Discovery of 4-anilino α-carbolines as novel Brk inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.03.002

Dysregulation of cell signalling processes caused by an enhanced activity of protein kinases mainly contributes to cancer progression. Protein kinase inhibitors have been established as promising drugs that inhibit such overactive protein kinases in cancer cells. The formation of metastases, which makes a therapy difficult, remains a great challenge for cancer treatment. Recently, breast tumor kinase (Brk) was discovered as novel and interesting target for a cancer therapy because Brk participates in both cell dysregulation and metastasis. We discovered 4-anilino substituted α-carboline compounds as a novel class of highly active Brk inhibitors. In the current work, structure-activity relationships are discussed including docking results obtained for 4-anilino α-carbolines. A first profiling of selective kinase inhibition and a proof of concept for the antiproliferative effects is demonstrated. These results qualify the compounds as a promising class of novel antitumor agents.

Full text of DOI:10.1016/j.bmcl.2014.03.002

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1948–1951
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-anilino a-carbolines as novel Brk inhibitors
Kazem Ahmed Mahmoud a, Martin Krug a, Tom Wersig a, Inna Slynko a, Christoph Schächtele b,
Frank Totzke b, Wolfgang Sippl a, Andreas Hilgeroth a,
⇑
a Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany
b ProQinase GmbH Freiburg, Breisacher Straße 117, 79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Dysregulation of cell signalling processes caused by an enhanced activity of protein kinases mainly con-
tributes to cancer progression. Protein kinase inhibitors have been established as promising drugs that
inhibit such overactive protein kinases in cancer cells. The formation of metastases, which makes a ther-
apy difficult, remains a great challenge for cancer treatment. Recently, breast tumor kinase (Brk) was dis-
covered as novel and interesting target for a cancer therapy because Brk participates in both cell
Received 10 October 2013
Revised 25 February 2014
Accepted 1 March 2014
Available online 12 March 2014
dysregulation and metastasis. We discovered 4-anilino substituted
class of highly active Brk inhibitors. In the current work, structure–activity relationships are discussed
including docking results obtained for 4-anilino -carbolines. A first profiling of selective kinase inhibi-
tion and a proof of concept for the antiproliferative effects is demonstrated. These results qualify the
compounds as a promising class of novel antitumor agents.
a-carboline compounds as a novel
Keywords:
a-Carbolines
a
Brk inhibitor
Structure–activity relationships
Anticancer activity
Ó 2014 Elsevier Ltd. All rights reserved.
The understanding of cell signalling processes in the last dec-
ades led to the identification of many protein kinases which were
found dysregulated in the various types of cancer.1–4 Being either
overexpressed or overactivated such protein kinases cause
increased cancer cell proliferation or antiapoptotic effects resulting
in progressive tumor growth. Protein kinase inhibitors have been
established as effective therapeutics for several forms of cancer.5–
7 However, they partly cause side effects because they do not only
inhibit protein kinases in cancer cells but also the protein kinases
found in normal cells.8–10
Therefore, it would be helpful to identify protein kinases in a
cancer cell that mainly contribute to the dysregulation to start a
personalized cancer treatment. Breast tumor kinase (Brk) was
identified as a non-receptor protein kinase. The cDNA of Brk was
originally cloned in a screen of tyrosine kinases which were found
expressed in a metastatic breast tumor.11 Brk occurred in a major-
ity of breast tumors.11,12 However, normal tissues show only low
and undetectable amounts of Brk.12,13 Thus, Brk became an ideal
target protein for a potential tumor therapy because selective tar-
geting of Brk promises no critical side effects for normal cells.
Meanwhile, several cellular substrates of Brk have been identi-
fied including RNA-binding proteins, transcription factors and cell
signalling molecules.14–20 Thus, Brk is discussed to increase onco-
genic signal events by phosphorylation of members of the ErbB
family.21 Additionally to a potential role in cancer progression
Brk is discussed to support the survival of detached cancer cells
by preventing an autophagic cell death.22 As a consequence Brk
may play an important role in tumor metastasis because of its con-
tribution to cellular survival.
b-Carbolines have recently been published to act as protein ki-
nase inhibitors.23 We have been interested to develop related
a-carbolines and to investigate their inhibitory effects for different
kinases. We discovered novel 4-anilino -carbolines in screening
a
studies as novel class of Brk inhibitors. A first profiling of selectiv-
ity towards other protein kinases is presented and structure–activ-
ity relationships are discussed. First nanomolar inhibitors have
been investigated in first breast cancer cell studies. They demon-
strate a successful antiproliferative activity as a result of a Brk
inhibition.
The synthesis started from benzotriazole 1 which was treated
with 2-bromopyridine in toluene under reflux conditions to give
the pyridin-2-yl benzotriazole 2. Compound 2 was heated in
polyphosphoric acid to cyclize to the
a-carboline scaffold 3
(Scheme 1).24
The 4-position of compound 3 was activated for the following
4-chlorination reaction by N-oxide formation in boiling acetic acid
using hydrogen peroxide solution. The 4-chlorination reaction suc-
ceeded with phosphorus(V) oxychloride at room temperature.
Then the different aniline substituents were introduced with an
excess of the respective aniline in a N-methylpyrrolidone solution
under reflux heating.
⇑
Corresponding author. Tel.: +49 345 55 25168; fax: +49 345 55 27207.
E-mail address: andreas.hilgeroth@pharmazie.uni-halle.de (A. Hilgeroth).
http://dx.doi.org/10.1016/j.bmcl.2014.03.002
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

K. A. Mahmoud et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1948–1951
1949
N
of the
actions with the hinge region, namely with backbone atoms of
Met267. Additionally, the -carboline and phenyl ring of these
a-carboline scaffold participate in two hydrogen bond inter-
N
N
a
N
+
N
a
N
N
H
N
Br
inhibitors are making a number of hydrophobic interactions with
residues in the ATP binding site and the adjacent gatekeeper pock-
et. Besides the above mentioned interactions, the 3-chloro group of
inhibitor 4e is suggested to interact with the carbonyl oxygen of
Ile262, acting as halogen bond donor. The putative binding mode
of compound 4e is shown in Figure 1.
The introduction of a 3-hydroxy function which is also able to
make a hydrogen bond to Ile262 led to best activities of the respec-
tive compound 4f with an IC50 value of 3.15 nM. We then produced
various 3-alkoxy aniline derivatives 4g–i to evaluate a combination
of favourable hydrogen bonding and observed hydrophobic substi-
tuent effects on Brk inhibition.
2
1
R2
R1
R3
R4
c - e
b
HN
N
H
N
N
H
3
N
4a - r
The methylated 3-hydroxy derivative 4g was inactive similar to
the 3-thioether compound 4d. The elongation of the methyl side
chain by one methylene group led to an IC50 value of 155 nM for
compound 4h. This increased activity was as a result of the partly
increased substituent lipophilicity. A benzylation of the 3-hydroxy
function in compound 4i further increased the activity with a
resulting IC50 value of 40.7 nM. This meant an activity similar to
that of the 2,3-phenyl ring annelated compound 4b.
Scheme 1. Reagents and conditions for the preparation of compounds: (a) toluene,
reflux, 18 h, 97%; (b) polyphosphoric acid, 160–175 °C, 3 h, 47%; (c) hydrogen
peroxide, acetic acid, reflux, 6.5 h, 73%; (d) phosporus oxychloride, dimethyl
formamide, rt, 24 h, 71%; (e) aniline, N-methylpyrrolidone, reflux, 6–36 h, 28–85%.
We first introduced substituents into the 2-position of the
aniline residue. Compound 4a25 with a 2-methyl aniline function
showed no inhibitory activity towards Brk. We then replaced the
2-methyl group with an annelated 2,3-phenyl residue in com-
pound 4b. We found a Brk inhibition with an IC50 value of
43.5 nM, which strongly indicated that the substitution of the
3-position of the aniline residue would be more promising com-
pared to the sole 2-position (Table 1).
The bulky and hydrophobic trifluoromethyl substituent instead
of the phenyl residue in compound 4c led to a slight decrease in
activity with an IC50 value of 59.4 nM. A lipophilic thioether func-
tion at the 3-position was found unfavourable because the respec-
tive compound 4d showed no Brk-inhibitory activity. We then
replaced the thioether function with a chloro substituent with less
space demand and the potential to undergo halogen bonding. The
resulting 3-chloro compound 4e showed a strong increase in inhib-
itory activity with an IC50 value of 4.75 nM. Thus, a first highly
When the most favourable 3-hydroxy group was replaced with
a nitro function in derivative 4j the activity maintained high with a
resulting IC50 value of 5.65 nM.
We then moved the aniline mono-substitution from the favour-
able 3- to the 4-position of the aniline residue. The corresponding
4-methoxy derivative 4k proved to be active with an IC50 value of
190 nM. However, the 3-methoxy aniline compound 4g has been
found inactive as Brk inhibitor. The 4-chloroaniline derivative 4l
was found to be inactive. So it could be concluded that a sole
4-substituent is unfavourable compared to the related 3-substi-
tuted inhibitors.
The best activities among the series of monosubstituted aniline
compounds were obtained for the 3-substituted derivatives with
potential hydrogen/halogen bonding properties as suggested by
the docking studies. However, lipophilic substituents also result
in compounds with promising nanomolar activities.
active compound was identified among the 4-anilino a-carbolines.
Subsequently, docking studies were performed for the most
active Brk inhibitors to analyze the potential binding mode. The
compounds were docked into the ATP-binding pocket of a Brk
homology model.26 Docking results suggest a common binding
The high sensitivity of the aniline substitution for the degree of
Brk inhibition encouraged us to investigate combined substituent
effects in the following disubstituted derivatives.
mode for the 4-anilino
a-carbolines, where the NH- and N-atoms
Table 1
Brk inhibition of target compounds 4a–r
Compound
R1
R2
R3
H
R4
IC50 valuea,b (nM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
n.a.c
43.5
59.4
n.a.c
4.75
3.15
n.a.c
155
–CH2ACH@CHACH2AR3
CF3
SMe
Cl
OH
OMe
OEt
OBn
NO2
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
OMe
Cl
Me
F
OBn
H
40.7
5.65
190
n.a.c
75.6
69.5
64.5
53.4
4.41
154
Cl
OMe
OEt
Br
Br
a
b
c
IC50 value determination follow the below described protocols.28
Standard errors are typically below 20%. In many cases standard errors below 10% are found.
n.a. = not active.

1950
K. A. Mahmoud et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1948–1951
Table 2
Antiproliferative activity of compound class-representing derivatives 4f and 4n in
various breast tumor cancer cell lines
Compound
MCF7
GI50 valuea
(lM)
HS-578/T
BT-549
4f
4n
0.99
8.12
1.02
10.2
1.58
>100
a
GI50 value determination was carried out using the sulfur rhodamine assay of
recent protocols.29,30
selective and thus they prove to be promising anticancer drug
candidates. We then determined the antiproliferative activity of
two selected compounds in various breast cancer cell lines. Beside
the involvement in signalling pathways Brk is discussed to play a
role in tumor progression by the phosphorylation of tumor-rele-
vant receptor tyrosine kinases of the ErbB family. It was suspected
that an inhibition of Brk might reduce such a receptor-mediated
proliferative effect in breast tumor cells where Brk has been found
overexpressed. We selected our most active Brk inhibitor 4f and
determined the growth inhibition (GI) of three breast tumor cell
lines under inhibitor application as shown in Table 2.
The highest antiproliferative activity was observed in the MCF7
cell line with a GI50 value of 0.99
cell line was only less increased with a GI50 value of 1.58
pound 4f was more active compared to lapatinib that is used as
l
M.27 The activity in the BT-549
Figure 1. Docking solution of compound 4e (cyan sticks) for Brk. The protein is
shown as pale green ribbon and important residues of the ATP binding site are
displayed. Hydrogen and halogen bonds between the inhibitor and the kinase are
displayed as dashed lines.
l
M. Com-
protein kinase inhibitor in breast cancer therapy with a reported
GI50 value of 2.0 lM in the MCF7 cell line.
We then investigated the disubstituted 3-chloro and 4-fluoro
substituted anilino derivative 4n with reduced Brk inhibition of
69.5 nM as determined IC50 value if compared to compound 4f.
The antiproliferative activity of derivative 4n was found reduced
First we combined the favourable 3-chloro substitution of the
aniline residue with varying 4-substitutions. An additional lipo-
philic 4-methyl function in derivative 4m decreased the compound
activity to an IC50 value of 75.6 nM. Similar results were found for
the 4-fluoro derivative 4n and the slightly more favourable 4-ben-
zyloxy substitution in derivative 4o. We also prepared one addi-
tionally 5-substituted compound with a 3,5-dichloro substitution
of the aniline residue in derivative 4p. Also this disubstitution
was less favourable than the sole 3-chloro function with a resulting
IC50 value of 53.4 nM for the respective compound 4p.
Then the combination of a 3-alkoxy compound with a 4-bromo
substitution was investigated. The 3-methoxy anilino compound
4g was found to be inactive. However, the additional bromo func-
tion in compound 4q led to highest activities with an IC50 value of
4.41 nM. If the 4-bromo substitution was combined with the
3-ethoxy function the activity remained unchanged compared to
the sole 3-ethoxy function with an IC50 value of 154 nM for deriv-
ative 4r.
in the MCF7 cell line with a GI50 value of 8.12 lM compared to that
of derivative 4f which means a similar range in the antiproliferative
activity reduction as in the Brk inhibition compared to compound
4f. A same tendency of both reduction of the antiproliferative activ-
ity and of the Brk inhibition was found in the HS-578/T cell line with
an IC50 value of 10.2 lM. However, the activity of derivative 4n in
the third breast cancer cell line BT-549 was only residual. The best
Brk inhibitor 4f also showed the poorest antiproliferative activity
in the BT-549 cell line. Thus, we present a first proof of concept
for a Brk inhibition that obviously correlates with the antiprolifera-
tive activity in relevant breast cancer cells.
In conclusion, the presented series of 4-anilino
a-carbolines
turned out as a highly promising class of anticancer agents. The
Brk inhibition depends on the kind and positioning of the aniline
substituents, which lead to nanomolar as well as to inactive inhib-
itors. The observed protein kinase inhibition profile documented a
first selectivity of Brk inhibition. The correlation of Brk inhibition
and mediated antiproliferative activity better than that of the
reported lapatinib qualifies the new compound class for further
preclinical studies.
Thus, we identified highly potent Brk inhibitors and decided to
determine the activity of the best 3-hydroxy aniline compound 4f
towards kinases from all kinase families of the human kinome.
With determined IC50 >10 lM we observed no activity against
the kinases PIM2 of the PIM kinase family, NEK1 and 2 of the NIMA
family, CAMK4, the isoforms MAPKAPK3 and 5 and MARK 1, 3 and
5 of the CAMK family. No activity was observed against the iso-
forms AKT1–3 and against DMPK of the AGC family, VRK1 of the
CK1 family, against the isoforms MST3 and 4 and PAK1–3 of the
STE family, against ERK1 and 2, GSK-3b, JNK1 and 3 and the HIPK
isoforms 1–3 of the CMGC family. No activity resulted for JAK3 of
Acknowledgements
Financial support of the work was given to Tom Wersig, Wolf-
gang Sippl and Andreas Hilgeroth by the German research founda-
tion (DFG) within the projects SI 868/10-1 and HI 687/10-1,
respectively.
the TK family and with IC50 values of 3.04
against Src of the TK family and against ALK1 of the TKL family,
respectively, the activity nearly reached the 10 M which means
lM and of 2.2 lM
References and notes
l
activities of a factor of thousand higher than that of 3.2 nM as
the determined Brk inhibitory activity of compound 4f.
This first protein kinase activity study including more than 30
kinases from all kinase families profiles the inhibitors of being
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active conformation (PDB code: 1Y57_A, resolution 1.91 Å) was chosen as a
template as it shows the highest sequence identity with Brk (around 53%). The
homology model was generated using MODELLER-9v11. The sequence
alignment between Brk and c-Src was made using align2d in MODELLER.
Hydrogen atoms and partial charges were assigned and the protein structure
was subjected for energy minimization in implicit solvent with RMSD
deviation of maximum 0.3 Å using the OPLS-AA 2005 forcefield (Maestro 9.3,
Schrödinger Inc.). The stereochemical analysis of Ramachandran plot with the
PROCHECK program confirmed that this model is reasonable, showing 88.4% of
the residues were in the favoured region, 11.3% were in the allowed region and
only 0.3% was in the disfavoured region. All ligands were prepared using the
LigPrep utility of the Schrödinger suite. MMFF force field was used for energy
minimization. Possible ionization states of each ligand were generated at pH
7.4 by using the Ionizer module. Options to generate tautomers, stereoisomers
and up to 10 low energy ring conformations were set on. Ligands were docked
into the ATP-binding pocket of Brk using GOLD version 5.1. The center of the
Brk binding site was set at Leu319 with a radius of 14 Å. Goldscore was chosen
as fitness functions and rescoring with Chemscore was applied, since this
protocol was validated on the available Src X-ray structures.
11. Mitchell, P. J.; Barker, K. T.; Martindale, J. E. Oncogene 1994, 9, 2383.
12. Barker, K. T.; Jackson, L. E.; Crompton, M. R. Oncogene 1997, 15, 799.
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27. Compared to the nanomolar Brk inhibition activity the cellular micromolar
activity may be plausible by the differences of in vitro activity of a direct
enzyme inhibition and the resulting cellular effects on proliferation which are
a result of following cell regulating effects so that excellent in vitro activities
consequently lead to lowered activities in a whole cellular system of growth
regulation.
28. The measuring of protein kinase activity was performed in 96-well
FlashPlates™ in a 50
20 L of assay buffer solution, 5
compound in a 10% dmso solution and finally a premixture of each 10
l
L reaction volume. The reaction mixture consisted of
L of ATP solution in water, 5 L of used test
L of
l
l
l
l
24. Witkop, B. J. Am. Chem. Soc. 1953, 75, 3361.
used substrate and enzyme solutions. The assay buffer solution contained
70 mM of HEPES-NAOH, each 3 mM of magnesium chloride and manganese(II)
25. Spectroscopical data of 4-anilino
compound 4a: Yield 0.206 g (61%); mp 215–218 °C; IR (ATR)
3252 (Arly-CH), 1692 (NH), 1593 (C@C) cmꢀ1 1H NMR (DMSO-d6) d = 11.52 (s,
a
-carboline compound class representing
m
= 3422 (NH),
chloride, 3
and finally 1
lM of sodium orthovanadate, 1.2 mM of DTT, 50 lg/mL of PEG20000
;
l
M of [
c
-
33P]-ATP making approximately 1.2 ꢁ 106 cpm per well.
1H, 9-NH), 8.15 (d, J = 7.9 Hz, 1H, 5-H), 8.00 (s, 1H, aniline NH), 7.94 (d,
J = 5.6 Hz, 1H, 2-H), 7.40 (d, J = 8.0 Hz, 1H, 8-H), 7.33 (d, J = 6.3 Hz, 1H. 30-H),
7.31 (dd, J = 8.0, 7.3 Hz, 1H, 7-H), 7.25–7.13 (m, 3H, 40-, 50-, 60-H), 7.09 (dd,
J = 7.9, 7.3 Hz, 1H, 6-H), 6.06 (d, J = 5.6 Hz, 1H, 3-H), 2.2 (s, 3H, CH3); m/z (EI)
273 (100, M+). Elem. Anal. Calcd (%) for C18H15N3: C, 79.10; H, 5.53; N 15.37.
Found: C, 78.88; H, 5.56; 15.48.
The Km for ATP to Brk was determined with 4.08 lM. The final Brk
concentration has been 6.1 nM. The used substrate was Poly(Glu,Tyr)4:1 in an
amount of 125 ng/50 L. The reaction mixtures were incubated at 30 °C for
60 min. The reaction was stopped with 50 of 2% (v/v) solution of
l
lL
a
phosphoric acid. Then the plates were aspirated and washed twice with 200 lL
of water or 0.9% solution of sodium chloride. The incorporation of 33Pi was
determined with a microplate scintillation counter. Ten different inhibitor
26. Computational methods: Since there is no crystal structure available for Brk, the
homology modeling methodology was used to predict the 3D structure. The
sequence of human protein kinase Brk (residues 13–450) was retrieved from
the UniProtKB repository (entry number Q13882). Following the NCBI Basic
Local Alignment Search Tool (BLAST) query for the search of template structure
in PDB, it was identified that members of the Src kinase family share the
highest sequence similarity (>50%) with Brk. Crystal structure of c-Src in the
concentrations were measured in
a range of 3 nM–100 lM. The residual
activity and the IC50 values were finally calculated.
29. Boyed, M. R. Pract. Oncol. 1989, 3, 1.
30. Monks, A.; Scudiero, D.; Skehan, P.; Boyed, M. Proc. Am. Assoc. Cancer Res. 1989,
30, 607.