Anticancer properties of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine derivatives; kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103055

Structure activity correlation revealed that the quinoxaline ring is a satisfactory backbone for anticancer activity and a specific functional group at position 1 and 2 can improve the activity. In this basis, besides quinoxaline, imidazoles as potential anticancer agents were used as a supplementary agents for cancer treatment. In this paper, a new series of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine derivatives were synthesized in a simple and efficient step. The products are fully characterized by 1H NMR, ¹³C NMR, FT-IR, HRMS, and CHN elemental analysis. Several starting materials with different functionalities have been used for the synthesis of the final products with high isolated yields. The biological activities of the synthesized compounds were evaluated in kinase inhibition and cytotoxic activity in several cancerous cell lines. All compounds (6) were evaluated for inhibition of the cell proliferation using 4 cancerous cell lines. Five of the more active compounds were studied for determination of IC50_%. Compounds 6(32–34) showed good activity on some of cancerous cell lines. The results showed that compound 6–32 has the highest biological activity (IC₅₀% 9.77 for K562 cell line). An IC₅₀% value of 15.84 μM was observed for 6–34. Furthermore 6–34 exhibited inhibition of ABL1 and c-Src kinases with an IC50% value of 5.25 μM and 3.94 μM respectively. Docking simulation was performed to position active synthesized compounds 6–32, 6–33, and 6–34 over the ABL1 active site in two different wild-type (DFG-in and DFG-out motif conformer) and T315I mutant to determine the probable binding orientation, conformation and mode of interaction. According to docking study, the docked location in wild type forms is similar and can be found near the P-loop region while in the case of T315I mutant form, the compounds have a distinct docked location which is close to the αC helix and activation loop. Also, it concluded the role of R₁ substituent on phenyl ring produced higher interaction energy. Additionally, the detailed inter-molecular energy and types of non-bonding interaction of these compounds over the wild-type and mutant form of ABL1.

Full text of DOI:10.1016/j.bioorg.2019.103055

Accepted Manuscript
Anticancer properties of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-
amine derivatives; kinase inhibitors
Zahra Rezaei, Mir Mehdi Didehvar, Mohammad Mahdavi, Homa Azizian,
Haleh Hamedifar, Eman H.M. Mohammed, Sayednaser Ostad, Mohsen Amini
PII:
S0045-2068(18)31297-5
https://doi.org/10.1016/j.bioorg.2019.103055
103055
DOI:
Article Number:
Reference:
YBIOO 103055
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 November 2018
27 March 2019
6 June 2019
Please cite this article as: Z. Rezaei, M. Mehdi Didehvar, M. Mahdavi, H. Azizian, H. Hamedifar, E.H.M.
Mohammed, S. Ostad, M. Amini, Anticancer properties of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-
amine derivatives; kinase inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103055
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Anticancer properties of N-alkyl-2, 4-diphenylimidazo [1, 2-a]
quinoxalin-1-amine derivatives; kinase inhibitors
Zahra Rezaeia, Mir Mehdi Didehvarb, Mohammad Mahdavic, Homa Aziziand, Haleh Hamedifare, Eman
H. M. Mohammedf, Sayednaser Ostadg, Mohsen Aminif*
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences,
14176 Tehran, Iran.
bSchool of Chemistry, University College of Science, University of Tehran, PO Box 14155-6455,
Tehran, Iran.
cEndocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute,
Tehran University of Medical Sciences, Tehran, Iran.
dDepartment of Medicinal Chemistry, School of Pharmacy-International Campus, Iran University of
Medical Sciences, Tehran, Iran.
eCinnaGen Medical Biotechnology Research Center, Alborz University of Medical Sciences, Karaj,
Iran.
fDepartment of Chemistry, Faculty of Sciences, Menoufia University, Shebin EI-Koam, Egypt.
gDepartment of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran.
fDepartment of Medicinal Chemistry, Faculty of Pharmacy and Drug Design and Development
Research Center, The Institute of Pharmaceutical Sciences (TIPS),
*Corresponding author: Mohsen Amini; moamini@tums.ac.ir
1

ABSTRACT
Structure activity correlation revealed that the quinoxaline ring is a satisfactory backbone for
anticancer activity and a specific functional group at 1st and 2nd position can improve the activity. In
this basis, besides quinoxaline, imidazoles as potential anticancer agents were used as a
supplementary agents for cancer treatment.
In this paper, a new series of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine derivatives
were synthesized in a simple and efficient step. The products are fully characterized by 1H NMR, 13
C
NMR, FT-IR, HRMS, and CHN elemental analysis. Several starting materials with different
functionalities have been used for the synthesis of the final products with high isolated yields. The
biological activities of the synthesized compounds were evaluated in kinase inhibition and cytotoxic
activity in several cancerous cell lines. All compounds (6) were evaluated for inhibition of the cell
proliferation using 4 cancerous cell lines. Five of the more active compounds were studied for
determination of IC50%. Compounds 6(32-34) showed good activity on some of cancerous cell lines.
The results showed that compound 6-32 has the highest biological activity (IC50% 9.77 for K562 cell
line). An IC50% value of 15.84 µM was observed for 6-34. Furthermore 6-34 exhibited inhibition of
ABL1 and c-Src kinases with an IC50% value of 5.25 µM and 3.94 µM respectively. Docking
simulation was performed to position active synthesized compounds 6-32, 6-33, and 6-34 over the
ABL1 active site in two different wild-type (DFG-in and DFG-out motif conformer) and T315I mutant to
determine the probable binding orientation, conformation and mode of interaction. According to
docking study, the docked location in wild type forms is similar and can be found near the P-loop
region while in the case of T315I mutant form, the compounds have a distinct docked location which
is close to the αC helix and activation loop. Also, it concluded the role of R1 substituent on phenyl ring
produced higher interaction energy. Additionally, the detailed inter-molecular energy and types of non-
bonding interaction of these compounds over the wild-type and mutant form of ABL1.
Keywords: Synthesis; Quinoxalin; Anticancer; Kinase inhibitor; Docking
1. Introduction
Cancer, as an abnormal cell growth disease, is one of the most important human diseases that is
reported as the second cause of death [1]. Between several clinical protocols for cancer treatment,
chemotherapy is interesting in aspect of molecular targeting and is a strategy in cancer treatment [2].
One way for cancer treatment is inhibition of cancerous cell growth in which Kinase inhibition is an
efficient approach due to its important role in cell growth [3]. Kinases are the largest and the most
important families of proteins that have been known as an enzyme for catalysis phosphate transfer,
are a key target for controlling the cell activities such as cell survival and death, cellular distinction,
transcription, metabolisms, and duplication processes [4]. This protein have been targeted for
different drug therapy in a wide range of diseases including inflammation and cardiovascular
diseases, CNS disorders and cancer. In cancer, the importance of kinase targeting could be referred
to recently identified over-express kinase receptors in various tumor but not in normal cells.
Therefore, it has been expected the chemical agents that have targeted kinases show low toxicity in
2

normal organs. In structural aspects, a high similarity could be observed in different human kinases
esp. in catalytic site, where is a connection part to bind with ATP. This part is including β-sheet in N-
terminal, α-helix in C-terminal and Hing region. The more kinases inhibitors act in Hing region. Based
on different substrates, protein kinases have been divided to several branches that may be
phosphorylated the phenolic group of tyrosine or react with other amino acids such as threonine and
serine. Recently, a new kinase has been identified that phosphorylates the nitrogen of histidine.
Among of them, the role of tyrosine kinases in pathogenesis of cancer has been extensively studied.
Mutation in the related gene has been distinguished in a large amounts of human tumors. Now today,
90 gene of tyrosine kinase have been discovered that most of them are related to receptor tyrosine
kinase. The position of this receptors is cell membrane and act both enzyme and receptors. Based on
the kinases domain, receptor tyrosine kinases have been categorized to several families. ErbB are
the first family of receptor tyrosine kinase that its function is related to EGFR. The decreasing activity
of EGFR causes to CNS disorders such as MS and Alzheimer’s disease and the increasing activity of
EFGR causes to cancer. This family includes four isoforms, ErbB1-4 that have been inhibited by
gefitinib, erlutinib, lapatiniband and vandetanib. All of them has 4-aminoquinazoline ring as core. The
second family including sorafenib, sunitinib, razopanib inhibit VEGF and in conclusion, act in
angiogenesis processes. Anaplastic lymphoma kinase inhibitors has been identified as third branches
of kinases including crizotinib, ceritinib, and alectinib. Src family kinases has been categorized as
non-receptor tyrosin kinases that are a family of nine diferent PTKs. In recent years development of
Src kinase inhibitors led to discovery of several drugs such as anti-cancer agents, among of them;
quinoxaline derivatives have gained considerable attention in several studies [5-8].
Quinoxaline is a heterocyclic compound containing benzene fused to a pyrazin ring. This class of
organic compounds is a core for a large number of candidate kinase inhibitor agents [9-13].
Compounds A and B (Fig. 1) have been introduced as type I1/2 and II Eph tyrosine kinase inhibitors
[16]. The development of quinoxaline core fused to pyrazole ring leaded to present compounds C
(Fig. 1) with considerable anti-cancer activity[42].
CONH2
NH2
NHCH3
N
N
N
N
N
N
N
H3C
N
N
N
R
R
H3C
A: R =
B: R =
C
D
t-Bu
t-Bu
OH
Fig. 1. Some quinoxaline derivatives having anticancer activity.
3

On the other hand imidazole derivatives have been studied vastly as anticancer agents. Some of the
imidazole derivatives showed a little or no drug resistance by cancerous cell lines that is a promising
source for cancer treatment [14-20]. Furthermore comound D (Fig.1), a derivative of imidazo[1,5-a]
quinoxaline showed a remarkable activity for IKK2 (IκB kinase) inhibition [15].
Docking methodologies asserted that binding imidazole derivatives to quinoxaline could increase its
biological activity drastically via enhancement affinity of ligand to kinase protein that has been
confirmed by experimental data [21, 22].
The most oncogenic receptor kinases have low activity in normal cells but they are hyper-activated in
malignant cells. Hence, targeting receptor kinase by inhibitors is very attractive in cancer drug
developments. Regarding the importance of the kinase inhibitors in cancer treatment, here by we
report the synthesis of a novel series of imidazo[1,2-a]quinoxalin-1-amine derivatives and evaluation
of their kinase inhibitory activities.
2. Results and Discussion
In the current work, a new simple and efficient process is suggested for producing imidazole-
quinoxaline derivatives. A glance at the previous literature revealed that there are different synthesis
route for quinoxaline derivatives synthesis. Most of the synthetic strategies are based on the reaction
of o-phenylenediamine with aldehydes, 2-nitroaniline, 1,2-diketone compounds, alkynes, and epoxide
compounds [23-31].
In this paper, N-methyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine derivatives were
synthesized in two steps. The first step consists of cyclization reaction of o-phenylenediamine with
sodium cyanide and benzaldehyde derivatives to form 3-phenylquinoxalin-2-amine intermediate (3).
The reaction was performed at 50 °C and was catalyzed with 4Ǻ molecular sieve (MS). Then, 3-
phenylquinoxalin-2-amine intermediate and isocyanate was prepared in the presence of a catalytic
amount of ammonium hydrochloride. The second step of the reaction is performed in the solvent free
conditions. Stirring the reaction mixture in high temperature can lead to higher isolated yields. The
reaction steps are presented in Scheme 1.
2.1. Chemistry
For finding the optimal reaction conditions, both of the reaction steps were studied in different
conditions. Optimization of the reactions was studied using different temperatures and reaction time in
several solvents. For the first step, the best yield was obtained in 50 °C for 5 h using DMF as solvent.
More details of the optimized results are presented in Table 1. The reaction has yielded the desired
product in all protic and aprotic solvents. Our experiences showed that increasing the reaction
temperature did not have any positive effects on the yields.
The second step of the reaction was performed in different reaction conditions using various solvents
to obtain the optimal reaction conditions. Using different solvents and in solvent free conditions
proved that, the best performance could be obtained in solvent free conditions. In addition, the
4

reaction was tested in different temperatures and reaction times. The results showed that 150 °C
temperature and 24 h reaction time are the best reaction conditions. Detailed results of the
optimization reactions are presented in Table 2.
R1
R2
CHO
CHO
NH2
NH2
N
N
NaCN
(4)
+
NH2
DMF, 50 °C
R1
(1)
(2)
(3)
R1
R3-N C (5)
N
N
N
NH4Cl
R1 = H, Cl, 3,4,5-trimethoxy
R2 = H, Cl, F, OCH3
R3 = Cyclohexyl, t-Buthyl
R3
N
(6)
H
R2
Scheme 1. Synthesis steps of N-alkyl-2,4-diphenylimidazo[1,2-a]quinoxalin-1-amine derivatives
Table 1. Optimization results for the preparation of 3-phenylquinoxalin-2-amine intermediatea
Entry
1
Solvent
Toluene
MeOH
EtOH
DMSO
DMF
Temperature (°C)
Time (h)
Yield (%) b
50
50
50
50
25
40
50
50
50
50
50
50
70
100
50
50
4
4
4
4
4
4
1
2
3
4
5
6
4
4
4
4
70
25
35
53
0
2
3
4
5
6
DMF
10
35
50
65
85
85
84
30
25
40
37
7
DMF
8
DMF
9
DMF
10
11
12
13
14
15
16
DMF
DMF
DMF
DMF
DMF
CH2Cl2
CH3Cl
5

aReaction conditions: o-phenylenediamine (5 mmol), benzaldehyde (5 mmol), sodium cyalide (6 mmol), 4Ǻ
b
molecular sieve (100 mg), solvent (10 mL); isolated yield.
Table 2. Optimization results for the preparation of 3-phenylquinoxalin-2-amine intermediatea
Entry
Solvent
Temperature
(°C)
Time (h)
Yield (%) b
1
2
DMF
150
150
150
150
150
150
150
20
24
24
24
24
24
24
24
24
24
24
24
6
19
15
8
Toluene
3
H2O
4
H2O-MeOH (1:1)
H2O- EtOH (1:1)
H2O-MeOH (2:1)
H2O- EtOH (2:1)
Solvent free
Solvent free
Solvent free
Solvent free
Solvent free
Solvent free
Solvent free
Solvent free
Solvent free
10
12
14
15
0
5
6
7
8
9
50
10
43
55
25
60
90
90
88
10
11
12
13
14
15
16
95
120
150
150
150
150
170
18
24
36
24
a
Reaction conditions: 3-phenylquinoxalin-2-amine (2 mmol), benzaldehyde (2 mmol), isocyanide (2 mmol),
ammonium chloride (0.25 mmol); b isolated yield.
Multicomponent reactions were investigated in optimal conditions. Various benzaldehydes with
electron-donating/electron-withdrawing functionalities were used in both reaction steps. As a result,
the desired products were achieved successfully. The structure of the products is shown in Table 3.
Table 3. The structure of compounds 6-(1-34) and their isolated yield.
R1
N
N
N
R3HN
R2
6

Compound
R1
R2
R3
MW
Isolated
yield (%)
6-1
6-2
4-Cl
4-Cl
3,4,5-(OMe)3
3,4-(OMe)2
4-OMe
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
t-butyl
487.43
543.06
513.04
483.01
470.98
517.03
487.00
456.97
426.95
452.99
461.39
598.70
572.66
568.67
542.64
538.65
512.61
508.62
508.62
482.58
478.60
452.56
418.54
85
90
88
91
84
89
87
82
85
86
89
88
90
91
87
90
88
89
92
89
90
86
83
4-Cl
6-3
4-Cl
6-4
4-Cl
6-5
4-Cl
4-F
6-6
4-Cl
3,4,5-(OMe)3
3,4-(OMe)2
4-OMe
6-7
4-Cl
t-butyl
6-8
4-Cl
t-butyl
6-9
4-Cl
H
t-butyl
6-10
6-11
6-12
6-13
6-14
6-15
6-16
6-17
6-18
6-19
6-20
6-21
6-22
6-23
4-Cl
H
Cyclohexyl
t-butyl
4-Cl
4-Cl
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4-(OMe)2
3,4-(OMe)2
4-OMe
Cyclohexyl
t-butyl
3,4,5-(OMe)3
3,4,5-(OMe)3
Cyclohexyl
t-butyl
3,4,5-(OMe)3
3,4,5-(OMe)3
Cyclohexyl
t-butyl
3,4,5-(OMe)3
4-OMe
3,4,5-(OMe)3
H
Cyclohexyl
Cyclohexyl
t-butyl
H
H
H
H
H
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4-(OMe)2
3,4-(OMe)2
H
Cyclohexyl
t-butyl
Cyclohexyl
6-24
6-25
H
H
H
t-butyl
392.51
436.53
84
85
4-F
Cyclohexyl
6-26
6-27
H
H
4-F
t-butyl
410.50
448.57
89
86
4-OMe
Cyclohexyl
6-28
6-29
H
H
4-OMe
4-Cl
t-butyl
422.53
452.99
84
85
Cyclohexyl
6-30
H
4-Cl
t-butyl
426.95
82
7

6-31
6-32
6-33
6-34
2-OH-3-OMe
2-OH-3-OMe
2-OH-3-OMe
2-OH-3-OMe
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4-(OMe)2
3,4-(OMe)2
Cyclohexyl
t-butyl
554.65
528.61
524.62
498.58
70
68
76
72
Cyclohexyl
t-butyl
2.2. Biological evaluation
The cytotoxic effect of the products was studied on three cancerous cell lines; HEK-293, MB-468 and
CCRF for 24 h and 72 h and normal cell lineas at a concentration of 50 µM. The results were
presented in table 4. The activity of the compounds was compared with Doxorubicin and Dasatinib (5
µM) as reference drugs. Compounds 6-32, 6-33, and 6-34 were the most active compounds in three
cancerous cell lines after 24 and 72 h. In addition, compounds 6-6 and 6-7 significantly inhibit the cell
proliferation of HEK-293 cells after 72 h incubation. For the three more active compounds, the
cytotoxcicity was studied on normal cell line, NIH-3T3, at 50 µM.
Table 4. The cytotoxicity of more active synthesized compounds in three cancerous cell lines.
Cell line
NIH-3T3
MB-468, MTS
(% Viability)
CCRF, MTS
(% Viability)
HEK-293, MTS
(% Viability)
(% Viability)
Comounds
Entry
24
72
24
72
24
72
24
72
Cell
Water
DMSO
6-5
100
98
31
-
100
97
27
-
100
107
37
93
120
In
100
121
21
12
73
In
100
103
21
In
100
96
31
In
100
101
12
In
100
96
19
In
6-6
-
-
In
In
103
106
In
12
12
In
6-7
-
-
In
In
6-12
-
-
In
In
95
In
74
In
6-13
-
-
80
93
62
95
97
In
21
21
72
66
69
In
In
In
6-14
-
-
In
In
In
In
6-16
-
-
In
In
In
In
6-20
-
-
In
In
In
In
6-22
-
-
In
In
In
In
6-32
73
74
98
79
91
55
61
77
27
66
14
16
30
89
85
31
29
48
71
75
43
27
31
66
95
31
22
18
20
126
6-33
60
66
78
104
60
21
29
86
6-34
DOX (5µM)
DAS (5 µM)
8

For cancerous cell lines, In the cases of more than 75% viability the results did not present or showed as “In”
(inactive) in table 4.
Cytotoxicity cell-based assays were conducted on human B leukemia cell lines (BV- 173, Creative
Bioarray # CSC-C0203), bone marrow lymphoma (K652, ATTC # CCL-243), breast epithelial (MCF
10A, ATCC # CRL-10317), and mammary gland/breast (MDA-MB-468, ATCC # HTB-132) cell lines.
BV-173 cell line is an example of Philadelphia chromosome (Ph1) + chronic myeloid leukemia derived
from the peripheral blood of a 45-year-old man with CML in blast crisis in 1980. K562 is an example of
bone marrow leukemia cells. As table 5, compounds 6-32, 6-33, and 6-34 exhibited anti-proliferative
activities in the concentration range tested with IC50 values of 9.77 to 15.84 µM against K-562 cells.
Other compounds did not show any significant anti-proliferative activities at the lower than 25 µM. The
effect of concentrations on the inhibitory activity of K-562 was presented in Figure 2. Values are
derives from 2 indipendent experiments, the standard deviation were also note.
Table 5. IC50 of the most active compounds in four cancerous cell lines.
Compounds IC50 (µM)
Cell Viability
Cell Line
BV-173
6-5
>25
>25
>25
>25
>25
6-14
>25
>25
>25
>25
>25
6-32
>25
6-33
6-34
Staurosporine
0.37 ± 0.02
>25
>25
K-562
9.77 ± 0.91
>25
12.02 ± 1.45 15.84 ± 1.15
0.047 ± 0.008
0.056 ± 0.004
0.15 ± 0.04
MCF 10A
MDA-MB-468
NIH-3T3
>25
>25
>25
>25
>25
>25
>25
>25
0.09 ± 0.003
9

K562 (B)
K562 (A)
150
100
50
150
100
50
0
0
-4.60 -4.70 -5.00 -5.30 -6.00 -6.70 -7.40
-4.60 -4.70 -5.00 -5.30 -6.00 -6.70 -7.40
Log[6-32] (M)
EC50%=9.77 µM
Log [6-33] (M)
EC50%=12.02
K562 (C)
150
100
50
0
-4.60 -4.70 -5.00 -5.30 -6.00 -6.70 -7.40
Log[6-34]
EC50%=15.84
Fig. 2. Inhibitory activity of compounds (A) 6-32; (B) 6-33; and (C) 6-34 against K-562
The inhibitory property of the most cytotoxic compounds was studied as kinase inhibitors. The
inhibitory activity of compounds 6-5, 6-14, 6-32, 6-33, and 6-34 were studied on three kinases: ABL1,
c-Src and LCK. The results are presented in Table 6. Compounds 6-5, 6-14, 6-32, and 6-33 did not
show any significant kinase inhibition in the lower than 125 µM. Compound 6-34 showed inhibition
against ABL1 and c-Src with IC50 values of about 5.25 and 3.93 µM, respectively. Compound 6-34 did
not show inhibition of LCK at a concentration of 125 µM, but inhibited the proliferation by
approximately 32% inhibition at 125 µM.
Table 6. Inhibitory activity of the products
Compound IC50 (μM)
Kinases
ABL1
c-Src
LCK
6-5
6-14
>125
>125
>125
6-32
>125
>125
>125
6-33
>125
>125
>125
6-34
Staurosporine
0.93 ± 0.01
>125
>125
>125
5.25 ± 0.08
3.94 ± 0.05
>125
0.038 ± .0045
0.036 ± 0.001
10

2.3. Molecular Docking
2.3.1. Evaluation of docking procedure
Before docking, it is necessary to verify the reliability of the docking study. The three native ligands
were extracted from the X-ray structures of ABL1 (PDB entry: 2gqg, 4zoq, 3qrj) and redocked into
their corresponding protein active site pockets according to the above docking protocol. The
superimposed lowest energy poses for redocking results over the co-crystalized ligands with their
corresponding interactions were shown in Figure 3(a-c). The RMSD of the re-docked and co-
crystalized ligands over related 2gqg, 4zoq, and 3qrj were 1.6 Å, 1.1 Å, and 0.6 Å, respectively, which
are considered as successfully docked procedure. As a result, the validity of docked parameters is
reasonable in order to predict the related co-crystalized structures. Table 6 indicates energy data and
types of interaction of each ligand- protein complex.
Fig. 3. Superimposed representation of redocking lowest energy poses into the co-crystalized ligands
dasatinib, VX-680, and DCC-2036 over 2gqg (a), 4zoq (b), 3qrj (c), respectively. Co-crystalized and
redock ligands were shown in green and gray carbon atom color. Dash line in green, orange, yellow,
pink, purple and pale pink are corresponding to H-bond, electrostatic, pi-sulfur, pi-pi and pi-alkyl
interaction, respectively.
2.3.2. Binding interaction and poses analysis of the active compounds
For study the molecular docking, the most active compounds (6-14, and 6-(32-34)) were chosen.
Furthermore, compounds 6-7 and 6-24 were selected for comparing the substitution effect on R1
position. In the predicted binding mode obtained by docking studies, all the studied compounds were
successfully docked into the corresponding binding pocket of ABL1. It can be found difference in
docked location and orientation of compounds over the active site of DFG-in, DFG-out and mutant
11

T315I forms of ABL1 (figure 4, table 7). The docked location in wild type forms (DFG-in and DFG-out)
is similar and can be found near the P-loop region of kinase active site (Figure 4a, b) while in the case
of T315I gatekeeper mutant form, the compounds have a distinct docked location which is close to
the αC helix and activation loop (figure 4c). The different binding location in ABL1 mutant conformer
could be a result of the long-range conformational impact of T315I mutation.
The docking results of synthesized compounds including; detailed intermolecular energy and the
residues involving in the types of nonbonding interaction over DFG-in, DFG-out and mutant T315I
form of ABL1 were presented in table 8, 9, and 10, respectively. It can be observed that active
compounds; 6-(32-34) with 2-hydroxy-3-methoxy group on phenyl ring (R1) have higher interaction
energy over ABL1 than compound 6-24 which does not have R1 group at mentioned position. As a
result, the presence of R1 substitution affects the ABL1 tyrosine kinase affinity. The 2-hydroxyl-3-
methoxy substituention has the ability to produce essential H-bond and polar interaction with
surrounding residues. Table 8 indicates that compounds 6-(32-34) have higher interaction energy
rather than dasatinib over DFG-in conformer of ABL1 which is quite compatible with the experimental
results of higher viability percentage of applied cell line after treating of dasatinib rather than
compounds 6-(32-34) over hek-293, MB-468, and CCRF cell line (Table 4). Furthermore, tables 8-10
show the interaction energy of active synthesized compounds 6-(32-34) which is higher than the
binding energy of staurosporin (-11.73, -13.12, -12.06, -9.41 kcalmol-1, respectively) over DFG-in
(2gqg) and mutant form (3qrj) (-13.01, -13.67, -12.49, -7.76 kcalmol-1, respectively), while the
electrostatic interaction energy of staurosporin is significantly higher than mentioned compounds (the
electrostatic energy for 6-(32-34), and staurosporin over 2gqg are -0.18, -0.07, -0.25, and -1.62 kcal
mol-1, respectively and over 3qrj are -0.06, -0.07, -0.07, and -0.77 kcal mol-1, respectively). This
finding can be used to propose the outstanding role of electrostatic and polar interaction in tyrosine
kinase binding affinity in active and mutant conformers of ABL1.
Table 7. Re-docking results for wild type (DFG-in and DFG-out) and mutant T315I ABL1
Ligand
Pdb
id
Estimated free binding Hydrogen bond
Electrostatic interaction
energy
(Donor-Acceptor)
(Kcalmol-1)
H-bond+ Vw
Electrostat
ic
Dasatinib 2gqg
-9.92
N9-Thr315:OG1
-
-
-10.65
-12.22
-16.31
-1.35
-0.81
-0.06
VX-680
4zoq
3qrj
-10.72
-14.29
MET318:N -N20
H26 - Met318:O
DCC-
2036
Met318:HN - N23
Asp381:HN - O63
N56 - Glu286:OE2
N60 - Glu286:OE2
N74 - Met318:O
Glu286:OE1 – quinazoline
ring
Glu286:OE2 – quinazoline
ring
Asp381:OD2 – quinazoline
ring
Asp381:OD2 – pyrazol ring
12

Fig. 4. Difference in docking location of synthesized compounds over DFG-in (a), DFG-out (b), and
mutant T315I (c) active sites of ABL1.
Table 8. Docking results of synthesized compounds over wild type (DFG-in) ABL1 (2gqg)
Ligand
Final Intermolecular Energy
(Kcal mol-1)
Hydrogen bond
(Donor-Acceptor)
Electrostatic
Hydrophobic
H-bond+ Vw Electrostatic
7
-11.76
Met318:HN-O34
Asn322:HD22-N15
-
-
Leu370, Tyr253,
Leu248, VAL256
ALA269
-11.77
-11.92
+0.01
-0.17
14
-12.09
LEU248, TYR253
PHE317, ALA269
LEU370
Gln252:HN - O31
Gln252:HN - O39
Tyr253:HN - O39
24
32
-11.21
-11.73
-
LEU370, TYR253,
PHE317, LEU248,
VAL256, ALA269
N15-Leu248:O
-11.28
-11.55
+0.07
-0.18
Asp325:OD2-
imidazol
TYR253, LEU248
LEU370, ARG367,
GLY321
Gln252:HN-O28
Tyr:HN-O28
33
34
-13.12
-12.06
Gln252:HN-O34
N15-Leu248:O
-
-
LEU248, TYR253,
LEU370, VAL256,
ALA269
-13.05
-11.81
-0.07
-0.25
Gln252:HN-O32
Tyr253:HN-O32
Met318:HN-O36
H2-N13
LEU248, VAL256
ALA269, LEU370
(intramolecular)
Staurosporin
-9.41
Asp325:OD2
Glu329:OD2
-methyl amine
TYR253, TYR320,
GLY321, LEU248
-7.79
-1.62
13

Table 9. Docking results of synthesized compounds over wild type (DFG-out) ABL1 (4zog)
Ligand
Final Intermolecular Energy
(Kcal mol-1)
Hydrogen bond
(Donor-Acceptor)
Electrostatic
hydrophobic
H-bond+ Vw Electrostatic
7
-11.23
LYS271:NZ-
LYS271:NZ-
TYR253, ALA269,
dimethoxy phenyl ring dimethoxy phenyl ring LYS271, VAL256
-11.22
-12.62
-0.02
-0.2
14
-12.82
LYS271:NZ-
dimethoxy phenyl ring
LYS271, ILE313,
VAL256, TYR253,
PHE382
ASN322:N -O37
GLY383:N-O41
24
32
33
-11.16
-12.31
-13.26
GLY383:HN-O31
GLY383:HN-O31
GLY383:HN-O31
LYS271:NZ-
dimethoxy phenyl ring VAL256, ALA269,
LYS271
LEU370, TYR253,
-11.13
-12.25
-0.03
-0.07
LYS271:NZ-
dimethoxy phenyl ring VAL256, ALA269,
LYS271
LEU370, TYR253,
LYS271:NZ-
LEU370, TYR253,
dimethoxy phenyl ring VAL256, ALA269,
LYS271, ILE313,
-13.19
-0.07
MET318, PHE317
34
-12.08
-10.54
GLY383:HN-O31
LYS271:NZ-
dimethoxy phenyl ring VAL256, ALA269,
LYS271, ALA380
LEU370, TYR253,
-12.03
-10.59
-0.05
Staurosporin
NH1 (from lactam
ring)- GLU316:O
C9-THR315:OG1
-
VAL299, LEU370,
ALA380, ALA269,
VAL256, TYR253,
LEU248,
+0.05
14

Table 10. Docking results of synthesized compounds over mutant T315I ABL1 (3qrj)
Ligand
7
Final Intermolecular Energy
(Kcal mol-1)
H-bond+ Vw Electrostatic
Hydrogen
bond
Electrostatic
Hydrophobic
-11.85
-
HIS361:NE2-quinoxaline ring
ASP381:OD2-imidazol ring
MET290, VAL299
HIS361, LYS285
LEU298, MET290
ALA380, VAL289
MET290, ILE315
-11.80
-11.25
-0.06
-0.07
14
24
32
33
-11.30
-11.53
-13.01
-13.67
-
-
-
-
Asp381:OD1-trimethoxy phenyl
ring
Asp381:OD2-imidazol ring
MET290, ILE293
LEU298, VAL299
ILE315, ALA380
Glu286:OE1-phenyl ring
Asp381:OD2-imidazolo ring
MET290, ILE293
LEU298, VAL299
ILE315, ALA380
-11.50
-12.96
-0.02
-0.06
GLU286:OE1-phenyl ring
ASP381:OD2-imidazolo ring
MET290, ILE293
LEU298, VAL299
ILE315, ALA380
GLU286:OE1-phenyl ring
ASP381:OD2-imidazolo ring
MET290, ILE293
LEU298, VAL299
ILE315, ALA380,
HIS361, PHE382
-13.61
-12.42
--6.96
-0.07
-0.07
-0.77
34
-12.49
-7.76
-
-
GLU286:OE1-phenyl ring
ASP381:OD2-imidazolo ring
ASP381, PHE382
MET290, ILE293
LEU298, VAL299
ILE315, ALA380
Staurosporin
-
LEU248
2.3.3. Binding pose comparison of compound 6-34 and co-crystalized inhibitors over DFG-in, DFG
out, and mutant variant of ABL1
Figure 5a shows binding interactions of compound 6-34 and dasatinib over ABL1 DFG-in. Compound
6-34 mainly occupies the adenine pocket by the two phenyl rings containing R1 and R2 substitutions,
while the quinoxaline and trimethyl amine (R3) group extend beyond the interlobe connector near the
mouth of the active site and are solvent exposed. A total of three bonds is formed between
compound 6-34 and the active ABL1. Two hydrogen bonds are formed to the Gln252 from P-loop
ATP binding pocket of kinase through the 2-hydroxy-3-methoxy phenyl moiety which is not observed
in ABL1:dasatinib. The other hydrogen bonds are made between a methoxy group of 3,4-dimethoxy
phenyl and the backbone NH of Met318 in the loop linking N-lobe and C-lobe together. The remaining
15

binding interactions are electrostatic interaction with Asp325 and Hydrophobic interaction of π-π T-
shape, π-sigma, and π-alkyl with Tyr253, Leu370, and Leu348, respectively.
Compound 6-34 and dasatinib share different functionalities that bind in the pocket of the DFG-in
motif. The main difference between compound 6-34 and dasatinib, therefore, is in their H-bonding
pattern. Dasatinib makes two H-bonds with Thr315 and Tyr320 which are not obsevable for
compound 6-34.
Figure 5b shows binding interactions of compound 6-34 and VX-280 over ABL1 DFG-out. Compound
6-34 with its dimethoxy phenyl part produced H-bonding interaction with the backbone NH of Gly383
from the DFG motif in the “out” conformation. Furthermore, the corresponding phenyl group stabilized
at specific pocket by side chain of Lys271 via electrostatic anion-π interaction.
Also, Ala380 from the activation loop, Tyr353 and Val256 from the P-loop participate in hydrophobic
interaction by π-alkyl, π-π stack and, π-alkyl interactions with compound 6-34. Comparison of the
ABL1: compound 6-34 and ABL1:VX-280 reveals that compound 6-34 can binds deeply into the
active site and it occupies the selectivity pocket behind the gatekeeper residue Thr315 and the
hydrophobic pocket induced by the DFG-out motif with the quinoxaline and 2-hydroxy-3-methoxy
moiety, while VX-268 could not reach to that specific pocket with its piperazine and pieridine rings are
near the mouth of the active site and are solvent exposed.
Figure 5c,d represent binding interactions of compound 6-34 and DCC-2036 over ABL1 mutant
variant. Compound 6-34 positioned in the kinase active site close to the αC helix motif in which the 2-
hydroxy-3-methoxy phenyl ring and trimethyl amine substituents are solvent assailable parts with
mentioned phenyl interacts Glu286 via π-anion electrostatic attraction. Additionally, the 3,4-dimethoxy
moiety put deeply into the active site and produced H-bond interaction with Lys271 through its
methoxy substitution. Also, the corresponding phenyl group and the quinoxaline ring stabilized
between αC helix and activation loop with Phe382 from DFG motif (π-π stack) and Ala380, Ile315,
Met290, Val299, and Leu298 via π-alkyl interaction. Similarly, DCC-2036 is in the same location as
compound 6-34 and interacts Lys271 and Glu286 via H-bonding (figure 5d).
16

Fig. 5. Structural comparison of compound 6-34 and ABL1 inhibitor co-crystalized ligand. (a) Overlay
of compound 6-34 (gray) with dasatinib (green). (b) Overlay of compound 6-34 with VX-680 (green).
(c) Overlay of compound 6-34 (gay) with DCC-2036 (green). Hydrogen bonds within ABL1: compound
6-34 are depicted as dashed yellow lines, whereas those in ABL1:dasatinib, ABL1:VX-680, and
ABL1:DCC-2036 are in black.
2.4 Molecular dynamic simulation
In order to carry out the movement of particles (atoms and molecules), molecular dynamics (MD)
simulation has been applied over the period of 40 ns. The total energy of the top ranked complex
accomplished by molecular dynamics simulation was discovered to be -95214.581 and -83268.453
kcal/mol for the wild type and T315I mutant variant, respectively.
17

Fig. 6. Backbone RMSD representation of ABL1 for (a) wild type and (b) T315I mutant during 40 ns of
simulation.
Root mean square deviation (RMSD) is used to reveal the stability of protein structure by measuring the
variation between the protein’s backbones from its original to final conformation over 40 ns which defined as
RMSD. The RMSD simulation showed that the wild type maintained an overall stability throughout the last 15
ns of simulation with higher fluctuation, stabilizing at an average of 2.7 Å, while the T315I mutant variant
displayed lower fluctuations over the same period and its equilibration was not obtained during that time (Figure
6).
3
2.5
2
1.5
Backbone-3qrj
Backbone-2qgq
1
0.5
0
233 253 273 293 313 333 353 373 393 413 433 453 473 493
Residue Number
Fig. 7. Backbone RMSF representation of ABL1 for wild type and T315I mutant system during 40 ns of
simulation.
18

the RMSF plot indicates the residues of both system fluctuate in low level of flexibility with the maximum
quantity at 2.5 Å. Also, it depictes both variants have the same flexibility pattern at N-loop residues, while the
wild-type shows higher flexibility at C-loop region specially at residue number 380 to 413 and 440 to 455 as
compared to the mutant variant which are related to the activation loop region (Figure 7).
Fig. 8. Ligand positional RMSD of compound 6-34 over (a) wild type and (b) T315I mutant of ABL1 during 40
ns of simulation.
In order to investigate binding stability of compound 6-34 in the active site of wild type and mutant variant,
ligand positional RMSD were generated and investigated. Compound 6-34 showed slightly more and continues
fluctuations in the size of 0.6– 0.8 Å (Figure 8a) for wild type and 0.6–1 Å for the mutant variant BCR-ABL
complex (Figure 8b). As shown in figure 8, the positional RMSD value of compound 6-34 in both types of
ABL1 during the MD run was less than 2.0 Å, so the ligand was judged to be stable and this MD run regarded
as a qualified run [32].
Comparing the structure of wild-type and the mutant variant ABL1 complexed with compound 6-34
after MD simulation revealed two important conformational differences. The first change has been
detected in the N-lobe which shows upward shifting of αC helix motif in mutant rather than wild-type
(Figure 10a,b). The other difference of the mutant variant structural feature is in the C-lobe and is
switching off Phe382 away from αC helix entrance gate and shifting toward the ATP binding loop
(Figure 9c, d). Consequently, the orientation of the proximal part of activation loop changed relative to
the αC helix. These conformational changes provided an open gate at the region between αC helix
and the activation loop, so compound 6-34 entered and stabilized from the active site open gate close
to the αC helix sides of the kinase.
19

Fig. 9. Representation of wild type and mutant variant structure of ABL1 after 40 ns MD simulation,
ABL1 wild-type (cyan) (a) and ABL1 T315I mutant (yellow) (b), 90 degrees left rotated of ABL1 active
site of wild type (c) and mutant variant (d). αC helix and activation loop are in red and orange,
respectively.
Fig. 10. Interaction frequencies of the non-bonding interactions between compound 6-34 and (a) wild
type ABL1 and (b) T315I mutant variant during 40 ns. H-bond, hydrophobic, and water bridge
interactions are in green, purple, and blue color, respectively.
20

Figure 10 shows the bar chart which gives the fraction of the simulation time that compound 6-34 is in
contact with various protein residues throughout the 40 ns MD trajectory of wild type and mutant
variant. By comparing the interaction strength which is quantified by the frequency of occurrences in the
trajectory it can revealed that during the course simulation, the most sustained interaction between 6-34 and
the wild-type is hydrogen bond with Tyr253 and Asn322 (85% and 80% of the time, respectively), while these
interactions is not seen with the mutant variant. As depicted in figure 12a and 12b however, compound 6-34 has
more solvent exposure and hydrophobic interaction over mutant form than wild type. Notably, Although Thr315
provides significant water molecule bridges interaction over the wild type ABL1, the Ile mutation at the
mentioned site contacts by less strength hydrophobic interaction with compound 6-34.
3. Materials and Methods
3.1. Characterization
IR spectra were taken using Nicolet FT-IR Magna 550 spectrographs. The samples were prepared for
analysis by spectroscopic grade potassium bromide. Progress of the reactions was followed by TLC
using silica gel SIL G/UV 254 plates. NMR spectra were run on a Bruker 500 MHz instrument
respectively in CDCl3 and DMSO-d6 with tetramethyl silane (TMS) as internal standard. Chemical
shifts are given in δ scale in parts per million (ppm). Singlet (s), doublet (d), triplet (t), doublet of
doublet (dd) and multiplet (m) are reported. Melting points were taken on a Kofler hot stage
apparatus.
3.1.1. General procedure for the synthesis of N-alkyl-2, 4-diphenylimidazo [1, 2-a] quinoxalin-1-amine
To a round bottom flask containing DMF (10 mL), o-phenylenediamine (5 mmol), related
benzaldehydes (5 mmol) and 4Ǻ molecular sieve (100 mg) were added. The reaction mixture stirred
at 50 °C for 4 h. and then, sodium cyanide (6 mmol) was added to the reaction mixture and stirred for
more 12 h. The completion of the reaction was monitored by TLC. The reaction mixture was poured
on ice water and allowed to precipitate. The precipitate was filtered and purified by column
chromatography (hexane/ethyl acetate, 90:10 v/v).
To a mixture of 3-phenylquinoxalin-2-amine (compound 3 in scheme 1) (2 mmol), related
benzaldehydes (2 mmol) and isocyanide (2 mmol) was added ammonium chloride (0.25 mmol). The
mixture was stirred at 150 °C for 24 h. The reaction performance was monitored by TLC. After the
reaction completion, the product was purified by column chromatography (hexane/ethyl acetate, 90:10
v/v).
2, 4-bis (4-chlorophenyl)-N-cyclohexylimidazo [1, 2-a] quinoxalin-1-amine (6-1)
M.p. = 202-203 °C ; IR (KBr): 3345, 2933, 1588 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.11-1.84 (10
H, m), 2.99 (1H, m), 3.59 (1H, d, J = 7.0 Hz), 7.47 (2H, d, J = 8.0 Hz), 7.53 (2H, d, J = 7.5 Hz), 7.58
(2H, t, J = 5.0 Hz), 7.86 (2H, d, J = 7.5 Hz), 8.12 (1H, d, J = 7.5 Hz), 8.78 (2H, d, J = 8.0 Hz), 9.14
(1H, d, J = 7.5 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.89, 25.58, 33.52, 57.34, 116.13, 126.13,
21

127.40, 128.43, 128.95, 130.38, 130.41, 130.92, 131.36, 131.53, 132.59, 133.69, 134.70, 136.49,
136.68, 137.19, 148.65, 159.33 ppm. Anal. calcd. for C28H24Cl2N4: C, 69.00; H, 4.96; N, 11.49; Found:
C, 69.11; H, 5.04; N, 11.57. HRMS: m/z [M+H]+calcd for C28H24Cl2N4: 487.1456, found: 487.0128.
4-(4-chlorophenyl)-N-cyclohexyl-2-(3, 4, 5-trimethoxyphenyl)imidazo[1,2-a]quinoxalin-1-amine (6-2)
1
M.p. = 171-172 °C ; IR (KBr): 3349, 2931, 1584 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.13-1.84
(10H, m), 3.04 (1H, m), 3.65 (1H, d, J = 6.5 Hz), 3.93-3.96 (9H, s), 7.12 (2H, s), 7.51 (2H, d, J = 8.0
Hz), 7.58 (2H, m), 8.12 (1H, d, J = 7.5 Hz), 8.79 (2H, d, J = 8.0 Hz), 9.13 (1H, d, J = 7.5 Hz) ppm ; 13
C
NMR (CDCl3, 125 MHz): δ = 24.81, 25.61, 33.51, 56.41, 57.16, 61.00, 105.47, 116.16, 126.05,
127.29, 128.36, 128.71, 129.57, 130.25, 130.36,131.29, 131.39, 131.57, 133.58, 134.78, 136.40,
136.68, 148.52, 153.58 ppm. Anal. calcd. for C31H31ClN4O3: C, 68.56; H, 5.75; N, 10.32; Found: C,
68.61; H, 5.84; N, 10.38. HRMS: m/z [M+H]+calcd for C31H31ClN4O3: 543.2163, found: 543.8993.
4-(4-chlorophenyl)-N-cyclohexyl-2-(3, 4-dimethoxyphenyl) imidazo [1, 2-a] quinoxalin-1-amine (6-3)
1
M.p. = 200-202 °C ; IR (KBr): 3348, 2937, 1610 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.10-1.81
(10H, m), 3.00 (1H, m), 3.65 (1H, d, J = 7.0 Hz), 3.95-3.99 (6H, s), 6.99 (1H, d, J = 7.0 Hz), 7.37 (1H,
s), 7.50 (2H, d, J = 8.0 Hz), 7.56 (2H, m), 8.11 (1H, d, J = 7.5 Hz), 8.29 (1H, d, J = 7.0 Hz), 8.80 (2H,
d, J = 8.0 Hz), 9.15 (1H, d, J = 7.5 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.59, 24.84, 33.45,
55.94, 56.06, 57.14, 111.28, 111.43, 116.19, 120.16, 125.91, 126.88, 127.17, 128.32, 128.72, 130.27,
131.08, 131.35, 131.56, 133.53, 134.82, 136.30, 136.62, 138.14, 148.38, 148.95 ppm. Anal. calcd. for
C30H29ClN4O2: C, 70.23; H, 5.70; N, 10.92; Found: C, 70.33; H, 5.81; N, 11.03. HRMS: m/z
[M+H]+calcd for C30H29ClN4O2: 513.2057, found: 513.9955.
4-(4-chlorophenyl)-N-cyclohexyl-2-(4-methoxyphenyl) imidazo [1, 2-a] quinoxalin-1-amine (6-4)
1
M.p. = 179-179 °C ; IR (KBr): 3333, 2936, 1609 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.08-1.83
(10H, m), 2.98 (1H, m), 3.63 (1H, d, J = 7.5 Hz), 3.88 (3H, s), 7.03 (2H, d, J = 8.0 Hz), 7.52 (2H, d, J =
8.5 Hz), 7.56 (2H, t, J = 5.0 Hz), 7.81 (2H, d, J = 8.0 Hz), 8.11 (1H, d, J = 8.0 Hz), 8.80 (2H, d, J = 8.5
Hz), 9.17 (1H, d, J = 8.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.87, 25.60, 33.44, 55.30, 57.20,
114.19, 116.22, 125.84, 126.58, 127.13, 128.34, 128.79, 129.06, 130.23, 130.95, 131.56, 133.59,
134.86, 136.29, 136.63, 138.15, 148.40, 159.33 ppm. Anal. calcd. for C29H27ClN4O: C, 72.11; H, 5.63;
N, 11.60.; Found: C, 72.19; H, 5.71; N, 11.69. HRMS: m/z [M+H]+calcd for C29H27ClN4O: 483.1951,
found: 483.1412.
4-(4-chlorophenyl)-N-cyclohexyl-2-(4-fluorophenyl) imidazo [1, 2-a] qyinoxalin-1-amine (6-5)
M.p. = 197-198 ; IR (KBr): 3336, 2925, 1595 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.10-1.82 (10H,
m), 2.97 (1H, m), 3.58 (1H, d, J = 7.5 Hz), 7.18 (2H, t, 3JH-H = 8.0 Hz, 3JH-F = 8.5 Hz), 7.52 (2H, d, J =
3
4
8.0 Hz), 7.55-7.58 (2H, m), 7.87 (2H, t, JH-H = 8.0 Hz, JH-F = 6.5 Hz), 8.11 (1H, d, J = 8.0 Hz), 8.77
(2H, d, J = 8.0 Hz), 9.13 (1H, d, J = 8.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.85, 25.57,
33.46, 57.24, 115.63, 115.80, 116.11, 126.04, 127.33, 128.40, 128.74, 129.53, 130.20, 131.19,
22

131.44 (2JC-F = 22.5 Hz), 133.73, 134.73, 136.43, 136.64, 137.44, 148.56, 162.45 (1JC-F = 250 Hz)
ppm. Anal. calcd. for C28H24ClFN4: C, 71.41; H, 5.14; N, 11.90; Found: C, 71.51; H, 5.24; N, 12.01.
HRMS: m/z [M+H]+calcd for C28H24ClFN4: 471.1752, found: 470.7887.
N-(tert-butyl)-4-(4-chlorophenyl)-2-(3, 4, 5-trimethoxyphenyl) imidazo [1, 2-a] quinoxalin-1-amine (6-6)
M.p. = 174-175 °C ; IR (KBr): 3352, 2974, 1585 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.02 (9H, s),
3.46 (1H, s), 3.91-3.96 (9H, s), 7.03 (2H, s), 7.52 (2H, d, J = 8.0 Hz), 7.54-7.57 (2H, m), 8.11 (1H, d, J
= 4.5 Hz), 8.81 (2H, d, J = 8.0 Hz), 9.60 (1H, d, J = 4.5 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ =
29.73, 56.50, 57.71, 61.03, 106.38, 117.15, 126.10, 126.81, 128.39, 129.15, 130.28, 130.37, 131.43,
131.60, 133.98, 134.73, 136.41, 136.71, 138.27, 141.78, 148.46, 153.54 ppm. Anal. calcd. for
C29H29ClN4O3: C, 67.37; H, 5.65; N, 10.84; Found: C, 67.43; H, 5.74; N, 10.92. HRMS: m/z
[M+H]+calcd for C29H29ClN4O3: 517.2006, found: 517.1574.
N-(tert-butyl)-4-(4-chlorophenyl)-2-(3, 4-dimethoxyphenyl) imidazo [1, 2-a] quinoxalin-1-amine (6-7)
M.p. = 239-241 °C ; IR (KBr): 3332, 2968, 1611 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 0.99 (9H, s),
3.48 (1H, s), 3.94-3.99 (6H, s), 6.95 (1H, d, J = 8.0 Hz), 7.29 (1H, d, J = 8.0 Hz), 7.41 (1H, s), 7.51-
7.51 (4H, m), 8.10 (1H, d, J = 7.5 Hz), 8.81 (1H, d, J = 8.0 Hz), 9.61 (1H, d, J = 7.5 Hz) ppm ; 13C
NMR (CDCl3, 125 MHz): δ = 29.69, 55.94, 56.15, 57.61, 111.14, 112.16, 117.17, 121.09, 125.95,
126.69, 127.64, 128.35, 129.19, 130.05, 130.19, 130.31, 131.43, 131.59, 133.95, 134.79, 136.34,
136.68, 141.66, 148.34 ppm. Anal. calcd. for C28H27ClN4O2: C, 69.06; H, 5.59; N, 11.50; Found: C,
69.16; H, 5.64; N, 11.58. HRMS: m/z [M+H]+calcd for C28H27ClN4O2: 487.1901, found: 486.8207.
N-(tert-butyl)-4-(4-chlorophenyl)-2-(4-methoxyphenyl) imidazo [1, 2-a] quinoxalin-1-amine (6-8)
M.p. = 176-178 °C ; IR (KBr): 3348, 2968, 1626 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.00 (9H, s),
3.46 (1H, s), 3.88 (3H, s), 7.03 (2H, d, J = 8.0 Hz), 7.52-7.55 (4H, m), 7.75 (2H, d, J = 8.0 Hz), 8.11
(1H, d, J = 7.5 Hz), 8.81 (2H, d, J = 8.0 Hz), 9.64 (1H, d, J = 7.5 Hz) ppm ; 13C NMR (CDCl3, 125
MHz): δ = 27.74, 29.73, 57.33, 117.23, 126.06, 126.73, 127.95, 128.44, 128.61, 129.35, 130.27,
130.31, 130.42, 131.44, 131.64, 134.14, 134.76, 134.82, 136.46, 136.78, 141.73 ppm. Anal. calcd. for
C27H25ClN4O: C, 70.97; H, 5.51; N, 12.26; Found: C, 71.11; H, 5.64; N, 12.38. HRMS: m/z
[M+H]+calcd for C27H25ClN4O2: 457.1795, found: 456.1406.
N-(tert-butyl)-4-(4-chlorophenyl)-2-phenylimidazo [1, 2-a] quinoxalin-1-amine (6-9)
M.p. = 212-213 °C ; IR (KBr): 3351, 2955, 1587 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 0.99 (9H, s),
3.54 (1H, s), 7.38 (1H, t, J = 7 Hz), 7.47-7.56 (6H, m), 7.82 (2H, d, J = 7.0 Hz), 8.11 (1H, d, J = 5.0
Hz), 8.82 (2H, d, J = 8.0 Hz), 9.64 (1H, d, J = 5.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 29.66,
57.74, 117.27, 126.03, 126.76, 127.98, 128.42, 128.64, 129.31, 130.23, 130.35, 131.46, 131.61,
134.18, 134.78, 134.84, 136.42, 136.73, 141.75, 148.57 ppm. Anal. calcd. for C26H23ClN4: C, 73.14;
H, 5.43; N, 13.12; Found: C, 73.23; H, 5.54; N, 13.23. HRMS: m/z [M+H]+calcd for C26H23ClN4:
427.1689, found: 426.4635.
23

4-(4-chlorophenyl)-N-cyclohexyl-2-phenyl) imidazo [1, 2-a] qyinoxalin-1-amine (6-10)
1
M.p. = 211-212 °C ; IR (KBr): 3341, 2927, 1594 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.08-1.83
(10H, m), 2.98 (1H, m), 3.70 (1H, d, J = 7.5 Hz), 7.38 (1H, t, J = 7.0 Hz), 7.48-7.56 (6H, m), 7.87 (2H,
d, J = 7.0 Hz), 8.10 (1H, d, J = 7.5 Hz), 7.80 (2H, d, J = 8.0 Hz), 9.15 (1H, d, J = 7.5 Hz) ppm ; 13C
NMR (CDCl3, 125 MHz): δ = 24.86, 25.57, 33.41, 57.26, 116.23, 125.94, 127.20, 127.76, 128.36,
128.73, 129.41, 130.13, 130.24, 131.39, 131.55, 133.71, 134.02, 134.80, 136.35, 136.63, 138.08,
148.55 ppm. Anal. calcd. for C28H25ClN4: C, 74.24; H, 5.56; N, 12.37; Found: C, 74.31; H, 5.64; N,
12.46. HRMS: m/z [M+H]+calcd for C28H25ClN4: 453.1846, found: 452.1356.
N-(tert-butyl)-2, 4-bis (4-chlorophenyl)-2-phenylimidazo [1, 2-a] quinoxalin-1-amine (6-11)
M.p. = 214-216 °C ; IR (KBr): 3378, 2923, 1592 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 0.99 (9H, s),
3.42 (1H, s), 7.45 (2H, d, J = 8.0 Hz), 7.51-7.56 (4H, m), 7.78 (2H, d, J = 8.0 Hz), 8.09 (1H, t, J = 5.0
Hz), 8.79 (2H, d, J = 8.0 Hz), 9.58 (1H, t, J = 5.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 29.75,
57.78, 117.12, 126.15, 126.88, 128.43, 128.83, 129.20, 129.78, 130.30, 130.39, 131.49, 133.36,
133.90, 134.26, 134.66, 136.52, 136.73, 140.59, 148.54 ppm. Anal. calcd. for C26H22Cl2N4: C, 67.68;
H, 4.81; N, 12.14; Found: C, 67.76; H, 4.89; N, 12.25. HRMS: m/z [M+H]+calcd for C26H22Cl2N4:
461.1300, found: 461.9944.
N-cyclohexyl-2,4-bis(3,4,5-trimethoxyphenyl)imadazo[1,2-a]quinoxalin-1-amine (6-12)
1
M.p. = 203-204 °C; IR (KBr): 3335, 2925, 1612 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.13-2.03
(10H, m), 3.09 (1H, m), 3.61 (1H, m), 3.94-4.03 (18H, s), 7.23 (2H, s), 7.57 (2H, m), 8.14 (1H, m),
8.27 (2H, s), 9.15 (1H, m) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.82, 25.65, 33.53, 56.15, 56.25,
57.17, 60.92, 61.01, 105.00, 107.73, 116.09, 125.96, 127.02, 128.68, 129.76, 130.26, 130.91, 131.52,
133.78, 136.68, 137.67, 138.12, 140.26, 148.79, 152.83, 153.49 ppm. Anal. calcd. for C34H38N2O6: C,
68.21; H, 6.40; N, 9.36; Found: C, 68.32; H, 6.49; N, 9.47. HRMS: m/z [M+H]+calcd for C34H38N4O6:
599.2869, found: 599.0741.
N-(tert-butyl)-2, 4-bis (3, 4, 5-trimethoxyphenyl)imidazo[1,2-a]quinoxalin-1-amine (6-13)
M.p. = 208.7-209.7 °C ; IR (KBr): 3387, 2925, 1586 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.05 (9H,
s), 3.44 (1H, s), 3.92-4.04 (18H, s), 7.10 (2H, s), 7.55 (2H, t, J = 4.0 Hz), 8.13 (1H, d, J = 5.5 Hz), 8.29
(2H, s), 9.61 (1H, d, J = 5.5 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 29.66, 56.14, 56.32, 57.74,
60.91, 61.02, 106.01, 107.73, 117.05, 126.00, 126.54, 129.11, 129.87, 130.29, 130.51, 131.45,
134.14, 136.68, 138.33, 140.23, 141.26, 148.74, 152.83, 153.39 ppm. Anal. calcd. for C32H36N4O6: C,
67.12; H, 6.34; N, 9.78; Found: C, 67.21; H, 6.42; N, 9.86. HRMS: m/z [M+H]+calcd for C32H36N4O6:
573.2713, found: 572.2761.
N-cyclohexyl-2-(3, 4-dimethoxyphenyl)-4-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-
amine (6-14)
24

1
M.p. = 171-172 °C ; IR (KBr): 3392, 2924, 1582 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.07-1.83
(10H, m), 3.01 (1H, m), 3.60 (1H, d, J = 6.5 Hz), 3.95-4.04 (15H, s), 6.95 (1H, d, J = 8.0 Hz), 7.40 (1H,
d, J = 8.0 Hz), 7.541-7.570 (2H, m), 7.58 (1H, s), 8.12 (1H, t, J = 4.5 Hz), 8.27 (2H, s), 9.15 (1H, t, J =
4.5 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.83, 25.60, 33.42, 55.80, 55.88, 56.11, 57.10, 60.88,
107.65, 111.01, 111.14, 116.12, 119.57, 125.79, 126.88, 127.07, 128.66, 130.09, 130.75, 131.58,
133.66, 136.59, 137.72, 140.11, 148.65, 148.77, 149.17, 152.77 ppm. Anal. calcd. for C33H36N4O5: C,
69.70; H, 6.38; N, 9.85; Found: C, 69.81; H, 6.47; N, 9.94. HRMS: m/z [M+H]+calcd for C33H36N4O5:
568.2686, found: 539.1565.
N-(tert-butyl)-2-(3, 4-dimethoxyphenyl)-4-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-
amine (6-15)
M.p. = 165-167 °C ; IR (KBr): 3341, 2921, 1586 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.02 (9H, s),
3.57 (1H, s), 3.95-4.04 (15H, s), 6.95 (1H, d, J = 8.0 Hz), 7.32 (1H, d, J = 8.0 Hz), 7.48 (1H, s), 7.54-
7.57 (2H, m), 8.13 (1H, d, J = 5.0 Hz), 8.28 (2H, s), 9.63 (1H, d, J = 5 Hz) ppm ; 13C NMR (CDCl3, 125
MHz): δ = 29.71, 55.92, 56.16, 57.69, 60.91, 61.03, 107.73, 111.01, 111.84, 117.13, 120.74, 125.89,
126.45, 127.84, 129.19, 129.81, 130.24, 131.55, 134.14, 136.70, 140.19, 141.24, 148.74, 148.98,
149.24, 152.85 ppm. Anal. calcd. for C31H34N4O5: C, 68.62; H, 6.32; N, 10.33; found: C, 68.71; H,
6.42; N, 10.42. HRMS: m/z [M+H]+calcd for C31H34N4O5: 543.2607, found: 542.3171.
N-cyclohexyl-2-(3, 4-methoxyphenyl)-4-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-
amine (6-16)
1
M.p. = 209-211 °C ; IR (KBr): 3336, 2926, 1612 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.09-1.85
(10H, m), 2.99 (1H, m), 3.62 (1H, d, J = 7.0 Hz), 3.88-4.04 (12H, s), 7.02 (2H, d, J = 8.0 Hz), 7.55 (2H,
t, J = 4.5 Hz), 7.84 (2H, d, J = 8.0 Hz), 8.13 (1H, d, J = 5.0 Hz), 8.24 (2H, s), 9.18 (1H, d, J = 5.0 Hz)
ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.91, 29.68, 33.47, 55.30, 56.24, 57.24, 60.91, 107.74,
114.17, 116.19, 125.79, 126.80, 126.89, 128.77, 030.07, 130.18, 130.74, 131.66, 133.77, 136.67,
137.86, 140.17, 148.94, 152.85, 159.24 ppm. Anal. calcd. for C33H36 N4O5: C, 69.70; H, 6.38; N, 9.85;
Found: C, 69.81; H, 6.47; N, 9.96. HRMS: m/z [M+H]+calcd for C32H34N4O4: 539.2658, found:
538.1494.
N-(tert-butyl)-2-(4-methoxyphenyl)-4-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-amine
(6-17)
1
M.p. = 216-217. °C ; IR (KBr): 3463, 2931, 1609 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.00 (9H,
s), 3.49 (1H, s), 3.86-4.04 (12H, s), 7.00 (2H, d, J = 8.0 Hz), 7.53 (2H, t, J = 5.0 Hz), 7.76 (2H, d, J =
8.0 Hz), 8.10-8.13 (1H, m), 8.26 (2H, s), 9.62 (1H, d, J = 8.0 Hz), ppm ; 13C NMR (CDCl3, 125 MHz): δ
= 29.67, 55.26, 56.23, 57.59, 60.89, 107.76, 114.01, 117.12, 125.81, 126.39, 127.52, 129.22, 129.50,
129.71, 130.21, 131.59, 134.17, 136.69, 140.17, 141.26, 148.85, 152.85, 159.35 ppm. Anal. calcd. for
C30H32N4O4: C, 70.29; H, 6.29; N, 10.93; Found: C, 70.41; H, 6.44; N, 11.08. HRMS: m/z [M+H]+calcd
for C30H32N4O4: 513.2502, found512.4182.
25

N-cyclohexyl-2-phenyl-4-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-amine (6-18)
1
M.p. = 220-221 °C ; IR (KBr): 3324, 2928, 1580 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.09-1.85
(10H, m), 3.01 (1H, m), 3.71 (1H, d, J = 7.5 Hz), 3.95-4.04 (9H, s), 7.37 (1H, t, J = 7.0 Hz), 7.49 (2H, t,
J = 7.0 Hz), 7.56 (2H, d, J = 3.5 Hz), 7.91 (2H, d, J = 7.0 Hz), 8.14 (1H, d, J = 4.5 Hz), 8.25 (2H, s),
9.17 (1H, d, J = 4.5 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.87, 25.59, 33.41, 56.22, 57.28,
60.89, 107.69, 116.18, 125.88, 126.96, 127.47, 127.62, 128.70, 129.74, 130.12, 131.57, 133.56,
133.88, 134.21, 136.66, 137.73, 140.18, 149.09, 152.85 ppm. Anal. calcd. for C31H32N4O3: C, 73.21;
H, 6.34; N, 11.02; Found: C, 73.32; H, 6.45; N, 11.13. HRMS: m/z [M+H]+calcd for C31H32N4O3:
509.2552, found: 508.2108.
N-cyclohexyl-4-phenyl-2-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-amine (6-19)
1
M.p. = 186-187 °C ; IR (KBr): 3315, 2930, 1579 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.13-1.86
(10H, m), 3.06 (1H, m), 3.66 (1H, d, J = 5.0 Hz), 3.92-3.96 (9H, s), 7.14 (2H, s), 7.52-7.57 (5H, m),
8.15 (1H, d, J = 5.0 Hz), 8.75 (2H, d, J = 7.0 Hz), 9.15 (1H, d, J = 5.0 Hz) ppm ; 13C NMR (CDCl3, 125
MHz): δ = 24.83, 25.65, 33.53, 56.42, 57.71, 61.00, 105.58, 116.13, 125.95, 127.08, 128.19, 128.88,
129.81, 130.14, 130.19, 130.34, 131.36, 131.59, 133.86, 136.32, 136.84, 138.51, 149.95, 153.58
ppm. Anal. calcd. for C31H32N4O3: C, 73.21; H, 6.34; N, 11.02; Found: C, 73.33; H, 6.48; N, 11.15.
HRMS: m/z [M+H]+calcd for C31H32N4O3: 509.2552, found: 508.8781.
N-(tert-butyl)-4-phenyl-2-(3, 4, 5-trimethoxyphenyl) imadazo [1, 2-a] quinoxalin-1-amine (6-20)
M.p. = 179-180 °C ; IR (KBr): 3335, 2926, 1581 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.02 (9H, s),
3.46 (1H, s), 3.90 (9H, s), 7.04 (2H, s), 7.54-7.55 (5H, m), 8.12 (1H, d, J = 5.0 Hz), 8.77 (2H, d, J =
7.0 Hz), 9.60 (1H, d, J = 5.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 29.73, 56.49, 57.64, 61.00,
106.45, 117.09, 125.33, 125.96, 126.56, 128.19, 129.14, 130.14, 130.19, 130.33, 130.53, 134.18,
136.33, 136.86, 138.46, 141.75, 149.96, 153.50 ppm. Anal. calcd. for C29H30N4O2: C, 72.18; H, 6.27;
N, 11.61; Found: C, 72.38; H, 6.48; N, 11.92. HRMS: m/z [M+H]+calcd for C29H30N4O3: 483.2396,
found: 482.7970.
N-cyclohexyl-2-(3, 4-dimethoxyphenyl)-4-phenylimadazo [1, 2-a] quinoxalin-1-amine (6-21)
1
M.p. = 171-173 °C ; IR (KBr): 3386, 2931, 1610 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.09-1.85
(10H, m), 3.02 (1H, m), 3.66 (1H, d, J = 7.0 Hz), 3.95-3.99 (6H, s), 6.98 (1H, d, J = 8.0 Hz), 7.39 (1H,
d, J = 8.0 Hz), 7.51-7.57 (6H, m), 8.14 (1H, d, J = 7.0 Hz), 8.76 (2H, d, J = 7.5 Hz), 9.16 (1H, d, J =
7.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.88, 25.65, 33.49, 55.97, 56.10, 57.18, 111.34,
111.60, 116.16, 120.22, 125.82, 126.98, 127.13, 128.17, 128.79, 130.14, 130.26, 131.00, 133.86,
136.47, 136.85, 138.16, 142.20, 148.94, 149.31, 149.98 ppm; MS (70 eV): m/z = 478.25 (M+). Anal.
calcd. for C30H30N4O2: C, 75.29; H, 6.32; N, 11.71; Found: C, 75.38; H, 6.41; N, 11.82. HRMS: m/z
[M+H]+calcd for C30H30N4O2: 478.2369, found: 478.9040.
N-(tert-butyl)-2-(3, 4-dimethoxyphenyl)-4-phenylimadazo [1, 2-a] quinoxalin-1-amine (6-22)
26

M.p. = 175-177 °C ; IR (KBr): 3340, 2934, 1577 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.00 (9H,s),
3.50 (1H, s), 3.94-3.99 (6H, s), 6.96 (1H, d, J = 8.0Hz), 7.30 (1H, d, J = 8.0 Hz), 7.43 (1H,s), 7.51-
7.57 (5H, m), 8.13 (1H, t, J =5.0 Hz), 8.77 (2H, d, J = 7.5 Hz), 9.63 (1H, t, J = 5.0 Hz) ppm ; 13C NMR
(CDCl3, 125 MHz): δ = 29.71, 55.95, 56.15, 57.61, 111.13, 112.23, 117.16, 121.11, 125.87, 126.50,
127.83, 128.20, 129.23, 129.98, 130.16, 130.29, 132.36, 134.19, 136.40, 136.87, 141.67, 149.07,
149.30, 149.92 ppm. Anal. calcd. for C28H28N4O2: C, 74.31; H, 6.24; N, 12.38; Found: C, 74.41; H,
6.34; N, 12.48. HRMS: m/z [M+H]+calcd for C28H28N4O4: 453.2990, found: 452.9129.
N-cyclohexyl-2, 4-diphenylimadazo [1, 2-a] quinoxalin-1-amine (6-23)
1
M.p. = 192-192 °C ; IR (KBr): 3333, 2924, 1605 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.07-1.84
(10H, m), 2.99 (1H, m), 3.71 (1H, d, J = 8.0 Hz), 7.37 (1H, t, J = 7.25 Hz), 7.47-7.56 (7H, m), 7.89
(2H, d, J = 7.0 Hz), 8.13 (1H, t, J = 5.0 Hz), 8.77 (2H, d, J = 7.0 Hz), 9.17 (1H, d, J= 5.0 Hz) ppm ; 13
C
NMR (CDCl3, 125 MHz): δ = 24.88, 25.61, 33.44, 57.27, 116.21, 125.85, 126.99, 127.66, 127.84,
128.19, 128.70, 128.83, 130.12, 130.17, 130.23, 131.53, 133.97, 134.21, 136.42, 136.84, 138.09,
150.15 ppm. Anal. calcd. for C28H26N4: C, 80.35; H, 6.26; N, 13.39; Found: C, 80.43; H, 6.37; N,
13.48. HRMS: m/z [M+H]+calcd for C28H26N4: 419.2235, found: 418.9160.
N-(tert-butyl)-2, 4-diphenylimadazo [1, 2-a] quinoxalin-1-amine (6-24)
M.p. = 197-199 °C ; IR (KBr): 3330, 2986, 1608 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 0.99 (9H, s),
3.55 (1H, s), 7.37 (1H, t, J = 7.25 Hz), 7.48 (2H, t, J = 7.25 Hz), 7.51-7.57 (5H, m), 7.83 (2H, d, J = 7.5
Hz), 8.13 (1H, t, J = 5.0 Hz), 8.78 (2H, d, J = 7.5 Hz), 9.65 (1H, t, J = 5.0 Hz) ppm ; 13C NMR (CDCl3,
125 MHz): δ = 29.67, 57.70, 117.22, 125.91, 126.53, 127.87, 128.22, 128.58, 128.65, 129.32, 130.17,
130.21, 130.31, 131.24, 135.00, 136.37, 136.91, 141.73, 150.10, 163.48 ppm. Anal. calcd. for
C26H24N4: C, 79.56; H, 6.16; N, 14.27; Found: C, 79.61; H, 6.24; N, 14.36. HRMS: m/z [M+H]+calcd for
C26H24N4: 393.2079, found: 393.2103.
N-cyclohexyl-2-(4-fluorophenyl)-4-phenylimadazo [1, 2-a] quinoxalin-1-amine (6-25)
1
M.p. = 201-203 °C ; IR (KBr): 3367, 2929, 1605 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.11-1.83
(10H ,m), 2.99 (1H, m), 3.58 (1H, d, J = 7.0Hz), 7.18 (2H, t, 3JH-H = 8.0 Hz, 3JH-F = 8.25 Hz), 7.50-7.61
(5H, m), 7.88 (2H, t, 3JH-H = 8.0 Hz, 4JH-F = 6.5 Hz), 8.15 (1H, d, J = 5.5 Hz), 8.74 (2H, d, J = 7.0 Hz),
9.14 (1H, d, J = 8.5Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.87, 25.61, 33.48, 57.24, 115.58,
115.75, 116.09, 125.94, 127.11, 128.21, 128.78, 129.52, 129.58, 130.13 (2JC-F = 22.5 Hz), 130.33,
131.10, 133.86, 136.35, 136.84, 137.45, 150.13, 162.43 (1JC-F = 250 Hz) ppm. Anal. calcd. for
C28H25FN4: C, 77.04; H, 5.77; N, 12.83; Found: C, 77.13; H, 5.84; N, 12.91. HRMS: m/z [M+H]+calcd
for C28H25FN4: 437.2141, found: 436.8382.
N-(tert-butyl)-2-(4-fluorophenyl)-4-phenylimadazo [1, 2-a] quinoxalin-1-amine (6-26)
M.p. = 207-209 °C ; IR (KBr): 3334, 2956, 1607 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 0.96 (9H, s),
3.38 (1H, s), 7.14 (2H, t, 3JH-H = 8.0 Hz, 3JH-F = 8.5 Hz), 7.49-7.56 (5H, m), 7.79 (2H, t, 3JH-H = 8.0 Hz,
27

4JH-F = 6.5 Hz), 8.11 (1H, d, J = 7.0 Hz), 8.77 (2H, d, J = 7.5 Hz), 9.54 (1H, d, J = 8.0 Hz) ppm ; 13C
NMR (CDCl3, 125 MHz): δ = 29.69, 57.52, 115.42, 115.59, 117.02, 125.91, 126.55, 128.20, 129.17,
129.97, 130.11, 130.23, 130.29, 131.09, 134.32, 136.28, 136.83, 140.81, 149.94, 162.50 (1JC-F = 250
Hz) ppm. Anal. calcd. for C26H23FN4: C, 76.08; H, 5.65; N, 13.65; Found: C, 76.16; H, 5.73; N, 13.74.
HRMS: m/z [M+H]+calcd for C26H23FN4: 411.1986, found: 410.3782.
N-cyclohexyl-2-(4-methoxyphenyl)-4-phenylimadazo [1, 2-a] quinoxalin-1-amine (6-27)
M.p. = 203.6-204.9 °C ; IR (KBr): 3333, 2928, 1610 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.09-1.82
(10H, m), 2.99 (1H, m), 3.63 (1H, d, J = 6.5 Hz), 3.87 (3H, s), 7.02 (2H, d, J = 7.5 Hz), 7.55 (5H, m),
7.83 (2H, d, J = 7.5 Hz), 8.13 (1H, d, J = 5.0 Hz), 8.76 (2H, d, J = 7.0 Hz), 9.18 (1H, d, J = 5.0 Hz)
ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.90, 25.64, 33.47, 55.31, 57.21, 114.16, 116.18, 125.75,
126.78, 126.92, 128.17, 128.83, 129.05, 130.13, 130.21, 130.87, 133.89, 136.49, 136.82, 138.16,
141.12, 149.96, 159.28 ppm. Anal. calcd. for C29H28N4O: C, 77.65; H, 6.29; N, 12.49; Found: C,
77.81; H, 6.44; N, 12.61. HRMS: m/z [M+H]+calcd for C29H28N4O: 449.2341, found: 448.9950.
N-(tert-butyl)-2-(4-methoxyphenyl)-4-phenylimadazo[1,2-a]quinoxalin-1-amine (6-28)
M.p. = 205-207 °C ; IR (KBr): 3330, 2960, 1612 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 0.99 (9H,
s), 3.46 (1H, s), 3.86 (3H, s), 7.01 (2H, d, J = 8.0 Hz), 7.51-7.56 (5H, m), 7.76 (2H, d, J = 8.0 Hz), 8.12
(1H, t, J = 4.5 Hz), 8.78 (2H, d, J = 7.5 Hz), 9.63 (1H, d, J = 5.0 Hz) ppm ; 13C NMR (CDCl3, 125
MHz): δ = 29.73, 55.31, 57.56, 114.05, 117.16, 125.80, 126.45, 127.54, 128.19, 128.83, 129.30,
129.81, 130.17, 130.27, 132.08, 134.26, 136.43, 136.88, 141.60, 149.91, 159.42 ppm. Anal. calcd.
For C27H26N4O: C, 76.75; H, 6.20; N, 13.26; Found: C, 76.83; H, 6.31; N, 13.38. HRMS: m/z
[M+H]+calcd for C27H26N4O4: 422.2182, found: 422.0198.
2-(4-chlorophenyl)-N-cyclohexyl-4-phenylimidazo[1,2-a]quinoxalin-1-amine (6-29)
1
M.p. = 203-204 °C ; IR (KBr): Ѵ =3333, 2928, 1610 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.09-1.71
(10H, m), 2.82-2.84 (1H, m), 3.78 (1H, d, J = 6.0 Hz), 7.12 (2H, d, J = 8.0 Hz), 7.19-7.21 (5H, m), 7.48
(2H, d, J = 8.0 Hz), 8.01 (1H, t, J = 5.0 Hz), 8.63 (2H, d, J = 5.0 Hz), 9.19 (1H, m) ppm ; 13C NMR
(CDCl3, 125 MHz): δ = 24.45, 25.29, 33.56, 57.70, 111.83, 116.88, 117.02, 120.29, 120.63, 121.07,
121.16, 123.53, 123.64, 124.46, 124.64, 127.03, 129.75, 132.04, 132.18, 136.37, 140.75, 148.07
ppm. Anal. calcd. For C28H25ClN4: C, 74.24; H, 5.56; N, 12.37; Found: C, 74.45; H, 6.82; N, 12.56.
N-(tert-butyl)-2-(4-chlorophenyl)-4-phenylimidazo[1,2-a]quinoxalin-1-amine (6-30)
1
M.p. = 210-214 °C ; IR (KBr): Ѵ =3345, 2960, 1618 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.01 (9H,
s), 3.62 (1H, s), 3.47 (1H, s), 7.07 (2H, d, J = 7.5 Hz), 7.31-7.36 (5H, m), 7.56 (2H, d, J = 5.5 Hz), 8.00
(1H, t, J = 5.0 Hz), 8.74 (2H, d, J = 7.5 Hz), 9.44 (1H, d, J = 4.0 Hz) ppm; 13C NMR (CDCl3, 125 MHz):
δ = 29.18, 57.90, 110.96, 115.11, 118.46, 121.36, 122.02, 123.00, 125.09, 127.66, 127.90, 129.57,
130.20, 130.47, 133.13, 142.57, 143.84, 150.07, 150.20, 151.47 ppm. Anal. calcd. For C26H23 ClN4: C,
73.14; H, 5.43; N, 13.12; Found: C, 73.38; H, 5.72; N, 13.31.
28

2-(1-(cyclohexylamino)-2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)-6-methoxyphenol (6-
31)
M.p. = 218-221 °C ; IR (KBr): Ѵ =3340, 2958, 1610 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.13-1.64
(10H, m), 2.95 (1H, m), 3.66 (1H, d, J = 6.0 Hz), 3.96-4.07 (12H, m), 5.90 (s, 1H, OH), 6.99 (1H, d, J =
8.0 Hz), 7.10 (2H, s), 7.41 (1H, d, J = 7.5 Hz), 7.55 (1H, m), 8.11 (1H, d, J = 5.0 Hz), 8.51 (2H, d, J =
7.5 Hz), 9.12 (1H, d, J = 5.0 Hz) ppm ; 13C NMR (CDCl3, 125 MHz): δ = 24.81, 29.70, 33.50, 55.92,
56.26, 57.11, 60.97, 105.15, 112.74, 114.19, 116.05, 119.11, 123.95, 124.41, 125.87, 126.63, 128.53,
128.62, 129.82, 130.00, 130.92, 133.76, 136.78, 137.63, 146.13 ppm. Anal. calcd. For C32H34N4O5: C,
69.30; H, 6.18; N, 10.10; Found: C, 69.48; H, 6.45; N, 10.24.
2-(1-(tert-butylamino)-2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)-6-methoxyphenol (6-
32)
M.p. = 210-214 °C ; IR (KBr): 3345, 2960, 1618 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.01 (9H, s),
3.62 (1H, s), 3.47 (1H, s), 3.91-4.07 (12H, m), 5.91 (1H, s, OH), 7.07-7.10 (3H, m), 7.51-7.56 (2H, m),
8.11 (1H, t, J = 4.0 Hz), 8.54-8.57 (2H, m), 9.60 (1H, d, J = 5.0 Hz) ppm; 13C NMR (CDCl3, 125 MHz):
δ = 29.70, 55.30, 56.24, 57.24, 60.91, 106.11, 112.60, 114.14, 117.04, 124.49, 125.93, 128.97,
130.02, 130.74, 131.66, 133.77, 136.67, 137.86, 140.17, 148.94, 152.85, 159.30 ppm. Anal. calcd. for
C30H32N4O5: C, 68.17; H, 6.10; N, 10.60; Found: C, 68.01; H, 5.98; N, 10.38. HRMS: m/z [M+H]+calcd
for C30H32N4O5: 529.2451, found: 528.0609.
2-(1-(cyclohexylamino)-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)-6-methoxyphenol
(6-
33)
1
M.p. = 214-218 °C ; IR (KBr): 3348, 2970, 1615 cm-1 ; H NMR (CDCl3, 500 MHz): δ = 1.12-1.85
(10H, m), 3.01 (1H, m), 3.72 (1H, d, J = 7.5 Hz), 3.97-4.06 (9H, m), 5.68 (s, 1H, OH), 7.12 (1H, s),
7.38 (2H, d, J = 7.0 Hz), 7.50 (1H, t, J = 7.0 Hz), 7.58 (2H, d, J = 7.0 Hz), 8.01 (1H, t, J = 5.0 Hz), 8.61
(2H, m), 9.21 (1H, m) ppm; 13C NMR (CDCl3, 125 MHz): δ = 25.61, 26.25, 32.62, 55.69, 56.11, 57.70,
110.98, 115.18, 115.55, 115.67, 118.48, 121.36, 122.02, 123.01, 124.69, 127.88, 128.08, 128.68,
135.89, 142.74, 143.88, 152.24, 152.43, 153.48, 159.29 ppm. Anal. calcd. for C31H32N4O4: C, 70.97;
H, 6.15; N, 10.68; Found: C, 71.15; H, 6.42; N, 10.80. HRMS: m/z [M+H]+calcd for C31H32N4O4:
525.2502, found: 524.0280.
2-(1-(tert-butylamino)-2-(3,4-dimethoxyphenyl)imidazo[1,2-a]quinoxalin-4-yl)-6-methoxyphenol (6-34)
M.p. = 210-214 °C ; IR (KBr): 3335, 2964, 1625 cm-1 ; 1H NMR (CDCl3, 500 MHz): δ = 1.00 (9H, s),
3.48 (1H, s), 3.95-4.07 (9H, m), 5.91 (1H, s, OH), 6.98 (1H, s), 7.10 (1H, m), 7.27-7.31 (2H, m), 7.45
(1H, t, J = 7.0 Hz), 7.53 (1H, d, J = 7.0 Hz), 8.10 (1H, t, J = 5.0 Hz), 8.55-8.58 (2H, m), 9.63 (1H, m)
ppm; 13C NMR (CDCl3, 125 MHz): δ = 29.70, 55.95, 56.20, 58.03, 111.05, 111.99, 112.68, 114.17,
117.12, 120.93, 124.49, 125.85, 126.12, 129.99, 135.89, 136.15, 147.76, 148.97, 156.16, 159.95
ppm. Anal. calcd. for C29H30N4O4: C, 69.86; H, 6.07; N, 11.24; Found: C, 71.15; H, 6.25; N, 11.43.
HRMS: m/z [M+H]+calcd for C29H30N4O4: 499.2345, found: 498.0599.
29