Discovery of a Nanomolar Multikinase Inhibitor (KST016366): A New Benzothiazole Derivative with Remarkable Broad-Spectrum Antiproliferative Activity

Article abstract of DOI:10.1002/cmdc.201600224

Herein we report the discovery of compound 6 [KST016366; 4-((2-(3-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-6-yl)oxy)picolinamide] as a new potent multikinase inhibitor through minor structural modification of our

Full text of DOI:10.1002/cmdc.201600224

DOI: 10.1002/cmdc.201600224
Communications
Discovery of a Nanomolar Multikinase Inhibitor
(KST016366): A New Benzothiazole Derivative with
Remarkable Broad-Spectrum Antiproliferative Activity
Ashraf Kareem El-Damasy,[a, b, c] Nam-Chul Cho,[a] Ghilsoo Nam,[a, b] Ae Nim Pae,[a, b] and
Gyochang Keum*[a, b]
Herein we report the discovery of compound 6 [KST016366; 4-
((2-(3-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phe-
cal cytotoxic drugs to targeted agents that modulate protein
kinases whose activities are more specifically linked with can-
cerous cells.[3]
nyl)ureido)benzo[d]thiazol-6-yl)oxy)picolinamide] as
a
new
potent multikinase inhibitor through minor structural modifica-
tion of our previously reported RAF kinase inhibitor A. In vitro
anticancer evaluation of 6 showed substantial broad-spectrum
antiproliferative activity against 60 human cancer cell lines. In
particular, it showed GI50 values of 51.4 and 19 nm against leu-
kemia K-562 and colon carcinoma KM12 cell lines, respectively.
Kinase screening of compound 6 revealed its nanomolar-level
inhibitory activity of certain oncogenic kinases implicated in
both tumorigenesis and angiogenesis. Interestingly, 6 displays
IC50 values of 0.82, 3.81, and 53 nm toward Tie2, TrkA, and ABL-
1 (wild-type and T315I mutant) kinases, respectively. Moreover,
6 is orally bioavailable with a favorable in vivo pharmacokinetic
profile. Compound 6 may serve as a promising candidate for
further development of potent anticancer chemotherapeutics.
In solid tumors, it is unusual for a single kinase aberration to
be the sole cause of disease and it is improbable that tumors
are dependent on only one dysregulated signaling pathway.[4]
In this regard, inhibition of a single kinase may not be suffi-
cient to achieve a clinical benefit, due to either the built-in re-
dundancy of signaling pathways, or the ability of tumors to ac-
quire resistance.[5,6] Therefore, multikinase Inhibitors have
emerged as a new paradigm in drug discovery to overcome
the resistance arising from selective kinase inhibitors.[7,8] Cur-
rently, a number of multitargeted kinase inhibitors are com-
mercially launched.
The absolute majority of kinase inhibitors are known as
type I inhibitors, which target the ATP-site of the kinase in its
active state (DFG-in). In contrast, type II inhibitors trap their
target kinases in the inactive (DFG-out) conformation, occupy-
ing a hydrophobic pocket adjacent to the ATP binding site.[9]
Sorafenib (Nexavarꢀ) is a multikinase type II inhibitor approved
by the Food and Drug Administration (FDA) for treatment of
advanced renal carcinoma.[10] Structural analysis of the co-crys-
tal structure of sorafenib[11] and other type II kinase inhibi-
tors[12,13] with their kinases (BRAF, c-KIT and ABL) defined the
prerequisite structural features to access type II binding confor-
mation.[14] Accordingly, sorafenib structure could be dissected
into the head pyridine moiety as hinge region binder, central
phenyl ring linker, urea as hydrogen bond donor/acceptor pair,
and a hydrophobic tail (4-chloro-3-trifluoromethylphenyl termi-
nal) that access the hydrophobic pocket created by the flip of
the DFG motif of the kinase activation loop.[15]
Cancer is still a prime leading cause of mortality worldwide, ac-
counting for 8.2 million deaths in 2012.[1] Cancer reflects a mul-
tistep process, arising from accumulation of hereditary and/or
acquired disorders in genes involved in the regulation of cell
propagation and survival. Activation or inactivation of four or
five different genes may be needed for the development of
a clinically detectable human cancer.[2] The conventional cyto-
toxic chemotherapeutics that directly interact with DNA are
one of the cornerstones for cancer treatment. However, their
severe side effects constitute a major obstacle that impedes
their efficacy. Over the past two decades, the attention of anti-
cancer drug development has shifted dramatically from classi-
Recently, we reported a series of 2-ureidoquinolines[16] and
ureidobenzothiazoles[17] derivatives as sorafenib congeners by
replacing the central phenyl linker of sorafenib with either qui-
nolone[16] or benzothiazole[17] scaffold, while conserving the
other structural features. Interestingly, such modifications led
to considerable improvement in the cellular anticancer poten-
cy as well as favorable inhibitory activity toward B-RafV600E and
C-Raf kinases.
[a] A. K. El-Damasy, Dr. N.-C. Cho, Prof. G. Nam, Prof. A. N. Pae, Prof. G. Keum
Center for Neuro-Medicine, Brain Science Institute,
Korea Institute of Science and Technology (KIST),
Hwarangro 14-gil 5, Seongbuk-gu, Seoul 136-791 (Republic of Korea)
E-mail: gkeum@kist.re.kr
[b] A. K. El-Damasy, Prof. G. Nam, Prof. A. N. Pae, Prof. G. Keum
Department of Biological Chemistry,
Korea University of Science and Technology (UST),
Gajungro 217, Youseong-gu, Daejeon 305-350 (Republic of Korea)
In the present study, we aimed at performing further struc-
tural modifications in terms of the hydrophobic tail, in an at-
tempt to improve the anticancer activity of the ureidobenzo-
thiazole A.[17] We thought that replacing the small lipophilic
chlorine atom of A with either (morpholin-1-yl)methyl 5 or (4-
ethylpiperazin-1-yl)methyl moiety 6 (Figure 1) may improve
the physicochemical properties of compound A and hence its
[c] A. K. El-Damasy
Department of Medicinal Chemistry,
Faculty of Pharmacy, University of Mansoura, Mansoura 35516 (Egypt)
Supporting information (detailed procedures for synthesis, cellular and
biochemical assays, and molecular docking) and the ORCID identification
number(s) for the author(s) of this article can be found under http://
dx.doi.org/10.1002/cmdc.201600224.
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Figure 1. Rational design of the target compounds.
cellular potency. From another perspective, it was worthy to in-
vestigate the impact of structural extension in the hydrophobic
tail fragment on the affinity toward various protein kinases. In
the light of these considerations, two new benzothiazoles 5
and 6 have been designed, synthesized, and evaluated for
their anticancer activities over a panel of 60 human cancer cell
lines. Moreover, the most promising compound 6 was further
tested against a panel of 50 oncogenic kinases.
drogen atmosphere to yield the corresponding anilines 3a and
3b. On the other hand, the 2-aminobenzothiazole derivative 4
was prepared adopting our previously reported method.[17,18]
Treatment of the benzothiazol-2-yl amine 4 with 1,1’-carbonyl-
diimidazole (CDI) in DMF at room temperature produced the
corresponding isocyanate. In the same pot, the appropriate
aniline 3a or 3b was added and the reaction mixture was
heated at 1008C for 3 h to afford the desired ureidobenzothia-
zoles 5 and 6.
The antiproliferative activity of compounds 5 and 6 was as-
sessed at 10 mm concentration against a panel of 60 human
cancer cell lines at National Cancer Institute (NCI, USA).[20] The
mean growth percentage (GP) and growth inhibition (GI) for
the full panel cell lines are presented in Table 1.
As illustrated in Scheme 1, in order to prepare the target
compounds, we synthesized the two main building blocks 3a,
3b and 4.[17,18] The anilines 3a and 3b were prepared in three
steps. First, a-bromination of 1-methyl-4-nitro-2-(trifluorome-
thyl)benzene with N-bromosuccinimide (NBS) in the presence
of azobisisobutyronitrile (AIBN) afforded the corresponding
bromo derivative 1 in 63% yield.[19] Nucleophilic substitution of
1 with morpholine or 4-ethylpiperazine using K2CO3 as a base
in dichloromethane produced the alkylated nitro derivatives
2a and 2b, which underwent reduction with Pd/C under hy-
By referring to the total number of sensitive cells, it could
be observed that all the tested compounds possess significant
broad spectrum antiproliferative activities (ꢀ53 cell lines). Re-
placing the lipophilic chlorine group of compound A with hy-
Scheme 1. Regents and conditions: a) NBS, AIBN, 1,2-dichloroethane, 908C, 18 h, 63%; b) morpholine or 4-ethylpiperazine, K2CO3, CH2Cl2, RT, 3 h; c) H2, Pd/C,
methanol, RT, 18 h, 99% (X=O, 3a), 78% (X=NꢁEt, 3b), over two steps; d) CDI, DMF, 24 h; e) DMF, 1008C, 3 h, 16.5% (X=O, 5), 16.2% (X=NꢁEt, 6), over
two steps.
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of the compound, as in 6, is associated with significant in-
crease in the anticancer activity. The aforementioned findings
point out the preponderance of ethylpiperazine than morpho-
line for achieving excellent antineoplastic activity. The GP of
NCI 60 cell lines upon treatment with compounds 5 and 6 as
well as the lead compound A at 10 mm are shown in Figure 2.
Inspection of the results illustrated in Figure 2 discloses the
similar anticancer activity of both the lead compound A and
its corresponding morpholinomethyl analogue 5. Moreover,
the sound lethal effects of compound 6 (minus values of GP)
over numerous hematological and solid cancer cell lines was
observed. Of special interest, compound 6 exerted GP=ꢁ60.0
against 24 cell lines. The melanoma UACC-62 cell line was the
most susceptible cell (GP=ꢁ93.0). Based on the impressive an-
tiproliferative activity of both compounds 5 and 6, they were
further evaluated in five-dose testing mode to determine their
GI50 (the molar concentration causing 50% GI), TGI (the molar
concentration producing 100% GI) and LC50 (the molar concen-
tration causing 50% lethality or tumor regression). The GI50
values, as a measure of compounds’ potency, of these two
compounds along with the lead compound A and sorafenib[21]
are listed in Table 2.
Table 1. Molar refractivity (MR) and cLogP values of compounds A, 5 and
6, and an overview of their preliminary anticancer assay results at
a single-dose concentration of 10 mm.
A
5
6
R
Cl
MR [cm3 molꢁ1
cLogP[a]
]
122.86
5.96
146.46
5.17
158.40
6.27
Mean growth [%][b]
Mean growth
inhib. [%][b]
8.49ꢂ30.5 19.9ꢂ19.9
91.51ꢂ30.4 80.13ꢂ21.9
ꢁ44.6ꢂ31.6
144.6ꢂ29.5
Positive cytostatic ef-
fect[c]
Positive cytotoxic ef-
fect[d]
33/59
21/59
54/59
51/58
5/58
5/53
48/53
53/53
No. sensitive cell lines
56/58
[a] Calculated partition coefficient; values were calculated by ChemDraw
Professional 15.0 software. [b] Data were obtained from the NCI in vitro
disease-oriented human tumor cell line screen (single-dose mean graph);
values were calculated and are presented as the meanꢂSD of 60 cell
lines. [c] Ratio of the number of cell lines with 0–50% growth over the
total number of cell lines. [d] Ratio of the number of cell lines with <0%
growth over the total number of cell lines.
As presented in Table 2, compounds 5 and 6 displayed sig-
nificant potency over almost all of the tested cell lines with
sub-micromolar or single-digit micromolar GI50 values. Upon
comparison with the lead candidate A, both 5 and 6 exhibited
superior potencies against 24 and 41 various cell lines, respec-
tively. Moreover, compounds 5 and 6 surpassed the antiproli-
ferative activity of sorafenib over 36 and 51 cancer cell lines,
respectively. In particular, the morpholine derivative 5 and its
drophilic morpholinomethyl moiety (compound 5) led to slight
decrease in the overall growth inhibitory activity (A, GP=8.49;
5, GP=19.9). Interestingly, incorporation of the (4-ethylpipera-
zin-1-yl)methyl moiety at para position neighboring to the m-
trifluoromethyl group led to remarkable improvement in the
activity (6, GP=ꢁ44.6). Furthermore, and in a stark contrast to
the cytostatic activity of compound 5 (51 out of 58), the corre-
sponding ethylpiperazine derivative 6 displayed a distinct posi-
tive cytotoxic effect (48 out of 53). In terms of MR, a steric
factor parameter, and the lipophilicity indicator cLogP, we can
observe that increasing both the lipophilic and steric character
ethylpiperazine analogue
6 exerted outstanding potency
against the colorectal carcinoma KM12 cell line (5, GI50 =
446 nm; 6, GI50 =19 nm), being 7.6 and 179.5-fold, respectively,
more potent than the chloro derivative A (GI50 =3410 nm).
Moreover, compound 6 showed a great growth inhibitory ac-
tivity toward the leukemia CCRF-CEM and K-562 cell lines with
GI50 values of 443 and 51.4 nm, respectively.
Apart from potency, the efficacies of 5 and 6 against certain
sensitive cell lines were examined and compared with those of
the lead compound A and sorafenib (Table 3). The high efficacy
Figure 2. Percent growth of the NCI 60-cell-line panel after treatment with compounds A (blue), 5 (red), and 6 (green) at 10 mm.
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Table 2. GI50 values of compounds A, 5, 6, and sorafenib over the NCI 60-cell-line panel.[a,b]
Cell line
GI50 [mm]
Cell line
GI50 [mm]
A
5
6
sorafenib
A
5
6
sorafenib
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung (NSCL) Cancer
A549/ATCC
EKVX
M14
1.96
1.90
5.69
1.56
ND
2.48
1.83
2.74
1.80
1.01
2.16
1.44
1.75
1.44
1.75
1.24
1.52
1.86
1.70
2.00
1.58
2.00
2.51
1.58
2.00
1.58
ND[c]
>100
ND[c]
ND[c]
2.22
2.73
2.70
1.53
2.57
1.72
1.90
0.443
2.01
0.051
2.13
2.10
2.81
2.00
1.58
3.16
3.16
1.58
3.16
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
ADR-RES
SK-OV-3
Renal Cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
3.37
2.06
0.412
1.93
1.75
1.94
1.15
2.69
2.48
2.62
1.79
2.54
2.15
2.10
2.82
1.44
1.73
1.70
2.33
2.01
1.97
1.99
2.01
1.95
1.94
1.45
1.83
2.24
1.91
1.66
3.16
2.51
2.00
1.58
2.00
2.00
2.51
2.51
2.00
2.02
2.11
1.59
2.46
2.34
2.43
1.89
2.77
1.79
1.72
2.44
2.63
2.07
2.18
1.76
1.89
1.69
2.12
2.11
1.95
1.82
2.51
3.16
3.16
3.16
2.51
2.51
2.51
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer
SF-268
1.79
2.49
1.85
2.54
1.93
2.44
2.29
2.17
2.22
1.90
1.68
2.05
1.81
2.58
4.45
1.65
1.03
1.83
1.39
1.94
1.67
1.80
2.50
1.83
3.16
2.51
2.51
3.16
3.16
2.51
3.98
2.51
ND[c]
ND[c]
1.89
1.95
ND[c]
3.41
2.99
2.10
3.00
1.78
2.09
2.14
0.446
3.31
1.80
2.05
2.00
3.16
1.58
2.51
2.00
1.58
2.51
1.73
1.65
1.27
0.019
2.07
Prostate Cancer
PC-3
DU-145
Breast Cancer
MCF7
MDA-MB-231
HS 578T
BT-549
1.42
2.24
2.04
2.73
1.87
1.76
2.00
3.16
2.34
1.79
1.78
2.77
1.52
1.79
3.22
1.90
1.58
3.82
1.74
1.77
2.00
1.82
1.54
2.33
1.37
1.85
2.51
1.58
1.58
3.16
3.16
2.00
SF-295
SF-539
SNB-19
SNB-75
1.72
1.43
1.53
2.64
0.72
1.36
1.75
2.14
2.55
3.02
1.53
1.45
1.23
1.74
2.01
2.32
1.99
1.66
2.51
1.26
2.51
3.16
1.58
2.00
U251
Melanoma
LOX IMVI
MALME-3m
T-47D
MDA-MB-468
2.68
1.61
1.82
1.44
1.71
1.20
1.58
2.00
[a] Data were obtained from the NCI in vitro disease-oriented human tumor cell line screen (five-dose–response curve). [b] Bold figures refer to superior po-
tency over the lead candidate A, bold italicized figures indicate sub-micromolar GI50 values for 5 and 6. [c] Not determined.
of compound 6 was demonstrated by its ability to trigger total
growth inhibition (TGI, Zero growth) in relatively small doses
(TGI<5.0 mm) than compound A and sorafenib over multiple
cell lines. For example, the leukemia SR cell line (6, TGI=
0.846 mm; A, TGI=9.10 mm; sorafenib, TGI=100 mm). The ethyl-
piperazine compound 6 was also able to induce 50% tumor
regression (LC50, 50% lethality) against numerous cell lines
with LC50 values <10.0 mm.
Furthermore, the cytotoxicity of compound 6 has been ex-
amined against the human foreskin fibroblast (HFF-1) normal
cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) assay. At 1.0 mm concentration, which is
close to the mean GI50 value over NCI-60 cancer cells, com-
pound 6 showed low growth inhibitory activity (10.36ꢂ0.19%
inhibition) against HFF-1 normal cell. Such finding may reveal
the differential cytotoxic activity of compound 6 toward
human cancer cells rather normal cell lines.
genic kinases at 10 mm concentration at Reaction Biology Cor-
poration (RBC, USA).[22] As illustrated in Table 4, compound 6
potently inhibited 23 kinases by more than 90%. For example,
ABL-1 (93.7%), LYN (99.6%), DDR1 (98.8%), FLT3 (96.8%), RET
(97%) and RAF1 (98.3%). Such findings disclose the distinct
multiple kinase inhibitory effect of compound 6 toward the
different classes of kinases, particularly the tyrosine protein kin-
ases (TKs). Relatively, the cyclin dependent kinase family
(CMGC) members, like JNK1, JNK3, and MAPK13 were moder-
ately inhibited by 6. Because those kinases inhibited by 6 are
involved in different cellular signaling pathways for tumorigen-
esis (such as Ras/Raf/Mek/Erk)[23] and angiogenesis (such as
VEGF),[24] it could be concluded that compound 6 may exert its
broad spectrum anticancer activity via multiple mechanism of
actions.
To further examine the potency of compound 6, its IC50
values were determined against a selected array (14 kinases) of
the most important kinases implicated in cancer pathogenesis
(Table 5). With the goal of comparative study, the lead com-
pound A was tested for its inhibitory activity over certain sensi-
Encouraged by the promising cellular potency of the ethylpi-
perazine derivative 6 and with the goal of investigating the
kinase inhibitory profile, it was tested over a panel of 50 onco-
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Table 3. TGI and LC50 [mm] values of compounds A, 5, 6 and sorafenib over a selected array of sensitive cell lines.[a,b]
Cell line
A
5
6
Sorafenib
TGI
LC50
TGI
LC50
TGI
LC50
TGI
LC50
Leukemia
CCRF-CEM
K-562
>100
>100
9.10
>100
>100
>100
>100
>100
ND[c]
>100
>100
>100
2.47
1.27
0.846
61.0
6.90
46.6
100
100
100
100
100
100
SR
NSCL Cancer
NCI-H226
NCI-H522
Colon Cancer
HCT-116
7.47
>100
>100
>100
4.30
8.36
12.8
62.9
3.16
3.59
6.91
7.67
7.94
6.31
31.6
25.1
5.39
>100
>100
>100
3.63
2.31
7.39
17.0
3.45
1.63
6.86
5.63
3.98
7.94
15.9
31.6
KM12
CNS Cancer
SF-539
SNB-75
3.90
3.24
ND[c]
6.88
3.37
4.95
7.22
21.9
2.97
2.73
5.73
5.46
10.0
10.0
50.1
39.8
Melanoma
LOX IMVI
UACC-257
Renal Cancer
A498
>100
>100
>100
>100
4.34
5.59
13.4
20.0
3.17
3.51
5.85
6.64
3.16
5.01
7.94
20.0
12.2
>100
>100
>100
4.05
9.36
8.65
67.8
3.54
3.66
6.87
7.43
6.31
10.0
31.6
39.8
SN12C
Breast Cancer
MDA-MB-231
BT-549
4.29
9.73
59.2
>100
5.80
9.18
45.9
36.3
3.40
4.25
6.65
7.79
3.98
5.01
20.0
15.8
[a] Data were obtained from the NCI in vitro disease-oriented human tumor cell line screen (five-dose–response curve). [b] Bold figures and italicized fig-
ures refer to superior efficacies over the lead candidate A in terms of TGI and LC50 values, respectively. [c] Not determined.
tive kinases potently inhibited by compound 6, and
sorafenib was used as a reference compound.
As shown in Table 5, compound 6 showed an
Table 4. Percent inhibition exerted by compound 6 over a panel of 50 oncogenic kin-
ases at a concentration of 10 mm.[a]
equipotent activity against both the wild-type ABL
kinase (IC50 =52.7 nm) and its most resistant gate-
keeper mutant ABLT315I (IC50 =53.3 nm), while sorafe-
nib was less active (ABL-1, IC50 =1133 nm; ABLT315I
IC50 =957 nm) and the lead compound A was quite
inactive (IC50 >10 mm). ABL generated by BCR-ABL
fusion is a non-receptor TK, and its constitutive acti-
vation is known to be the etiologic cause for chronic
myeloid leukemia (CML).[27] In this regard, it is note-
worthy mentioning that compound 6 exerted a mag-
nificent anticancer activity against the ABL depen-
dent leukemia K-562 cell line with GI50 value of
51.4 nm.
c-Src family kinases (SFK), like c-Src, FYN, LYN, YES
and LCK, are cytoplasmic TKs which possess a high
degree of structural similarity to ABL. While the
chloro compound A showed an unpretentious activi-
ty against LCK and LYN kinases (IC50 >10 mm), its cor-
responding ethylpiperazine 6 displayed a potent in-
hibitory activity surpassing sorafenib over certain
SFK members, such as LCK (IC50 =6.66 nm), LYN
(IC50 =19.9 nm), and FYN (IC50 =160 nm).
Kinase class/name
Inhibition [%]
Kinase class/name
Inhibition [%]
Non-receptor tyrosine kinase
PDGFRa
RET
TIE2
TRKA
TRKB
97.3
97.0
98.5
98.3
98.4
ABL1
93.7
ABL1T315I
93.1
83.1
75.0
84.6
67.4
18.3
88.4
90.0
95.3
56.5
98.5
93.6
99.6
ABL2
BMX
CSK
c-Src
FAK
FER
FES
FYN
JAK1
LCK
YES
LYN
Serine/threonine kinases
Aurora A
MAP4K2
LCK2
LOK
PIM1
PIM2
SGK1
TAK1
TNIK
27.5
48.5
ꢁ87.2
88.0
14.4
1.4
14.0
90.7
90.3
87.0
Receptor tyrosine kinase
ZAK
ALK
15.7
26.2
98.8
91.6
30.1
97.8
97.7
96.8
90.4
82.7
98.3
98.9
Tyrosine kinase like kinases (TKL)
c-MET
DDR1
DDR2
EGFR
EPHA2
EPHA3
FLT3
BRAF
71.4
95.2
98.3
BRAFV600E
RAF1
CMGC group[b]
JNK1
66.5
83.5
60.1
79.4
44.3
87.8
JNK2
JNK3
FMS
MAPK12
MAPK13
MAPK14
Apart from the cytoplasmic TKs, the discoidin
domain containing receptor (DDR1) is a receptor TK
whose deregulation is associated with the progres-
sion of breast,[28] and colon[29] cancers. Interestingly,
compound 6 was able to inhibit the DDR1 kinase on
the enzymatic level (IC50 =26.5 nm), being sixfold
FGFR3
VEGFR1
VEGFR2
[a] Compound 6 was tested in single-dose duplicate mode at a concentration of
10 mm. [b] CMGC: cyclin-dependent kinase family.
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Table 5. IC50 values of compounds 6, A, and sorafenib against the most sensitive kinases.[a,b]
Kinase
6
A
Sorafenib
Inhibition [%]
IC50 [nm]
Inhibition [%]
IC50 [nm]
IC50 [nm]
ABL1
ABLT315I
BRAFV600E
DDR1
VEGFR1
VEGFR2
LCK
LYN
RAF1
TrkA
93.7
93.1
71.4
98.8
98.3
98.9
98.5
99.6
98.3
98.3
98.4
98.5
90.4
95.3
52.7
53.3
133
31.3
NT
NT
87.9
ꢁ3
22.6
31
>10000
NT
NT
1133[25]
957[25]
38.0[10]
160[26]
26.0[10]
90.0[10]
1495[25]
510[26]
6.0[10]
218[25]
832[25]
–
26.5
42.8
43.1
6.66
19.9
61.7
3.81
4.42
0.82
102
NT
>10000
>10000
>10000
>10000
111
>10000
NT
>10000
NT
12
94.8
ꢁ0.2
NT
7.0
NT
TrkB
Tie2
FMS
FYN
29.0[25]
–
160
NT
NT
[a] Compound 6 was tested in a 10-dose IC50 mode with 3-fold serial dilution starting at 20 mm; NT: not tested [b] Bold figures refer to potent activity (sub-
nanomolar, single- or double-digit nanomolar IC50 values).
more potent than sorafenib (IC50 =160 nm). In addition, it
showed anticancer activity against the DDR1 relevant cell lines
(colon HCT-116, GI50 =1.73 mm; breast T-47D, GI50 =1.99 mm).
Also, the lead compound A showed a considerable activity
toward DDR1 kinase (88% inhibition at 10 mm).
By referring to the angiogenesis mediating kinases,
VEGFR,[24] it is interesting to report the equipotent activity of
compound 6 over both VEGFR1 and VEGFR2 (IC50 =43 nm).
Most importantly, Tie2 kinase, the other key regulator for
tumor angiogenesis,[30] was potently suppressed by compound
6 (IC50 =0.82 nm). Such promising biochemical results of com-
pound 6 provide a strong evidence for its prospective anti-an-
giogenic activity. In contrast, the lead compound A was ex-
tremely inactive (IC50 >10 mm) against VEGFR1, VEGFR2, and
Tie2 kinases, while sorafenib was relatively more potent
toward VEGFR1 (IC50 =26 nm) than VEGFR2 kinase (IC50 =
90 nm).
On the other hand, tropomyosin related kinases (Trk) are re-
ceptor TKs for the neurotrophin family of ligands that play
a pivotal role for growth and differentiation in normal neuronal
cells.[31] Pathological overexpression of TrkA is linked to malig-
nant transformation and invasion signaling in certain human
cancers including breast,[32] colon,[33] and neuroblastoma.[34]
Moreover, the overexpression of TrkB has been reported in var-
ious malignancies.[35] In this sense, compound 6 showed a dis-
tinct inhibitory effects against both TrkA (IC50 =3.81 nm) and
TrkB (IC50 =4.42 nm). Such great activity was also observed on
the TrkA relevant colorectal KM12 cell line[36] (GI50 =19 nm).
In respect to the tyrosine kinase like (TKL) kinases, exempli-
fied by RAF kinases, both compounds A and 6 showed favora-
ble inhibitory effects (A, IC50 =111 nm; 6, IC50 =61.7 nm) against
C-RAF (RAF1) kinase. In comparison with sorafenib (IC50 =
6.0 nm), both compounds A and 6 were relatively less potent.
C-RAF kinase is implicated in the abnormal proliferation in mel-
anoma[37] as well as being overexpressed in renal cell carcino-
ma (RCC),[38] and colon cancers.[39] The nanomolar activity of
compound 6 over C-RAF kinase suggest its possible effects on
the Ras/Raf/Mek/Erk pathway.
Based on the aforementioned findings, we can conclude
that both compounds A and 6 are able to target the DDR1
and RAF kinases. Moreover, replacing the small chlorine atom
of the lead compound A with (4-ethylpiperazin-1-yl)methyl
moiety has resulted in a substantial kinase inhibitory activity
toward a number of oncogenic kinases which could not be in-
hibited by the original lead A.
To better understand and justify the obtained kinase inhibi-
tory activities of both compounds A and 6 from a 3D structur-
al perspective, a molecular docking study was conducted. First-
ly, we aimed at addressing the binding affinity of compounds
A and 6 toward the DDR1 kinase (PDB ID: 4CKR).[40] As illustrat-
ed in Figure 3, both compounds A and 6 are well bound into
the ATP binding site of DDR1 kinase through forming the cru-
cial hydrogen bond (HB) with the Met704 residue in the kinase
hinge region via their pyridine nitrogen. Moreover, the urea
moiety of benzothiazoles was engaged in three additional HBs
with Asp784 and Glu672. In addition, the m-trifluoromethyl-
phenyl group of compounds A and 6 was involved in hydro-
phobic interactions with the backbone Asp784 residue in the
DFG region. An additional ionic interaction was observed be-
tween the positively charged nitrogen of ethylpiperazine in
compound 6 and the negatively charged carbonyl oxygen of
Asp784, which may explain the superior binding affinity of 6
(Gscore=ꢁ11.972 kcalmolꢁ1) rather the chloro compound A
(Gscore=ꢁ10.418 kcalmolꢁ1).
Secondly, we focused our efforts to recognize the significant
role of (4-ethylpiperazin-1-yl)methyl moiety in compound 6 for
targeting certain kinases not inhibited by the original lead A,
like VEGFR2, LYN, TrkA and Tie2. Therefore, a molecular dock-
ing study for both compounds A and 6 was performed using
the crystal structures of VEGFR2[41] and TrkA[42] as representa-
tive examples. Inspection of the predicted binding modes of
compounds A and 6 in VEGFR2[41] (Figure 4) revealed that both
compounds could contribute in significant binding interactions
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Figure 4. The putative binding mode of compounds A (orange, top) and 6
(green, bottom) in the catalytic kinase domain of VEGFR2 in its DFG-out con-
formation.
Figure 3. The predicted binding mode of compounds A (orange, top) and 6
(green, bottom) in the catalytic kinase domain of DDR1 in its DFG-out con-
formation.
with the hinge region residue (Cys919) via their N-methylpicoli-
namide head, DFG loop (Asp1046) and C-helix (Glu885) resi-
dues through their urea spacer, and hydrophobic pocket via
their m-trifluoromethylphenyl moiety. However, only com-
pound 6 was able to access the deep hydrophobic pocket in
the VEGFR2 allosteric site through its piperazinyl group, which
is expected to create bifurcated hydrogen bonds with the
backbone -CO groups of VEGFR2 residues (His1026). These in-
teractions are in accordance with those observed in several
crystal structures of type II kinase inhibitors and could provide
a logical justification for the superior potency of compound 6
than its original lead A toward VEGFR2.
6 with TrkA. The missing of such hydrophobic and HB interac-
tion in the original lead A could explain its low TrkA inhibitory
activity. Taken together, we can conclude that the insertion of
(4-ethylpiperazin-1-yl)methyl moiety instead of chlorine in
compound A plays an important role in increasing the binding
affinity toward multiple oncogenic kinases. Moreover, the puta-
tive binding modes of compound 6 suggest its possibility to
be a type II kinase inhibitor.
Furthermore, the in vivo pharmacokinetic (PK) properties of
compound 6 was evaluated (Table 6). The AUC values of com-
1
pound 6 are 4.24 mghmLꢁ1 and 1.07 mghmLꢁ1 in intravenous
and oral administration, respectively. Compound 6 was slowly
On the other hand, regarding TrkA kinase, the putative bind-
ing mode illustrated in Figure 5 revealed that both compounds
A and 6 lacked HB interactions with Met592 in the TrkA hinge
region. However, this deficiency in hinge region binding was
compensated, in compound 6, by the significant hydrophobic
interactions between its ethylpiperazinyl moiety and the allo-
steric binding site around a-C segment of TrkA kinase. The eth-
ylpiperazinyl group is expected to be deeply inserted within
the hydrophobic pocket (Phe646 and His648) adjacent to the
ATP binding site, in addition to the formation of HB with
Asp668 and hence increasing the binding affinity of compound
distributed to reach the blood after oral administration (tmax =
4.67 h), and exhibited slow clearance (21 mLkgꢁ1 minꢁ1). In ad-
dition, it showed good oral exposure as indicated by its Cmax
value (64.6 ngmLꢁ1). Taken together, compound 6 possesses
a favorable PK properties with reasonable oral bioavailability
(F) value of 25.1%.
In conclusion, a new potent benzothiazole multikinase inhib-
itor 6 (KST016366) has been discovered, through changing the
hydrophobic tail of our previously reported RAF kinase inhibi-
tor A. The ethylpiperazinyl derivative 6 exerted broad spec-
trum antiproliferative activity against NCI-60 cancer cell lines,
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KST016366 showed favorable PK profile with good oral bio-
availability. Therefore, KST016366 could be considered as
a promising candidate for further preclinical and clinical inves-
tigations or a lead compound for development of potent anti-
cancer chemotherapeutics.
Acknowledgements
This research was supported by the Korea Institute of Science
and Technology (KIST) Institutional Program (2E26650 and
2E26663). We express our sincere appreciation and gratitude to
the National Cancer Institute (NCI, Bethesda, MD, USA) for per-
forming the anticancer evaluation of the new compounds.
Keywords: anticancer
activity
·
benzothiazoles
·
ethylpiperazine · multikinase inhibitors
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Parameter
max [h]
i.v.
p.o.
[b]
t
–
–
4.67ꢂ1.15
64.6ꢂ0.0124
11.4ꢂ6.98
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Cmax [ngmLꢁ1
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t1/2 [h]
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4.24ꢂ1.5
AUCt [mghmLꢁ1
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]
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0.704ꢂ0.392
1.07ꢂ0.893
CL [mLkgꢁ1 minꢁ1
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21.0ꢂ0.445
6.97ꢂ1.84
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[b]
Vss [Lkgꢁ1
F [%]
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[b]
25.1
[a] Dosed at 5 mgkgꢁ1; values are the meanꢂSD (n=3). AUC: area under
plasma concentration time curve; Cmax: peak plasma concentration; tmax
:
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Published online on && &&, 0000
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A. K. El-Damasy, N.-C. Cho, G. Nam,
A. N. Pae, G. Keum*
&& – &&
Discovery of a Nanomolar Multikinase
Inhibitor (KST016366): A New
Benzothiazole Derivative with
Remarkable Broad-Spectrum
Antiproliferative Activity
A potent oral multikinase inhibitor
was identified: KST016366. Slight modi-
fication of the hydrophobic tail of RAF
kinase inhibitor A has a dramatic impact
on cellular and enzymatic activity.
KST016366 shows nanomolar antiproli-
ferative activity against both leukemia
K-562 (ABL-kinase-dependent) and
colon carcinoma KM12 (TrkA-depen-
dent) cells. Moreover, KST016366 inhib-
its two key angiogenic kinases: VEGFR2
and Tie2.
KST016366 exerts distinct inhibitory ef-
fects on multiple oncogenic kinases, in
contrast to its lead compound A.
&
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