 14 (1.2 mmol, 0.70 g) in DCM (4 mL) was added trifluoroacetic acid
(10 mmol, 0.74 mL), and the solution was stirred at room temperature for 4 h
(monitored by TLC). The reaction solution was alkalized to pH 9 with saturated
sodium bicarbonate solution and the aqueous phase was extracted with DCM (3×10
mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and
purified by flash column chromatography to give 15 as a yellow solid in 68% yield,
mp: 125-127°C. ESI-MS: m/z 481.3 [M + 1]+. C19H21IN4OS (480.05).
4-((4-((4-(4-iodo-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)

piperidin-1-yl)methyl)benzenesulfonamide (16)
Compound 15 (1.0 mmol, 0.48 g), 4-(bromomethyl)benzenesulfonamide (1.1
mmol, 0.27 g) and anhydrous K2CO3 (1.2 mmol, 0.16 g) were dissolved in 10 mL
anhydrous DMF. The reaction mixture was stirred at room temperature for 5 h and
then 50 mL water was added, extracted with ethyl acetate (3×10 mL), and the organic
phase was dried over anhydrous Na2SO4, purified by flash column chromatography to
afford compound 16 as a white solid in 75% yield, mp: 279-181°C. ESI-MS: m/z
650.5 [M + 1]+. C26H28IN5O3S2 (649.07).
4-((4-((4-(4-ethynyl-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)
piperidin-1-yl)methyl)benzenesulfonamide (A6)
Compound 16 (1.0 mmol, 0.45 g), PdCl2(PPh3)2 (0.05 mmol, 0.04 g), CuI (0.2
mmol, 0.04 g) and Et3N (4.0 mmol, 0.5 mL) were added to 10 mL THF. Then
trimethylsilylacetylene (2.2 mmol, 0.2 g) was added to the solution under N2 and
stirred at 50°C for 8 h. Then the mixture was filtered, concentrated and purified by
flash column chromatography to afford the key intermediate 17. Then 17 was treated
with K2CO3 (7.0 mmol, 1.1 g) in 5 mL MeOH for 1 h at room temperature. The
reaction mixture was added 10 mL saturated NaHCO3 solution, filtered and washed
the solid with water. Then the crude product was recrystallized in MeOH to get the
target product A6 as white solid with 54% yield, mp: 283-286 . 1H NMR (400 MHz,
DMSO-d6, ppm): δ 8.12 (d, J = 5.6 Hz, 1H, C6-thienopyrimidine-H), 7.78 (d, J = 8.2
Hz, 2H, C3,C5-Ph-H), 7.70 (s, 2H, C3,C5-Ph’-H), 7.47 (d, J = 8.1 Hz, 2H, C2,C6-Ph-H),
7.27 (s, 2H, SO2NH2), 7.16 (d, J = 5.3 Hz, 1H, C7-thienopyrimidine-H), 6.74 (s, 1H,

NH), 4.15 (s, 1H), 3.77-3.74 (m, 1H), 3.48 (s, 2H, N-CH2), 2.72 (s, 2H), 2.11 (s, 6H),
2.03-1.91 (m, 2H), 1.80-1.40 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 165.4,
162.0, 160.2, 153.4, 143.3, 136.7, 133.5, 132.2, 129.8, 126.4, 123.8, 119.5, 109.2,
82.5, 81.0, 61.9, 52.8, 31.6, 16.2. ESI-MS: m/z 548.2 [M + 1]+. C28H29N5O3S2
(547.17).
General procedure for the preparation of AXNX
Compound A1-A6 (0.5 mmol) and a variety of azide substituents (0.6 mmol)
were added in a mixed solution of water and DMF (v/v= 1:1, 10 mL). Then a solution
of 1 M freshly prepared sodiumascorbate (0.195 mmol, 0.04 g) and 7.5%
CuSO4·5H2O (0.065 mmol, 0.017 g) in water were added to the mixture. The
heterogeneous mixture was stirred vigorously at 50 for 4-12 h (monitored by TLC).
Then 20 mL water was added to the mixture and extracted with ethyl acetate (3 × 10
mL). The organic phase was dried over anhydrous Na2SO4, filtered, and purified by
flash column chromatography. The product was recrystallized from ethyl
acetate/petroleum ether to afford the target compound AXNX.
4-((2-((1-((1-(2-cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperidin-4-yl)
amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (A3N1)
1
White solid, 92% yield, mp: 149-151°C. H NMR (400 MHz, DMSO-d6) δ 8.20
(d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 8.08 (s, 1H, triazol-H), 7.92 (dd, J = 7.7,
1.4 Hz, 1H, C3-Ph-H), 7.73 (d, J = 1.5 Hz, 1H, C5-Ph-H), 7.72 (s, 2H, C3,C5-Ph’-H),
7.60-7.55 (m, 1H, C6-Ph-H), 7.36 (d, J = 7.8 Hz, 1H, C4 -Ph-H), 7.26-7.25 (m, 1H,
C7-thienopyrimidine-H), 6.88 (s, 1H, NH), 5.80 (s, 2H, triazol-CH2), 3.71 (s, 1H),

3.56 (s, 2H, N-CH2), 2.80 (s, 2H), 2.11 (s, 6H), 1.72-1.53 (m, 3H), 1.45-1.39 (m, 3H).
13C NMR (100 MHz, DMSO-d6) δ 162.4, 153.4, 139.4, 134.3, 133.8, 133.2, 133.0,
129.8, 129.6, 124.9, 119.0, 117.4, 111.6, 109.0, 52.9, 52.3, 51.4, 39.6, 31.5, 16.2.
ESI-MS: m/z 576.6 [M + 1]+. C31H29N9OS (575.22).
4-((2-((1-((1-(4-cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperidin-4-yl)
amino)thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (A3N3)
1
White solid, 90% yield, mp: 140-142°C. H NMR (400 MHz, DMSO-d6) δ 8.20
(d, J = 5.3 Hz, 1H, C6-thienopyrimidine-H), 8.13 (s, 1H, triazol-H), 7.86 (d, J = 8.0
Hz, 2H, C3,C5-Ph-H), 7.72 (s, 2H, C3,C5-Ph’-H), 7.44 (d, J = 7.5 Hz, 2H, C2,C6-Ph-H),
7.27 (s, 1H, C7-thienopyrimidine-H), 6.87 (s, 1H, NH), 5.73 (s, 2H, triazol-CH2),
3.72-3.71 (s, 1H), 3.54 (s, 2H, N-CH2), 2.79 (s, 2H), 2.11 (s, 6H), 1.91-1.57 (m, 4H),
1.45-1.39 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.4, 153.4, 139.4, 134.3,
133.8, 133.2, 133.0, 129.8, 129.6, 124.9, 119.0, 117.4, 111.6, 109.0, 52.9, 52.3, 51.4,
39.6, 31.5, 16.2. ESI-MS: m/z 576.6 [M + 1]+. C31H29N9OS (575.22).
3,5-dimethyl-4-((2-((1-((1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)
piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (A3N4)
1
White solid, 93% yield, mp: 155-157°C. H NMR (400 MHz, DMSO-d6) δ 8.21
(d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 8.19-8.17 (m, 1H), 8.01 (s, 1H, triazol-H),
7.72 (s, 2H, C3,C5-Ph’-H), 7.62-7.55 (m, 2H), 7.32-7.31 (m, 2H), 7.26-7.24 (m, 1H,
C7-thienopyrimidine-H), 6.88 (s, 1H, NH), 5.80 (s, 2H, triazol-CH2), 3.70 (s, 1H),
3.52 (s, 2H, N-CH2), 2.80 (s, 2H), 2.11 (s, 6H), 1.85-1.60 (m, 4H), 1.42-1.31 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 162.4, 153.4, 139.7, 134.3, 133.8, 133.2, 133.0,

129.8, 124.9, 119.0, 117.4, 111.6, 109.0, 52.9, 52.3, 51.3, 39.6, 31.5, 16.2. ESI-MS:
m/z 596.4 [M + 1]+. C30H29N9O3S (595.21).
3,5-dimethyl-4-((2-((1-((1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)
piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (A3N10)
1
White solid, 88% yield, mp: 114-116°C. H NMR (400 MHz, DMSO-d6) δ 8.20
(d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.91 (s, 1H, triazol-H), 7.71 (s, 2H,
C3,C5-Ph’-H), 7.28-7.18 (m, 4H), 7.05 (d, J = 7.4 Hz, 1H), 6.87 (s, 1H, NH), 5.58 (s,
2H, triazol-CH2), 3.68 (s, 1H), 3.52 (s, 2H, N-CH2), 2.77 (s, 2H), 2.30 (s, 3H), 2.11 (s,
6H), 1.90-1.39 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 162.4, 153.4, 136.6, 134.8,
133.2, 130.8, 128.9, 128.7, 126.7, 124.9, 119.0, 117.4, 111.6, 109.0, 53.0, 52.3, 51.3,
39.6, 31.5, 19.1, 16.2. ESI-MS: m/z 565.6 [M + 1]+. C31H32N8OS (564.24).
3,5-dimethyl-4-((2-((1-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)
piperidin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (A3N12)
1
White solid, 84% yield, mp: 112-114°C. H NMR (400 MHz, DMSO-d6) δ 8.20
(d, J = 5.4 Hz, 1H, C6-thienopyrimidine-H), 7.98 (s, 1H, triazol-H), 7.71 (s, 2H,
C3,C5-Ph’-H), 7.25-7.12 (m, 5H), 6.87 (s, 1H, NH), 5.51 (s, 2H, triazol-CH2), 3.68 (s,
1H), 3.50 (s, 2H, N-CH2), 2.76 (s, 2H), 2.28 (s, 3H), 2.11 (s, 6H), 1.87-1.62 (m, 3H),
1.50- 1.39 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.4, 153.4, 139.4, 137.8,
134.3, 133.6, 133.2, 129.7, 128.3, 124.9, 119.0, 117.8, 109.0, 52.9, 52.3, 31.5, 21.1,
16.2. ESI-MS: m/z 565.6 [M + 1]+. C31H32N8OS (564.24).
4-((4-((4-((4-(4-cyano-2,6-dimethylphenoxy)thieno[3,2-d]pyrimidin-2-yl)
amino)piperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzenesulfonamide