9-substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles as highly selective and potent anaplastic lymphoma kinase inhibitors

Article abstract of DOI:10.1021/jm200652u

9-Substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles were discovered as highly selective and potent anaplastic lymphoma kinase (ALK) inhibitors by structure-based drug design. The high target selectivity was achieved by introducing a substituent close to the E₀ region of the ATP binding site, which has a unique amino acid sequence. Among the identified inhibitors, compound 13d showed highly selective and potent inhibitory activity against ALK with an IC₅₀ value of 2.9 nM and strong antiproliferative activity against KARPAS-299 with an IC₅₀ value of 12.8 nM. The compound also displayed significant antitumor efficacy in an established ALK fusion gene-positive anaplastic large-cell lymphoma (ALCL) xenograft model in mice without body weight loss.

Full text of DOI:10.1021/jm200652u

ARTICLE
pubs.acs.org/jmc
9-Substituted 6,6-Dimethyl-11-oxo-6,11-dihydro-5H-
benzo[b]carbazoles as Highly Selective and Potent Anaplastic
Lymphoma Kinase Inhibitors
Kazutomo Kinoshita,*,† Takamitsu Kobayashi,† Kohsuke Asoh,† Noriyuki Furuichi,† Toshiya Ito,†
Hatsuo Kawada,† Sousuke Hara,† Jun Ohwada,† Kazuo Hattori,† Takuho Miyagi,† Woo-Sang Hong,‡
Min-Jeong Park,‡ Kenji Takanashi,† Toshiyuki Tsukaguchi,† Hiroshi Sakamoto,† Takuo Tsukuda,† and
Nobuhiro Oikawa†
†Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
‡C&C Research Laboratories, 146-141, Annyeong-dong, Hwaseong-si, Gyeonggi-do 445-380, Republic of Korea
S Supporting Information
b
ABSTRACT: 9-Substituted 6,6-dimethyl-11-oxo-6,11-dihy-
dro-5H-benzo[b]carbazoles were discovered as highly selective
and potent anaplastic lymphoma kinase (ALK) inhibitors by
structure-based drug design. The high target selectivity was
achieved by introducing a substituent close to the E0 region of
the ATP binding site, which has a unique amino acid sequence.
Among the identified inhibitors, compound 13d showed highly
selective and potent inhibitory activity against ALK with an IC50
value of 2.9 nM and strong antiproliferative activity against
KARPAS-299 with an IC50 value of 12.8 nM. The compound also displayed significant antitumor efficacy in an established ALK
fusion gene-positive anaplastic large-cell lymphoma (ALCL) xenograft model in mice without body weight loss.
’ INTRODUCTION
The inhibition of particular kinases is considered to have risk
of adverse events. For example, inhibitions of KIT and KDR have
been elucidated as associated with bone marrow suppression15
and hypertension,16 respectively. The most developed (as of this
writing) ALK inhibitor 1 (crizotinib, Pfizer, Figure 1), which has
been tested clinically for treatment of EML4-ALK-positive
NSCLC, is a MET/ALK dual inhibitor.17 On the other hand,
our reported ALK inhibitor 218 (IC50 = 1.5 nM, Figure 1) did not
have potent inhibitory activity against MET (IC50 = 7200 nM)
but showed submicromolar IC50 values against several tyrosine
kinases including KDR. Therefore, further modification was
performed to address this issue.
To achieve high target selectivity, on the basis of the binding
mode of 2 with ALK homology models, we designed 9-substi-
tuted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles
which recognize a unique amino acid sequence of ALK in the
ATP binding site. For an efficient derivatization at the 9-position
of 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole, we
modified 13a(Figure 1) as a scaffold instead of 2, using a common
intermediate. Eventually we identified the 9-ethyl derivative 13d
as a highly selective ALK inhibitor.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine
kinase (RTK) belonging to the insulin receptor superfamily,
which includes insulin-like growth factor-1 receptor (IGF-1R)
and leukocyte tyrosine kinase (LTK). The function of ALK in
the normal human body is unclear. A possible role of ALK is in
physiological development, and its expression in normal tissues
is very restricted to the central nervous system.1,2 The kinase
was originally identified as a part of the fusion oncogene
nucleophosmin (NPM)-ALK resulting from a t(2;5) chromo-
somal translocation in anaplastic large cell lymphomas
(ALCL).3 NPM-ALK is detected in approximately 75% of all
ALK-positive ALCL and is implicated in the pathogenesis of
ALCL.4 Not only NPM-ALK but also other ALK fusion genes
have been identified in ALCL,5 inflammatory myofibroblastic
tumor (IMT),6 diffuse large B-cell lymphoma (DLBCL),7
squamous cell carcinoma (SCC),8 and nonsmall cell lung
cancer (NSCLC).9 It is noteworthy that echinoderm micro-
tubule-associated protein-like 4(EML4)-ALK in NSCLC was
the first fusion gene found to be an oncogenic factor in solid
tumors.10 EML4-ALK has been detected in approximately 5%
of NSCLC patients11 and is mutually exclusive for known
oncogenic mutated-EGFR and KRAS.12 In addition, genetic
amplification and overexpression of ALK have recently been
discovered to cause childhood neuroblastoma.13,14 These find-
ings indicate that ALK is an attractive drug target for treatment
of various ALK-positive cancers in blood and solid tumors.
Herein, we wish to report the discovery of a highly selective
and potent ALK inhibitor, 13d, as well as its pharmacokinetics
and in vivo efficacy.
Received: May 23, 2011
Published: August 08, 2011
r
2011 American Chemical Society
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Figure 1. Structure of ALK inhibitors.
Scheme 1. Synthesis of Compound 13aa
a Reagents and conditions: (a) N-Boc-piperazine, NMP, 140 °C; (b) TFA, CH2Cl2, rt; (c) 3-oxetanone, NaBH(OAc)3, THF, 40 °C.
’ CHEMISTRY
13c, 13h, 13i, and 13j with catalytic palladium on charcoal
The preparation of compound 13a from intermediate 318,19 is
shown in Scheme 1. Aromatic nucleophilic substitution of 3 with
N-Boc-piperazine gave compound 4. After removal of Boc group
with trifluoroacetic acid, reductive alkylation of 5 with 3-oxeta-
none and sodium triacetoxyborohydride in tetrahydrofuran
afforded the target compound 13a.
afforded the corresponding 9-alkyl derivatives 13d, 13e, 13f,
and 13g.
’ RESULTS AND DISCUSSION
Molecular Design. Since our previous study18 revealed that
the ALK inhibitors bearing a 6,6-dimethyl-11-oxo-6,11-dihydro-
5H-benzo[b]carbazole scaffold were ATP competitive, we in-
vestigated the amino acid sequences surrounding the ATP-
binding site of several kinases. We found that the amino acid
residues of the E0 region21 of ALK (A1200 and G1201) were
characteristically small (Chart 1) and that only 14 out of 490
kinases have the same amino acid sequence in the region as that
of ALK (existence ratio is less than 3%).22 The region often
serves as an entrance for ligand binding, and ALK is considered to
have a wide-open surface for binding of a ligand. Therefore, we
considered that the steric repulsion between a ligand and the
amino acid residues of the E0 region of off-target proteins should
afford high ALK selectivity.
The docking study of 2 with our ALK homology models
indicates that the 9-position of 6,6-dimethyl-11-oxo-6,11-dihy-
dro-5H-benzo[b]carbazole scaffold would be closest to A1200 of
the E0 region of ALK protein. During this work, we used ALK
homology models for the docking study because no ALK crystal
structure was available at that time. On the basis of the binding
model, we designed 9-substituted 6,6-dimethyl-11-oxo-6,11-di-
hydro-5H-benzo[b]carbazoles to know the influence on kinase
selectivity. Because the residues of amino acids in the E0 region of
ALK are hydrophobic (A1200 and G1201), we chose several
The synthesis of 8,9-substituted 6,6-dimethyl-11-oxo-6,11-
dihydro-5H-benzo[b]carbazoles (13bÀj) is summarized in
Scheme 2. After bromination of compound 620 with N-bromo-
succinimide, the Fischer indole ring formation of 7 gave a 1:1
mixture of regio isomers (1-CN- and 3-CN-derivatives). The
undesired regio isomer (1-CN-derivative) was precipitated
more easily than the desired one (3-CN-derivative) in organic
solvents. Therefore, most of the undesired indole was removed
by filtration. After washing with AcOEt/n-hexane, DDQ oxida-
tion was conducted without further purification. Washing the
resulting extract with AcOEt afforded the desired ketone 8 as a
single product. A methoxy group at 8-position was converted
into a triflate group by demethylation with pyridinium hydro-
chloride at 190 °C followed by treatment with triflic anhydride.
The triflate 10 was treated with piperazine in NMP to give
8-piperazinyl compound 11. The reductive alkylation of 11 with
3-oxetanone and sodium cyanoborohydride in tetrahydrofur-
an/methanol afforded 12. A versatile intermediate 12 allows
efficient derivatization at the 9-position. The intermediate was
treated with corresponding building blocks under conventional
Pd-catalyzed cross-coupling conditions to give compounds
13b, 13h, 13i, and 13j. 9-TIPS-acetylene derivative was depro-
tected with TBAF to give compound 13c. Hydrogenation of
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Scheme 2. Synthesis of Compound 13bÀga
a Reagents and conditions: (a) N-bromosuccinimide, CH3CN, rt; (b) 3-cyanophenylhydrazine, TFA, 100 °C; (c) DDQ, THF/H2O, 0 °C; (d)
pyridinium hydrochloride, 190 °C; (e) trifluoromethanesulfonic anhydride, pyridine, rt; (f) piperazine, NMP, 120 °C; (g) 3-oxetanone, NaBH3CN,
AcOH, THF/MeOH, rt; (h) trimethylboroxine, Pd(PPh3)4, K2CO3, DMF, 100 °C; (i) TIPS-acetylene, Pd(CH3CN)2Cl2, X-Phos, Cs2CO3, CH3CN,
80 °C, and then TBAF, THF, rt; (j) propyne, Pd(CH3CN)2Cl2, X-Phos, Cs2CO3, CH3CN, 80 °C; (k) isopropenylboronic acid pinacol ester,
Pd(PPh3)2Cl2, Na2CO3, DME/H2O, 80 °C; (l) cyclopentylacetylene, Pd(CH3CN)2Cl2, X-Phos, Cs2CO3, CH3CN, 80 °C; (m) 10% Pd/C, H2 gas,
THF/MeOH, rt.
Chart 1. Amino Acid Sequences of ALK and Off-Target
Proteins
coordinates (Figure 2) overlapped well with that of 2 based on
our ALK homology models. The result indicates that our docking
study is highly reliable.
StructureÀActivity Relationship (SAR). The inhibitory
activity against the four kinases and against KARPAS-299
(an NPM-ALK-positive ALCL cell line),25 and the in vitro
metabolic stability are summarized in Table 1. As expected, a
substituent at 9-position affected inhibitory activity against
both ALK and off-target proteins. The 9-methyl group did not
remarkably affect inhibitory activity against any proteins
(13b). In contrast, bigger substituents than the methyl group
notably weakened inhibitory activity against off-target pro-
teins (13cÀ13g). These results indicate that steric bulkiness is
a critical factor for inhibitory activity against off-target pro-
teins. Among 13cÀ13g, inhibitory activity against ALK was
inversely proportional to bulkiness of the 9-substituent. The
results suggest that C2-unit (ethyl and ethynyl groups) is
optimum to occupy the space of the E0 region and would make
a hydrophobic interaction with the amino acid residues there.
On metabolic stability, on the other hand, there was no
impact made by a substituent at 9-position except for the most
bulky derivative 13g, which showed relatively higher in vitro
clearance (CL) value in mouse liver microsome in the presence
of NADPH.
substituents which are expected to make a hydrophobic interac-
tion with these residues. However, the regioselective CÀC bond
formation of intermediate 10 at either 8- or 9-position was not
successful under palladium-catalyzed cross-coupling conditions
(Scheme 2). Therefore, the CÀN bond was formed regioselec-
tively at the 8-position (from 10 to 11), and an efficient
derivatization at the 9-position was conducted from the common
intermediate 12.
Recently, we obtained an X-ray cocrystal structure of another
6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole deri-
vative 14 (CH5424802, Figure 2)23 with human ALK (PDB
code 3AOX). The binding model24 of 13a derived from the X-ray
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Figure 2. Binding model of 13a in complex with human ALK derived from the X-ray cocrystal structure with 14 (PDB code 3AOX). Compound 13a is
shown as a stick model colored by element type (C in green, O in red, and N in blue). Overall structure of human ALK is shown as a cartoon diagram in
pale blue. Residues L1198 through G1202 of human ALK are shown as a stick model colored by element type as the ligand except C in white and S in yellow.
A hydrogen bond between the carbonyl oxygen of 13a and the backbone amide of M1199 (hinge) is depicted by a yellow dashed line. Hydrogen atoms
and solvent molecules are not displayed for clarity. The distance between C9 and alfa carbon of A1200 is 4.2 Å. The figure was prepared with PyMol
(http://www.pymol.org).
Among the derivatives, 13d showed both strong inhibitory
activity against ALK and antiproliferative activity against KAR-
PAS-299 with high target selectivity and favorable in vitro
metabolic stability.
calculated. Compound 13d showed strong antitumor efficacy
dose-dependently. A significant tumor regression (TGI = 119%)
was observed at the dose of 20 mg/kg without apparent body
weight loss. This efficacy is greater than that of the first genera-
tion ALK inhibitor 2.18
Kinase Selectivity Profile. Compound 13d was further
evaluated for inhibitory activity against 17 tyrosine and serine/
threonine kinases (Table 2). Compound 13d showed improved
target selectivity compared to that of our first generation ALK
inhibitor 2. The amino acid sequences in the E0 region of 17
kinases support our hypothesis. It is noteworthy that 13d showed
a relatively low IC50 value against INSR and that the kinase has
the same amino acid in a corresponding position to that of A1200
in ALK. This fact indicates that A1200 would be a key amino acid
for kinase selectivity.
Pharmacokinetics. The pharmacokinetic properties of 13d
were evaluated in monkeys (Table 3). Compound 13d demon-
strated favorable plasma clearance in monkeys, which is consis-
tent with in vitro CL values in human and mouse liver
microsomes in the presence of NADPH. In addition, 13d showed
moderate bioavailability and T1/2 value. A preliminary pharma-
cokinetic study was conducted in mice. The result is provided in
Supporting Information.
’ CONCLUSION
By modifying our previously reported ALK inhibitor 2
bearing a unique 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo-
[b]carbazole scaffold, we identified highly selective ALK in-
hibitors which eliminate the concerns of adverse events caused
by off-target kinase modulation. Among them, highly selective
and potent ALK inhibitor 13d, bearing an ethyl group at the
9-position, was discovered. The location of the ethyl group
around the E0 region at the binding sites of kinases might have
afforded high ALK selectivity over off-target kinases. Com-
pound 13d showed inhibitory activity against ALK with an IC50
value of 2.9 nM and antiproliferative activity against KARPAS-
299 with an IC50 value of 12.8 nM. The pharmacokinetic
properties of 13d were favorable. Oral administration of 13d
at 20 mg/kg demonstrated significant tumor regression (TGI =
119%) without body weight loss in an established NPM-ALK-
positive ALCL xenograft model in mice.
Efficacy in Tumor Xenograft Studies. Compound 13d was
also evaluated in an established NPM-ALK-positive ALCL
xenograft model in mice (Figure 3). Mice bearing KARPAS-
299 were administered 13d orally once daily for 11 days at 2, 6,
and 20 mg/kg, their body weight and the volume of the tumor
were measured twice a week, and the relative body weight was
’ EXPERIMENTAL SECTION
Chemistry. All solvents and reagents were obtained commercially.
1H and 13C NMR spectra were recorded on a VARIAN 400-MR or a
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Table 1. In Vitro Inhibitory Activity and Metabolic Stability of 9-Substituted Derivatives
a The values are averages of 2 separate determinations. The values in each assay are provided in Supporting Information.
a Waters LCMS system with a SunFire C18 (5 μm, 4.6 mm  50 mm)
column or a Wakosil-II 3C18 AR (3.0À3.7 μm, 4.6 mm  30 mm)
column (mobile phase, CH3CN/H2O with 0.05% TFA; flow rate,
2.0 mL/min). High resolution mass spectra (HRMS) were measured
with a Thermo Fisher Scientific LTQ Orbitrap XL MS spectrometer
using an ESI source coupled to a Waters HPLC system operating in
reversed phase with an ACQUITY UPLC BEH C18 (1.7 μm, 2.1 mm Â
50 mm) column. Flash column chromatography was performed with
Biotage SNAP cartridges or SILICYCLE SiliaSep packed columns.
Preparative HPLC was conducted with a SunFire Prep C18 OBD
(5 μm, 30 mm  50 mm) column (mobile phase, CH3CN/H2O with
0.05% TFA). All tested compounds are g95% except for 13e (93%).
The purity was determined by LCMS analysis.
Table 2. Inhibitory Activity of 2 and 13d against 17 Kinases,
and Amino Acids in the E0 Regiona
6,6-Dimethyl-8-(1-oxetan-3-yl-piperidin-4-yl)-11-oxo-6,11-dihydro-
5H-benzo[b]carbazole-3-carbonitrile (2). The synthetic procedure of 2
was described in a previous report.18 LCMS (ESI), >95% pure; m/z, 426
[M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.74 (1H, bs), 8.32 (1H,
d, J = 8.4 Hz), 8.13 (1H, d, J = 7.9 Hz), 8.00 (1H, s), 7.74 (1H, s), 7.61
(1H, d, J = 7.9 Hz), 7.40 (1H, d, J = 8.4 Hz), 4.56 (2H, t, J = 6.4 Hz), 4.46
(2H, t, J = 6.1 Hz), 3.46À3.39 (1H, m), 2.85À2.82 (2H, m), 2.71À2.64
(1H, m), 1.92À1.86 (2H, m), 1.82À1.79 (4H, m), 1.77 (6H, s). 13C
NMR (100 MHz, DMSO-d6) δ 179.0, 160.1, 150.9, 148.1, 135.6, 129.3,
127.5, 125.7, 125.3, 125.0, 124.7, 121.5, 119.9, 116.3, 109.2, 104.5, 74.6,
58.5, 49.8, 41.9, 36.5, 32.2, 29.7. HRMS (ESI), m/z calcd for
C27H28N3O2 [M + H]+ 426.2176, found 426.2179.
a The experimental details for each kinase are available in Supporting
Information.
JEOL JNM-EX270 spectrometer, and chemical shifts are expressed as δ
units using tetramethylsilane as an internal standard. The spectral
splitting patterns are described as follows: s, singlet; d, doublet; dd,
double doublet; t, triplet; q, quartet; m, multiplet; and bs, broad singlet
peak. Liquid chromatography/mass spectra (LCMS) were measured by
4-(3-Cyano-6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazol-
8-yl)-piperazine-1-carboxylic Acid tert-Butyl Ester (4). To a solution of
318 (25 mg, 0.058 mmol) in NMP (1.25 mL) was added N-Boc-
piperazine (302 mg, 28 equiv). After stirring for 27 h at 140 °C, the
reaction mixture was purified by preparative HPLC to give 4 (15 mg,
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Table 3. Pharmacokinetic Profiles of 13d in Monkeys (Male and Female)a
po
iv
Fb (%)
AUC (ng h/mL)
Cmax (ng/mL)
T1/2 (h)
CLtot (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
3
no. 1
no. 2
mean
21.6
34.7
28.2
2510
4030
3270
288
366
327
6.27
6.22
6.25
0.808
0.652
0.730
0.298
0.294
0.296
4.6
5.9
5.3
a Dose: po and iv at 0.5 mg/kg (n = 2). b Oral bioavailability.
AcOEt. After addition of water, stirring of the resulting solution for 0.5 h
at 0 °C afforded a precipitate. The precipitate was filtered off and washed
with AcOEt/water to give 13a as a light-yellow solid (27 mg, 90%).
1
LCMS (ESI), >95% pure; m/z, 427 [M + H]+. H NMR (400 MHz,
DMSO-d6) δ 8.29 (1H, dd, J = 8.2, 0.59 Hz), 8.02 (1H, d, J = 9.0 Hz),
7.97 (1H, d, J = 0.59 Hz), 7.56 (1H, dd, J = 8.0, 1.4 Hz), 7.22 (1H, d, J =
2.3 Hz), 7.04 (1H, dd, J = 8.8, 2.2 Hz), 4.56À4.59 (2H, m), 4.47À4.50
(2H, m), 3.43À3.48 (1H, m), 3.39À3.42 (4H, m), 2.40À2.42 (4H, m),
1.74 (6H, s).
6-Bromo-7-methoxy-1,1-dimethyl-3,4-dihydro-1H-naphthalen-2-one
(7). To a solution of 620 (2.00 g, 9.79 mmol) in CH3CN (40 mL) was
added N-bromosuccinimide (1.92 g, 1.1 equiv). After stirring for 2.5 h at
room temperature, the reaction mixture was poured into water (40 mL)
to give a white precipitate. This was collected by filtration and air-dried
to give 7 (2.55 g, 92%). 1H NMR (400 MHz, DMSO-d6) δ: 7.43 (1H, s),
7.04 (1H, s), 3.87 (3H, s), 2.98 (2H, t, J = 6.8 Hz), 2.59 (2H, t, J = 6.8
Hz), 1.34 (6H, s). HRMS (ESI), m/z calcd for C13H16O2Br [M + H]+
283.0328, found 283.0333.
9-Bromo-8-methoxy-6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo-
[b]carbazole-3-carbonitrile (8). A mixture of 7 (7.89 g, 27.8 mmol),
3-cyanophenylhydrazine (4.45 g, 1.2 equiv), and trifluoroacetic acid
(250 mL) was heated at 100 °C for 2 h. The reaction mixture was cooled
and then evaporated under reduced pressure. The residue was neutra-
lized by saturated aqueous NaHCO3 and extracted with AcOEt. The
combined organic layers were washed with brine, and precipitated solid
(undesired regioisomer) was filtered off. The filtrate was evaporated
under reduced pressure. The resulting solid was washed with AcOEt/n-hexane
to give the desired compound roughly purified, which was used in the
next reaction without further purification. To a solution of the com-
pound in THF (120 mL) and water (12 mL) was gradually added DDQ
(4.55 g, excess amount). After stirring for 3 h at 0 °C, the reaction
mixture was evaporated under reduced pressure. The residue was
dissolved with CPME. The solution was washed with aqueous NaOH,
water, and brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was washed with AcOEt to yield 8 (1.90 g,
17% in two steps from 7). 1H NMR (400 MHz, DMSO-d6) δ12.82 (1H, s),
8.28À8.32 (2H, m), 8.03 (1H, s), 7.61 (1H, d, J = 8.4 Hz), 7.49 (1H, s),
4.04 (3H, s), 1.81 (6H, s). HRMS (ESI), m/z calcd for C20H16BrN2O2
[M + H]+ 395.039, found 395.0392.
9-Bromo-8-hydroxy-6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo-
[b]carbazole-3-carbonitrile (9). A mixture of 8 (270 mg, 0.68 mmol)
and pyridinium hydrochloride (1.62 g, 22 equiv) was heated to 190 °C in
the microwave for 1.5 h. The reaction mixture was dissolved in water,
neutralized by saturated aqueous NaHCO3, and extracted with AcOEt/
THF (Â3). The combined organic layer was washed with aqueous
NaHCO3 and brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash column chromatog-
raphy (n-hexane/AcOEt) to yield 9 as a brown solid (140 mg, 54%).
1H NMR (400 MHz, DMSO-d6) δ 12.77 (1H, s), 11.13 (1H, s), 8.31
(1H, d, J = 7.9 Hz), 8.25 (1H, s), 8.01 (1H, s), 7.61 (1H, d, J = 7.9 Hz),
7.28 (1H, s), 1.74 (6H, s). HRMS (ESI), m/z calcd for C19H14BrN2O2
[M + H]+ 381.0233, found 381.0239.
Figure 3. Growth inhibitory effect of 13d on KARPAS-299 tumor
growth in mouse xenograft model. SCID mice bearing KARPAS-299
were administered compound 13d orally once daily for 11 d (days
8À18) at 2 mg/kg (tumor growth inhibition TGI = 49%), 6 mg/kg
(TGI = 103%), and 20 mg/kg (TGI = 119%).
55%). 1H NMR (270 MHz, CDCl3) δ 8.52 (1H, d, J = 8.1 Hz), 8.32 (1H,
d, J = 9.7 Hz), 7.72 (1H, s), 7.53 (1H, d, J = 8.1 Hz), 6.94À7.03 (2H, m),
3.59À3.67 (4H, m), 3.32À3.42 (4H, m), 1.50 (9H, s). MS (ESI) m/z:
471 [M + H]+.
6,6-Dimethyl-11-oxo-8-piperazin-1-yl-6,11-dihydro-5H-benzo[b]carba-
zole-3-carbonitrile (5). To a solution of 4 (15 mg, 0.032 mmol) in
CH2Cl2 (2.0 mL) was added trifluoroacetic acid (0.4 mL, excess
amount). After stirring for 2 h at room temperature, the reaction mixture
was evaporated under reduced pressure. The residue was purified by
preparative HPLC to give 5 as a light-yellow powder (10 mg, 89%). 1H
NMR (400 MHz, DMSO-d6) δ: 8.32 (1H, d, J = 8.5 Hz), 8.03 (1H, d, J =
9.1 Hz), 7.99 (1H, s), 7.59 (1H, dd, J = 8.2, 1.5 Hz), 7.20 (1H, d, J =
2.4 Hz), 7.04 (1H, dd, J = 8.8, 2.1 Hz), 3.30À3.32 (4H, m), 2.87À2.88
(4H, m), 1.77 (6H, s). MS (ESI) m/z: 371 [M + H]+.
6,6-Dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-6,11-dihydro-
5H-benzo[b]carbazole-3-carbonitrile (13a). To a solution of 5 (26 mg,
0.07 mmol) in THF (2.0 mL) were added 3-oxetanone (15 mg, 3 equiv)
and sodium triacetoxyborohydride (59 mL, 4 equiv). After stirring for
7 h at 40 °C, the reaction mixture was cooled to room temperature and
evaporated under reduced pressure. The residue was dissolved with
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Trifluoromethanesulfonic Acid 9-Bromo-3-cyano-6,6-dimethyl-11-
oxo-6,11-dihydro-5H-benzo[b]carbazol-8-yl Ester (10). To a solution
of 9 (140 mg, 0.37 mmol) in pyridine (2 mL) was added trifluorometha-
nesulfonic anhydride (188 μL, 3 equiv) at 0 °C. After stirring for
1.5 h at room temperature, the reaction mixture was poured into aqueous
NH4Cl and then extractedwith AcOEt (Â 2). The combined organic layer
was washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash column chro-
matography (n-hexane/AcOEt) to yield 10 as an off-white solid (120 mg,
64%). 1H NMR (400 MHz, DMSO-d6) δ 12.99 (1H, s), 8.51 (1H, s), 8.31
(1H, d, J = 8.2 Hz), 8.17 (1H, s), 8.07 (1H, s), 7.67 (1 H, d, J = 8.2 Hz),
1.81 (6H, s). HRMS (ESI), m/z calcd for C20H13BrF3N2O4S [M + H]+
512.9726, found 512.9728.
under reduced pressure. The residue was triturated with MeOH
(1.0 mL). The precipitated solid was filtered off and washed with n-
hexane to afford 13c as a brown solid (10.7 mg, 48%). LCMS (ESI), 95%
1
pure; m/z 451 [M + H]+. H NMR (400 MHz, DMSO-d6) δ: 12.77
(1H, s), 8.31 (1H, d, J = 7.9 Hz), 8.16 (1H, s), 8.02 (1H, s), 7.61 (1H, d,
J = 7.9 Hz), 7.27 (1H, s), 4.55À4.63 (2H, m), 4.46À4.53 (3H, m),
3.47À3.56(1H, m), 3.35À3.43 (4H, m), 2.43À2.50 (4H, m), 1.78 (6H, s).
HRMS (ESI), m/z calcd for C28H27N4O2 [M + H]+ 451.2129, found
451.2127.
9-Ethyl-6,6-dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (13d). To a solution of
13c (34.0 mg, 0.08 mmol) in MeOH/THF (1.6 mL/2.4 mL), 10%
palladium on charcoal (20 mg, 60% w/w) was added at room tempera-
ture. After being stirred vigorously under hydrogen gas for 2 h at room
temperature, the reaction mixture was filtered through Celite. The
filtrate was concentrated under reduced pressure. The residue was
purified by flash column chromatography (CH2Cl2/MeOH) to yield
13d as a white solid (6.9 mg, 19%). LCMS (ESI), 95% pure; m/z 455
[M + H]+.; 1H NMR (400 MHz, DMSO-d6) δ 12.70 (1H, s), 8.29 (1H,
d, J = 8.4 Hz), 8.05 (1H, s), 8.00 (1H, s), 7.61 (1H, d, J = 8.4 Hz), 7.38
(1H, s), 4.55À4.62 (2H, m), 4.45À4.52 (2H, m), 3.48À3.55 (1H, m),
2.98À3.05 (4H, m), 2.71 (2H, q, J = 7.5 Hz), 2.43À2.51 (4H, m), 1.74
(6H, s), 1.26 (3H, t, J = 7.5 Hz). 13C NMR (100 MHz, DMSO-d6) δ
179.1, 159.9, 155.0, 146.8, 136.5, 135.8, 127.8, 126.4, 126.0, 124.5, 121.6,
119.9, 116.7, 116.2, 109.5, 104.7, 74.3, 58.6, 51.5, 49.4, 36.6, 30.1, 22.7,
14.5. HRMS (ESI), m/z calcd for C28H31N4O2 [M + H]+ 455.2442,
found 455.2440.
9-Bromo-6,6-dimethyl-11-oxo-8-piperazin-1-yl-6,11-dihydro-5H-
benzo[b]carbazole-3-carbonitrile (11). To a solution of 10 (6.0 g, 11.7
mmol) in NMP (75 mL) was added piperazine (10.1 g, 10 equiv). After
stirring for 30 min at 120 °C, the reaction mixture was cooled and
poured into water to afford precipitation. The precipitate was filtered off
and dried under reduced pressure to yield 11 as a white solid (3.9 g,
1
74%). H NMR (400 MHz, DMSO-d6) δ 8.30 (1H, d, J = 7.9 Hz),
8.28 (1H, s), 8.00 (1H, s), 7.61 (1H, d, J = 7.9 Hz), 7.41 (1H, s), 3.32
(2H, bs), 3.01À3.10 (4H, m), 2.85À2.91 (4H, m), 1.76 (6H, s). HRMS
(ESI), m/z calcd for C23H22BrN4O [M + H]+ 449.0972, found
449.0973.
9-Bromo-8-(4-cyclopropyl-piperazin-1-yl)-6,6-dimethyl-11-oxo-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (12). To a solution of
11 (50 mg, 0.11 mmol) in MeOH/THF/AcOH (1.5 mL/1.5 mL/
0.25 mL) were added 3-oxetanone (39.4 μL, 7 equiv) and sodium
cyanoborohydride (36.8 mg, 5 equiv). To the mixture, an excess amount
of 3-oxetanone (total 25 equiv) was added gradually at room tempera-
ture. The reaction was monitored by HPLC until the starting material
was consumed (stirred for 24 h). The resulting reaction mixture was
poured into water to give a precipitate. The precipitated solid was
filtered off and washed with water to afford 12 as an off-white solid (41.2
mg, 73%). 1H NMR (400 MHz, DMSO-d6) δ 12.83 (1H, s), 8.30 (1H, d,
J=7.9Hz), 8.28(1H, s), 8.02(1H, s), 7.62(1H, d, J=7.9Hz), 7.48(1H, s),
4.56À4.61 (2H, m), 4.46À4.51 (2H, m), 3.47À3.56 (1H, m), 3.15À3.24
(4H, m), 2.44À2.54 (4H, m), 1.78 (6H, s). HRMS (ESI), m/z calcd for
C26H26BrN4O2 [M + H]+ 505.1234, found 505.1235.
6,6-Dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-9-prop-1-ynyl-
6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (13h). A mixture
of 12 (210 mg, 0.42 mmol), cesium carbonate (609 mg, 4.5 equiv),
X-Phos (29.7 mg, 0.15 equiv), CH3CN (8.0 mL), and Pd(CH3CN)2Cl2
(5.39 mg, 0.05 equiv) was stirred under propyne gas for 16 h at 80 °C.
The reaction mixture was cooled and then triturated with water (12 mL).
The precipitated solid was filtered off and washed with AcOEt/n-hexane
1
to yield 13h as a brown solid (153 mg, 79%). H NMR (400 MHz,
CD3OD) δ 8.37 (1H, d, J = 8.2 Hz), 8.18 (1H, s), 7.84 (1H, s), 7.53 (1H,
d, J = 8.2 Hz), 7.19 (1H, s), 4.70À4.77 (2H, m), 4.62À4.68 (2H, m),
3.57À3.63 (1H, m), 3.38À3.45 (4H, m), 2.54À2.61 (4H, m), 2.10 (3H,
s), 1.79 (6H, s). MS (ESI) m/z: 465 [M + H]+.
6,6,9-Trimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-6,11-dihydro-
5H-benzo[b]carbazole-3-carbonitrile (13b). A mixture of 12 (30 mg,
0.06 mmol), trimethylboroxine (16.5 μL, 2 equiv), potassium carbonate
(24.6 mg, 3 equiv), DMF (0.9 mL), and Pd(PPh3)4 (6.86 mg, 0.1 equiv)
was stirred for 10 h at 100 °C. The reaction mixture was then cooled,
diluted with AcOEt, washed with brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by
preparative HPLC to give 13b (11.2 mg, 42%). LCMS (ESI), >95%
6,6-Dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-9-propyl-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (13e). Compound 13e
was prepared from 13h following the same procedure as described for
13d (48%, an off-white solid). LCMS (ESI), 93% pure; m/z 469 [M +
1
H]+. H NMR (270 MHz, DMSO-d6) δ 12.75 (1H, s), 8.30 (1H, d,
J = 8.2 Hz), 7.97À8.01 (2H, m), 7.59 (1H, d, J = 8.2 Hz), 7.38 (1H, s),
4.53À4.61 (2H, m), 4.43À4.51 (2H, m), 3.49À3.55 (1H, m),
2.96À3.02 (4H, m), 2.63 (2H, t, J = 7.3 Hz), 2.41À2.47 (4H, m),
1.73 (6H, s), 1.61À1.70 (2H, m), 0.94 (3H, t, J = 7.4 Hz).
1
pure; m/z, 441 [M + H]+. H NMR (270 MHz, DMSO-d6) δ 12.71
(1H, s), 8.32 (1H, d, J = 8.1 Hz), 8.01 (1H, s), 7.97 (1H, s), 7.60 (1H, d,
J = 8.1 Hz), 7.32 (1H, s), 4.57À4.61 (2H, m), 4.47À4.51 (2H, m),
3.49À3.55 (1H, m), 2.99À3.11(4H, m), 2.42À2.50 (4H, m), 2.33 (3H, s),
1.76 (6H, s).
9-Isopropenyl-6,6-dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-
6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (13i). A mixture of
12 (56 mg, 0.11 mmol), isopropenylboronic acid pinacol ester (29.3 μL,
1.4 equiv), sodium carbonate (35.2 mg, 3 equiv), DME/water (2.5 mL/
0.5 mL), and Pd(PPh3)2Cl2 (3.88 mg, 0.05 equiv) was stirred for 38 h at
80 °C. The reaction mixture was cooled and then triturated with water
(9.0 mL). The precipitated solid was filtered off and purified by flash
column chromatography (CH2Cl2/MeOH) to yield 13i as a white solid
(19.0 mg, 37%). 1H NMR (270 MHz, CD3OD + CDCl3) δ 8.44 (1H, d,
J = 7.8 Hz), 8.09 (1H, s), 7.83 (1H, s), 7.54 (1H, d, J = 7.8 Hz), 7.18 (1H,
s), 5.20À5.24 (2H, m), 4.68À4.81 (4H, m), 3.56À3.70 (1H, m),
3.24À3.34 (4H, m), 2.52À2.61 (4H, m), 2.21 (3H, s), 1.82 (6H, s).
MS (ESI) m/z: 467 [M + H]+.
9-Ethynyl-6,6-dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (13c). A mixture of 12
(26.7 mg, 0.05 mmol), TIPS-acetylene (17.8 μL, 1.5 equiv), cesium
carbonate (81.5 mg, 4.5 equiv), X-Phos (7.56 mg, 0.3 equiv), CH3CN
(1.0 mL), and Pd(CH3CN)2Cl2 (1.37 mg, 0.1 equiv) was stirred for 4 h
at 80 °C. The reaction mixture was then cooled, diluted with AcOEt,
washed with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was used in the next reaction without
further purification. To a solution of the residue in THF (2.0 mL) was
added TBAF (74.2 μL, 1.5 equiv). After stirring for 1 h at room
temperature, the reaction mixture was diluted with AcOEt, washed with
water (Â 6) and brine, dried over Na2SO4, filtered, and concentrated
9-Isopropyl-6,6-dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-11-oxo-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (13f). Compound 13f
was prepared from 13i following the same procedure as described for
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ARTICLE
13d (38%, a white solid). LCMS (ESI), >95% pure; m/z 469 [M + H]+.
1H NMR (270 MHz, CD3OD + CDCl3) δ 8.44 (1H, d, J = 7.8 Hz), 8.27
(1H, s), 7.84 (1H, s), 7.54 (1H, d, J = 7.8 Hz), 7.36 (1H, s), 4.70À4.82
(4H, m), 3.63À3.73 (1H, m), 3.35À3.52 (1H, m), 3.09À3.13 (4H, m),
2.55À2.72 (4H, m), 1.81 (6H, s), 1.31 (6H, d, J = 7.0 Hz).
volume of acetonitrile, the reaction mixture was centrifuged at 1500 rpm
for 10 min. The resultant supernatant was used as a test sample to
measure the stability in human (or mouse) liver microsome by quantitat-
ing the compound in the sample using LC/MS.
Determination of Pharmacokinetic Parameters in Mon-
keys. Fasted cynomolgus monkeys (n = 2 per treatment group) were
given 13d by oral (po) or intravenous (iv) route at a dose of 0.5 mg/kg.
Compound13d was dissolvedina vehicle of20%EtOH, 10% Cremophor
EL, 15% PEG400, 15% HPCD (2-hydroxypropyl-β-cyclodextrin), and
0.02 mol/L HCl in water. Blood samples were collected with heparin as
an anticoagulant at 0.08, 0.25, 1, 2, 4, 7, 24, and 48 h following iv dosing
and at 0.50, 1, 2, 4, 7, 24, and 48 h following oral dosing. Samples were
centrifuged and the plasma collected and stored at À80 °C until analysis.
Samples were analyzed by LC-MS/MS technique. The pharmacokinetic
parameters were calculated by noncompartmental analysis.
9-Cyclopentylethynyl-6,6-dimethyl-8-(4-oxetan-3-yl-piperazin-1-yl)-
11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (13j). A
mixture of 12 (38 mg, 0.08 mmol), cyclopentylacetylene (26 μL, 3
equiv), cesium carbonate (97.9 mg, 4 equiv), X-Phos (5.37 mg, 0.15
equiv), CH3CN (2.0 mL), and Pd(CH3CN)2Cl2 (0.97 mg, 0.05 equiv)
was stirred for 14 h at 80 °C. The reaction mixture was cooled and then
triturated with water (2.0 mL). The precipitated solid was filtered off and
washed with n-hexane to yield 13j as a light-brown solid (25 mg, 66%). 1H
NMR (400 MHz, DMSO-d6) δ 12.74 (1H, s), 8.31 (1H, d, J = 8.4 Hz),
8.04 (1H, s), 8.00 (1H, s), 7.60 (1H, d, J = 8.4 Hz), 7.22 (1H, s),
4.54À4.63 (2H, m), 4.43À4.53 (2H, m), 3.45À3.53 (1H, m), 3.34À3.43
(4H, m), 2.89À2.98 (1H, m), 2.41À2.50 (4H, m), 1.92À2.05 (2H, m),
1.76 (6H, s), 1.51À1.81 (6H, m). MS (ESI) m/z: 519 [M + H]+.
9-(2-Cyclopentyl-ethyl)-6,6-dimethyl-8-(4-oxetan-3-yl-piperazin-1-
yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (13g).
Compound 13g was prepared from 13j following the same procedure
as described for 13d (29%, a white solid). LCMS (ESI), >95% pure; m/z
523 [M + H]+. 1H NMR (270 MHz, CD3OD) δ 8.40 (1H, d, J = 7.8 Hz),
8.13 (1H, s), 7.86 (1H, s), 7.55 (1H, d, J = 7.8 Hz), 7.40 (1H, s),
4.62À4.78 (4H, m), 3.52À3.68 (1H, m), 3.07À3.11 (4H, m), 2.72À2.78
(2H, m), 2.56À2.60 (4H,m), 1.80 (6H, s), 1.20À1.90 (11H, m).
In Vitro Kinase Enzyme Assay. ALK protein was purchased from
Carna Biosciences. The inhibitory ability was evaluated by examining
their ability to phosphorylate substrate peptide (Biotin-EGPWLEEEEE-
AYGWMDF) in the presence of 30 μM ATP and 10 mM MgCl2 using
time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
The inhibitoryactivityagainst Raf-1was evaluated byexamining the ability
of the kinase to phosphorylate MEK1/2 using europium-antiphospho-
MEK1/2 (Ser217/221) antibody. The quantity of enzyme and ATP and
the kind of substrate and cation species added in each assay for evaluation
of other kinases are given in the Supporting Information.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental conditions for
b
enzyme assays. IC50 values of Table 1 in each assay. Amino acid
sequences of the 14 kinases having AG at the E0 region.
Pharmacokinetic study of 13d in mice. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +81(467)47-6050. Fax: +81(467)47-2248. E-mail:
kinoshitakzt@chugai-pharm.co.jp.
’ ACKNOWLEDGMENT
We thank Y. Tachibana, K. Sakata, and T. Fujii for biological
assays, K. H. Ahn for chemical synthesis, S. Kuramoto and N.
Shibahara for PK analysis, Y. Akada, M. Hiramoto and K.
Yoshinari for physicochemical assays, and H. Suda, F. Matsuno
and Y. Ishiguro for mass spectrometry measurements.
In Vitro Cell Growth Assay. KARPAS-299 cells were treated with
various concentrations of assay compounds for 96 h. Cell growth
inhibition was determined by Cell Counting Kit-8 assay.
’ ABBREVIATIONS USED
In Vivo Antitumor Activity on KARPAS-299 in SCID Mice.
Cell line (KARPAS-299) was used to evaluate antitumor activity of 13d
in vivo. It was grown as sc tumors in SCID mice (CLEA Japan, Inc.).
Therapeutic experiments were started (day 0) when the tumor reached
around 200 mm3. Mice (four mice per group) were randomized to
treatment groups to receive vehicle or 13d (oral, QD) on days 0 to 10.
Final concentration of vehicle was 10% DMSO, 10% Cremophor EL,
15% PEG400, and 15% HPCD (2-hydroxypropyl-β-cyclodextrin). Com-
pound 13d was given to mice by forced oral administration using a sonde
tube. The length (L) and width (W) of the tumor mass were measured,
and the tumor volume (TV) was calculated as: TV = (L Â W2)/2. Body
weight changerate (BW) wascalculatedusing thefollowing formula: BW
= W/W0 Â 100, where W and W0 are the body weight on a specific
experimental day and on the first day of treatment, respectively. Tumor
growth inhibition (TGI) was calculated using the following formula:
TGI = [1 À (T À T0)/(C À C0)] Â 100, where T and T0 are the mean
tumor volumes on a specific experimental day and on the first day of
treatment, respectively, for the experimental groups and likewise, where C
and C0 are the mean tumor volumes for the control group.
ALCL, anaplastic large cell lymphomas; ALK, anaplastic lymphoma
kinase; Boc, t-butoxycarbonyl; CL, clearance; CPME, cyclopro-
pyl methyl ether; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone;
DLBCL, diffuse large B-cell lymphoma; DME, 1,2-dimethox-
yethane; DMF, N,N-dimethylformamide; DMSO, dimethylsulfox-
ide; EGFR, epidermal growth factor receptor; EML4, echinoderm
microtubule-associated protein-like 4;IGF-1R, insulin-like growth
factor-1 receptor; IMT, inflammatory myofibroblastictumor;INSR,
insulin receptor; KDR, kinase insert domain receptor; KRAS, v-
Ki-ras2 Kirsten rat sarcoma viral oncogene homologue; LTK,
leukocyte tyrosine kinase; NADPH, nicotinamide adenine dinu-
cleotide phosphate; NMP, N-methyl pyrrolidone; NSCLC, non-
small cell lung cancer; PDB, Protein Data Bank; RTK, receptor
tyrosine kinase; SCC, squamous cell carcinoma; TBAF, tetrabu-
tylammonium fluoride;TFA, trifluoroacetic acid; TGI, tumor
growth inhibition; THF, tetrahydrofuran;TIPS, triisopropylsilyl
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(22) This is the result of our kinase database search. The names and
amino acid sequences of ATP binding pocket of the 14 kinases are
described in Supporting Information.
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construct complex models of compound 13a. Compound 13a was
derived by modifying the original ligand. The missing atoms of human
ALK were constructed with reference to other human ALK crystal
structures (PDB codes 3LCS and 2XP2). Solvent molecules within a
distance of 5.0 Å around the original ligand were kept. All hydrogen
atoms and modified parts of ligand were subjected to energy minimiza-
tion using MMFF94x force field parameters in combination with GBSA
solvent model. The final root-mean-square gradient was set to 0.001.
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6294
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