Synthesis, Biological Activities and Docking Studies of Novel 4-(Arylaminomethyl)benzamide Derivatives as Potential Tyrosine Kinase Inhibitors

Article abstract of DOI:10.3390/molecules24193543

A number of new compounds containing the 4-(aminomethyl)benzamide fragment as a linker were designed and synthesized, and their biological activities were evaluated as potential anticancer agents. The cytotoxicity activity of the designed compounds was studied in two hematological and five solid cell lines in comparison with the reference drugs. Targeted structures against eight receptor tyrosine kinases including EGFR, HER-2, HER-4, IGF1R, InsR, KDR, PDGFRa, and PDGFRb were investigated. The majority of the compounds showed a potent inhibitory activity against the tested kinases. The analogues 11 and 13 with the (trifluoromethyl)benzene ring in the amide or amine moiety of the molecule were proven to be highly potent against EGFR, with 91% and 92% inhibition at 10 nM, respectively. The docking of synthesized target compounds for nine protein kinases contained in the Protein Data Bank (PDB) database was carried out. The molecular modeling results for analogue 10 showed that the use of the 4-(aminomethyl)benzamide as a flexible linker leads to a favorable overall geometry of the molecule, which allows one to bypass the bulk isoleucine residue and provides the necessary binding to the active center of the T315I-mutant Abl (PDB: 3QRJ).

Full text of DOI:10.3390/molecules24193543

molecules
Article
Synthesis, Biological Activities and Docking Studies
of Novel 4-(Arylaminomethyl)benzamide Derivatives
as Potential Tyrosine Kinase Inhibitors
Elena Kalinichenko *, Aliaksandr Faryna, Viktoria Kondrateva, Alena Vlasova,
Valentina Shevchenko, Alla Melnik, Olga Avdoshko and Alla Belko
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, BY-220141 Minsk, Belarus;
farynaa@yandex.ru (A.F.); kondrateva@iboch.by (V.K.); alenavlasova@iboch.by (A.V.);
vas_57iboch@mail.ru (V.S.); al_melk50@mail.ru (A.M.); olga.avdoshko@iboch.by (O.A.);
alyabelko@iboch.by (A.B.)
* Correspondence: kalinichenko@iboch.by; Tel./Fax: +375-17-265-06-11
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 July 2019; Accepted: 25 September 2019; Published: 30 September 2019
Abstract: A number of new compounds containing the 4-(aminomethyl)benzamide fragment as a
linker were designed and synthesized, and their biological activities were evaluated as potential
anticancer agents. The cytotoxicity activity of the designed compounds was studied in two
hematological and five solid cell lines in comparison with the reference drugs. Targeted structures
against eight receptor tyrosine kinases including EGFR, HER-2, HER-4, IGF1R, InsR, KDR, PDGFRa,
and PDGFRb were investigated. The majority of the compounds showed a potent inhibitory activity
against the tested kinases. The analogues 11 and 13 with the (trifluoromethyl)benzene ring in the
amide or amine moiety of the molecule were proven to be highly potent against EGFR, with 91% and
92% inhibition at 10 nM, respectively. The docking of synthesized target compounds for nine protein
kinases contained in the Protein Data Bank (PDB) database was carried out. The molecular modeling
results for analogue 10 showed that the use of the 4-(aminomethyl)benzamide as a flexible linker
leads to a favorable overall geometry of the molecule, which allows one to bypass the bulk isoleucine
residue and provides the necessary binding to the active center of the T315I-mutant Abl (PDB: 3QRJ).
Keywords: 4-(aminomethyl)benzamide; Bcr-Abl; protein kinase inhibitors; receptor tyrosine kinases;
molecular docking; molecular dynamics; anticancer activity
1. Introduction
Protein kinases serve as therapeutic targets for a range of clinical indications and represent the
largest category of drug targets in current clinical trials [
imatinib achieved great clinical success and became the first-line drug for the treatment of chronic
myeloid leukemia (CML) [ ]. Despite advances in treatment with imatinib, some patients are initially
insensitive to the drug, or, amid ongoing therapy with imatinib, they lose the achieved response [ ].
1]. The first-generation Bcr-Abl inhibitor
2
3–5
After the introduction of imatinib, the therapeutic armamentarium in solid malignancies was expanded
by the registration of several other tyrosine kinase inhibitors (TKIs). These include the epidermal
growth factor receptor (EGFR) inhibitors erlotinib and gefitinib, the dual EGFR and human epidermal
growth factor receptor (HER)-2 inhibitor lapatinib, and the VEGFR (vascular endothelial growth
factor receptor) inhibitors sunitinib, sorafenib, and, recently, pazopanib. Importantly, none of the
TKIs are entirely specific for one target. In particular, the VEGFR inhibitors target a wide spectrum of
kinases, including the platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor
receptor (FGFR).
Molecules 2019, 24, 3543; doi:10.3390/molecules24193543
www.mdpi.com/journal/molecules

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The progress in kinase structural biology offers a conceptual framework for understanding many
aspects of kinase biology and accelerating drug discovery programs targeting protein kinases [ ]. The
structure of human Abl in complex with imatinib shows that the molecule binds in the cleft between the
N- and C-terminal lobes [ ]. A number of approved drugs which are type I inhibitors, such as gefitinib,
6,7
8
target the ATP binding pocket and are termed as ATP competitive inhibitors. Type II inhibitors, which
include imatinib, nilotinib, and ponatinib, bind to the hydrophobic pocket adjacent to the ATP binding
pocket, which is accessible only in a DFG (Asp-Phe-Gly)-out conformation of the enzyme. Although
the occupancy at the allosteric site is characteristic of type II inhibitors, they also extend past the
“gatekeeper” into the adenine pocket and form hydrogen bonds with the “hinge” residues [9–11].
Imatinib joins in the ATP pocket of the Bcr-Abl protein mainly with its pyridine–pyrimidine
fragment, next to which there is a methylphenyl group located opposite the “gatekeeper” residue,
Thr-315, while the methyl piperazinyl group of the molecule is located superficially mostly in the
pocket of specificity (Figure 1). A hydrogen-bond interaction forms between the hydroxyl group of
Thr-315 and the secondary amine linking the methylphenyl group to the pyrimidine moiety. The
benzamide moiety packs against the residue Leu-293 in this pocket, and the carbonyl group in the
benzamide moiety forms a hydrogen bond with the amide group of the residue Asp-381. In addition,
an amine in the piperazine group forms a hydrogen bond with the carbonyl group of Ile-360 [12].
Figure 1. Inhibitors of the tyrosine kinase type II and target structure. Interactions between imatinib
and Brc-Abl tyrosine kinase.
Despite high response rates, CML patients treated with imatinib often develop resistance to the
drug from point mutations in the kinase domain of Bcr-Abl that disrupt imatinib binding directly [13].
An additional structure–activity relationship study around the core phenylaminopyrimidine scaffold
of imatinib led to a more potent Bcr-Abl inhibitor, nilotinib. The higher binding affinity of nilotinib for
Abl derives from van der Waals interactions with the trifluoromethyl and imidazole substituents on
the phenyl ring, which are located in the pocket of specificity [12].
One key mutation against which nilotinib is inactive is T315I, the gatekeeper mutation. Ponatinib
was designed to overcome this type of mutation [14,15]. The key feature of the drug is a carbon–carbon
triple bond between the methylphenyl and purine groups that can accommodate the isoleucine side
chain without steric interference [11].
However, resistance to ponatinib was observed in some patients, making the development of new
T315I-targeted inhibitors a necessity. The new drug rebastinib demonstrates the potential for treating
patients with relapsed chronic myeloid leukemia and acute myeloid leukemia [16].

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Being a drug of new generation, rebastib is a “switch control” inhibitor of several tyrosine kinases,
including ABL1 (Abelson tyrosine kinase 1), FLT3 (FMs-related tyrosine kinase 3), and TIE2 (tunica
interna endothelial cell kinase 2). Rebastinib binds as a type II inhibitor that additionally penetrates into
and binds the switch control pocket of the ABL1 kinase domain, key amino-acid residues that mediate
the change from inactive to active conformation. Through occupancy of this switch control pocket,
rebastinib blocks the conformational process required for kinase activation. This unique binding mode
enables rebastinib to maintain the ABL1 kinase domain in the inactive conformation, independent of
the state of phosphorylation of the regulatory Tyr-393. [17].
Sorafenib is a clinically approved drug that is a type II as well. While this drug inhibits several
kinases, including many receptor tyrosine kinases, VEGFR was implicated as the primary target, in the
context of the approved drug indications for renal cell carcinoma and colorectal cancer.
In a crystal structure of sorafenib-bound VEGFR kinase domain, the kinase domain adopts a
“DGF-out”, activation loop-in conformation, resembling the inactive conformation of Abl. Sorafenib
forms extensive interactions with VEGFR in this conformation. Notably, the trifluoromethylphenyl
ring is tightly packed into the specificity pocket. This binding mode of the trifluoromethylphenyl ring
also resembles the one in the nilotinib-bound Abl kinase domain [18].
Compounds shown in Figure 1 are ATP-competitive inhibitors, classified as type II inhibitors, by
reference to the conformation of the highly conserved aspartate–phenylalanine–glycine (DFG) motif in
the beginning of the activation loop in the C-lobe of the kinase domain [19].
Based on the structural analysis of imatinib, nilotinib, and their analogues (Figure 1), we started the
development of new Bcr-Abl inhibitors by applying a combination strategy of bio-isosteric replacement
and conformational restriction. The structural modification focused on two parts: the heterocyclic
fragment binding to the adenine pocket and forming a hydrogen bond with the amino acids Met-318 and
Thr-315 and the modification of the “tail area”, introducing various aromatic and acyclic substituents.
As a “linker” between adenine and allosteric pockets the methylphenyl group is used as a rule (Figure 1).
We proposed to use a flexible “linker” 4-(aminomethyl)benzamide to avoid a steric collision with the
remainder of Ile-315 Bcr-Abl. In general, the structure suggested the possibility of developing specific
inhibitors of protein kinase type II binding, in which the allosteric pocket of the protein active site is
involved in ligand binding. Considering these assumptions, we designed and synthesized 22 novel
target compounds as potential TKIs.
2. Results and Discussion
2.1. Chemistry
Two general strategies were developed to obtain target compounds starting from initial methyl
4-formylbenzoate (1) by introducing other functional moieties (Scheme 1).
Scheme 1. Cont.

Molecules 2019, 24, 3543
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.
Scheme 1. Reagents and conditions: (i) NaBH4, MeOH; (ii) KOH, MeOH/H2O; (iii) SOCl2, CHCl3,
reflux; (iv) R2NH2 (a), Et3N, CHCl3; (v) R1NH2 (a/b/c/e/g), DMF, reflux; (vi) R1NH2 (a–d), NaBH(OAc)3,
AcOH, CHCl3; (vii) HCl (18–27%), 80 ◦C; (viii) (1) SOCl2, CHCl3, (2) R2NH2 (a/b/g/h), NEt3, CHCl3;
(ix) R2NH2 (a/c/f/g/h), DCC (CDI), BtOH, NEt3, DMF.
In the first approach, the initial methyl 4-formylbenzoate (
with a quantitative yield by known methods [20 21]. A key intermediate product
the interaction of with amine in chloroform with 60% yield. Compounds 12 were synthesized by
) with moderate yields
1) was converted to benzoyl chloride
4
,
5
was obtained by
4
a
8–
the reaction of primary amines a/b/c/e/g with the 4-(chloromethyl)benzamide (
5
(10–25%).
A previously described synthetic method of the reductive amination [22] was used in the
second approach. The reaction between aldehyde and primary amines in the presence of
triacetoxyborohydride sodium and acetic acid proceeded at room temperature with the formation
of the amines 6a 6d with high yield (80–95%). Treatment of 6a 6d with an aqueous solution of HCl
(18–27%) at 80–90 ◦C allowed obtaining derivatives 7a–7d with high yield (80–85%).
Two ways were studied to obtain the targeted compounds 13 25 with the aim of the yield
optimizing. The first way was to obtain the intermediate acyl chlorides by the action of thionyl
chloride on carboxyl derivatives 7a 7d, which were then directly introduced into the reaction with
the corresponding amine a/b/g/h in the presence of triethylamine. Yields of target amides 10, 13 15,
and 22 25 were 20–50% after isolation and purification by column chromatography. The second
way was to activate the carboxyl group of compounds 7a 7d with the use of condensing agents
1
a–d
–
–
–
–
–
–
–
N,N’-dicyclohexylcarbodiimide (DCC) or 1,1’-carbonyldiimidazole (CDI) in the presence of NEt3/BtOH
and the subsequent addition of primary amines a/c/f/g/h. This approach made it possible to obtain
target amides 16–21 with a fairly good yield (34–82%) (Table 1).
Table 1. Synthesis methods and structures of obtained compounds 8–25.
Method
Yield (%)
Method
Yield (%)
№
Structure
№
Structure
v
15
ix
58
8
17

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Table 1. Cont.
Method
Yield (%)
Method
Yield (%)
№
Structure
№
Structure
v (ix)
10 (21)
ix
38
9
18
v (viii)
19 (20)
ix
34
10
19
v
17
ix
80
11
12
20
21
v
25
ix
82
viii
25
viii
50
13
14
22
23
viii
40
viii
31
CF3
N
N
H
viii
30
viii
37
H
15
16
24
25
N
N
O
NO2
ix
76
viii
27
The synthesis of compounds 28i
prepared by the method of reductive amination. Amidation of the corresponding intermediates 27i
with amine gave targeted compounds 28i 28l. The selected amines were chosen on the basis of their
–
28l is summarized in Scheme 2. Derivatives 26i
–
26l were
–
27l
a
–
lipophilicity and limited number of possible conformations, providing more lipophilic and rigid final
compounds 28i–28l.

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Scheme 2. Reagents and conditions: (i) R1H (i/j/k/l), NaBH(OAc)3, AcOH, CHCl3; (ii) HCl (18–27%),
80 ◦C; (iii) (1) SOCl2, CHCl3, (2) RNH2 (a), NEt3, CHCl3.
All target compounds were isolated as free bases. In most cases, crystallization or column
chromatography was required for the purification. The structures of most synthetic intermediates
and products were established by spectroscopy techniques (1H-, 13C-NMR) and high-resolution mass
spectrometry (HRMS). The purities of compounds were 95–99.00% (by HPLC).
2.2. Biological Studies
2.2.1. Anti-Proliferation Activity
To determine the selectivity of the synthesized compounds 8–25 and 28i–28l toward various cancer
cells and normal human cells, the anti-proliferative activities against K-562 (chronic myelogenous
leukemia), HL-60 (promyelocytic leukemia), MCF-7 (breast adenocarcinoma), HeLa (cervix carcinoma),
A549 (lung carcinoma), OCD-GS (carcinoma of the kidney), and normal cells RPMI 1788 (B lymphocyte,
the human cell line), were compared with imatinib, nilotinib, and sorafenib as the positive control.
All new compounds showed moderate to significant activity against two or more hematologic and/or
solid tumor cell lines in 100 µM concentration. Compounds 5, 8–11, 13–15, 19, 23 and 28i–28l, which
showed activity against more than two cell lines, were further studied in vitro for the anti-proliferative
activity at a concentration of 0.1–100 µM (Table 2).
It was found that most compounds displayed a moderate suppression on the growth of the
positive human chronic myelogenous leukemia K562 cells expressing a high level of Bcr-Abl protein
and Bcr-Abl-negative leukemia cells (line HL60). Generally, they moderately inhibited solid tumor
cell cultures. As shown in Table 2, compounds 10 and 15 turned out to be more active against
HL60 (IC50 values 8.2
Analog 13 showed activity against K562 with the value IC50 = 5.6
heterocyclic amines, compounds 28j 28l exhibited the highest potency against K562 cells with IC50
values 6.9 M, 3,6 M, and 4.5 M, respectively. Promyelocytic leukemia cell line (HL60) survived at
much higher concentrations of 28j 28l indicating the specificity of these compounds. We supposed
µM and 5.6
µM, respectively) compared to K562 cells (40
µM and 31 µM).
µM. Among the series of tertiary
–
µ
µ
µ
–
that the introduction of the [4-(benzoyl)piperazin-1-yl]methyl group including tertiary heterocyclic
amine could improve the lipophilicity which led to the high permeability [23]. Log P of imatinib was
3.89, while amides 28i–28l had log P values between 2.65 and 4.02 (Table 2).

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Table 2. In vitro anti-proliferative activities of targets compounds against cancer cells (K-562, HL-60,
MCF-7, Hela, A-549, OKP-GS) and human normal cells (RPMI 1788).
Anti-Proliferative Activities in Different Cancer Cell Lines
(IC50 (µM)a
Inhibition
(%) 100 µM
Log P b (TPSA)
Comp.
K-562
HL-60
MCF-7
Hela
A-549
OKP-GS
RPMI 1788
5
8
9
4.20 (46.92)
5.68 (76.78)
4.30 (97.63)
5.00 (110.17)
5.75 (58.95)
5.75 (58.95)
0.75 (133.55)
5.22 (104.78)
3.74 (150.27)
−0.63 (154.40)
3.94 (79.70)
4.00 (70.47)
4.02 (70.47)
2.65 (83.36)
69
n/a
51
40
53
5.6
50
31
64
66
45
n/a
57
8.2
51
46
45
5.8
>100
49
54
9.5
>100
>100
57
51.8
87
n/a
>100
54
57.5
n/a
78.5
n/a
n/a
11.5
n/a
n/a
53.5
85
>100
>100
61
55
n/a
73
n/a
70
>100
>100
81
54.3
52
n/a
>100
n/a
n/a
78
n/a
n/a
<0.1
19.32
n/a
10.42
15.53
9.36
10.45
15.93
10
11.52
6.32
6.44
3.14
n/a
10
11
13
14
15
19
23
28i
28j
28k
28l
69
n/a
>100
n/a
n/a
63
71
n/a
77
n/a
30.7
6.9
3.6
4.5
38
>100
>100
n/a
30.6
38.5
53.5
>100
>100
>100
Reference Compounds
Imatinib
Nilotinib
Sorafenib
3.89 (86.28)
4.99 (97.63)
3.76 (92.35)
0.41
<0.1
n/a
44.5
55
n/a
49
n/a
55
45.8
n/a
n/a
54
n/a
58
48
55
51
13.37
<0.1
n/a
a IC50, the concentration that causes 50% growth inhibition; b logPo/w values were calculated based on Molinspiration
program predictions (http://www.molinspiration.com/cdi-bin/properties); TPSA—topological polar surface area;
n/a—not applicable.
2.2.2. Kinase Inhibitory Assays
We investigated the kinase selectivity of targeted compounds against eight receptor tyrosine
kinases including EGFR (epidermal growth factor receptor), HER-2 and HER4 (human epidermal
growth factor receptor), IGF1R (insulin like growth factor 1 receptor), InsR (gene insulin receptor), KDR
(kinase insert domain receptor), and PDGFRa and PDGFRb (platelet-derived growth factor receptors).
The assay was performed using the Kinase-Glo Plus luminescence kinase assay kit (TK-1+ADP-Glo™,
Promega, San Diego, CA, USA). It measures kinase activity by quantitating the amount of ATP
remaining in solution following a kinase reaction. The inhibition percentage of the enzymatic activities
caused by our compounds was evaluated at the single concentration of 10 nM against imatinib. The
obtained results are given in Table 3.
As follows from the obtained data, analogues 11 and 13 with N-(trifluoromethyl)benzene moiety
in the amide or amine moiety revealed their high potency against EGFR, 91% and 92%, respectively.
Compounds 10
21% and 65%, as illustrated in Table 3. Target products 10
,
20
,
22, and 24 also showed significant inhibitory activity EGFR, which was between
11 13 18 20 22 24 28k, and 28l exhibited
,
,
,
,
,
,
,
inhibitory activity comparable to imatinib in relation to HER-4, a receptor tyrosine kinase of the
EGFR family.
Vascular endothelial growth factor receptor 2 (VEGFR2), also called kinase insert domain receptor
(KDR), is a tyrosine kinase receptor for VEGFs that plays a central role in tumor angiogenesis; therefore,
the inhibition of VEGFR2 is a promising therapeutic strategy for inhibiting angiogenesis and tumor
growth. Compounds 10, 11, 13, 18, 20, 22, 28k, and 28l showed inhibitory activity of VEGFR2 in the
range of 16–48% at a concentration of 10 nM.
Compounds 18 and 20 containing the N-[3-(trifluoromethyl)phenyl]benzamide fragment exhibited
a high inhibitory activity against PDGFRa, 67% and 77%, respectively, despite various substituents in
the amine fragment. The influence of the difference in ATP binding sites between various tyrosine
kinase receptors is obvious, yet not very clear. Perhaps these features can explain why compounds
5,
14 15 17 28i, and 28j showed high selectivity for the gene insulin receptor, whereas synthetic products
,
,
,

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10
,
11,
13
,
18
,
20,
22
,
24,
28k, and 28l had significant influence on the biochemical activities of EGFR,
HER-4, VEGFR-2, and PDGFRa.
Table 3. Inhibition of receptor tyrosine kinases by compounds 5, 10, 11, 13–15, 17, 18, 20, 22–24, 28i–28l,
and imatinib.
% Inhibition at 10 nM
Compound
EGFR
HER-2
HER-4
IGF1R
InsR
KDR
PDGFRa PDGFRb
5
10
11
13
14
15
17
18
20
0
65
91
92
0
0
0
0
56
21
0
24
0
0
0
0
0
0
0
0
34
0
0
0
14
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18
0
1
0
3
0
0
0
0
57
46
0
0
27
16
20
0
0
0
24
48
28
0
2
0
0
23
17
0
0
66
55
59
0
0
0
67
77
0
0
0
0
8
0
0
56
0
0
0
8
0
0
0
13
28
0
0
0
0
2
2
7
75
51
49
54
0
0
0
56
57
49
12
44
0
0
23
43
49
0
0
0
49
0
52
50
33
0
22
23
24
28i
28j
28k
28l
Imatinib
0
43
51
42
36
2.3. Docking and Molecular Dynamics
An in-depth in silico study of the inhibitory activity of synthesized compounds
8–
25 and 28i–28l
to various protein kinases was implemented in comparison with some known inhibitors aiming the
investigation of a possible binding mechanism.
Initially, docking was performed to obtain protein–ligand complexes of the studied structures
8–25 and 28i–28l with a variety of tyrosine kinases. AutoDock Vina was used as a docking tool [24].
Structures of receptors were taken from Protein Data Bank (PDB) complexes of known protein kinase
inhibitors: human ABL kinase (PDB record 3CS9), human ABL kinase (2HYY), VEGF-receptor (3WZE),
and T315I-mutant Abl kinase (3QRJ). Imatinib (the original ligand in the complex 2HYY), nilotinib
(3CS9), rebastinib (3QRJ), sorafenib (3WZE), and ponatinib were used as reference structures. Docking
results showed that AutoDock Vina correctly reproduced the experimental binding models of imatinib,
nilotinib, sorafenib, and rebastinib with RMSDs (root mean squared deviation of heavy atoms) of less
than 2 Å. Nilotinib (
The obtained protein–ligand complexes having docking scores better than
molecular dynamics (MD) simulations using GROMACS [25].
−
13.2) showed the best docking score and sorafenib (
−
11.5) was the worst (Table 4).
11.0 were taken to the 2-ns
−
Table 4. AutoDock Vina’s docking scores for targets (8–25, 28i–28l) and reference compounds.
Protein Data Bank (PDB) Code Protein Kinases
Ligand
2HYY
2PL0
3GCS
3WZE
3CS9
3QRJ *
4AG8
4ASD
5HI2
8
9
−10.7
−11.4
−8.9
−7.9
−7.8
−8.8
−7.0
−7.2
−9.5
−7.3
−8.4
−8.1
−10.3
−9.4
−8.4
−9.8
−7.8
−10.5
−8.9
−10.2
−7.8
−8.9
−8.1
−8.1
−9.0
−4.2
−8.8
−6.7
−8.1
−4.4
−9.4
−8.3
−8.9
−8.8
−10.5
−11.1
−10.8
−10.6
−8.5
−9.1
−11.0
−12.3
−12.2
−9.7
−3.0
−7.2
−6.3
−8.6
−6.4
−9.9
−8.1
−9.0
−8.7
−9.1
−10.3
−2.4
−8.7
−6.7
−8.6
−5.2
−8.6
−7.9
−9.2
−8.6
−11.4
−10.8
−5.9
−7.8
−3.9
−8.3
−8.5
−10.9
−7.8
−9.6
−8.4
−9.4
−10.8
10
11
12
13
14
15
16
17
18
−11.6
−7.7
−11.9
−7.9
−11.4
−10.1
−10.4
−9.2
−11.7
−10.5
−10.7
−9.6
−11.5
−9.4
−10.9
−7.5
−10.9
−11.1
−10.1
−11.7
−10.2
−10.1

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Table 4. Cont.
Protein Data Bank (PDB) Code Protein Kinases
Ligand
2HYY
2PL0
3GCS
3WZE
3CS9
3QRJ *
4AG8
4ASD
5HI2
19
20
21
22
23
24
25
28i
28j
28k
28l
Imatinib
Nilotinib
−8.8
−8.0
−11.0
−8.6
−8.0
−8.8
−10.4
−9.0
−7.8
−8.2
−9.1
−8.9
−8.4
−9.5
−8.3
−9.5
−8.5
−9.7
−8.7
−4.4
−6.6
−7.1
−6.3
−8.6
−11.5
−9.2
−8.6
−9.4
−11.5
−9.2
−4.5
−8.3
−10.1
−9.8
−8.8
−10.9
−9.4
−8.8
−11.2
−9.9
−8.5
−9.4
−9.1
−4.2
−9.3
−10.4
−8.3
−6.2
1.2
−8.8
−11.3
−9.3
−7.0
−9.3
−11.3
−9.2
−9.3
−8.7
−10.1
−9.9
−8.7
−10.4
−8.5
−9.3
−8.5
−10.7
−8.9
6.3
−11.6
−9.8
−11.0
−9.4
−11.0
−9.8
−11.7
−9.2
−11.5
−10.5
−7.3
−11.3
−10.3
−3.2
−10.9
−9.1
−10.8
−9.1
−9.8
−10.9
−10.2
−11.2
15.5
9.8
3.8
−8.7
−11.0
−10.5
−4.9
−9.7
−6.6
−11.9
−13.2
−11.6
Ponatinib −11.6
Rebastinib
−12.6
Sorafenib
−11.5
* Abl tyrosine kinase with T315I mutation.
Based on the molecular dynamics trajectories, the binding energy for each complex was estimated
by the MM-PBSA method with decomposition to polar interactions, apolar van der Waals (VdW)
interactions, and polar solvation energies. Energy calculations were carried out using g_mmpbsa [26].
In terms of binding energy, the studied structures showed generally worse results but in some cases
comparable ones with known inhibitors. For reference, the structure with rebastinib showed the best
binding energy of
values of binding energies were obtained for imatinib (
(−149.3 ± 20.2 kJ/mol, 2HYY). The results of binding energy estimations are summarized in Table 5.
−
178.0
±
14.2 kJ/mol in complex with T315I-mutant tyrosine kinase. The lowest
154.7 12.3 kJ/mol, 2HYY) and ponatinib
−
±
Table 5. Calculated binding affinities for complexes of target structures with protein kinases and
reference compounds.
AutoDock
Vina Score
PDB Code for
Protein Kinases
Polar
Solvation
Binding
Energy
Comp.
VdW
Electrostatic
9
−11.4
−12.3
−11.6
−12.2
−11.9
−11.7
−10.7
−11.2
−11.5
−11.5
−11.2
−11.5
−12.0
2HYY
3QRJ *
2HYY
3QRJ *
3CS9
−
−
−
−
−
−
−
−
−
−
−
−
−
270.9
±
±
±
±
±
±
±
±
±
±
±
±
±
10.7
11.8
11.6
14.4
10.3
14.9
11.1
12.6
13.0
17.3
12.3
14.7
14.6
−68.5 ± 11.1
−18.2 ± 14.1
−38.4 ± 10.2
−20.7 ± 7.6
−39.4 ± 15.6
−34.7 ± 8.2
−65.9 ± 8.5
−69.8 ± 14.3
−41.9 ± 14.1
−51.8 ± 12.9
−23.0 ± 8.6
−26.9 ± 8.0
−69.0 ± 20.3
222.2 ± 14.4
175.7 ± 21.0
187.6 ± 14.8
168.1 ± 17.1
173.2 ± 17.4
200.3 ± 17.8
239.0 ± 16.0
208.2 ± 22.0
203.3 ± 19.8
202.8 ± 17.2
161.9 ± 18.0
172.0 ± 8.3
238.9 ± 41.2
−
−
−
−
−
−
−
144.1
160.0
137.6
139.2
151.5
117.9
122.3
±
±
±
±
±
±
±
12.5
13.7
13.9
15.6
11.8
17.6
17.1
10
11
11
11
13
15
18
20
22
24
24
28l
289.1
261.1
261.2
259.0
257.5
269.2
215.1
271.5
272.3
250.5
238.3
290.5
3CS9
3CS9
2HYY
3WZE
2HYY
2HYY
3WZE
3CS9
−99.1 ± 18.0
−136.2
−148.2
−135.3
−115.9
−148.6
±
±
±
±
±
14.7
15.7
17.9
14.5
19.5
Reference
imatinib
ponatinib
ponatinib
nilotinib
rebastinib
sorafenib
−11.9
−11.6
−11.6
−13.2
−12.6
−11.5
2HYY
2HYY
3CS9
3CS9
3QRJ *
3WZE
−
−
−
−
−
−
282.1
290.9
273.2
304.1
322.1
254.3
±
±
±
±
±
±
10.0
10.9
13.1
12.4
10.7
12.4
−42.4 ± 7.4
−32.9 ± 8.7
−47.7 ± 15.2
−63.2 ± 10.6
−77.0 ± 13.0
−73.9 ± 8.4
195.6 ± 10.4
191.0 ± 20.0
198.4 ± 24.1
216.4 ± 13.2
250.3 ± 13.8
181.0 ± 9.8
−154.7
−159.4
−149.3
−177.7
−178.0
−170.0
±
±
±
±
±
±
12.3
18.4
20.2
11.6
14.2
12.0
* Abl tyrosine kinase with T315I mutation.

Molecules 2019, 24, 3543
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To further analyze the interactions between the inhibitors and Abl-wt kinase, more specific
docking details are given in Figures 2–4.
Thr-315
Thr-315
Met-318
Glu-286
Met-318
H-bond
Glu-286
Asp-381
Asp-381
a
b
Thr-315
Thr-315
Met-318
H-bond
Met-318
H-bond
Glu-286
Glu-286
Asp-381
Asp-381
c
d
Figure 2. Binding models of inhibitor
bonds (H-bonds) are indicated by red lines to key amino acids. (
(Protein Data Bank (PDB): 3CS9; H-bond—non-detected). ( ) Binding model of
H-bond—Met-318). ( ) Binding model of 15 with Abl (PDB: 3CS9; H-bond—Met-318). (
model of 15 with Abl (PDB: 2HYY; H-bond—Met-318).
9
and 15 to Abl-wt kinase (wild-type Abl structure). Hydrogen
) Binding model of with Abl
with Abl (PDB: 2HYY;
) Binding
a
9
b
9
c
d
The comparable binding modes for the structures having the same substituents but attached to the
benzyl group via amine or amide bonds, as in the case with pairs of compounds and 15 or 11 and 13
to Abl-wt kinases, are presented in the Figure 2. As follows from the results of molecular docking, the
analogue containing the 2-methyl-5-nitroaniline fragment exhibited a higher binding affinity ( 11.4)
to Abl (PDB 2HYY) compared to the N-(2-methyl-5-nitrophenyl)benzamide derivative 15 10.4). In
9
9
−
(−
both cases, AutoDock Vina considered a binding advantage to place the trifluoromethyl and imidazole
substituents of the phenyl ring in the allosteric pocket, regardless of amide and amine bonds placement
(Figure 2). In addition, the nitro group in the 2-methyl-5-nitrophenyl ring formed a hydrogen bond
with the amino group of Met-318 (Figure 2b,d).
The docking studies of compounds 11 and 13 and nilotinib were carried out. As in the case of
nilotinib, the N-arylimidazole group of the abovementioned analogues was located in the allosteric
pocket, regardless of the connection type (Figure 3). Unlike the rigid methylphenyl group, the flexible
linker avoids the clash with the gatekeeper residue which often occurs in kinase resistance mutations.
The geometry of molecules 11 and 13 is more flattened due to the conformational mobility of the
4-(aminomethyl)benzamide, which apparently leads to a closer interaction with the ABL kinase active
site (Figure 3a,c).

Molecules 2019, 24, 3543
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Thr-315
Thr-315
H-bond
Glu-286
Met-318
Glu-286
H-bond
Met-318
H-bond
Asp-381
Asp-381
a
b
Thr-315
Thr-315
Met-318
Glu-286
Glu-286
Met-318
Asp-381
Asp-381
c
d
Figure 3. Binding models of inhibitor 11 and 13 to Abl-wt kinase. Hydrogen bonds are indicated by
red lines to key amino acids. Experimental binding model of nilotinib is shown in white (a, c). (
Binding model of 11 with Abl (PDB: 3CS9; H-bond—Thr-315, Glu-286, Asp-381). ( ) Binding model
of 11 with Abl (PDB: 2HYY; H-bond—non-detected). ( ) Binding model of 13 with Abl (PDB: 3CS9;
a
)
b
c
H-bond—non-detected). (d) Binding model of 13 with Abl (PDB: 2HYY; H-bond—non-detected).
The experimental binding model shows that analog 11 can form three hydrogen bonds: between
the secondary amine of the 3-(trifluoromethyl)aniline fragment and Thr-315 residue, and between the
residues Glu-286 and Asp-381 and the amide fragment of linker (Figure 3a). Compound 11 (Table 4) is
characterized by a higher estimated binding energy (
−
151.5
±
11.8 kJ/mol) in relation to Abl-wt kinase
17.6 kJ/mol) and completely comparable
(PDB: 3CS9) compared to the isomeric molecule 13
to ponatinib (−149.3 ± 20.2 kJ/mol, PDB 3CS9).
(
−
117.9
±
Thr-315 residue is a critical point for the binding of most Bcr-Abl inhibitors, regardless of whether
they interact with the active or inactive conformation of the kinase [27
a high estimated binding energy against T315I-mutant Abl ( 160.0
inferior to rebastinib ( 178.0 14.2) (Table 5). Docking of designed analog 10 with T315I Bcr-Abl (PDB
–
30]. Compound 10 demonstrated
−
±
13.7 kJ/mol), although slightly
−
±
code 3QRJ) was performed with rebastinib as the control (Figure 4a,c).
Our computational research shows that the use of the 4-(aminomethyl)benzamide as a flexible
linker leads to a favorable overall geometry of the molecule comparable to rebastinib, which allows
one to bypass the bulk isoleucine residue and provides the necessary binding to the active center of the
T315I-mutant Abl (PDB 3QRJ) (Figure 4a). The docking-simulated conformation of the compound 10
in the active center of the mutant protein suggests the possibility of forming hydrogen bonds with
amino acids Glu-286 and Asp-381. MD simulations showed high level of van der Waals interactions of
trifluoromethyl and imidazole groups in the allosteric fragment. In dynamics, there is also possibility
of formation of several hydrogen bonds in the ATP site due to the presence of a large number of
heteroatoms (Figure 4c, Table 5).
Compounds 28i–28l are characterized by the presence of a tertiary amino group of piperazine as
a part of the linker. In the molecular dynamics experiment, structure 28l showed a decrease in total

Molecules 2019, 24, 3543
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binding energy (
and non-polar components (
−
148.6
±
19.5) compared to nilotinib and comparable values of polar (
−
290.5
±
14.6)
−
69.0 20.3) in the complex with the human ABL kinase (PDB 3CS9). As
±
seen in Figure 4b, analog 28l binds to Abl in an imatinib/nilotinib-like (DFG-out) mode and makes
similar interactions, including hydrogen bonds with the main chain of Met-318, side chain of Glu-286,
and main chain amide of Asp-381. The two-dimensional interaction map of 28l with Abl-wt (PDB
3CS9) reveals the presence of four hydrogen bonds (Met-318, Asp-381, Glu-286, and Tyr-253) and the
non-covalent interaction between the pyridine fragment and the aromatic ring Tyr-253 (Figure 4d).
Thr-315
Ile-315
Met-318
Glu-286
H-bond
Glu-286
H-bond
H-bond
Met-318
H-bond
H-bond
Asp-381
Asp-381
a
b
c
d
Figure 4. Binding models of inhibitor 10 and 28l to Abl kinase. Hydrogen bonds are indicated by red
lines to key amino acids. ( ) Binding model of 10 with T315I-mutant Abl (PDB: 3QRJ; H-bond—Asp-381,
Glu-286). Experimental binding model of rebastinib is shown in white. ( ) Binding model of 28l with
Abl-wt (PDB: 3CS9; H-bond—Met-318, Asp-381, Glu-286). Experimental binding model of nilotinib
is shown in white. ( ) Two-dimensional interactions map of 10 with T315L-mutant Abl (PDB: 3QRJ;
H-bond—Asp-381, Glu-286). ( ) Two-dimensional interactions map of 28l with Abl-wt (PDB: 3CS9;
a
b
c
d
H-bond—Met-318, Asp-381, Glu-286, Tyr-253).
2.4. Structure–Activity Relationship Analysis
Molecular modeling showed that for compounds 11 and 13 or
9 and 15, the placement of the
trifluoromethyl and imidazole substitutes on the phenyl ring in the allosteric pocket is the most
favorable. The aniline fragment of molecules containing strong electronegative groups, CF3 or NO2, is
located in the adenine pocket.

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According to Tables 2 and 3, the EGFR protein inhibitory activity of compounds 11 and 13 is
positively correlated with the anti-tumor cell proliferation activity in contrast to compounds and 15
9
containing the NO2 group. Consequently, we believe that the inhibitory activity against tumor cells
of target compounds 11 and 13 containing (trifluoromethyl)aniline fragments was closely related to
the affinity of these compounds to EGFR, thus inhibiting EGFR protein binding competitively. Its
phosphorylation process is probably the main reason of the anti-tumor effect of these compounds.
The results also indicated that there was a moderate correlation between inhibition of receptor
tyrosine kinases and the inhibition of cancer cellular proliferation. Comparing compounds 28i–28l,
the priority order of R1 substituents was established as follows: (3-fluorobenzoyl)piperazin >
(4-nicotinoylpiperazin) > (2-fluorobenzoyl)piperazin >> (4-methoxybenzoyl)piperazin (Table 2). In
addition, the results indicated that the chemical space position of the fluor atom could have slight
influences on the inhibitory activity of compounds; the meta-position was better than the ortho-position.
The docking results revealed that introduction of the 4-(aminomethyl)benzamide as a flexible
linker leads to a flattening of the structure, which allows the target molecules to be located in the
allosteric pocket and penetrate into the adenine pocket, providing a more favorable location in the
active center of the protein kinases. Perhaps this can explain the appearance of the high inhibitory
activity of target compounds against some of protein kinase receptors. Taking into account the results
obtained, further structural modifications of the studied compounds may be promising for discovering
new inhibitors of protein kinases.
3. Experimental Section
3.1. General Information
Organic solvents were purified and dried by standard methods before usage in the synthesis. The
reactions were monitored and the purities of the compounds were checked by ascending thin-layer
chromatography (TLC) on silica gel-coated aluminum plates (60 F254, 0.25 mm, Merck, Darmstadt,
Germany). The following solvent systems were used: mixture of chloroform and methanol (10:1
or 4:1) or n-hexane–EtOAc (3:2 or 1:1); the spots were visualized under ultraviolet light at 254 nm.
Preparative column chromatography was performed on silica gel Merck 60 (70–230 mesh). NMR
spectra were registered on Bruker Avance 500 MHz spectrometer at 500 MHz (1H) and 125 MHz
(13C). Chemical shifts (
δ) are given in ppm related to internal SiMe4 and coupling constants (J) in Hz.
The signals were designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
High-resolution mass spectra (HRMS) were recorded on an Agilent 1290 Accurate-Mass 6500 Series
Q-TOF using ESI (electrospray ionization). The purity of the synthesized compounds was determined by
high-performance liquid chromatography (HPLC) (Waters, 996, USA) with an EC 250/4,6 NUCLEODUR
100-5 C18ec column using KH2PO4(0.02 M, pH = 6.8) with formic acid/acetonitrile 45:55 mobile phase
(1.0 mL/min). Melting points were determined on an electrically heated melting point (m.p.) apparatus
and were uncorrected.
3.2. Chemistry
The synthesis of the starting compounds
below [20,21].
2–4 according to the previously reported method is given
4-(Chloromethyl)-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide (5). To a solution
of amine 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (a) (one equivalent) and Et3N
(1.5 equivalents) in dry CHCl3 cooled to 0 ◦C was added dropwise to a solution of
4-(chloromethyl)benzoyl chloride (4) (one equivalent) in dry CHCl3. The reaction mixture was
stirred at room temperature. The progress was monitored by TLC. Cold water was added to the
reaction mixture. The organic layer was separated, and the water layer was extracted by CHCl3 three
times. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum.
The product was purified by column chromatography on silica gel. The white solid
5 was given

Molecules 2019, 24, 3543
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with 60% yield; m.p. 80–82 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 10.74 (s, 1H), 8.32 (s, 1H), 8.26 (d,
J = 1.0 Hz, 1H), 8.17 (s, 1H), 8.03 (d, J = 8.3 Hz, 2H), 7.77 (s, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.52 (s, 1H),
4.89 (s, 2H), 2.21 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 166.1, 142.2, 141.7, 139.4, 138.4, 135.5, 134.4,
129.4, 128.6, 115.4, 114.7, 112.2, 45.8, 14.0; HRMS (ESI+) m/z calculated for C19H16ClF3N3O [M + H]+
394.0929, found 394.0927; purity: 97.93% (by HPLC).
3.2.1. General Methods for the Synthesis of 8–12
Amixtureof
(DMF) was stirred at reflux. The progress of reaction was monitored by TLC. DMF was removed
under reduced pressure. The crude was diluted with water, and NH3 H2O was added to obtain
5(oneequivalent), amineRNH2 (a/b/c/e/g) (1.1equivalents)inN,N-Dimethylformamide
·
pH = 10–11. The resulting solid was filtered and washed with water. The product was purified by
column chromatography on silica gel.
N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-4-(((3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)
phenyl)amino)methyl)benzamide (8) was synthesized from compound
5 and 3-(4-methyl-1H-imidazol
-1-yl)-5-(trifluoromethyl)aniline (a) as a white solid. M.p. 200–202 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ
:
11.06 (s, 1H), 10.05 (d, J = 1.6 Hz, 1H), 8.47 (s, 1H), 8.28 (s, 1H), 8.23 (d, J = 1.3 Hz, 1H), 8.18 (d, J = 8.3 Hz,
2H), 8.15 (s, 1H), 7.76 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.51 (s, 1H), 7.24 (s, 1H), 7.11 (s, 1H), 7.07 (s, 1H),
6.28 (s, 1H), 5.67 (s, 2H), 2.24 (d, J = 0.6 Hz, 2H), 2.20 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 165.9,
151.7, 141.8, 139.4, 138.3, 136.9, 136.6, 135.4, 134.5, 132.1, 129.1, 128.4, 119.3, 115.5, 114.6, 109.8, 105.1,
50.1, 14.0, 9.4; HRMS (ESI+) m/z calculated for C30H25F6N6O [M + H]+ 599.1989, found 599.1993.
N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-4-(((2-methyl-5-nitrophenyl)amino)methyl)benzamide
(9) was synthesized from compound
5 and 2-methyl-5-nitroaniline (b) using the general method in
Section 3.2.1 or from compound 7b and 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (a)
using the general method in Section 3.2.4.2 as a yellow solid. M.p. 113–115 ◦C; 1H-NMR (DMSO-d6,
500 MHz) δ: 10.69 (s, 1H), 8.31 (s, 1H), 8.29 (d, J = 1.0 Hz, 1H), 8.16 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.75
(s, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.52 (s, 1H), 7.38 (dd, J = 8.0, 2.2 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H),
7.09 (d, J = 2.2 Hz, 1H), 6.54 (t, J = 6.1 Hz, 1H), 4.58 (d, J = 6.1 Hz, 2H), 2.33 (s, 3H), 2.21 (s, 3H);
13C-NMR (DMSO-d6, 125 MHz)
δ: 166.4, 147.4, 147.3, 144.5, 141.9, 139.0, 138.3, 135.4, 133.2, 131.0, 130.8,
128.5, 127.4, 115.4, 114.8, 111.1, 103.2, 46.3, 18.5, 13.8; HRMS (ESI+) m/z calculated for C26H23F3N5O3
[M + H]+ 510.1748, found 510.1747; purity: 96.63% (by HPLC).
N-methyl-4-(4-((4-((3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)carbamoyl)benzyl)amino)phenoxy)
picolinamide (10) was synthesized from compound 5 and 4-(4-aminophenoxy)-N-methylpicolinamide
(c) using the general method in Section 3.2.1 or from compound 7c and 3-(4-methyl-
1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (a) using the general method in Section 3.2.4.1.
as a white solid. M.p. 153–156 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 10.68 (s, 1H), 8.76 (q, J = 4.7 Hz,
1H), 8.47 (d, J = 5.4 Hz, 1H), 8.31 (s, 1H), 8.23 (d, J = 1.3 Hz, 1H), 8.17 (s, 1H), 8.00 (d, J = 8.3 Hz, 1H),
7.74 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.33 (d, J = 2.6 Hz, 1H), 7.09 (dd, J = 5.8, 2.6 Hz, 1H),
6.95 (d, J = 8.7 Hz, 1H), 6.69 (d, J = 9.0 Hz, 1H), 6.59 (t, J = 5.9 Hz, 1H), 4.43 (d, J = 5.8 Hz, 1H), 2.79 (d,
J = 4.8 Hz, 3H), 2.20(s, 2H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 167.2, 166.4, 164.4, 152.8, 150.7, 147.1,
145.3, 143.6, 141.9, 139.4, 138.4, 135.4, 133.1, 128.4, 127.7, 122.2, 115.3, 114.7, 114.3, 113.8, 112.0, 108.7,
46.9, 26.5, 14.0; HRMS (ESI+) m/z calculated for C32H28F3N6O3 [M + H]+ 601.2170, found 601.2167;
purity: 99.27% (by HPLC).
N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-4-(((3-(trifluoromethyl)phenyl)amino)methyl)benzamide
(11) was synthesized from compound
83–85 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
1H), 7.75 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.27 (t, J = 7.9 Hz, 1H), 6.91 (t, J = 6.1 Hz, 1H), 6.88 (s, 1H), 6.84
(s, 1H), 6.83 (s, 1H), 4.46 (d, J = 6.1 Hz, 1H), 2.21 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
: 166.4, 149.4,
5
and 3-(trifluoromethyl)aniline (g) as a white solid. M.p.
δ: 10.67 (s, 1H), 8.31 (s, 1H), 8.16 (s, 1H), 7.98 (d, J = 8.3 Hz,
δ

Molecules 2019, 24, 3543
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144.8, 141.8, 138.4, 133.1, 130.4, 128.4, 127.7, 116.1, 115.4, 114.8, 112.4, 108.7, 46.3, 13.9; HRMS (ESI+) m/z
calculated for C26H21F6N4O [M + H]+ 519.1614, found 519.1613; purity: 97.02% (by HPLC).
Methyl-4-amino-3-((4-((3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)carbamoyl)benzyl)amino)benzoate
(12) was synthesized from compound
5
and 3,4-diaminobenzoic acid (e) as a brown solid. M.p.
: 11.88 (brs, 1H), 10.68 (s, 1H), 8.34 (s, 1H), 8.31 (s, 1H),
130–132 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ
8.25 (d, J = 1.3 Hz, 1H), 8.16 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.75 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.51 (s,
1H), 7.14 (dd, J = 8.2, 1.8 Hz, 1H), 6.90 (d, J = 1.9 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 5.47 (brs., 1H), 4.47 (d,
J = 1.0 Hz, 2H), 2.20 (s, 3H), 2.11 (s, 1H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 168.4, 166.5, 145.3, 141.9,
141.2, 139.2, 138.4, 135.4, 134.3, 133.1, 128.3, 127.5, 121.1, 118.8, 115.4, 114.7, 112.8, 112.1, 111.5, 79.7, 47.0,
14.0; HRMS (ESI+) m/z calculated for C26H23F3N5O3 [M + H]+ 510.1748, found 510.1752.
3.2.2. General Procedure for the Synthesis of 6a–d
A mixture of methyl 4-formylbenzoate (1) (1.1 equivalents) and amine R1NH2 (a–d) (1.0 equivalent)
was dissolved in dry CHCl3. Sodium triacetoxyborohydride (2.8 equivalents) and AcOH (six
equivalents) were added to this solution, and the resulting solution was stirred at room temperature
for 2–3 h. The reaction mixture was quenched with an ice-cold saturated solution of NaHCO3 (to
pH 7), and extracted with CHCl3. The combined organic layers were dried over Na2SO4, filtered,
and concentrated under vacuum. The residue was dissolved in ethyl acetate, and the product was
precipitated with hexane. The solid was filtered and washed with ethyl acetate/hexane.
Methyl 4-({[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]amino}methyl)benzoate (6a) was
synthesized from methyl 4-formylbenzoate (
1H-NMR (DMSO-d6, 500 MHz)
: 8.13 (d, J = 1.0 Hz, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 8.3 Hz,
2H), 7.42 (s, 1H), 7.14 (s, 1H), 7.04 (s, 1H), 6.96 (s, 1H), 6.86 (s, 1H), 4.54 (d, J = 6.1 Hz, 2H), 4.51–4.56 (m,
1) and compound a as a white solid. The yield was 81%.
δ
2H), 3.85 (s, 3H), 2.15 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 166.6, 150.7, 145.6, 139.1, 138.9, 135.3,
129.9, 127.9, 114.6, 107.6, 106.1, 103.8, 52.5, 46.2, 14.0; HRMS (ESI+) m/z calculated for C20H19F3N3O2
[M + H]+ 390,1424, found 390.1425.
Methyl-4-{[(2-methyl-5-nitrophenyl) amino) methyl} benzoate (6b) was synthesized from
1 and
1
2-methyl-5-nitroaniline (b) as a yellow solid. The yield was 93%. H-NMR (DMSO-d6, 500 MHz)
δ:
7.95 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.37 (dd, J = 8.0, 2.2 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.04
(d, J = 2.2 Hz, 1H), 6.50 (t, J = 6.1 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H), 3.84 (s, 3H), 2.31 (s, 3H); 13C NMR
(DMSO-d6, 125 MHz) δ: 166.6, 147.4, 147.3, 145.8, 131.0, 130.8, 129.9, 128.8, 127.5, 111.1, 103.1, 67.5, 52.5,
46.4, 25.6, 18.5; HRMS (ESI+) m/z calculated for C16H17N2O4 [M + H]+ 301.1183, found 301.1185.
Methyl-4-{[(4-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}phenyl)amino]methyl}benzoate (6c) was synthesized
from
1H-NMR (DMSO-d6, 500 MHz)
(m, 2H), 7.31–7.35 (m, 1H), 7.06–7.10 (m, 1H), 6.90–6.96 (m, 2H), 6.64–6.68 (m, 2H), 6.52–6.57 (m, 1H),
4.38–4.43 (m, 2H), 3.86 (s, 3H), 2.79 (d, J = 4.8 Hz, 3H); 13C-NMR (DMSO-d6, 500 MHz)
: 221.7, 221.5,
1
and 4-(4-aminophenoxy)-N-methylpicolinamide (c) as a white solid. The yield was 88%.
δ: 8.73–8.79 (m, 1H), 8.44–8.49 (m, 1H), 7.93–7.98 (m, 2H), 7.53–7.58
δ
221.0, 218.0, 217.5, 216.6, 216.5, 215.8, 212.3, 212.0, 211.8, 210.4, 208.4, 208.2, 207.0, 192.8, 191.5, 186.3;
HRMS (ESI+) m/z calculated for C22H22N3O4 [M + H]+ 392,1605, found 392.1602.
Methyl 4-({[4-(pyridin-3-yl)pyrimidin-2-yl]amino}methyl)benzoate (6d) was synthesized from
4-(pyridin-3-yl)pyrimidin-2-amine (
) as a white solid. The yield was 81%. 1H-NMR (DMSO-d6,
500 MHz) : 9.23 (br.s., 1H), 8.70 (br. s., 1H), 8.43 (d, J = 5.1 Hz, 2H), 8.03 (t, J = 6.1 Hz, 1H), 7.93 (d,
J = 8.3 Hz, 2H), 7.53 (br.s., 3H), 7.28 (d, J = 5.1 Hz, 1H), 4.67 (d, J = 5.8 Hz, 2H), 3.84 (s, 3H); 13C-NMR
(DMSO-d6, 125 MHz) : 166.6, 162.9, 151.7, 148.5, 146.9, 134.7, 132.8, 129.7, 128.4, 127.7, 124.3, 52.5, 44.5;
HRMS (ESI+) m/z calculated for C18H17N4O2 [M + H]+ 321.1346, found 321.1347.
1 and
d
δ
δ

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3.2.3. General Procedure for the Synthesis of 7a–d
Aqueous 18–27% HCl (6 ml) was added to 2.5 mmol of ester 6a
–
d
, and the reaction mixture heated
for 4–6 h at 80–90 ◦C. The reaction mixture was cooled to room temperature. The solid was filtered and
washed with water. The product 7 was purified by precipitation from organic solvents.
4-({[3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]amino}methyl)benzoic acid (7a) was
1
synthesized from compound 6a as a white solid. The yield was 82%. H-NMR (DMSO-d6, 500 MHz)
δ
:
8.90 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.73 (s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 5.9 Hz, 1H), 7.14 (s,
1H), 7.06 (s, 1H), 6.98 (s, 1H), 4.54 (d, J = 5.4 Hz, 1H), 2.26 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ
: 167.6, 150.8, 144.8, 138.1, 134.8, 130.0, 127.8, 116.3, 108.9, 106.9, 104.6, 46.1, 12.0; HRMS (ESI+) m/z
calculated for C19H17F3N3O2 [M + H]+ 376,1267, found 376.1271.
4-{[(2-Methyl-5-nitrophenyl) amino)methyl}benzoic acid (7b) was synthesized from compound 6b as a
1
yellow solid. The yield was 83%. H-NMR (DMSO-d6, 500 MHz)
δ
: 7.92 (d, J = 8.3 Hz, 2H), 7.50 (d,
J = 8.3 Hz, 2H), 7.37 (dd, J = 8.0, 2.2 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.05 (d, J = 2.6 Hz, 1H), 6.48 (t,
J = 6.1 Hz, 1H), 4.55 (d, J = 6.1 Hz, 2H), 2.31 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 167.7, 147.4,
147.3, 145.2, 131.0, 130.8, 130.0, 129.9, 127.3, 111.1, 103.1, 46.4, 18.5; HRMS (ESI+) m/z calculated for
C15H15N2O4 [M + H]+ 287.1026, found 287.1030.
4-{[(4-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}phenyl)amino]methyl}benzoic acid (7c) was synthesized from
1
compound 6c as a white solid. The yield was 79%. H-NMR (DMSO-d6, 500 MHz)
δ: 8.76 (q, J = 4.5 Hz,
1H), 8.46 (d, J = 5.8 Hz, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 2.6 Hz, 1H),
7.07 (dd, J = 5.6, 2.7 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 6.66 (d, J = 8.7 Hz, 2H), 6.55 (brs., 1H), 4.38 (d,
J = 4.8 Hz, 2H), 2.79 (d, J = 4.8 Hz, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 221.8, 221.7, 221.0, 218.0,
217.5, 216.6, 216.3, 215.7, 212.3, 211.7, 210.4, 208.4, 208.2, 207.0, 191.5, 186.3; HRMS (ESI+) m/z calculated
for C21H20N3O4 [M + H]+ 378,1448, found 378.1454.
4-({[4-(Pyridin-3-yl)pyrimidin-2-yl]amino}methyl)benzoic acid (7d) was synthesized from compound 6d as
1
a white solid. The yield was 68%. H-NMR (DMSO-d6, 500 MHz)
δ
: 11.72–13.85 (m, 1H), 9.34 (brs.,
1H), 8.83 (brs., 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.48 (brs., 1H), 8.21 (brs., 1H), 7.91 (d, J = 8.0 Hz, 2H),
7.43–7.56 (m, 4H), 4.69 (brs., 2H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 167.7, 159.6, 148.9, 146.0, 145.8,
137.9, 133.9, 129.8, 129.6, 127.6, 125.6, 44.5; HRMS (ESI+) m/z calculated for C17H15N4O2 [M + H]+
307.1190, found 307.1190.
3.2.4. General Methods for the Synthesis of 9, 10, and 13–25
3.2.4.1. Method viii
Step 1 (synthesis of acyl chloride). To a solution of acid 7a–d in dry CHCl3 was added dropwise SOCl2
(four equivalents). The reaction mixture was stirred at 60 ◦C. The reaction progress was monitored by
TLC. The excess thionyl chloride and solvent were removed under reduced pressure. The obtained
acyl chlorides were immediately taken to the next step.
Step 2 (synthesis of benzamide). To a solution of chloroanhydride in dry CHCl3 was added
amine R2NH2 (one equivalent) and Et3N (1.5 equivalents). The reaction mixture was stirred at
room temperature. The reaction progress was monitored by TLC. Cold water was added to the
reaction mixture. The organic layer was separated from the water. The combined organic layers were
dried over Na2SO4, filtered, and concentrated under vacuum. The product was purified by column
chromatography on silica gel.
3.2.4.2. Method ix
A mixture of acid 7b (or 7c) (1.3 equivalents), 1-hydroxybenzotriazole (1.3 equivalents), and
N,N’-dicyclohexylcarbodiimide or 1,1’-carbonyldiimidazole (1.3 equivalents) was dissolved in dry
DMF. Et3N (2.5 equivalents) and amine R2NH2 (one equivalent) were added. The resulting mixture

Molecules 2019, 24, 3543
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was stirred at room temperature. The reaction progress was monitored by TLC. The solvent was
removed under reduced pressure. The crude was diluted with water, and NH4OH was added to obtain
pH = 10–11. The resulting solid was filtered and washed with water. The product was purified by
column chromatography on silica gel.
4-({[3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]amino}methyl)-N-[3-(trifluoromethyl)phenyl]benzamide
(13) was synthesized from compound 7a and 3-(trifluoromethyl)aniline (g) using the general method
in Section 3.2.4.1 as a white solid. M.p. 129–131 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 10.59 (s, 1H),
8.84 (brs., 1H), 8.28 (s, 1H), 8.08 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.72 (s, 1 H), 7.59–7.63 (m,
1H), 7.57 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 6.1 Hz, 1H), 7.14 (s, 1H), 7.08 (s, 1H),
7.00 (s, 1H), 4.52–4.60 (m, 2H), 2.25 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 166.2, 150.8, 143.8, 140.5,
138.2, 134.9, 133.6, 130.3, 128.5, 127.7, 124.2, 120.3, 116.8, 116.8, 116.1, 108.8, 106.9, 104.5, 60.2, 46.1, 14.6,
12.3; HRMS (ESI+) m/z calculated for C26H21F6N4O [M + H]+ 519.1614, found 519.1611; purity: 97.11%
(by HPLC).
Diethyl(4-(((3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)amino)methyl)benzoyl)glutamate (14)
was synthesized from compound 7a and diethyl glutamate (
h
) using the general method in Section 3.2.4.1
as a white solid. M.p. 125–127 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 8.70 (d, J = 7.4 Hz, 1H), 8.15 (brs.,
1H), 7.86 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.45 (brs., 1H), 7.12 (t, J = 5.9 Hz, 1H), 7.04 (s,
1H), 6.97 (s, 1H), 6.87 (s, 1H), 4.51 (d, J = 5.8 Hz, 2H), 4.44 (ddd, J = 9.4, 7.3, 5.4 Hz, 1H), 4.08–4.17
(m, 2H), 4.05 (q, J = 7.1 Hz, 2H), 2.45 (t, J = 7.7 Hz, 2H), 2.16 (s, 3H), 1.94–2.14 (m, 2H), 1.12–1.24 (m,
6H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 172.7, 172.3, 167.0, 150.7, 143.5, 139.0, 132.9, 128.2, 127.6, 107.6,
106.2, 103.8, 61.0, 60.4, 52.5, 46.2, 30.6, 26.2, 14.5, 14.1; HRMS (ESI+) m/z calculated for C28H32F3N4O5
[M + H]+ 561.2319, found 561.2321; purity: 97.13% (by HPLC).
4-({[3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]amino}methyl)-N-{[(2-methyl-5-nitrophenyl)
benzoate (15) was synthesized from compound 7a and 2-methyl-5-nitroaniline (
b
) using the general
method in Section 3.2.4.1 as a white solid. M.p. 205–208 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 10.11 (s,
1H), 8.37 (d, J = 2.2 Hz, 1H), 8.23 (s, 1H), 8.04 (dd, J = 8.5, 2.4 Hz, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.56–7.60
(m, 3H), 7.48 (s, 1H), 7.19 (t, J = 6.3 Hz, 1H), 7.06 (s, 1H), 6.99 (s, 1H), 6.91 (s, 1H), 4.56 (d, J = 6.1 Hz,
2H), 2.40 (s, 3H), 2.17 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 166.0, 150.7, 146.2, 144.0, 142.0, 138.9,
138.5, 137.9, 135.3, 133.2, 132.0, 128.6, 127.7, 120.8, 120.7, 114.8, 107.8, 106.2, 103.8, 46.1, 18.7, 13.9; HRMS
(ESI+) m/z calculated for C26H23F3N5O3 [M + H]+ 510.1748, found 510.1745; purity: 99.06% (by HPLC).
Diethyl (4-(((2-methyl-5-nitrophenyl)amino)methyl)benzoyl)glutamate (16) was synthesized from compound
7b and diethyl glutamate (
h
) using the general method in Section 3.2.4.2 as a yellow solid. M.p.
: 8.70 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.50 (d,
127–129 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ
J = 8.3 Hz, 2H), 7.37 (dd, J = 8.0, 2.2 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 6.49 (t,
J = 6.1 Hz, 1H), 4.54 (d, J = 6.1 Hz, 2H), 4.44 (ddd, J = 9.5, 7.5, 5.4 Hz, 1H), 4.12 (qd, J = 7.1, 2.2 Hz, 2H),
4.05 (q, J = 7.1 Hz, 2H), 2.43–2.48 (m, 2H), 2.31 (s, 3H), 2.07–2.16 (m, 1H), 2.01 (dd, J = 9.6, 7.4 Hz, 1H),
1.18 (dt, J = 15.4, 7.2 Hz, 6H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 172.7, 172.3, 167.1, 147.4, 147.3, 143.6,
132.9, 130.9, 130.7, 128.2, 127.1, 111.0, 103.2, 79.7, 61.0, 60.4, 52.5, 46.3, 30.6, 26.2, 18.5, 14.5; HRMS (ESI+)
m/z calculated for C24H30N3O7 [M + H]+ 472.2078, found 472.2080; purity: 97.32% (by HPLC).
4-{[(2-Methyl-5-nitrophenyl)amino]methyl}-N-(4-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}phenyl) benzoate (17)
was synthesized from compound 7b and 4-(4-aminophenoxy)-N-methylpicolinamide (c) using the
general method in Section 3.2.4.2 as a yellow solid. M.p. 231–233 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ
: 10.38 (brs, 1H), 8.80 (brs, 1H), 8.53 (brs, 1H), 7.93 (brs, 4H), 7.56 (brs, 2H), 7.41 (brs, 2H), 7.04–7.32
(m, 5H), 6.53 (brs, 1H), 4.58 (brs, 2H), 2.81 (brs, 3H), 2.33 (brs, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ:
166.4, 165.9, 164.3, 153.1, 150.9, 149.1, 147.4, 147.3, 144.0, 137.7, 133.9, 131.0, 130.8, 128.4, 127.2, 122.6,
121.7, 114.3, 111.1, 109.2, 103.3, 46.2, 26.5, 18.5; HRMS (ESI+) m/z calculated for C28H26N5O5 [M + H]+
512.1928, found 512.1926; purity: 95.23% (by HPLC).

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4-{[(2-Methyl-5-nitrophenyl)amino]methyl}-N-[3-(trifluoromethyl)phenyl]benzamide (18) was synthesized
from compound 7b and 3-(trifluoromethyl)aniline ( ) using the general method in Section 3.2.4.2 as a
yellow solid. M.p. 169–171 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
: 10.53 (s, 1H), 8.26 (brs, 1H), 8.04
g
δ
(d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.46 (d,
J = 7.4 Hz, 1H), 7.38 (d, J = 6.7 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H), 7.09 (s, 1H), 6.52 (brs, 1H), 4.57 (d,
J = 5.8 Hz, 2H), 2.32 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz):
δ = 166.3, 147.4, 147.3, 144.2, 140.5, 133.6,
131.0, 130.8, 130.3, 128.5, 127.3, 124.2, 120.3, 116.8, 111.1, 103.2, 46.3, 18.5; HRMS (ESI+) m/z calculated
for C22H19F3N3O3 [M + H]+ 430.1373, found 430.1374; purity: 98.18% (by HPLC).
Methyl 4-amino-3-[(4-{[(2-methy-5-nitrophenyl)amino]methyl}benzoyl)amino]benzoate (19) was synthesized
from compound 7b and methyl 3,4-diaminobenzoate (
f
) using the general method in Section 3.2.4.2 as
a yellow solid. M.p. 208–210 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 9.63 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H),
7.79 (d, J = 1.6 Hz, 1H), 7.60 (dd, J = 8.7, 1.9 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.38 (dd, J = 8.0, 2.2 Hz,
1H), 7.27 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 2.2 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.50–6.55 (m, 1H), 5.86 (s,
2H), 4.54–4.59 (m, 2H), 3.77 (s, 3H), 2.33 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz) δ: 166.6, 166.1, 148.9,
147.4, 147.4, 143.6, 133.6, 131.0, 130.8, 129.4, 128.8, 128.6, 127.0, 122.3, 116.6, 115.0, 111.0, 103.2, 51.8, 46.3,
18.5; HRMS (ESI+) m/z calculated for C23H23N4O5 [M + H]+ 435.1663, found 435.1656; purity: 95.87%
(by HPLC).
4-{[(4-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}phenyl)amino]methyl}-N-[3-(trifluoromethyl)phenyl]benzamide
(20) was synthesized from compound 7c and 3-(trifluoromethyl)aniline (
g
) using the general method
in Section 3.2.4.2 as a white solid. M.p. 170–171 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ
: 10.54 (s, 1H),
8.77 (d, J = 4.8 Hz, 1H), 8.47 (d, J = 5.4 Hz, 1H), 8.28 (s, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.3 Hz,
2H), 7.60–7.67 (m, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 2.6 Hz, 1H), 7.09 (dd,
J = 5.8, 2.6 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.69 (d, J = 9.0 Hz, 2H), 6.58 (s, 1H), 4.42 (d, J = 5.4 Hz, 2H),
2.79 (d, J = 4.8 Hz, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 167.2, 166.3, 164.4, 152.8, 150.7, 147.1, 145.0,
143.6, 140.5, 133.5, 130.3, 128.4, 127.6, 124.2, 122.2, 120.3, 116.8, 116.7, 114.0, 108.7, 79.7, 46.9, 26.5; HRMS
(ESI+) m/z calculated for C28H24F3N4O3 [M + H]+ 521.1795, found 521.1800; purity: 98.85% (by HPLC).
Diethyl (4-(((4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)amino)methyl)benzoyl)glutamate (21) was
synthesized from compound 7c and diethyl glutamate (
h
) using the general method in Section 3.2.4.2
as a white solid. M.p. 162–164 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 8.76 (q, J = 4.8 Hz, 1H), 8.70
(d, J = 7.4 Hz, 1H), 8.47 (d, J = 5.4 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.33 (d,
J = 2.6 Hz, 1H), 7.08 (dd, J = 5.4, 2.6 Hz, 1H), 6.91–6.95 (m, 2 H), 6.65–6.68 (m, 2 H), 6.53 (t, J = 5.9 Hz,
1 H), 4.45 (ddd, J = 9.6, 7.4, 5.1 Hz, 1H), 4.38 (d, J = 6.1 Hz, 2H), 4.12 (qd, J = 7.1, 2.4 Hz, 2H), 4.06 (q,
J = 7.1 Hz, 2H), 2.79 (d, J = 4.8 Hz, 3H), 2.44–2.48 (m, 2H), 2.08 - 2.17 (m, 1H), 1.97–2.07 (m, 1H), 1.19
(dt, J = 13.1, 7.1 Hz, 6H); 13C-NMR (DMSO-d6, 125 MHz):
δ = 172.7, 172.3, 167.2, 167.1, 164.4, 152.8,
150.7, 147.1, 144.4, 143.6, 132.7, 128.1, 127.4, 122.1, 114.2, 113.8, 108.8, 61.0, 60.4, 52.5, 47.0, 33.8, 30.7,
24.9, 14.6; HRMS (ESI+) m/z calculated for C30H35N4O7 [M + H]+ 563.2500, found 563.2502; purity:
98.00% (by HPLC).
N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-4-(((4-(pyridin-2-yl)pyrimidin-2-yl)amino)methyl)
benzamide (22) was synthesized from compound 7dand 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)
aniline (
(DMSO-d6, 500 MHz)
a
) using the general method in Section 3.2.4.1. as a white solid. M.p. 150–152 ◦C; 1H-NMR
δ
: 10.70 (s, 1H), 9.26 (brs., 1H), 8.71 (brs., 1H), 8.44 (d, J = 4.8 Hz, 2H), 8.34 (brs.,
1H), 8.31 (brs., 1H), 8.18 (s, 1H), 8.05 (t, J = 6.1 Hz, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.74 (s, 1H), 7.55 (d,
J = 13.1 Hz, 4H), 7.29 (d, J = 5.1 Hz, 1H), 4.69 (d, J = 5.1 Hz, 2H), 2.21 (s, 3H); 13C-NMR (DMSO-d6,
125 MHz): δ: 166.4, 162.9, 151.8, 148.5, 141.9, 138.3, 134.7, 132.8, 128.3, 127.6, 124.3, 115.5, 114.8, 112.1,
49.1, 44.5, 13.8; HRMS (ESI+) m/z calculated for C28H23F3N7O [M + H]+ 530.1911, found 530.1913;
purity: 96.67% (by HPLC).
Diethyl (4-(((4-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)benzoyl)glutamate (23) was synthesized from
compound 7d and diethyl glutamate (h) using the general method in Section 3.2.4.1 as a white solid.

Molecules 2019, 24, 3543
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M.p. 130–132 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ
: 9.23–9.30 (m, 1H), 8.69–8.75 (m, 1H), 8.65–8.69 (m,
1H), 8.43 (d, J = 5.1 Hz, 2H), 7.98–8.06 (m, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.53–7.61 (m, 1H), 7.42–7.52
(m, 2H), 7.28 (d, J = 5.1 Hz, 1H), 4.61–4.69 (m, 2H), 4.40–4.46 (m, 1H), 4.01–4.19 (m, 4H), 2.44 (s, 2H),
1.95–2.19 (m, 2H), 1.12–1.25 (m, 6H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 172.7, 172.3, 167.1, 151.6, 148.3,
134.9, 132.9, 132.5, 127.9, 127.4, 124.4, 61.0, 60.4, 52.4, 44.4, 30.6, 26.2, 14.5; HRMS (ESI+) m/z calculated
for C26H30N5O5 [M + H]+ 492.2241, found 492.2238; purity: 98.89% (by HPLC).
4-({[4-(Pyridin-3-yl)pyrimidin-2-yl]amino}methyl)-N-[3-(trifluoromethyl)phenyl]benzamide (24) was
synthesized from compound 7d and 3-(trifluoromethyl)aniline (
g
) using the general method in
Section 3.2.4.1. as a white solid. M.p. 138–141 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
δ: 10.51 (s, 1H), 9.29
(brs., 1H), 8.75 (br.s., 1H), 8.52 (d, J = 7.7 Hz, 1H), 8.46 (d, J = 4.8 Hz, 1H), 8.26 (s, 1H), 8.09 (brs., 1H),
8.05 (d, J = 8.3 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.52–7.66 (m, 4H), 7.46 (d, J = 7.7 Hz, 1H), 7.32 (d,
J = 5.1 Hz, 1H), 4.69 (br.s., 2H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 166.3, 150.9, 147.7, 145.2, 140.5,
135.7, 133.2, 130.3, 128.3, 127.5, 124.7, 124.2, 120.3, 116.7; HRMS (ESI+) m/z calculated for C24H19F3N5O
[M + H]+ 450.1536, found 450.1535; purity: 99.00% (by HPLC).
4-({[4-(Pyridin-3-yl)pyrimidin-2-yl]amino}methyl)-N-{[(2-methyl-5-nitrophenyl) benzoate (25) was
synthesized from compound 7d and 2-methyl-5-nitroaniline (b) using the general method in
Section 3.2.4.1 as a white solid. M.p. 175–176 ◦C; 1H NMR (DMSO-d6, 500 MHz) δ: 10.08 (s, 1H), 9.27
(brs., 1H), 8.71 (brs., 1H), 8.44 (d, J = 4.5 Hz, 2H), 8.36 (d, J = 1.9 Hz, 1H), 8.02–8.08 (m, 2H), 7.97 (d,
J = 8.0 Hz, 2H), 7.58 (d, J = 8.3 Hz, 4H), 7.29 (d, J = 5.1 Hz, 1H), 4.69 (d, J = 4.2 Hz, 2H), 2.39 (s, 3H); 13
C
NMR (DMSO-d6, 125 MHz): δ: 166.1, 151.7, 148.4, 146.2, 141.9, 137.9, 134.8, 132.9, 132.8, 132.0, 128.3,
127.6, 124.4, 120.8, 120.7, 44.5, 18.7; HRMS (ESI+) m/z calculated for C24H21N6O3 [M + H]+ 441.1670,
found 441.1670; purity: 99.42% (by HPLC).
3.2.5. Compounds 26i–l Prepared According to Section 3.2.2 Described for 6a–d
Methyl 4-((4-(4-methoxybenzoyl)piperazin-1-yl)methyl)benzoate
(
26i
) and methyl 4-formylbenzoate (
: 7.94 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.3 Hz,
)
was synthesized from
(4-methoxyphenyl)(piperazin-1-yl)methanone (
solid. The yield was 75%. H-NMR (DMSO-d6, 500 MHz)
i
1) as a white
1
δ
2H), 7.36 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), 3.84–3.89 (m, 3H), 3.80 (s, 3H), 3.60 (s, 2H),
3.43–3.58 (m, 4H), 2.32–2.46 (m, 4H); 13C-NMR (DMSO-d6, 125 MHz)
δ
: 169.4, 166.6, 160.6, 144.2, 129.7,
129.5, 128.9, 128.2, 114.1, 79.6, 61.8, 55.7, 53.1, 52.6.
Methyl
4-((4-(2-fluorobenzoyl)piperazin-1-yl)methyl)benzoate
(
26j)
was
synthesized
from
(2-fluorophenyl)(piperazin-1-yl)methanone (
j
) and compound
1
as a white solid. The yield
1
was 22%. H-NMR (DMSO-d6, 500 MHz)
δ: 7.94 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.3 Hz, 3H), 7.37–7.42
(m, 1H), 7.27–7.33 (m, 2H), 3.86 (s, 3H), 3.63–3.73 (m, 2H), 3.60 (s, 2H), 3.19–3.28 (m, 2H), 2.40–2.50 (m,
2H), 2.30–2.39 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 166.6, 164.4, 159.0, 157.0, 144.1, 131.9, 129.6,
129.5, 129.2, 128.9, 125.4, 116.3, 61.7, 53.2, 52.7, 52.6, 47.0, 41.8.
Methyl 4-((4-(3-fluorobenzoyl)piperazin-1-yl)methyl)benzoate
(
26k
)
was synthesized from
(3-fluorophenyl)(piperazin-1-yl)methanone (
k
) and compound
1
as a white solid. The yield
1
was 19%. H-NMR (DMSO-d6, 500 MHz)
δ: 7.94 (d, J = 8.3 Hz, 2H), 7.46–7.53 (m, 3H), 7.28–7.33 (m,
1H), 7.21–7.26 (m, 2H), 3.86 (s, 3H), 3.64 (brs, 2H), 3.60 (s, 2H), 3.33 (brs, 2H), 2.45 (brs, 2H), 2.37 (brs,
2H); 13C-NMR (DMSO-d6, 125 MHz)
: 167.9, 166.6, 163.3, 161.3, 144.2, 131.2, 129.7, 129.5, 128.9, 123.4,
116.9, 114.4, 61.7, 53.2, 52.5, 47.5, 42.0.
δ
Methyl
4-((4-nicotinoylpiperazin-1-yl)methyl)benzoate
(26l
)
was
synthesized
from
piperazin-1-yl(pyridin-3-yl)methanone (
l
) and compound
1
as a white solid. The yield was
1
83%. H-NMR (DMSO-d6, 500 MHz)
δ
: 8.65 (dd, J = 4.8, 1.6 Hz, 1H), 8.61 (d, J = 1.3 Hz, 1H), 7.94 (d,
J = 8.3 Hz, 2H), 7.84 (dt, J = 7.7, 1.9 Hz, 1H), 7.47–7.51 (m, 3H), 3.86 (s, 3H), 3.67 (brs, 2H), 3.61 (s, 2H),

Molecules 2019, 24, 3543
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3.36 (brs, 2H), 2.47 (brs, 2H), 2.39 (brs, 2H); 13C-NMR (DMSO-d6, 125 MHz)
δ
: 167.2, 166.6, 150.9, 148.0,
144.2, 135.3, 132.2, 129.7, 129.5, 128.9, 124.0, 61.7, 53.2, 52.6, 47.7, 42.1.
3.2.6. Compounds 27i–l Prepared According to Section 3.2.3 Described for 7a–d
4-((4-(4-Methoxybenzoyl)piperazin-1-yl)methyl)benzoic acid (27i) was synthesized from compound 26i as a
1
white solid. The yield was 60%. H-NMR (DMSO-d6, 500 MHz)
δ
: 7.92 (d, J = 8.3 Hz, 2H), 7.46 (d,
J = 8.3 Hz, 2H), 7.34–7.39 (m, 2H), 6.96 - 7.02 (m, 2H), 3.80 (s, 3H), 3.53–3.70 (m, 6H), 2.41 (brs, 4H);
13C-NMR (DMSO-d6, 125 MHz)
δ: 169.4, 167.7, 160.6, 143.6, 130.1, 129.8, 129.5, 129.4, 128.2, 114.1, 79.6,
61.9, 55.7, 53.1.
4-((4-(2-Fluorobenzoyl)piperazin-1-yl)methyl)benzoic acid (27j) was synthesized from compound 26j as
1
a white solid. The yield was 60%. H-NMR (DMSO-d6, 500 MHz)
δ
: 7.93 (s, 2H), 7.48–7.54 (m, 1H),
7.45 (d, J = 8.3 Hz, 2H), 7.40 (td, J = 7.3, 1.8 Hz, 1H), 7.27–7.33 (m, 2H), 3.67 (brs, 2H), 3.58–3.61 (m,
2H), 3.22–3.26 (m, 2H), 2.45 (brs, 2H), 2.36 (d, J = 9.0 Hz, 2H); 13C-NMR (DMSO-d6, 125 MHz)
: 167.7,
164.4, 159.0, 157.0, 143.5, 131.9, 130.1, 129.8, 129.4, 129.2, 125.4, 116.3, 61.8, 53.2, 52.7, 47.0, 41.8.
δ
4-((4-(3-Fluorobenzoyl)piperazin-1-yl)methyl)benzoic acid (27k) was synthesized from compound 26k as a
1
white solid. The yield was 53%. H-NMR (DMSO-d6, 500 MHz)
δ: 7.92 (d, J = 8.3 Hz, 2H), 7.50 (td,
J = 7.9, 5.9 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.27–7.34 (m, 1H), 7.21–7.26 (m, 2H), 3.64 (brs, 2H), 3.59 (s,
2H), 3.33 (brs, 2H), 2.45 (brs, 2H), 2.38 (brs, 2H); 13C-NMR (DMSO-d6, 125 MHz)
: 167.9, 167.7, 163.3,
161.3, 143.6, 131.2, 130.2, 129.8, 129.4, 123.4, 116.9, 114.4, 61.8, 53.1, 52.6, 47.5, 42.1.
δ
4-((4-Nicotinoylpiperazin-1-yl)methyl)benzoic acid 27l was synthesized from methyl
4-((4-nicotinoylpiperazin-1-yl)methyl)benzoate 26l as a white solid. The yield was 72%. 1H-NMR
(DMSO-d6, 500 MHz) : 8.65–8.67 (m, 1H), 8.61 (d, J = 1.6 Hz, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.84 (dt,
(
)
δ
J = 7.7, 1.9 Hz, 1H), 7.47–7.50 (m, 1H), 7.46 (d, J = 8.3 Hz, 2H), 3.67 (brs, 2H), 3.60 (s, 2H), 3.36 (brs, 2H),
2.47 (brs, 2H), 2.39 (brs, 2H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 167.7, 167.2, 150.9, 148.0, 143.6, 135.3,
132.2, 130.1, 129.8, 129.4, 124.0, 61.8, 53.2, 52.6, 47.7, 42.1.
3.2.7. Compounds 28i–28l Prepared According to Method viii (Section 3.2.4.1)
4-{[4-(4-Methoxybenzoyl)piperazin-1-yl]methyl}-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]
benzamide (28i) was synthesized from compound 27i and amine
a
as a white solid. M.p. 98–102 ◦C;
1H-NMR (DMSO-d6, 500 MHz)
δ: 10.69 (s, 1H), 8.30 (s, 1H), 8.22 (d, J = 1.0 Hz, 1H), 8.18 (s, 1H), 7.98
(d, J = 8.3 Hz, 2H), 7.75 (s, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.50 (s, 1H), 7.34–7.40 (m, J = 8.7 Hz, 2H),
6.97–7.02 (m, J = 9.0 Hz, 2H), 3.80 (s, 3H), 3.62 (s, 2H), 3.45–3.60 (m, 4H), 2.37 - 2.47 (m, 4H), 2.20 (s, 3H);
13C-NMR (DMSO-d6, 125 MHz)
δ: 169.4, 166.4, 160.7, 142.9, 141.8, 139.4, 138.4, 135.5, 133.3, 129.5, 129.4,
128.3, 115.4, 114.7, 114.1, 61.8, 55.7, 14.0; HRMS (ESI+) m/z calculated for C31H31F3N5O3 [M + H]+
578.2374, found 578.2362; purity: 95.81% (by HPLC).
4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]methyl}-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]
benzamide (28j) was synthesized from compound 27j and amine
a
as a white solid. M.p. 106–109 ◦C;
1H-NMR (DMSO-d6, 500 MHz)
δ: 10.68 (s, 1H), 8.31 (s, 1H), 8.22 (d, J = 1.0 Hz, 1H), 8.17 (s, 1H), 7.99 (d,
J = 8.3 Hz, 2H), 7.75 (s, 1H), 7.49–7.55 (m, 4H), 7.38–7.43 (m, 1H), 7.28–7.34 (m, 2H), 3.69 (brs, 2H), 3.63
(s, 2H), 3.26 (t, J = 4.3 Hz, 2H), 2.48 (brs, 2H), 2.39 (d, J = 4.5 Hz, 2H), 2.20 (s, 3H); 13C-NMR (DMSO-d6,
125 MHz) δ: 166.4, 164.4, 159.1, 157.0, 142.9, 141.8, 139.4, 138.4, 135.5, 133.4, 132.0, 129.4, 128.3, 125.4,
116.4, 116.2, 115.4, 114.7, 112.1, 79.7, 61.7, 53.3, 52.7, 47.1, 41.8, 14.1; HRMS (ESI+) m/z calculated for
C30H28F4N5O2 [M + H]+ 566.2174, found 566.2176; purity: 95.05% (by HPLC).
4-{[4-(3-Fluorobenzoyl)piperazin-1-yl]methyl}-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]
benzamide (28k) was synthesized from compound 27k and amine
90–93 ◦C; 1H-NMR (DMSO-d6, 500 MHz)
: 10.69 (s, 1H), 8.31 (s, 1H), 8.22 (d, J = 1.3 Hz, 1H), 8.18
(s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.75 (s, 1H), 7.48–7.55 (m, 4H), 7.29–7.34 (m, 1H), 7.22–7.28 (m, 2H),
a as a light yellow solid. M.p.
δ

Molecules 2019, 24, 3543
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3.61–3.71 (m, 4H), 3.33–3.38 (m, 2H), 2.35–2.50 (m, 4H), 2.20 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ:
167.9, 166.4, 163.3, 161.3, 142.9, 141.8, 139.4, 138.4, 135.5, 133.4, 131.2, 129.4, 128.3, 123.4, 116.9, 116.8,
115.3, 114.7, 112.1, 79.7, 61.8, 53.2, 52.6, 47.6, 42.0, 14.1; HRMS (ESI+) m/z calculated for C30H28F4N5O2
[M + H]+ 566,2174, found 566.2176; purity: 94.86% (by HPLC).
4-{[4-(Pyridin-3-yl-carbonyl)piperazin-1-yl]methyl}}-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]
benzamide (28l) was synthesized from compound 27l and amine
a
as a white solid. M.p. 96–100 ◦C;
1H-NMR (DMSO-d6, 500 MHz)
δ: 10.68 (s, 1H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),
8.31 (s, 1H), 8.22 (d, J = 1.3 Hz, 1H), 8.17 (s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.85 (dt, J = 7.8, 1.9 Hz, 1H),
7.75 (s, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.49–7.52 (m, 2H), 3.66–3.72 (m, 2H), 3.64 (s, 2H), 3.37–3.42 (m, 2H),
2.50 (brs, 2H), 2.42 (brs, 2H), 2.20 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz)
δ: 167.2, 166.4, 150.9, 148.1,
142.9, 141.8, 139.4, 138.4, 135.5, 135.3, 133.4, 132.2, 129.4, 128.3, 124.0, 115.4, 114.7, 112.1, 61.7, 53.2, 52.6,
49.1, 42.1, 14.0; HRMS (ESI+) m/z calculated for C29H28F3N6O2 [M + H]+ 549.2221, found 549.2242;
purity: 98.12% (by HPLC).
3.3. Cytotoxicity Assay
Each in vitro experiment was performed at least in triplicate, and the standard deviation of
absorbance was less than 10% of the mean. For the in vitro assays, a stock solution (1% DMSO in the
appropriate buffer with the tested compound diluted under sonication) was prepared from which
several dilutions were made with the appropriate buffer.
Human cell lines were obtained from the Institute of Cytology, Russian Academy of Sciences.
The cell lines studied were maintained in RPMI 1640 medium (AppliChem, Darmstadt, Germany)
supplemented with 20% (HL-60, RPMI 1788) fetal calf serum (HyClone, Cramlington, UK), in DMEM
(A 549) and Eagle’s MEM (AppliChem) (MCF7, HeLa) with 10% fetal calf serum (HyClone). Cells were
cultivated at 37 ◦C in a humidified atmosphere of 5% CO2. Cellular sensitivities to clofarabine and its
lipid derivatives were measured with the MTT assay. Cells were plated in triplicate in 96-well plates
(105 cells/well for suspended cell cultures and 5
day (suspended cell cultures) or on the following day (monolayer cultures), compounds were added
at the appropriate dilutions. Plates were incubated under standard conditions for 48 h. Thereafter,
×
103 cells/well for monolayer cultures); on the same
10
µL of MTT (Sigma) in phosphate-buffered saline (5 mg/mL) was added. The plates were incubated
for an additional 4 h, and 150
µL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan
crystals. The optical density was read on a HALO MPR-95 Microplate Reader (Dynamica Scientific
Ltd., Australia) at 570 nm. The antiproliferative effects were determined as degrees of inhibition of
tumor cells (%).
3.4. Kinase Inhibitory Assays
The assay was performed using a Kinase Selectivity Profiling System (TK-1+ADP-Glo
and TK-2+ADP-Glo Assay, Promega), where the kinase activity was measured by quantifying the
amount of ADP produced during a kinase reaction. The assay is performed in two steps; firstly, after
the kinase reaction, an equal volume of ADP-Glo Reagent is added to terminate the kinase reaction
™ Assay
™
™
and deplete the remaining ATP. Secondly, the Kinase Detection Reagent is added to simultaneously
convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin
reaction. The luminescent signal generated is proportional to the ADP concentration produced and is
correlated with kinase activity. Our compounds were diluted with kinase buffer (5% DMSO), and 1
of the dilution was added to a 5- L reaction so that the final concentration of DMSO was 1% in all of
reactions. All of the enzymatic reactions were conducted at 23 ◦C for 60 min. The 5-
L reaction mixture
contained 1 L of the compound solution, 2 L of the enzyme solution, and 2 L of kinase substrate.
After the enzymatic reaction, 5 L of ADP-Glo Reagent (Promega, USA) was added to each reaction,
and the plate was incubated for 40 min at 23 ◦C. Then, 10
L of Kinase Detection Reagent (Promega,
µL
µ
µ
µ
µ
µ
µ
™
µ

Molecules 2019, 24, 3543
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USA) was added to each reaction, and the plate was incubated for 30 min at 23 ◦C. Luminescence
signal was measured using an Infinite M 200 plate reader (Tecan, Switzerland).
Kinase activity assays were performed in duplicate at each concentration. The luminescence
data were analyzed using the computer software, Magellan
intensities in the absence of Kinase (Lut) and in the presence of Kinase (Luc) was defined as 100%
activity (Lut Luc). Using luminescence signal (Lu) in the presence of the compound, percentage
inhibition of kinase was calculated as follows:
™. The difference between luminescence
−
% inhibition = [1 − (Lut − Lu)(Lut − Luc)] × 100%
where Lu is the luminescence intensity in the presence of the compound.
3.5. Docking
AutoDock Vina was used for docking studies [24]. Ligands from PDB complexes were extracted by
copying the HEATATM section of the PDB file. Two-dimensional structures were created using Marvin
Sketch [31]. Three-dimensional (3D) structures were generated using molconvert [32]. Conformation
generation and structure minimization were performed with Open Babel [33]. Missing protein residues
were restored with MODELLER [34]. GROMACS with the AMBER FF99SB-ILDN force field was used
for obtaining minimized receptors. Binding site coordinates of receptors were determined by the size
of the ligand in the PDB file and were scaled up by 20% in each dimension. Docking results were
limited by only one docking pose having the best docking score. Preparation of ligands and receptors
for docking was carried out in MGL Tools [35]. Visualization of docking results and the H-bond search
were done in Chimera [36]. Two-dimensional interaction maps were generated by PoseView [37].
3.6. Molecular Dynamics
Molecular dynamics was carried out using the GROMACS package with the AMBER FF99SB-ILDN
force field. All ligands were parametrized with ACPYPE [38]. The simulation workflow was as
follows: solvation, neutralizing by adding NaCl ions up to a concentration of 0.15 mol, energy
minimization, NPT and NVT equilibration steps (200 ps each), and a final 2-ns production run at 300 K.
A dodecahedron box of 1.2 nm and periodic bounding conditions were used. The Berendsen thermostat
was used for equilibration. Long electrostatic coupling was treated according to the PME method.
The g_mmpbsa tool was used for MM-PBSA binding energy calculations [26]. To perform energy
calculations, every 20th frame of the final molecular dynamics trajectory was extracted, skipping the
first 100 frames.
Experimental data of compounds, biological assays and computational methods are available in
Supplementary Materials.
4. Conclusions
In summary, a series of novel compounds, containing as a linker the 4-(aminomethyl)benzamide,
were successfully prepared and examined for their anti-tumor activities. The tertiary heterocyclic
amines 28j
,
28k
,
28l, and analog 13 demonstrated significant activity against K562 cells (IC50 of 6.9
µM,
3.6 M, 4.5
µ
µM, and 5.6 µM, respectively). The biological evaluation indicated that most of compounds
displayed multitarget profile and high inhibitory activity toward EGFR, KDR, PDGFRa, HER-4, and
InsR kinases.
The docking results showed that the studied compounds exhibited a similar binding mode to type II
inhibitors of tyrosine kinase. We established for analog 10 that the use of the 4-(aminomethyl)benzamide
as a flexible linker leads to a favorable overall geometry of the molecule, allowing one to bypass the bulk
isoleucine residue, and providing the necessary binding to the active center of the T315I-mutant Abl.
The results gleaned from this investigation have important potential applications in the development
of new anticancer drugs overcoming the clinical acquired resistance.

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Supplementary Materials: The following are available online.
Author Contributions: A.F., working on this research, designed the target compounds and performed the
chemistry experiments, docking, MD studies, as well as biologically evaluating the new compounds as a part of his
PhD thesis; E.K. supervised the research and contributed in the writing, design, synthesis, biological evaluation,
and analysis of the data; V.K., A.V., V.S., and A.M. performed parts of the synthetic experiments; O.A. and A.B.
performed the biological assay.
Funding: This work was supported by the National Academy of Sciences of Belarus within the research project
number 5.20 of State Program “Chemical Technologies and Materials”, subprogram 5 “Pharmacology and
Pharmacy”.
Acknowledgments: The authors are greatly indebted to V.F. Baranovsky and the Laboratory of Physical and
Chemical Research Methods for their contributions to the determination of HRMS, 1H-NMR, and 13C-NMR. The
authors are grateful to A.V. Yantsevich for contributions to the determination of HRMS.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).