Design, synthesis, and biological evaluation of novel Bcr-AblT315I inhibitors incorporating amino acids as flexible linker

Article abstract of DOI:10.1016/j.bmc.2021.116398

Despite the success of imatinib in CML therapy through Bcr-Abl inhibition, acquired drug resistance occurs over time in patients. In particular, the resistance caused by T315I mutation remains a challenge in clinic. Herein, we embarked on a structural optimization campaign aiming at discovery of novel Bcr-Abl inhibitors toward T315I mutant based on previously reported dibenzoylpiperazin derivatives. We proposed that incorporation of flexible linker could achieve potent inhibition of Bcr-Abl^T315I by avoiding steric clash with bulky sidechain of Ile315. A library of 28 compounds with amino acids as linker has been developed and evaluated. Among them, compound AA2 displayed the most potent activity against Bcr-Abl^WT and Bcr-Abl^T315I, as well as toward Bcr-Abl driven K562 and K562R cells. Further investigations indicated that AA2 could induce apoptosis of K562 cells and down regulate phosphorylation of Bcr-Abl. In summary, the compounds with amino acid as novel flexible linker exhibited certain antitumor activities, providing valuable hints for the discovery of novel Bcr-Abl inhibitors to overcome T315I mutant resistance, and AA2 could be considered as a candidate for further optimization.

Full text of DOI:10.1016/j.bmc.2021.116398

Journal Pre-proofs
Design, Synthesis, and Biological Evaluation of Novel Bcr-AblT315I Inhibitors
Incorporating Amino Acids as Flexible Linker
Xiaoyan Pan, Nanxin Liu, Qingqing Zhang, Kai Wang, Yanchen Li,
YuanYuan Shan, Zilong Li, Jie Zhang
PII:
S0968-0896(21)00406-5
https://doi.org/10.1016/j.bmc.2021.116398
BMC 116398
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 July 2021
31 August 2021
2 September 2021
Please cite this article as: X. Pan, N. Liu, Q. Zhang, K. Wang, Y. Li, Y. Shan, Z. Li, J. Zhang, Design, Synthesis,
and Biological Evaluation of Novel Bcr-AblT315I Inhibitors Incorporating Amino Acids as Flexible Linker,
Bioorganic & Medicinal Chemistry (2021), doi: https://doi.org/10.1016/j.bmc.2021.116398
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Design, Synthesis, and Biological Evaluation of Novel Bcr-AblT315I
Inhibitors Incorporating Amino Acids as Flexible Linker
Xiaoyan Pan1, Nanxin Liu1, Qingqing Zhang1, Kai Wang1, Yanchen Li1, YuanYuan Shan2, Zilong
Li1, Jie Zhang1*
1School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China
2Department of Pharmacy, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an,
China
Abstract
Despite the success of imatinib in CML therapy through Bcr-Abl inhibition,
acquired drug resistance occurs over time in patients. In particular, the resistance
caused by T315I mutation remains a challenge in clinic. Herein, we embarked on a
structural optimization campaign aiming at discovery of novel Bcr-Abl inhibitors
toward T315I mutant based on previously reported dibenzoylpiperazin derivatives.
We proposed that incorporation of flexible linker could achieve potent inhibition of
Bcr-AblT315I by avoiding steric clash with bulky sidechain of Ile315. A library of 28
compounds with amino acids as linker has been developed and evaluated. Among
them, compound AA2 displayed the most potent activity against Bcr-AblWT and
Bcr-AblT315I, as well as toward Bcr-Abl driven K562 and K562R cells. Further
investigations indicated that AA2 could induce apoptosis of K562 cells and down
regulate phosphorylation of Bcr-Abl. In summary, the compounds with amino acid as
novel flexible linker exhibited certain antitumor activities, providing valuable hints
for the discovery of novel Bcr-Abl inhibitors to overcome T315I mutant resistance,
and AA2 could be considered as a candidate for further optimization.
Keywords: Bcr-Abl, T315I mutant, Flexible linker, Amino acids, Biological activity,
Modeling study
1. Introduction
Protein kinases are crucial in the regulation of critical cellular processes, and
deregulation of protein kinases function has been implicated in cancer and other
disorders, such as immunological and metabolic disease [1,2]. Bcr-Abl, resulting from
a reciprocal chromosome translocation between chromosomes 9 and 22, known as
Philadelphia (Ph) chromosome, plays a vital role in the pathogenesis and progression
of chronic myeloid leukemia (CML) [3]. It is found in >90% of patients with CML.
Meanwhile, it is also presented in 25% of adult patients with acute lymphocytic
leukemia (ALL) [4]. This fusion protein, characterized by constitutively active
tyrosine kinase activity, promotes growth and replication through downstream

pathways such as JAK/STAT, RAS/RAF/ MEK/ERK, PI3K/AKT, and BAD/BCL-XI
[5]. Thus, it represents potential therapeutic target for the treatment of CML. Imatinib,
the first Bcr-Abl inhibitor, could induce apoptosis of Bcr-Abl positive CML cells, and
it has been used as a first-line treatment for CML [6].
Despite the great success in clinical use of imatinib, many patients eventually
develop acquired resistance. Several possible mechanisms of imatinib resistance are
suggested, such as amplification of the fusion gene, mutation in Bcr-Abl, activation of
compensatory pathways, binding of α1 acid glycoprotein, and so on [7]. Among these
mechanisms, point mutation in Bcr-Abl especially in the kinase domain that interferes
with imatinib binding is most important, which accounts for 60-80% of the case of
imatinib resistance [8]. To date, over 100 mutations have been reported in resistance
to imatinib which lead to >50 different amino acid substitutions, primarily centered
around or within the P-loop, ATP binding site and activation loop [9]. The degree of
resistance ranges from a few fold for some of the A-loop mutants, up to complete
resistance for the T315I mutation.
To overcome imatinib-resistance, four second generation Bcr-Abl tyrosine kinase
inhibitors were developed, including dasatinib (Sprycel), nilotinib (Tasigna),
bosutinib (Bosulif), and bafetinib [10-13]. These inhibitors retain efficacy against the
majority of Bcr-Abl mutant forms. However, none is capable of inhibiting T315I
mutation, which accounts for 15-20% in all clinical required resistance [14]. Ponatinib
(Iclusig), a third generation Bcr-Abl inhibitor, was then developed for the treatment of
resistant or intolerant CML and Ph+ ALL patients against imatinib, especially those
harboring T315I mutation [15]. It is presently the only clinically available Bcr-Abl
inhibitor effective against T315I. However, due to its serious side effects seen in ~12%
of the patient population, ponatinib has been permitted to resume sales to a limited
patient population with an expanded black box warning [16]. Thus, there is still an
urgent need to develop novel kinase inhibitors to overcome Bcr-AblT315I drug
resistance.
T315 is referred as the “gatekeeper” residue, which controls the type II inhibitors’
access to the back cavity generated in inactive conformation [17]. However, the
substitution of T315 with a bulkier and more hydrophobic isoleucine results in the
loss of an important hydrogen bond with inhibitors and creates a steric hindrance that
could disrupt the inhibitor binding [18]. In our previous research, pharmacophores for
type II Bcr-Abl inhibitors are summarized in three parts: the hinge binding moiety
(HBM), linker, and the selective site binding moiety (SBM) [19]. Under this analysis,
flexible linker strategy is proposed based on the hypotheses that improving linker
flexibility might avoid the steric clash with Ile315 to make inhibitor access to the back

cavity. Meanwhile, to compensate the decreased affinity caused by T315I mutation,
hydrogen bond network strategy is applied to HBM modification to improve the
affinity with hinge region which is critical for inhibitor binding. (Figure 1)
Figure 1. The design of Bcr-Abl inhibitors with amino acids as flexible linker.
The feasibility of flexible linker strategy for Bcr-Abl inhibitors design has been
verified in the modification with diethylamine or piperazine as linker [20]. As a
continuous to our work, the further exploration is to expand the diversity of flexible
linker, developing Bcr-Abl inhibitors with new scaffold. Studies have indicated that
the introduction of amino acid could enhance the lipid solubility and relieve the
toxicity for antitumor drugs, as well as improving the selectivity to tumor cells [21,
22]. Hence, based on bioelectronics isometric strategy, amino acids are expected to be
used as flexible linker. Meanwhile, phenylpyridine with various amide side chain,
generating hydrogen bonds with hinge region, is incorporated as HBM. As for
selectivity binding site moiety, 4-chloro-3-trifluoromethylaniline is used according to
our previous biological results. (Figure 1)
Herein, a series of compounds were designed and synthesized with two amino
acids, alanine and hydroxyproline, as flexible linker. The biological activities were
evaluated through enzyme inhibition experiment and MTT assay. Furthermore, the
compound with the highest activity was studied to reveal the anticancer mechanisms
by western blot. In addition, molecular docking and molecular dynamics simulations
were performed using the most potent compounds to study the dynamic behavior of
this complex involved in the inhibition process and investigate the possible binding
interactions between synthesized compounds and Bcr-Abl active site.
2. Chemistry
The general synthetic procedures for two classes of title compounds were
outlined in Scheme 1 and 2. All of the title compounds were prepared using
Pd-catalyzed Suzuki coupling and amide condensation as key reactions. The
preparation of compounds AA1-AA14 started with the construction of key

intermediates 3a-3d, 4a-4d, 5a-5c and 6a-6c from commercially available
5-bromopyridin-2-amine and 5-bromonicotinic acid, respectively.
5-bromopyridin-2-amine was firstly acylated using various acyl chloride [23],
followed by coupling with 4(3)-carboxyphenyl-boronic acid, to generate
intermediates 3a-3d and 4a-4d [24, 25]. For other intermediates 5a-5c and 6a-6c,
5-bromonicotinic acid was activated by thionyl chloride to generate active benzoyl
chloride, then reacted with various aliphatic amines [26], followed by coupling with
4(3)-carboxyphenyl-boronic acid to obtain 5a-5c and 6a-6c. L-alanine was used as
starting material to generate another key intermediate 9. At first, the amine group of
L-alanine was protected by (Boc)2O [27]. Next, Boc-Ala-OH was reacted with
5-amino-2-chlorobenzotrifluoride using a mixed anhydride method with isobutyl
chloroformate as condensation reagent [28], and then deprotected by trifluoroacetic
acid to yield compound 9 [29]. Consequently, treatment of 9 with various biphenyl
carboxylic acid 3a-3d, 4a-4d, 5a-5c and 6a-6c in the presence of
N-methylmorpholine and isobutyl chloroformate afforded title compounds
AA1-AA14 [30]. (Scheme 1)
Br
N
COOH
H2N
R1
N
COOH
COOH
R1
Br
N
H
N
HO
HO
Br
Br
a
b
B
N
3a-3d, 4a-4d
N
OH
H
c
H2N
+
1a-1c
N
O
N
COOH
R2
B
R2
HOOC
Br
OH
N
O
2a-2c
5a-5c, 6a-6c
3a: p-COOH, R1=acetyl
3b: p-COOH, R1=pivaloyl
3c: p-COOH, R1=methanesulfonyl
3d: p-COOH, R1=H
1a: R1=acetyl
1b: R1=pivaloyl
1c: R1=methanesulfonyl
5a: p-COOH, R2=N-cyclopropylamino
5b: p-COOH, R2=N,N- diethylamino
5c: p-COOH, R2=N-[2-(dimethylamino)ethyl]amino
2a: R2=N-cyclopropylamino
2b: R2=N,N- diethylamino
2c: R2=N-[2-(dimethylamino)ethyl]amino
6a: m-COOH, R2=N-cyclopropylamino
6b: m-COOH, R2=N,N- diethylamino
6c: m-COOH, R2=N-[2-(dimethylamino)ethyl]amino
4a: m-COOH, R1=acetyl
4b: m-COOH, R1=pivaloyl
4c:m-COOH, R1=methanesulfonyl
4d: m-COOH, R1=H
CF3
CF3
O
O
Cl
Cl
O
O
e
f
d
OH
OH
NH2
N
N
HN
H
H
Boc
7
HN
NH2
8
Boc
9
O
AA1: p-, R1=acetyl
AA2: p-, R1=pivaloyl
AA3: p-, R1=methanesulfonyl
AA4: p-, R1=H
H
N
CF3
Cl
N
H
AA9: p-, R2=N-cyclopropylamino
AA10: p-, R2=N,N- diethylamino
N
O
R1
N
AA1-AA8
AA5: m-, R1=acetyl
AA11: p-, R2=N-[2-(dimethylamino)ethyl]amino
AA12: m-, R2=N-cyclopropylamino
H
AA6: m-, R1=pivaloyl
AA7: m-, R1=methanesulfonyl
AA8: m-, R1=H
AA13: m-, R2=N,N- diethylamino
AA14: m-, R2=N-[2-(dimethylamino)ethyl]amino
g
O
H
O
N
CF3
Cl
N
H
R2
O
AA9-AA14
N
Scheme 1. Synthesis route of the title compounds AA1-AA14.

Reagents and conditions: a. acyl chloride, Et3N, CH2Cl2, 0ºC→r.t.; b. SOCl2, reflux, amine,
CH2Cl2, 0ºC→r.t.; c. Pd(PPh3)4, Cs2CO3, CH3CN/H2O (V:V=3:2), 100ºC; d. (Boc)2O, NaOH/H2O,
THF, 25ºC; e. ClCOO-iBu, Et3N, CH2Cl2, -20ºC, 5-amino-2-chlorobenzotrifluoride; f. CH2Cl2,
TFA; g. ClCOO-iBu, 9, NMM, THF, 0ºC→r.t.
Title compounds AA15-AA28, with proline as linker, were readily prepared using
the synthetic route described in Scheme 2. Commercially available
L-(4S)-hydroxyproline was firstly NH2-protected by (Boc)2O [27], and reacted with
4-chloro-3-(trifluoromethyl)aniline using ethyl chloroformate as condensation reagent
[31]. Then, the hydroxyl group of compound 11 was mesylated, followed by reaction
with sodium azide to convert into azide group. Meanwhile, the chirality of C4 was
reversed from S to R. Next, the azide group was hydrated by Pd/C under H2, to
generate the key intermediate 14 [32]. Consequently, using the same condensation
method as described for the preparation of title compounds in Scheme 1, compounds
3a-3d, 4a-4d, 5a-5c and 6a-6c were reacted with compound 14 to get corresponding
compounds 15a-15d, 16a-16d, 17a-17c and 18a-18c, which were deprotected by
trifluoroacetic acid to obtain target compounds AA15-AA28.
3a: p-COOH, R1=acetyl
COOH
5a: p-COOH, R2=N-cyclopropylamino
5b: p-COOH, R2=N,N- diethylamino
5c: p-COOH, R2=N-[2-(dimethylamino)ethyl]amino
3b: p-COOH, R1=pivaloyl
3c: p-COOH, R1=methanesulfonyl
3d: p-COOH, R1=H
N
R1
N
H
4a: m-COOH, R1=acetyl
4b: m-COOH, R1=pivaloyl
4c:m-COOH, R1=methanesulfonyl
4d: m-COOH, R1=H
6a: m-COOH, R2=N-cyclopropylamino
6b: m-COOH, R2=N,N- diethylamino
6c: m-COOH, R2=N-[2-(dimethylamino)ethyl]amino
3a-3d, 4a-4d
O
COOH
Boc
R2
H
Cl
Boc
O
N
N
COOH
N
COOH
a
N
b
CF3
N
5a-5c, 6a-6c
H
HO
HO
10
11
HO
Boc
c
Cl
Cl
CF3
Cl
CF3
Boc
O
Boc
N
O
O
e
N
d
N
CF3
N
H
N
H
N
H
12
H2N
N3
MsO
14
13
Boc
O
N
H
O
NH
H
N
CF3
Cl
N
CF3
Cl
N
N
N
H
N
O
H
g
O
R1
R1
N
15a-15d, 16a-16d
N
H
AA15-AA22
H
f
Boc
N
H
O
NH
H
O
O
N
CF3
Cl
O
N
CF3
Cl
N
H
g
N
R2
O
R2
H
O
N
AA23-AA28
N
17a-17c, 18a-18c
15a, AA15: p-, R1=acetyl
15b, AA16: p-, R1=pivaloyl
15c, AA17: p-, R1=methanesulfonyl
15d, AA18: p-, R1=H
17a, AA23: p-, R2=N-cyclopropylamino
17b, AA24: p-, R2=N,N- diethylamino
17c, AA25: p-, R2=N-[2-(dimethylamino)ethyl]amino
18a, AA26: m-, R2=N-cyclopropylamino
16a, AA19: m-, R1=acetyl
16b, AA20: m-, R1=pivaloyl
16c, AA21: m-, R1=methanesulfonyl
16d, AA22: m-, R1=H
18b, AA27: m-, R2=N,N- diethylamino
18c, AA28: m-, R2=N-[2-(dimethylamino)ethyl]amino
Scheme 2. Synthesis route of the title compounds AA15-AA28.
Reagents and conditions: a. (Boc)2O, NaOH/H2O, THF, 25ºC; b. ClCOOEt, Et3N, CH2Cl2, 0ºC,
5-amino-2-chlorobenzotrifluoride; c. methylsulfonyl chloride, Et3N, CH2Cl2, 0ºC→r.t.; d. NaN3,

DMF, 65-70ºC; e. 10% Pd/C, MeOH; f. ClCOO-iBu, 14, NMM, THF, 0ºC→r.t.; g. CH2Cl2, TFA.
1
All of the title compounds were characterized by H-NMR, 13C-NMR, LC-MS,
HRMS and melting point. Detailed synthetic procedures are described in
Experimental Section, and the NMR, MS and MP data are listed in Supplementary
Material. In addition, to standard the 13C NMR resonance signals of CF3 group and
nearby
C
atom
in
phenyl
ring,
the
13C-NMR
data
of
4-chloro-3-trifluoromethyl-aniline, used as starting material in synthesis of AA series,
19
is provided. Furthermore, HSQC-NMR date and F-NMR data of two representative
compounds (AA2 and AA19) are depicted in Supplementary Material.
3. Results and discussion
3.1 In Vitro kinases inhibitory activity and SAR
Enzymatic inhibition evaluation of the title compounds against Bcr-AblWT and
Bcr-AblT315I was performed using ADP-Glo kinase assay kit (Promega, Wisconsin,
USA) [33, 34]. The results were depicted in Table 1 and Table 2, with imatinib as
positive control. The majority of the title compounds exhibited moderate to good
inhibitory activity. As shown in Table 1, with alanine as linker, HBM was diversified,
including various pyridinyl amide and nicotinamide at m- or p- position of A ring.
The biological results indicated that p- substitution of heterocycle on A ring was more
favorable than m- substitution for Bcr-AblWT inhibition, especially for Bcr-AblT315I
inhibition. With pyridine on p- position of phenyl ring as hinge binding group,
compound AA4 displayed good activity toward Bcr-AblWT and Bcr-AblT315I kinases
with IC50 of 0.77 μM and 7.51 μM, respectively. Furthermore, the pivaloylation of
pyridine yielded the most potent compound, AA2, with IC50 of 0.041 μM and 0.53
μM against Bcr-AblWT and Bcr-AblT315I, respectively, comparable to that of imatinib.
When the hinge binding group turned out to be nicotinamide, the inhibitory potency
against Bcr-AblT315I was enhanced, while the Bcr-AblWT inhibition activity was
retained. However, compounds AA10 and AA13 displayed poor activity against
T315I mutant, which may be attributed to the bulky and basic sidechain, N,N-
diethylamino.
Table 1 Structures and kinase inhibitory activities of title compounds AA1-AA14.
Compd
Het
Py
R1/R2
Bcr-AblWT IC50 (μM)
Bcr-AblT315I IC50 (μM)
AA1
AA2
p-
p-
acetyl
0.20
22.45
0.53
pivaloyl
0.041

AA3
AA4
p-
p-
methanesulfonyl
H
3.77
0.77
0.60
4.48
3.34
4.40
9.38
0.011
6.62
1.46
0.29
0.64
0.054
9.95
7.51
AA5
m-
m-
m-
m-
p-
acetyl
38.64
243.91
129.31
86.84
4.99
AA6
pivaloyl
AA7
methanesulfonyl
H
AA8
AA9
N-cyclopropylamino
N,N- diethylamino
N-[2-(dimethylamino)ethyl]amino
N-cyclopropylamino
N,N-diethylamino
N-[2-(dimethylamino)ethyl]amino
AA10
AA11
AA12
AA13
AA14
Imatinib
p-
59.87
2.28
p-
m-
m-
m-
7.83
86.01
2.41
0.28
For compounds with proline as linker, the results are depicted in Table 2.
Compared with the series above, the majority of these compounds exhibited decreased
Bcr-AblWT and Bcr-AblT315I inhibitory activities. Compound AA17 showed the most
potent inhibition against Bcr-Abl with IC50 of 0.15 μM. As for Bcr-AblT315I inhibition,
most compounds exhibited poor inhibitory potency with IC50 up to dozens micromolar.
The results indicated that alanine, branched chain amino acid, might be more
favorable than proline as flexible linker for Bcr-Abl inhibitors. Besides, in compounds
(AA15-AA22) with pyridinyl amide as hinge binding group, the same phenomenon
was observed that compounds bearing pyridine on p- position of A ring had better
potency than compounds with pyridine on m- position. It may be concluded that the
heterocycle in p-position of linker was more beneficial for improving Bcr-Abl
inhibition than m-position of each other, when heterocyclic biphenyl group was
adopted as HBM. Interestingly, it was also found that compounds AA24 and AA27,
bearing N,N- diethylamino as a sidechain, had poor potency against T315I mutant. We
suspected that the bulky diethylamino group resulted in large spatial conformation,
which was not preferred for binding to the ATP pocket of Bcr-AblT315I. Overall,
compound AA2 was the most potent against Bcr-AblWT and Bcr-AblT315I, which can
be used for further modification.
Table 2 Structures and kinase inhibitory activities of title compounds AA15-AA28.
Compd
Het
Py
R1/R2
Bcr-AblWT IC50 (μM)
Bcr-AblT315I IC50 (μM)
AA15
AA16
p-
p-
acetyl
34.47
2.64
12.99
50.26
pivaloyl

AA17
AA18
AA19
AA20
AA21
AA22
AA23
AA24
AA25
AA26
AA27
AA28
Imatinib
p-
p-
methanesulfonyl
H
0.15
9.18
0.49
4.63
6.48
11.96
61.21
7.40
4.23
16.00
3.75
2.41
0.054
36.92
14.10
24.59
147.17
16.16
103.11
39.03
50.47
22.10
26.61
42.67
21.75
0.28
m-
m-
m-
m-
p-
acetyl
pivaloyl
methanesulfonyl
H
N-cyclopropylamino
N,N- diethylamino
-[2-(dimethylamino)ethyl]amino
N-cyclopropylamino
N,N-diethylamino
-[2-(dimethylamino)ethyl]amino
p-
p-
N
N
m-
m-
m-
3.2 Anti-proliferative activity against CML cell lines
Based on the inhibition data from kinases assay, all of title compounds were
screened for their antiproliferative potency against Bcr-Abl positive K562 cells, using
MTT method with imatinib as positive control [35]. The results were presented in
Table 3. Meanwhile, for further understanding of these compounds’ antiproliferative
activity, the determined ClogP values were also provided. As shown in Table 3, the
majority of these compounds displayed good antiproliferative activities with IC50
value at micromolar level. Compared with imatinib, most compounds had an
increased ClogP value, which indicated that the amino acids motif has indeed
improved the lipophilicity for these compounds. In particular, compounds AA7, AA11,
and AA25 accounted for the most significant effect with IC50 values of 4.66 μM, 4.65
μM and 4.56 μM, respectively, comparable to imatinib with IC50 of 4.26 μM. In
addition, the antiproliferative activities of compounds (AA1-AA14), with alanine as
linker, were generally better than that of compounds (AA15-AA28) with proline as
linker. These results were consistent with their enzymatic inhibition against Bcr-Abl
kinase. Moreover, the potent activities of Ala-based inhibitors might also be attributed
to their general higher ClogP values compared with Pro-based series. Since increased
ClogP values meant enhanced lipid solubility, compounds with Ala-linker could easier
penetrate the cell membrane and enter into cytoplasm to interact with Bcr-Abl kinase.
Consequently, they exhibited good activity in cell assays, in accordance with the
results shown in Table 3. More importantly, compound AA2, the most potent
compound in kinase assay, also had a good antiproliferative activity with IC50 of 7.45
μM, confirming its rationality as a lead compound for next optimization.
Table 3 Antiproliferative activities of title compounds against K562 cell lines and ClogP values.

Compd
K562 IC50(μM)
ClogP
Compd
K562 IC50(μM)
ClogP
AA1
AA2
106.77
7.45
4.23
5.45
3.64
4.15
4.34
5.57
3.75
4.26
3.79
3.80
3.46
3.90
3.91
3.57
3.28
AA15
AA16
AA17
AA18
AA19
AA20
AA21
AA22
AA23
AA24
AA25
AA26
AA27
AA28
Imatinib
22.80
11.55
44.99
9.13
3.39
4.61
2.84
3.95
4.13
4.72
2.95
4.06
3.63
3.59
3.33
3.74
3.70
3.44
3.28
AA3
7.24
AA4
5.95
AA5
42.81
18.40
4.66
8.71
AA6
28.03
24.62
20.04
65.30
55.48
4.56
AA7
AA8
8.09
AA9
396.00
20.21
4.65
AA10
AA11
AA12
AA13
AA14
Imatinib
15.12
29.93
7.92
27.29
24.89
19.76
4.26
4.26
3.3 In virto activity of AA2 against K562R cells and its cytotoxicity
According to the Bcr-Abl inhibitory potency and anti-proliferative activity,
compound AA2 was further evaluated for its activity toward K562R cells expressing
T315I mutation [36], with ponatinib and imatinib as positive control. The result was
displayed in Figure 2A, and imatinib was less potent against K562R cells, exhibiting
about 45% inhibition rate at the concentration of 100 μM. For compound AA2, it was
found to moderately inhibit the growth of K562R cells with IC50 value of 9.10 μM,
while ponatinib had an IC50 value of 0.093 μM toward K562R cells. In addition, we
identified the cytotoxicity of compound AA2 on two normal cells, including Hek293
(human embryonic kidney 293 cells) and EA.hy 926 (human vascular endothelial
cells), with imatinib as control. As shown in Figure 2B and 2C, compound AA2
exhibited less toxicity toward these two cell lines when its concentration increased up
to 100 μM. In brief, compound AA2 displayed selectivity growth inhibitory activity
against Bcr-Abl positive K562 and K562R cells compared to normal human cell lines.
A
B
C
Figure 2. Anti-proliferative activity of compound AA2 against K562R cells (A) expressing T315I
mutation, and cytotoxicity of compound AA2 toward two normal human cells, including Hek293
cells (B) and EA.hy926 cells (C).

3.4 Effects of compound AA2 on apoptosis in K562 cell lines
Compound AA2 was also selected to do the flow cytometry analysis to
investigate its effect on cell proliferation. K562 cells was used, and the apoptosis
assay was performed by Annexin V/PI staining [37]. A substantial and dose-dependent
apoptotic activity in K562 cells at various concentrations for 48 h was observed
(Figure 3A). The apoptotic K562 cells (15.58%) of negative control group were
depicted in Figure 3B. After treatment of compound AA2 at the concentration of 1,
10, 20 μM, the percentage of cell apoptosis was 24.5%, 58.99%, and 62.42%,
respectively. These data indicated that compound AA2 could induce K562 cells
apoptosis in a dose-dependent manner. Next, western blot assay of compound AA2
was performed to explore its inhibitory mechanism. K562 cells were treated with
compound AA2 at 1, 5, 10, 15 μM for 48 h, and the phosphorylation status of Bcr-Abl
in CML cells was determined. As expected, we observed that compound AA2
dose-dependently decreased tyrosine phosphorylation of Bcr-Abl in K562 cells
compared with the control group (Figure 3C). Meanwhile, as illustrated in Figure 3D,
at the concentration of 10 μM, compound AA2 begin to remarkably suppress the
phosphorylation of Bcr-Abl. This result suggested that compound AA2 may inhibit
the growth of K562 cells and further induce K562 cells apoptosis through down
regulation of phosphorylation level of Bcr-Abl.
Control
AA2
A
1 μM
10 μM
20 μM
0 μM
Annexin V
C
B
AA2
D
Imatinib (IM)
0.1
AA2
0
1
10
(μM)
0
1
5
10
15
(μM)
p‐Bcr‐Abl
GADPH

Figure 3. Apoptosis analysis of K562 cells after treatment with AA2 (A. Flow cytometry
analysis, B. Apoptotic ratio), and effect of AA2 on the phosphorylated level of Bcr-Abl in K562
cells (C. p-Bcr-Abl/GADPH ratio, D. western blotting results).
3.5 Assessment of the simulation stability via RMSD analysis
For further structural optimization and investigation of the potential binding
mode, the most potent compound AA2 were applied to do the molecular modeling
study with Bcr-AblWT and Bcr-AblT315I kinases, respectively [15, 38]. In addition,
compound AA19, with proline as linker, was also employed in the modeling studies.
Molecular docking was conducted using Sybyl-X to generate the initial conformations
of protein-ligand complex, and then the constructed systems were subjected to 20 ns
MD simulation to check for their structural stability and movements during the course
of simulation [39]. The RMSD values of the backbone atoms of protein, and the
heavy atoms of ligands were used to monitor the dynamic stabilities of the studied
systems.
A
B
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
C
D
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
Figure 4. Protein-Ligand RMSD plot for Abl kinases and its inhibitors as a function of time in
MD simulations (A. Bcr-AblWT and AA2, B. Bcr-AblWT and AA19, C. Bcr-AblT315I and AA2, D.
Bcr-AblT315I and AA19). The protein backbone is presented in blue, and the ligand is presented in
red.
As shown in Figure 4A, in AA2-1IEP (Bcr-AblWT) complex, protein RMSD
started from 1.2 Å and later stabilized at 2.4 Å, whereas, ligand RMSD stabilized
around 1.0 Å. As for AA2-3IK3 (Bcr-AblT315I) complex (Figure 4B), the protein
RMSD fluctuated at around 2.2 Å while the ligand RMSD at about 0.9 Å when the
system reached to its equilibrium state. The dynamic study of AA19 with Bcr-Abl
kinases were depicted in Figure 4C and 4D. The system of AA9 with 1IEP was stable

during the MD process, with the average values of protein RMSD and ligand RMSD
at 2.0 Å and 1.4 Å, respectively. In terms of AA19-3IK3 complex, the average value
for protein RMSD was 2.5 Å with a maximum of 4.0 Å at 18 ns, and the ligand
RMSD was stable around 2.5 Å. Changes of the order of 1-3 Å are perfectly
acceptable for small, globular proteins. Overall spectrum of RMSD for above studied
complexes did not show any considerable structural shifts during MD process, which
explains the stability of the kinases structure and strength of ligand attachment inside
the active site pocket.
3.6 The interaction analysis between target compounds and Bcr-Abl kinases
The interaction mode of compounds AA2 and AA19 with Bcr-Abl kinases after
MD processed were applied and presented by PyMOL softwore. The interaction
analysis of AA2 with Bcr-Abl kinases were displayed in Figure 5. Favorable binding
interactions of AA2 with Bcr-AblWT included three hydrogen bonds (Figure 5A):
N-(pyridin-2-yl)pivalamide in AA2 formed one hydrogen bond with Met318 in the
hinge region of Bcr-Abl with distance of 2.4Å. In addition, the terminal NH of linker
part generated one hydrogen bond with conserved Glu286 for the bond length of 2.1Å,
while CO formed a hydrogen bond with Asp381 of DFG-motif with distance of 2.1Å.
A
B
D
C
E
Figure 5. Docking results of compound AA2 (Orange). A and C, Bcr-AblWT docking with
imatinib (Green) as control. B and D, Bcr-AblT315I docking with ponatinib (Green) as control. A
and B are represented as Ribbon model, while C and D are shown as surface for the binding site.
E, Bcr-AblT315I docking with Ile315 presented as CPK models.
As for Bcr-AblT315I, the binding mode was depicted in Figure 5B. The CO of
pivalamide generated one hydrogen bond with Tyr253 for the bond length of 2.2Å,
while the terminal NH of linker part also formed one hydrogen bond with conserved
Glu286 with distance of 1.9Å. Compared with Bcr-AblWT docking, the hydrogen bond

with DFG motif has been diminished. Meanwhile, as shown in 5D, the channel, where
HBM occupied, came to be smaller when it went to the Gatekeeper residue part,
compared with that of Bcr-AblWT (Figure 5C). This narrowed channel made the
phenyl ring in HBM a little crash with its around residues when it was binding,
resulting in a certain loss of interaction between HBM and adenine pocket.
Consequently, the decreased affinity might lower the activity of AA2 against
Bcr-AblT315 observed in enzymatic assays.
The above molecular docking results indicated that alanine could generate
hydrogen bond with conserved Glu286 or DFG-motif, which was important for
Bcr-Abl binding, and avoid steric clash with Ile315 in Bcr-AblT315I seen in Figure 5E,
thus providing novel scaffolds for developing anti-leukemia agents. Besides, as we
expected, N-(pyridin-2-yl)pivalamide generated hydrogen bonds with hinge region,
beneficial for improving affinity with Bcr-Abl.
A
B
E
C
D
Figure 6. Docking results of compound AA19 (Orange). A and C, Bcr-AblWT docking with
imatinib (Green) as control. B and D, Bcr-AblT315I docking with ponatinib (Green) as control. A
and B are represented as Ribbon model, while C and D are shown as surface for the binding site.
E, Bcr-AblT315I docking with Ile315 presented as CPK models.
For compound AA19, the docking results with Bcr-Abl kinases (WT and T315I)
were depicted in Figure 6. Similar as Ala-linker, Pro-linker could avoid Ile315 bulky
steric clash (Figure 6E), attributing to its flexibility. Compound AA19 fitted the ATP
pocket of Bcr-AblWT well, and formed five hydrogen bonds with kinase protein
(Figure 6A). As for Bcr-AblT315I, although there were four hydrogen bonds generated
(Figure 6B), the HBM of AA19 pointed almost out of the pocket and was exposed to
the solvent compared with that of ponatinib, which may result in the deletion of

hydrophobic interaction with hinge region (Figure 6D). The decreased affinity with
Bcr-AblT315I might explain the decreased activity of compound AA19 toward T315I
mutant.
In addition, we also calculated the CScore of these two compounds, AA2 and
AA19, which was used as criterion to assess the inhibitors’ binding affinity in
Sybyl-X. The CScore of AA2 was 11.8549, while AA19 had a CScore value of 9.1113.
It was indicated that compound AA2, with Ala-linker, had better affinity with Bcr-Abl,
than Pro-based compound AA19. We suspected that Pro-linker, containing a
five-membered ring, had a certain degree of rigidity, making Pro-based compounds
requiring more energy to fit in the ATP pocket. This may explain why most Ala-based
compounds had better enzymatic activity than compounds with Pro-linker.
4. Conclusion
In this study, a series of twenty-eight compounds was developed to overcome
drug resistance caused by T315I mutation in Bcr-Abl. Amino acids were firstly
introduced as flexible linker to avoid steric clash with the isoleucine gatekeeper
residue in Bcr-AblT315I, with heterocyclic structure applied as HBM to generate
hydrogen bonds with hinge region. The biological results indicated that compound
AA2 displayed the most potent inhibition of Bcr-AblWT and Bcr-AblT315I, comparable
to that of imatinib. Meanwhile, it displayed potent antiproliferative activity against
Bcr-Abl positive K562 cells. In addition, compound AA19, with proline as linker, also
exhibited promising activity against Bcr-AblWT and Bcr-AblT315I as well as toward
K562 cells. Meanwhile, compound AA2 exhibited the potent antiproliferative activity
toward K562R cells with T315I mutant. These compounds, especially compound
AA2, might be considered as novel lead compounds for further development of
Bcr-Abl inhibitors as anti-leukemia agents. Furthermore, cytometry analysis
suggested that the representative compound AA2 could induce the apoptosis of K562
cells, and western blot revealed that AA2 exerted the growth inhibition of K562 cells
through down regulation of Bcr-Abl phosphorylation. Moreover, molecular docking
studies identified the rationality of amino acids as linker for Bcr-Abl inhibitor design,
and phenyipyridine with amide sidechain was beneficial for potency of these
inhibitors. Together with these prior studies, our study strongly supports the notion
that introducing flexible linker can be a general strategy for developing inhibitors of
protein kinases containing a bulky gatekeeper residue. Specially, our study raises the
interesting possibility that applying amino acids as linker lead to promising inhibitors
for Bcr-AblT315I. This study is currently ongoing in our laboratory.
5. Experimental section

5.1. Chemistry: General procedure
Chemicals and solvents were purchased from commercial suppliers and used
without further purification unless otherwise indicated. All anhydrous solvents were
dried according to the standard methods and parts of them were freshly distilled prior
1
to use. Melting points were measured on an X-4 micromelting apparatus. H NMR
and 13C NMR spectra were measured on a Bruker Advance 400 at 400 and 100 HZ,
respectively. Chemical shifts (δ) in DMSO-d6 were referenced to tetra-methyl silane
(TMS 0.0 ppm). The solvent peak and H2O peak were set to 2.50 ppm and 3.33 ppm.
Mass spectra were partly measured using liquid chromatography mass spectrometry
(LC-MS) with electrospray ionization (ESI), while others were measured using gas
chromatography mass spectrometry (GC-MS) with electron impact ionization (EI).
All reactions progress was monitored by TLC (GF254), and visualized with UV (254
nm) except Boc-protection of amino acids using ninhydrin reaction. All compounds
were purified by chromatography with silica gel (300-400 mesh).
5.1.1 N-(5-bromopyridin-2-yl)acetamide (1a)
5-bromopyridin-2-amine (5.19 g, 30 mmol) and Et3N (20 mL) were dissolved in
anhydrous CH2Cl2 (100 mL), cooled to 0 , followed by dropwise addition of acetyl
chloride (2.54 mL, 36 mmol). The reaction solution was warmed to room temperature
and stirred overnight. Then the solution was diluted with CH2Cl2, and washed with
water (30 mL×3), saturated NaHCO3 aq (30 mL×3), and brine (30 mL). The organic
phase was dried over Na2SO4, filtered, and purified by column chromatography (PE/
EtOAc=5:1) to give a white solid (5.65 g, 88%). Mp 78-81 . EI-MS (m/z) 214[M]+.
5.1.2 N-(5-bromopyridin-2-yl)pivalamide (1b)
The method described above was used with 4.43 mL pivaloyl chloride. Elution with
petroleum ether gave a white solid (7.37 g, 96%). Mp 42-44 . EI-MS (m/z) 256[M]+.
5.1.3 N-(5-bromopyridin-2-yl)methanesulfonamide (1c)
Same method as compound 1a was used with 2.78 mL methanesulfonyl chloride.
Elution with PE/EtOAc (5/1, v/v) yield a white solid (5.52 g, 86%). Mp 159-160 .
EI-MS (m/z) 250[M]+.
5.1.4 5-bromo-N-cyclopropylnicotinamide (2a)
Sulfoxide chloride (36 mL) was added dropwise into 5-bromonicotinoc acid (5.0 g,
24.7 mmol) under N2 at room temperature. The resulting mixture was refluxed for 2 h

till the solution was clear, and then the volatiles were removed under vacuum. The
generated acid chloride was dissolved in anhydrous CH2Cl2 (30 ml), and the solution
was added slowly at 0 to a solution of cyclopropylamine (3.77 mL, 54.4 mmol) in
CH2Cl2 (30 mL). Stirring was continued overnight. K2CO3 aq (2 M, 20 mL) was
added and the aqueous layer was extracted with CH2Cl2 (10 mL×3). The combined
organic layer was washed with brine (30 mL), dried over Na2SO4, filtered,
concentrated in vacuo, and purified by chromatography column (PE/EtOAc=1:1) to
obtain a white solid (5.27 g, 89%). Mp 140-142 . EI-MS (m/z) 240[M]+.
5.1.5 5-bromo-N,N-diethylnicotinamide (2b)
Same method as described above was used with 5.60 mL diethylamine. Elution
with PE/EtOAc (1/1, v/v) gave an oil (5.50 g, 87%). EI-MS (m/z) 256[M]+.
5.1.6 5-bromo-N-(2-(dimethylamino)ethyl)nicotinamide (2c)
Same
method
as
compound
2a
was
used
with
4.80
mL
N,N-dimethyl-1,2-ethanediamine. Elution with ethyl acetate gave a colorless oil (5.35
g, 80%). EI-MS (m/z) 271[M]+.
5.1.7 4-(6-(acetylamino)pyridin-3-yl)benzoic acid (3a)
N-(5-bromopyridin-2-yl)acetamide (1a) (4.30 g, 20 mmol), 4-boronobenzoic acid
(3.66 g, 22 mmol), Cs2CO3 (13.0 g, 40 mmol), and Pd (PPh3)4 (1.2 g, 1 mmol) were
suspended in a mixture of CH3CN/H2O (200 mL, v/v=3:2) under N2. The mixture was
heated in oil bath at 90 for 48 h. Then the resulting solution was filtered
immediately after the reaction completed. The filtrate was adjusted to pH4 using HCl
aq (6M). The precipitate was filtered out and dried overnight to get the product as
white solid (3.89 g, 76%). Mp 156-158 . EI-MS (m/z) 256[M]+.
Compounds 3b-3d, 4a-4d, 5a-5c, and 6a-6c were prepared using the same method
as described above.
5.1.8 4-(6-pivalamidopyridin-3-yl)benzoic acid (3b).
N-(5-bromopyridin-2-yl)pivalamide (1b) (5.14 g) was reacted with
4-boronobenzoic acid, giving a solid (4.77 g, 80%). Mp 276-277 . EI-MS (m/z)
298[M]+.
5.1.9 4-(6-(methylsulfonamido)pyridin-3-yl)benzoic acid (3c)
N-(5-bromopyridin-2-yl)methanesulfonamide (1c) (5.02 g) was reacted with
4-boronobenzoic acid, giving a solid (4.32 g, 74%). Mp 294-295 . EI-MS (m/z)

292[M]+.
5.1.10 4-(6-aminopyridin-3-yl)benzoic acid (3d)
5-bromopyridin-2-amine (3.46 g) was reacted with 4-boronobenzoic acid, giving a
solid (3.12 g, 73%). Mp >300 . EI-MS (m/z) 216.0765[M+H]+, 214.0622[M-H]-.
5.1.11 3-(6-acetamidopyridin-3-yl)benzoic acid (4a)
N-(5-bromopyridin-2-yl)acetamide (1a) (4.30 g) was reacted with 3-boronobenzoic
acid, giving a solid (3.32 g, 65%). Mp 238-240 . EI-MS (m/z) 255.30[M-H]+.
5.1.12 3-(6-pivalamidopyridin-3-yl)benzoic acid (4b)
N-(5-bromopyridin-2-yl)pivalamide (1b) (5.14 g) was reacted with
3-boronobenzoic acid, giving a solid (4.29 g, 72%). Mp 222-223 . EI-MS (m/z)
297.25[M-H]+.
5.1.13 3-(6-(methylsulfonamido)pyridin-3-yl)benzoic acid (4c)
N-(5-bromopyridin-2-yl)methanesulfonamide (1c) (5.02 g) was reacted with
3-boronobenzoic acid, giving a solid (3.67 g, 63%). Mp 264-265 . EI-MS (m/z)
291.20[M-H]+.
5.1.14 4-(6-aminopyridin-3-yl)benzoic acid (4d)
5-bromopyridin-2-amine (3.46 g) was reacted with 3-boronobenzoic acid, giving a
solid (2.60g, 61%). Mp 240-241 . EI-MS (m/z) 215.10[M]+.
5.1.15 4-(5-(cyclopropylcarbamoyl)pyridin-3-yl)benzoic acid (5a)
5-bromo-N-cyclopropylnicotinamide (2a) (4.82 g) was reacted with
4-boronobenzoic acid, giving a solid (4.79 g, 85%). Mp 258-260 . EI-MS (m/z)
282[M]+.
5.1.16 4-(5-(diethylcarbamoyl)pyridin-3-yl)benzoic acid (5b)
5-bromo-N,N-diethylnicotinamide (2b) (5.12 g) was reacted with 4-boronobenzoic
acid, giving a solid (4.53 g, 76%). Mp 235-236 . EI-MS (m/z) 297[M-H]-.
5.1.17 4-(5-((2-(dimethylamino)ethyl)carbamoyl)pyridin-3-yl)benzoic acid (5c)
5-bromo-N-(2-(dimethylamino)ethyl)nicotinamide (2c) (5.44 g) was reacted with
4-borono-benzoic acid, giving a solid (4.38 g, 70%). Mp 160-162 . EI-MS (m/z)

313.95[M+H]+.
5.1.18 3-(5-(cyclopropylcarbamoyl)pyridin-3-yl)benzoic acid (6a)
5-bromo-N-cyclopropylnicotinamide (2a) (4.82 g) was reacted with
3-boronobenzoic acid, giving a solid (4.39 g, 78%). Mp 263-264 . EI-MS (m/z)
281.10[M-H]+.
5.1.19 3-(5-(diethylcarbamoyl)pyridin-3-yl)benzoic acid (6b)
5-bromo-N,N-diethylnicotinamide (2b) (5.12 g) was reacted with 3-boronobenzoic
acid, giving a solid (3.93 g, 66%). Mp 176-178 . EI-MS (m/z) 297.25[M-H]+.
5.1.20 3-(5-((2-(dimethylamino)ethyl)carbamoyl)pyridin-3-yl)benzoic acid (6c)
5-bromo-N-(2-(dimethylamino)ethyl)nicotinamide (2c) (5.44 g) was reacted with
3-borono-benzoic acid, giving a solid (3.63 g, 58%). Mp 167-168 . EI-MS (m/z)
299.15[M-CH3]-.
5.1.21 (tert-butoxycarbonyl)-L-alanine (7)
L-alanine (7.14 g, 80 mmol) was dissolved in NaOH aq (40 M, 80 mL), followed
by adding 80 mL THF, and cooled to 0 . (Boc)2O (19.2 g, 88 mmol) was added
dropwise into the above solution, and stirring was continued at room temperature
untill L-alanine was consumed completely by TLC (ninhydrin reaction). Then THF
was removed under vacuum, and the concentrated solution was adjusted to pH 2-3
under water-ice bath using citric acid, extracted with EtOAc (60 mL×3). The
combined organic phase was washed with brine (60 mL), dried over Na2SO4. The
crude product was recrystallized using PE/EtOAc, yielding Boc-L-alanine (13.6 g,
90%). Mp 81-83 .
5.1.22
tert-butyl-(R)-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-
2-yl)carbamate (8)
(tert-butoxycarbonyl)-L-alanine (7) (1.9 g, 10 mmol) was dissolved in 50 mL
anhydrous CH2Cl2, cooled to -20 , and stirred for 5-10 min, followed by adding
triethylamine (2.77 mL, 20 mmol). Then isobutyl chloroformate (1.95 mL, 15 mmol)
in CH2Cl2 (3 mL) was added slowly, and stirred for 10-20 min to generate activated
anhydride. Next, 4-chloro-3-(trifluoro-methyl)aniline (1.95 g, 10 mmol) in CH2Cl2
(10 mL) was added dropwise to the above solution, and stirring was continued for 2 h.
After that, 5% NaHCO3 aq (50 M, 100 mL) was added, and the reaction solution was
warmed to room temperature, extracted with CH2Cl2 (30 mL×3). The combined

organic layer was washed with 5% NaHCO3 aq, 5% HCl aq, brine, and dried over
Na2SO4. The crude product was purified by column chromatography (PE/EtOAc=20:1)
to obtain compound 8 (3.23 g, 88%). EI-MS (m/z): 367 [M+H]+.
5.1.23 (R)-2-amino-N-(4-chloro-3-(trifluoromethyl)phenyl)propanamide (9)
Under ice bath, compound 8 (3.23 g, 8.8 mmol) was dissolved in 100 mL
anhydrous CH2Cl2, and trifluoroacetic acid (6.5 mL, 88 mmol) in CH2Cl2 (10 mL)
was added slowly to the above solution. The reaction solution was stirred under room
temperature overnight. Then water (150 mL) was added, stirred for 20 min, and the
organic layer was discarded. The aqueous phase was adjusted to pH 8 using saturated
NaHCO3 aq, and extracted with EtOAc (60 mL×3). The combined EtOAc was
washed with brine, dried over Na2SO4, filtered, concentrated, and the residue was
obtained, which was used next without purification. EI-MS (m/z) 267.05 [M+H]+,
265.00[M-H]-.
5.1.24
(R)-4-(6-acetamidopyridin-3-yl)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)
amino)-1-oxopropan-2-yl)benzamide (AA1)
In a 100 mL flask, compound 3a (2.5 mmol) was dissolved in anhydrous THF (60
mL), followed by adding 4-methylmorpholine (0.85 mL, 7.5 mmol). At 0 , isobutyl
chloroformate (0.55 mL, 3.75 mmol) in anhydrous THF (5 mL) was dropped slowly
into the above suspension. Then the reaction mixture was reacted under 0 for 1 h.
Next, the THF (10 mL) solution of compound 9 (0.8 g, 3 mmol) and
4-methylmorpholine (0.85 mL) was added dropwise into the above solution, and
stirring was continued under room temperature overnight. THF was removed in vacuo,
and the residue was dissolved with EtOAc (60 mL). The organic layer was washed
with water (15 mL), brine (15 mL), dried over Na2SO4. The crude product was
purified by column chromatography (PE/EtOAc=1:1) to get a white solid (0.34 g,
27%). Mp 224-225 . EI-MS (m/z): 505 [M+H]+, 503 [M-H]-. HRMS (ESI): calcd for
[M+H]+ C24H21ClF3N4O3: 505.12543, found 505.12735. 1H NMR (400 MHz,
DMSO-d6) δ 10.67 (s, 1H), 10.55 (s, 1H), 8.81 (d, J = 6.6 Hz, 1H), 8.74 (s, 1H), 8.25
(d, J = 2.4 Hz, 1H), 8.19 (s, 2H), 8.04 (d, J = 8.4 Hz, 2H), 7.90 (dd, J = 8.8, 2.3 Hz,
1H), 7.86 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 4.63 – 4.56 (m, 1H), 2.13 (s,
3H), 1.47 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.71, 169.87,
166.43, 152.33, 146.47, 140.07, 139.04, 136.79, 133.08, 132.60, 130.33, 128.85 (2C),
127.63/127.32/ 127.02/126.71 (q, J = 30.3 Hz, Cphenyl-CF3), 126.42 (2C),
127.26/124.55/121.84/119.13 (q, J = 273.7 Hz, CF3), 124.44, 124.32, 118.33/118.28
(CHphenyl-CF3), 113.61, 50.64, 24.41, 17.89.

Compound AA2-AA14 was prepared using the same method as described above.
5.1.25
(R)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)-4-
(6-pivalamido-pyridin-3-yl)benzamide (AA2)
Compound 3b was reacted with compound 9, and the crude product was
purification with PE/EtOAc (3/1, v/v) to give a white solid (0.29 g, 21%). Mp
179-181 . EI-MS (m/z): 547 [M+H]+, 545 M-H]-. HRMS (ESI): calcd for [M+H]+
1
C27H27ClF3N4O3: 547.117238, found 547.17132. H NMR (400 MHz, DMSO-d6) δ
10.56 (s, 1H), 9.99 (s, 1H), 8.82 (d, J = 6.6 Hz, 1H), 8.76 (s, 1H), 8.25 (d, J = 2.2 Hz,
1H), 8.19 (s, 2H), 8.05 (d, J = 8.3 Hz, 2H), 7.90 (dd, J = 9.0, 2.2 Hz, 1H), 7.87 (d, J =
8.3 Hz, 2H), 7.68 (d, J = 8.7 Hz, 1H), 4.64 – 4.57 (m, 1H), 1.48 (d, J = 7.1 Hz, 3H),
1.27 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 177.78, 172.72, 166.42, 152.59,
146.17, 140.04, 139.04, 136.68, 133.12, 132.59, 130.45, 128.86 (2C),
127.64/127.33/127.03/126.72 (q,
J
=
30.3Hz, Cphenyl-CF3), 126.45 (2C),
127.26/124.55/121.84/119.13 (q, J = 273.7 Hz, CF3), 124.42, 124.32, 118.33/118.27,
114.38, 50.64, 39.91, 27.34 (3C), 17.90.
5.1.26
(R)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)-4-
(6-(methyl-sulfonamido)pyridin-3-yl)benzamide (AA3)
Compound 3c was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/1, v/v) to give a white solid (0.63 g, 47%). Mp
155-157 . EI-MS (m/z): 541 [M+H]+, 539 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C23H21ClF3N4O4S: 541.09241, found 541.09284. H NMR (400 MHz, DMSO-d6) δ
10.87 (s, 1H), 10.54 (s, 1H), 8.80 (d, J = 6.7 Hz, 1H), 8.68 (s, 1H), 8.24 (d, J = 2.3 Hz,
1H), 8.16 (dd, J = 8.6, 2.4 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.90 (dd, J = 8.8, 2.3 Hz,
1H), 7.83 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 4.66
13
– 4.54 (m, 1H), 1.47 (d, J = 7.1 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 172.70,
166.45, 152.55, 145.67, 139.91, 139.03, 137.57, 133.15, 132.59, 129.08, 128.86 (2C),
127.63/127.32/127.02/126.71 (q, J = 30.3 Hz, Cphenyl-CF3), 126.41 (2C),
127.27/124.55/121.84/119.10 (q, J = 273.7 Hz, CF3), 124.43, 124.32, 118.34/118.28
(CHphenyl-CF3), 112.73, 50.64, 42.23, 17.89.
5.1.27
(R)-4-(6-aminopyridin-3-yl)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)
amino)-1-oxopropan-2-yl)benzamide (AA4)
Compound 3d was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/2, v/v) to give a white solid (0.52 g, 45%). Mp

137-138 . EI-MS (m/z): 464 [M+H]+, 462 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C22H19ClF3N4O2: 464.12269, found 464.11249. H NMR (400 MHz, DMSO-d6) δ
10.54 (s, 1H), 8.76 (d, J = 6.6 Hz, 1H), 8.68 (s, 2H), 8.24 (d, J = 2.5 Hz, 1H), 8.00 (d,
J = 8.3 Hz, 2H), 7.90 (dd, J = 8.8, 2.5 Hz, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.68 (d, J =
8.8 Hz, 1H), 6.91 (s, 2H), 4.62 – 4.55 (m, 1H), 1.47 (d, J = 7.2 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 172.75, 166.50, 163.64, 156.66 (3C), 139.03, 138.67, 132.55,
132.34, 128.84 (2C), 127.63/127.32/127.02/126.71 (q, J = 30.3 Hz, Cphenyl-CF3),
125.12 (2C), 127.26/124.54/121.83/ 119.12 (q, J = 273.7 Hz, CF3), 124.42, 124.31,
121.45, 118.33/118.28 (CHphenyl-CF3), 50.63, 17.88.
5.1.28
(R)-3-(6-acetamidopyridin-3-yl)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)
amino)-1-oxopropan-2-yl)benzamide (AA5)
Compound 4a was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/1, v/v) to give a white solid (0.30 g, 24%). Mp
128-131 . EI-MS (m/z): 505 [M+H]+, 503 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C24H21ClF3N4O3: 505.12543, found 505.12689. H NMR (400 MHz, DMSO-d6) 1H
NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 10.57 (s, 1H), 8.89 (d, J = 6.6 Hz, 1H),
8.74 (s, 1H), 8.29 – 8.23 (m, 2H), 8.23 – 8.16 (m, 2H), 7.92 – 7.89 (m, 3H), 7.69 (d, J
= 8.8 Hz, 1H), 7.62 – 7.58 (m, 1H), 4.65 – 4.58 (m, 1H), 2.13 (s, 3H), 1.48 (d, J = 7.1
13
Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 172.68, 169.84, 166.63, 152.16, 146.41,
139.01, 137.30, 136.77, 134.95, 132.58, 130.81, 129.73, 129.59, 127.46, 127.64/
127.33/127.02/126.71 (q, J = 30.3 Hz, Cphenyl-CF3), 125.69, 124.45, 124.34,
127.25/124.55/121.83/119.11 (q, J = 273.7 Hz, CF3), 118.35/118.28 (CHphenyl- CF3),
113.62, 50.68, 24.40, 17.92.
5.1.29
(R)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)-3-
(6-pivalamido-pyridin-3-yl)benzamide (AA6)
Compound 4b was reacted with compound 9, and the crude product was
purification with PE/EtOAc (3/1, v/v) to give a white solid (0.45 g, 33%). Mp
115-116 . EI-MS (m/z): 547 [M+H]+, 545 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C27H27ClF3N4O3: 547.17238, found 547.17277. H NMR (400 MHz, DMSO-d6) δ
10.57 (s, 1H), 9.97 (s, 1H), 8.90 (d, J = 6.6 Hz, 1H), 8.75 (s, 1H), 8.25 (d, J = 2.5 Hz,
2H), 8.20 (d, J = 1.3 Hz, 2H), 7.92 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.64
– 7.59 (m, 2H), 4.65 – 4.58 (m, 1H), 1.49 (d, J = 7.2 Hz, 3H), 1.27 (s, 9H). 13C NMR
(101 MHz, DMSO-d6) δ 177.75, 172.68, 166.65, 152.40, 146.10, 139.02, 137.25,
136.65, 134.98, 132.00, 130.90, 129.59, 129.27, 127.48, 127.64/127.33/127.03/126.72
(q, J = 30.3 Hz, Cphenyl-CF3), 125.71, 124.45, 124.36, 127.26/124.55/121.83/ 119.12 (q,

J = 273.7 Hz, CF3), 118.34/118.25 (CHphenyl-CF3), 114.41, 50.69, 39.89, 27.35 (3C),
17.92.
5.1.30
(R)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)-3-
(6-(methyl-sulfonamido)pyridin-3-yl)benzamide (AA7)
Compound 4c was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/1, v/v) to give a white solid (0.33 g, 24%). Mp
246-247 . EI-MS (m/z): 541 [M+H]+, 539 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C23H21ClF3N4O4S: 541.09241, found 541.09142. H NMR (400 MHz, DMSO-d6) δ
10.78 (s, 1H), 10.57 (s, 1H), 8.87 (d, J = 6.7 Hz, 1H), 8.69 (s, 1H), 8.28 – 8.22 (m,
2H), 8.17 – 8.14 (m, 1H), 7.92 – 7.87 (m, 3H), 7.69 (d, J = 8.8 Hz, 1H), 7.62 – 7.58
(m, 1H), 7.11 (d, J = 8.6 Hz, 1H), 4.67 – 4.55 (m, 1H), 3.34 (s, 3H), 1.48 (d, J = 7.1
13
Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 172.67, 166.57, 152.39, 139.01, 137.59,
137.11, 134.93, 132.61 (2C), 129.69, 129.62 (2C), 127.46, 127.62/127.32/
127.01/126.70 (q, J = 30.3 Hz, Cphenyl-CF3), 125.73, 127.27/124.55/ 121.84/119.13 (q,
J = 273.7 Hz, CF3), 124.46, 124.34, 118.33/118.29 (CHphenyl-CF3), 112.75, 50.66, 42.28,
17.93.
5.1.31
(R)-3-(6-aminopyridin-3-yl)-N-(1-((4-chloro-3-(trifluoromethyl)phenyl)
amino)-1-oxopropan-2-yl)benzamide (AA8)
Compound 4d was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/2, v/v) to give a white solid (0.43 g, 37%). Mp
215-216 . EI-MS (m/z): 464 [M+H]+, 462 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C22H19ClF3N4O2: 464.12269, found 464.11103. H NMR (400 MHz, DMSO-d6) δ
10.56 (s, 1H), 8.84 (d, J = 6.2 Hz, 1H), 8.70 (s, 2H), 8.25 (s, 1H), 8.19 (s, 1H), 7.91
–7.81 (m, 3H), 7.67 (d, J = 8.7 Hz, 1H), 7.56 – 7.52 (m, 1H), 6.88 (s, 2H), 4.68 – 4.57
(m, 1H), 1.49 (d, J = 6.8 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.69, 166.65,
163.57, 156.61 (3C), 139.01, 135.76, 134.86, 132.57, 129.53, 128.57,
127.63/127.33/127.02/127.62 (q, J = 30.3 Hz, Cphenyl-CF3), 126.78, 124.51, 124.45,
124.36, 127.26/124.54/121.83/119.11 (q, J = 273.7 Hz, CF3), 121.93, 118.35/118.30
(CHphenyl-CF3), 50.67, 17.93.
5.1.32
(R)-5-(4-((1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)
carbamoyl)phenyl)-N-cyclopropylnicotinamide (AA9)
Compound 5a was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/1→1/2, v/v) to give a white solid (0.59 g, 44%). Mp
207-209 . EI-MS (m/z): 531 [M+H]+, 529 [M-H]-. HRMS (ESI): calcd for [M+H]+

1
C26H23ClF3N4O3: 531.14108, found 531.14209. H NMR (400 MHz, DMSO-d6) δ
10.57 (s, 1H), 9.10 (d, J = 2.1 Hz, 1H), 8.99 (d, J = 1.9 Hz, 1H), 8.87 (d, J = 6.6 Hz,
1H), 8.77 (d, J = 3.9 Hz, 1H), 8.49 – 8.48 (m, 1H), 8.25 (d, J = 2.4 Hz, 1H), 8.10 (d, J
= 8.4 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.69 (d, J = 8.8
Hz, 1H), 4.68 – 4.53 (m, 1H), 2.93 – 2.88 (m, 1H), 1.48 (d, J = 7.2 Hz, 3H), 0.79 –
0.72 (m, 2H), 0.66 – 0.58 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 172.67, 166.36,
166.28, 150.30, 148.35, 139.63, 139.03, 134.55, 133.95, 133.16, 132.58, 130.37,
128.92 (2C), 127.30 (2C), 127.64/127.33/ 127.03/126.72 (q, J = 30.3 Hz, Cphenyl-CF3),
127.26/124.55/121.84/ 119.12 (q, J = 273.7 Hz, CF3), 124.43, 124.35, 118.34/118.28
(CHphenyl-CF3), 50.68, 23.54, 17.89, 6.23 (2C).
5.1.33
(R)-5-(4-((1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)
carbamoyl)phenyl)-N,N-diethylnicotinamide (AA10)
Compound 5b was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/1, v/v) to give a white solid (0.38 g, 28%). Mp
104-106 . EI-MS (m/z): 547 [M+H]+, 545 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C27H27ClF3N4O3: 547.17238, found 547.17580. H NMR (400 MHz, DMSO-d6) δ
10.56 (s, 1H), 9.05 (d, J = 2.0 Hz, 1H), 8.87 (d, J = 6.6 Hz, 1H), 8.60 (d, J = 1.7 Hz,
1H), 8.25 (d, J = 2.1 Hz, 1H), 8.18 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 8.3
Hz, 2H), 7.90 (dd, J = 8.8, 2.0 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 4.65 – 4.54 (m, 1H),
3.50 (d, J = 6.4 Hz, 2H), 3.25 (d, J = 6.6 Hz, 2H), 1.47 (d, J = 7.1 Hz, 3H), 1.19 (d, J
= 6.9 Hz, 3H), 1.09 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.67, 167.87,
166.36, 148.59, 146.48, 139.49, 139.03, 134.74, 133.95, 133.71, 132.58, 132.36,
128.91 (2C), 127.31 (2C), 127.63/127.32/127.02/126.72 (q, J = 30.3 Hz, Cphenyl-CF3),
127.26/124.55/121.84/119.13 (q, J = 273.7 Hz, CF3), 124.44, 124.32, 118.34/118.29
(CHphenyl-CF3), 50.67, 43.49, 39.56, 17.87, 14.52, 13.28.
5.1.34
(R)-5-(4-((1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)
carbamoyl)phenyl)-N-(2-(dimethylamino)ethyl)nicotinamide (AA11)
Compound 5c was reacted with compound 9, and the crude product was
purification with EtOAc/MeOH (7/1, v/v) to give a white solid (0.26 g, 18%). Mp
126-129 . EI-MS (m/z): 562 [M+H]+, 560 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C27H28ClF3N5O3: 562.18328, found 562.18362. H NMR (400 MHz, DMSO-d6) δ
10.62 (s, 1H), 9.11 (s, 1H), 9.02 (s, 1H), 8.90 (s, 1H), 8.81 (s, 1H), 8.53 (s, 1H), 8.26
(s, 1H), 8.10 (d, J = 6.4 Hz, 2H), 7.96 (d, J = 7.0 Hz, 2H), 7.91 (d, J = 7.3 Hz, 1H),
7.69 (d, J = 8.4 Hz, 1H), 4.61 (s, 1H), 3.32 – 3.26 (m, 2H), 2.48 (s, 2H), 2.22 (s, 6H),
13
1.49 (d, J = 5.5 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 172.71, 166.38, 165.01,

150.32, 148.39, 139.62, 139.04, 134.59, 133.97, 133.23, 132.60, 130.46, 128.93 (2C),
127.32 (2C), 127.62/127.32/127.01/126.71 (q, J = 30.3 Hz, Cphenyl-CF3),
127.25/124.56/121.86/119.14 (q, J = 273.7 Hz, CF3), 124.48, 124.34, 118.34/118.30
(CHphenyl-CF3), 58.54, 50.71, 45.67 (2C), 37.91, 17.89.
5.1.35
(R)-5-(3-((1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)
carbamoyl)phenyl)-N-cyclopropylnicotinamide (AA12)
Compound 6a was reacted with compound 9, and the crude product was purified
with PE/EtOAc (1/1→1/2, v/v) to give a white solid (0.79 g, 60%). Mp 191-192 .
EI-MS (m/z): 531 [M+H]+, 529 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C26H23ClF3N4O3: 531.14108, found 531.14293. H NMR (400MHz, DMSO-d6) δ
10.58 (s, 1H), 9.12 (d, J = 2.1 Hz, 1H), 8.99 (d, J = 1.9 Hz, 1H), 8.94 (d, J = 6.7 Hz,
1H), 8.78 (d, J = 4.0 Hz, 1H), 8.50 – 8.49 (m, 1H), 8.34 (s, 1H), 8.25 (d, J = 2.4 Hz,
1H), 7.99 (d, J = 7.8 Hz, 2H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.70 – 7.64 (m, 2H), 4.70
– 4.56 (m, 1H), 2.94 – 2.85 (m, 1H), 1.49 (d, J = 7.2 Hz, 3H), 0.78 – 0.73 (m, 2H),
0.64 – 0.62 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 172.65, 166.50, 166.35,
150.38, 148.08, 139.01, 136.92, 135.08, 135.03, 133.29, 132.61, 130.50, 130.43,
129.72, 128.30, 127.63/127.33/127.02/126.72 (q, J = 30.3 Hz, Cphenyl-CF3), 126.49,
127.27/124.55/121.83/ 119.12 (q, J = 273.7 Hz, CF3), 124.46, 124.36, 118.34/118.29
(CHphenyl-CF3), 50.71, 23.55, 17.93, 6.22 (2C).
5.1.36
(R)-5-(3-((1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)
carbamoyl)phenyl)-N,N-diethylnicotinamide (AA13)
Compound 6b was reacted with compound 9, and the crude product was
purification with PE/EtOAc (1/1, v/v) to give a white solid (1.05 g, 77%). Mp
114-116 . EI-MS (m/z): 547 [M+H]+, 545 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C27H27ClF3N4O3: 547.17238, found 547.17466. H NMR (400 MHz, DMSO-d6) δ
10.57 (s, 1H), 9.08 (d, J = 2.2 Hz, 1H), 8.94 (d, J = 6.7 Hz, 1H), 8.61 (d, J = 1.9 Hz,
1H), 8.32 (s, 1H), 8.25 (d, J = 2.4 Hz, 1H), 8.20 – 8.19 (m, 1H), 8.01 – 7.96 (m, 2H),
7.90 (dd, J = 8.8, 2.4 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.66 – 7.62 (m, 1H), 4.67 –
4.60 (m, 1H), 3.50 (d, J = 6.8 Hz, 2H), 3.25 (d, J = 6.6 Hz, 2H), 1.49 (d, J = 7.2 Hz,
3H), 1.19 (d, J = 7.1 Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 172.63, 167.93, 166.48, 148.67, 146.24, 139.01, 136.71, 135.18, 135.06,
133.66, 132.57, 132.37, 130.45, 129.74, 128.38, 127.64/ 127.33/127.03/ 126.72 (q, J
= 30.3 Hz, Cphenyl-CF3), 126.43, 127.26/ 124.54/121.83/119.11 (q, J = 273.7 Hz, CF3),
124.45, 124.37, 118.35/118.30 (CHphenyl-CF3), 50.67, 43.50, 39.60, 17.94, 14.52, 13.27.

5.1.37
(R)-5-(3-((1-((4-chloro-3-(trifluoromethyl)phenyl)amino)-1-oxopropan-2-yl)
carbamoyl)phenyl)-N-(2-(dimethylamino)ethyl)nicotinamide (AA14)
Compound 6c was reacted with compound 9, and the crude product was
purification with EtOAc/MeOH (7/1, v/v) to give a white solid (0.43 g, 30%). Mp
114-117 . EI-MS (m/z): 562 [M+H]+, 560 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C27H28ClF3N5O3: 562.18328, found 562.18576. H NMR (400 MHz, DMSO-d6) δ
10.62 (s, 1H), 9.13 (d, J = 1.9 Hz, 1H), 9.02 (d, J = 1.7 Hz, 1H), 8.97 (d, J = 6.6 Hz,
1H), 8.85 – 8.82 (m, 1H), 8.56 (s, 1H), 8.37 (s, 1H), 8.26 (d, J = 2.3 Hz, 1H), 8.00 (d,
J = 7.6 Hz, 2H), 7.91 (dd, J = 8.8, 2.1 Hz, 1H), 7.71 – 7.63 (m, 2H), 4.68 – 4.57 (m,
1H), 3.45 – 3.41 (m, 2H), 2.49 (s, 2H), 2.24 (s, 6H), 1.50 (d, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 172.68, 166.49, 165.08, 150.37, 148.15, 139.04,
136.91, 135.09, 135.03, 133.33, 132.55, 130.45 (2C), 129.70, 128.31,
127.63/127.32/127.02/126.71 (q,
J
=
30.3 Hz, Cphenyl-CF3), 126.52,
127.26/124.54/121.83/119.12 (q, J =273.7 Hz, CF3), 124.45, 124.33, 118.35/118.29
(CHphenyl-CF3), 58.42, 50.79, 45.48 (2C), 37.74, 17.93.
5.1.38 (2R,4S)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (10)
The prepare procedure was same as compound 7.
5.1.39
tert-butyl(2R,4S)-2-((4-chloro-3-(trifluoromethyl)phenyl)carbamoyl)-4-
hydroxypyrrolidine-1-carboxylate (11)
Compound 10 (3.48 g, 15.31 mmol) was dissolved in anhydrous CH2Cl2 (60 mL),
and triethylamine (1.5 mL, 15.31 mmol) was added. Under 0 , ethyl chloroformate
(2 mL, 15.31 mmol) in CH2Cl2 (5 mL) was dropped into the solution. Stirring was
continued
for
30
min
to
generate
activated
anhydride.
Then
4-chloro-3-(trifluoromethyl)aniline (2.7 g, 13.92 mmol) in CH2Cl2 (15 mL) was added
dropwise into the above reaction solution. The reaction was performed overnight
under room temperature. After that, the resulting solution was diluted with CH2Cl2 (30
mL), and washed with saturated NaHCO3 (30 mL), water (30 mL), and brine (30 mL).
The organic layer was dried over Na2SO4, filtered, concentrated, and purified by
column chromatography (PE/EtOAc=3:1) to provide product (4.26 g, 75%). EI-MS
(m/z): 407 [M-H]-.
5.1.40
tert-butyl(2R,4S)-2-((4-chloro-3-(trifluoromethyl)phenyl)carbamoyl)-4-
((methylsulfonyl)oxy)pyrrolidine-1-carboxylate (12)
In a 100 mL flask, compound 11 (3.79 g, 9.27 mmol) was dissolved in anhydrous

CH2Cl2, and cooled to 0 , followed by adding triethylamine (1.55 mL, 11.12 mmol).
Stirring was continued for 15 min, and methylsulfonyl chloride (0.86 mL, 11.12 mmol)
was dropped slowly into the solution. Then the reaction was performed overnight
under room temperature. Water was added to quench the reaction, and the organic
layer was washed with brine, dried over Na2SO4. Solvents was removed in vacuo to
get product (3.87 g, 86%). EI-MS (m/z): 485 [M-H]-.
5.1.41 tert-butyl(2R,4R)-4-azido-2-((4-chloro-3-(trifluoromethyl)phenyl)carbamoyl)
pyrrolidine-1-carboxylate (13)
Compound 12 (3.78g, 7.76 mmol) was dissolved in anhydrous DMF (10 mL), and
sodium azide (0.99g, 15.52 mmol) was added. Then the reaction was continued at
65-70 under N2 protection for 16 h. Next, the reaction solution was poured into 100
mL ice-water, and extracted with EtOAc (80 mL×3). The combined organic phase was
washed with brine (50 mL), dried over Na2SO4. The crude product was purified by
column chromatography (PE/EtOAc=5:1) to give a white solid (2.69 g, 80%). EI-MS
(m/z): 434 [M+H]+, 432 [M-H]-.
5.1.42 tert-butyl(2R,4R)-4-amino-2-((4-chloro-3-(trifluoromethyl)phenyl)carbamoyl)
pyrrolidine-1-carboxylate (14)
Compound 13 (2.77 g) was dissolved in anhydrous methanol (25 mL), and 5%
Pd/C (0.54g) was added. The solution was purged with H2 for 3 times, and the
reaction was continued under H2 overnight. Then the reaction was filtered, and
washed with methanol. The combined methanol was concentrated under vacuum, and
the residue was obtained for next step. EI-MS (m/z): 408 [M+H]+.
5.1.43 Compounds 15a-15d, 16a-16d, 17a-17c, and 18a-18c
These compounds were prepared using the same method as compound AA1. All
the compounds were purified by column chromatography. The elution solvents for
17c and 18c was EtOAc/MeOH (7/1, v/v), while others were eluted with PE/EtOAc
(1/1 or 1/2, v/v).
5.1.44
(2R,4R)-4-(4-(6-acetamidopyridin-3-yl)benzamido)-N-(4-chloro-3-
(trifluoromethyl)phenyl)pyrrolidine-2-carboxamide (AA15)
Compound 15a was used as starting material, and then deprotection of Boc group
was performed using the method same as compound 9. The overall yield of last two
steps was 14%. Mp 249-251 . EI-MS (m/z): 546 [M+H]+. HRMS (ESI): calcd for
[M+H]+ C26H24ClF3N5O3: 546.15198, found 546.17135. 1H NMR (400 MHz,

DMSO-d6) δ 10.65 (s, 1H), 10.42 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H), 8.45 (d, J = 6.6 Hz,
1H), 8.21 – 8.10 (m, 3H), 7.95 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.73 (d, J
= 8.5 Hz, 2H), 7.58 – 7.54 (m, 1H), 7.41 (d, J = 7.7 Hz, 1H), 4.39 – 4.34 (m, 1H),
3.87 – 3.83 (m, 1H), 3.23 – 3.18 (m, 1H), 2.90 – 2.86 (m, 1H), 2.48 – 2.44 (m, 1H),
2.12 (s, 3H), 2.02 – 1.95 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 174.37, 169.87,
166.20, 152.28, 146.36, 139.81, 139.79, 136.71, 133.66, 130.37, 130.36,
130.32/130.01/129.69/129.38 (q, J = 30.3 Hz, Cphenyl-CF3), 128.49 (2C), 126.32 (2C),
128.67/125.96/123.25/120.54 (q, J = 273.3 Hz, CF3), 123.47, 120.16/120.11
(CHphenyl-CF3), 116.02/115.98 (CHphenyl-CF3), 113.61, 60.05, 51.97, 51.67, 36.77, 24.40.
5.1.45
(2R,4R)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-(4-(6-pivalamidopyridin-
3-yl)benzamido)pyrrolidine-2-carboxamide (AA16)
Compound 15b was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two
steps was 57%. Mp 178-180 . EI-MS (m/z): 588 [M+H]+. HRMS (ESI): calcd for
[M+H]+ C29H30ClF3N5O3: 588.19893, found 588.20236. 1H NMR (400 MHz,
DMSO-d6) δ 10.72 (s, 1H), 9.98 (s, 1H), 8.70 (d, J = 1.2 Hz, 1H), 8.57 (d, J = 6.5 Hz,
1H), 8.19 – 8.15 (m, 3H), 7.92 (d, J = 8.4 Hz, 3H), 7.77 (d, J = 8.4 Hz, 2H), 7.60 –
7.56 (m, 1H), 7.44 (d, J = 7.8 Hz, 1H), 4.49 – 4.44 (m, 1H), 4.11 – 4.07 (m, 1H), 3.32
– 3.29 (m, 1H), 3.08 – 3.04 (m, 1H), 2.67 – 2.55 (m, 1H), 2.10 – 2.05 (m, 1H), 1.26 (s,
9H). 13C NMR (101 MHz, DMSO-d6) δ 177.77, 171.99, 166.31, 152.53, 146.08,
139.89, 139.73, 136.60, 133.47, 130.46, 130.42, 130.37/130.06/129.74/129.43 (q, J =
30.3 Hz, Cphenyl-CF3), 128.56 (2C), 126.39 (2C), 128.61/125.90/123.19/120.48 (q, J=
273.3 Hz, CF3), 123.51, 120.37/120.34 (CHphenyl-CF3), 116.07/116.03 (CHphenyl-CF3),
114.39, 59.73, 51.11, 50.76, 36.25, 27.32 (3C), 21.59.
5.1.46 (2R,4R)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-(4-(6-(methylsulfonamido)
pyridin-3-yl)benzamido)pyrrolidine-2-carboxamide (AA17)
Compound 15c was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two
steps was 29%. Mp 130-133 . EI-MS (m/z): 582[M+H]+. HRMS (ESI): calcd for
[M+H]+ C25H24ClF3N5O4S: 582.11896, found 582.13965. 1H NMR (400MHz,
DMSO-d6) δ 10.43 (s, 1H), 8.61 (s, 1H), 8.45 (d, J = 6.6 Hz, 1H), 8.21 (s, 1H), 8.09 (d,
J = 8.6 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.2 Hz,
2H), 7.58 – 7.54 (m, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 4.39 –
4.34 (m, 1H), 3.88 – 3.84 (m, 1H), 3.33 (s, 3H), 3.23 – 3.18 (m, 1H), 2.90 – 2.86 (m,
1H), 2.48 – 2.44 (m, 1H), 2.04 – 1.94 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ

174.31, 166.23, 152.59, 145.60, 139.81, 139.65, 137.48, 133.71, 130.34,
130.32/130.02/129.70/129.39 (q, J = 30.3 Hz, Cphenyl-CF3), 129.05, 128.49 (2C),
126.30 (2C), 128.67/125.96/123.25/120.54 (q, J = 273.3 Hz, CF3), 123.47,
120.16/120.12 (CHphenyl-CF3), 116.03/115.98 (CHphenyl-CF3), 112.76, 60.04, 51.95, 51.65,
42.20, 36.78.
5.1.47
(2R,4R)-4-(4-(6-aminopyridin-3-yl)benzamido)-N-(4-chloro-3-
(trifluoromethyl)phenyl)pyrrolidine-2-carboxamide (AA18)
Compound 15d was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two
steps was 35%. Mp 170-172 . EI-MS (m/z): 504 [M+H]+. HRMS (ESI): calcd for
[M+H]+ C24H22ClF3N5O2: 504.14141, found 504.14154. 1H NMR (400 MHz,
DMSO-d6) δ 10.45 (s, 1H), 9.02 (s, 1H), 8.62 (s, 2H), 8.20 (s, 1H), 7.95 – 7.91 (m,
2H), 7.85 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.58 – 7.54 (m, 1H), 7.42 (d, J
= 7.8 Hz, 1H), 6.90 (s, 2H), 4.40 – 4.35 (m, 1H), 3.90 – 3.87 (m, 1H), 3.24 – 3.20 (m,
1H), 2.94 – 2.85 (m, 1H), 2.48 – 2.41 (m, 1H), 2.04 – 1.94 (m, 1H). 13C NMR (101
MHz, DMSO-d6) δ 172.63, 166.26, 156.57 (2C), 139.79, 138.39, 132.87, 130.38,
130.33/130.02/129.70/129.39 (q, J = 30.3 Hz), 128.57, 128.47 (2C), 126.37, 125.04
(2C), 128.65/125.94/123.24/120.53 (q, J = 273.3 Hz, CF3), 123.48, 121.45,
120.20/120.17 (CHphenyl-CF3), 116.03/115.99 (CHphenyl-CF3), 59.97, 51.78, 51.43, 36.65.
5.1.48
(2R,4R)-4-(3-(6-acetamidopyridin-3-yl)benzamido)-N-(4-chloro-3-
(trifluoromethyl)phenyl)pyrrolidine-2-carboxamide (AA19)
Compound 16a was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two
steps was 37%. Mp 149-151 . EI-MS (m/z): 546 [M+H]+. HRMS (ESI): calcd for
[M+H]+ C26H24ClF3N5O3: 546.15198, found 546.15245. 1H NMR (400 MHz,
DMSO-d6) δ 10.63 (s, 1H), 10.43 (s, 1H), 8.66 (s, 1H), 8.50 (d, J = 5.9 Hz, 1H), 8.17
(d, J = 9.1 Hz, 2H), 8.08 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 7.9 Hz, 1H), 7.84 – 7.79 (m,
2H), 7.50 (d, J = 3.9 Hz, 2H), 7.37 (d, J = 7.5 Hz, 1H), 4.40 – 4.36 (m, 1H), 3.91 –
3.82 (m, 1H), 3.25 – 3.20 (m, 1H), 2.91 – 2.87 (m, 1H), 2.50 – 2.45 (m, 1H), 2.13 (s,
3H), 2.08 – 1.96 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 174.33, 169.83, 166.40,
152.09,
146.33,
139.76,
137.32,
136.66,
135.52,
130.80,
130.24,
130.30/129.99/129.68/129.37 (q, J = 30.3 Hz, Cphenyl-CF3), 129.46, 129.41, 127.11,
128.60/125.90/123.19/ 120.49 (q, J = 273.7 Hz, CF3), 125.30, 123.42, 120.12/120.09
(CHphenyl-CF3), 115.98/115.94 (CHphenyl-CF3), 113.58, 59.99, 51.93, 51.72, 36.74, 24.39.

5.1.49
(2R,4R)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-(3-(6-pivalamidopyridin-
3-yl)benzamido)pyrrolidine-2-carboxamide (AA20)
Compound 16b was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two
steps was 25%. Mp 155-157 . EI-MS (m/z): 588 [M+H]+. HRMS (ESI): calcd for
[M+H]+ C29H30ClF3N5O3: 588.19893, found 588.21917. 1H NMR (400 MHz,
DMSO-d6) δ 10.45 (s, 1H), 9.94 (s, 1H), 8.68 (s, 1H), 8.52 (d, J = 5.9 Hz, 1H), 8.15 (d,
J = 8.8 Hz, 2H), 8.10 (d, J = 7.0 Hz, 2H), 7.91 (d, J = 8.3 Hz, 1H), 7.85 (d, J = 8.2 Hz,
1H), 7.80 (d, J = 7.1 Hz, 1H), 7.51 (s, 2H), 7.38 (s, 1H), 4.41– 4.39 (m, 1H), 3.98 –
3.84 (m, 1H), 3.26 – 3.22 (m, 2H), 3.00 – 2.86 (m, 1H), 2.10 – 1.99 (m, 1H), 1.27 (s,
9H). 13C NMR (101 MHz, DMSO-d6) δ 177.73, 172.69, 166.45, 152.32, 146.04,
139.75, 137.28, 136.56, 135.54, 130.91, 130.28, 130.31/130.00/129.68/129.37 (q, J =
30.3 Hz, Cphenyl-CF3), 129.51, 129.42, 127.11, 128.60/125.89/123.19/120.48 (q, J =
273.7 Hz, CF3), 125.37, 123.44, 120.16/120.12 (CHphenyl-CF3), 115.99/115.95
(CHphenyl-CF3), 114.36, 59.95, 51.80, 51.60, 36.64, 27.35 (3C), 21.65.
5.1.50 (2R,4R)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-(3-(6-(methylsulfonamido)
pyridin-3-yl)benzamido)pyrrolidine-2-carboxamide (AA21)
Compound 16c was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two
steps was 38%. Mp 140-142 . EI-MS (m/z): 582 [M+H]+. HRMS (ESI): calcd for
1
[M+H]+ C25H24ClF3N5O4S: 582.11896, found 582.13934. H NMR (400 MHz,
DMSO-d6) δ 10.43 (s, 1H), 8.60 (s, 1H), 8.48 (d, J = 5.9 Hz, 1H), 8.18 (s, 1H), 8.06 (s,
2H), 7.91 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 7.1 Hz, 2H), 7.50 (d, J = 7.3 Hz, 2H), 7.38
(d, J = 6.9 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H), 4.44 – 4.31 (m, 1H), 3.91 – 3.81 (m, 1H),
3.34 (s, 3H), 3.27 – 3.13 (m, 2H), 2.94 – 2.85 (m, 1H), 2.05 – 1.96 (m, 1H). 13C NMR
(101 MHz, DMSO-d6) δ 174.36, 166.34, 152.35, 145.58, 139.75, 137.47, 137.13,
135.49, 130.28, 130.27/129.97/129.67/129.37 (q, J = 30.3 Hz, Cphenyl-CF3), 129.50,
129.44 (2C), 127.11, 128.63/125.91/123.20/120.49 (q, J = 273.7 Hz, CF3), 125.30,
123.44, 120.14/120.10 (CHphenyl-CF3), 115.96/115.92 (CHphenyl-CF3), 112.72, 59.98,
51.95, 51.71, 42.24, 36.76.
5.1.51
(2R,4R)-4-(3-(6-aminopyridin-3-yl)benzamido)-N-(4-chloro-3-
(trifluoromethyl)phenyl)pyrrolidine-2-carboxamide (AA22)
Compound 16d was used as starting material, and then deprotection of Boc group
was performed using the same method as compound 9. The overall yield of last two