Discovery of New Imidazo[2,1- b]thiazole Derivatives as Potent Pan-RAF Inhibitors with Promising in Vitro and in Vivo Anti-melanoma Activity

Article abstract of DOI:10.1021/acs.jmedchem.1c00230

BRAF is an important component of MAPK cascade. Mutation of BRAF, in particular V600E, leads to hyperactivation of the MAPK pathway and uncontrolled cellular growth. Resistance to selective inhibitors of mutated BRAF is a major obstacle against treatment of many cancer types. In this work, a series of new (imidazo[2,1-b]thiazol-5-yl)pyrimidine derivatives possessing a terminal sulfonamide moiety were synthesized. Pan-RAF inhibitory effect of the new series was investigated, and structure-activity relationship is discussed. Antiproliferative activity of the target compounds was tested against the NCI-60 cell line panel. The most active compounds were further tested to obtain their IC50 values against cancer cells. Compound 27c with terminal open chain sulfonamide and 38a with a cyclic sulfamide moiety showed the highest activity in enzymatic and cellular assay, and both compounds were able to inhibit phosphorylation of MEK and ERK. Compound 38a was selected for testing its in vivo activity against melanoma. Cellular and animal activities are reported.

Full text of DOI:10.1021/acs.jmedchem.1c00230

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Article
Discovery of New Imidazo[2,1‑b]thiazole Derivatives as Potent Pan-
RAF Inhibitors with Promising In Vitro and In Vivo Anti-melanoma
Activity
Mohammed S. Abdel-Maksoud, Mohammed I. El-Gamal, Bong S. Lee, Mahmoud M. Gamal El-Din,
Hong R. Jeon, Dow Kwon, Usama M. Ammar, Karim I. Mersal, Eslam M. H. Ali, Kyung-Tae Lee,
Kyung Ho Yoo, Dong Keun Han, Jae Kyun Lee, Garam Kim, Hong Seok Choi, Young Jik Kwon,
Kwan Hyi Lee, and Chang Hyun Oh*
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ABSTRACT: BRAF is an important component of MAPK
cascade. Mutation of BRAF, in particular V600E, leads to
hyperactivation of the MAPK pathway and uncontrolled cellular
growth. Resistance to selective inhibitors of mutated BRAF is a
major obstacle against treatment of many cancer types. In this
work, a series of new (imidazo[2,1-b]thiazol-5-yl)pyrimidine
derivatives possessing a terminal sulfonamide moiety were
synthesized. Pan-RAF inhibitory effect of the new series was
investigated, and structure−activity relationship is discussed.
Antiproliferative activity of the target compounds was tested
against the NCI-60 cell line panel. The most active compounds
were further tested to obtain their IC50 values against cancer cells.
Compound 27c with terminal open chain sulfonamide and 38a
with a cyclic sulfamide moiety showed the highest activity in enzymatic and cellular assay, and both compounds were able to inhibit
phosphorylation of MEK and ERK. Compound 38a was selected for testing its in vivo activity against melanoma. Cellular and animal
activities are reported.
poor prognosis in ovarian18 and androgen-insenstive prostate
INTRODUCTION
■
cancer.19
The MAPK/ERK pathway is a cascade of events that is
Sorafenib (I, Figure 1) was initially introduced as a CRAF
considered as a fundamental action for cell growth and
inhibitor to treat RAS-mutated cancer20 prior to the emerging
survival.1 Any mutation of one or more members of this
of BRAF mutation in cancer and it is binding to the ATP
binding pocket. It is considered as the first RAF inhibitor to be
pathway leads to hyperactivation and ultimately cancer.2 RAF
kinases are serine/threonine kinases that are closely related to
used clinically. Recently, two new entries, vemurafenib (II,
cancer since discovered by Rapp et al.3 Three distinct subtypes
of RAF kinases are known,4,5 in addition to homologues of
BRAF, which were characterized in Drosophila melanogaster
(D-RAF) and Caenorhabditis elegans.6,7 RAF is the first
downstream effector of RAS, and both kinases are important
regulators of ERK−MAPK.
Figure 1)21 and dabrafenib (III, Figure 1),22 were introduced
for treatment of metastatic melanoma.
Although the breakthrough that occurred by the introduc-
tion of selective V600E-BRAF in the treatment of melanoma,
resistance to selective BRAF arises after 8 months of
treatment.23−26 The acquired resistance occurs by reactivation
of the MAPK pathway as a result of either genetic or epigenetic
changes.27−30 Genetic causes exist in 52% of resistant
Identification of BRAF mutation paved the way for a new era
of cancer therapy.8 There are more than 30 identified
mutations of BRAF kinase,9,10 out of which V600E-RAF is
the most abundant type of BRAF mutation. BRAF point
mutation occurs in 60% of malignant melanoma cases,11
thyroid cancer,1 colorectal carcinoma, lung cancer, papillary
craniopharyngioma,12 hairy cell leukemia,13,14 and metanephric
kidney adenoma.15 C-RAF kinase is overexpressed in renal cell
carcinoma,16 hepatocellular carcinoma,17 and associated with
Received: February 5, 2021
Published: May 17, 2021
https://doi.org/10.1021/acs.jmedchem.1c00230
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 6877−6901
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Figure 1. Chemical structures of selective V600E-BRAF and Pan-RAF inhibitors and rational design of final target compounds.
melanoma and include BRAF amplification, MAP2K1 and
MAP2K2 mutations, RAS mutation, and, to a less extent,
mutations in the PI3K pathway.30,31 Melanoma cells subjected
to selective V600E-BRAF inhibitors can switch to another RAF
isoform to maintain MAPK activity. In addition, over-
expression of CRAF can also lead to MAPK reactivation.32
The RAF dimer dilemma is another obstacle against the
successfulness of first generation selective V600E-BRAF
inhibitors such as vemurafenib. Pan-RAF inhibition is one of
the potential approaches toward fixing this issue.33
After the discovery of resistance to selective V600E-BRAF
inhibitors, a hypothesis was established that the usage of pan-
RAF inhibitors could be useful in the case of patients having
oncogenic V600E-BRAF and NRAS in which wild-type BRAF
and CRAF play a critical role in tumor growth. In addition,
using pan-RAF inhibitors could overcome resistance to
traditional selective V600E-BRAF and could be used as
monotherapy for treatment of melanoma instead of combined
therapy.34 Two pan-RAF inhibitors, TAK632 (IV, Figure
1)34,35 and LY3009120 (V, Figure 1),36 were introduced and
showed high success to produce minimum paradoxical
activation and good activity in both mutated BRAF and RAS
cases. Recently, pan-RAF inhibitors exhibited antitumor effect
on lung cancer37 and were able to induce apoptosis in acute
myeloid leukemia cells.38 Despite the clinical significance of
pan-RAF inhibitors, the number of candidates and scaffolds
that act as pan-RAF inhibitors are still unsatisfactory. Also, the
potency of pan-RAF inhibitors is lower when compared to
selective V600E-BRAF (IC50 of pan-RAF inhibitors are still
high when compared to vemurafenib and dabrafenib).
In our previous work, we reported the antiproliferative and
kinase inhibitory effects of imidazo[2,1-b]thiazole and imidazo-
[2,1-b]oxazole with different terminal substitutions such as
urea, amide, or sulfonamide39−43 as part of a comprehensive
program to produce selective mutated BRAF inhibitors.
According to the results obtained, we reported that compound
VI possesses inhibitory effect against both V600E-BRAF and
CRAF (IC50 values are 40 and 19 nM, respectively) (Figure 1).
Although the design and synthesis of selective inhibitors of
V600E-BRAF are more difficult compared with broad-
spectrum kinase inhibitors, obtaining highly selective pan-
RAF inhibitors is more challenging. Fine tuning of compound
VI structure yielded compound VII, which exhibits higher
potency against wild-type BRAF, V600E-BRAF, and CRAF
with IC50 values of 22, 9, and 18 nM, respectively. In order to
optimize the obtained structure over RAF kinases, we replaced
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a
Scheme 1. Synthesis of Compounds 14−16
a
Reagents and conditions: (i) ethanol, reflux, 16 h, 70−80%; (ii) 4-chloro-2-(methylthio)pyrimidine, Pd(OAc)2, Cs2CO3, PPh3, DMF, 80 °C, 16 h,
9−15%; (iii) Oxone, MeOH, H2O, rt, 16 h, 90−95%.
a
Scheme 2. Alternative Pathway for Synthesis of Compound 12
a
Reagents and conditions: (i) Br2, dichloromethane, rt, 24 h, 60%; (ii) potassium acetate, bis-(pinacolate)diboron, Pd(dppf)Cl2 DMSO, 80 °C, 12
h, 70%; (iii) 4-chloro-2-(methylthio)pyrimidine, Pd(dppf)Cl2, Cs2CO3, DME, H2O, 80 °C, 12 h, 30%.
Scheme 3. Synthesis of Sulfonamide Side Chains 22 and 23
imidazo[2,1-b]thiazole with imidazo[2,1-b]oxazole. Imidazo-
[2,1-b]oxazole derivatives showed moderate kinase and cellular
activity. So, we retained imidazo[2,1-b]thiazole as a main
scaffold and started molecular modification based on the
crystal structure of V600E-BRAF. Bioisosteric replacement of
m-fluoro with m-hydroxyl on the phenyl ring attached to
position 6 of the imidazothiazole nucleus and testing both
open chain sulfonamide and cyclic sulfamide were the main
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a
Scheme 4. Synthesis of Final Target Compounds 24 and 27
a
Reagents and conditions: (i) N,N-diisopropylethylamine, DMSO, 80 °C, 8 h, 20−62%; (ii) BBr3, dichloromethane, −78 °C, 10−50%.
a
Scheme 5. Synthesis of Cyclic Sulfamide Side Chain 36a−g
a
Reagents and conditions: (i) appropriate ethylenediamine, pyridine, reflux, 3 h, 32−52%; (ii) 34, NaH (60% in mineral oil), DMF, 42−76%; (iii)
H2/Pd-C, MeOH, rt, 1 h; (iv) benzyl chloroformate, TEA, CH2Cl2, 0 °C, 59%; (v) methanesulfonyl chloride, TEA, CH2Cl2, 0 °C, 53%.
a
Scheme 6. Synthesis of Final Target Compounds 37a−g and 38a−g
a
Reagents and conditions: (i) N,N-diisopropylethylamine, DMSO, 80 °C, 8 h, 29−64%; (ii) BBr3, dichloromethane, −78 °C, 16−27%.
structural modifications to be done on compound VII. In
addition to the hydroxyl group, we decided to add the methoxy
group at position 3 in the phenyl ring at position 6 to
investigate the effect of a hydrogen bond acceptor and
hydrogen bond donor on kinase activity. According to our
previous work and molecular modeling study performed at the
beginning of our anticancer project, we decided to limit the
spacer between the pyrimidine ring and sulfonamide terminal
side chain to be ethylene or propylene only. Synthetic
procedures as well as in vitro and in vivo results are reported
herein in detail.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of the final compounds was
accomplished by adopting the synthetic pathways illustrated
in Schemes 1−6. Our strategy is divided into two main steps:
the first step is the synthesis of key methyl sulfonyl
intermediates 14−16, while the second step is the synthesis
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Table 1. In Vitro Enzyme Activity of Compounds 24a−f
BRAFwt
V600E-BRAF
CRAF
a
b
a
b
a
b
compound no.
n
R
%
IC50
%
IC50
%
IC50
24a
24b
24c
24d
24e
24f
1
1
1
2
2
2
4-F
96.62 1.02
95.41 0.95
92.51 0.81
98.50 0.75
97.26 0.98
96.51 1.32
375 2.25
459 3.51
623 3.28
276 1.10
299 4.21
521 4.42
97.42 0.64
96.32 0.46
94.86 0.98
99.21 0.42
98.64 0.54
97.72 0.82
69.1 2.13
77.5 1.52
89.3 2.05
57.7 0.41
66.1 1.27
85.3 1.72
97.01 0.12
95.67 0.55
93.21 0.37
99.13 0.31
98.92 0.24
96.99 0.96
112 1.95
172 2.15
251 2.71
87.8 1.51
98.3 2.34
201 5.01
4-OCH3
4-CH3
4-F
4-OCH3
4-CH3
a
b
Mean %inhibition at a single dose (10 μM). Data are represented as mean value
S.D. IC50 (nM). Values are presented as mean value
(duplicate test) S.D.
Table 2. In Vitro Enzyme Activity of Compounds 25a−m
BRAFwt
V600E-BRAF
CRAF
a
b
a
b
a
b
compound no.
n
R
%
IC50
%
IC50
%
IC50
25a
25b
25c
25d
25e
25f
25g
25h
25i
1
1
1
1
1
1
1
2
2
2
2
2
2
H
4-F
3-F
4-OCH3
4-CH3
1-naphthyl
4-CF3
H
4-F
3-F
90.21 0.81
95.25 0.95
94.92 0.79
92.11 0.82
89.59 0.96
90.64 0.88
93.96 0.97
92.41 0.69
97.53 1.12
95.22 1.02
94.32 0.93
90.57 0.71
91.92 1.01
941 3.01
522 5.26
561 6.12
745 4.55
987 8.21
921 9.32
687 10.51
721 4.21
430 5.64
526 7.22
589 6.66
894 5.26
842 10.35
95.61 0.81
96.86 0.71
96.69 0.52
94.33 0.54
92.67 0.77
93.66 0.87
98.21 1.56
96.11 2.10
98.37 0.89
97.65 0.78
95.55 1.25
94.71 0.45
94.92 0.38
120 2.11
115 3.12
112 2.85
160 4.32
177 3.68
169 4.33
98.1 1.27
109 5.12
90.3 1.13
102 1.58
156 6.32
168 7.16
165 5.54
92.22 0.31
97.32 1.15
97.14 0.69
95.62 1.06
94.35 0.44
94.65 0.74
97.31 0.44
93.55 0.68
98.12 1.01
97.16 0.99
95.27 0.33
92.35 0.54
93.95 0.99
220 2.56
100 1.58
106 3.21
151 2.28
163 5.21
190 2.99
142 2.11
129 3.26
95.2 1.21
111 1.31
164 4.21
209 2.55
172 1.98
25j
25k
25l
4-OCH3
4-CH3
1-naphthyl
25m
a
b
Mean %inhibition at a single dose (10 μM). Data are represented as mean value
S.D. IC50 (nM). Values are presented as mean value
(duplicate test) S.D.
of sulfonamide side chains 22a−g, 23a−f, and 36a−d
possessing the free amino group. The last synthetic step
involves the reaction of the amines with mesyl intermediates to
get the target compounds 24−27 (Scheme 4) in addition to 37
and 38 (Scheme 6).
Synthesis of mesyl intermediates is presented in Scheme 1.
Refluxing of 2-aminothiazole (4) with appropriate 2-bromo-1-
phenylethan-1-one derivative 5−7 in absolute ethanol followed
by basification with ammonium hydroxide led to cyclization
and formation imidazothiazole intermediates 8−10. Coupling
reaction of compounds 8−10 with 4-chloro-2-(methylthio)-
pyrimidine using palladium acetate, cesium carbonate, and
triphenylphosphine in anhydrous dimethylformamide at 80 °C
produced arylated products 11−13.44 Oxidation of the
methylthio group of compounds 11−13 using Oxone in a
mixture of methanol and water gave the corresponding mesyl
analogues 14−16.
The low yield of step 2 in Scheme 1 encouraged us to look
for an alternative pathway to synthesize compound 12. Scheme
2 shows the alternative strategy to produce compound 12.
Bromination of 6-(3-methoxyphenyl)imidazo[2,1-b]thiazole
(9) using bromine in dichloromethane formed the correspond-
ing 5-bromo analogue 17 in quantitative yield. Reaction of
compound 17 with bis(pinacolato)diboron in the presence of a
catalytic amount of [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II) and potassium acetate as a base gave 6-
(3-methoxyphenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)imidazo[2,1-b]thiazole (18). Suzuki coupling of boronic
acid ester (18) with 4-chloro-2-(methylthio)pyrimidine using
cesium carbonate as a strong base and [1,1′-bis-
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Table 3. In Vitro Enzyme Activity of Compounds 26a−d
BRAFwt
V600E-BRAF
CRAF
a
b
a
b
a
b
compound no.
R
%
IC50
%
IC50
%
IC50
26a
26b
26c
26d
4-F
4-OCH3
4-CH3
60.21 0.92
51.65 0.77
35.25 0.33
33.25 1.52
9210 260
9890 650
19,250 370
20,780 450
45.36 0.48
29.62 1.02
31.32 0.84
21.63 0.66
13,250 130
26,310 420
26,560 630
25.52 0.23
32.68 0.54
39.89 0.71
26.88 0.92
32,220 550
24,320 1210
21,330 1320
28,920 1010
1-naphthyl
>35,000
a
b
Mean %inhibition at a single dose (10 μM). Data are represented as mean value
S.D. IC50 (nM). Values are presented as mean value
(duplicate test) S.D.
Table 4. In Vitro Enzyme Activity of Compounds 27a−i
BRAFwt
V600E-BRAF
CRAF
a
b
a
b
a
b
compound no.
n
R1
R2
%
IC50
%
IC50
65
19
1
55
20
%
IC50
27a
27b
27c
27d
27e
27f
27g
27h
27i
1
1
1
1
2
2
2
1
1
H
4-OH
H
4-F
4-CH3
H
3-F
4-CH3
4-OH
4-CH3
99.56 0.51
97.89 0.26
100 0.11
98.21 0.09
98.91 0.15
100 0.08
100 0.23
51 0.62
97 1.2
35 2.5
99.25 0.33
98.64 0.51
100 0.11
97.89 0.64
99.35 0.69
100 0.25
100 0.31
35.32 0.22
39.51 0.12
3
98.59 0.62
98.12 0.35
99.14 0.87
98.32 0.44
98.75 0.74
99.87 0.67
99.92 0.51
39.57 0.27
40.29 0.36
75 5.2
21 2.3
0.5
62 2.1
23 1.5
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
4-OH
4-OH
1
0.2
5
2.7
64
29
1
3
1
6
2
4.2 0.12
18
0.98 0.012
0.8
25,120 1560
5
0.13
13 1.6
3
9
9710 260
11,320 1020
19,360 1350
16,510 2150
47 0.44
21,330 2430
a
b
Mean %inhibition at a single dose (10 μM). Data are represented as mean value
S.D. IC50 (nM). Values are presented as mean value
(duplicate test) S.D.
(diphenylphosphino)ferrocene]dichloropalladium(II) as a cat-
alyst produced compound 12.43
ethanolamine (31) was protected with benzyl chloroformate to
give benzyl (2-hydroxyethyl)carbamate (32). Reaction of
compound 32 with methanesulfonyl chloride in the presence
of triethylamine gave Cbz-protected methanesulfonate inter-
mediate 33. Coupling of compounds 33 and 29/30 in
dimethylformamide and using sodium hydride as a base
produced compounds 34a−c and 35a−d, which, upon
deprotection with palladium/C under a hydrogen atmosphere,
resulted in cyclic sulfamide side chain 36a−g.
Synthesis of the open chain sulfonamide side chains was
achieved through single step pathway through reaction of
ethylenediamine (19) or propylenediamine (20) with
appropriate sulfonyl chloride 21a−g in the presence of
triethylamine (TEA) [Scheme 3]. Compounds 19 and 20
were used in large excess (30 equivalents), and the sulfonyl
chlorides were diluted with dichloromethane and added
dropwise at 0 °C.
The final target compounds 24−27 were synthesized as
shown in Scheme 4. Reaction of mesyl intermediates 14−16
with amino reactants 22 or 23 in dimethyl sulfoxide and using
N,N-diisopropylethylamine as a base at 80 °C led to the
formation of 24a−f, 25a−m, and 26a−d. Demethylation of
methoxy compounds 25 and 26 using boron tribromide
solution in dichloromethane at −78 °C produced the target
hydroxyl compounds 27a−i.
In a similar manner, synthesis of the target compounds 37a−
g was performed through the coupling of compound 15 and
̈
cyclic sulfamide side chains 36a−g in the presence of Hnig’s
base. Demethylation of compounds 37a−g using boron
tribromide in dichloromethane led to the formation of
corresponding hydroxyl analogues 38a−g.
Biological Screening. Kinase Profiling. The primary
biological screening was to test the ability of the final
compounds to inhibit wild-type BRAF, V600E-BRAF and
CRAF (Tables 1−5). Both mean inhibition percentage and
IC50 values were determined for each group of final
compounds. For N-(2-((4-(6-phenylimidazo[2,1-b]thiazol-5-
yl)pyrimidin-2-yl derivatives (24a−f), compounds with a
The synthesis of cyclic sulfamide side chains was done as
illustrated in Scheme 5. Synthesis of compounds 29a−c and
30a−d was done via the condensation of sulfamide (28) with
an appropriate diamine in refluxed pyridine. Moreover, 2-
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Figure 2. Percent inhibition of 38a over 52 kinases at 10 μM.
propylene linker between the pyrimidine and sulfonamide
terminal moiety 24d−f showed higher activity in terms of both
inhibition percentage and IC50 compared to ethylene linker
24a−c. Compound 24d with the p-fluoro substituent is the
most potent against the three isoforms of RAF kinase with IC50
values of 276, 57, and 87 nM over wild-type BRAF, V600E-
BRAF, and CRAF, respectively. The activity over mutated
BRAF was higher than CRAF and finally wild-type BRAF. The
order of activity is compounds having p-F (24a and 24d)
followed by p-OMe (24b and 24e) and finally compounds
possessing the p-Me group (24c and 24f) [Table 1].
The second modification step was to change the position of
the methoxy group from meta to para (compounds 26a−d,
Table 3). This modification dramatically reduced the
inhibitory effect against all RAF isoforms. The IC50 values
over V600E-BRAF and CRAF are more than 10 μM. The best
potency among these derivatives was that of compounds 26a
and 26b against wild-type B-RAF (IC50 = 9.21 and 9.89 μM,
respectively).
The next modification was to change the hydrogen bond
acceptor methoxy group to the hydroxyl group, which has both
a hydrogen bond acceptor and donor properties in addition to
a smaller size and higher hydrophilicity compared to the
methoxy group (Table 4). The demethylated compounds
showed a great increase in enzyme inhibitory effect over the
three enzymes for both unsubstituted phenyl and meta-
substituted phenyl imidazo[2,1-b]thiazole, while the para-
substituted derivative did not show any improvement toward
the investigated enzymes. Compounds 27b−g possessing the
3-hydroxyphenyl moiety at position 6 of the imidazo[2,1-
b]thiazole nucleus have greater activities compared to
unsubstituted and 4-hydroxyl analogues. Against wild-type
BRAF, compound 27c (p-fluorobenzenesulfonamide and an
ethylene linker) is the most potent compound with an IC50 of
2.7 nM followed by 27f (m-fluorobenzenesulfonamide and
propylene) with an IC50 equals 4.2 nM, 27g (p-toluene
sulfonamide and propylene) with an IC50 of 18 nM, and
compound 27e (unsubstituted benzenesulfonamide and
propylene linker) with an IC50 value of 29 nM. Compound
27f had the lowest IC50 of 0.98 nM followed by 27c with an
IC50 of 1 nM and 27g with IC50 equals 9 nM. Compound 27h
and 27i with 4-hydroxyphenyl showed the highest IC50s, 25.12
and 21.33 μM, respectively. Finally, CRAF activities were
ranging from 5 nM for compound 27f and 6 nM for compound
27c to 19.36 μM for compound 27h. Compound 27a with
unsubstituted phenyl at position 6 showed a higher IC50
compared to 3-hydroxyl derivatives with an IC50 of 75 nM.
We finally decided to check the effect of using the cyclic
sulfamide moiety instead of open chain sulfonamide and
keeping the meta-hydroxyphenyl moiety at position 6 of
imidazo[2,1-b]thiazole. Table 5 shows the activity of cyclic
sulfamide derivatives 37a−g and 38a−g against wild-type
BRAF, V600E-BRAF, and CRAF. The phenolic compounds
The first modification to optimize the enzyme activity was
the addition of the methoxy group at the phenyl ring at
position 6 of the imidazo[2,1-b]thiazole scaffold. Table 2
shows the activity of compounds 25a−m over wild-type BRAF,
V600E-BRAF, and CRAF. Generally, the compounds had
higher potency over V600E-BRAF and CRAF compared to
wild-type BRAF. Compounds 25h−m with a propylene linker
are more potent over wild-type BRAF and mutated BRAF. On
the other hand, compounds 25a−g with an ethylene spacer
show insignificant potency difference against CRAF compared
to propylene spacer compounds 25h−m. In the case of wild-
type BRAF, compounds 25i with p-fluorobenzenesulfonamide
and a propylene linker and 25j with m-fluorophenyl
sulfonamide and a propylene linker showed the highest activity
against wild-type BRAF with IC50 values of 0.43 and 0.52 μM,
respectively. Regarding the activity against V600E-BRAF,
compound 25i showed an IC50 of 0.09 μM followed by 25g
(p-CF3 and ethylene) with an IC50 of 0.098 μM and finally 25j
with an IC50 of 0.10 μM. The CRAF inhibitory effect of
compounds 25a−m is ranging from 0.095 μM for 25i to 0.22
μM for 25a. The order of activity over CRAF started with
compound 25b (p-fluorobenzenesulfonamide and an ethylene
spacer) with an IC50 of 0.10 μM, 25c (m-fluorobenzenesulfo-
namide and ethylene linker) with an IC50 of 0.106 μM, 25j
with an IC50 of 0.11 μM, and 25h (unsubstituted
benzenesulfonamide and propylene) with an IC50 of 0.12
μM. Compounds having a small electron-withdrawing group
25b, 25c, 25i, and 25j showed no significant difference in
potency over both V600E-BRAF and CRAF and only
compound 25i exerted higher potency over wild-type BRAF
(Table 2).
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Table 6. IC50 Values (μM) of Compounds 25g, 25j, 27c, 27f, and 38a over the Most Sensitive Kinases
enzyme
25g
25j
27c
27f
38a
ABL1
ERN1
FGFR
FLT3
HIPK1
MAPK8
MAPK11
MAPK14
ZAK
9.85 0.32
>20
7.98 0.48
15.65 0.97
11.80 76
2.98 0.45
3.84 0.36
0.36 0.01
0.095 0.005
0.088 0.007
11.25 0.79
7.58 0.65
10.37 0.77
9.54 0.41
9.58 0.92
1.99 0.31
5.13 0.24
1.35 0.11
1.21 0.12
2.01 0.19
7.58 0.69
8.71 0.45
13.58 0.36
10.25 0.29
10.61 0.89
1.48 0.43
4.88 0.75
1.87 0.17
0.98 0.04
1.78 0.16
10.29 0.34
10.27 0.91
12.37 0.67
11.32 0.49
13.52 0.76
1.51 0.06
3.78 0.24
0.91 0.08
0.82 0.09
1.02 0.12
9.01 0.72
8.04 0.97
15.56 1.01
2.17 0.10
4.58 0.51
0.42 0.019
0.12 0.007
0.098 0.003
8.11 0.88
9.25 0.74
NTRK2
38a−g are proved to be more active against all RAF isoforms
compared to methoxy derivatives 37a−g. In addition, five-
membered ring cyclic sulfamides 37a and 38a were more
potent than six-membered ring cyclic sulfamides 37d and 38d.
Compound 38a with N-Me cyclic sulfamide and 3-hydrox-
yphenyl exhibited the highest activity over wild-type BRAF,
V600E-BRAF, and CRAF with IC50 values of 2, 0.07, and 1.12
nM, respectively. The methoxy analogue of 38a, compound
37a, demonstrated lower potency toward the three enzymes
with an IC50 of 6.12 nM over wild-type BRAF, 1.51 nM over
V600E-BRAF, and 2.52 nM over CRAF. Replacement of the
methyl group with a bulkier group such as benzyl reduced the
activity for both methoxy and hydroxyl compounds. The N-
benzyl derivative of methoxyphenyl 37b showed a higher IC50
against the three RAF isoforms with a value of 17.32 nM for
wild-type BRAF, 8.36 nM for V600E-mutated BRAF, and 4.32
nM for CRAF. The hydroxyl analogue 38b exhibited slightly
higher potency over the three enzymes with an IC50 of 15.55
nM over wild-type BRAF, 7.25 nM over V600E-BRAF, and
3.91 nM over CRAF. Removal of the methyl group decreased
the activity compared to methyl-containing compounds but to
a less degree compared to benzyl derivatives. Replacement of
five-membered ring sulfonamide with a six-membered ring led
to lower activity compared to the N-methyl five-membered
ring but was higher compared to an unsubstituted five-
membered ring and N-benzyl derivative. Unsubstituted cyclic
sulfamides and bulky tert-butylcarbonyl substitutions (37e, 37f,
37g, 38e, 38f, and 38g) are significantly less active compared
to derivatives with small alkyl substitution (37a and 38a).
Compound 38a with the highest enzyme activity against the
three RAF isozymes was tested over a panel of 52 kinases of
diversity of families at a single dose concentration of 10 μM to
check the selectivity of the compound. In addition to the three
RAF isozymes, compound 38a showed strong inhibitory
activity against FLT3, MAPK8, MAPK14, and MAPK11. In
addition, moderate inhibitory effect was observed over ABL1,
ERN1, HIPK1, FGFR1, NTRK2, and ZAK (Figure 2). The
potency of compound 38a over the most sensitive enzymes
was determined by measuring of its IC50 over these sensitive
enzymes (Table 6). Compound 38a showed sub-micromolar
activity against MAPK8 and MAPK11 with IC50 values equal
910 and 820 nM, respectively. Compound 38a had one-digit
micromole over FLT3, HIPK1, MAPK14, ZAK, and NTRK2
with IC50 values of 1.51, 3.78, 1.02, 9.01, and 8.04 μM,
respectively. Therefore, it can be concluded that compound
38a is relatively selective against RAF kinases (V600E-BRAF,
CRAF, and wild-type BRAF) compared to the other tested
kinases, i.e., pan-RAF potent inhibitor. In addition to
compound 38a, two compounds belong to methoxy derivatives
(25g and 25j) and two hydroxyl derivatives (27c and 27f)
were tested against the same kinases. Compounds 27c and 27f
showed similar selectivity to compound 38a. Interestingly,
compounds 25g and 25j showed high activity over MAPK8,
MAPK11, and MAPK14, which was almost equipotent to their
RAF isoform inhibitory activity (Table 6).
Antiproliferative Activity. The final compounds were
submitted to the National Cancer Institute (NCI, USA) and
selected for one-dose testing against 60 human cancer cell line
assay (breast, CNS, colon, leukemia, melanoma, non-small cell
lung, ovarian, prostate, and renal cancer). Compounds 25g,
27f, and 37d showed more than 80% mean inhibition
percentages over the 60 cell lines. In addition, compounds
24b and 25i have mean inhibition percentages over 60%
(Figure 3).
Profound investigation of each cancer sub-type is presented
in Figure 4. In general, compounds 25 and 27 exhibited a
higher inhibition percentage over most cell lines. In the case of
leukemia cell lines, compounds 25g, 25j, and 27f had the
highest %inhibition with mean values of 93.20%, 93.80%, and
93.81%, respectively, followed by 24b and 35i with mean %
inhibition values of 81.30% and 79.50%, respectively. For non-
small cell lung cancer cell lines, compounds 25g, 25j, and 27f
showed the highest activity with mean inhibition percentage
81.3%, 83.3%, and 83.1%. In the case of colon cancer,
compound 25g had the strongest inhibitory effect with a mean
inhibition percentage of 86.2% followed by 25f and 24b with
mean inhibition percentages of 83.09% and 76.7%, respec-
tively. The activity of final target compounds over CNS cancer
cell lines was lower compared to other subpanels. The highest
mean inhibition percentages are encountered with compounds
25g and 27f with values 91.40% and 83.09%, respectively. The
inhibitory effect of final compounds over melanoma cell lines
was higher than the other cancer types. Compound 24b was
the most active amongst derivatives with the unsubstituted
phenyl ring at position 6 of the imidazothiazole nucleus with a
mean percentage inhibition of 83.8% followed by 24d with
percentage inhibition equals 59.7%. Within the 3-methox-
yphenyl derivatives, compounds 25g and 25i exhibited a lethal
effect over melanoma cell lines with mean inhibition
percentages of 107.8% and 101.2%, respectively. Compound
27f is the most potent among the hydroxyl group-containing
compounds with a mean percentage inhibition of 101.29%
followed by 27c with a mean percentage inhibition of 75.51%.
Compounds 38a, 38d, and 38c are the most active among
cyclic sulfamide-possessing compounds with mean percentage
inhibition values of 97.91%, 84.78%, and 79.71%, respectively.
Finally, the activity over ovarian cell lines showed that
compounds 25g is the most potent with mean %inhibition =
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Figure 3. (A) Mean inhibition percentage of the target compounds over cancer 60 cell lines at a 10 μM concentration. (B) Heat map of final target
compounds over NCI 60 cell lines: (B1) final target compounds 24a−f, (B2) final target compounds 25a−m and 26a−d, (B3) final target
compounds 27a−i, and (B4) final target compounds 37a−g and 38a−g.
80% followed by 27f with mean %inhibition around 75% and
finally 24b with mean inhibition percentage = 73.2%.
Compounds 24b, 25g, 25j, 27c, 27f, 38a, and 38d were
selected to be tested at 5-dose mode in the NCI to determine
their IC50 over NCI-60 cell lines (Table 7). Compound 24b
shows potency ranging from 2.19 μM over colon cancer cell
lines, 13.3 μM over breast cancer cell lines, to 4.03 μM against
melanoma cell lines. Compound 25g exhibited the highest
activity over leukemia cell lines with a mean IC50 of 0.51 μM
followed by prostate cancer with an IC50 of 0.9 μM. The lowest
activity for 25g was 3.85 μM against ovarian cell lines.
Compounds 27c, 27f, 38a, and 38d exhibited the highest
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Figure 4. Mean inhibition percentages of the target compounds over different types of cancers.
in 5-dose testing mode against melanoma cell lines are
presented in Table 8. The cyclic sulfamide derivatives 38a and
38d are obviously the most potent amid this series against
melanoma cell lines. Both compounds exerted strong potency
with IC50 values ranging from 2-digit nanomolar to one-digit
micromolar scale. Compound 38a with N-methyl 5-membered
cyclic sulfamide is generally more potent than the N-methyl-
substituted 6-membered analogue 38d. It seems that ring
expansion in this part of the molecule is not very favorable for
activity. The promising results of compound 38a encouraged
us to extend our biological investigations on it.
Immunoblot Assay. Compounds 27c as the most potent
open sulfonamide derivative over melanoma cell lines and 38a
as the most potent cyclic sulfamide derivative over melanoma
cell lines were chosen to check their ability to inhibit the
MAPK pathway inside melanoma cells (Figure 5). To assess
the effect of 27c and 38a on the RAF/MEK/ERK signaling
pathway, A375 cells were treated with the tested compounds at
three different concentrations (0.1, 0.5, and 1 μM) for 24 h
and immunoblotted with antibodies against phospho-MEK1/2,
phospho-ERK1/2, ERK, and MEK, respectively. Both
compounds showed remarkable reduction on phosphorylation
of both MEK and ERK at all tested concentrations. Dose-
dependent inhibition is noticed in western blotting of both
compounds against phospho-MEK1/2, phospho-ERK1/2.
Compounds 27c and 38a showed higher inhibitory activity
compared to vemurafenib at the same concentration (5 μM)
(Figure 5).
Table 7. Mean IC50 of the Test Compounds over In Vitro
Subpanel Cancer Cell Lines
a
compound no.
subpanel cancer cell
line
24b
25g
27c
27f
38a
38d
leukemia
non-small lung
colon
2.99
5.42
2.19
6.46
4.03
3.57
3.70
2.45
13.3
0.51
2.76
2.75
2.73
2.24
3.85
1.76
0.90
1.17
2.82
3.26
5.42
7.58
1.49
14.18
5.38
7.30
4.10
2.04
2.55
3.02
3.19
2.03
2.97
2.93
2.38
2.41
12.17
14.56
10.15
24.50
0.85
20.67
5.77
14.30
11.10
11.87
13.55
8.08
16.41
1.79
12.05
5.98
11.57
11.93
CNS
melanoma
ovarian
renal
prostate
breast
a
Mean IC50 values were calculated by dividing the summation of IC50
values of the compound over cell lines of the same cancer type by the
number of cell lines in the subpanel.
potency over melanoma cell lines with mean IC50 values of
2.03, 1.49, 2.03, 0.85, and 1.79 μM, respectively. Compound
27f is the most potent over leukemia, non-small cell lung
cancer, CNS, ovarian, renal, prostate, and breast cancer. On
the other hand, compounds 38a and 38d possessing the cyclic
sulfamide terminal moiety exhibited high selectivity toward
melanoma cell lines compared to the other cell lines.
Compound 38a is 6.79 times more selective against the
melanoma subpanel than the second most sensitive subpanel,
renal cancer cell lines.
MTT Assay against L132 Normal Cell Line, Normal Skin
Fibroblast Cell Line (BJ1), and Acute In Vivo Toxicity. In
order to move to the next step and check the in vivo activity of
The IC50 and total growth inhibition (TGI) values of
compounds 24b, 25g, 27c, 27f, 38a, and 38d that were tested
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Table 8. IC50 and Total Growth Inhibition (TGI) Values (μM) of the Most Potent Compounds over NCI Melanoma Cancer
Cell Lines
comp.
24b
25g
27c
27f
38a
38d
cell line
IG50
TG
IG50
TG
IG50
TG
20
IG50
TG
5.82
7.86
10.2
6.51
6.57
6.86
2.28
IG50
TG
IG50
TG
LOX-IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
3.9
8.7
2.7
1.2
4.9
5.7
1.7
6.4
1.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.26
2.35
0.69
1.44
3.04
3.68
1.24
4.41
0.48
19.2
5.22
3.25
5.88
9.26
55.5
2.52
>100
3.40
4.11
1.91
2.6
2.15
19.2
0.77
13.3
>100
9.21
>100
17
4.36
28.2
0.408
12.9
51.6
7.36
52
10.3
>100
3.14
0.279
0.567
0.939
4.49
0.66
1.61
0.072
0.302
0.381
2.63
0.176
1.73
0.036
0.174
0.490
2.26
0.240
0.348
8.21
15
2.54
2.00
2.32
2.29
1.06
2.85
0.75
11.6
20.2
24
3.98
16.5
10.3
a
0.568
0.270
>100
2.73
0.170
0.059
ND
5.0
0.058
a
ND: not determined.
showed 61.6-fold higher selectivity toward melanoma than
normal cells. On the other hand, compound 38a is 688-fold
more selective to the MALME-3M melanoma cell line
compared to the BJ1 cell line. The acute toxicity of the most
potent compound 38a was adopted by injection of the tested
compound in male Hsd:Athymic Nude-Foxn1nu (Harlan Co.
(USA)) mice at five different doses (50, 100, 250, 500, and
1000 mg/kg). At doses 50, 100, and 250 mg/kg, there was no
sign of toxicity over a group of seven animals for each
concentration. At 500 mg/kg, one animal was lost, while at a
dose of 1000 mg/kg, three animals died out of the seven-
animal group. It can be concluded that compound 38a is safe
enough up to a 250 mg/kg dose.
In Vivo Antitumor Activity of Compound 38a. The next
step was to test the ability of compound 38a to inhibit tumor
growth in the animal model. The animal model was generated
using A375 melanoma xenograft in male BALB/C nude mice
using vemurafenib as a standard drug and DMSO as a negative
control (Figure 6). Our candidate 38a was injected daily to
two groups of mice in two different doses (25 and 50 mg/kg) 5
days following subcutaneous injection of a human-derived
malignant melanoma cell line A375 cell line to nude mice (2 ×
106 in 200 μL). The effects of compound 38a on tumor
volume and weight were measured. At the end of the
experiment, the negative control (DMSO group) showed an
average tumor volume of 400 mm3 and an average tumor
weight of 3.15 g, while the vemurafenib-treated group showed
reduction of average tumor size to 126 mm3 and average tumor
weight to 1.2 g. The animals that received 25 mg/kg
compound 38a showed reduction of tumor size by 68.2%
(150 mm3) and reduction of tumor weight by 46.8% (2.7 g).
On the other hand, animals received 50 mg/kg exhibited
reduction of tumor size by 75% (100 mm3) and that of tumor
weight by 68.75% (1.00 g). Thus, compound 38a at a dose of
50 mg/kg exhibited more promising results than the standard
drug, vemurafenib. That dose is well tolerated by the animals
as per the in vivo toxicity experiment.
Figure 5. Effect of 27c and 38a on phosphorylation of MEK and ERK
on different concentrations (0, 0.1, 0.5, and 1 μM) and comparing
both compounds’ phosphorylation activity compared to vemurafenib
at 5 μM.
the promising pan-RAF inhibitor, both normal cell cytotoxicity
and acute animal toxicity were tested. The antiproliferative
activity of tested compounds over human embryonic
pulmonary epithelial cells (L132) and normal skin fibroblast
cell line (BJ1) are presented in Table 9. All the tested
compounds showed a high IC50 over L132 and BJ1 cell lines,
which indicate a high therapeutic index for these compounds.
Comparison of the activities of compound 38a over both
melanoma cell lines (0.73 μM) and L132 cell line (45.00 μM)
Table 9. IC50 Values (μM) of Representative Compounds
over L132 and BJ1
a
compound no.
L132 IC50
BJ1 IC50
selectivity
24b
25g
25j
26c
27f
27c
38a
38d
60.25 3.01
74.51 2.14
49.02 2.35
465.85 3.46
56.30 1.57
52.37 2.89
45.00 1.36
93.59 4.65
55.64 2.71
62.36 2.33
42.32 1.52
125.65 3.11
45.22 2.67
50.62 2.96
49.56 3.15
75.64 2.66
6.4
26.5
ND
ND
In Vivo Pharmacokinetic and Caco-2 Cell Permeability
Test of Compound 38a. Compound 38a showed high potency
over RAF kinases and cancer cell lines. In addition, it had
remarkable in vivo anti-melanoma activity. The above-
mentioned results encouraged us to investigate the pharmaco-
kinetic of compound 38a and its permeability and plasma
stability. Pharmacokinetic parameters for compound 38a were
determined in rat and are summarized in Table 10 and Figure
17.4
181.5
688.33
2101.11
a
Selectivity was determined by dividing IC50 value against normal skin
fibroblast/IC50 value against the MALME-3M melanoma cell line.
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Figure 6. In vivo antitumor activity of compound 38a in mice bearing the A375 xenograft. (A) 38a was administered daily by i.p. injection at the
doses indicated (25 or 50 mg/kg). Vemurafenib was administered at a dose of 50 mg/kg daily by i.p. injection. Tumor volumes were measured
every 2 days. Data are represented as mean S.E.M. for each treatment group. (B) Tumor weights measured after 3 weeks for the 38a-treated
group, vehicle, and vemurafenib-treated group. The graph shows the mean S.E.M. calculated from tumors in each group. (C) Representative
tumor images of A375 xenografts.
Table 10. Pharmacokinetic Parameters of Compound 38a
after Administration of 5 mg/kg by Both IV and PO
Table 11. Permeability and Efflux Ratio (ER) of Compound
38a (Papp, ×10−5 cm/sec)
parameter
Tmax (h)
Cmax (μg/mL)
T1/2 (h)
IV (5 mg/kg)
PO (5 mg/kg)
compound
A to B
B to A
efflux ratio (ER)
2.08 0.44
0.09687 0.0025
3.35 0.57
0.433 0.021
0.196 0.035
38a
1.421
2.605
0.584
0.013
2.175
2.484
0.727
0.014
1.53
0.95
1.25
1.08
caffeine
ofloxacin
atenolol
1.72 0.41
6.57 0.81
6.59 0.82
2.07 0.25
0.578 0.05
AUC
AUC
⃗
0
T
⃗
0
∞
CL (mL min−1 kg−1
)
absorption with a permeability value of 1.4 × 10−5 cm/sec and
efflux ratio (ER) of 1.53. The plasma stability test for
compound 38a was performed in both human plasma and
rate plasma. Compound 38a has high stability profile in both
human and rat plasma. After 30 min, 97.2% of compound 38a
remained unchanged after 30 min, which decreased to 95.4%
after 120 min, while that of procaine was 1.2% after 30 min and
0.2% after 2 h, and the percentage for diltiazem was 91.5% after
30 min and 89.3% after 2 h (Table 12).
Vd (L/kg)
F (%)
6.59%
7. The compound was given in a dose of 5 mg/kg for both IV
and PO. Compound 38a reached its highest concentration
Table 12. Plasma Stability of Compound 38a (%
Remaining)
human
rat
compound
30 min
120 min
30 min
120 min
38a
procaine
diltiazem
97.2
1.2
91.5
95.4
0.2
89.3
90.6
84.9
86.3
76.6
42.7
45.4
Figure 7. Plasma concentration after intravenous and oral
administration at a dose of 5 mg/kg (n = 3).
Molecular Docking of Compound 38a. In order to gain
a better understanding of the potency of the synthesized
compounds and guide further SAR studies, molecular docking
into the domain of V600E-BRAF kinase for compound 38a
was performed. All calculations were done using MOE 2008.10
software installed on 2.0G Core 2 Duo. The crystal structure of
V600E-BRAF kinase in complex with vemurafenib (PDB code:
1UWJ) was obtained from the Protein Data Bank (PDB)
(http://www.rcsb.org/pdb). The automated docking program
after 2.08 h, its half-life is 1.72 and 3.35 h following IV and oral
administration, respectively, and its bioavailability is 6.59%. IV
clearance was 2.07 (mL min−1 kg−1) with volume of
distribution equal to 0.578 L/kg. The Caco-2 permeability
test was applied to check 38a human absorption. The obtained
data in Table 11 revealed that more than 80% human
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Figure 8. Molecular docking and 2D presentation of binding mode for (A) vemurafenib and (B) 38a into the V600E-BRAF kinase domain (PDB
code: 1UWJ).
of MOE 2008.10 was used for docking of 38a into the domain
of V600E-BRAF kinase. The complex was energy-minimized
with a MMFF94x force field till the gradient convergence of
0.01 kcal/mol was reached. The docking study has revealed
that the ligands have bound in the active site of one of the
protomers in the protein dimer through the formation of
strong hydrogen bonds between the binding site and the
ligand. For compound 38a, the molecular docking data are
illustrated in Figure 8. Compound 38a showed four hydrogen
bonding interactions in the binding site (one hydrogen bond
between the 3-hydroxyphenyl group at position 6 of the
imidazothiazole nucleus and Ser 455, two hydrogen bonds
between one oxygen from the sulfamide group and Asp 594
and Lys 483, and one hydrogen bond between protonated
sulfur of the imidazothiazole nucleus and Ser 535). In addition
to hydrogen bonding, the aromatic interaction between the
pyrimidine ring and Val 471 was recorded.
propylene spacer between the pyrimidine ring and terminal
sulfonamide moiety gave the highest activity (compound 24d)
over the three RAF isoforms with IC50 values of 256, 57, and
87 nM over wild-type BRAF, V600E-BRAF, and CRAF,
respectively.
The second and third groups of open chain sulfonamide
compounds contain the methoxy group on the phenyl ring at
position 6 of the imidazo[2,1-b]thiazole nucleus. Compounds
25a−m have 3-methoxyphenyl, and compounds 26a−d
contain the 4-methoxyphenyl moiety. Changing the position
of the methoxy group from meta to para showed a dramatic
decrease in enzyme activity, and compounds 26a−d showed
much higher IC50 values compared to compounds 25a−m.
Upon designing of meta-methoxy compounds and based on
our previous work, only para-substituted aryl sulfonamides and
small electron-withdrawing meta aryl sulfonamide were
synthesized. In these compounds, both ethylene and propylene
spacers showed insignificant difference in activity over RAF
isoforms. The most potent compound in this group is 25i,
which carries 4-fluorobenzenesulfonamide and a propylene
spacer with IC50 values of 430, 90, and 95 nM over BRAF,
V600E-BRAF, and CRAF, respectively, followed by compound
25g that possesses 4-CF3 and an ethylene spacer with IC50
values of 681, 98, and 142 nM over BRAF, V600E-BRAF, and
CRAF, respectively.
CONCLUSIONS
■
In this study, a new series of rationally designed imidazo[2,1-
b]thiazole was synthesized and screened for antiproliferative
activity as well as kinase inhibitory effect. The newly
synthesized compounds were divided into two distinct groups:
open chain sulfonamide (24a−f, 25a−m, 26a−d, and 27a−i)
and cyclic sulfamide (37a−g and 38a−g).
The last group of open chain sulfonamide derivatives is
27a−i, which carry hydroxyl (either para or meta) on phenyl at
position 6 of the imidazo[2,1-b]thiazole scaffold. Compounds
with 3-OH showed higher activity compared to 4-OH
analogues. In these derivatives, compounds with an electron-
withdrawing group on the terminal sulfonamide moiety exerted
Open chain sulfonamide final target compounds were
divided into four distinct groups. The first group possesses
the unsubstituted phenyl ring at position 6 of the imidazo[2,1-
b]thiazole scaffold (compounds 24a−f). These compounds
had a lower IC50 over V600E-BRAF and CRAF compared to
wild-type BRAF. A small electron-withdrawing group and
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the highest activity. Moreover, both ethylene and propylene
analogues showed similar potency over RAF isoforms.
Compound 27f (m-F and a propylene spacer) showed the
highest activity with IC50 values of 4, 0.98, and 5 nM over
BRAF, V600E-BRAF, and CRAF, respectively, followed by 27c
(p-F and an ethylene spacer) with IC50 values equal 2.7, 1, and
6 nM over BRAF, V600E-BRAF, and CRAF, respectively.
The main structural modification applied to open chain
sulfonamide series was to make cyclic sulfamide derivatives
37a−g and 38 a−g. Compounds 37a−g contain m-
methoxyphenyl at position 6 of the imidazothiazole nucleus
and either the five or six-membered ring cyclic sulfamide
terminal moiety. The cyclic sulfamide moiety was substituted
with H, small alkyl, tert-butylcarbonyl, or benzyl terminals.
Small alkyl-substituted compounds are more potent compared
to other substitutes. In addition, m-OH derivatives are more
active compared to their methoxy congeners. The most potent
compound is 38a with IC50 values of 2, 0.07, and 1.2 nM over
BRAF, V600E-BRAF, and CRAF, respectively.
The kinase panel screening for compound 38a showed high
activity over ABL1, ERN1, HIPK1, FGFR1, NTRK2, and ZAK
in addition to RAF isoform activity. To check the selectivity of
the most potent pan-RAF inhibitors, IC50 values of 25g and
25j (as representatives of methoxy-containing derivatives), 27c
and 27f (as representatives of hydroxyl-containing analogues),
and 38a (the most potent compound and cyclic sulfamide
representative) were calculated over the most sensitive kinases.
Compounds 27c, 27f, and 38a showed high selectivity for RAF
isoforms compared to the other sensitive kinases. On the other
hand, methoxy-containing compounds showed high activity
over MAPK8, MAPK11, and MAPK 14 besides their RAF
inhibitory effect.
being considered. The structure modifications include
replacement of the OH group with NH2 to improve the
solubility and microsomal stability, changing the substitution
on the cycle sulfamide moiety (ethyl, propyl, or direct aryl) to
improve both enzyme and cellular activity, and checking the
effect of multisubstituents on both the phenyl ring at position 6
and terminal sulfamide. Improving the oral bioavailability is
also one of our top goals in the next phase of this study.
EXPERIMENTAL SECTION
■
General. All solvents and reagents were commercially available
and used without further purification. The target compounds and
intermediates were purified by column chromatography using silica
gel (0.040−0.063 mm, 230−400 mesh) and technical grade solvents.
Analytical thin layer chromatography (TLC) was performed on silica
gel 60 F254 plates from Merck. IR spectra (KBr disks) were recorded
with a Bruker FT-IR instrument (Bruker Bioscience, Billerica, MA,
USA). 1H-NMR and 13C-NMR spectra were recorded on Bruker
Avance 400 or 300 spectrometers using tetramethylsilane as an
internal standard, and signals are described as s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), brs (broad singlet), or dd
(doublet of doublets). LC−MS analysis was carried out 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
CH3OH; flow rate = 3.0 mL/min; the AUC was calculated using
Waters MassLynx 4.1 software. Solvents and liquid reagents were
transferred using hypodermic syringes. Purity % of all the target
compounds were determined by HPLC and found to be >95%. All
animal experiments were conducted in compliance with institutional
guidelines.
The antiproliferative activity over NCI-60 cell lines for final
target compounds showed that compound 24b, 25g, 27c, 27f,
38a, and 38d had the highest activity among all final targets.
Compounds 38a and 38d exhibited the lowest IC50 values over
melanoma cell lines (72, 302, 381, 176, 170, and 59 nM for
38a over MALME-3M, M14, MDA-MB-435, SK-MEL-28,
UACC-257, and UACC-62, respectively).
General Procedure for Synthesis of Compounds 8−10. A
mixture of 2-aminothiazole (4, 4.74 g, 47.4 mmol) and appropriate 2-
bromoacetopenone derivative (5−7, 47.4 mmol) in absolute ethanol
(60 mL) was refluxed for 16 h with vigorous stirring. The reaction
mixture was concentrated to 30 mL under reduced pressure. To the
remaining suspension, 50 mL of ice water was added followed by 30%
ammonia solution (100 mL). The mixture was stirred for an
additional 2 h, and the formed precipitate was filtered off and dried
under reduced pressure at 50 °C to obtain the titled compounds.
6-Phenylimidazo[2,1-b]thiazole (8). Yield 78%; solid; mp 143−
144 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 8.63 (d, 1H, J = 7.2 Hz),
8.29 (t, 1H, J = 5.7 Hz), 7.77 (t, 3H, J = 6.7 Hz), 7.49−7.36 (m, 3H);
13C-NMR (DMSO-d6, 75 MHz) δ 148.1, 138.4, 129.9, 129.6, 127.7,
125.7, 121.9, 119.6, 111.5; IR (KBr) cm−1: 3139, 3120, 1955, 1884,
1602; LC−MS: m/z calculated for C11H8N2S: 200.04, found 201.0
(M + 1)+.
6-(3-Methoxyphenyl)imidazo[2,1-b]thiazole (9). Yield 80%; solid;
mp 136−137 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 8.64 (d, 1H, J =
1.2 Hz), 8.27 (dd, 1H, J = 1.3, 4.2 Hz), 7.70 (dd, 1H, J = 1.2, 4.2 Hz),
7.42 (d, 3H, J = 6.0 Hz), 7.00 (dd, 1H, J = 1.6, 6.00 Hz), 3.83 (s, 3H);
13C-NMR (DMSO-d6, 75 MHz) δ 160.2, 148.3, 139.45, 130.9, 129.9,
121.9, 118.7, 117.9, 115.4, 111.6, 111.2, 55.9; IR (KBr) cm−1: 3131,
3118, 2996, 2940, 1677, 1597; LC−MS: m/z calculated for
C12H10N2OS: 230.05, found 231.0 (M + 1)+.
Immunoblot assay of both compounds 27c and 38a proved
that their antiproliferative activities came from their potential
inhibitory effect against the ERK pathway. Both compounds
are able to inhibit phosphorylation of both MEK and ERK at
different dose intervals. At a 5 μM concentration, both
compounds are stronger than vemurafenib regarding inhibition
of both MEK and ERK phosphorylation. Normal cell line
cytotoxicity revealed high selectivity toward cancer cell lines
over normal cell lines with selectivity index reaching 600 for
38a and 2000 for 38d. In vivo investigations show stronger
anticancer activity at a 50 mg/kg dose compared to
vemurafenib as a positive control at the same dose range,
and no toxicity was encountered at the same dose. Compound
38a showed high plasma stability and good permeability, and
its pharmacokinetic profile showed moderate half-life with oral
bioavailability equals 6.59%. Molecular docking of compound
38a into the binding site of V600E-BRAF showed that the 3-
hydroxyl group on the phenyl ring at position 6 is important
for binding and added additional hydrogen bonding. This
explains the previously discussed higher potency of hydroxyl
derivatives than the methoxy analogues.
6-(4-Methoxyphenyl)imidazo[2,1-b]thiazole (10). Yield 70%;
1
solid; mp 145−146 °C; H-NMR (DMSO-d6, 300 MHz) δ 8.08 (s,
1H), 7.88 (d, 1H, J = 3.0 Hz), 7.74 (d, 2H, J = 9.0 Hz), 7.21 (d, 1H, J
= 6.0 Hz), 6.95 (d, 2H, J = 9.0 Hz), 3.76 (s, 3H); 13C-NMR (DMSO-
d6, 75 MHz) δ 158.9, 149.4, 146.7, 127.3, 126.5, 120.4, 114.5, 113.0,
108.6, 55.5; IR (KBr) cm−1: 3133, 3109, 2990, 1683, 1603; LC−MS:
m/z calculated for C12H10N2OS: 230.05, found 231.0 (M + 1)+.
General Procedure for Preparation of Compounds 11−13.
In a three-neck flask, 4-chloro-2-(methylthio)pyrimidine (6.5 g, 41.3
mmol), cesium carbonate (13.4 g, 41.3 mmol), palladium acetate
This study ends up with a promising pan-RAF drug lead 38a
for treatment of melanoma. It can be further investigated in
clinical studies, and further tuning of the structure is currently
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(1.22 g, 5.5 mmol), and triphenylphosphine (2.896 g, 11.04 mmol)
were mixed with appropriate 6-phenylimidazo[2,1-b]thiazole deriva-
tive (8−10, 41.3 mmol). The system was evacuated from air and
replaced by nitrogen twice. Anhydrous DMF (60 mL) was added, and
the mixture was purged with nitrogen thrice. The reaction mixture
was stirred at 80 °C for 16 h. The mixture was cooled to room
temperature, and 300 mL of distilled water was added followed by
200 mL of ethyl acetate. The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate (2 x 200 mL). The
combined organic solvent was dried using anhydrous sodium sulfate
and evaporated. The residue was subject to column chromatography
using hexane:ethyl acetate (5:1 v/v).
5-(2-(Methylthio)pyrimidin-4-yl)-6-phenylimidazo[2,1-b]thiazole
(11). Yield 15%; white solid; mp 143−144 °C; 1H-NMR (CDCl3, 300
MHz) δ 8.62 (dd, 1H, J = 0.7, 4.5 Hz), 8.23 (dd, 1H, J = 0.7, 5.4 Hz),
7.65−7.62 (m, 2H), 7.47−7.45 (m, 3H), 6.97 (dd, 1H, J = 0.7, 4.5
Hz), 6.89 (dd, 1H, J = 0.7, 4.8 Hz), 2.64 (s, 3H); 13C-NMR (CDCl3,
75 MHz) δ 172.4, 156.2, 156.2, 152.7, 151.1, 134.6, 129.2, 128.9,
128.8, 122.2, 120.1, 112.7, 112.0, 14.2; IR (KBr) cm−1: 3054, 6103,
1538, 1685; LC−MS: m/z calculated for C16H12N4S2: 324.05, found
325.0 (M + 1)+.
Hz), 7.57 (d, 1H, J = 9.0 Hz), 7.42 (d, 1H, J = 6.0 Hz), 7.04 (d, 4H, J
= 9.0 Hz), 3.92 (s, 3H), 3.39 (s, 3H); 13C-NMR (CDCl3, 75 MHz) δ
165.7, 160.6, 157.6, 156.7, 154.4, 153.4, 130.4, 126.4, 123.3, 119.3,
117.3, 114.5, 113.5, 55.4, 39.3; IR (KBr) cm−1: 3185, 3113, 1647,
1610, 1567, 1449, 1348; LC−MS: m/z calculated for C17H14N4O3S2:
386.05, found 387.1 (M + 1)+.
Preparation of 5-Bromo-6-(3-methoxyphenyl)imidazo[2,1-
b]thiazole (17). A solution of bromine (1.01 g, 6.4 mmol) in
dichloromethane (30 mL) was added over 1 h to a solution of
compound 9 (2.00 g, 6.4 mmol) in dry dichloromethane (50 mL) at 0
°C. The reaction mixture was stirred for 24 h at room temperature.
Water was added, and the organic layer was separated, dried over
anhydrous sodium sulfate, and evaporated under reduced pressure.
The residue was subjected to column chromatography using
hexane:ethyl acetate 10:1 v/v to give 1.60 g (60%) of the desired
product as a pale yellow solid. mp 90−91 °C; 1H-NMR (CDCl3, 300
MHz) δ 7.15 (d, 1H, J = 9.0 Hz), 7.12 (d, 1H, J = 3.0 Hz), 7.30−7.35
(m, 2H), 6.92 (dd, 1H, J = 3.0, 9.0 Hz), 6.86 (d, 1H, J = 3.0 Hz), 3.90
(s, 3H); 13C-NMR (CDCl3, 75 MHz) δ 159.7, 148.7, 143.6, 134.4,
129.4, 119.3, 117.5, 113.9, 113.0, 111.9, 90.3, 55.3; IR (KBr) cm−1:
1911, 1574, 1455, 564; LC−MS: m/z calculated for C12H9BrN2OS:
307.18, found 308.10 (M + 1)+.
6-(3-Methoxyphenyl)-5-(2-(methylthio)pyrimidin-4-yl)imidazo-
1
[2,1-b]thiazole (12). Yield 9%; white solid; mp 108−109 °C; H-
Preparation of 6-(3-Methoxyphenyl)-5-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)imidazo[2,1-b]thiazole (18). To a
solution of compound 17 (1 g, 3.2 mmol) in DMSO (6.8 mL),
potassium acetate (1.27 g, 12.94 mmol), bis-(pinacolato)diboron
(1.62 g, 6.2 mmol), and 1,10-bis(diphenylphosphino)ferrocene
palladium dichloride (Pd(dppf)Cl2, 0.23 g, 0.32 mmol) were added.
The mixture was degassed and charged with nitrogen and stirred at 80
°C for 12 h. The reaction was cooled to ambient temperature, diluted
with ethyl acetate (100 mL), and filtered through celite. The ethyl
acetate extract was washed with water (5 x 100 mL) and dried over
anhydrous sodium sulfate. The organic layer was filtrated and
concentrated. The residue was purified on a silica gel column eluting
with hexane:ethyl acetate 6:1 v/v to provide 800 mg (70%) as a white
solid. mp 140−141 °C; 1H-NMR (CDCl3, 300 MHz) δ 7.71 (s, 1H),
7.44 (s, 1H), 7.39 (t, 2H, J = 4.5 Hz), 7.34 (t, 1H, J = 6.0 Hz), 6.85
(d, 1H, J = 6.0 Hz), 6.79 (d, 2H, J = 3.0 Hz), 3.88 (s, 3H), 1.27 (d,
12H, J = 12.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ 159.9, 150.1,
147.5, 135.4, 129.6, 118.5, 117.6, 113.5, 112.6, 110.3, 108.3, 82.6,
75.0, 55.3, 24.8, 24.6; IR (KBr) cm−1: 3133, 3109,1768, 1603, 1467.
Synthesis of 6-(3-Methoxyphenyl)-5-(2-(methylthio)-
pyrimidin-4-yl)imidazo[2,1-b]thiazole (12). To a solution of 4-
chloro-2-(methylthio)pyrimidine (45 mg, 0.28 mmol) and 6-(4-
methoxyphenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
imidazo[2,1-b]thiazole (18,100 mg, 0.28 mmol) in DME (3 mL) was
added an aqueous solution of Cs2CO3 (210 mg, 0.65 mmol) in water
(1 mL) followed by the addition of 1,10-bis(diphenylphosphino)-
ferrocene palladium dichloride (Pd(dppf)Cl2,16.1 mg, 0.022 mmol).
The reaction flask was degassed and charged with nitrogen and then
heated at 80 °C for 12 h. The reaction mixture was diluted with ethyl
acetate and washed with water, and the organic layer was separated,
dried over anhydrous sodium sulfate, and evaporated. The residual oil
was purified by column chromatography using hexane:ethyl acetate
5:1 v/v to give 30 mg of the product (30%) as a white solid.
General Procedure for Synthesis of N-(2-Aminoethyl)-
benzene (Substituted Benzene) Sulfonamide (22a−g) and
N-(2-Aminopropyl)benzene (Substituted Benzene) Sulfona-
mide (23a−f). To a solution of ethylenediamine (19) or 1,3-
diaminopropane (20) (75 mmol) and triethylamine (1.07 mL, 7.6
mmol) in anhydrous dichloromethane (5 mL), a solution of
appropriate sulfonyl chloride (21a−g) (2.5 mmol) in anhydrous
dichloromethane (5 mL) was added dropwise at 0 °C. The reaction
mixture was stirred overnight. Ethyl acetate and a saturated solution
of Na2CO3 were added, and the organic layer was separated, washed
with distilled water, dried over Na2SO4, and evaporated. The residue
was purified by flash column using dichloromethane:methanol 10:1 v/
v.
NMR (CDCl3, 300 MHz) δ 8.61 (d, 1H, J = 3.4 Hz), 8.24 (d, 1H, J =
4.1 Hz), 7.35 (t, 1H, J = 5.9 Hz), 7.18 (d, 2H, J = 5.8 Hz), 4.78 (d,
2H, J = 3.4 Hz), 6.92(d, 1H, J = 4.1 Hz), 3.82 (s, 3H), 2.24 (s, 3H);
13C-NMR (CDCl3, 75 MHz) δ 172.4, 159.9, 156.3, 156.1, 152.7,
150.9, 135.8, 129.8, 122.2, 121.5, 120.2, 115.2, 114.0, 112.8, 112.2,
55.4, 14.2; IR (KBr) cm−1: 3153, 3114, 2919, 1602, 670; LC−MS: m/
z calculated for C17H14N4OS2: 354.06, found 355.10 (M + 1)+.
6-(4-Methoxyphenyl)-5-(2-(methylthio)pyrimidin-4-yl)imidazo-
1
[2,1-b]thiazole (13). Yield 15%; white solid; mp 151−152 °C; H-
NMR (CDCl3, 300 MHz) δ 8.57 (d, 1H, J = 2.7 Hz), 8.21 (d, 1H, J =
3.2 Hz), 7.53 (dd, 2H, J = 2.0, 6.8 Hz), 6.98−6.89 (m, 4H), 3.86 (s,
3H), 2.60 (s, 3H); 13C-NMR (CDCl3, 75 MHz) δ 172.3, 160.1,
156.2, 152.6, 151.1, 130.4, 126.9, 122.3, 119.8, 114.2, 112.5, 111.8,
55.3, 14.2; IR (KBr) cm−1: 1906, 1607, 1556, 630; LC−MS: m/z
calculated for C17H14N4OS2: 354.06, found 355.10 (M + 1)+.
General Procedure for Preparation of Compounds 14−16.
To a solution of compound 11−13 (6 mmol) in methanol (250 mL),
a solution of Oxone (12.3 g, 18 mmol) in water (50 mL) was added.
The mixture was stirred at room temperature for 16 h. The organic
solvent was removed under reduced pressure, and the remaining
aqueous solution was extracted with CH2Cl2 (50 mL). The organic
layer was separated, and the aqueous layer was extracted with CH2Cl2
(3 x 25 mL). The combined organic layer extracts were washed with
brine, dried over anhydrous sodium sulfate, and filtered. The organic
solvent was evaporated under reduced pressure, and the residue was
purified by flash column chromatography using hexane:ethyl acetate
5:3 v/v.
5-(2-(Methylsulfonyl)pyrimidin-4-yl)-6-phenylimidazo[2,1-b]-
1
thiazole (14). Yield 95%; white solid; mp 176−177 °C; H-NMR
(CDCl3, 300 MHz) δ 8.99 (d, 1H, J = 4.4 Hz), 8.58 (d, 1H, J = 5.4
Hz), 7.73 (d, 2H, J = 2.5 Hz), 7.66−7.47 (m, 3H), 7.45 (d, 1H, J =
5.2 Hz), 7.17 (d, 1H, J = 4.4 Hz), 3.47 (s, 3H); 13C-NMR (CDCl3, 75
MHz) δ 165.7, 157.5, 156.8, 153.3, 134.1, 133.3, 130.0, 129.5, 129.1,
129.0, 128.3, 119.5, 117.5, 113.8, 39.3; IR (KBr) cm−1: 3137, 3110,
2931, 1979, 1902, 1797, 1444, 1373; LC−MS: m/z calculated for
C16H12N4O2S2: 356.05, found 357.1 (M + 1)+.
6-(3-Methoxyphenyl)-5-(2-(methylsulfonyl)pyrimidin-4-yl)-
imidazo[2,1-b]thiazole (15). Yield 90%; white solid; mp 125−126
°C; 1H-NMR (CDCl3, 300 MHz) δ 8.86 (d, 1H, J = 3.4 Hz), 8.49 (d,
1H, J = 4.2 Hz), 7.39 (d, 2H, J = 3.3 Hz), 7.17 (d, 2H, J = 5.5 Hz),
7.07−7.02 (m, 2H), 3.84 (s, 3H), 3.36 (s, 3H); 13C-NMR (CDCl3, 75
MHz) δ 165.6, 160.1, 157.4, 156.9, 156.7, 154.3, 153.0, 135.3, 130.2,
123.1, 121.2, 119.54, 117.8, 115.6, 114.0, 55.4, 39.3; IR (KBr) cm−1:
3159, 3088, 1601, 1574, 1487, 1317; LC−MS: m/z calculated for
C17H14N4O3S2: 386.05, found 387.1 (M + 1)+.
Compound 16. Yield 85%; white solid; mp 192−193 °C; 1H-NMR
(CDCl3, 300 MHz) δ 8.88 (d, 1H, J = 3.0 Hz), 8.51 (d, 1H, J = 6.0
N-(2-Aminoethyl)benzenesulfonamide (22a). Yield 80% as a
1
white solid; mp 80−81 °C; H-NMR (CDCl3, 300 MHz) δ 7.77 (d,
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2H, J = 9.0 Hz), 7.46−7.38 (m, 3H), 3.99 (brs, 3H), 3.86 (t, 2H, J =
3.0 Hz), 2.67 (t, 2H, J = 3.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ
140.0, 132.4, 129.1, 126.8, 45.2, 40.9.
129.6, 127.0, 42.1, 40.1, 31.4, 21.4; LC−MS: m/z calculated for
C10H16N2O2S: 228.07, found: 229.10 (M + 1)+.
N-(3-Aminopropyl)naphthalene-2-sulfonamide (23f). Yield 79%
1
N-(2-Aminoethyl)-4-fluorobenzenesulfonamide (22b). 72% as a
as a white solid; mp 114−115 °C; H-NMR (CDCl3,400 MHz) δ
1
8.41 (s, 1H), 7.94−7.83 (m, 4H), 7.58 (d, 2H, J = 8.0 Hz), 3.12 (brs,
1H), 3.06 (t, 2H, J = 6.0 Hz), 2.73 (t, 2H, J = 6.0 Hz), 1.56 (t, 2H, J =
6.4 Hz); 13C-NMR (CDCl3, 100 MHz) δ 136.9, 134.7, 132.1, 129.4,
129.1, 128.5, 128.4, 128.1, 127.8, 127.4, 122.3, 42.5, 40.4, 31.2; LC−
MS: m/z calculated for C13H16N2O2S: 264.09, found: 265.10 (M +
1)+.
white solid; mp 108−109 °C; H-NMR (CDCl3, 300 MHz) δ 7.84−
7.79 (m, 2H), 7.11 (t, 2H, J = 6.0 Hz), 2.89 (t, 2H, J = 6.0 Hz),
2.71(t, 2H, J = 6.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ 166.7, 136.0,
129.7, 116.3, 45.2, 40.9; LC−MS: m/z calculated for C8H11FN2O2S:
218.06, found: 219.1 (M + 1)+.
N-(2-Aminoethyl)-3-fluorobenzenesulfonamide (22c). Yield 65%
1
Synthesis of 4-Methoxy-N-(2-((4-(6-phenylimidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(24a). A mixture of compound 14 (0.32 g, 0.92 mmol), 22b (0.54 g,
2.48 mmol), and N,N-diisopropylethylamine (0.57 mL, 3.3 mmol) in
DMSO (10 mL) was stirred at 80 °C for 8 h. The mixture was cooled
to room temperature, quenched with water (20 mL), and extracted
with ethyl acetate (3 x 20 mL). The combined organic layer was
washed with brine, dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography. The title product was obtained as an
as a viscous light brown oil; H-NMR (CDCl3, 300 MHz) δ 7.65−
7.44 (m, 3H), 7.24−7.19 (m, 1H), 4.29 (brs, 2H), 2.59 (t, 2H, J = 6.0
Hz), 2.76 (t, 2H, J = 6.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ 163.5,
161.0, 142.1 (J = 18 Hz), 131.1, 122.7 (J = 39 Hz), 114.3 (J = 69 Hz),
45.3, 41.0.
N-(2-Aminoethyl)-4-methoxybenzenesulfonamide (22d). Yield
1
82% as a white solid; mp 90−91 °C; H-NMR (CDCl3, 300 MHz)
δ 7.78 (d, 2H, J = 9.0 Hz), 6.96 (d, 2H, J = 6.0 Hz), 3.85 (s, 3H), 3.10
(brs, 2H), 2.93 (t, 2H, J = 6.0 Hz), 2.76 (t, 2H, J = 6.0 Hz); 13C-
NMR (CDCl3, 75 MHz) δ 163.3, 132.2, 129.6, 114.8, 56.2, 46.0, 41.6;
LC−MS: m/z calculated for C9H14N2O3S: 230.07, found: 231.10 (M
+ 1)+.
1
orange solid (0.24 g, 52%). mp 121−22 °C; H NMR (400 MHz,
CDCl3) δ 8.15 (d, J = 4.4 Hz, 1H), 7.93 (d, J = 5.2 Hz, 1H), 7.87 (d, J
= 7.2 Hz, 2H), 7.62 (dd, J = 7.2, J = 4.0 Hz, 2H), 7.52 (d, J = 7.2,Hz,
2H), 7.48−7.44 (m, 6H), 6.55 (d, J = 4.8 Hz, 1H), 6.39 (s, 1H, NH),
3.64 (s, 2H), 3.25 (d, J = 6.0 Hz, 2H), 13C-NMR (DMSO-d6, 75
MHz) δ 164.2, 162.5, 160.9, 158.2, 156.2, 151.6, 148.0, 140.9, 132.7,
131.7 (J = 7.5 Hz), 129.6, 126.8, 121.0, 115.9, 114.5, 105.7, 38.7,
29.5; LC−MS: m/z calculated for C23H19FN6O2S2, 494.10, found
495.10 (M + 1)+; HRMS calculated for C23H19FN6O2S2, is 494.0994,
found 495.1072 (M + 1)+.
N-(2-Aminoethyl)-4-methylbenzenesulfonamide (22e). Yield
74% as a white solid; mp 120−121 °C; 1H-NMR (CDCl3, 300
MHz) δ 7.75 (d, 2H, J = 9.0 Hz), 7.39 (d, 2H, J = 9.0 Hz), 3.45 (s,
2H), 2.89 (t, 2H, J = 6.0 Hz), 2.66 (t, 1H, J = 6.0 Hz), 2.44 (s, 3H);
13C-NMR (CDCl3, 75 MHz) δ 143.3, 137.0, 129.7, 127.0, 45.5, 41.0,
21.5; LC−MS: m/z calculated for C9H14N2O2S: 214.08, found:
215.10 (M + 1)+.
N-(2-Aminoethyl)naphthalene-2-sulfonamide (22f). Yield 90% as
a white solid; mp 132−133 °C; 1H-NMR (CDCl3, 300 MHz) δ 8.43
(s, 1H), 8.15 (t, 2H, J = 6.0 Hz), 8.04 (d, 1H, J = 9.0 Hz), 7.82 (d,
1H, J = 9.0 Hz), 7.70 (d, 2H, J = 6.0 Hz), 3.32 (s, 2H), 2.75 (t, 2H, J
= 6.0 Hz), 2.50 (t, 2H, J = 3.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ
136.8, 134.7, 132.1, 129.5, 129.2, 128.7, 128.3, 127.8, 127.5, 122.3,
45.5, 41.0; LC−MS: m/z calculated for C12H14N2O2S: 250.08, found:
251.10 (M + 1)+.
4-Methoxy-N-(2-((4-(6-phenylimidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (24b). Ob-
tained from the reaction of compounds 14 and 22d using the same
procedure for compound 24a to yield 65% as a white solid. mp 199−
1
200 °C; H-NMR (CDCl3, 400 MHz) δ 8.11 (d, J = 1.6 Hz, 1H),
7.93 (d, J = 5.8 Hz, 1H), 7.73 (d, J = 9.2 Hz, 2H), 7.63−7.60 (m,
2H), 7.44−7.42 (m, 4H), 7.24 (d, J = 6.8 Hz, 2H, Ar-H), 6.54 (d, J =
2.8 Hz, 1H, Ar-H), 6.27 (s, 1H, NH), 5.90 (s, 1H, NH), 3.93 (s, 3H),
3.61 (d, J = 5.2 Hz, 2H), 3.22 (d, J = 5.6 Hz, 2H); 13C-NMR
(DMSO-d6, 100 MHz) δ 163.8, 162.5, 161.4, 158.1, 156.2, 151.6,
148.1, 142.9, 138.0, 131.7, 130.1, 127.0, 121.0, 115.9, 114.4, 105.7,
55.3, 41.7, 29.5; LC−MS: m/z calculated for C24H22N6O2S2, 506.12,
found 507.10 (M + 1)+; HRMS calculated for C24H22N6O2S2,
506.1194, found 507.1272 (M + 1)+.
N-(2-Aminoethyl)-4-(trifluoromethyl)benzenesulfonamide (22g).
1
Yield 60% as a viscous oil; H-NMR (CDCl3, 300 MHz) δ 7.99 (d,
1H, J = 6.0 Hz), 7.64 (d, 1H, J = 9.0 Hz), 7.52 (t, 2H, J = 6.0 Hz),
3.15 (s,1H), 2.91 (s, 2H), 2.67 (s, 2H); 13C-NMR (CDCl3, 75 MHz)
δ 138.7, 132.6, 130.6, 128.2, 128.1, 44.9, 40.8.
N-(3-Aminopropyl)benzenesulfonamide (23a). Yield 84% as a
1
buff solid; mp 71−72 °C; H-NMR (CDCl3, 300 MHz) δ 7.90 (d,
4-Methyl-N-(2-((4-(6-phenylimidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (24c). Ob-
tained from the reaction of compounds 14 and 22e using the same
procedure for compound 24a to yield 60% as a light brown solid. mp
2H, J = 6.0 Hz), 7.60−7.51 (m, 3H), 3.11 (t, 2H, J = 6.0 Hz), 2.82 (t,
2H, J = 6.0 Hz), 1.62 (q, 2H, J = 6.0 Hz); 13C-NMR (CDCl3, 75
MHz) δ 139.8, 132.4, 129.1, 126.9, 41.1, 38.7, 30.2.
1
N-(3-Aminopropyl)-4-fluorobenzenesulfonamide (23b). Yield
82% as a white solid; mp 104−105 °C; 1H-NMR (CD3OD, 300
MHz) δ 7.90 (dd, 2H, J = 5.1, 8.9 Hz), 7.32 (t, 2H, J = 8.7 Hz), 2.92
(t, 2H, J = 6.0 Hz), 2.67 (t, 2H, J = 6.0 Hz), 1.61 (q, 2H, J = 6.0 Hz);
13C-NMR (CDCl3, 75 MHz) δ 162.9, 136.2, 129.4, 116.1, 41.1, 39.2,
31.5.
N-(3-Aminopropyl)-3-fluorobenzenesulfonamide (23c). Yield
55% as a viscous oil; 1H-NMR (CDCl3, 300 MHz) δ 7.52 (brs,
3H), 7.27 (s, 1H), 3.28 (s, 2H), 3.08 (s, 2H), 2.79 (s, 2H), 1.62
(s,2H); 13C-NMR (CDCl3, 75 MHz) δ 160.7, 142.3, 130.8 (J = 30
Hz), 122.6, 119.5, 114.3, 42.7, 40.5, 31.0.
N-(3-Aminopropyl)-4-methoxybenzenesulfonamide (23d). Yield
75% as a white solid; mp 76−77 °C; 1H-NMR (CD3OD, 300 MHz) δ
7.79 (d, 2H, J = 8.9 Hz), 7.08 (d, 2H, J = 8.9 Hz), 3.88 (s, 3H), 2.88
(t, 2H, J = 6.8 Hz), 2.66 (t, 2H, J = 6.9 Hz), 1.60 (t, 2H, J = 6.9 Hz);
13C-NMR (CDCl3, 75 MHz) δ 162.6, 131.6, 129.0, 114.2, 55.6, 41.6,
39.7, 31.1.
N-(3-Aminopropyl)-4-methylbenzenesulfonamide (23e). Yield
70% as a white solid; mp 119−120 °C; 1H-NMR (CDCl3, 400
MHz) δ 7.64 (d, 2H, J = 7.6 Hz), 7.19 (d, 2H, J = 8.0 Hz), 3.27 (brs,
3H), 2.89 (t, 2H, J = 6.00 Hz), 2.64 (t, 2H, J = 6.0 Hz), 2.31 (s, 3H),
1.48 (t, 2H, J = 6.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ 143.1, 137.1,
242−243 °C; H-NMR (CDCl3, 400 MHz) δ 8.14 (s, 1H), 7.96 (s,
1H), 7.71−7.62 (m, 4H), 7.49 (s, 4H), 7.44 (s, 2H), 6.54 (s, 1H),
6.18 (s, 1H), 5.77 (s, 1H), 3.47 (s, 2H), 3.04 (s, 2H), 2.35 (s, 3H);
13C-NMR (CDCl3, 75 MHz) δ 162.7, 162.2, 157.1, 152.2, 150.2,
134.8, 131.7, 129.3, 129.0, 128.6, 128.6, 122.0, 120.6, 114.2, 112.7,
107.2, 40.2, 38.2, 29.9; LC−MS: m/z calculated for C24H22N6O2S2,
490.12, found 491.10 (M + 1)+; HRMS calculated for C24H22N6O2S2,
490.1245, found 491.1323 (M + 1)+.
4-Fluoro-N-(3-((4-(6-phenylimidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)benzenesulfonamide (24d). Ob-
tained from the reaction of compounds 14 and 23b using the same
procedure for compound 24a to yield 68% as a buff solid. mp 188−
1
189 °C; H-NMR (CDCl3, 400 MHz) δ 8.06 (d, 1H, J = 5.2 Hz),
7.85 (dd, 3H, J = 5.6, 8.8 Hz), 7.59 (dd, 2H, J = 1.6, 7.6 Hz), 7.48−
7.37 (m, 7H), 6.31 (d, 1H, J = 5.2 Hz), 2.87 (t, 2H, J = 6.8 Hz), 2.51
(t, 2H, J = 2.0 Hz), 1.70 (t, 2H, J = 6.4 Hz); 13C-NMR (CDCl3, 75
MHz) δ 166.0, 164.7, 159.6, 158.7, 147.2, 140.1, 139.3, 131.0, 129.7
(J = 21 Hz), 128.8, 127.6, 125.1, 122.7, 119.9, 116.0 (J = 22.5 Hz),
114.8, 112.2, 106.0, 40.0, 38.2, 30.0; IR (KBr) cm−1: 3252, 3152,
3113, 2957, 1728, 1549, 1491, 1455, 1336; LC−MS: m/z calculated
for C23H21FN6O2S2: 508.12, found 509.10 (M + 1)+; HRMS
calculated for C23H21FN6O2S2: 508.1151, found 509.1229 (M + 1)+.
6893
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J. Med. Chem. 2021, 64, 6877−6901

Journal of Medicinal Chemistry
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Article
4-Methoxy-N-(3-((4-(6-phenylimidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)benzenesulfonamide (24e). Ob-
tained from the reaction of compounds 14 and 23d using the same
procedure for compound 24a to yield 45% as a white solid. mp 88−89
°C; 1H-NMR (CDCl3, 300 MHz) δ 8.55 (d, 1H, J = 6.0 Hz), 8.02 (d,
1H, J = 6.0 Hz), 7.75 (d, 2H, J = 9.0 Hz), 7.67 (dd, 2H, J = 3.0, 6.0
Hz), 7.45 (t, 3H, J = 3.0 Hz), 6.98 (d, 1H, J = 6.0 Hz), 6.92 (d, 2H, J
= 9.0 Hz), 6.50 (d, 1H, J = 9.0 Hz), 5.53 (brs, 1H), 3.83 (s, 3H), 3.57
(q, 2H, J = 6.0 Hz), 3.05 (q, 2H, J = 6.0 Hz), 1.80 (t, 2H, J = 6.0 Hz);
13C-NMR (CDCl3, 75 MHz) δ 162.7, 162.2, 157.1, 152.2, 150.2,
LC−MS: m/z calculated for C25H24N6O4S2: 526.13, found: 527.10
(M + 1)+.
N-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4-methylbenzenesulfonamide
(25e). Obtained from the reaction of 15 and 22e using the same
1
procedure for compound 24a to yield 46% as a viscous oil; H-NMR
(CDCl3, 300 MHz) δ 8.25 (s, 1H), 8.01 (t, 1H, J = 3.0 Hz), 7.73 (dd,
2H, J = 3.0, 6.0 Hz), 7.56 (t, 2H, J = 4.2 Hz), 7.00−6.92 (m, 5H),
6.57 (t, 1H, J = 3.0 Hz), 5.52 (brs, NH), 3.90 (s, 3H), 3.63 (brs, 2H),
3.26 (brs, 2H), 2.39 (s, 3H); LC−MS: m/z calculated for
C25H24N6O3S2: 520.14, found: 521.10 (M + 1)+.
N-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)naphthalene-2-sulfonamide (25f).
Obtained from the reaction of 15 and 22f using the same procedure
for compound 24a to yield 55% as a white solid; mp 136−137 °C;
1H-NMR (CDCl3, 300 MHz) δ 8.40 (s, 2H), 7.94 (d, 2H, J = 6.0 Hz),
7.84 (d, 3H, J = 9.0 Hz), 7.77 (d, 1H, J = 9.0 Hz), 7.32 (d, 1H, J = 9.0
Hz), 7.17 (d, 2H, J = 3.0 Hz), 6.97 (d, 1H, J = 9.0 Hz), 6.56 (d, 1H, J
= 6.0 Hz), 6.48 (d, 1H, J = 3.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ
161.9, 159.7, 156.9, 152.0,150.0, 136.7, 136.1, 134.6, 132.0, 129.6,
129.4, 129.0, 128.6, 128.2, 127.7, 127.4, 122.1, 121.7, 114.8, 114.3,
112.5, 107.5, 55.3, 43.5, 41.2.; IR (KBr) cm−1: 3600, 3254, 3129,
3087, 1672, 1601, 1451, 1428, 1322; LC−MS: m/z calculated for
C28H24N6O3S2: 556.14, found: 557.10 (M + 1)+.
N-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4-(trifluoromethyl)-
benzenesulfonamide (25g). Obtained from the reaction of 15 and
22g using the same procedure for compound 24a to yield 20% as a
colorless viscous oil; 1H-NMR (DMSO-d6, 400 MHz) δ 8.05 (d, 1H, J
= 5.2 Hz), 7.73 (t, 2H, J = 7.2 Hz), 7.59 (dd, 2H, J = 2.0, 8.0 Hz),
7.47 (d, 4H, J = 7.2 Hz), 7.06 (d, 2H, J = 8.8 Hz), 6.31 (d, 1H, J = 5.2
Hz), 3.79 (s, 3H), 3.39 (brs, 2H), 2.94 (brs, 2H); LC−MS: m/z
calculated for C25H21F3N6O3S2: 574.1, found: 575.10 (M + 1)+;
HRMS calculated for C25H21F3N6O3S2: 574.1069 found: 575.1148
(M + 1)+.
N-(3-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)benzenesulfonamide (25h). Ob-
tained from the reaction of 15 and 23a using the same procedure for
compound 24a to yield 55% as a white solid; mp 136−137; 1H-NMR
(CDCl3, 300 MHz) δ 8.55 (d, 1H, J = 6.0 Hz), 8.02 (d, 1H, J = 6.0
Hz), 7.75 (d, 2H, J = 9.0 Hz), 7.62 (q, 2H, J = 3.0 Hz), 7.45 (t, 3H, J
= 3.0 Hz), 6.98 (d, 1H, J = 6.0 Hz), 6.92 (d, 2H, J = 9.0 Hz), 6.50 (d,
1H, J = 6.0 Hz), 5.53 (brs, NH), 3.83 (s, 3H), 3.57 (q, 2H, J = 6.0
Hz), 3.05 (q, 2H, J = 6.0 Hz), 1.80 (t, 2H, J = 6.0 Hz); 13C-NMR
(CDCl3, 75 MHz) δ 162.7, 162.2, 157.1, 152.2, 150.2134.8, 131.7,
129.3, 129.0, 128.6, 128.6, 122.0, 120.6, 114.2, 112.7, 107.2, 55.5,
40.2, 38.2, 29.9; LC−MS: m/z calculated for C25H24N6O3S2: 520.14;
found: 521.10 (M + 1)+.
134.8, 131.7, 129.3, 129.0, 128.6, 128.6, 122.0, 120.6, 114.2, 112.7,
107.2, 55.5, 40.2, 38.2, 29.9; LC−MS: m/z calculated for
C25H24N6O3S2: 520.14, found: 521.10 (M + 1)+.
4-Methyl-N-(3-((4-(6-phenylimidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)benzenesulfonamide (24f). Ob-
tained from the reaction of compounds 14 and 23e using the same
procedure for compound 24a to yield 30% as a viscous oil; 1H-NMR
(CDCl3, 400 MHz) δ 8.52 (d, 1H, J = 4.0 Hz), 7.99 (s, 1H), 7.72 (d,
2H, J = 8.0 Hz), 7.29 (t, 1H, J = 8.0 Hz), 7.16 (s, 1H), 6.94−6.88 (m,
5H), 6.51 (d, 1H, J = 5.2 Hz), 5.50 (brs, 1H), 3.52 (brs, 2H), 3.00
(brs, 2H), 2.04 (s, 3H), 1.75 (d, 2H, J = 4.8 Hz).
N-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (25a). Ob-
tained from the reaction of compounds 15 and 22a using the same
procedure for compound 24a to yield 40% as a white solid; mp152−
1
153 °C; H-NMR (CDCl3, 300 MHz) δ 8.53 (d, 1H, J = 6.0 Hz),
8.04 (d, 1H, J = 6.0 Hz), 7.87 (d, 2H, J = 6.0 Hz), 7.57−7.49 (m,
4H), 7.36 (t, 1H, J = 7.5 Hz), 7.20 (s, 2H), 6.99 (d, 1H, J = 9.0 Hz),
6.96 (d, 1H, J = 6.0 Hz), 6.60 (d, 1H, J = 6.0 Hz), 5.50 (t, 1H, J = 6.0
Hz), 3.86 (s, 3H), 3.64 (t, 2H, J = 6.0 Hz), 3.28 (t, 2H, J = 6.0 Hz);
13C-NMR (CDCl3, 75 MHz) δ 162.1, 159.7, 157.1, 152.1, 139.9136.7,
132.6, 129.6, 129.1, 126.9, 122.1, 121.7, 114.9, 114.2, 112.6, 107.9,
55.4, 41.4, 39.9; IR (KBr) cm−1: 3246, 3126, 3061, 2859, 1603, 1430,
1325; LC−MS: m/z calculated for C24H22N6O3S2: 506.12, found
507.10 (M + 1)+.
4-Fluoro-N-(2-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(25b). Obtained from the reaction of compounds 15 and 22b using
the same procedure for compound 24a to yield 35% as a white solid;
mp163−164 °C; 1H-NMR (CDCl3, 400 MHz) δ 8.51 (d, 1H, J = 4.0
Hz), 8.00 (d, 1H, J = 4. Hz), 7.87 (dd, 2H, J = 4.0, 8.0 Hz), 7.34 (d,
1H, J = 8.0 Hz), 7.31 (d, 1H, J = 16.0 Hz), 7.19−7.13 (m, 4H), 7.01−
6.95 (m, 2H), 6.58 (d, 1H, J = 8.0 Hz), 5.33 (brs, 1H), 3.85 (s, 3H),
3.64 (dd, 2H, J = 4.0, 12.0 Hz), 3.26 (dd, 2H, J = 4.0, 12.0 Hz); 13C-
NMR (CDCl3, 100 MHz) δ 166.2, 163.7, 161.6, 159.7, 157.2, 156.3,
152.3, 150.3, 135.0, 129.7 (J = 9.6 Hz), 122.3, 121.7, 120.6, 116.3,
114.6, 107.5. 55.4, 43.1, 41.3; LC−MS: m/z calculated for
C24H21FN6O3S2: 524.12, found: 525.10 (M + 1)+.
4-Fluoro-N-(3-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]-
t h i a z o l - 5 - y l ) p y r i m i d i n - 2 - y l ) a m i n o ) p r o p y l ) -
benzenesulfonamide (25i). Obtained from the reaction of
compounds 15 and 23b using the same procedure for compound
3-Fluoro-N-(2-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(25c). Obtained from the reaction of 15 and 22c using the same
procedure for compound 24a to yield 30% as a white solid; mp 146−
1
24a to yield 59% as a white solid; mp 76−77 °C; H-NMR (CDCl3,
1
147 °C; H-NMR (CDCl3, 400 MHz) δ 8.43 (s, 1H), 7.94 (s, 1H),
400 MHz) δ 8.53 (d, 1H, J = 4.4 Hz), 8.03 (d, 1H, J = 5.6 Hz), 7.82
(dd, 2H, J = 1.6, 5.2 Hz), 7.35 (t, 1H, J = 8.0 Hz), 7.18−7.11 (m,
4H), 7.00−6.96 (m, 2H), 6.55 (d, 2H, J = 5.2 Hz), 5.53 (brs, NH),
3.84 (s, 3H), 3.56 (q, 2H, J = 6.40 Hz), 3.05 (q, 2H, J = 6.40 Hz),
1.79 (p, 2H, J = 6.00 Hz); 13C-NMR (CDCl3, 100 MHz) δ 166.1,
163.6, 162.2, 159.7, 157.2, 157.0, 152.1, 150.0, 136.3, 136.0, 129.6,
129.5, 121.9, 121.7, 120.6, 116.3, 114.7, 114.4, 112.7, 107.4, 55.3,
40.1, 38.1, 30.0; IR (KBr) cm−1: 3390, 3264, 3116, 2933, 1574, 1522,
1430, 1330; LC−MS: m/z calculated for C25H23FN6O3S2: 538.13,
found: 539.10 (M + 1)+.
7.59 (s, 1H), 7.51 (s, 1H), 7.39 (s, 1H), 7.27 (d, 1H, J = 3.6 Hz), 7.13
(brs, 3H), 6.93 (s, 1H), 6.88 (s, 1H), 6.50 (s, 1H). 5.66 (s, 1H), 3.80
(s, 3H), 3.55 (s, 2H), 3.20 (s, 2H), 1.25 (s, 1H); 13C-NMR (CDCl3,
100 MHz) δ 163.6, 162.0, 161.1, 159.7, 157.0, 152.1, 150.0, 142.2,
135.9, 130.9, 129.7, 122.6, 122.3, 122.0, 121.8, 120.6, 119.7, 114.7,
114.5, 114.2, 114.1, 112.6, 107.7 (J = 12 Hz), 55.3, 43.4, 41.3; IR
(KBr) cm−1: 3294, 3085, 2866, 1946, 1574, 1455, 1331; LC−MS: m/
z calculated for C24H21FN6O3S2.:524.11, found: 525.10 (M + 1)+.
4-Methoxy-N-(2-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(25d). Obtained from the reaction of 15 and 22d using the same
procedure for compound 24a to yield 35% as a white solid; mp 88−
89 °C; 1H-NMR (CDCl3, 300 MHz) δ 8.55 (d, 1H, J = 4.4 Hz), 8.04
(d, 1H, J = 5.4 Hz), 7.79 (d, 2H, J = 8.8 Hz), 7.37 (s, 1H), 7.30 (s,
1H), 7.21 (s, 1H), 6.94 (d, 4H, J = 9.0 Hz), 6.60 (d, 1H, J = 5.4 Hz),
5.46−5.44 (brs, NH), 3.86 (s, 3H), 3.83 (s, 3H), 3.64 (brs, 2H), 3.25
(brs, 2H); (KBr) cm−1: 3585, 3249, 3082, 2855, 1736, 1455, 1322;
3-Fluoro-N-(3-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]-
t h i a z o l - 5 - y l ) p y r i m i d i n - 2 - y l ) a m i n o ) p r o p y l ) -
benzenesulfonamide (25j). Obtained from the reaction of
compounds 15 and 23c using the same procedure for compound
1
24a to yield 30% as a white solid; mp 78−79 °C; H-NMR (CDCl3,
400 MHz) δ 8.42 (d, J = 4.0 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.76
(d, J = 12.0 Hz, 2H), 7.75−7.35 (m, 3H), 7.30 (d, J = 4.0 Hz, 1H),
7.18−6.95 (m, 2H), 6.53 (t, J = 8.0 Hz, 2H), 5.51 (d, J = 8.0 Hz, 1H),
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Article
3.81 (s, 3H), 3.56 (q, J = 8.0 Hz, 2H), 3.04 (q, J = 8.0 Hz, 2H), 1.84
(t, J = 8.0 Hz, 2H); LC−MS: m/z calculated for C25H23FN6O3S2:
538.13, found: 539.10 (M + 1)+; HRMS calculated for
C25H23FN6O3S2: 538.1257, found: 539.1335 (M + 1)+.
(brs, 2H), 2.39 (s, 3H); IR (KBr) cm−1: 3253, 3078, 2841, 1954,
1736, 1372, 1324.
N-(2-((4-(6-(4-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)naphthalene-1-sulfonamide
(26d). Obtained from the reaction of compounds 16 and 33 using the
same procedure for compound 41 to yield 37% as a yellowish white
4-Methoxy-N-(3-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]-
t h i a z o l - 5 - y l ) p y r i m i d i n - 2 - y l ) a m i n o ) p r o p y l ) -
benzenesulfonamide (25k). Obtained from the reaction of
compounds 15 and 23d using the same procedure for compound
1
solid; H-NMR (CDCl3, 300 MHz) δ 8.40 (brs, 1H), 7.93−7.82 (m,
4H), 7.35 (brs, 3H), 7.34−7.11 (m, 3H), 6.97 (d, 1H, J = 9.0 Hz),
1
6.47 (brs, 1H), 3.83 (s, 3H), 3.58 (brs, 2H), 3.30 (brs, 2H).
24a to yield 56% as a white solid; mp 96−97 °C; H-NMR (CDCl3,
Synthetic Procedure for 4-Hydroxy-N-(2-((4-(6-
phenylimidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)-
ethyl)benzenesulfonamide (27a). To a solution of compound 24a
(50 mg, 0.1 mmol) in anhydrous dichloromethane (1 mL), BBr3
(0.02 mL of 1 M solution in dichloromethane, 0.3 mmol) was added
dropwise at −78 °C under N2. The reaction mixture was stirred at the
same temperature for 30 min. The mixture was allowed to warm to
room temperature and stirred for 1 h. The reaction mixture was
quenched with saturated aqueous Na2CO3. Ethyl acetate (10 mL) was
added, and the organic layer was separated. The aqueous layer was
extracted with ethyl acetate (3 x 3 mL). The combined organic layer
extract was washed with brine and dried over anhydrous Na2SO4.
After evaporation of the organic solvent, the residue was purified by
column chromatography using hexane:ethyl acetate 1:2 v/v to give 25
300 MHz) δ 8.55 (d, 1H, J = 6.0 Hz), 6.03 (d, 1H, J = 6.0 Hz), 7.74
(d, 2H, J = 9.0 Hz), 7.35−7.30 (m, 2H), 7.20−7.18 (m, 3H), 6.94
(dd, 2H, J = 6.0, 9.0 Hz), 6.54 (t, 1H, J = 3.0 Hz), 5.32 (brs, NH),
3.84 (s, 3H), 3.83 (s, 3H), 3.56 (q, 2H, J = 6.0 Hz), 3.04 (d, 2H, J =
6.0 Hz), 1.79 (t, 2H, J = 6.0 Hz); 13C-NMR (CDCl3, 75 MHz) δ
162.7, 162.2, 159.7, 157.1, 152.1, 149.9, 136.0, 131.7, 129.6, 129.0,
122.1, 121.7, 120.7, 114.8, 114.3, 114.2, 112.7, 107.3, 55.5, 55.3, 40.2,
38.2. 29.8; IR (KBr) cm−1: 3382, 3264, 3117, 2938, 1903, 1574, 1455,
1328; LC−MS: m/z calculated for C26H26N6O4S2: 550.15, found:
551.10 (M + 1)+.
N-(3-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)-4-methylbenzenesulfonamide
(25l). Obtained from the reaction of compounds 15 and 23e using
the same procedure for compound 24a to yield 42% as a white solid;
1
1
mg (51.4%) as a buff solid. mp 208−209 °C; H-NMR (DMSO-d6,
mp 78−79 °C; H-NMR (CDCl3, 300 MHz) δ 8.57 (d, 1H, J = 3.0
300 MHz) δ 10.33 (s, 1H), 8.83 (s, NH), 8.05 (d, 1H, J = 3.0 Hz),
7.63−7.57 (m, 4H), 7.46 (s, 5H), 6.87 (t, 2H, J = 4.5 Hz), 6.32 (d,
1H, J = 6.0 Hz), 3.18 (s, 2H), 2.90 (brs, 2H); 13C-NMR (DMSO-d6,
75 MHz) δ 163.8, 162.5, 161.4, 158.1, 156.2, 151.6, 148.1, 142.9,
138.0, 131.7, 130.1, 127.0, 121.0, 116.1, 115.9, 115.7, 114.4, 105.7
49.0, 29.5; LC−MS: m/z calculated for C23H20N6O3S2:492.10, found
493.10 (M + 1)+; HRMS calculated for C23H20N6O3S2:492.1038,
found 493.1116 (M + 1)+.
N-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (27b). The
compound was prepared by the same demethylation reaction as
compound 27a. Yielding 44% of a dark orange solid; mp 70−71 °C;
1H-NMR (CD3OD, 400 MHz) δ 9.64 (s, 1H, exchangeable), 8.64 (bs,
1H, exchangeable), 8.19 (s, 1H), 8.10 (d, J = 5.2 Hz, 1H), 7.85 (d, J =
7.0 Hz, 3H), 7.62 (d, J = 7.3 Hz, 3H), 7.31 (t, J = 7.5 Hz, 1H), 7.03
(d, J = 7.6 Hz, 2H), 6.88 (d, J = 7.3 Hz, 1H), 6.50 (d, J = 5.2 Hz, 1H),
3.40 (s, 2H), 2.99 (s, 2H); IR (KBr) cm−1: 3115, 2854, 1639, 1574,
1445, 1328; LC−MS: m/z calculated for C23H20N6O3S2: 492.10,
found 493.10 (M + 1)+.
4-Fluoro-N-(2-((4-(6-(3-hydroxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(27c). The compound was prepared by demethylation of compound
25d using the same procedure to prepare compound 27a. Yield 32%;
dark orange solid; mp 70−71 °C; 1H-NMR (DMSO-d6, 300 MHz) δ
10.35 (s, 1H), 9.00 (brs, NH), 7.16 (d, 4H, J = 9.0 Hz), 7.45 (brs,
2H), 7.30 (t, 2H, J = 9.0 Hz), 7.86 (d, 2H, J = 9.0 Hz), 6.29 (d, 1H, J
= 9.0 Hz), 3.33 (brs, 2H), 2.90 (d, 2H, J = 9.0 Hz); IR (KBr) cm−1:
3389, 3065, 2927, 1731, 1614, 1552, 1499, 1446, 1326, 1157; LC−
MS: m/z calculated for C23H19FN6O3S2: 510.09, found: 511.10 (M +
1)+; HRMS calculated for C23H19FN6O3S2: 510.0944 found:
511.1022 (M + 1)+.
Hz), 7.71 (d, 2H, J = 9.0 Hz), 7.36 (d, 3H, J = 9.0 Hz), 7.22 (d, 2H, J
= 6.0 Hz), 7.03 (s, 2H), 6.59 (d, 1H, J = 6.0 Hz), 5.56 (brs, NH), 3.87
(s, 3H), 3.63 (t, 2H, J = 6.0 Hz), 1.63 (t, 2H, J = 6.0 Hz); LC−MS:
m/z calculated for C26H26N6O3S2: 534.15, found: 535.10 (M + 1)+.
N-(3-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)naphthalene-2-sulfonamide
(25m). Obtained from the reaction of compounds 15 and 23f using
the same procedure for compound 24a to yield 40% as a white solid;
mp 98−99 °C; 1H-NMR (CDCl3, 400 MHz) δ 8.38 (s, 1H), 8.06 (s,
1H), 7.97 (d, J = 4.0 Hz,1H), 7.88−7.76 (m, 4 H), 7.60−7.52 (m,
2H), 7.41 (s, 1H), 7.31 (dd, J = 4.0, 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
2H), 6.59 (dd, J = 4.0 Hz, 1H), 6.54 (d, J = 4.0 Hz, 1H), 5.68 (t, J =
8.0 Hz, 1H), 3.81 (s, 3H), 3.52 (q, J = 8.0 Hz, 2H), 3.07 (q, J = 8.0
Hz, 2H), 1.78 (t, J = 8.0 Hz, 2H),; 13C-NMR (CDCl3, 75 MHz) δ
162.1, 159.7, 157.0, 152.1, 149.9, 136.9, 136.0, 134.6, 132.1, 129.7,
129.4, 129.4, 129.1, 129.0, 128.7, 128.5, 128.2, 128.0, 127.8, 127.7,
127.4, 122.2, 121.9, 120.7, 114.7, 114.4, 114.2, 107.1, 55.3, 40.3, 38.2,
29.7; IR (KBr) cm−1: 3377, 3259, 3117, 1574, 1450, 1369, 1327;
LC−MS: m/z calculated for C29H26N6O3S2: 570.15, found: 571.10
(M + 1)+.
4-Fluoro-N-(2-((4-(6-(4-methoxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(26a). Obtained from the reaction of compounds 16 and 22b using
the same procedure for compound 24a to yield 29%; 1H-NMR
(DMSO-d6, 300 MHz) δ 8.05 (d, 1H, J = 6.0 Hz), 7.67 (d, 4H, J = 6.0
Hz), 7.50 (d, 2H, J = 9.0 Hz), 7.33 (d, 2H, J = 6.0 Hz), 7.02 (d, 2H, J
= 9.0 Hz), 6.34 (d, 1H, J = 3.0 Hz), 3.82 (s, 3H), 2.93 (t, 2H, J = 6.0
Hz), 2.38 (brs, 2H); IR (KBr) cm−1: 3364, 3304, 3063, 2924, 2861,
1924, 1597, 1455, 1318; LC−MS: m/z calculated for
C24H21FN6O3S2: 524.13, found 525.10 (M + 1)+.
4-Methoxy-N-(2-((4-(6-(4-methoxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(26b). Obtained from the reaction of compounds 16 and 22d using
the same procedure for compound 24a to yield 31%; 1H-NMR
(DMSO-d6, 300 MHz) δ 8.04 (d, 1H, J = 6.0 Hz), 7.71 (d, 2H, J = 9.0
Hz), 7.60 (brs, 1H), 7.49 (d, 2H, J = 8.4 Hz), 7.43 (s, 1H), 7.03 (t,
4H, J = 9.0 Hz), 6.34 (d, 1H, J = 3.0 Hz), 3.81 (s, 3H), 3.77 (s, 3H),
3.30 (brs, 2H), 2.94 (brs, 2H); LC−MS: m/z calculated for
C25H24N6O4S2: 536.13, found 537.10 (M + 1)+.
N-(2-((4-(6-(4-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4-methylbenzenesulfonamide
(26c). Obtained from the reaction of compounds 16 and 22e using
the same procedure for compound 24a to yield 33% as a buff solid;
mp 195−196 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 8.53 (d, 1H, J =
6.0 Hz), 8.02 (d, 1H, J = 6.0 Hz), 7.73 (t, 2H, J = 4.5 Hz), 7.57 (t,
2H, J = 3.0 Hz), 7.31−7.26 (m, 2H), 6.96 (t, 2H, J = 4.5 Hz), 6.58 (t,
1H, J = 3.0 Hz), 5.56 (brs, NH), 3.90 (s, 3H), 3.64 (brs, 2H), 3.26
N-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4-methylbenzenesulfonamide
(27d). The compound was prepared by demethylation of compound
25e using the same procedure to prepare compound 27a. Yield 10%;
1
buff solid; mp 115−116 °C; H-NMR (DMSO-d6, 300 MHz) δ 8.02
(brs, 1H), 7.69 (brs, 2H), 6.67 (d, 6H, J = 6.0 Hz), 6.40 (d, 5H, J =
9.0 Hz), 3.10 (t, 2H, J = 6.0 Hz), 2.74 (d, 2H, J = 6.0 Hz), 2.38 (s,
3H); LC−MS: m/z calculated for C24H22N6O3S2: 506.12, found:
507.10 (M + 1)+; HRMS calculated for C24H22N6O3S2: 506.1195,
found: 507.1273 (M + 1)+.
N-(3-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)pbopyl)benzenesulfonamide (27e). The
compound was prepared by demethylation of compound 25h using
the same procedure to prepare compound 27a. Yield 33%; brown
1
solid; mp 77−78 °C; H-NMR (DMSO-d6, 300 MHz) δ 8.71 (brs,
1H), 7.91 (d, 1H, J = 3.0 Hz), 7.83 (d, 1H, J = 9.0 Hz), 7.52 (d, 4H, J
6895
https://doi.org/10.1021/acs.jmedchem.1c00230
J. Med. Chem. 2021, 64, 6877−6901

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
= 6.0 Hz), 7.32 (d, 2H, J = 9.0 Hz), 7.00 (s, 1H), 6.92 (d, 3H, J = 6.0
Hz), 6.46 (d, 1H, J = 3.0 Hz), 3.49 (brs, 2H), 2.98 (d, 2H, J = 6.0
Hz), 1.82 (d, 2H, J = 6.0 Hz); LC−MS: m/z calculated for
C24H22N6O3S2: 506.12, found 507.10 (M + 1)+.
Hz), 7.33 (t, 1H, J = 7.8 Hz), 7.23−7.19 (m, 2H), 6.98−6.90 (m,
2H), 6.58 (d, 1H, J = 5.4 Hz), 3.83 (s, 3H), 3.78 (q, 2H, J = 6.1 Hz),
3.43−3.30 (m, 6H), 2.78 (s, 3H); LC−MS: m/z calculated for
C29H26F3N7O3S: 485.13, found: 486.10 (M + 1)+.
3-Fluoro-N-(3-((4-(6-(3-hydroxyphenyl)imidazo[2,1-b]-
t h i a z o l - 5 - y l ) p y r i m i d i n - 2 - y l ) a m i n o ) p r o p y l ) -
benzenesulfonamide (27f). The compound was prepared by
demethylation of compound 25j using the same procedure to prepare
2-Benzyl-5-(2-((4-(6-(3-methoxyphenyl)imidazo[2,1-b]thiazol-5-
yl)pyrimidin-2-yl)amino)ethyl)-1,2,5-thiadiazolidine 1,1-Dioxide
1
(37b). Yield 64%; white solid; mp 151−152 °C; H-NMR (CDCl3,
300 MHz) δ 8.59 (d, 1H, J = 4.4 Hz), 8.06 (d, 1H, J = 5.4 Hz), 7.39−
7.31 (m, 6H), 7.22−7.19 (m, 2H), 6.98−6.94 (m, 2H), 6.59 (d, 1H, J
= 5.4 Hz), 4.22 (s, 2H), 3.83−3.77 (m, 5H), 3.39 (q, 4H, J = 6.7 Hz),
3.20 (t, 2H, J = 6.5 Hz); LC−MS: m/z calculated for C27H27N7O3S2:
561.16, found: 562.10 (M + 1)+.
1
compound 27a. Yield 37%; light yellow solid; mp 111−112 °C; H-
NMR (CD3OD, 400 MHz) δ 8.29 (s, 1H), 7.93 (d, 1H, J = 5.5 Hz,),
7.81 (d, 1H, J = 1.5 Hz), 7.66 (d, 1H, J = 7.9 Hz), 7.62−7.51 (m,
2H), 7.45−7.43 (m, 1H), 7.42−7.24 (m, 3H), 7.20−7.16 (m, 1H),
6.38 (d, 1H, J = 5.4 Hz), 3.45 (s, 2H), 2.99 (t, 2H, J = 6.4 Hz), 1.80
(t, 2H, J = 6.7 Hz); 13C-NMR (CD3OD, 101 MHz) δ 163.8, 162.1,
161.4, 156.4, 155.5, 144.5, 142.6, 139.1, 136.6, 130.9, 130.8, 125.1,
122.5, 119.1, 118.9, 117.0, 115.9, 115.1, 113.7, 104.8, 40.3, 38.1, 29.2;
LC−MS (m/z) calculated for C24H21FN6O3S2 (m/z): 524.11, found:
525.0 (M + 1)+; HRMS calculated for C24H21FN6O3S2 (m/z):
524.1101, found: 525.1179 (M + 1)+.
2-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4,4-dimethyl-1,2,5-thiadiazolidine 1,1-
1
Dioxide (37c). Yield 54%; white solid; mp 186−187 °C; H NMR
(CDCl3, 300 MHz) δ 8.57 (d, 1H, J = 4.5 Hz), 8.06 (d, 1H, J = 5.4
Hz), 7.33 (t, 1H, J = 8.0 Hz), 7.26−7.19 (m, 2H), 6.96−6.91 (m,
2H), 6.58 (d, 1H, J = 5.4 Hz), 5.49 (m, 1H), 3.83−3.77 (m, 4H), 3.35
(t, 2H, J = 5.9 Hz), 3.21 (s, 2H), 1.43 (s, 6H); LC−MS: m/z
calculated for C22H25N7O3S2: 498.15, found: 499.10 (M + 1)+.
2-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-6-methyl-1,2,6-thiadiazinane 1,1-Di-
oxide (37d). Yield 54%; white solid; mp 105−106 °C; 1H-NMR
(CDCl3, 300 MHz) δ 8.61 (d, 1H, J = 5.3 Hz), 8.10 (d, 1H, J = 5.3
Hz), 7.41−7.34 (m, 1H), 7.26−7.23 (m, 2H), 7.01−6.99 (m, 2H),
6.61 (d, 1H, J = 5.5 Hz), 5.47 (m, 1H), 3.86 (s, 3H), 3.77−3.71 (m,
2H), 3.55 (t, 2H, J = 5.5 Hz), 3.46−3.36 (m, 4H), 2.81 (s, 3H),
1.87−1.85 (m, 2H); LC−MS: m/z calculated for C22H25N7O3S2:
499.13, found: 500.00 (M + 1)+.
N-(3-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)propyl)-4-methylbenzenesulfonamide
(27g). It was prepared by demethylation of compound 25l using the
same procedure to prepare compound 27a. Yield 40%; mp 171−172
°C; 1H-NMR (400 MHz, MeOD) δ 8.40 (s, 1H), 8.02 (d, 1H, J = 5.4
Hz), 7.89 (d, 1H, J = 1.4 Hz), 7.72 (d, 2H, J = 8.2 Hz), 7.55−7.49 (m,
1H), 7.45 (d, 1H, J = 7.7 Hz), 7.37−7.35 (m, 3H), 7.24 (m, 1H), 6.47
(d, 1H, J = 5.4 Hz), 3.48 (s, 2H), 2.98 (t, 2H, J = 6.7 Hz), 2.39 (s,
3H), 1.81−1.78 (m, 2H); 13C-NMR (CD3OD, 101 MHz) δ 162.1,
157.1, 155.7, 143.5, 137.3, 130.1, 129.3, 126.5, 125.2, 123.6, 116.1,
115.3, 106.2, 104.7, 40.1, 38.3, 29.8, 19.9; LC−MS (m/z) calculated
for C25H24N6O3S2 (m/z): 520.14, found: 521.10 (M + 1)+.
4-Hydroxy-N-(2-((4-(6-(4-hydroxyphenyl)imidazo[2,1-b]-
thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide
(27h). The compound was prepared by demethylation of compound
26b using the same procedure to prepare compound 27a. Yield 15%;
light yellow solid; mp 160−161 °C; 1H-NMR (DMSO-d6, 300 MHz)
δ 8.04 (d, 1H, J = 5.5 Hz), 7.95 (s, 3H), 7.61 (d, 2H, J = 6.0 Hz), 7.41
(d, 1H, J = 3.5 Hz), 7.37 (d, 1H, J = 8.6 Hz), 6.85 (t, 4H, J = 8.9 Hz),
6.36 (d, 2H, J = 5.3 Hz), 2.89 (s, 2H), 2.73 (s, 2H); IR (KBr) cm−1:
3391, 3294, 3089, 2929, 1914, 1452, 1330; LC−MS: m/z calculated
for C23H20F3N6O4S2: 508.10, found 509.00 (M + 1)+.
2-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-1,2,5-thiadiazolidine 1,1-Dioxide
1
(37e). White solid (49%); mp 100−101 °C; H NMR (CDCl3, 300
MHz) δ 8.61 (d, 1H, J = 4.5 Hz), 8.10 (d, 1H, J = 5.4 Hz), 7.37 (t,
1H, J = 7.6 Hz), 7.25−7.22 (m, 2H), 7.01−6.99 (m, 2H), 6.63 (d,
1H, J = 5.4 Hz), 5.55 (t, 1H, J = 6.1 Hz), 4.44 (s, 1H), 3.87 (s, 3H),
3.85−3.80 (q, 2H, J = 6.0 Hz), 3.59−3.53 (m, 4H), 3.40 (t, 2H, J =
6.1 Hz); LC−MS: m/z calculated for C20H21N7O3S2: 471 Found: 472
(M + 1)+.
2-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-1,2,6-thiadiazinane 1,1-Dioxide (37f).
White solid (38%); mp 102−103 °C; 1H NMR (CDCl3, 300 MHz) δ
8.49 (d, 1H, J = 4.5 Hz), 7.86 (s, 1H), 7.28 (t, 1H, J = 8.2 Hz), 7.12−
7.10 (m, 2H), 6.93−6.90 (m, 2H), 6.52 (d, 2H, J = 5.8 Hz), 3.76 (s,
3H), 3.66 (q, 2H, J = 5.9 Hz), 3.51−3.44 (m, 2H), 3.38 (t, 2H, J = 5.5
Hz), 3.3 (t, 2H, J = 6.2 Hz), 1.90−1.82 (m, 2H); LC−MS: m/z
calculated for C21H23N7O3S2: 485 Found: 486 (M + 1)+.
N-(2-((4-(6-(4-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4-methylbenzenesulfonamide
(27i). The compound was prepared by demethylation of compound
26c using the same procedure to prepare compound 27a. Yield 33%;
1
buff solid; mp 163−164 °C; H-NMR (DMSO-d6, 300 MHz) δ 9.73
(s, 1H), 8.03 (d, 1H, J = 6.0 Hz), 7.66 (d, 2H, J = 8.1 Hz), 7.38 (s,
1H), 7.34 (d, 3H, J = 3.2 Hz), 6.83 (d, 2H, J = 6.3 Hz), 6.35 (d, 1H, J
= 5.3 Hz), 2.92 (d, 4H, J = 5.3 Hz), 2.31 (s, 3H); LC−MS: m/z
calculated for C24H22N6O3S2: 506.12, found 507.10 (M + 1)+.
Synthesis of Compounds 29a−c and 30a−d. Compounds
29a−c and 30a−d were prepared according to a well-known
procedure.45
tert-Butyl 6-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-
yl)pyrimidin-2-yl)amino)ethyl)-1,2,6-thiadiazinane-2-carboxylate
1
1,1-Dioxide (37g). White solid (29%); mp 102−103 °C; H NMR
(CDCl3, 300 MHz) δ 8.56(d, 1H, J = 3.4 Hz), 8.06 (d, 1H, J = 4.1
Hz), 7.31 (t, 1H, J = 5.7 Hz), 7.24−7.19 (m, 2H), 7.01−6.99 (m,
2H), 6.62 (s, 1H), 5.47 (s, 1H), 3.99 (t, 2H, J = 5.7 Hz), 3.87 (s, 3H),
3.75 (q, 2H, J = 5.9 Hz), 3.68 (t, 2H, J = 5.8 Hz), 3.51 (t, 2H, J = 6.1
Hz), 1.90−1.82 (m, 2H), 1.55 (s, 9H); LC−MS: m/z calculated for
C26H31N7O5S2: 584 Found: 585 (M + 1)+.
Synthesis of Compound 33. Compound 33 was performed
according to reported procedures.46
Synthetic Procedure for 2-(2-((4-(6-(3-Hydroxyphenyl)-
imidazo[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-5-
methyl-1,2,5-thiadiazolidine 1,1-Dioxide (38a). To a solution of
compound 37a (50 mg, 0.1 mmol) in dichloromethane (1 mL), BBr3
(0.02 mL of a 1 M solution in dichloromethane, 0.3 mmol) was added
dropwise at −78 °C under N2. The reaction mixture was stirred at the
same temperature for 30 min. The mixture was allowed to warm to
room temperature and stirred for another 1 h. The reaction mixture
was quenched with saturated aqueous sodium carbonate. Ethyl acetate
(5 mL) was added, and the organic layer was separated. The aqueous
layer was extracted with ethyl acetate (3 × 3 mL). The combined
organic layer extract was washed with brine and dried over anhydrous
sodium sulfate. After evaporation of the organic solvent, the residue
was purified by column chromatography using hexane:ethyl acetate
1:2 v/v to give 12.71 mg (27%) of the pure product as a light yellow
solid. mp 151−152 °C; 1H-NMR (DMSO-d6, 300 MHz) δ 8.65 (brs,
Synthesis of Cyclic Sulfamide Side Chains 36a−g. Com-
pounds 36a−g were prepared by adopting the reported procedures.47
General Procedure for Preparation of the Target Com-
pounds (37a−g). A mixture of compound 15 (355.52 mg, 0.92
mmol), and selected compound from 36a−g (2.48 mmol), and
diisopropylethylamine (DIPEA, 0.57 mL, 3.3 mmol) in DMSO (10
mL) was stirred at 80 °C for 8 h. The mixture was cooled to room
temperature, quenched with water (20 mL), and then extracted with
ethyl acetate (3 × 20 mL). The combined organic layer extracts were
washed with brine, dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography.
2-(2-((4-(6-(3-Methoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-5-methyl-1,2,5-thiadiazolidine 1,1-Di-
oxide (37a). Yield 50%; white solid; mp 144−145 °C; 1H-NMR
(CDCl3, 300 MHz) δ 8.58 (d, 1H, J = 4.5 Hz), 8.06 (d, 1H, J = 5.3
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1H), 7.96 (d, 1H, J = 5.3 Hz), 7.29−7.21 (m, 7H), 6.43 (d, 1H, J =
5.3 Hz), 3.67 (t, 2H, J = 5.6 Hz), 3.44 (t, 2H, J = 5.4 Hz) 3.33−3.28
(m, 4H), 2.70 (s, 3H); 13C-NMR (CD3OD, 101 MHz) δ 159.9,
157.4, 154.4, 152.6, 135.4, 134.5, 114.7, 113.8, 113.6, 104.4, 54.4,
46.6, 45.8, 39.7, 25.5; LC−MS: m/z calculated for C20H21N7O3S2:
471.11, found: 472.1 (M + 1)+; HRMS calculated for C20H21N7O3S2:
471.1147, found: 472.1225 (M + 1)+.
Compounds 38b−g Were Synthesized by the Same
Procedure Utilized for Demethylation and Synthesis of
Compound 38a. 2-Benzyl-5-(2-((4-(6-(3-hydroxyphenyl)imidazo-
[2,1-b]thiazol-5-yl)pyrimidin-2-yl)amino)ethyl)-1,2,5-thiadiazoli-
dine 1,1-Dioxide (38b). Yield 23%; white solid; mp 155−156 °C; 1H-
NMR (DMSO-d6, 300 MHz) δ 9.56 (s, 1H), 8.17 (d, 1H, J = 5.4 Hz),
7.37−7.25 (m, 9H), 6.98 (d, 2H, J = 6.5 Hz), 6.82 (d, 1H, J = 8.7
Hz), 6.41 (d, 1H, J = 8.7 Hz), 4.12 (s, 2H), 3.57 (s, 4H), 3.21−3.13
(m, 4H); LC−MS: m/z calculated for C26H25N7O3S2: 547.15; found:
548.1 (M + 1)+.
In Vitro Kinase Panel Assay. Kinase-tagged T7 phage strains
were grown in parallel in 24-well blocks in an E. coli host derived from
the BL21 strain. E. coli were grown to log-phase and infected with T7
phage from a frozen stock (multiplicity of infection = 0.4) and
incubated with shaking at 32 °C until lysis (90−150 min). The lysates
were centrifuged (6000g) and filtered (0.2 μm) to remove cell debris.
The remaining kinases were produced in HEK-293 cells and
subsequently tagged with DNA for qPCR detection. Streptavidin-
coated magnetic beads were treated with biotinylated small molecule
ligands for 30 min at room temperature to generate affinity resins for
kinase assays. The liganded beads were blocked with excess biotin and
washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05%
Tween 20, 1 mM DTT) to remove the unbound ligand and to reduce
non-specific phage binding. Binding reactions were assembled by
combining kinases, liganded affinity beads, and test compounds in 1x
binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, and 6
mM DTT). Test compounds were prepared as 40x stocks in 100%
DMSO and directly diluted into the assay. All reactions were
performed in polypropylene 384-well plates in a final volume of 0.04
mL. The assay plates were incubated at room temperature with
shaking for 1 h, and the affinity beads were washed with wash buffer
(1x PBS, 0.05% Tween 20). The beads were then re-suspended in
elution buffer (1x PBS, 0.05% Tween 20, and 0.5 μM non-biotinylated
affinity ligand) and incubated at room temperature with shaking for
30 min. The kinase concentration in the eluates was measured by
qPCR.
2-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-4,4-dimethyl-1,2,5-thiadiazolidine 1,1-
1
Dioxide (38c). Yield 25%; white solid; mp 196−197 °C; H-NMR
(CDCl3, 400 MHz) δ 8.55 (d, 1H, J = 5.4 Hz), 8.04 (d, 1H, J = 5.4
Hz), 7.30−7.28 (m, 1H), 7.15−7.13 (m, 2H), 6.95 (d, H, J = 8.1 Hz),
3.76 (t, 2H, J = 6.1 Hz), 3.33 (t, 2H, J = 5.9 Hz), 3.25 (s, 2H), 1.45 (s,
6H); LC−MS: m/z calculated for C21H23N7O3S2: 485.13, found:
486.10 (M + 1)+.
2-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-6-methyl-1,2,6-thiadiazinane 1,1-Di-
oxide (38d). Yield 21%; white solid; mp 184−185 °C; 1H-NMR
(CDCl3, 400 MHz) δ 8.57 (d, 1H, J = 4.5 Hz), 8.01 (d, 1H, J = 5.4
Hz), 7.28−7.26 (m, 1H), 7.15−7.11 (m, 2H), 6.88 (d, H, J = 7.7 Hz),
6.59 (d, 1H, J = 5.3 Hz) 3.67 (t, 2H, J = 5.4 Hz), 3.48 (t, 2H, J = 5.2
Hz), 3.37 (t, 2H, J = 6.0 Hz), 2.75 (s, 3H), 1.78−1.72 (m, 2H); LC−
MS: m/z calculated for C21H23N7O3S2: 485, found: 489 (M + 1)+.
2-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-1,2,5-thiadiazolidine 1,1-Dioxide
(38e). White solid (22%); mp 195−196 °C; 1H NMR (CD3OD,
300 MHz) δ 8.65 (s, 1H), 7.90 (d, 1H, J = 5.4 Hz), 7.21−7.16 (m,
2H), 6.93−6.89 (m, 2H), 6.78 (s, 1H), 3.6 (t, 2H, J = 5.6 Hz), 3.3 (t,
2H, J = 5.5 Hz), 3.15 (t, 2H, J = 6.3 Hz); LC−MS: m/z calculated for
C19H19N7O3S2: 457 Found: 458 (M + 1)+.
2-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
pyrimidin-2-yl)amino)ethyl)-1,2,6-thiadiazinane 1,1-Dioxide (38f).
White solid (25%); mp 183−184 °C; 1H NMR (CDCl3, 400 MHz) δ
8.85 (s, 1H), 8.14 (d, 1H, J = 5.9 Hz), 7.78 (d, 1H, J = 4.1 Hz), 7.47
(t, 1H, J = 7.7 Hz), 7.17−7.09 (m, 3H), 6.74 (d, H, J = 6.7 Hz),
3.88−3.85 (m, 2H), 3.60 (t, 2H, J = 7.7 Hz), 3.44−3.41 (m, 4H),
1.78 (m, 2H); LC−MS: m/z calculated for C20H21N7O3S2: 471
Found: 472 (M + 1)+.
NCI-60 Cancer Cell Line Screening. Screening against a panel of
60 cancer cell lines was applied at the National Cancer Institute
(NCI), Bethesda, Maryland, USA (www.dtp.nci.nih.gov), applying
their standard protocol (https://dtp.cancer.gov/discovery_
development/nci-60/methodology.htm).
MTT Assay against L132 (Human Embryonic Pulmonary
Epithelial Cells). The human lung normal cell line (L132; human
embryonic pulmonary epithelial cells) was obtained from the Korean
cell line bank (KCLB, Seoul, Korea). Cells were cultured in RPMI
1640 supplemented with 10% heat-deactivated FBS, penicillin (100
units/mL), and streptomycin sulfate (100 μg/mL). Cells were
cultured at 37 °C in an atmosphere of 5% CO2. MTT assay was
used to determine sample cytotoxicity. Cells (5 × 104) were seeded in
each well containing 100 μL of the medium supplemented with 10%
FBS in a 96-well plate. After 24 h, various concentrations of the tested
samples were added. After 48 h, 20 μL of MTT (5 mg/mL stock
solution in phosphate-buffered saline (PBS)) was added and the
plates were incubated for an additional 4 h. The medium was
discarded, and formazan blue, which was formed in the cells, was
dissolved with 200 μL of DMSO. The optical density was measured at
540 nm using microplate readers (Molecular Devices, CA, USA).
MTT Assay against BJ1 Cells (Normal Skin Fibroblast Cell
Line). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide) assay developed by Mosmann was modified by Miura
and used to determine the in vitro inhibitory effects of the test
compound on cell growth. A medium containing 10 × 103 cells (BJ1
cells) in a fresh complete growth medium was seeded into each well
of a 96-well microplate, with the compound solution added
simultaneously to triplicate wells, before the final volume was made
up to 100 mL. The plate was incubated at 37 °C for 72 h in a
humidified atmosphere of 5% CO2 using a water- jacketed carbon
dioxide incubator (TC2323; Sheldon, Cornelius, OR). The medium
was aspirated, fresh medium (without serum) was added, and cells
were incubated, with different concentrations of the sample, to give
final concentrations of 500, 100, 50, 25, 12.5, 6.25, 3.125, and 0.78
mg/mL. Cells were suspended in DMEM-F12, 1% antibiotic−
antimycotic mixture (10,000 U/mL potassium penicillin, 10,000
mg/mL streptomycin sulfate, and 25 mg/mL amphotericin B), and
1% L-glutamine in a 96-well flat-bottom microplate at 37 °C under 5%
CO2. After 48 h of incubation, the medium was aspirated; 200 mL of
10% sodium dodecyl sulfate (SDS) in deionized water was added to
each well and further incubated overnight at 37 °C under 5% CO2.
Then, 200 mL of 10% SDS in deionized water was added to each well
to stop the reaction and to solubilize any MTT formazan that had
tert-Butyl 6-(2-((4-(6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazol-5-
yl)primidin-2-yl)amino)ethyl)-1,2,6-thiadiazinane-2-carboxylate
1
1,1-Dioxide (38g). White solid (16%); mp 180−181 °C; H NMR
(CDCl3, 400 MHz) δ 8.57 (d, 1H, J = 4.5 Hz), 8.04 (d, 1H, J = 5.6
Hz), 7.26−7.19 (m, 2H), 6.96−6.91 (m, 2H), 6.58 (d, H, J = 5.4 Hz),
3.72 (t, 2H, J = 5.9 Hz), 3.54 (t, 2H, J = 5.8 Hz), 3.45 (t, 2H, J = 5.1
Hz), 3.36 (t, 2H, J = 5.1 Hz), 1.78 (m, 3H), 1.61 (s, 9H); LC−MS:
m/z calculated for C25H29N7O5S2: 571 Found: 572 (M + 1)+.
In Vitro Enzyme Assay. The enzymatic assays of wild-type BRAF,
V600E-BRAF, and CRAF were performed in Reaction Biology Corp.
(http://www.reactionbiology.com) using a standard protocol and at a
1 μM ATP concentration and 3-fold dilution factor. In a final reaction
volume of 25 μL, kinase (5−10 mU) is incubated with 25 mM Tris
pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic protein, 10 mM
magnesium acetate, and [γ33P-ATP] (specific activity approx. 500
cpm/pmol, concentration as required). The reaction is initiated by
the addition of the Mg-ATP mix. After incubation for 40 min at room
temperature, the reaction is stopped by the addition of 5 μL of a 3%
phosphoric acid solution. A 10 μL solution of the reaction is then
spotted onto a P30 filtermat and washed three times for 5 min in 75
mM phosphoric acid and once in methanol prior to drying and
scintillation counting.
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formed, and then it was incubated overnight at 37 °C. Also, 100 mL
of 0.02 N HCl/50% N,N-dimethylformamide, 20% SDS was added to
solubilize any MTT formazan that had formed. The optical density of
each well was measured at 575 nm (OD575) using a microplate
multiwell reader (model 3350; Bio-Rad Laboratories Inc., Hercules,
CA), and the inhibition of cell growth (%) was calculated as (1 − T/
C) × 100, where C is the mean OD575 of the control group and T is
that of the treated group. The IC50 value was determined from the
dose−response curve.
Caco-2 Cell Permeability Test of Compound 38a. Caco-2
cells were seeded in 12-well Transwell plates with 5 × 105 cells and
cultured for 3 weeks. Compound 38a was diluted with a transport
buffer and 1% DMSO (transport buffer: 10 mM glucose, 4 mM
sodium bicarbonate, and 1 mM HEPES in HBSS (pH 7.4)). The
Caco-2 monolayer was placed between partition A and partition B.
Compound 38a in transport buffer was placed on side A, and only
transport buffer only on side B (to calculate A to B). Compound 38a
in transport buffer was placed on side B, and only transport buffer
only on side A (to calculate B to A). For each test, the temperature
was kept at 37 °C and the sample was taken every 15 min for 1 h. In
the sample obtained from each side, acetonitrile and an internal
standard used for mass spectrometry were added to reach a final
concentration of 5 μM. The concentration of each sample was
measured by LC/MS/MS.
humidity of 50 5%. Animals were kept under a 12 h light and dark
cycle. Animals were allowed to freely reach food and water. Animals
were fasted 16 h before oral dose. Compound 38a was dissolved in
5% DMSO, 40% PEG400, and 55% distilled water. For oral dose, 500
μL was used and 250 μL was used for intravenous injection. Serum
samples were collected each 30 min. The blood concentration of the
test compounds was determined by LC/MS/MS (Agilent 1290
infinity II series equipped with an on-line degasser, binary pump,
thermostated well-plate autosampler, and column compartment,
Waters Atlantis HSS T3 (2.1 × 100 mm, 1.9 μm) column, and
mobile phase linear gradient from 95% A (0.1% formic acid in water)/
5% B (0.1% formic acid in acetonitrile) to 5% A/95% B, 6500+
QTRAP LC/MS/MS/MS system, Turbo Spray Ion Drive as an ion
source, and carbamazepine as an internal standard. Pharmacokinetic
parameters were obtained by non-compartmental analysis of the
plasma concentration−time profiles using Kinetica (Thermo Fisher
Scientific, Inc., Woburn, MA, USA).
Plasma Stability. About 10 μM compound 38a was mixed with
human plasma and rat plasma, and the mixture was shaken at 37 °C
for 30 min and 120 min. After each time interval, acetonitrile was
added and the mixture was centrifuged (14,000 rpm, 4 °C). The
supernatant was injected into the LC−MS to detect the remaining
amount of compound 38a.
Molecular Docking of Compound 38a. The X-ray crystal
structure of V600E-BRAF oncogenic mutant kinase in complex with
vemurafenib (PDB ID: 1UWJ) was downloaded from the Protein
Data Bank (www.rcsb.org) in PDB format. The 2D structure of
compound 38a was drawn using ChemDraw software. Molecular
Operating Environment (MOE, 2014.0901) software was used for the
molecular docking operation of the target compound 38a with
V600E-BRAF kinase domain (PDB ID: 1UWJ). The kinase was
prepared for the molecular docking procedure by applying 3D
protonation of enzyme amino acids and the native ligands. In
addition, water of crystallization was removed from V600E-BRAF
kinase domains. The active site of the enzyme was isolated. The
docking simulation of the native ligand with the active site of V600E-
BRAF kinase was investigated in order to validate the docking
protocol. Both 3D protonation and energy minimization were
performed for the target compounds using MOE software.
Immunoblot Assay. A375 cells were disrupted in RIPA buffer
containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.25% sodium
deoxycholate, 1 mM EDTA, 1% NP40, 1 mM NaF, 0.2 mM
phenylmethyl sulfonyl fluoride, 0.1 mM sodium orthovanadate, and a
protease inhibitor cocktail (Roche Life Science, Indianapolis, IN,
USA). The proteins were resolved by sodium dodecyl sulfate−
polyacrylamide gel electrophoresis and transferred to polyvinylidene
difluoride membranes (Millipore, Burlington, MA, USA) blocked in
5% skim milk and probed with the indicated antibodies. The
immunoblots were visualized using a SuperSignal West Femto
chemiluminescence substrate (Thermo Fisher Scientific, Waltham,
MA, USA) and detected using the LAS 4000-mini biomolecular
imaging system (FUJIFILM, Tokyo, Japan).
In Vivo Antitumor Activity. The anticancer activity of compound
38a was performed by using A375 melanoma xenograft in male
Hsd:Athymic Nude-Foxn1nu (Harlan Co. (USA)) mice (8−12 weeks
old and 25−40 g weight). The test animals were divided into four
groups, and each group composed of seven mice. Group 1: control
group, which took placebo and did not take any drug; Group 2: each
animal received 25 mg/kg/day of compound 38a; Group 3: each
animal received 50 mg/kg/day of compound 38a; Group 4: each
animal received 50 mg/kg/day of vemurafenib. The four groups were
monitored for 21 days, and both body weight and tumor size were
measured every 3 days. The animals were kept under controlled
weather and feeding conditions.
In Vivo Pharmacokinetic of Compound 38a. Compound 38a
was administered intravenously and orally at a dose of 5 mg/kg in rats.
Each group composed of three animals (7−8 weeks old male SD rat
(Core Tech, Hana Corporation). The weight of animals was 250−300
g. Animals were kept at a fixed temperature of 22 2 °C and relative
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00230.
■
sı
Tables summarizing the inhibition percentage values of
the target compounds over the NCI-60 cell line panel
(CSV)
Figures showing heat maps of NCI results of the target
compounds, raw NCI results, characterization charts
(1H-NMR, 13C-NMR, LC−MS, and HRMS), HPLC
traces, and 2D/3D docking files (PDF)
Molecular formula strings file including the target
compound SMILES and IC50 values against BRAF
(wild-type), V600E-BRAF, and CRAF (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Chang Hyun Oh − Center for Biomaterials, Korea Institute of
Science & Technology (KIST), 136-791 Seoul, Republic of
Korea; Phone: +82 2 958 5160; Email: choh@kist.re.kr
Authors
Mohammed S. Abdel-Maksoud − Medicinal &
Pharmaceutical Chemistry Department, Pharmaceutical and
Drug Industries Research Division, National Research Centre
(NRC), Dokki, Giza 12622, Egypt
Mohammed I. El-Gamal − Department of Medicinal
Chemistry, College of Pharmacy and Sharjah Institute for
Medical Research, University of Sharjah, Sharjah 27272,
United Arab Emirates; Department of Medicinal Chemistry,
Faculty of Pharmacy, University of Mansoura, Mansoura
35516, Egypt; orcid.org/0000-0002-4269-5264
Bong S. Lee − CTC SCIENCE, Gyeonggi-do 18576, Republic
of Korea
Mahmoud M. Gamal El-Din − Medicinal & Pharmaceutical
Chemistry Department, Pharmaceutical and Drug Industries
Research Division, National Research Centre (NRC), Dokki,
Giza 12622, Egypt
Hong R. Jeon − CTCBIO Inc., Gyeonggi-do 18576, Republic
of Korea
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Dow Kwon − CTC SCIENCE, Gyeonggi-do 18576, Republic
of Korea
Cancer Institute; q, quartet; s, singlet; t, triplet; TEA,
triethylamine; TGI, total growth inhibition
Usama M. Ammar − Strathclyde Institute of Pharmacy and
Biomedical Sciences, University of Strathclyde, Glasgow G4
0NR, Scotland, United Kingdom
Karim I. Mersal − Center for Biomaterials, Korea Institute of
Science & Technology (KIST), 136-791 Seoul, Republic of
Korea; Department of Biomolecular Science, University of
Science & Technology (UST), Daejeon, Yuseong-gu 34113,
Republic of Korea
Eslam M. H. Ali − Center for Biomaterials, Korea Institute of
Science & Technology (KIST), 136-791 Seoul, Republic of
Korea; Department of Biomolecular Science, University of
Science & Technology (UST), Daejeon, Yuseong-gu 34113,
Republic of Korea
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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We would like to thank CTCBIO Inc., Korea Institution of
Science and Technology (KIST) (projects #2E30341 and
2E31130) and University of Sharjah (project #16011101018-
P) for financial and technical support. The authors are grateful
to the National Cancer Institute (NCI), Bethesda, Maryland,
USA, for testing the antiproliferative activity of the target
compounds against the NCI-60 cancer cell line panel of nine
different cancer types.
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ABBREVIATIONS USED
■
brs, broad singlet; d, doublet; dd, doublet of doublets; DIPEA,
N,N-diisopropylethylamine; ER, efflux ratio; HRMS, high-
resolution mass spectrometry; m, multiplet; NCI, National
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