Anticancer profile of newly synthesized BRAF inhibitors possess 5-(pyrimidin-4-yl)imidazo[2,1-b]thiazole scaffold

Article abstract of DOI:10.1016/j.bmc.2019.03.062

In this work, a new series of imidazo[2,1-b]thiazole was designed and synthesized. The new compounds are having 3-fluorophenyl at position 6 of imidazo[2,1-b]thiazole and pyrimidine ring at position 5. The pyrimidine ring containing either amide or sulphonamide moiety attached to a linker (ethyl or propyl) at position 2 of the pyrimidine ring. The final compounds were selected by NCI for in vitro cytotoxicity screening. Most derivatives showed cytotoxic activity against colon cancer and melanoma cell lines. In addition, IC₅₀s of the target compounds were determined over A375 and SK-MEL-28 cell lines using sorafenib as positive control. Compounds12b, 12c, 12e, 12f, 15a, 15d, 15f, 14g and 15h exhibited superior activity when compared to sorafenib. The most potent compounds were tested against wild type BRAF, v600e BRAF, and CRAF. Compound 15h exhibited a potential inhibitory effect against^V600EBRAF (IC₅₀ = 9.3 nM).

Full text of DOI:10.1016/j.bmc.2019.03.062

Accepted Manuscript
Anticancer profile of newly synthesized BRAF inhibitors possess 5-(pyrimi-
din-4-yl)imidazo[2,1-b]thiazole scaffold
Mohammed S. Abdel-Maksoud, Usama M. Ammar, Chang-Hyun Oh
PII:
S0968-0896(19)30147-6
https://doi.org/10.1016/j.bmc.2019.03.062
BMC 14854
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 January 2019
18 March 2019
31 March 2019
Please cite this article as: Abdel-Maksoud, M.S., Ammar, U.M., Oh, C-H., Anticancer profile of newly synthesized
BRAF inhibitors possess 5-(pyrimidin-4-yl)imidazo[2,1-b]thiazole scaffold, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.03.062
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Anticancer profile of newly synthesized BRAF inhibitors possess 5-
(pyrimidin-4-yl)imidazo[2,1-b]thiazole scaffold
Mohammed S. Abdel-Maksouda,*, Usama M. Ammarb-e,*, Chang-Hyun Ohb-d,**
aMedicinal& Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division,
National Research Centre (NRC), Dokki, Giza, 12622, Egypt
bCenter for Biomaterials, Korea Institute of Science & Technology (KIST School), Seoul, Seongbuk-gu, 02792,
Republic of Korea
cUniversity of Science & Technology (UST), Daejeon, Yuseong-gu, 34113, Republic of Korea
dDepartment of Biomolecular Science, University of Science & Technology (UST), Daejeon, Yuseong-gu, 34113,
Republic of Korea
ePharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza, 12566, Egypt
* Both author contributed equally in this work
** Corresponding author (choh@kist.re.kr)
Author
Mohammed S. Abdel-Maksoud
Usama M. Ammar
ORCID ID
http://orcid.org/0000-0002-8763-5345
https://orcid.org/0000-0002-7218-641X
Chang-Hyun Oh
1

Abstract
In this work, a new series of imidazo[2,1-b]thiazole was designed and synthesized. The new
compounds are having 3-fluorophenyl at position 6 of imidazo[2,1-b]thiazole and pyrimidine
ring at position 5. The pyrimidine ring containing either amide or sulphonamide moiety attached
to a linker (ethyl or propyl) at position 2 of the pyrimidine ring. The final compounds were
selected by NCI for in vitro cytotoxicity screening. Most derivatives showed cytotoxic activity
against colon cancer and melanoma cell lines. In addition, IC50s of the target compounds were
determined over A375 and SK-MEL-28 cell lines using sorafenib as positive control.
Compounds12b, 12c, 12e, 12f, 15a, 15d, 15f, 14g and 15h exhibited superior activity when
compared to sorafenib. The most potent compounds were tested against wild type BRAF, v600e
BRAF, and CRAF. Compound 15h exhibited a potential inhibitory effect againstV600EBRAF
(IC50 = 9.3 nM).
Keywords
Imidazo[2,1-b]thiazole; Anticancer; Colon cancer; Melanoma; BAF inhibitors
Introduction
Cancer is a type of disease that causes threat to human health as well as human life.1-6 In recent
years, the morbidity rate of cancer is increased.2-4, 7 Cell proliferation is triggered through the
activation of MAPK signalling pathway.8-10The activation of MAPK signalling pathway is
regulated by a cascade of protein kinases phosphorylated successively and control a number of
cellular activities such as; cell proliferation, differentiation and migration.10-13
RAF kinases (ARAF, BRAF and CRAF) play an important role in MAPK signalling pathway
activation.14 High frequency of RAF kinases mutation was investigated to cause cancer disease.15
Among the RAF kinase subtypes, BRAF is the major activator of MAPK signalling pathway. 16-
2

18The most common mutation in BRAF is the substitution of valine (V) by glutamate (E) at
residue 600 in the BRAF kinase domain (V600EBRAF)14, 19, 20 V600E
.
BRAF was identified in 8% of
all cancer types, such as; thyroid cancer (30-70%), melanoma (60%) and colorectal cancer
(10%).15,20-25 Therefore, BRAF kinase enzyme is considered a crucial biological target in treating
and controlling the cancer disease.26, 27
Both vemurafenib (I) and sorafenib (II) (Figure 1) are FDA-approved drugs in the treatment of
melanoma through inhibition of mutated BRAF kinase enzyme (V600EBRAF).28-33On the other
hand, imidazothiazole-based scaffold (compounds III, and IV) (Figure 1) has an attracted
significant interest as lead compounds for molecular optimization to have potent anticancer
activity. Where, they exert their action through inhibiting V600EBRAF kinase enzyme.34-36
In the present research, based on the aforementioned data, a new series of imidazo[2,1-b]thiazole
derivatives was designed and synthesized. The final target compounds possess the sub-structures
found in compounds III and IV in order to enhance their anticancer activity and increase their
BRAF inhibitory effect to add more drug candidates to the medicinal chemistry library in the
field of anticancer agents especially in the area of BRAF kinase inhibitors. The new compounds
are having 3-fluorophenyl at position 6 of imidazo[2,1-b]thiazole and pyrimidine ring at position
5. The pyrimidine ring containing either amide or sulphonamide moiety attached to a linker
(ethyl or propyl) at position 2 of the pyrimidine ring.
3

Cl
H
H
N
N
N
F
O
F
H
H
N
O
N
O
Cl
S
N
O
O O
N
F
F
F
H
I
II
(Vemurafenib)
(Sorafenib)
S
N
N
R1
R2
N
NH
H2N
NH
III (R1=H, R2=NO2)
IV (R1=NO2, R2=Cl)
S
N
N
R
F
H
N
N
X
n
N
N
H
Target compounds
X=CO, SO2
n= 1 or 2
R = H, p-Br, p-Cl, p-f, p-OMe, p-Me, p-Cf3, m-f, phenyl
Fig.1. Chemical structure of reported BRAF inhibitors and final target compounds
2. Results & discussion
2.1. Chemistry
The final target compounds 12a-f, 13a-e, 14a-i and 15a-j were synthesized as depicted in
Scheme 1. The first step is condensation and cyclization of 2-bromo-1-(3-fluorophenyl)ethan-1-
one(6) with 2-aminothiazole (7) in absolute ethanol under refluxed condition to provide6-(3-
4

1
fluorophenyl)imidazo[2,1-b]thiazole(8). H NMR showed an additional signals at δ 6.99 and
6.85 ppm attributed to the thiazole protons. In addition to disappearance of methyl protons
signals of compound 6 at δ 4.56 ppm. C. Mizoroki-Heck cross-coupling40 of compound 8 with 4-
chloro-2-(methylthio)pyrimidine(9) was accomplished in presence of palladim acetate as a
catalyst, triphenyl phosphine as a ligand and potassium carbonate as a base and at 80 °C to give
6-(3-fluorophenyl)-5-(2-(methylthio)pyrimidin-4-yl)imidazo[2,1-b]thiazole (10). Oxidation of
thiomethyl group of compound 10 was achieved by reaction with oxone to afford 6-(3-
fluorophenyl)-5-(2-(methylsulfonyl)pyrimidin-4-yl)imidazo[2,1-b]thiazole(11). 1H NMR showed
deshielded signals at δ 3.47 ppm attributed to the methyl sulfonyl group (-SO2CH3). The second
part was the synthesis of the side chain. Reaction of excess (10 mole equivalent) ethylene
diamine (1a) or propylene diamine (1b) with appropriate acid chloride 2a-k or benzene sulfonyl
chloride 3a-s lead to formation of amide side chain 4a-k and sulfonamide side chain 5a-s.Final
target compounds 12a-f, 13a-e, 14a-i and 15a-j were provided by reaction of compound 11 with
appropriate side chains 4a-k and 5a-s in DMSO and in presence of DIPEA at 80 °C .
5

H
N
n
Ar
a
NH2
Ar
X
Cl
n
X
+
H2N
H2N
2a-k X=CO
3a-s X=SO2
1a n=1
1b n=2
4a-k X=CO
5a-s X=SO2
S
S
O
N
N
N
N
F
Br
H2N
b
F
F
c
S
+
N
N
Cl
N
7
8
6
S
N
N
9
S
10
S
S
N
e
N
N
F
d
N
12a-f n=1 X=CO
13a-e n=2 X=CO
14a-i n=1 X=SO2
15a-j n=2 X=SO2
F
N
H
H
N
n
Ar
Ar
N
N
X
X
S
n
N
H2N
N
O
O
N
H
4a-k X=CO
5a-s X=SO2
11
Reagents and conditions: a) MC, 0 °C-rt, 18 h., 60-80%; b) EtOH, reflux, 18 h., 75%; c) Pd(OAc)2,
K2CO3, Ph3P, DMF, 80 °C, 18 h., 30%; d) Oxone, MeOH/H2O, rt, 18 h., 90%; e) DIPEA, DMSO, 100 °C,
18 h., 45-80%
Scheme 1. Synthesis of compounds 12a-f, 13a-e, 14a-i and 15a-j.
2.2. Biological evaluation
2.2.1. NCI 60 cell lines one dose test
The final target compounds were submitted to the NCI, USA for in vitro antiproliferative
screening against a 60 cell lines panel belonging to nine cancer types (leukemia, non-small cell
lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer
and breast cancer) and compounds 12a,b,d-f, 13a-c,e, 14a, b, d, f-i and 15a-e, g-j were selected
to be tested at single dose of 10 µM. The mean % inhibition of each of the tested compounds
against the cancer cell lines are shown in Figure 2 and Table 2s.
6

Fig. 2. Mean growth percent of the final target compounds against NCI 60 cell lines.
All final target compounds reduced the mean growth of cancer cell lines. In the amide series,
compounds with ethyl linker between pyrimidine ring and amide group were more potent
compared to compounds with propylene linker. Compounds 12b (unsubstituted phenyl) and 12f
(2,6-diflourophenyl) inhibited the mean growth percentage to half the control value. Compound
12e (p-Cl phenyl) reduced cell growth by 40%. Regarding the final compounds possess
sulfonamide moiety, compounds 14a (unsubstitutedphenyl) showed the highest percent
inhibition among all compound with value 60% followed by 15b (m-f phenyl) with 55% growth
inhibition. Similarly, to amide containing compounds, sulfonamide derivatives with ethylene
linker were more potent comparing to their propylene analogues. Also, compounds with electron
withdrawing group 14f, 14g,14h and 15b or unsubstituted 14a were stronger compared to one
with bulky group such as naphthyl14i Figure 2.
7

The sub-panel deep analysis (Table 2s) showed interesting results, the highest percent inhibition
was exhibited against two sub-panel colon cancer sub-panel and melanoma sub-panel.
Compounds 12b, 12e, 12f, 15b and 15h exhibited the highest percent inhibition against
melanoma cell lines with mean percent inhibition 63%, 55%, 65%, 62% and 53%, respectively.
On the other hand, compounds 14a, 14f, 14g, 14h and 15j had the highest percent inhibition
against colon cancer cell lines with mean percent inhibition 66%, 52%, 56%, 53%, and 77%,
respectively.
The individual cell line activities of compounds 12b, 12f, 14a, 15b, and 15j are represented in
Table 1. Compound 12b showed the highest percent inhibition against COLO 205, MALME-3M
and M14 with percent inhibitions 89.59%, 76.29% and 71.56%. Compound 12f exhibited 82.85%
inhibition against MDA-MB-435. Compound 14a inhibited 95.1% of IGROV1 growth and 82.5 %
against MDA-MB-435. Compound 15b showed a lethal effect on T-47D cell line with percent
inhibition 102% and inhibited UACC-62, SK-MEL-5 and MOLT-4 with percent inhibition
93.64%, 86.49%, and 90.06%, respectively. Finally, compound 15j inhibited 98.03% and 95.95%
of HT29 and MDA-MB-231/ATCC cell lines.
Table 1: In vitro cytotoxic activity (%inhibition) of the most active compounds 12b,f, 14a and
15b,j against different human cancer cells.
Cell name
Compound No.
14a
12b
12f
15b
15j
Leukemia
CCRF-CEM
HL-60(TB)
K-562
59.07
16.32
55.98
53.49
57.25
13.59
59.79
64.12
66.42
36.91
76.06
83.43
75.33
59.53
79.92
90.06
61.54
46.1
84.75
70.7
MOLT-4
8

Non-Small Cell Lung Cancer
A549/ATCC
NCI-H226
NCI-H460
73.27
55.69
71.18
70.86
59.33
75.23
74.83
65.49
74.11
50.73
76.76
41.68
54.38
63.23
80.15
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
89.59
26.15
74.39
65.37
74.19
80.76
29.63
75.13
68.91
79.63
75.85
33.65
77.29
74.20
76.84
76.59
ND
78.46
83.67
67.58
81.95
98.03
52.03
77.45
69.40
HT29
Melanoma
LOX IMVI
MALME-3M
M14
64.92
76.29
71.56
73.04
74.40
88.82
67.62
68.43
70.70
82.65
82.85
89.70
63.70
31.49
58.60
82.58
80.92
75.06
61.01
36.76
71.37
75.96
86.49
93.64
79.08
21.26
57.32
77.38
68.17
75.67
MDA-MB-435
SK-MEL-5
UACC-62
Ovarian Cancer
87.93
IGROV1
OVCAR-4
83.48
44.57
53.16
35.08
95.10
60.01
66.12
53.74
81.11
73.22
81.57
82.39
77.73
59.85
61.50
52.90
39.14
55.83
43.09
OVCAR-8
NCI/ADR-RES
Renal Cancer
786-0
ACHN
CAKI-1
UO-31
52.65
70.95
63.60
77.25
55.81
73.99
65.45
79.17
52.61
74.02
60.94
84.64
33.73
57.46
58.46
71.76
47.14
65.32
70.89
79.97
9

Prostate Cancer
42.97 66.86
Breast Cancer
82.53
PC-3
37.14
72.91
58.80
MCF7
MDA-MB-
231/ATCC
T-47D
76.29
34.93
78.26
34.03
66.32
24.14
62.63
18.80
95.95
65.49
73.30
81.18
102.25
81.63
2.2.2. Antiproliferative activity against A375 and SK-MEL-28
In order to determine the potency of the final target compounds,12b-f, 13d-f, 14a-h, 15a-d and
15f-I were evaluated for their antiproliferative (IC50) effect against melanoma cell lines (A375
and SK-MEL-28) using sorafenib as positive control. The IC50 values (µM) of the tested
compounds against melanoma cell lines are showed in Table 2. The tested compounds exhibited
significant inhibitory effect against both A375 melanoma cell line (2.57-15.44 µM) and Skmel28
melanoma cell line (6.60-19.02 µM) compared to sorafenib (7.88 and 9.45 µM, respectively).
Regarding A375 cell line, compounds 12b (unsubstituted phenyl), 12c (p-methyl), 12e (p-Cl),
12f (2,6-diflouro), 14d (p-CF3), 15a (unsubstitutedphenyl), 15d (p-CF3), 15f (p-OMe), 15g (p-
Cl) and 15h(p-Br) were more potent thensorafenib with IC50s 5.06, 5.88, 2.57, 2.70, 7.44, 6.60,
4.09, 5.51, 5.90 and 7.04 µM. Generally, compounds with amide moiety were more potent then
compounds with sulfonamide. Among amide derivatives, compounds with ethylene linker
showed lower IC50s then compounds with propylene linker and compounds with electron
withdrawing substitution were more potent then electron donating group. Compound 12e is the
most active compound with IC50 2.57 µM. For sulfonamide derivatives, compounds with
propylene linker were more potent then compounds containing ethylene linker. Similarly,
10

compounds with electron withdrawing groups were more potent then compounds having electron
donating group and compound 15d was the most potent compound among these derivatives with
IC50 4.09 µM.
On the other hand, compounds 12d, 12e, 14d, 14h, 15a, 15b, 15f, 15g and 15h were more potent
thensorafenib against SK-MEL-28 cell line with IC50s 9.26, 8.16, 8.45, 9.45, 6.85, 8.55, 8.98,
8.45 and 9.40 µM. As in A375 cell line compounds with amide moiety and ethylene linker were
more potent compared to propylene derivatives. While for sulfonamide compounds, propylene
derivatives were more potent then ethylene compounds. There was no significant difference
between both amide and sulfonamide derivatives on SK-MEL-28 cell line.
Table 2: In vitro cytotoxic activity (IC50 µM) of target compounds 12b-f, 13d-f, 14a-h, 15a-d
and 15f-i against human melanoma cell lines (A375 and SK-MEL-28) using sorafenib as
standards.
Compound
Sorafenib
12b
A375
7.88
5.06
5.88
8.71
2.57
2.70
13.72
14.90
SK-MEL-28 Compound
A375
12.63
18.57
13.52
9.45
SK-MEL-28
12.63
18.57
13.52
9.45
9.45
17.04
18.77
9.26
14e
14f
12c
14g
14h
15a
15b
15c
15d
12d
12e
8.16
6.60
6.85
12f
11.44
> 20
> 20
13.72
8.38
8.55
13d
9.50
13e
4.09
11.65
11

13f
14a
14b
14c
14d
11.21
11.02
14.91
15.44
7.44
13.67
19.02
18.28
> 20
15f
15g
15h
15i
5.51
5.90
8.98
8.45
9.40
> 20
7.04
14.48
8.45
Finally, in order to check the toxicity of the new synthesized compounds, the most potent
compounds were tested over L132 cell line (as normal human embryonic epithelial cells). All
tested compounds had higher IC50 over normal cell line then cancer cell lines. All compounds
had an IC50 more then 150 uM. The most potent compound against normal cell line was
compound 12f with IC50 198 uM , which is 18 folds compared to its IC50 over A375 and SK-
MEL-28 cell lines (Table 3)
Table 3: IC50 of most potent compound over the normal cell line (L132)
Compound
12b
IC50 (uM)
200
12c
245
12f
198
15a
>400
320
15d
2.2.3. In vitro kinase assay
As the imidazthiazole-based derivatives were reported as BRAF kinase inhibitors35, 36, 40, among
the most potent antiproliferative compounds, compounds 12b, 15c,d and 15f-h were evaluated
12

for their in vitro kinase assay against WTBRAF, V600EBRAF and CRAF. The results are depicted
in Table 4.
The results revealed that the tested compounds exhibited more inhibitory effect against
V600EBRAF (9.30-89.00 nM) than that of WTBRAF (22.90-182.00 nM) and CRAF (18.60-312.00
nM).Moreover, all tested compound showed nanomolar IC50 and compound 15h showed that
most potential inhibitory effect against V600EBRAF (9.30 nM). Compounds with sulfonamide
moiety were more potent then compounds with amide side chain over the three enzymes. In
addition , the presence of electron withdrawing groups enhanced the inhibitory effect compared
to unsubstituted derivatives. The order of activity was bromo>chloro>triflouromethyl.
Compound 15f with para methoxy group showed similar activity against both v600e BRAF and
CRAF. Amide containing derivatives exhibited high selectivity for mutant BRAF compared to
wild type and CRAF.
Table 4: Kinase inhibitory effect (IC50nM) of target compounds 12b, 15c,d and 15f-h against
WTBRAF, V600EBRAF and CRAF.
Compound
12b
WTBRAF
162.00
105.02
95.30
V600EBRAF
37.00
CRAF
312.00
120.1
12e
27.20
12f
33.10
102.00
88.40
14d
85.20
22.30
15c
182.00
63.00
89.00
143.00
40.00
15d
26.00
15f
55.10
25.10
26.10
13

15g
15h
49.60
22.90
16.20
9.30
34.00
18.60
2.3. Molecular docking
The X-ray crystal structure of BRAF kinase enzyme domain with dabrafenib (PDB ID:
5CSW)was identified (Figure 3). Analysis of the docking results revealed that the dabrafenib-
BRAF kinase domain complex had low root mean square deviation, RMSD (0.4737) that proved
valid docking protocol with dock score (-17.8199 kcal/mol, Table 5).The native ligand
(dabrafenib) showed H-bonding with Lys 483, Cys 532 and Phe 595 amino acids. In addition, it
exhibited both arene-cation and arene-arene interactions with Lys 483 and Phe 583 amino acids,
respectively. The binding affinity of the ligand was evaluated with energy score (S, Kcal/mol)
(Table 5). Low dock score energy indicates good affinity.
Table 5: Docking results of target compounds 12b, 15c, 15d, 15f, 15gand 15h with BRAF
kinase enzyme domain (PDB ID: 5CSW):
Compound Energy score
Amino
acid
Binding group
Interaction
Hydrogen bond
length (Å)
S (Kcal/mol)
Native
ligand
-17.8199
Lys 483
Lys 483
Cys 532
Cys 532
Phe 583
Phe 595
Lys 483
SO2
H-bond
2.74
Phenyl ring
NH2
Arene cation
H-bond
2.95
2.87
N pyrimidine
Pyrimidine ring
SO2
H-bond
Arenearene
H-bond
3.04
12b
-27.9025
Phenyl ring
Arene cation
14

Gly 534
Phe 583
Cys 532
Gly 534
Lys 483
Phe 583
Phe 595
Lys 483
Phe 583
Asp 594
Trp 531
Phe 583
Asp 594
Asn 580
Phe 583
NH amide
Pyrimidine ring
N pyrimidine
NH amide
SO2
H-bond
1.89
Arenearene
H-bond
3.15
1.76
2.86
H-bond
15c
15d
-25.1258
-20.6763
H-bond
Pyrimidine ring
SO2
Arenearene
H-bond
2.65
2.74
SO2
H-bond
Pyrimidine ring
SO2
Arenearene
H-bond
2.77
15f
-23.6009
-24.4202
Phenyl ring
Pyrimidine ring
SO2
Arenearene
Arenearene
H-bond
15g
2.43
1.58
15h
-27.4217
NH sulphonamide
Imidazole ring
H-bond
Arenearene
The docking study revealed that most of the target compounds mad molecular interactions with
Lys 483, Leu 514, Trp 531, Cys 532, Gly 534, Lys 578, Asn 580, Phe 583, Asp 594 and Phe 595
amino acids. Moreover, all the target compounds shared the same binding groups towards BRAF
kinase enzyme active site with H-bonding interaction (amide, sulphonamide and N pyrimidine
chemical moieties) as well as arene-arene and arene-cation interactions (phenyl, naphthyl,
imidazolyl and pyrimidinyl rings).
Analysis of the docking results revealed that most of the target compounds showed more
molecular interactions with the BRAF active site than the native ligand. Moreover, target
15

compounds 12e, 13a, 14b, 14h, 14i, 15e, 15i and 15j exhibited four to five visible interactions
with BRAF active site (-29.4200, -27.2361, -23.4021, -30.2736, -26.8178, -30.89.75, -25.4579
and -20.4652 kcal/mol) (Figure 3). In addition, it was revealed that the target compounds
bearing sulphonamide chemical moiety (14a-i and 15a-j) showed additional molecular
interactions with the BRAF active site than that bearing amide group (12a-f and 13a-e).
Fig.3. 2D representation of docking study output (PDB ID: 5CSW) A) 2D interaction of 14h
(native ligand) with BRAF kinase enzyme domain; B) 2D interaction of 15e (native ligand) with
BRAF kinase enzyme domain C) 2D interaction of dabrafenib (native ligand) with BRAF kinase
enzyme domain.
3. Experimental
3.1. Chemistry
3.1.1. General
The intermediate compounds 8 and 11 as well as the target compounds 12a-f, 13a-e, 14a-i and
15a-j were purified by flash column chromatography using silica gel 60 (0.040-0.063 mm, 230-
16

1
13
400 mesh ASTM) and technical grade solvents. H NMR and C NMR analyses were carried
out on a Bruker Avance 400 spectrometer using tetramethylsilane (TMS) as an internal standard.
Melting points were measured on a Walden Precision Apparatus Electrothermal 9300 apparatus
and were uncorrected. LC-MS analysis was conducted using the following system: Waters 2998
photodiode array detector, Waters 3100 mass detector, Waters SFO system fluidics organizer,
Waters 2545 binary gradient module, Waters reagent manager, Waters 2767 sample manager,
Sunfire™ C18 column (4.6 × 50 mm, 5 µm particle size); Solvent gradient = 95% A at 0 min, 1%
A at 5 min; solvent A: 0.035% trifluoroacetic acid (TFA) in water; solvent B: 0.035% TFA in
MeOH; flow rate = 3.0 mL/min; the AUC was calculated using Waters MassLynx 4.1 software.
The solvents and liquid reagents were transferred using hypodermic syringes. All the solvents
and reagents were purchased from commercial companies, and used as such.
3.1.2. Synthesis of N1-sustituted ethane(propane)-1,2(3)diamine(4a-k and 5a-s):
Synthesis of side chains (4a-k and 5a-s) was carried out through a pathway previously reported37.
3.1.3. Synthesis of 6-(3-fluorophenyl)imidazo[2,1-b]thiazole (8)
A mixture of 2-bromo-1-(3-fluorophenyl)ethan-1-one (6, 9.6 g, 44.6 mmol) and 2-aminothiazole
(5, 4.46 g, 44.6 mmol, 1 Eq) in absolute ethanol (60 ml) was dissolved, stirred and refluxed for
18 h. The reaction mixture was concentrated under reduced pressure. Ice-cold water (50 ml) was
added followed by ammonia solution (30%, 100 ml). The reaction mixture was stirred at rt for 2
h. The formed precipitate was filtered off, washed with water (2 × 20 ml) and dried to give the
crude solid product. The crude product was purified by column chromatography (hexane:ethyl
acetate; 5:1) to give the titled product 8.
1
Yield: 75%. m.p.: 91-2 °C. H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H, Ar-H), 7.59 (d, J =
8.0 Hz, 1H, Ar-H), 7.56 (d, J = 8.0 Hz, 1H, Ar-H), 7.44 (d, J = 4.0 Hz, 1H, Ar-H), 7.37
17

(q, J = 8.0 Hz, 1H, Ar-H), 6.99 (t, J = 8.0 Hz, 1H, Ar-H), 6.85 (d, J = 4.0 Hz, 1H, Ar-H).
13C NMR (100 MHz, CDCl3) δ 164.48 (Ar-C), 162.04 (Ar-C), 150.29 (Ar-C), 146.52
(Ar-C), 136.25 (Ar-C), 130.15 (Ar-C), 120.76 (Ar-C), 118.51 (Ar-C), 114.14 (Ar-C),
112.45 (Ar-C), 111.97 (Ar-C), 108.5 (Ar-C).LC-MS (m/z) calculated for C11H7FN2S (m/z):
218.03 found: 218.9 (M + H)+.
3.1.4. Synthesis of 6-(3-fluorophenyl)-5-(2-(methylthio)pyrimidin-4-yl)imidazo[2,1-b]thiazole
(10)
In a three neck flask, 4-chloro-2-(methylthio)pyrimidine (7, 70.7 mg, 0.44 mmol, 1 Eq),
potassium carbonate (60.8 mg, 0.44 mmol, 1 Eq), palladium acetate (19.8 mg, 0.09 mmol, 0.2 Eq)
and triphenylphosphine (34.6 mg, 0.13 mmol, 0.3 Eq) were mixed with compound 8 (96 mg,
0.44 mmol, 1 Eq). Air was replaced by nitrogen. Anhydrous DMF (10 ml) was added and the
mixture was purged with nitrogen several times. The reaction mixture was stirred at 80 °C for 18
h. The reaction mixture was cooled and extracted between EA (20 ml) and water (10 ml). The
organic layer was separated, dried over anhydrous sodium sulfate and evaporated. The crude
residue was used in the next step without further purification.
3.1.5.
Synthesis
of
6-(3-fluorophenyl)-5-(2-(methylsulfonyl)pyrimidin-4-yl)imidazo[2,1-
b]thiazole (11)
To a solution of compound 10 (103 mg, 0.44 mmol) in methanol (10 ml), a solution of oxone
(0.9 g, 1.32 mmol) in water (10 ml) was added dropwise at rt. The mixture was stirred at rt for 48
h. The reaction mixture was concentrated under reduced pressure. The reaction mass was
extracted with dichloromethane (20 ml) and the organic layer was separated. The aqueous layer
was extracted with dichloromehane (3 × 10 ml). The combined organic layers were dried over
18

anhydrous sodium sulfate and evaporated under reduced pressure. The crude residue was
purified by column chromatography (hexane:ethyl acetate; 2:1) to give the titled products 11.
Yield: 90%. m.p.: 188-90 °C. 1H NMR (400 MHz, DMSO- d6) δ 8.83 (d, J = 5.2 Hz, 1H, Ar-H),
8.63 (d, J = 4.4 Hz, 1H, Ar-H), 7.65 (d, J = 4.4 Hz, 1H, Ar-H), 7.55 (m, 3H, Ar-H), 7.43 (d, J =
13
5.6 Hz, 1H, Ar-H), 7.37 (t, J = 8.8 Hz, 1H, Ar-H), 3.47 (s, 3H, SO2CH3). C NMR (100 MHz,
DMSO-d6) δ 165.92 (Ar-H), 163.96 (Ar-H), 161.53 (Ar-H), 158.89 (Ar-H), 156.74 (Ar-H),
153.75 (Ar-H), 150.23 (Ar-H), 136.72 (Ar-H), 131.58 (Ar-H), 125.58 (Ar-H), 122.24 (Ar-H),
119.95 (Ar-H), 118.91 (Ar-H), 116.62 (Ar-H), 116.51 (Ar-H), 116.41 (Ar-H), 116.26 (Ar-H),
116.04 (Ar-H), 40.01 (SO2CH3). LC-MS (m/z) calculated for C16H11FN4S2(m/z):374.03 found:
375.0 (M + H)+.
3.1.6. General procedure for synthesis of 4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)-N-
substituted pyrimidin-2-amine (12a-f, 13a-e, 14a-i and 15a-j):
To a solution of compound 11 (0.26 mmol) in DMSO (3 ml), compounds 4a-k and 5a-s (0.39
mmol) (Scheme 1, Table 1s) and DIPEA (300 mg, 2.34 mmol) were added. The reaction
mixture was stirred at 100 °C for 18 h. The reaction mixture was cooled and extracted between
EA (20 ml) and water (10 ml)). The organic layer was dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The crude residue was purified by column chromatography
(hexane:ethyl acetate; 1:1) to give the titled products 12a-f, 13a-e, 14a-i and 15a-j.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
yl)amino)ethyl)cyclohexanecarboxamide (12a);Yield: 75%. m.p.: 202-3 °C. 1H NMR (400 MHz,
CDCl3) δ 8.51 (d, J = 2.3 Hz, 1H, Ar-H), 8.04 (d, J = 1.2 Hz, 1H, Ar-H), 7.35 (m, 3H, Ar-H),
7.07 (m, 1H, Ar-H), 6.95 (d, J = 2.2 Hz, 1H, Ar-H), 6.73 (s, 1H, NH), 6.48 (d, J = 3.1 Hz, Ar-H),
6.05 (s, 1H, NH), 3.61 (m, 2H, Aliph-H), 3.49 (m, 2H, Aliph-H), 2.02 (m, 1H, cyclohexyl), 1.70
19

(m, 4H, cyclohexyl), 1.23 (m, 2H, cyclohexyl), 1.19 (m, 4H, cyclohexyl).13C NMR (100 MHz,
CDCl3) δ 177.06 (C=O), 162.54 (Ar-C), 157.55 (Ar-C), 156.77 (Ar-C), 152.08 (Ar-C), 148.30
(Ar-C), 136.90 (Ar-C), 130.18 (Ar-C), 124.97 (Ar-C), 122.04 (Ar-C), 121.02 (Ar-C), 116.15
(Ar-C), 115.44 (Ar-C), 112.94 (Ar-C), 107.39 (Ar-C), 45.41 (Aliph-C), 40.89 (Aliph-C), 29.62
(Aliph-C), 25.69 (Aliph-C).LC-MS (m/z) calculated for C24H25FN6OS (m/z):464.18 found: 465.0
(M + H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzamide
(12b); Yield: 70%. m.p.: 177-8 °C. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 4.1 Hz, 1H, Ar-
H), 8.05 (d, J = 4.2 Hz, 1H, Ar-H), 7.83 (d, J = 16.0 Hz, 1H, NH), 7.73 (d, J = 8.0 Hz, 2H, Ar-H),
7.38 (m, 3H, Ar-H), 7.32 (m, 3H, Ar-H), 7.09 (m, 1H, Ar-H), 6.91 (d, J = 4.4 Hz, 1H, A-H), 6.48
(d, J = 5.2 Hz, 1H, Ar-H), 6.30 (s, 1H, NH), 3.72 (s, 4H, Aliph-H).13C NMR (100 MHz, CDCl3)
δ 168.07 (C=O), 183.95 (Ar-C), 162.65 (Ar-C), 161.50 (Ar-C), 157.53 (Ar-C), 152.07 (Ar-C),
148.32 (Ar-C), 136.84 (Ar-C), 134.31 (Ar-C), 131.35 (Ar-C), 130.16 (Ar-C), 128.31 (Ar-C),
126.92 (Ar-C), 124.95 (Ar-C), 121.94 (Ar-C), 120.97 (Ar-C), 116.10 (Ar-C), 115.42 (Ar-C),
112.93 (Ar-C), 41.42 (Aliph-C), 40.85 (Aliph-C).LC-MS (m/z) calculated for C24H19FN6OS
(m/z):458.13 found: 459 (M+H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-4-
1
methylbenzamide (12c);Yield: 70%. m.p.: 180-1 °C. H NMR (400 MHz, CDCl3) δ 8.49 (d, J =
8.0 Hz, 1H, Ar-H), 8.05 (d, J = 8.0 Hz,1H, Ar-H), 7.70 (t, J = 4.0 Hz, 1H, Ar-H), 7.60 (d, J = 8.0
Hz, 2H, Ar-H), 7.36 (m, 3H, Ar-H), 7.09 (m, 2H, Ar-H), 6.89 (d, J = 4.0 Hz, 1H, Ar-H), 6.48 (d,
J = 8.0 Hz, 1H, Ar-H), 6.25 (s, 1H, NH), 3.71 (s, 4H, Aliph-H), 2.29 (s, 3H, CH3).13C NMR (100
MHz, CDCl3) δ 168.09 (C=O), 163.96 (Ar-C), 162.66 (Ar-C), 161.52 (Ar-C), 152.09 (Ar-C),
148.35 (Ar-C), 141.81 (Ar-C), 136.82 (Ar-C), 131.39 (Ar-C), 130.19 (Ar-C), 129.09 (Ar-C),
20

127.00 (Ar-C), 124.99 (Ar-C), 116.18 (Ar-C), 115.51 (Ar-C), 112.92 (Ar-C), 107.47 (Ar-C),
41.45 (Aliph-C), 40.86 (Aliph-C), 21.34 (CH3).LC-MS (m/z) calculated for C25H21FN6OS
(m/z):472.15 found: 473.3 (M + H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-4-
1
(trifluoromethyl)benzamide (12d);Yield: 65%. m.p.: 167-8 °C. H NMR (400 MHz, CDCl3) δ
8.48 (d, J = 4.0 Hz, 1H, Ar-H), 8.10 (d, J = 8.0 Hz, 1H, Ar-H), 7.83 (d, J = 8.0 Hz, 2H, Ar-H),
7.59 (d, J = 4.0 Hz, 2H, Ar-H), 7.40 (m, 2H, Ar-H), 7.33 (m, 1H, Ar-H), 7.12 (m, 1H, Ar-H),
6.93 (d, J = 4.0 Hz, 1H, Ar-H), 6.56 (d, J = 8.0 Hz, 1H, Ar-H), 3.76 (m, 4H, Aliph-H).13C NMR
(100 MHz, CDCl3) δ 166.66 (C=O), 164.01 (Ar-C), 162.75 (Ar-C), 161.56 (Ar-C), 152.25 (Ar-
C), 148.73 (Ar-C), 137.60 (Ar-C), 136.73 (Ar-C), 133.26 (Ar-C), 132.94 (Ar-C), 130.26 (Ar-C),
127.33 (Ar-C), 125.44 (Ar-C), 124.93 (CF3), 121.76 (Ar-C), 120.78 (Ar-C), 116.13 (Ar-C),
115.62 (Ar-C), 113.02 (Ar-C), 107.88 (Ar-C), 42.49 (Aliph-C), 41.22 (Aliph-C).LC-MS (m/z)
calculated for C25H18F4N6OS (m/z): 526.12 found: 527.5 (M + H)+.
4-Chloro-N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
yl)amino)ethyl)benzamide (12e);Yield: 80%. m.p.: 170-1 °C. 1H NMR (400 MHz, CDCl3) δ 8.50
(d, J = 4.0 Hz, 1H, Ar-H), 8.06 (d, J = 8.0 Hz, 1H, Ar-H), 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 7.34,
(m, 5H, Ar-H), 7.10 (m, 1H, Ar-H), 6.91 (d, J = 4.0 Hz, 1H, Ar-H), 6.49 (d, J = 4.0 Hz, 1H, Ar-
13
H), 6.18 (s, 1H, NH), 3.73 (s, 4H, Aliph-H). C NMR (100 MHz, CDCl3) δ 163.99 (C=O),
162.69 (Ar-C), 161.54 (Ar-C), 157.56 (Ar-C), 152.12 (Ar-C), 148.42 (Ar-C), 136.85 (Ar-C),
134.29 (Ar-C), 131.41 (Ar-C), 130.20 (Ar-C), 128.35 (Ar-C), 126.89 (Ar-C), 124.99 (Ar-C),
121.92 (Ar-C), 120.95 (Ar-C), 116.13 (Ar-C), 115.55 (Ar-C), 112.97 (Ar-C), 107.55 (Ar-C),
41.44 (Aliph-C), 40.88 (Aliph-C).;LC-MS (m/z) calculated for C24H18ClFN6OS (m/z):
492.09 found: 493.3 (M+H)+.
21

2,6-Difluoro-N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
yl)amino)ethyl)benzamide (12f);Yield: 70%. m.p.: 185-7 °C. 1H NMR (400 MHz, CDCl3) δ 8.48
(d, J = 8.0 Hz, 1H, Ar-H), 8.05 (d, J = 8.0 Hz, 1H, Ar-H), 7.41 (m, 3H, Ar-H), 7.34 (m, 1H, Ar-
H), 7.13 (m, 1H, Ar-H), 6.95 (d, J = 4.0 Hz, 1H, Ar-H), 6.87 (d, J = 8.0 Hz, 2H, Ar-H), 6.52 (d, J
= 4.0 Hz, 1H, Ar-H), 5.82 (s, 1H, NH), 3.76 (s, 4H, Aliph-H).13C NMR (100 MHz, CDCl3) δ
164.01 (C=O), 162.52 (Ar-C), 161.56 (Ar-C), 160.85 (Ar-C), 152.09 (Ar-C), 148.35 (Ar-C),
136.84 (Ar-C), 131.64 (Ar-C), 130.20 (Ar-C), 124.92 (Ar-C), 121.68 (Ar-C), 120.86 (Ar-C),
116.18 (Ar-C), 115.55 (Ar-C), 112.96 (Ar-C), 111.84 (Ar-C), 107.87 (Ar-C), 41.10 (Aliph-C),
40.94 (Aliph-C).LC-MS (m/z) calculated for C24H17F3N6OS (m/z): 494.11 found: 495.2
(M+H)+.
N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)propyl)cyclohexanecarboxamide(13a);Yield: 75%. m.p.: 187-9 °C. H NMR (400
MHz, CDCl3) δ 8.56 (s, 1H, NH), 8.09 (d, J = 8.0 Hz, 1H, Ar-H), 7.41 (m, 3H, Ar-H), 7.12 (m,
1H, Ar-H), 6.98 (d, J = 8.0 Hz, 1H, Ar-H), 6.54 (d, J = 4.0 Hz, 1H, Ar-H), 6.34 (s, 1H, NH), 5.73
(t, J = 8.0 Hz, 1H, Ar-H), 3.55 (m, 2H, Aliph-H), 3.40 (m, 2H, Aliph-H), 2.11 (m, 8H,
Cyclohexyl), 2.79 (m, 2H, Aliph-H), 1.68 (m, 1H, Cyclohexyl), 1.45 (m, 2H, Cyclohexyl).13C
NMR (100 MHz, CDCl3) δ 176.58 (C=O), 164.03 (Ar-C), 162.42 (Ar-C), 161.59 (Ar-C), 157.57
(Ar-C), 152.05 (Ar-C), 148.38 (Ar-C), 137.00 (Ar-C), 130.18 (Ar-C), 125.02 (Ar-C), 122.02
(Ar-C), 121.08 (Ar-C), 116.20 (Ar-C), 115.41 (Ar-C), 112.80 (Ar-C), 107.27 (Ar-C), 45.68
(Aliph-C), 38.31 (Aliph-C), 36.11 (Aliph-C), 35.20 (Aliph-C), 30.02(Aliph-C), 29.80 (Aliph-C),
25.76 (Aliph-C).LC-MS (m/z) calculated for C25H27FN6OS (m/z):478.20 found: 479.4 (M
+ H)+.
22

N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)propyl)benzamide
(13b);Yield: 65% m.p.: 190-1 °C. 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 4.0 Hz, 1H, Ar-H),
8.02 (d, J = 8.0 Hz, 1H, Ar-H), 7.78 (d, J = 8.0 Hz, 2H, Ar-H), 7.35 (m, 5H, Ar-H), 7.06 (m, 1H,
Ar-H), 6.87 (d, J = 4.0 Hz, 1H, Ar-H), 6.44 (d, J = 8.0 Hz, 1H, Ar-H), 6.01 (t, J = 7.75 Hz, 1H,
Ar-H), 3.56 (m, 4H, Aliph-H), 1.89 (t, J = 7.75 Hz, 2H, Aliph-H).13C NMR (100 MHz, CDCl3) δ
167.82 (C=O), 163.95 (Ar-C), 162.48 (Ar-C), 161.50 (Ar-C), 151.97 (Ar-C), 134.74 (Ar-C),
131.28 (Ar-C), 130.14 (Ar-C), 128.38 (Ar-C), 126.97 (Ar-C), 125.00 (Ar-C), 122.04 (Ar-C),
116.12 (Ar-C), 115.14 (Ar-C), 112.78 (Ar-C), 107.05 (Ar-C), 40.89 (Aliph-C), 38.39 (Aliph-C),
29.66 (Aliph-C). LC-MS (m/z) calculated for C25H21FN6OS (m/z):472.15 found: 473.4
(M+H)+.
N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)propyl)-4-
1
(trifluoromethyl)benzamide (13c);Yield; 60%. m.p.: 173-4 °C. H NMR (400 MHz, CDCl3) δ
8.51 (d, J = 4.0 Hz, 1H, Ar-H), 8.00 (d, J = 4.0 Hz, 1H, Ar-H), 7.92 (d, J = 8.0 Hz, 2H, Ar-H),
7.61 (d, J = 8.0 Hz, 2H, Ar-H), 7.35 (m, 3H, Ar-H), 7.05 (m, 1H, Ar-H), 6.89 (d, J = 4.0 Hz, 1H,
Ar-H), 6.43 (d, J = 4.0 Hz, 1H, Ar-H), 3.55 (m, 4H, Aliph-H), 1.89 (m, 2H, Aliph-H).13C NMR
(100 MHz, CDCl3) δ 166.42 (C=O), 163.92 (Ar-C), 162.45 (Ar-C), 161.47 (Ar-C), 157.40 (Ar-
C), 151.99 (Ar-C), 138.10 (Ar-C), 136.87 (Ar-C), 132.93 (Ar-C), 130.18 (Ar-C), 127.58 (Ar-C),
125.32 (CF3), 124.96 (Ar-C), 122.01 (Ar-C), 116.11 (Ar-C), 115.50 (Ar-C), 112.82 (Ar-C),
107.05 (Ar-C), 40.85 (Aliph-C), 38.37 (Aliph -C), 29.41 (Aliph -C).LC-MS (m/z) calculated
for C26H20F4N6OS (m/z):540.14 found: 541.0 (M+H)+.
4-Chloro-N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)propyl)benzamide (13d); Yield; 75%. m.p.: 104-6 °C. H NMR (400 MHz, CDCl3) δ
8.51 (d, J = 4.0 Hz, 1H, Ar-H), 8.01 (d, J = 4.0 Hz, 1H, Ar-H), 7.78 (d, J = 8.0 Hz, 2H, Ar-H),
23

7.37 (m, 5H, Ar-H), 7.06 (m, 1H, Ar-H), 6.88 (d, J = 4.0 Hz, 1H, Ar-H), 6.44 (d, J = 8.0 Hz, 1H,
Ar-H), 6.02 (s, 1H, NH), 3.55 (m, 4H, Aliph-H), 1.90 (t, J = 7.74 Hz, 2H, Aliph-H).LC-MS
(m/z) calculated for C25H20ClFN6OS (m/z):506.11 found: 507.2 (M+H)+.
4-Fluoro-N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)propyl)benzamide(13e);Yield: 70%. m.p.: 161-3 °C. H NMR (400 MHz, CDCl3) δ
8.52 (d, J = 4.0 Hz, 1H, Ar-H), 8.09 (d, J = 4.0 Hz, 1H, Ar-H), 7.82 (s, 2H, Ar-H), 7.43 (m, 3H,
Ar-H), 7.10 (m, 3H, Ar-H), 6.92 (d, J = 4.0 Hz, 1H, Ar-H), 6.53 (d, J = 4.0 Hz, 1H, Ar-H), 3.62
(m, 4H, Aliph-H), 1.93 (m, 2H, Aliph-H). 13C NMR (100 MHz, CDCl3) δ 166.68 (C=O), 164.03
(Ar-C), 163.37 (Ar-C), 162.55 (Ar-C), 161.58 (Ar-C), 157.08 (Ar-C), 152.14 (Ar-C), 148.58
(Ar-C), 136.88 (Ar-C), 130.25 (Ar-C), 129.13 (Ar-C), 125.00 (Ar-C), 121.87 (Ar-C), 120.81
(Ar-C), 116.22 (Ar-C), 115.63 (Ar-C), 112.83 (Ar-C), 107.43 (Ar-C), 38.34 (Aliph-C), 36.90
(Aliph-C), 29.97 (Aliph-C).LC-MS (m/z) calculated for C25H20F2N6OS (m/z): 590.14 found:
591.6 (M+H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)ethyl)benzenesulfonamide (14a);Yield: 50%. m.p.: 225-7 °C. H NMR (400 MHz,
DMSO-d6) δ 8.15 (s, 1H, NH), 7.85 (d, J = 7.2 Hz, 2H, Ar-H), 7.78 (d, J = 8.1 Hz, 3H, Ar-H),
7.68 (d, J = 6.8 Hz, 2H, Ar-H), 7.62 (m, 5H, Ar-H), 7.45 (t, J = 9.2 Hz, 1H, Ar-H), 7.32 (t, J =
7.6 Hz, 1H, Ar-H), 6.40 (s, 1H, NH), 3.45 (s, 2H, Aliph-H), 3.02 (t, J = 4.8 Hz, 2H, Aliph-H).13C
NMR (100 MHz, DMSO-d6) δ 168.08 (Ar-C), 163.96 (Ar-C), 162.66 (Ar-C), 161.51 (Ar-C),
157.53 (Ar-C), 152.07 (Ar-C), 148.32 (Ar-C), 136.86 (Ar-C), 134.31 (Ar-C), 131.36 (Ar-C),
130.18 (Ar-C), 128.31 (Ar-C), 126.92 (Ar-C), 124.95 (Ar-C), 121.95 (Ar-C), 120.97 (Ar-C),
116.15 (Ar-C), 115.45 (Ar-C), 112.93 (Ar-C), 107.43 (Ar-C), 41.42 (Aliph-C), 40.86 (Aliph-C);
HRMS (m/z) calculated for C23H19FN6O2S2(m/z): 494.0995 found: 495.1073 (M+H)+.
24

3-Fluoro-N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)ethyl)benzenesulfonamide (14b);Yield: 65%. m.p.: 188-90 °C. H NMR (400 MHz,
CDCl3) δ 8.44 (d, J = 4.4 Hz, 1H, Ar-H), 8.04 (d, J = 5.2 Hz, 1H, Ar-H), 7.62 (d, J = 7.6 Hz, 1H,
Ar-H), 7.52 (d, J = 7.6 Hz, 1H, Ar-H), 7.39 (t, J = 16.4 Hz, 3H, Ar-H), 7.24 (d, J = 17.2 Hz, 2H,
Ar-H), 7.10 (s, 1H, Ar-H), 6.94 (d, J = 4.4 Hz, 1H, Ar-H), 6.54 (d, J = 5.2 Hz, 1H, Ar-H), 5.45 (s,
1H, Ar-H), 3.62 (s, 2H, Aliph-H), 3.25 (d, J = 4.8 Hz, 2H, Aliph-H). LC-MS (m/z) calculated
for C23H19F2N6O2S2(m/z): 512.09 found: 513.0 (M+H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-4-
1
methylbenzenesulfonamide (14c);Yield: 60%. m.p.: 200-1 °C. H NMR (400 MHz, MeOH) δ
8.36 (s, 1H, Ar-H), 8.01 (d, J = 4.0 Hz, 1H, Ar-H), 7.89 (d, J = 4.0 Hz, 1H, Ar-H), 7.72 (d, J =
8.0 Hz, 2H, Ar-H), 7.52 (m, 1H, Ar-H), 7.44 (d, J = 8.0 Hz, 1H, Ar-H), 7.35 (d, J = 8.4 Hz, 1H,
Ar-H), 7.31 (d, J = 8.0 Hz, 2H, Ar-H), 7.23 (t, J = 8.0 Hz, 1H, Ar-H), 6.47 (d, J = 4.0 Hz, 1H,
Ar-H), 3.53 (s, Aliph-H), 3.14 (t, J = 4.0 Hz, 2H, Aliph-H), 2.36 (s, 3H, CH3). HRMS (m/z)
calculated for C24H21FN6O2S2(m/z): 508.1151 found: 509.1229 (M+H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-4-
1
(trifluoromethyl)benzenesulfonamide(14d);Yield: 55%. m.p.: 73-5 °C. H NMR (400 MHz,
CDCl3) δ 8.35 (d, J = 4.0 Hz, 1H, Ar-H), 8.89 (t, J = 8.0 Hz, 3H, Ar-H), 7.63 (d, J = 8.0 Hz, 2H,
Ar-H), 7.27 (t, J = 8.0 Hz, 3H, Ar-H), 7.18 (s, 1H, Ar-H), 6.83 (d, J = 4.0 Hz, 1H, Ar-H), 6.40 (d,
J = 4.0 Hz, 1H, Ar-H), 5.60 (s, 1H, NH), 3.52 (s, 2H, Aliph-H), 3.14 (d, J = 4.0 Hz, 2H, Aliph-
H).13C NMR (101 MHz, CDCl3) δ 162.10 (Ar-C), 157.30 (Ar-C), 156.90 (Ar-C), 152.26 (Ar-C),
148.56 (Ar-C), 143.71 (Ar-C), 136.77 (Ar-C), 130.25 (Ar-C), 127.45 (Ar-C), 126.23 (Ar-C),
124.97 (CF3), 121.94 (Ar-C), 116.11 (Ar-C), 115.66 (Ar-C), 113.03 (Ar-C), 107.76 (Ar-C),
25

43.50 (Ar-C), 41.47 (Ar-C). HRMS calculated for C24H18F4N6O2S2(m/z): 562.0869 found:
563.0948 (M+H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-4-
1
methoxybenzenesulfonamide(14e);Yield: 55%. m.p.: 203-4 °C. H NMR (400 MHz, CDCl3) δ
8.53 (s, 1H, Ar-H), 7.99 (d, J = 4.4 , 1H, Ar-H), 7.81 (m, 2H, Ar-H), 7.41 (m, 3H, Ar-H), 7.13
(m, 1H, Ar-H), 7.99 (d, J = 4.0 Hz, 1H, Ar-H), 6.93 (m, 2H, Ar-H), 6.51 (d, J = 4.0 Hz, 1H, Ar-
H), 6.10 (s, 1H, NH), 3.82 (s, 3H, OCH3), 3.65 (d, J = 8.0 Hz, 2H, Aliph-H), 3.24 (d, J = 8.0 Hz,
2H, Aliph-H).LC-MS (m/z) calculated for C24H24FN6O3S2(m/z): 524.11 found: 525.3
(M+H)+.
4-Chloro-N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)ethyl)benzenesulfonamide(14f);Yield: 75%. m.p.: 183-5 °C. H NMR (400 MHz,
DMSO-d6) δ 8.86 (s, 1H, NH), 8.49 (s, 1H, NH), 8.14 (d, J = 4.8 Hz, 1H, Ar-H), 7.85 (d, J = 7.2
Hz, 2H, Ar-H), 7.64 (q, J = 6.8 Hz, 3H, Ar-H), 7.55 (t, J = 6.8 Hz, 2H, Ar-H), 7.47 (t, J = 8.4 Hz,
2H, Ar-H), 7.32 (s, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 3.02 (s, 2H, Aliph-H), 2.56 (s, 2H, Aliph-H).
LC-MS (m/z) calculated for C23H18ClFN6O2S2(m/z): 528.06 found: 529.08 (M+H)+.
4-Bromo-N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)ethyl)benzenesulfonamide(14g);Yield: 70%. m.p.: 160-2 °C. H NMR (400 MHz,
MeOH) δ 8.37 (s, 1H, Ar-H), 8.01 (d, J = 4.0 Hz, 1H, Ar-H), 7.65 (t, J = 6.8 Hz, 1H, Ar-H), 7.55
(m, 5H, Ar-H), 7.44 (d, J = 8.0 Hz, 1H, Ar-H), 7.36 (d, J = 8.0 Hz, 1H, Ar-H), 7.24 (t, J = 8.0 Hz,
1H, Ar-H), 6.48 (d, J = 8.0 Hz, 1H, Ar-H), 3.55 (s, 2H, Aliph-H), 3.15 (t, J = 4.0 Hz, 2H, Aliph-
H).; LC-MS (m/z) calculated for C23H18BrFN6O2S2(m/z): 572.01 found: 573 (M + 1)+.
4-Fluoro-N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)ethyl)benzenesulfonamide(14h);Yield: 70%. m.p.: 220-2 °C. H NMR (400 MHz,
26

CDCl3) δ 8.56 (d, J = 8.0 Hz, 1H, Ar-H), 7.97 (s, 1H, Ar-H), 7.91 (m, 2H, Ar-H), 7.53 (m, 1H,
Ar-H), 7.47 (m, 2H, Ar-H), 7.37 (m, 1H, Ar-H), 7.18 (d, J = 8.0 Hz, 3H, Ar-H), 7.12 (s, 1H,
NH), 6.58 (d, J = 8.0 Hz, 1H, Ar-H). HRMS (m/z) calculated for C23H18F2N6O2S2(m/z):
512.0901 found: 513.0978 (M+H)+.
N-(2-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)naphthalene-
2-sulfonamide (14i):Yield: 45%. m.p.: 166-8 °C. 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 4.8
Hz, 2H, Ar-H), 7.94 (d, J = 5.2 Hz, 1H, Ar-H), 7.78 (m, 4H, Ar-H), 7.51 (m, 2H, Ar-H), 7.32 (m,
3H, Ar-H), 6.85 (d, J = 4.0 Hz, 1H, Ar-H), 6.41 (d, J = 5.2 Hz, 1H, Ar-H), 5.68 (s, 1H, NH), 3.58
(d, J = 5.2 Hz, 2H, Aliph-H), 3.28 (t, J = 4.8 Hz, 2H, Aliph-H).13C NMR (100 MHz, CDCl3) δ
157.24 (Ar-C), 156.71 (Ar-C), 152.13 (Ar-C), 148.48 (Ar-C), 136.67 (Ar-C), 134.67 (Ar-C),
132.05 (Ar-C), 130.19 (Ar-C), 129.22 (Ar-C), 128.42 (Ar-C), 127.51 (Ar-C), 124.98 (Ar-C),
122.15 (Ar-C), 116.2 (Ar-C), 115.51 (Ar-C), 112.80 (Ar-C), 107.52 (Ar-C), 43.50 (Aliph-C),
41.23 (Aliph-C).LC-MS (m/z) calculated for C27H21FN6O2S2(m/z): 544.12 found: 545.2
(M+H)+.
N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)propyl)benzenesulfonamide(15a);Yield: 60%. m.p.: 144-5 °C. H NMR (400 MHz,
CDCl3) δ 8.49 (d, J = 4.4 Hz, 1H, Ar-H), 8.01 (d, J = 5.2 Hz, 1H, Ar-H), 7.81 (t, J = 8.0 Hz, 4H,
Ar-H), 7.50 (m, 6H, Ar-H), 6.45 (d, J = 5.2 Hz, 1H, Ar-H), 5.57 (s, 1H, NH), 3.54 (q, J = 6.4 Hz,
2H, Aliph-H), 3.04 (d, J = 5.6 Hz, 2H, Aliph-H), 1.78 (t, J = 6.0 Hz, 2H, Aliph-H).13C NMR
(100 MHz, CDCl3) δ 162.3 (Ar-C), 157.4 (Ar-C), 156.91 (Ar-C), 152.23 (Ar-C), 148.43 (Ar-C),
140 (Ar-C), 136.92 (Ar-C), 132.6 (Ar-C), 130.21 (Ar-C), 129.15 (Ar-C), 126.89 (Ar-C), 125.02
(Ar-C), 121.95 (Ar-C), 120.91 (Ar-C), 116.22 (Ar-C), 115.51 (Ar-C), 113.13 (Ar-C), 107.29
27

(Ar-C), 40.28 (Aliph-C), 39.82 (Aliph-C), 29.87 (Aliph-C). HRMS (m/z) calculated for
C24H21FN6O2S2(m/z): 508.1151 found: 509.1229 (M+H)+.
3-Fluoro-N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-
1
yl)amino)propyl)benzenesulfonamide (15b);Yield: 65%. m.p.: 155-7 °C. H NMR (400 MHz,
CDCl3) δ 8.49 (d, J = 4.4 Hz, 1H, Ar-H), 8.04 (d, J = 5.2 Hz, 1H, Ar-H), 7.60 (d, J = 7.6 Hz, 1H,
Ar-H), 7.53 (s, 1H, Ar-H), 7.42 (m, 3H, Ar-H), 7.36 (m, 1H, Ar-H), 7.23 (q, J = 8.0 Hz, 1H, Ar-
H), 7.11 (q, J = 7.6 Hz, 1H, Ar-H), 6.97 (s, 1H, Ar-H), 6.48 (d, J = 5.6 Hz, 1H, Ar-H), 3.55 (d, J
= 6.0 Hz, 2H, Aliph-H), 3.06 (s, 2H, Aliph-H), 1.80 (d, J = 5.6 Hz, 2H, Aliph-H).13C NMR (100
MHz, CDCl3) δ 162.33 (Ar-C), 157.69 (Ar-C), 152.19 (Ar-C), 147.99 (Ar-C), 146.79 (Ar-C),
136.61 (Ar-C), 130.89 (Ar-C), 130.19 (Ar-C), 125.01 (Ar-C), 122.62 (Ar-C), 121.93 (Ar-C),
119.51 (Ar-C), 116.22 (Ar-C), 115.66 (Ar-C), 114.21 (Ar-C), 112.99 (Ar-C), 107.33 (Ar-C),
40.33 (Aliph-C), 38.03 (Aliph-C), 30.28 (Aliph-C).LC-MS (m/z) calculated for
C24H20F2N6O2S2(m/z): 526.11 found: 527.2 (M+H)+.
N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)propyl)-4-
1
methylbenzenesulfonamide (15c);Yield: 60%. m.p.: 171-3 °C. H NMR (400 MHz, CDCl3) δ
8.41 (d, J = 4.4 Hz, 1H, Ar-H), 7.93 (d, J = 5.2 Hz, 1H, Ar-H), 7.59 (d, J = 7.6 Hz, 2H, Ar-H),
7.25 (m, 3H, Ar-H), 7.13 (d, J = 7.6 Hz, 2H, Ar-H), 7.00 (t, J = 7.6 Hz, 1H, Ar-H), 6.87 (s, 1H,
Ar-H), 6.37 (d, J = 5.6 Hz, 1H, Ar-H), 5.63 (s, 1H, NH), 3.44 (d, J = 6.0 Hz, 2H, Aliph-H), 2.93
(d, J = 5.2 Hz, 2H, Aliph-H), 2.27 (s, 3H, CH3), 1.69 (t, J = 5.2 Hz, 2H, Aliph-H).13C NMR (100
MHz, CDCl3) δ 162.31 (Ar-C), 161.53 (Ar-C), 157.42 (Ar-C), 156.81 (Ar-C), 152.16 (Ar-C),
148.31 (Ar-C), 143.23 (Ar-C), 137.17 (Ar-C), 136.91 (Ar-C), 130.18 (Ar-C), 129.66 (Ar-C),
126.90 (Ar-C), 125.01 (Ar-C), 122.01 (Ar-C), 120.97 (Ar-C), 116.30 (Ar-C), 115.41 (Ar-C),
28

113.07 (Ar-C), 107.14 (Ar-C), 40.30 (Aliph-C), 38.32 (Aliph-C), 29.77 (Aliph-C), 21.44 (CH3).
LC-MS (m/z) calculated for C25H23FN6O2S2(m/z): 522.13 found: 523.4 (M+H)+.
N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)propyl)-4-
1
(trifluoromethyl)benzenesulfonamide (15d):Yield: 55%. m.p.: 107-8 °C. H NMR (400 MHz,
CDCl3) δ 8.48 (s, 1H, Ar-H), 7.95 (m, 3H, Ar-H), 7.70 (d, J = 7.2 Hz, 2H, Ar-H), 7.31 (m, 3H,
Ar-H), 7.09 (s, 1H, Ar-H), 6.96 (s, 1H, Ar-H), 6.47 (d, J = 4.8 Hz, 1H, Ar-H), 5.60 (s, 1H, NH),
3.55 (s, 2H, Aliph-H), 3.06 (s, 2H, Aliph-H), 1.79 (s, 2H, Aliph-H). 13C NMR (100 MHz, CDCl3)
δ 164.00 (Ar-C), 162.32 (Ar-C), 157.38 (Ar-C), 156.94 (Ar-C), 152.26 (Ar-C), 148.48 (Ar-C),
144.04 (Ar-C), 130.26 (Ar-C), 127.36 (Ar-C), 126.23 (Ar-C), 124.98 (CF3), 121.85 (Ar-C),
116.13 (Ar-C), 115.65 (Ar-C), 113.11 (Ar-C), 107.38 (Ar-C), 40.24 (Aliph-C), 38.19 (Aliph-C),
29.96 (Aliph-C).LC-MS (m/z) calculated for C25H20F4N6O2S2(m/z): 576.10 found: 577.2
(M+H)+.
N-(3-((4-(6-(3-fluorophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)propyl)-4-
1
hydroxybenzenesulfonamide(15e);Yield: 75%. H NMR (400 MHz, MeOH) δ 8.75 (s, 1H, OH),
7.95 (d, J = 4.0 Hz, 1H, Ar-H), 7.68 (d, J = 8.0 Hz, 3H, Ar-H), 7.52 (m, 2H, Ar-H), 7.48 (m, 1H,
Ar-H), 7.34 (m, 1H, Ar-H), 7.25 (t, J = 8.0 Hz, 1H, Ar-H), 6.86 (d, J = 8.0 Hz, 2H, Ar-H), 6.43
(d, J = 4.0 Hz, 1H, Ar-H), 3.52 (s, 2H, Aliph-H), 2.98 (t, J = 4.0 Hz, 2H, Aliph-H), 1.87 (t, J =
8.0 Hz, 2H, Aliph-H).13C NMR (100 MHz, MeOH) δ 163.99 (Ar-C), 161.42 (Ar-C), 158.72 (Ar-
C), 153.92 (Ar-C), 150.60 (Ar-C), 136.13 (Ar-C), 130.61 (Ar-C), 129.71 (Ar-C), 128.97 (Ar-C),
125.34 (Ar-C), 124.97 (Ar-C), 120.55 (Ar-C), 116.04 (Ar-C), 115.82 (Ar-C), 115.26 (Ar-C),
105.42 (Ar-C), 40.13 (Aliph-C), 38.66 (Aliph-C), 28.60 (Aliph-C).LC-MS (m/z) calculated for
C24H21FN6O3S2(m/z): 524.11 found: 525.2 (M+H)+.
29