Structural optimization of imidazothiazole derivatives affords a new promising series as B-Raf V600E inhibitors; synthesis, in vitro assay and in silico screening

Article abstract of DOI:10.1016/j.bioorg.2020.103967

BRAF mutation is commonly known in a number of human cancer types. It is counted as a potential component in treating cancer. In this study, based on structural optimization of previously reported inhibitors (3-fluro substituted derivatives of imidazo[2,1-b]thiazole-based scaffold), we designed and synthesized sixteen new imidazo[2,1-b]thiazole derivatives with m-nitrophenyl group at position 6. The electron withdrawing properties was reserved while the polarity was modified compared to previously synthesized compounds (-F). Furthermore, the new substituted group (–NO₂) provided an additional H-bond acceptor(s) which may bind with the target enzyme through additional interaction(s). In vitro cytotoxicity evaluation was performed against human cancer cell line (A375). In addition, in vitro enzyme assay was performed against mutated B-Raf (B-Raf V600E). Compounds 13a, 13g and 13f showed highest activity on mutated B-Raf with IC₅₀ 0.021, 0.035 and 0.020 μM. All target compounds were tested for in vitro cytotoxicity against NCI 60 cell lines. Compounds 13a and 13g were selected for 5 doses test mode. Moreover, in silico molecular simulation was explored in order to explore the possible interactions between the designed compounds and the B-Raf V600E active site.

Full text of DOI:10.1016/j.bioorg.2020.103967

Bioorganic Chemistry 100 (2020) 103967
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Structural optimization of imidazothiazole derivatives affords a new
promising series as B-Raf V600E inhibitors; synthesis, in vitro assay and in
silico screening
a,b,c
, Mohammed S. Abdel-Maksoud , Eslam M.H. Alia,b, Karim I. Mersala,b
d
Usama M. Ammar
,
e
a,b,⁎
Kyung Ho Yoo , Chang-Hyun Oh
a
b
c
d
Center for Biomaterials, Korea Institute of Science & Technology (KIST School), Seoul, Seongbuk-gu 02792, Republic of Korea
University of Science & Technology (UST), Daejeon, Yuseong-gu 34113, Republic of Korea
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza 12566, Egypt
Medicinal & Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (NRC), Dokki, Giza 12622, Egypt
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
BRAF mutation is commonly known in a number of human cancer types. It is counted as a potential component
in treating cancer. In this study, based on structural optimization of previously reported inhibitors (3-fluro
substituted derivatives of imidazo[2,1-b]thiazole-based scaffold), we designed and synthesized sixteen new
imidazo[2,1-b]thiazole derivatives with m-nitrophenyl group at position 6. The electron withdrawing properties
was reserved while the polarity was modified compared to previously synthesized compounds (-F). Furthermore,
Anti-cancer
B-Raf V600E
Cancer
Imidazo[2,1-b]thiazole
In silico screening
the new substituted group (–NO ) provided an additional H-bond acceptor(s) which may bind with the target
2
enzyme through additional interaction(s). In vitro cytotoxicity evaluation was performed against human cancer
cell line (A375). In addition, in vitro enzyme assay was performed against mutated B-Raf (B-Raf V600E).
Compounds 13a, 13g and 13f showed highest activity on mutated B-Raf with IC50 0.021, 0.035 and 0.020 µM.
All target compounds were tested for in vitro cytotoxicity against NCI 60 cell lines. Compounds 13a and 13g were
selected for 5 doses test mode. Moreover, in silico molecular simulation was explored in order to explore the
possible interactions between the designed compounds and the B-Raf V600E active site.
1
. Introduction
Cancer remains the main cause of high mortality rate in the globe
[19,20], dabrafenib(III) [21] and encorafenib(IV) [22,23] (Fig. 1). Al-
though a diversity of anti-cancer agents have been developed, it is es-
sential to enhance the anti-cancer properties of the existing drugs and
discovery of new candidates for specific types of human cancer [1,24].
Heteroatoms-bearing molecules are considered a substantial structural
motifs for drug development as they exhibit additional connectivity
with the target biological receptor through H-bonding interaction
[25–27]. The bi- and tri-cyclic fused heterocyclic rings show a sig-
nificant platform for a wide range of anti-cancer agents [27,28]. Among
these heterocyclic scaffolds, imidazothiazole is becoming an interesting
molecular scaffold in pharmaceutical chemistry field due to its broad
spectrum pharmacological properties. We previously reported the
synthesis of imidazothiazole derivatives with B-Raf V600E inhibitory
activity [29]. In the present work, structural optimization of imida-
zothiazole derivatives has been considered to afford a new series of
after cardiac diseases [1–4]. It claims > 6 million human death per a
year and still growing based on the WHO records [1]. Around 200
cancers were reported in different vital organs in human body [1].
Recent genetic screening showed that malignant tumors display a huge
molecular events which can be used as a significant and potential
therapeutic target for the treatment of cancer [5–12]. Mutation in BRAF
have been reported as the major component in a number of human
cancer types such as melanoma, thyroid and colon cancer [5,11,13–15].
This component is considered an important key in the MAPK pathway
and play a major role in its the activation [5,15–17]. Mutation in BRAF
occurs due to a single glutamate substitution for valine amino acid at
position 600 (BRAF V600E) [5]. A number of drugs have been reported
as mutated B-Raf inhibitors such as sorafenib (I) [18], vemurafenib(II)
NO -bearing compounds in which the fluoro group of previously
2
⁎
Corresponding author at: Center for Biomaterials, Korea Institute of Science & Technology (KIST School), Seoul, Seongbuk-gu 02792, Republic of Korea.
E-mail address: choh@kist.re.kr (C.-H. Oh).
https://doi.org/10.1016/j.bioorg.2020.103967
Received 10 February 2020; Received in revised form 13 May 2020; Accepted 20 May 2020
Available online 22 May 2020
0045-2068/ © 2020 Published by Elsevier Inc.

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Fig. 1. Reported B-Raf V600E inhibitors.
Fig. 2. The rational design of new synthesized derivatives (12a-h and 13a-h).
reported compounds V (Fig. 2)[29] was replaced with NO
2
group. The
range at δ 14.23 ppm. Oxidation of SCH of compound 10 was ac-
3
electron withdrawing property was reserved while additional H-bond
acceptor(s) were incorporated in the designed derivatives which may
increase the binding to the B-Raf V600E active site.
complished by reaction with potassium peroxymonosulfate to afford the
1
key intermediate (11). H NMR showed downfield-shift signal at δ
1
3
3.42 ppm attributed to SO
2
CH protons. Moreover, C NMR showed
3
downfield-shift signal at δ 39.28 ppm. Final target compounds 12a-h
and 13a-h (Table 1) were provided by reaction between intermediate
2
. Results and discussion
(
11) and different appropriate N-based side chain (3a-h and 4a-h) in
1
presence of base. H NMR showed an additional signals in both aro-
matic and aliphatic ranges attributed to the protons of built side chain.
2.1. Chemistry
Final compounds 12a-h and 13a-h were synthesized as depicted in
Scheme 1. N-based side chain (3a-h and 4a-h) were prepared through
nucleophilic substitution reaction between diamines (1a,b) and dif-
ferent substituted benzene sulfonyl chlorides (2a-h) [30]. α-bromina-
tion of 3′-nitroacetophenone (5) have been accomplished using N-bro-
2.2. Biological evaluation
2.2.1. In vitro enzyme assay
All synthesized compounds were tested against mutated B-Raf (B-
Raf V600E). Final compounds were tested for both % inhibition at
10 µM (Table 2) and in 10-dose IC50 with a 3-fold serial dilution at 1 µM
(Table 3). The assays were carried out at 1 µM ATP. In general, all new
compounds showed higher activity to B-Raf V600E compared to wild-
type B-Raf. Compounds 12e and 13f completely inhibited the mutated
enzyme at the tested concentration.
1
mosuccinamide in DMF to give compound 6. H NMR spectrum showed
a singlet downfield signal at 5.06 ppm assigned for CH Br. Condensa-
2
tion of compound 6 with 2-aminothiazole (7) in MeOH provided
1
compound 8. H NMR exhibited an additional signals at aromatic range
at δ 8.25, 8.09 and 7.32 ppm attributed to the imidazothiazole protons.
Moreover, disappearance of CH Br protons signals at δ 5.06 ppm.
2
Coupling of compound 8 with compound 9 is accomplished through
The results showed that all compounds showed sub-micromolar
IC50s. Compounds 12e, 13a, 13 g and 13f showed higher inhibitory
activities among the synthesized compounds (0.027, 0.021, 0.035 and
0.020 µM, respectively). Compounds 12b, 12 g, 13c and 13e had
higher IC50s with values 0.072, 0.096, 0.082 and 0.063 µM. Finally,
compounds 12a, 12c, 12d, 12f, 13b, and 13d exhibited moderate in-
hibitory effect with IC50 0.158, 0.297, 0.103, 0.148, 0.216, and
carbon–carbon bond- forming reaction (Heck reaction) [4,31] in pre-
sence of a catalytic amount of palladium acetate, Ph
3
P and K
2
CO to
3
1
provide compound 10. H NMR showed an additional aromatic protons
signals at δ 6.86 and 7.06 ppm attributed the pyrimidine ring. Fur-
thermore, additional signals at δ 2.67 ppm contributed to the SCH
3
1
3
protons. In addition, C NMR showed an additional signal at aliphatic
2

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Scheme 1. Reagents and conditions. a) TEA, DCM, 0 °C-rt, 8 h; b) NBS, DMF, 60 °C, 3 h; c) MeOH, reflux, 18 h; d) Pd(OAc)
2
, Ph
3
P, K
2
3
CO , DMF, 80 °C, 8 h; e) Oxone,
MeOH/H O, rt, 9 h; f) DIPEA, DMSO, 90 °C, 8 h.
2
Table 1
Table 3
IC50 values (µM) of target compounds (12a-h and 13a-h) against B-Raf V600E.
Key structure of target compounds 12a-h and 13a-h.
Compd
B-Raf V600E IC50 (µM)
Compd
B-Raf V600E IC50 (µM)
1
1
1
1
1
1
1
1
2a
2b
2c
0.158 ± 0.0011
0.096 ± 0.0009
0.297 ± 0.0013
0.103 ± 0.0021
0.027 ± 0.0014
0.148 ± 0.0008
0.072 ± 0.0012
0.634 ± 0.0021
0.031
13a
13b
13c
13d
13e
13f
0.021 ± 0.0007
0.216 ± 0.0016
0.082 ± 0.0015
0.131 ± 0.0021
0.063 ± 0.0006
0.020 ± 0.0003
0.035 ± 0.0021
0.259 ± 0.0019
2d
2e
2f
2 g
2 h
13 g
13 h
Vemurafenib
Compd
n
Ar
Compd
n
Ar
1
1
1
1
1
1
1
1
2a
2b
2c
1
1
1
1
1
1
1
1
3-FPh
13a
13b
13c
13d
13e
13f
2
2
2
2
2
2
2
2
3-FPh
0
.131 µM. Compounds 12e, 13a and 13f had IC50s lower than vemur-
4-CH
3
Ph
Ph
4-CH
3
Ph
Ph
afenib
4-CF
3
4-CF
3
2d
2e
2f
4-OCH
4-ClPh
3
Ph
4-OCH
3
Ph
The structure activity relationship for ethylene bridged compounds
4-ClPh
12a-h revealed that moderate size electron-withdrawing group such as
Cl 12e had the highest activity over the mutated enzyme followed by
small size electron-withdrawing group such as F in 12 g. Compound
that had small size electron donating group such as methyl in 12b gave
an 2 digits nano-molar IC50 . large size electron withdrawing group like
4-BrPh
4-FPh
4-BrPh
4-FPh
2 g
2 h
13 g
13 h
1-Naph
1-Naph
Table 2
Mean % inhibition of target compounds (12a-h and 13a-h) against B-Raf V600E and wild-type B-Raf.
Compd
B-Raf V600E
B-Raf (WT)
Compd
B-Raf V600E
B-Raf (WT)
1
1
1
1
1
1
1
1
2a
2b
2c
96.72 ± 0.12
98.62 ± 0.22
95.87 ± 0.21
96.99 ± 0.34
100
64.12 ± 1.23
71.52 ± 0.99
78.25 ± 1.11
72.63 ± 1.35
76.14 ± 0.81
72.51 ± 0.98
70.25 ± 0.96
59.81 ± 0.86
13a
13b
13c
13d
13e
13f
99.64 ± 0.22
97.26 ± 0.16
98.32 ± 0.24
96.88 ± 0.17
98.97 ± 0.33
100
66.31 ± 1.36
69.36 ± 2.22
71.66 ± 0.97
70.35 ± 0.55
77.91 ± 1.65
75.16 ± 1.19
69.26 ± 1.09
65.33 ± 2.01
2d
2e
2f
97.52 ± 0.41
99.14 ± 0.19
94.68 ± 0.11
2 g
2 h
13 g
13 h
95.21 ± 0.31
96.45 ± 0.28
3

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Fig. 3. Mean % inhibition of final target compounds against different cancer sub-types.
triflouromethyl and bromo derivatives 12c and 12f were less active
compared to small size electron with-drawing group but more active
compared to large planer naphthyl derivative 12 h. On the other hand,
compounds with propylene bridge 13a-h showed less IC50 in general
compared to ethylene analogues. Compound with large electron with
drawing bromine atom13f had the highest activity in this group fol-
lowed by unsubstituted benzene ring 13a and small electron with
drawing substituted derivative 12 g. In similar manner to ethylene
compounds, large aryl group such as naphthyl ring 13 h had the lowest
activity in this group.
exhibited 100% against BT-549, MCF7, MDA-MB-468 and T-47D cell
lines. Compounds 13f, 13d and 13e had % inhibition 98.2, 96.69, and
93.45 against T-47D cell line and 70.77, 83.80 and 86.87% against
MDA-MB-468. Compounds 12f and 12 g showed their highest effect
against T-47D with % inhibition 85.31 and 88, respectively.
On the other hand, the activity of the new compounds against CNS
cancer cell lines (Table 3s) was moderate to low with highest % in-
hibition 94.43% for 13 g against SF-295 cell line followed by 13a with
94.14% against the same cell line.
The activity of the new series against colon cancer cell lines was
presented at Table 4s. Compound 13a completely inhibited the growth
of HCT-15 and KM12 and inhibited COLO 205 by 95.48% and HCT-116
with % inhibition 95.57% and SW-620 with 81.68%. While 13a in-
hibited COLO205 and KM12 completely at 10 µM. Also 13a inhibited
HCT-116, and HCT-15 with mean % inhibition 92.23 and 97.79, re-
spectively. Compounds 13c, 13d, 13e, and 13f showed > 50% inhibi-
tion against COLO205, HCT-16, HT29 and KM12 cell lines.
Compounds 13a and 13 g were able to cease the growth of HL-60,
MOLT-4 and RPMI-8226 cell lines. Compound 12b showed > 50% in-
hibition against CCRF-CEM, K562, MOLT-4 and RPMI-8226. While
compound 12c had > 50% inhibition against all leukemia cell lines.
Compounds 13b, 13c, 13d, 13e, and 13f with propyl bridge between
pyrimidine ring and terminal sulfonamide moiety showed higher %
inhibition against the leukemia cell lines compared to their ethyl ana-
logues (Table 5s).
2
.2.2. In vitro cytotoxicity
2.2.2.1. NCI-60 human cell lines
2.2.2.1.1. One-dose 60 cell line assay. All final derivatives were
accepted by the National Cancer Institute (NCI), and selected for one-
dose 60 human cancer cell line assay (breast, colon, leukemia,
melanoma, non-small cell lung, ovarian, prostate and renal cancer).
All derivatives exhibited broad spectrum cytotoxic inhibitory effect
against all NCI human cancer cell lines (Fig. 3 and Table 2s).
In general, compounds 13a and 13 g showed the highest % in-
hibition against nine cancer sub-types. 13a and 13 g showed 100%
inhibition for melanoma cell lines and 90% inhibition against leukemia
and prostate cancer. In addition, both compounds inhibited colon
cancer, NSCLC, ovarian cancer and breast cancer with % inhibition of
8
0%.
Compounds 13a and 13 g inhibited all melanoma cell lines at tested
dose. In addition, compounds 13c, 13d, 13e and 13f successfully in-
hibited LOX IMVI, M14, MDA-MB-435, SK-MEL-2, and UACC-62 cell
lines with % inhibition of 50%.
The in deep results study showed that all compounds had different
%
inhibition against NCI-60 human cancer cell lines. Compounds 12c,
1
3b-d and 13f showed moderate mean % inhibition against all cancer
cell lines. Compounds 13a and 13 g showed potent % inhibition against
NCI cancer cell lines. Compounds with propyl spacer showed higher
inhibitory activities than that of ethyl spacer.
In addition to the above mentioned cell lines, compounds 13a and
1
3 g completely inhibited NCI-H226, NCI-H23 and NCI-H522 non-small
cell lung cancer cell lines, NCI/ADRRES, OVCAR-3, OVCAR-4 and
Regarding breast cancer (Table 2s), compounds 13 g and 13a
4

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Table 4
Table 5
%
inhibition of most potent compounds (12a, 12c, 13a and 13 g) against NCI
GI50 and TGI values (µM) of compounds 13a and 13 g against NCI-60 cancer
6
0 cell lines at single dose (10 µM).
Cell line
cell lines.
NCI-cell lines
12b
12c
13a
13g
GI50
13a
TGI
13a
Breast Cancer
BT-549
74.35 73.58
17.10 33.56
64.59 78.07
27.01 39.80
100.00 100.00
13 g
13g
HS 578 T
MCF7
68.82 77.85
100.00 100.00
52.36 55.13
Breast Cancer
BT-549
MDA-MB-231/
ATCC
2.18
4.73
2.16
6.41
0.83
0.39
2.14
3.25
2.53
4.98
1.50
3.03
11.90
47.30
8.48
27.2
6.10
4.64
7.03
HS 578 T
MCF7
> 100
ND
MDA-MB-468
T-47D
56.63 71.71
100.00 100.00
86.42 100.00 100.00 100.00
MDA-MB-231/ATCC
MDA-MB-468
T-47D
> 100
7.11
CNS cancer
SF-268
54.23 60.24
34.34 62.59
12.89 25.46
24.26 38.59
29.94 48.61
42.12 62.80
88.17
94.17
65.23
58.80
60.22
90.68
94.43
73.54
70.67
73.22
SF-295
2.82
SF-539
CNS Cancer
SF-268
SNB-19
3.21
2.52
4.04
4.34
4.56
2.92
2.62
3.61
4.01
3.73
21.9
7.79
20.8
61.8
39.40
> 100
8.15
U251
SF-295
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
100.00 95.48
SF-539
> 100
> 100
> 100
ND
34.80
34.40
92.23
97.79
78.66
41.70
95.57
100.00
81.56
SNB-19
42.91 64.36
50.49 73.27
59.30 60.01
81.11 77.49
U251
Colon Cancer
COLO 205
HCC-2998
HCT-116
HT29
2.46
6.79
2.74
2.30
3.12
2.39
4.12
2.44
7.29
2.85
2.08
2.90
2.59
3.95
8.11
8.24
KM12
100.00 99.01
> 100
> 100
12.30
> 100
8.14
> 100
> 100
9.79
SW-620
CCRF-CEM
HL-60(TB)
K-562
ND
30.18
77.13
96.96
81.68
98.05
Leukemia
Melanoma
63.04 79.75
36.30 84.00
58.59 78.15
62.24 81.85
63.72 95.20
49.47 60.81
46.03 69.58
39.08 62.22
10.83 58.89
56.29 79.28
39.52 73.10
HCT-15
100.00 100.00
HT29
> 100
7.91
95.74
92.49
KM12
MOLT-4
RPMI-8226
SR
100.00 100.00
100.00 100.00
SW-620
> 100
> 100
Leukemia
CCRF-CEM
HL-60(TB)
K-562
86.78
90.99
1.30
2.97
1.73
1.17
9.84
3.10
1.20
2.55
1.62
1.23
0.68
2.53
> 100
8.57
7.81
LOX IMVI
M14
100.00 100.00
100.00 99.98
100.00 100.00
100.00 98.34
100.00 100.00
100.00 100.00
6.69
> 100
ND
> 100
5.52
MALME-3 M
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
A549/ATCC
EKVX
MOLT-4
RPMI-8226
SR
4.18
2.94
> 100
> 100
7.02
41.20
Melanoma
LOX IMVI
M14
81.90 100.00 100.00 100.00
2.58
2.82
2.14
2.98
2.04
2.63
0.82
2.71
1.16
2.57
2.72
2.00
2.55
2.06
2.75
1.06
2.54
1.20
11.00
12.00
7.06
11.6
5.98
7.76
2.10
8.27
4.26
1.04
> 100
6.76
7.93
5.43
7.92
2.45
7.96
4.21
29.58 50.88
53.68 86.79
53.94 68.11
34.58 60.48
100.00 99.98
100.00 100.00
MALME-3 M
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
Non-small cell lung
cancer
92.73
80.89
86.94
53.67
90.33
81.75
87.54
53.77
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
IGROV1
NCI/ADR-RES
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
DU-145
9.57
26.98
36.78 55.26
46.07 59.70
23.39 56.74
30.81 38.20
39.96 66.63
40.57 65.73
54.63 72.86
51.53 73.09
41.68 59.46
64.60 68.76
100.00 100.00
100.00 100.00
54.79
83.66
73.24
89.55
2.68
2.77
3.50
2.80
1.66
1.98
3.99
3.34
2.02
2.73
2.90
3.16
1.74
1.95
2.42
3.21
2.94
1.59
24.30
52.70
14.10
30.30
8.22
> 100
> 100
9.61
100.00 99.88
Ovarian cancer
96.82
94.97
HOP-62
100.00 100.00
100.00 95.88
100.00 100.00
HOP-92
> 100
7.60
NCI-H226
NCI-H23
7.34
9.16
ND 12.64
54.87 73.90
3.42 26.04
33.80
40.87
NCI-H322M
NCI-H460
NCI-H522
Ovarian Cancer
IGROV1
285
> 100
> 100
6.020
100.00 96.56
> 100
7.97
55.35
90.23
62.88
89.44
Prostate cancer
Renal cancer
49.90 51.53
64.70 82.99
36.21 39.66
45.75 63.41
59.56 77.54
50.20 69.75
44.24 43.98
56.80 67.01
17.51 42.09
74.34 82.72
PC-3
100.00 100.00
2.71
1.72
2.53
1.64
7.83
1.89
5.82
2.90
1.85
2.31
1.65
5.63
1.74
4.17
11.60
6.49
26.8
786–0
56.58
66.29
93.64
89.99
47.99
81.35
47.09
55.79
70.29
94.62
90.21
46.57
89.45
55.43
NCI/ADR-RES
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
7.31
A498
11.90
7.64
8.49
ACHN
6.55
CAKI-1
> 100
17.70
30.50
> 100
22.20
> 100
RXF 393
SN12C
TK-10
Prostate Cancer
DU-145
UO-31
100.00 100.00
2.60
1.37
2.65
1.14
12.90
6.62
12.90
4.82
PC-3
Renal Cancer
7
86–0
7.97
1.56
2.19
2.33
8.13
2.71
5.38
1.25
7.39
1.87
2.19
2.33
6.95
2.61
4.34
1.40
45.60
30.50
11.20
14.10
41.60
44.70
52.10
9.04
> 100
> 100
10.80
OVCAR-8 ovarian cancer cell lines, PC-3 prostate cancer cell line and
UO-31 renal cancer cell line (Tables 4, 1s-10s)
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
2
.2.2.1.2. Five-dose 60 cell line assay. Compounds 13a and 13 g (the
> 100
> 100
> 100
> 100
8.39
most active compounds in one-dose assay) were selected by the NCI to
be evaluated in five-dose assay in order to identify their GI50 values NCI
human cancer cell lines (Table 5 and 6).
UO-31
Compounds 13a and 13g showed mean GI50 of range 1.90 –
3
.94 µM against all NCI human cancer cell lines. They exhibited potent
mean GI50 against prostate cancer cell lines (mean GI50 1.99 and
.90 µM, respectively) and melanoma cell lines (mean GI50 2.21 and
1
5

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Table 6
Table 9
Mean GI50 and TGI of compounds 13a and 13 g against NCI-60 cancer cell lines.
IC50 (µM) of target compounds 12a-h and13a-h, human embryonic pulmonary
epithelial cells (L132).
Compd
NCI-cell lines
Mean GI50 (µM)
Mean TGI (µM)
L132 IC50 (µM)
Compd
L132 IC50 (µM)
1
3a
13g
13a
13g
1
2a
91
101
69
75
80
90
82
94
13a
13b
13c
13d
13e
13f
80
95
70
77
88
82
76
86
Breast Cancer
CNS Cancer
2.78
3.73
3.42
3.35
2.21
2.75
3.45
1.99
3.94
2.91
3.38
3.44
1.64
2.16
2.52
2.89
1.90
3.64
17.60
30.34
9.52
5.65
8.15
8.65
5.74
5.46
8.10
14.27
8.86
9.60
12b
12c
12d
12e
12f
Colon Cancer
Leukemia
6.38
Melanoma
7.78
Non-Small Cell Lung Cancer
Ovarian Cancer
Prostate Cancer
Renal Cancer
53.74
14.31
9.76
12 g
12h
13 g
13h
31.11
2
.2.2.2. In vitro assay against A375 melanoma cell line. Compounds 13a-
Table 7
h were selected for testing their cytotoxic inhibitory activity against
melanoma cell line (A375) (Table 8). Their activities were compared to
both vemurafenib and sorafenib as standards using MTT (3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)assay [33].
Mean GI50 and TGI of compounds 13a and 13 g compared to different singling
agents.
Compounds
Mean GI50 (µM)
Mean TGI (µM)
The results revealed that NO -bearing derivatives showed different
2
1
1
3a
3g
3.07
2.72
0.33
5.5
20.06
8.28
8.9
59
inhibitory activities against melanoma cell line (A375). In addition,
most of compounds had comparable inhibitory activities to sorafenib
(IC50 10.28 µM). Furthermore, compounds 13a and 13b exhibited po-
tent inhibitory activities (IC50 2.99 and 4.51 µM, respectively) com-
pared to sorafenib (IC50 10.28 µM).
Dasatinib
Erlotinib
Gefitinib
Imatinib
Lapatinib
Nilotinib
3.2
19
15
43
2.9
20
In addition to the ability of new target compounds to inhibit A375
proliferation, compounds 13a and 13 g were tested for their ability to
inhibit phosphorylation of both MEK and ERK in A375 cells. Both
compounds showed significant reduction in phosphorylation of both
enzyme starting from 1uM concentration. Both compounds were in-
cubated with A375 cells and phosphorylated MEK and ERK were in-
vestigated using western plot analysis at three different doses 1, 3 and
2.9
13
Table 8
IC50 (µM) of target compounds 13a-h, vemurafenib and sorafenib against
human melanoma cell line (A375).
1
0 uM (Fig. 4) .
Compd
A375 IC50 (µM)
Compd
A375 IC50 (µM)
In order to check the effect of the newly synthesized compounds
1
1
1
1
1
3a
3b
3c
3d
3e
2.99
13f
> 10
7.96
over normal cell line to determined they toxicity, all final target com-
pounds were tested over human embryonic pulmonary epithelial cells
4.51
13g
7.87
13h
> 10
10.28
5.9
(
L132). The IC50s over L132 cell line is presented in Table 9. All final
> 10
10.68
Sorafenib
Vemurafenib
target compounds showed at least 5 folds selectivity for cancer cell lines
compared to normal cell line. For series 12a-h, compounds 12b had the
highest IC50 with value 101 uM followed by 12 h and 12a with values
94 and 91uM. On the other hand, compound 13b showed the highest
IC50 with value 95 uM, followed by 13e, 13 h and 13f with IC50s 88, 86
and 82 uM, respectively.
2
.16 µM, respectively). Moreover, compound 13 g exhibited higher
inhibitory activities against all NCI human cancer cell lines than that of
3a (mean GI50 2.72 and 3.07 µM, respectively). The results revealed
1
that compounds with para substituted phenyl ring in side chain with F
group (13 g) were more active that that with meta F (13a).
Compounds 13a and 13 g showed activities that are more potent
compared to Erlotinib, Gefitinib and Imatinib [32] in term of mean GI50
over NCI 60 cell lines. Compound 13 g had lower GI50 compared to
Erlotinib, Gefitinib, Imatinib, Lapatinib, Nilotinib but higher compared
to Dasatinib. The total growth inhibition of 13 g was lower compared to
six singling agents while 13a was more potent than Erlotinib, and Im-
atinib.(see Table 7)
2.3. Molecular docking
The X-ray structural complex of PLX3203 (native ligand, PDB ID:
325) [34] with the B-Raf V600E protein (PDB ID: 4FK3) [33] was used
in the molecular docking study of target compounds (12a-h and 13a-h).
The docking protocol used for the docking of native ligand was valid
with virtual ligand screening (VLS) score of −6.6343 kcal/mol and
route mean square deviation (RMSD) of 1.2458 (Fig. 5).
Fig. 4. The ability of compounds 13a and 13g to inhibit phosphorylation of ERK and MEK on A375 cell line.
6

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Fig. 5. 2D interaction (A) and 3D interaction (B) of native ligand (PLX2303) with B-Raf V600E (PDB ID: 4FK3) active site.
Most of target compounds (12a-h and 13a-h) showed a number of
purification. All reactions were monitored by TLC using Hexane/Ethyl
acetate. All spots were visualized at 365 and 254 nm by Spectroline UV
ENF-240C/FE (Spectronics Co., Westbury, New York, USA). All melting
points were determined on Thomas-Hoover (Uni-Melt) Capillary
Melting Point Apparatus (Arthur H. Thomas Company, Philadelphia
PA., USA). The Nuclear Magnetic Resonance (NMR) were recorded on
Bruker ARX-400, 400 MHz spectrometer (Bruker Bioscience, Billerica,
MA, USA). Samples were prepared using deuterated Cambridge NMR
interactions with the conserved amino acid residues of the active site
(
Val 471, Lys 483, Cys 532 and Asp 594). In addition, they exhibited
VLS score range of −8.7141 to −6.2267 kcal/mol with different in-
teractions (arene-cation and H bonding interactions) (Table 9s).
The results showed that most of synthesized compounds made ad-
ditional interaction between substituted NO and the amino acid re-
2
sidues in the active site of B-Raf V600E (Lys 483 and Asp 594).
Moreover, compounds 12a, 12d, 13b and 13g exhibited higher binding
scores with the active site among the synthesized derivatives (S = -
solvents; CDCl , DMSO and MeOD. Chemical shift were quoted in ?? as
3
parts per million (ppm) downfield from TMS as internal standard. Mass
spectra (MS) were determined by LC-MS analysis using the following
system: Water 2998 photodiode array detector, Water 3100 mass de-
tector, Water SFO system fluidics organizer, Water 2545 binary gra-
8
5
1
.3757, −8.7141, −8.0109 and −8.0237 kcal/mol, respectively) with
or 6 visible interactions (Fig. 6). Furthermore, these compounds (12a,
2d, 13b and 13 g) showed additional H-bond interaction(s) through
incorporated NO
2
group.
dient module, Water reagent manager, Waters 2767 sample manager,
TM
From the molecular docking studies, it was investigated that sub-
Sunfire
C188 column (4.6 × 50 mm, 5 μm particle size); solvent
stituted NO group has a significant effect in the binding mode with the
2
gradient = 95% A at 0 min, 1% A at 5 min; solvent A = 0.04% tri-
fluoroacetic acid (TFA) in water; solvent B = 0.04% TFA in MeOH; flow
rate = 3.0 ml/min; AUC was calculated using Waters MassLynx 4.1
software.
B-Raf V600E active site through additional H-bond(s) with Lys 483 and
5
94.
3
. Conclusion
4.1. Chemistry
A new series of imidazo[2,1-b]thiazole-based compounds deriva-
tives was designed, synthesized and evaluated for their cytotoxic ac-
tivity and enzyme assay. Compounds 13a and 13 g were the most active
derivatives in cytotoxicity among the synthesized compounds.
Compound 13a showed potent in vitro enzyme inhibitory activity.
Generally, compounds with halogenated substitution were more potent
compared to other substitution or unsubstituted phenyl derivatives.
Moreover, compounds with propyl linker between aryl sulfonamide
terminal group and pyrimidine ring at position 5 of the imidazo[2,1-b]
thiazole ring were more potent compared to that with ethyl linker. The
4.1.1. General procedure for synthesis of N-(2-aminoethyl and 3-
aminopropyl)unsubstituted and substituted phenyl sulfonamide (3a-h and
4a-h):
A solution of appropriate sulfonyl chloride (2a-h, 5 mmol) in di-
chloromethane (2 ml) was added dropwise to a solution of proper
diamine (1a,b, 10 mmol) and trimethylamine (300 mg, 30 mmol) in
dichloromethane (20 ml) at 0 °C. The reaction mixture was allowed to
stir at rt for 18 h. The reaction mixture was washed with saturated
solution of NaHCO (20 ml). The organic layer was dried over anhy-
3
molecular docking studies revealed that the incorporated NO
2
group in
drous sodium sulfate and evaporated under vacuum to give the titled
the terminal phenyl ring had a significant effect in the binding to the B-
products 3a-h and 4a-h [30].
Raf V600E active site through additional H-bond interaction(s) with
conserved amino acid residues. Finally, these results afforded NO -
2
4
.1.2. Synthesis of 2-bromo-1-(3-nitrophenyl)ethan-1-one (6):
bearing imidazothiazole derivatives as a key scaffold for further mole-
cular and structural optimization.
N-bromosuccinamide (1.3 g, 7.2 mmol, 1.2 Eq) was added portion-
wise to a solution of compound 5 (1 g, 6 mmol, 1 Eq) in anhydrous DMF
(
5 ml) at rt. The reaction mixture was stirred at 60 °C for 3 h. The
4
. Experimental
reaction mixture was cooled and extracted between EtOAc (40 ml) and
water (60 ml). The organic layer was washed with water (3 × 50 ml).
All chemicals, including starting compounds, reagents and solvents,
The organic layer was dried over anhydrous Na
2
SO and evaporated
4
were obtained from Sigma-Aldrich Co., Tokyo Chemical Industry (TCI)
Co. and Daejung Chemicals & Metals Co. and used without any
under vacuum. The crude residue was purified through column chro-
matography (hexane, EtOAc; 100, 1) to give the titled product 6. Yield:
7

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
Fig. 6. 2D interaction of compounds 12a (A), 12d (B), 13b (C) and 13g (D) with B-Raf V600E (PDB ID: 4FK3) active site.
1
7
5%. m.p.: 95–6 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.71 (t,
4.1.4. Synthesis of 5-(2-(methylthio)pyrimidin-4-yl)-6-(3-nitrophenyl)
imidazo[2,1-b]thiazole (10):
J = 2.0 Hz, 1H, Ar-H), 8.51–8.48 (m, 1H, Ar-H), 8.44–8.41 (m, 1H, Ar-
1
3
H), 7.87 (t, J = 8.0 Hz, 1H, Ar-H), 5.06 (s, 1H, CH
100 MHz, DMSO‑d ) δ 190.75 (Ar-C), 148.46 (Ar-C), 135.67 (Ar-C),
35.33 (Ar-C), 131.13 (Ar-C), 128.37 (Ar-C), 123.46 (Ar-C), 34.61
2
Br). C NMR
A solution compound 9 (0.7 g, 4.1 mmol, 1 Eq) in anhydrous DMF
(5 ml) was added dropwise to a mixture of compound 8 (1 g, 4.1 mmol,
(
6
1
1 Eq), Ph
3
P (0.3 g, 1.3 mmol, 0.3 Eq), K
2
CO (0.6 g, 4.1 mmol, 1 Eq)
3
+
(
CH2Br). LC/MS: 245 (M + 1)
.
and Pd(OAc) (0.2 g, 0.8 mmol, 0.2 Eq) in anhydrous DMF (10 ml). The
2
reaction mixture was stirred at 80 °C for 8 h. The reaction mixture was
cooled and stirred with crushed ice (30 g). The crude ppt was filtered.
The crude solid residue was stirred in MeOH (50 ml) at 60 °C for 1 h.
The solid product was filtered and washed with MeOH (3 × 20 ml) and
4
.1.3. Synthesis of 6-(3-nitrophenyl)imidazo[2,1-b]thiazole (8):
A solution of compound 6 (1 g, 4.1 mmol, 1 Eq) and compound 7
1
(
0.5 g, 4.9 mmol, 1.2 Eq) in MeOH (50 ml) was stirred under reflux for
dried to give the titled product 10. Yield: 30%. m.p.: 187–9 °C. H NMR
1
8 h. The organic solvent was evaporated under vacuum. The crude
(400 MHz, CDCl ) δ 8.58 (d, J = 4.4 Hz, 2H, Ar-H), 8.34–8.31 (m, 2H,
3
solid ppt was stirred in NH
4
OH solution at rt for 2 h. The crude solid
Ar-H), 8.02 (d, J = 8.0 Hz, 1H, Ar-H), 7.66 (t, J = 8.0 Hz, 1H, Ar-H),
product was filtered and washed with cold water (3 × 100 ml) and
7.06 (d, J = 4.4 Hz, 1H, Ar-H), 6.86 (d, J = 5.6 Hz, 1H, Ar-H), 2.66 (s,
1
13
dried to give the titled product 8. Yield: 90%. m.p.: 167–9 °C. H NMR
3H, SCH
3
). C NMR (100 MHz, CDCl
3
) δ 173.15 (Ar-C), 156.83 (Ar-C),
(
400 MHz, DMSO‑d
6
) δ 8.63 (t, J = 2.2 Hz, 1H, Ar-H), 8.46 (s, 1H, Ar-
155.66 (Ar-C), 153.01 (Ar-C), 148.57 (Ar-C), 147.65 (Ar-C), 136.32
(Ar-C), 135.04 (Ar-C), 129.83 (Ar-C), 124.14 (Ar-C), 123.53 (Ar-C),
121.96 (Ar-C), 120.82 (Ar-C), 113.65 (Ar-C), 112.00 (Ar-C), 14.23
H), 8.27–8.24 (m, 1H, Ar-H), 8.09–8.06 (m, 1H, Ar-H), 7.98 (d,
J = 4.4 Hz, 1H, Ar-H), 7.67 (t, J = 8.2 Hz, 1H, Ar-H), 7.32 (d,
1
3
). LC/MS 371 (M + 1)+
.
J = 4.4 Hz, 1H, Ar-H). C NMR (100 MHz, DMSO‑d
6
) δ 150.25 (Ar-C),
(SCH
3
1
48.78 (Ar-C), 144.40 (Ar-C), 136.46 (Ar-C), 131.26 (Ar-C), 130.61
(
Ar-C), 121.87 (Ar-C), 120.52 (Ar-C), 119.32 (Ar-C), 114.39 (Ar-C),
4.1.5. Synthesis of 5-(2-(methylsulfonyl)pyrimidin-4-yl)-6-(3-nitrophenyl)
imidazo[2,1-b]thiazole (11):
+
1
11.44 (Ar-C). LC/MS 246 (M + 1)
.
A solution of potassium peroxymonosulfate (5 g, 8.1 mmol, 3 Eq) in
8

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
water (20 ml) was added dropwise to a solution of compound 10 (1 g,
(Ar-C), 130.68 (Ar-C), 130.47 (Ar-C), 130.15 (Ar-C), 129.58 (Ar-C),
2
.7 mmol, 1 Eq) in MeOH (50 ml). The reaction mixture was stirred at rt
125.25 (CF3), 123.78 (Ar-C), 123.41 (Ar-C), 55.37 (Aliph-C), 42.35
for 9 h. The organic solvent was evaporated under vacuum. The crude
residue was extracted between DCM (70 ml) and water (30 ml). The
organic layer was washed with brine solution (3 × 30 ml). The organic
(Aliph-C). LC/MS 591 (M + 1)+
.
4.1.6.4. 4-Methoxy-N-(2-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-
layer was dried over anhydrous Na
2
SO
4
and evaporated under reduced
yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (12d):. Yield: 80%.
1
pressure. The crude solid residue was purified through column chro-
m.p.: 96–9 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.44 (s, 1H, Ar-H),
matography (hexane, EtOAc; 2, 1) to give the titled product 11. Yield:
8.28 (d, J = 8.0 Hz, 1H, Ar-H), 8.39 (d, J = 5.2 Hz, 1H, Ar-H), 8.08 (d,
J = 8.0 Hz, 1H, Ar-H), 7.73 (d, J = 8.8 Hz, 3H, Ar-H), 7.66 (d,
J = 8.8 Hz, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.08 (t, J = 4.4 Hz, 2H, Ar-
1
8
1
8
0%. m.p.: 215–6 °C. H NMR (400 MHz, CDCl
3
) δ 8.91 (d, J = 4.4 Hz,
H, Ar-H), 8.61 (t, J = 5.6 Hz, 2H, Ar-H), 8.39–8.37 (m, 1H, Ar-H),
.04 (d, J = 8.0 Hz, 1H, Ar-H), 7.75 (t, J = 7.6 Hz, 1H, Ar-H), 7.35 (d,
H), 6.42 (s, 1H, NH), 3.79 (s, 3H, OCH ), 3.40 (s, 2H, Aliph-H), 2.94 (d,
3
1
3
J = 5.6 Hz, 1H, Ar-H), 7.17 (d, J = 4.8 Hz, 1H, Ar-H), 3.42 (s, 3H,
J = 6.0 Hz, 2H, Aliph-H). C NMR (100 MHz, DMSO‑d ) δ 167.63(Ar-
6
1
3
SO
2
CH
3
). C NMR (100 MHz, CDCl
3
) δ 166.13 (Ar-C), 157.49 (Ar-C),
C), 167.52 (Ar-C), 148.30 (Ar-C), 136.65 (Ar-C), 135.19 (Ar-C), 132.45
(Ar-C), 132.23(Ar-C), 130.69 (Ar-C), 129.10 (Ar-C), 129.00(Ar-C),
1
54.59 (Ar-C), 149.59 (Ar-C), 148.70 (Ar-C), 135.71 (Ar-C), 130.32
(
Ar-C), 124.21 (Ar-C), 123.16 (Ar-C), 120.01 (Ar-C), 117.48 (Ar-C),
123.22 (Ar-C), 122.53(Ar-C), 114.83 (Ar-C), 114.22(Ar-C), 56.07
+
+
1
14.79 (Ar-C), 39.27 (SO
2
CH
3
). LC/MS 403 (M + 1)
.
(OCH
3
), 42.57 (Aliph-C), 42.36 (Aliph-C). LC/MS 553 (M + 1)
.
4
.1.6. General procedure for synthesis of N-substituted-4-(6-(3-
4.1.6.5. 4-Chloro-N-(2-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
nitrophenyl)imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-amine (12a-h and
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (12e):. Yield: 45%.
1
13a-h):
m.p.: 150–1 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.44 (s, 1H, Ar-H),
A mixture of compound 11 (0.2 g, 0.5 mmol, 1 Eq), appropriate
8.28 (d, J = 7.6 Hz, 1H, Ar-H), 8.14 (d, J = 5.2 Hz, 1H, Ar-H), 8.08 (d,
J = 8.0 Hz, 1H, Ar-H), 7.81 (d, J = 6.8 Hz, 2H, Ar-H), 7.76 (t,
J = 8.0 Hz, 1H, Ar-H), 7.57–7.63 (m, 3H, Ar-H), 7.52 (s, 1H, Ar-H),
6.43 (s, 1H, NH), 3.41 (s, 2H, Aliph-H), 2.97 (d, J = 5.2 Hz, 2H, Aliph-
amine (3a-h and 4a-h, 0.6 mmol, 1.2 Eq) and N,N-diisopropylethyla-
mine (0.6 g, 1.3 ml, 4.5 mmol, 9 Eq) in anhydrous DMSO (2 ml). The
reaction mixture was stirred at 90 °C for 9 h. The reaction mixture was
cooled and extracted between EtOAc (10 ml) and water (30 ml). The
organic layer was washed with water (3 × 30 ml). The organic layer
H). LC/MS 557 (M + 1)+
.
was dried over anhydrous Na
SO
2 4
and evaporated under reduced
4.1.6.6. 4-Bromo-N-(2-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
pressure. The crude residue was purified through column chromato-
graphy (hexane, EtOAc; 1, 1) to give the titled product 12a-h and 13 a-
h, respectively.
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (12f):. Yield: 60%. m.p.:
1
118–9 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.44 (s, 1H, Ar-H), 8.28 (d,
J = 7.6 Hz, 1H, Ar-H), 8.13 (d, J = 4.8 Hz, 1H, Ar-H), 8.08 (d,
J = 8.0 Hz, 1H, Ar-H), 7 (s, 1H, Ar-H), 7.73–7.77 (m, 5H, Ar-H), 7.52
(s, 1H, Ar-H), 6.42 (s, 1H, Ar-H), 3.36 (s, 2H, Aliph-H), 3.00 (t,
4
.1.6.1. 3-Fluoro-N-(2-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
1
3
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (12a):. Yield: 60%.
J = 6.4 Hz, 2H, Aliph-H). C NMR (100 MHz, DMSO‑d ) δ 163.12 (Ar-
6
1
m.p.: 161–3 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.44 (s, 1H, Ar-H),
C), 162.98(Ar-H), 148.37 (Ar-C), 140.16 (Ar-C), 136.66 (Ar-C), 132.68
(Ar-C), 130.69 (Ar-C), 128.98 (Ar-C), 126.6 (Ar-C), 123.74 (Ar-C),
119.54 (Ar-C), 114.37 (Ar-C), 114.16 (Ar-C), 49.45 (Aliph-C), 42.31
8
.28 (d, J = 8.0 Hz, 1H, Ar-H), 8.13 (d, J = 5.2 Hz, 1H, Ar-H), 8.08 (d,
J = 4.0 Hz, 1H, Ar-H), 7.93 (s, 1H, NH), 7.78 (s, 1H, Ar-H), 7.56–7.65
m, 4H, Ar-H), 7.48–7.55 (m, 3H, Ar-H), 3.41 (s, 2H, Aliph-H), 3.02 (t,
(
(Aliph-C). LC/MS 601 (M + 1)+
.
1
3
J = 4.0 Hz, 2H, Aliph-H). C NMR (100 MHz, DMSO‑d
C), 160.98 (Ar-C), 148.38 (Ar-C), 143.04 (Ar-C), 136.64 (Ar-C), 135.76
Ar-C), 132.08 (Ar-C), 130.69 (Ar-C), 123.51 (Ar-C), 123.17 (Ar-C),
6
) δ 163.44 (Ar-
4.1.6.7. 4-Fluoro-N-(2-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
(
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (12 g):. Yield: 65%.
1
1
20.27 (Ar-C), 120.09 (Ar-C), 119.88 (Ar-C), 114.08 (Ar-C), 113.84
m.p.: 160–2 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.44 (s, 1H, Ar-H),
(
Ar-C), 42.65 (Aliph-C), 42.37 (Aliph-C). LC/MS 541 (M + 1)+
.
8.28 (d, J = 7.2 Hz, 1H, Ar-H), 8.14 (d, J = 5.2 Hz, 1H, Ar-H), 8.08 (d,
J = 8.0 Hz, 1H, Ar-H), 7.85–7.89 (m, 2H, Ar-H), 7.81 (d, J = 6.8 Hz,
1H, Ar-H), 7.76 (d, J = 8.0 Hz, 1H, Ar-H), 7.54–7.61 (m, 1H, Ar-H),
7.52 (s, 1H, NH), 7.38–7.43 (m, 2H, Ar-H), 6.43 (s, 1H, Ar-H), 3.41 (s,
4
.1.6.2. 4-Methyl-N-(2-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (12b):. Yield: 80%.
1
13
m.p.: 98–100 °C.
H
NMR (400 MHz, DMSO‑d
6
)
δ
8.44 (t,
2H, Aliph-H), 2.98 (d, J = 5.2 Hz, 2H, Aliph-H). C NMR (100 MHz,
J = 1.6 Hz, 1H, Ar-H), 8.28 (d, J = 7.6 Hz, 1H, Ar-H), 8.13 (d,
J = 4.8 Hz, 1H, Ar-H), 8.08 (d, J = 7.6 Hz, 1H, Ar-H), 7.78 (s, 1H, Ar-
H), 7.68 (d, J = 8.4 Hz, 3H, Ar-H), 7.62 (d, J = 8.4 Hz, 1H, Ar-H), 7.52
DMSO‑d ) δ 163.67 (Ar-C),148.38 (Ar-C), 136.64 (Ar-C), 130.69 (Ar-C),
6
130.00 (Ar-C), 129.64 (Ar-C), 126.92 (Ar-C), 123.77 (Ar-C), 123.53
(Ar-C), 116.87 (Ar-C), 116.64 (Ar-C), 42.59 (Aliph-C), 42.34 (Aliph-C).
(
s, 1H, NH), 7.34–7.43 (m, 3H, Ar-H), 6.42 (s, 1H, NH), 3.40 (s, 2H,
LC/MS 541 (M + 1)+
.
1
3
Aliph-H), 2.95 (d, J = 6.0 Hz, 2H, Aliph-H), S.34 (s, 3H, CH
3
). C NMR
(
100 MHz, DMSO‑d
6
) δ 149.39 (Ar-C), 143.18 (Ar-C), 143.09 (Ar-C),
4.1.6.8. N-(2-((4-(6-(3-Nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
1
37.89 (Ar-C), 137.83 (Ar-C), 136.66 (Ar-C), 135.77 (Ar-C), 130.69
pyrimidin-2-yl)amino)ethyl)naphthalene-1-sulfonamide (12 h):. Yield:
1
(
Ar-C), 130.11 (Ar-C), 130.04 (Ar-C),126.95 (Ar-C), 124.93 (Ar-C),
70%. m.p.: 148–9 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.27 (s, 2H,
1
24.91(Ar-C), 123.74 (Ar-C), 124.52 (Ar-C), 42.6 (Aliph-C), 42.37
Ar-H), 8.06 (d, J = 8.4 Hz, 1H, Ar-H), 7.95 (s, 4H, Ar-H), 7.82 (d,
J = 8.4 Hz, 1H, Ar-H), 7.74 (d, J = 7.6 Hz, 1H, Ar-H), 7.62 (d,
J = 9.2 Hz, 1H, Ar-H), 7.63 (d, J = 8.4 Hz, 3H, Ar-H), 7.51 (s, 1H, Ar-
+
(
Aliph-C), 21.4 (CH
3
). LC/MS 537 (M + 1)
.
1
3
4
.1.6.3. N-(2-((4-(6-(3-Nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
H), 6.33 (s, 1H, Ar-H), 3.39 (s, 2H, Aliph-H), 3.04 (s, 2H, Aliph-H).
C
pyrimidin-2-yl)amino)ethyl)-4-(trifluoromethyl)benzenesulfonamide
NMR (100 MHz, DMSO‑d ) δ167.27 (Ar-C), 163.45 (Ar-C), 148.37 (Ar-
6
1
(
12c):. Yield: 85%. m.p.: 145–7 °C. H NMR (400 MHz, DMSO‑d
6
) δ
C), 136.66 (Ar-C), 134.52 (Ar-C), 132.12 (Ar-C), 130.68 (Ar-C), 129.78
(Ar-C), 129.53 (Ar-C), 128.99 (Ar-C), 128.15 (Ar-C), 127.85 (Ar-C),
127.77 (Ar-C), 122.68 (Ar-C), 114.26 (Ar-C), 108.06(Ar-C), 42.58
8
8
.44 (s, 1H, Ar-H), 8.28 (d, J = 7.6 Hz, 1H, Ar-H), 8.13 (s, 2H, Ar-H),
.08 (d, J = 6.4 Hz, 3H, Ar-H), 8.01 (d, J = 8.0 Hz, 1H, Ar-H), 7.83 (t,
J = 8.4 Hz, 1H, Ar-H), 7.76 (t, J = 8.0 Hz, 1H, Ar-H), 7.51 (s, 1H, Ar-
H), 6.43 (s, 1H, Ar-H), 3.41 (s, 2H, Aliph-H), 3.03 (t, J = 6.4 Hz, 2H,
(Aliph-C), 42.38 (Aliph-C). LC/MS 573 (M + 1)+
.
1
3
Aliph-H). C NMR (100 MHz, DMSO‑d
6
) δ 162.32 (Ar-C),148.38 (Ar-
4.1.6.9. 3-Fluoro-N-(3-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
C), 142.16 (Ar-C), 136.64 (Ar-C), 135.75 (Ar-C), 131.31 (Ar-C), 131.64
pyrimidin-2-yl)amino)propyl)benzenesulfonamide (13a):. Yield: 65%.
9

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
1
m.p.: 155–7 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.44 (s, 1H, Ar-H), 8.27
4.1.6.14. 4-Bromo-N-(3-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-
(
d, J = 8.0 Hz, 1H, Ar-H), 8.13 (d, J = 5.2 Hz, 1H, Ar-H), 8.12 (d,
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (13f):. Yield: 70%.
1
J = 7.6 Hz, 1H, Ar-H), 7.76 (q, 2H, Ar-H), 7.64 (s, 2H, Ar-H), 7.58 (d,
J = 8.4 Hz, 1H, Ar-H), 7.52 (d, J = 4.6 Hz, 2H, Ar-H), 6.41 (s, 1H, Ar-
H), 3.31 (t, J = 6.4 Hz, 2H, Aliph-H), 2.91 (s, 2H, Aliph-H), 1.71 (t,
m.p.: 105–6 °C. H NMR (400 MHz, CDCl
3
) δ 8.58 (s, 1H, Ar-H), 8.53
(d, J = 4.0 Hz, 1H, Ar-H), 8.33 (d, J = 8.0 Hz, 2H, Ar-H), 8.03 (d,
J = 8.0 Hz, 2H, Ar-H), 7.72 (t, J = 8.4 Hz, 3H, Ar-H), 7.63 (d,
J = 8.4 Hz, 3H, Ar-H), 7.16 (s, 1H, NH), 6.50 (d, J = 5.6 Hz, 1H, Ar-H),
3.69 (s, 2H, Aliph-H), 3.14 (d, J = 5.2 Hz, 2H, Aliph-H), 1.92 (s, 2H,
1
3
J = 6.8 Hz, 2H, Aliph-H). C NMR (100 MHz, DMSO‑d ) δ 163.45 (Ar-
6
C), 162.64 (Ar-C), 160.99 (Ar-C), 151.89 (Ar-C), 148.36 (Ar-C), 143.08
Ar-C), 136.69 (Ar-C), 135.77 (Ar-C), 132.04 (Ar-C), 130.63 (Ar-C),
1
3
(
Aliph-H). C NMR (100 MHz, CDCl ) δ 162.33 (Ar-C), 157.65 (Ar-C),
3
1
23.33 (Ar-C), 120.03 (Ar-C), 119.81 (Ar-C), 113.91 (Ar-C), 41.12
152.51 (Ar-C), 148.38 (Ar-C), 146.91 (Ar-C), 139.27 (Ar-C), 136.41
(Ar-C), 132.42 (Ar-C), 129.65 (Ar-C), 128.42 (Ar-C), 124.15 (Ar-C),
(
Aliph-C), 38.7 (Aliph-C), 29.47 (Aliph-C). LC/MS 555 (M + 1)+
.
1
23.29 (Ar-C), 121.44 (Ar-C), 113.71 (Ar-C), 107.26 (Ar-C), 40.22
+
4
.1.6.10. 4-Methyl-N-(3-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-
(Aliph-C), 38.18 (Aliph-C), 29.93 (Aliph-C). LC/MS 616 (M + 1)
.
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (13b):. Yield: 75%.
1
m.p.: 103–5 °C. H NMR (400 MHz, DMSO‑d
6
) δ 8.17 (s, 1H, NH), 8.11
4.1.6.15. 4-Fluoro-N-(3-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-
(
(
d, J = 5.2 Hz, 1H, Ar-H), 7.66 (d, J = 7.6 Hz, 2H, Ar-H), 7.55–7.51
m, 2H, Ar-H), 7.46 (d, J = 8.0 Hz, 1H, Ar-H), 7.41 (d, J = 8.8 Hz, 1H,
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (13 g):. Yield: 60%.
1
m.p.: 158–160 °C. H NMR (400 MHz, CDCl
3
) δ 8.57 (s, 1H, Ar-H), 8.47
Ar-H), 7.35 (d, J = 6.8 Hz, 3H, Ar-H), 7.29 (t, J = 8.8 Hz, 1H, Ar-H),
(s, 1H, Ar-H), 8.27 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 8.03 (s, 1H, Ar-H),
7.85 (s, 2H, Ar-H), 7.63 (s, 1H, Ar-H), 7.04–7.30 (m, 3H, Ar-H), 6.47 (s,
6
2
2
1
.41 (d, J = 4.0 Hz, 1H, Ar-H), 3.35 (s, 3H, CH3), 3.31 (d, J = 9.6 Hz,
H, Aliph-H), 2.83 (t, J = 6.0 Hz, 2H, Aliph-H), 1.69 (t, J = 6.8 Hz,
1H, Ar-H), 5.59 (s, 1H, NH), 3.59 (s, 2H, Aliph-H), 3.09 (s, 2H, Aliph-
1
3
13
H,). C NMR (100 MHz, DMSO‑d
6
) δ 163.699 (Ar-C), 162.56 (Ar-C),
H), 1.83 (s, 2H, Aliph-H). C NMR (100 MHz, CDCl
3
) δ 166.21 (Ar-C),
61.27 (Ar-C), 155.63 (Ar-C), 142.94 (Ar-C), 138.03 (Ar-C), 137.60
163.68 (Ar-C), 162.44 (Ar-C), 157.81 (Ar-C), 156.67 (Ar-C), 152.44
(Ar-C), 148.36 (Ar-C), 14,678 (Ar-C), 136.35 (Ar-C), 135.17 (Ar-C),
129.59 (Ar-C), 124.14 (Ar-C), 121.45 (Ar-C), 116.33 (Ar-C), 113.62
(Ar-C), 107.25 (Ar-C), 40.23 (Aliph-C), 38.19 (Aliph-C), 29.91 (Aliph-
(
Ar-C), 131.05 (Ar-C), 130.97 (Ar-C), 126.953 (Ar-C), 125.79 (Ar-C),
1
16.34 (Ar-C), 115.89 (Ar-C), 41.09 (Aliph-C), 29.52 (Aliph-C), 21.36
+
(
Aliph-C). LC/MS 551 (M + 1)
.
C). LC/MS 555 (M + 1)+
.
4
.1.6.11. N-(3-((4-(6-(3-Nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
pyrimidin-2-yl)amino)propyl)-4-(trifluoromethyl)benzenesulfonamide
4.1.6.16. N-(3-((4-(6-(3-Nitrophenyl)imidazo[2,1-b]thiazol-5-yl)
1
(
13c):. Yield: 70%. m.p.: 98–100 °C. H NMR (400 MHz, CDCl
3
) δ 8.57
pyrimidin-2-yl)amino)propyl)naphthalene-1-sulfonamide (13 h):. Yield:
1
(
(
s, 1H, Ar-H), 8.46 (s, 1H, Ar-H), 8.27 (d, J = 7.2 Hz, 1H, Ar-H), 8.11
d, J = 3.2 Hz, 1H, Ar-H), 2.02 (d, J = 6.4 Hz, 1H, Ar-H), 7.95 (d,
80%. m.p.: 95–7 °C. H NMR (400 MHz, CDCl
3
) δ 8.52 (s, 1H, Ar-H),
8.40 (d, J = 8.6 Hz, 2H, Ar-H), 8.23 (d, J = 5.2 Hz, 1H, Ar-H),
7.87–8.01 (m, 4H, Ar-H), 7.49–7.71 (m, 3H, Ar-H), 7.28–7.36 (m, 1H,
Ar-H), 7.00 (s, 1H, Ar-H), 6.40 (s, 1H, Ar-H), 5.72 (s, 1H, Ar-H), 3.58 (s,
J = 6.0 Hz, 2H, Ar-H), 7.75 (d, J = 6.8 Hz, 2H, Ar-H), 7.63 (d,
J = 7.2 Hz, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 6.48 (s, 1H, Ar-H), 5.54 (s,
1
3
1
H, NH), 3.61 (d, J = 3.6 Hz, 2H, Aliph-H), 3.14 (s, 2H, Aliph-H), 1.84
2H, Aliph-H), 3.13 (s, 2H, Aliph-H), 1.83 (s, 2H, Aliph-H). C NMR
1
3
(
s, 2H, Aliph-H). C NMR (100 MHz, CDCl
Ar-C), 156.75 (Ar-C), 152.50 (Ar-C), 148.37 (Ar-C), 146.87 (Ar-C),
44.01 (Ar-C), 136.36 (Ar-C), 135.13 (Ar-C), 129.64 (Ar-C), 127.38
3
) δ 162.47 (Ar-C), 157.81
(100 MHz, CDCl ) δ 162.38 (Ar-C), 157.77 (Ar-C), 156.52 (Ar-C),
3
(
152.38 (Ar-C), 148.32 (Ar-C), 146.64 (Ar-C), 136.62 (Ar-C), 134.67
(Ar-C), 132.08 (Ar-C), 129.48 (Ar-C), 127.67 (Ar-C), 124.11 (Ar-C),
123.18 (Ar-C), 122.11 (Ar-C), 121.71 (Ar-C), 113.56 (Ar-C), 107.05
(Ar-C), 40.40 (Aliph-C), 38.27 (Aliph-C), 29.81 (Aliph-C). LC/MS 587
1
(
Ar-C), 126.26 (Ar-C), 124.12 (CF
3
), 123.28 (Ar-C), 121.49 (Ar-C),
1
13.66 (Ar-C), 107.37 (Ar-C), 40.21 (Aliph-C), 38.12 (Aliph-C), 30.1
+
(M + 1)+
.
(
Aliph-C). LC/MS 605 (M + 1)
.
4
.1.6.12. 4-Methoxy-N-(3-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-
4.2. Biological evaluation
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (13d):. Yield: 80%.
1
m.p.: 153–5 °C. H NMR (400 MHz, CDCl
3
) δ 8.57 (s, 1H, Ar-H), 8.49 (s,
4.2.1. In vitro enzyme assay
1
H, Ar-H), 8.26 (d, J = 6.4 Hz, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 8.03 (d,
J = 6.4 Hz, 1H, Ar-H), 7.77 (s, 2H, Ar-H), 7.63 (s, 1H, Ar-H), 6.93–7.00
m, 4H, Ar-H), 6.46 (s, 1H, Ar-H), 5.61 (s, 1H, Ar-H), 3.84 (s, 3H,
OCH ), 3.60 (s, 2H, Aliph-C), 3.08 (s, 2H, Aliph-C), 1.84 (s, 2H, Aliph-
Reaction Biology Corp. Kinase HotSpotSM service was used for
screening of final compounds. Assay protocol: as reported on Reaction
Biology Corp. website using 1 µM concentration of ATP [35]. Isolated
human BRAF (V599E) was used and MEK1 was used as substrate at 1
(
3
1
3
33
C). C NMR (100 MHz, CDCl
3
) δ 162.80 (Ar-C), 157.67 (Ar-C), 156.60
uM concentration and 1uM ATP concentration ( P labeled ATP was
3
3
(
Ar-C), 152.42 (Ar-C), 148.37 (Ar-C), 146.71 (Ar-C), 136.51 (Ar-C),
used to produce P-Substrate which was a measure for enzyme ac-
tivity).
1
31.44 (Ar-C), 129.11 (Ar-C), 124.15 (Ar-C), 123.21 (Ar-C), 121.71
(
Ar-C), 114.31 (Ar-C), 113.61 (Ar-C), 107.15 (Ar-C), 55.59 (OCH
3
),
4
0.15 (Aliph-C), 38.29 (Aliph-C), 29.79 (Aliph-C). LC/MS 567
4.2.2. In vitro cytotoxicity
+
(
M + 1)
.
Screening against the cancer cell lines was carried out for com-
pounds 12a-h and 13a-h at the National Cancer Institute (NCI),
Bethesda, Maryland, USA, applying the standard protocol of the NCI. In
addition, 13a-h were evaluated for their antiproliferative activity
against melanoma cell lines (A375) using MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay [33].
4
.1.6.13. 4-Chloro-N-(3-((4-(6-(3-nitrophenyl)imidazo[2,1-b]thiazol-5-
yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (13e):. Yield: 75%.
1
m.p.: 100–2 °C. H NMR (400 MHz, CDCl
3
) δ 8.58 (s, 1H, Ar-H), 8.49 (s,
1
7
H, Ar-H), 8.27 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 8.04 (s, 1H, Ar-H),
.85 (s, 2H, Ar-H), 7.63 (s, 2H, Ar-H), 7.55 (s, 1H, Ar-H), 7.49 (s, 1H,
Ar-H), 7.04 (s, 1H, NH), 6.47 (s, 1H, Ar-H), 5.57 (s, 1H, NH), 3.60 (s,
4.3. Molecular docking
1
3
2
H, Aliph-H), 3.10 (s, 2H, Aliph-H), 1.85 (s, 2H, Aliph-H). C NMR
) δ 162.44 (Ar-C), 157.85 (Ar-C), 156.65 (Ar-C),
52.43 (Ar-C), 148.38 (Ar-C), 146.76 (Ar-C), 140.11 (Ar-C), 136.49
(
100 MHz, CDCl
3
The X-ray structure of mutated B-Raf (B-Raf V600E) with PLX3203
(native ligand) was downloaded from the protein databank RCSB PDB
(PDB ID: 4FK3) [34,36]. The X-ray structure of B-Raf V600E was vi-
sualized and manipulated by MOE 2014.13 (Molecular Operating En-
vironment) [37]. The PDB file consist of one domain in form of five
1
(
Ar-C), 132.58 (Ar-C), 129.21 (Ar-C), 126.85 (Ar-C), 124.16 (Ar-C),
1
23.24 (Ar-C), 121.52 (Ar-C), 113.61 (Ar-C), 107.21 (Ar-C), 40.26
(
Aliph-C), 38.22 (Aliph-C), 29.93 (Aliph-C). LC/MS 571 (M + 1)+
.
10

U.M. Ammar, et al.
Bioorganic Chemistry 100 (2020) 103967
chains; two chains of amino acid residues belonging to the mutated B-
Raf, one ligand (PLX3203, PDB ID: 325) and two chains of water mo-
lecules of crystallization.
103349.
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.3.1. Preparing the B-Raf protein structure for molecular docking
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enzyme. The Rigid Protein Docking protocol was used in the validation
step. Triangle Matcher method (bond rotation method) was used in
order to generate the native ligand conformations. The produced con-
formations were ranked with the London dG scoring function. The
minimization of conformations energies was accomplished with
Forcefield functional form. The identified conformations were rescored
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(
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.3.3. Docking of target compounds
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