Design, synthesis and biological evaluation of β-carboline derivatives as novel inhibitors targeting B-Raf kinase

Article abstract of DOI:10.1016/j.bmcl.2012.05.053

β-Carboline family of compounds is a large group of alkaloids widely distributed in nature and exhibits broad-spectrum anti-tumor activities. We designed and synthesized two series of novel 1-carboxamide- and 6-sulfonamide-substituted β-carboline derivatives 7a-p and 12a-b, and their wild type B-Raf kinase inhibitory activities were described. Most compounds showed moderate to excellent inhibitory activities. Among them, 1-carboxamide-6-(N-(3-(dimethylamino)propyl)-sulfamoyl)-β-carboline, 7e exhibited potent activity (IC₅₀ = 1.62 μM), showing the potential for further investigation as a lead compound.

Full text of DOI:10.1016/j.bmcl.2012.05.053

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4783–4786
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of b-carboline derivatives
as novel inhibitors targeting B-Raf kinase
Botao Xin a,b, , Weifang Tang b, , Yue Wang b, Guowu Lin b, Haichun Liu a, Yu Jiao b, Yong Zhu b,
b,c,
Haoliang Yuan a, Yadong Chen a, , Tao Lu
⇑
⇑
a Laboratory of Molecular Design and Drug Discovery, School of Basic Sciences, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Organic Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
c State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Carboline family of compounds is a large group of alkaloids widely distributed in nature and exhibits
broad-spectrum anti-tumor activities. We designed and synthesized two series of novel 1-carboxamide-
and 6-sulfonamide-substituted b-carboline derivatives 7a–p and 12a–b, and their wild type B-Raf kinase
inhibitory activities were described. Most compounds showed moderate to excellent inhibitory activities.
Among them, 1-carboxamide-6-(N-(3-(dimethylamino)propyl)-sulfamoyl)-b-carboline, 7e exhibited
Received 29 March 2012
Revised 27 April 2012
Accepted 14 May 2012
Available online 24 May 2012
potent activity (IC50 = 1.62
l
M), showing the potential for further investigation as a lead compound.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
B-Raf kinase inhibitor
b-Carboline derivatives
Kinase inhibitory activities
B-Raf is a member of the Raf kinase family and serves as a crit-
ical component of the MAPK signal-transduction pathway which
regulates cell proliferation, differentiation and survival.1 Mutant
B-Raf induces a constitutive activation of this pathway and is cru-
cial to the development of various tumor types.2 Therefore, B-Raf
qualifies as an excellent molecular target for anti-cancer therapy.3
A number of small molecular B-Raf kinase inhibitors with various
scaffolds have been reported in the recent past.4 Vemurafenib, also
known as PLX4032, and Sorafenib have been developed as B-Raf ki-
nase inhibitors and used for the treatment of cancer.
Harmine can be found in a wide variety of different organisms
and most of them are plants. It belongs to the b-carboline family
of compounds (Fig. 1). Recently, it has been discovered that b-carb-
oline derivatives may exert anti-tumor activity through multiple
mechanisms, such as intercalating into DNA,5,6 inhibiting topoiso-
merase I and II,7,8 CDK,9 and monoamine oxidase.10,11
to make classic hydrogen bond interactions with kinase hinge
backbone residues.12 Therefore, it has provided the evidence that
the carboline scaffold may serve as a valuable template for the de-
sign and development of specific inhibitors of various kinases,
including B-Raf kinase.
The b-carboline ring was used as the new hinge binder to design
novel class of B-Raf kinase inhibitors, which could serve as an
inspiration for rational drug design in this research. Meanwhile,
the crystal structure of Sorafenib bound to wild type B-Raf kinase
reveals that the A ring of Sorafenib typically forms hydrogen bonds
with the hinge residues of wild type B-Raf kinase. While maintain-
ing the hydrogen bond interaction to the hinge region, structure
modification was carried out to form a tricyclic scaffold through
a carbon–carbon single bond between A ring and B ring (Fig. 2).
The resulting scaffold was replaced by b-carboline ring through
scaffold hopping. Furthermore, it has been reported that the sul-
fonamide moiety of PLX4032 makes crucial hydrogen bond inter-
actions with the DFG loop of B-Raf kinase.13,14 We also integrated
the sulfonamide moiety onto the 6-position of the b-carboline ring
as the DFG loop binder to form hydrogen bonds. Therefore, we
designed and synthesized a series of 1-carboxamide- and 6-sulfon-
amide-substituted b-carboline derivatives (Table 1). We tested
their inhibitory activities against wild type B-Raf kinase and IC50
values of these compounds are shown in Table 1.
Specifically, related carbolines such as compound 1 (Fig. 1),
1-cyclohexylamino-7-(1-methyl-1H-pyrazol-4-yl)-5Hpyrido[4,3-
b]indole-4-carboxamide recently have been reported as JAK2 inhib-
itors and it has been suggested that the c-carboline scaffold is able
Abbreviations: MAPK, mitogen-activated protein kinase; IC50, half maximal
inhibitory concentration; JAK2, janus kinase 2; CDK, cyclin-dependent kinase.
⇑
Corresponding authors. Tel.: +86 25 86185163; fax: +86 25 86185179 (Y.C.);
tel.: +86 25 86185140; fax: +86 25 86185179 (T.L.).
We prepared two series of b-carboline derivatives as shown in
Table 1. Compounds 7a–7p were prepared in a similar way.15 Trypt-
amine was used as the starting material and compound 2 was
E-mail addresses: ydchen@cpu.edu.cn (Y. Chen), lutao@cpu.edu.cn (T. Lu).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.053

4784
B. Xin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4783–4786
Table 1
The structures and IC50 values of the target compounds
HN
N
N
O
N
H
N
N
N
H
O
NH2
Harmine
Compound 1
Figure 1. The structures of Harmine and compound 1.
prepared through Pictet–Spengler reaction (yield, 94.7%). Com-
pound 4 was prepared through esterification (yield, 95.4%) and
dehydrogenation with potassium permanganate (yield, 86.7%).
After ammonolysis (yield, 95.1%), the resulting compound 5 reacted
with chlorosulfonic acid to give compound 6 (yield, 85.0%). Com-
pound 6 reacted with various amines to yield the target compounds
(Scheme 1).
Compounds 12a and 12b were prepared by another method
(Scheme 2). L-Tryptophan was used as the starting material and
compound 9 was also prepared through Pictet–Spengler reaction
(yield, 83.0%). Compound 9 was dehydrogenated and decarboxyl-
ated with selenium dioxide in glacial acetic acid to give compound
10 (yield, 75.0%). Compound 11 was prepared in a similar way to
that of compound 6 (yield, 86.0%) and then reacted with ammonia
and acetamide to give compounds 12a and 12b, respectively,
(yield, 80.0% and 63.0%, respectively).
We improved the reaction conditions for preparing compounds
2 and 6. In the preparation of compound 2, according to the meth-
od reported in the literature,16,17 glyoxylic acid was added to the
aqueous solution of tryptamine hydrochloride, followed by the
addition of aqueous potassium hydroxide and the yield was only
65.0%. The improved method was to add glyoxylic acid and aque-
ous potassium hydroxide at the same time and pH was kept
around 4 during the reaction. The yield was improved to 94.0%.
The reported reaction condition for the preparation of b-carbo-
line-6-sulfonylchloride derivatives was 90 °C for 5 h and the yield
of the crude product was only 48%.18 It was probably because the
product was not stable at high temperature. In our method, the
reaction was carried out at 0–5 °C and completed within 30 min.
The yield of the crude product was improved to 85.0% and the
workup procedure was also simplified.
a B-Raf IC50 values are means of two experiments and individual values are within
50% of the reported mean value.
b The biological evaluation of compound 7p and other compounds is not carried out
at the same time.
All new b-carboline derivatives were evaluated for their inhibi-
tory activities against wild type B-Raf.19 We determined IC50 values
for all b-carboline derivatives and the results are summarized in
Table 1. Compound 7e was the most potent inhibitor with an
IC50 value of 1.62
replaced by ethyl groups, the resulting compound 7b was a little
less potent than 7e with an IC50 value of 2.81 M. The inhibitory
activity was decreased when the N,N-dimethyl group was replaced
by methoxyl (compound 7i, IC50 = 22.65 M) and hydroxyl group
(compound 7c, IC50 = 20.5 M). The different positions of the hy-
droxyl group did not affect the activity (7c and 7d, IC50 = 20.5
and 21.99 M, respectively) and the change of carbochain length
did not affect the activity either (7h, IC50 = 23.74 M). When two
hydroxyl groups were introduced onto the carbochain, the inhibi-
tory activity was significantly decreased (7g, IC50 = 45.09 M).
lM (entry 5). When both methyl groups were
l
l
l
l
M
l
l
l
The activity was also greatly decreased when N,N-dimethyl group
Cl
the hinge binder
O
O
Cl
N
H
O
A
the DFG loop binder
B
O
C
F
N
N
H
N
H
CF3
H
O
N
N
S
Sorafenib
N
H
F
O
PLX 4032
Ring fusion
O
O
N
H
N
H2N
Scaffold hopping
O
A
H
B
N
N
O
S
H
N
7a
β-carboline scaffold
O
Figure 2. Design of b-carboline derivatives as B-Raf inhibitors.

B. Xin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4783–4786
4785
c
a
b
NH
NH
NH2
N
H
3
N
H
N
H
1
COOMe
COOH
2
ClO2S
e
d
N
N
N
H
N
H
N
N
H
COOMe
CONH2
CONH2
4
5
6
O
O
R1
S
f
N
H
N
N
H
CONH2
7
Scheme 1. Synthetic route of compounds 7a–7p. Reagents and conditions: (a) concd HCl, glyoxylic acid, 30% KOH, rt; (b) SOCl2, CH3OH, reflux, 6 h; (c) KMnO4, DMF, rt 24 h;
(d) MeOH, NH3ÁH2O, rt; (e) ClSO3H, 0 °C; (f) NH3ÁH2O, rt.
COOH
COOH
NH
a
b
NH2
N
N
H
8
N
H
9
N
H
10
O
O
R1
S
ClO2S
d
c
N
H
N
N
N
N
H
H
12
11
Scheme 2. Synthetic route of compounds 12a–12b. Reagents and conditions: (a) HCHO, NaOH, rt 3 h and then reflux 3 h; (b) SeO2, AcOH, reflux 12 h; (c) ClSO3H, 0 °C; (d)
NH3ÁH2O, rt.
was removed (7a, IC50 = 28.51
inhibitor with an IC50 value of 91.55
group was just a hydrogen atom, compound 7p was also a potent
inhibitor with an IC50 value of 2.12 M.
Aromatic ring was also introduced as the R1 substituent group.
Compound 7k was a potent inhibitor with an IC50 value of 2.74 M.
The activity was slightly decreased with fluoro group on the ben-
zene ring (7m, IC50 = 8.89 M) and the activity was also decreased
with methyl group on the benzene ring (7l, IC50 = 20.5 M). When
aromatic heterocycles were used, the inhibitory activity could be
obtained without much decrease (7n and 7o, IC50 = 3.76 M and
7.48 M, respectively). When cyclopropyl group was introduced,
the activity was moderate (7j, IC50 = 9.07 M).
l
M). Compound 7f was a poor
Bank. Binding sphere was selected from the active site, using the
binding site tools. For all tested compounds, hydrogens were added
and CHARMm force fields were employed. The initial geometries
were fully minimized by Dreiding Minimize tool. CDOCKER (Discovery
Studio 2.5) was used for the docking simulation. To validate the pos-
sible binding model derived from CDOCKER, a superimposition of
docked BAY439006 onto the crystallographic geometry yielded
RMSD of 0.37 Å, which revealed that CDOCKER program performed
well in reproducing experimentally observed binding conformation
of Sorafenib. Therefore, CDOCKER is suitable for molecular docking
l
M. When the R1 substituent
l
l
l
l
l
l
l
The results indicated that N,N-dimethylamino group is benefi-
cial to increase the inhibitory activity. In addition, aromatic rings
and aromatic heterocycles are also acceptable but there should
be no substitution on the benzene ring. However, the introduction
of hydroxyl and alkyl groups decreased the inhibitory activity.
We also removed the carboxamide group on the carboline ring
to investigate the potential effect. Two compounds were prepared.
Compound 12a was also a potent inhibitor with an IC50 value of
2.49 lM, which is similar to that of compound 7p (IC50 = 2.12 lM).
Therefore, the carboxamide group on the carboline ring is not
essential to the inhibitory activity. When an acetyl group was
introduced onto the nitrogen, the inhibitory activity was dramati-
cally decreased (12b, IC50 = 59.11 lM).
To better understand the binding model of these compounds
with wild type B-Raf kinase, a docking study of compound 7e was
carried out on the most potent compound 7e, with an IC50 value of
1.62
lM. The X-ray crystal structure of wild type B-Raf kinase bound
to Sorafenib (PDB code: 1UWH) was retrieved from the Protein Data
Figure 3. Docked binding model of 7e with wild type B-Raf kinase.

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B. Xin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4783–4786
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study. Then, compound 7e was docked into the wild type B-Raf
kinase crystal structure (Fig. 3).
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We observe that the NH of the carboxamide and the nitrogen
atom of the pyridine ring can form two hydrogen bonds with the
hinge backbone carbonyl oxygen and amide of CYS531, respec-
tively. In addition, the oxygen atom of sulfonamide can form two
hydrogen bonds with the amine of LYS482. The NH and nitrogen
atom of sulfonamide can form two hydrogen bonds with carbonyl
oxygen and amide of ASP593. This is similar to the binding models
observed for the hinge region and DFG loop in the crystal struc-
tures in complex with the B-Raf inhibitor Sorafenib and PLX4032,
respectively. This model is also consistent with our design idea that
the b-carboline ring can be used as the hinge binder to design novel
B-Raf inhibitors.
In this Letter, we discovered a new scaffold, b-carboline ring, as
novel B-Raf kinase inhibitors. Eighteen 1-carboxamide-6-sulfon-
amide-substituted b-carboline derivatives were synthesized and
these compounds exhibited inhibitory effect against wild type
B-Raf kinase. Our work just paved the way for further investigation
of these b-carboline derivatives as B-Raf kinase inhibitors and fur-
ther chemical modification and biological evaluation of these com-
pounds are still being undertaken in our group.
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15. Experimental procedures for the synthesis of compound 6 and 7a: (1) Compound
6: In a 25 mL single-necked, round-bottomed flask equipped with a magnetic
stirrer, 4.0 mL of chlorosulfonic acid (60.20 mmol) was added. Cool the
reaction mixture to 5 °C and compound 5 (0.53 g, 2.51 mmol) was added
through several times. After addition, the reaction mixture was stirred at rt for
0.5 h. Then the reaction mixture was slowly poured into 200 mL of cooled
water. The yellow solid precipitated and collected on a filter, washed with
water (20 mL Â 2). The crude product can be used in next step without further
purification (0.66 g, yield 85.0%); (2) compound 7a: In a 100 mL three-necked
bottle equipped with
a magnetic stirrer, the crude compound 6 (0.17 g,
0.55 mmol) and 40 mL of DMF were added, followed by the addition of
propylamine (0.011 mL, 1.38 mmol). The reaction mixture was stirred at rt for
3 h and then poured into 100 mL of cooled water. The pH was adjusted to 6–7
with glacial acetic acid. The mixture was extracted with EtOAc (40 mLÂ3). The
combined organic extract was washed with 40 mL of water and saturated
brine, respectively and dried over anhydrous MgSO4. Remove the solvent
under vacuo and chromatograph the crude product over silica gel, eluting with
10% methanol in chloroform to afford the title compound (0.15 g, 83.3%).
16. Panosyan, F. B.; Still, I. W. Can. J. Chem. 2001, 79, 1110.
17. Cao, R.; Chen, H.; Peng, W.; Ma, Y.; Hou, X.; Guan, H.; Liu, X.; Xu, A. Eur. J. Med.
Chem. 2005, 40, 991.
18. Rinehart, K. L.; Kobayashi, J.; Harbour, G. C.; Gilmore, J.; Mascal, M.; Holt, T. G.;
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Acknowledgments
This work was financially supported by Natural Science Foun-
dation of Jiangsu Province (No. BK2009303), National Nature Sci-
ence Foundation of China (No. 30973609), Fundamental Research
Funds for the Central Universities (No. JKZ2011004) and Special-
ized Research Fund for the Doctoral Program of Higher Education
(No. 20100096110007).
19. B-Raf kinase assay: Kinase activity was measured as the percent of ATP
consumed following the kinase reaction using luciferase–luciferin-coupled
chemiluminescence. Reactions were conducted in the 384-well plate
(PerkinElmer). B-Raf kinase reactions were initiated by adding test
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