Novel tetracyclic benzo[ b ]carbazolones as highly potent and orally bioavailable ALK inhibitors: Design, synthesis, and structure - activity relationship study

Article abstract of DOI:10.1016/j.ejmech.2015.10.005

Four series of tetracyclic benzo[b]carbazolone compounds possessing more rotatable bonds and higher molecular flexibility were designed by either inserting a linker within the C8-side chain or by opening the middle ketone ring on the basis of compound 5 (Alectinib, CH5424802). Compound 15b was identified showing nearly identical high potency against both wild-type and the gatekeeper mutant ALK kinase (3.4 vs 3.9 nM). This compound has favorable PK profile with an oral bioavailability of 67.1% in rats. Moreover, compound 15b showed significant growth inhibition against ALK driven cancer cells and KARPAS-299 xenograft model.

Full text of DOI:10.1016/j.ejmech.2015.10.005

Accepted Manuscript
Novel tetracyclic benzo[b]carbazolones as highly potent and orally bioavailable ALK
inhibitors: design, synthesis, and structure—activity relationship study
Xiaolong Jiang, Ji Zhou, Jing Ai, Zilan Song, Xia Peng, Li Xing, Yong Xi, Junfeng
Guo, Qizheng Yao, Jian Ding, Meiyu Geng, Ao Zhang
PII:
S0223-5234(15)30287-7
10.1016/j.ejmech.2015.10.005
EJMECH 8140
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 May 2015
Revised Date: 1 October 2015
Accepted Date: 4 October 2015
Please cite this article as: X. Jiang,, J. Zhou,, J. Ai,, Z. Song,, X. Peng,, L. Xing,, Y. Xi,, J. Guo,, Q.
Yao,, J. Ding,, M. Geng,, A. Zhang, Novel tetracyclic benzo[b]carbazolones as highly potent and orally
bioavailable ALK inhibitors: design, synthesis, and structure—activity relationship study, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.005.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Novel tetracyclic benzo[b]carbazolones as highly potent and orally bioavailable
ALK inhibitors: design, synthesis, and structure—activity relationship study
Xiaolong Jiang,a,c,1 Ji Zhou,b,1 Jing Ai,b, Zilan Song,a Xia Peng,b Li Xing,a Yong Xi,b
Junfeng Guo,a Qizheng Yao,c, Jian Ding,b Meiyu Geng,b, Ao Zhanga,

ACCEPTED MANUSCRIPT
Novel tetracyclic benzo[b]carbazolones as highly potent and orally bioavailable
ALK inhibitors: design, synthesis, and structure—activity relationship study
Xiaolong Jiang,a,c,1 Ji Zhou,b,1 Jing Ai,b, Zilan Song,a Xia Peng,b Li Xing,a Yong Xi,b
Junfeng Guo,a Qizheng Yao,c, Jian Ding,b Meiyu Geng,b, Ao Zhanga,
aCAS Key Laboratory of Receptor Research, Synthetic Organic & Medicinal Chemistry Laboratory,
Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai, China
201203.
bDivision of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203.
cDepartment of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China.
1These two authors contributed equally to this work.
ABSTRACT
Four series of tetracyclic benzo[b]carbazolone compounds possessing more
rotatable bonds and higher molecular flexibility were designed by either inserting a
linker within the C8-side chain or by opening the middle ketone ring on the basis of
compound 5 (Alectinib, CH5424802). Compound 15b was identified showing nearly
identical high potency against both wild-type and the gatekeeper mutant ALK kinase
(3.4 vs 3.9 nM). This compound has favorable PK profile with an oral bioavailability
of 67.1% in rats. Moreover, compound 15b showed significant growth inhibition
against ALK driven cancer cells and KARPAS-299 xenograft model.
Keywords: Benzo[b]carbazolones; ALK inhibitors; Anti-cancer; Anti-resistance;
Non-small-cell lung cancer.
Corresponding Authors:
For Ao Zhang: e-mail, aozhang@simm.ac.cn; for Meiyu Geng: e-mail, mygeng@simm.ac.cn; for Jing
Ai: e-mail, jai@simm.ac.cn; for Qi-zheng Yao: e-mail, qz_yao@163.com.
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1. Introduction
The treatment of lung cancer, especially non-small cell lung cancer (NSCLC) has
been impressively revolutionized in recent years triggered by the emergence of
molecular targeted therapy (MTT). By targeting distinct genetic alternations including
chromosomal translocation, point mutation and gene amplification, MTT can
recognize genotype-specific subsets of patients and exert optimum clinical outcomes.
The orphan receptor tyrosine kinase - anaplastic lymphoma kinase (ALK) is a recently
identified molecular target that exists in 3-5% of NSCLC patients harboring the fusion
gene of ALK with echinoderm microtubule-associated protein-like 4 (EML4) [1,2].
Clinically, the EML4-ALK fusion gene-bearing patients has been found to have no
overlap with patients harboring EGFR mutation or KRAS mutations, supporting that
EML4-ALK positive cancer represents a distinct subclass of NSCLC [3,4]. Therefore,
since the identification of EML4-ALK as a tumor oncogene in 2007, targeting
EML4-ALK has been on the high-speed train as a novel MTT for the subclass of
NSCLC patients, for whom currently available treatments are poorly effective [5-10].
The first-generation ALK inhibitor Crizotinib (1, PF2341066, Xalkori) [11,12]
(Figure 1) was approved by FDA in 2011 as the proof-of-concept of the
EML4-ALK-targeting therapy. Although this first-in-class ALK inhibitor offers
patients significant objective response rates and progression-free survival times,
resistance has been identified generally within one year of starting therapy [13-15].
Therefore, novel and second-generation ALK inhibitors with capacity to overcome the
resistance of Crizotinib are highly needed.
Several mechanisms underlying the acquired resistance to Crizotinib have been
proposed including target gene secondary mutation/amplification, up-regulation of
alternative signalling pathways (e.g. c-Kit, EGFR) [3,16]. The point mutations in the
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kinase domain are known as an important mechanism of acquired resistance to
small-molecule kinase inhibitors. In particular, the gatekeeper mutations, such as
T790M in EGFR and T351I in ABL, are one of the most frequent causes of resistance
[17]. Similarly, the gatekeeper mutation L1196M was also identified in the resistant
patients clustering around the ATP binding site of EML4-ALK [14]. In addition,
dozens of other mutations within the ALK tyrosine kinase domain have been
identified as well from the biopsy samples of patients who relapsed on Crizotinib
treatment, including L1196M, G1269A, F1174L, C1156Y, T1151ins, L1152R,
G1202R, and so on [3,8,19].
<Insert Figure 1 here>
Despite the importance of ALK secondary mutations to the resistance of
Crizotinib has been well recognized, it remains largely unclear how and to what
extent these mutations affect the drug-kinase interaction and subsequently reduce
antitumor efficacy [20,21]. Therefore, the current strategy to the development of
second-generation ALK inhibitors to combat the acquired resistance of Crizotinib is
rather dependent on the fine structural tuning on the earlier ALK inhibitors either to
elevate the antitumor potency or to identify new structural scaffolds capable of
interacting with the secondary mutations, especially the gatekeeper L1196M (Figure
1). Several structurally distinct small molecules have been developed as the
second-generation ALK inhibitors, including PF-06463922 (2) [22-24], Ceritinib (3,
LDK-378) [25-27], Alectinib (5, CH5424802) [10,28-32], AP26113 [33,34], X-396
[35], TSR-011 [36,37], and so on [7,38]. Among these, Ceritinib and Alectinib have
received drug approval respectively from the Food and Drug Administration of the
United States (FDA) and the Japanese Ministry of Health, Labor and Welfare (MHLW)
in the same year of 2014 for the treatment of patients with ALK-positive metastatic
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NSCLC who experience disease progression on or who are intolerant to Crizotinib
(Figure 1).
The second-in-class ALK inhibitor 2,4-diaminopyrimidine Ceritinib (3) [25,26]
and the third-in-class ALK inhibitor benzo[b]carbazolone Alectinib (5) [28-30]
represent the two major chemotypes of second-generation ALK inhibitors possessing
distinct structural characteristics. Compared to many medicinal chemistry efforts on
the 2,4-diaminopyrimidine scaffold (as in 3) [7,9], limited structural optimization on
the benzo[b]carbazolone scaffold (as in 5) has been reported, likely due to the
difficulty in chemical synthesis. The currently available SAR on this tetracyclic
framework was mostly reported by scientists at the Japanese company Chugai (a
subsidiary of Roche) [28-30]. From the X-ray co-crystal structure (PDB ID: 3AOX)
[31] of the catalytic domain of ALK in complex with 5 (Alectinib) (Figure 2), the
tetracylic benzo[b]carbazolone core exists as a planar conformation where the
carbonyl group interacts with the backbone NH of Met1199 in the kinase hinge region.
Meanwhile, more H-bondings were formed from the NH and cyano moieties in 5 with
several amino acids in the kinase domain. In addition, the morpholinyl substituted
piperidine fragment in 5 points to the solvent interaction region. In view of the
structural novelty of 5, we decided to conduct an additional medicinal chemistry
campaign by either inserting a linker within the side chain (series I, II and IV) or by
opening the middle ketone ring (series III), thus leading to four series of new
compounds with relatively more rotatable bonds and higher molecular flexibility.
<Insert Figure 2 here>
2. Chemistry
As
illustrated
in
Scheme
1,
commercially
available
6-methoxy-1,1-dimethyl-3,4-dihydronaphthalen-2(1H)-one was used as the starting
4

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material to synthesize the intermediate 6a, which was then converted to the key
precursor 7a in additional five steps by following literature procedures [28,29].
Condensation of 7a with corresponding N-Boc acids in the presence of HATU and
HOAt followed by deprotection with TFA provided amides 8a and 8b in 70% and
64% overall yields (two steps), respectively [39-41]. Meanwhile, following the
literature
procedures
[42],
iodide
6b
was
obtained
from
2-(4-bromophenyl)-2-methylpropanoic acid in six steps. Suzuki coupling of 6b with
appropriate boronic esters in the presence of Pd(PPh3)4 and Na2CO3 followed by
N-deprotection provided the key intermediate 7b in 75% overall yield [28,43].
Amides 9a-c were prepared in 62-84% overall yields from 7b and corresponding
acids under the similar reaction condition as that for preparation of 8a and 8b.
<Insert Scheme 1 here>
As described in Scheme 2, treating 7a with appropriate bromides in the presence of
DIPEA, K2CO3 and KI gave (heterocycle-substituted)methyl piperazines 10a, 10b and
12 in 45-60% yields [44]. Similarly, alkylation of 7b with 4-(bromomethyl)tetrahydro
-2H-pyran afforded compound 13 in 67% yield. The key intermediates 7c and 7d
were prepared in 49% and 65% overall yields, respectively by coupling iodide 6b
with appropriate heterocyclic amines in the presence of Pd2(dba)3, S-Phos and
NaHMDS followed by N-deprotection with TFA [42,45]. Alkylation of 7c with
4-(bromomethyl)tetrahydro-2H-pyran under the similar reaction condition as that for
preparation of 12 and 13 provided compound 11 in 54% yield. Meanwhile, compound
15f was prepared by reductive amination of 7d with oxetan-3-one in 46% overall
yield [28,29].
<Insert Scheme 2 here>
5

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Bromide 6c was prepared by following procedures similar to that for preparation
of 6b. Subsequent C-N coupling of 6c with tert-butyl piperazine-1-carboxylate
followed by removal of the N-Boc protective group provided the key intermediate 7f
in
49%
overall
yield
[42,45].
Amidation
of
7f
with
1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid followed by N-deprotection
afforded amide 14 in 74% overall yield (Scheme 3). Intermediate 7e was prepared in
56% overall yield by Suzuki coupling of 6c with corresponding boronic ester.
Subsequent condensation of 7e with 4-(tert-butoxycarbonyl)morpholine-3-carboxylic
acid followed by reduction [28] and N-deprotection gave 15a in 45% overall yield.
Moreover, compounds 15b, 15d and 15e were prepared in 30-47% overall yields by
alkylation of 7e with corresponding bromides or p-toluenesulfonates followed by
reduction. Meanwhile, treating 7e with 1,6-dioxaspiro[2.5]octane in the presence of
DIPEA followed by palladium-catalyzed hydrogenation generated compound 15c in
37% overall yield (two steps) [46].
<Insert Scheme 3 here>
As described in Scheme 4, Pd(PPh3)4-catalyzed Suzuki coupling of 6c with
appropriate borates 16 under microwave (MW) heating afforded pyrazoles 17 and 19a
in 65% and 62% yields, respectively. N-Deprotection of 19a with TFA provided 19b
in 91% yield. Similarly, substituted pyrazoles 18a-d were prepared by Suzuki
coupling of 6b with corresponding borates 16 in 41-69% yields. In addition,
N-deprotection of 18b with TFA afforded compound 20 in 92% yield. N-Alkylation of
18d with appropriate alkyl chlorides led to substituted pyrazoles 21a-c in 58-71%
yields [47].
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<Insert Scheme 4 here>
The preparation of ring-opening analogues was illustrated in Scheme 5. The key
intermediate 23 was prepared from compound 22 by following the literature
procedures [42]. Suzuki coupling of 23 with appropriate N-Boc boronic esters
followed by N-deprotection with TFA, decarboxylation in basic condition, and
subsequent acetylation [48] provided compounds 24 and 25 in 37% and 29% overall
yields, respectively. Meanwhile, compound 26 was obtained in 25% overall yield by
C-N coupling of 23 with 4-(piperidin-4-yl)morpholine followed by decarboxylation in
basic condition. Compound 28 was obtained in 52% overall yield by following the
similar procedures as that for the preparation of 21a. Subsequent removal of the
esteric group using TFA or hydrolysis using TMSCl gave amides 27 or 29 in 85% and
80% overall yields, respectively [42].
<Insert Scheme 5 here>
The preparation of compounds 31-35, 37 and 38 was described in Scheme 6. The
key intermediate 30 was obtained in 30% yield by coupling of 6b with ammonia in
the presence of Fe(acac)3, CuI and NaOH [49]. Treating 30 with corresponding
amines in the presence of triphosgene and Et3N gave the final products 31-35 in
34-63% yields [50]. Meanwhile, compound 30 was acylated with chloroacetyl
chloride followed by nucleophilic displacement [51,52] with corresponding amines
provided compounds 37 and 38 in 65% and 43% overall yields, respectively.
<Insert Scheme 6 here>
3. Results and discussion
3.1. Structure—activity relationship (SAR).
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All the new compounds were screened for their inhibitory effects against ALK
kinase [40]. As shown in Table 1, compounds bearing a C-8 side chain where the two
heterocycles (A and B) were linked through a carbonyl or a methylene unit were
tested first. Compared to parent compound 5 (CH5424802, Alectinib) [28], all these
compounds retained high inhibitory potency against ALK. As to the two
C8-(4-acyl)piperazinyl analogues 8a and 8b, morpholine-3-carbonyl 8b displayed a
3.5-fold higher potency than the azetidine-3-carbonyl 8a, with IC50 values of 0.8 and
2.8 nM, respectively. Nearly equally high potency (1.0-2.1 nM) was observed for
C8-(N-acyl-dihydropyridin-1-yl)
substituted
analogues
9a-c,
however,
tetrahydro-2H-pyran-4-carbonyl 9b has a reduced lipophilicity. Compounds 10-13
contain relatively flexible C8-substituents where the two heterocycles (A and B) were
joined through a methylene linkage. Although 3-(oxetan-3-ylmethanol) methyl
substituted compound 10b was expected to show improved aqueous solubility and
bioavailability, it was 5-fold less potent than the pyrrolidin-2-one-5-methy congener
10a (6.9 vs 1.3 nM). High potency was observed for the
tetrahydro-2H-pyran-4-methyl substituted analogues 11-13, but they have much
higher lipophilicity. Notably, the absence of C9-ethyl substituent helped to reduce the
lipophilicity, but the potency was reduced as well. Compared to the ethyl substituted
compound 8a, 14 was more than 3-fold less potent (2.8 vs 10.4 nM). Similarly, lower
potency was observed for the C9-nonsubstituted 15a (15.5 nM) and 15c-e (8.1-33
nM). Compound 15c was a potential oxidative metabolite of 15b and showed a 5-fold
reduced potency (16.4 vs 3.3 nM). Adding an additional methylene to 15d led to
8

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compound 15e with a 4-fold reduction in ALK potency (8.1 vs 33 nM). Interestingly,
compound 15f bearing a unique methylamino linkage retained high ALK potency
with an IC50 values of 9.5 nM.
<Insert Table 1 here>
Compounds 17-21 represent a series of analogues bearing a (1-substituted)-1H-
pyrazo-4-yl moiety as the C8-substituent (Table 2). Again, the C9-ethyl was an
important contributor to the ALK potency, since compound 17 was nearly 4-fold less
potent than the corresponding C7-ethyl congener 18a (40.3 vs 11.2 nM). Interestingly,
compared to the tetrahydro-pyran-4-yl substituted compound 18a, the tetrahydrofuran
-3-yl substituted analog 18c was equally potent (11.2 vs 11.8 nM); however, much
higher potency (0.9 nM) was observed for the piperidin-4-yl substituted pyrazole 20,
indicating that the nature other than the size of the substituent on the pyrazole was
important for the ALK interaction. Surprisingly, compound 19b lacking the C9-ethyl
group was more than 50-fold less potent than 20 (53 vs 0.9 nM). Compounds 21a-c
bearing a more flexible acetamide or propane-1,2-diol moiety also showed good
potency against ALK with IC50 values of 4-8 nM, although they were 5- to 10-fold
less potent than compound 20.
<Insert Table 2 here>
To obtain conformationally more flexible compounds and to explore the
importance of the planar conformation [31] of the central benzo[b]carbazolone core,
multi-substituted indoles 24-29 were prepared and evaluated for their inhibitory
effects against ALK kinase. As shown in Table 3, all the 6-cyano-2-substituted indoles
24-27 were completely inactive against ALK, even for compound 26, the direct
ring-opening congener of parent compound 5. Further addition of a 3-carboxylic acid
(29) or 3-carboxylic ester (28) to indole 27 did not offer any improvement. These
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results confirmed that the central tetracyclic planar structure is crucial to the
interaction with ALK.
<Insert Table 3 here>
Included in Table 4 were a series of compounds bearing a flexible aminocarbonyl
moiety as the C8-substituent. All these compounds have reduced lipophilicity than
parent compound 5 indicating that these compounds may have better pharmacokinetic
properties. Direct insertion of the aminocarbonyl linkage into parent compound 5
yielded compound 31 showing an IC50 value of 32 nM, which was only 3-fold less
potent. Replacement of the terminal morpholinyl moiety with an isopropanol group
slightly improved the ALK potency (32, 21 nM), whereas fusing a heterocycle to the
piperazine afforded compound 33 (38 nM) possessing nearly identical potency as that
of 31. Significant reduction in potency was observed by replacing the six-membered
piperidine moiety in 31 with a four-membered azetidine moiety (34, 381 nM).
Interestingly, a pronounced discrepancy in potency was observed between the fused
bicyclic analogues 35 and 37. Compound 37 bearing a methylene unit was 8-fold
more potent than 35 (23 vs 182 nM). Similarly, high potency was also observed for
compound 38 bearing a C8-(bicyclic amino)acetamido fragment (24 nM).
<Insert Table 4 here>
3.2. Inhibition on gatekeeper mutation and cellular antiproliferation.
Since drug resistance to the first-generation ALK inhibitor Crizotinib (1) is the
major clinical concern [20,21], new ALK inhibitors with the capacity to address
kinase mutation-triggered resistance are expected. In this regard, the newly
synthesized structurally more flexible compounds with higher ALK potency than that
of the parent compound 5 were selected for further testing their inhibitory potency
against the gatekeeper mutation L1196M. Meanwhile, antiproliferative effects of
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these compounds in ALK-dependent H3122 cell were also evaluated [40]. As shown
in Table 5, the parent compound 5 was nearly equally potent against wild-type ALK
and the gatekeeper mutant with IC50 values of 11.5 and 15.2 nM respectively. This
compound also showed similar inhibitory potency (19 nM) against the proliferation of
ALK-dependent H3122 cells. The C8-(4-acylpiperazin-1-yl) analogues 8a and 8b
showed identical potency against the L1196M gatekeeper mutation (4.1 vs 4.3 nM),
while morpholine-3-carbonyl compound 8b was nearly 9-fold more potent than the
azetidine-3-carbonyl compound 8a in the cellular assay with IC50 values of 19.9 and
174 nM, respectively. Although equally potent against wild-type ALK, the
C8-(N-acyl-dihydropyridin-1-yl) substituted analogues 9a-c showed much difference
in
the
inhibition
of
the
gatekeeper
mutation,
amongst
which
tetrahydro-2H-pyran-4-carbonyl 9b was 5-fold less potent than 9a and 9c. All three
compounds showed significantly reduced potency against the proliferation of
ALK-dependent H3122 cells. The slightly higher cellular potency on 8b and 9a than
their congeners may indicate that the morpholinyl moiety in both compounds is
beneficial to the cell penetration. Although the alcoholic analogue 10b was 5-fold less
potent against ALK than its analogue 10a, it was more potent against the gatekeeper
mutation and in the cellular assay as well showing IC50 values of 1.8 and 15.3 nM,
respectively. Compounds 11-13 containing an alkyl linkage within the C8-side chain,
despite possessing comparable potency against both wild-type ALK and the
gatekeeper mutant, were much less potent in the cellular assay with IC50 values
ranging between 20.6-185 nM.
<Insert Table 5 here>
Compound 15a bearing a carbonyl moiety in the C8-side chain and lacking the
C9-ethyl moiety showed nearly identical potencies against both wild-type ALK and
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the gatekeeper mutation with IC50 values of 15.5 and 16.8 nM, respectively; whereas
high potency was observed in the H3122 cellular assay (2.7 nM). It was not clear
whether the higher cellular potency of 15a was caused by off-target effects due to the
lack
of
C9-ethyl
substituent.
Compound
15b
bearing
a
1-((tetrahydropyran-4-yl)methyl) piperidin-4-yl as the C8-substituent showed nearly
identical high potency against both wild-type ALK and the gatekeeper mutant (3.4 vs
3.9 nM), whereas slightly lower potency (17.9 nM) was observed in the cellular assay.
The potential metabolite 15c was less potent in all the assays, whereas compound 15d
bearing the 1-(oxetan-3-ylmethyl) piperidin-4-yl moiety as the C8-substituent was
highly potent in all the assays with IC50 values of 8.1 nM against wild-type ALK, 5.6
nM against L1196 mutant, and 2.4 nM against the proliferation of H3122 cells,
respectively. Compound 15f containing a unique methylamino linkage in the C8-side
chain showed much reduced potency against L1196M mutant, although good cellular
potency was observed in the H3122 cells with an IC50 value of 16.3 nM. In spite of
the high potency against the wild ALK, pyrazole 20 was 15-fold less potent against
the L1196M mutant and was 60-fold less potent in the cellular assay. Amongst the
acetamide- and propane-1,2-diol substituted compounds 21a-c, only 21a containing
an N,N-dimethyl acetamide moiety showed high potencies in both enzymatic and
cellular assays with IC50 values of 8.1 nM against wild-type ALK, 17.3 nM against
L1196 mutant, and 5.9 nM against the proliferation of H3122 cells, respectively. It has
to be mentioned that the first-generation ALK inhibitor 1 was much less potent
against L1196M mutant (972 nM) and in the cell proliferative inhibition assays (261
nM).
<Insert Table 6 here>
3.3. Pharmacokinetics.
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From the results above, several new compounds showed equally high or much
higher potency than the parent compound 5 not only against both wild-type ALK and
the L1196M mutant but also against the cell proliferation in ALK-addicted H3122 cell
lines. Since these compounds contain structurally more flexible side chain at the
C8-position, optimal pharmacokinetic (PK) properties would be expected. Therefore,
these compounds were selected for further investigation in the PK study in rats
[40,51,53]. As shown in Table 6, despite their similar enzymatic and cellular potencies,
these compounds showed significantly different PK parameters. Compared to the
reported data on parent compound 5 [28], both compound 10a containing a methyl
unit and compound 15a bearing a carbonyl moiety within the C8-side chain displayed
disappointingly low plasma exposure and oral bioavailability (< 10%). However,
morpholinylcarbonyl substituted analog 15b showed optimal PK parameters with a
long half-life of 15.8 h, high plasma exposure of 7286 ngꢀh/mL, and high oral
bioavailability of 67.1%. Much lower exposure was observed for the hydroxylated
analog 15c. Compound 15d bearing the 1-(oxetan-3-ylmethyl)piperidin-4-yl moiety
as the C8-substituent displayed a reasonable half-life of 4.2 h and a good
bioavailability of 56.9%. Pyrazole 21a showed moderate plasma exposure and much
poor bioavailability (7.1 %).
3.4. Tumor growth inhibition and lead identification.
To identify the best compound for further profiling, the new ALK inhibitors 15b
and 15d with both potent target inhibition and optimal PK properties were further
evaluated for their antitumor efficacy in the NPM-ALK-positive human lymphoma
KARPAS-299 SCID xenograft model [29]. Mice bearing KARPAS-299 cells were
administered the selected compound orally once daily for 11 days at indicated doses,
and their body weight and the volume of the tumor were measured twice a week.
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Compound 21a with structural novelty though having poor PK parameters, and the
known ALK inhibitor 1 were also tested as comparison. As summarized in Table 7,
morpholin-4-ylmethyl analog 15b caused complete tumor regression in all tested mice
at the dose of 20 mg/kg with a slightly body weight increase. The oxetan-3-ylmethyl
analog 15d also showed significant tumor growth inhibition at the dose of 50 mg/kg;
however, body weight loss and mice casualty were observed indicating its toxicity.
Pyrazole 21a showed poor tumor growth inhibition even at the dose of 100 mg/kg
(T/C = 71.0%), which correlated quite well with its poor plasma exposure and
bioavailability (7.1 %). From the results above, compound 15b stands out as a new
orally available ALK inhibitor with high in vitro and in vivo activity. Therefore,
compound 15b was elected for further evaluation for its candidacy as a
second-generation ALK inhibitor in our early preclinical testings.
<Insert Table 7 here>
4. Conclusion
In summary, we have designed four series of tetracyclic compounds bearing the
benzo[b]carbazolone skeleton and containing relatively more rotatable bonds and
higher molecular flexibility by either inserting a linker within the C8-side chain or by
opening the middle ketone ring on the basis of compound 5 (Alectinib, CH5424802).
An extensive structure—activity study was conducted and compound 15b bearing a
1-((tetrahydropyran-4-yl)methyl)piperidin-4-yl as the C8-substituent was identified
showing nearly identical high potency against both wild-type and the gatekeeper
mutant ALK kinase (3.4 vs 3.9 nM). This compound showed favorable PK profile in
rats with high volume of distribution and good oral bioavailability (67.1%). 15b
significantly inhibited ALK driven growth of cancer cells and KARPAS-299 xenograft
model, with efficacy higher than that of 1. Based on the optimal in vitro and in vivo
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antitumor efficacy, the kinase selectivity and anti-resistant effects of compound 15b
are being profiled further in our early preclinical settings, and the data will be
presented elsewhere.
5. Experimental
5.1. General methods
NMR spectral data were recorded on a Varian Mercury 300, 400 or 500 NMR
spectrometer. Low-resolution mass spectra (MS) and high-resolution mass spectra
(HRMS) were recorded at anionizing voltage of 70 eV on a Finnigan/MAT95
spectrometer. Column chromatography was carried out on silica gel (200−300 mesh).
All reactions were monitored using thin layer chromatography (TLC) on silica gel
plates. All the reagents were obtained from commercial sources, and used without
further purification unless otherwise stated.
5.2. General procedure for the synthesis of compounds 8a and 8b
To a solution of 7a (0.2 mmol) and an appropriate acid (0.3 mmol) in DMF (2
mL) at 0 oC, HATU (0.4 mmol) and HOAt (0.2 mmol) were added. The reaction was
stirred for 10 min and DIPEA (0.8 mmol) was dropped slowly. The resulting mixture
was warmed to room temperature and stirred until the completion of the reaction. The
mixture was diluted with EtOAc (120 mL) and washed successively with water and
brine. The organic layer was dried over sodium sulfate, filtered, and evaporated. The
resulting intermediate was purified by chromatograph (petroleum/acetoacetate) and
then treated with TFA (100 equiv) in DCM at room temperature overnight. The
mixture was concentrated in vacuum, alkalified with saturated sodium bicarbonate to
15

ACCEPTED MANUSCRIPT
pH > 7, and then extracted with DCM. The combined organic layer was washed with
brine, dried (MgSO4) and filtered. After evaporation of the solvent, the residue was
purified by chromatograph (CHCl3/MeOH) to afford the corresponding target product.
5.2.1. 8-(4-(Azetidine-3-carbonyl)piperazin-1-yl)-9-ethyl-6,6-dimethyl-11-oxo-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (8a)
Pale yellow solid (70% yield in two steps). 1H NMR (300 MHz, CDCl3+ CD3OD)
δ 8.38 (d, J = 8.1 Hz, 1H), 8.18 (s, 1H), 7.71 (s, 1H), 7.43 (dd, J = 8.1, 1.2 Hz, 1H),
7.09 (s, 1H), 4.04 – 3.93 (m, 2H), 3.85 – 3.69 (m, 5H), 3.47 – 3.39 (m, 2H), 2.98 –
13
2.86 (m, 4H), 2.67 (q, J = 7.4 Hz, 2H), 1.70 (s, 6H), 1.24 (t, J = 7.5 Hz, 3H). C
NMR (126 MHz, CDCl3+ CD3OD) δ 180.8, 170.6, 160.0, 154.3, 146.7, 137.4, 136.0,
128.2, 127.5, 127.2, 125.0, 122.3, 120.3, 116.2, 116.1, 110.4, 105.2, 52.4, 51.8, 48.8,
45.6, 42.3, 36.6, 36.0, 30.3, 23.1, 14.3. MS (ESI) m/z 482 [M + H]+. HRMS: calcd for
C29H32N5O2 [M + H]+, 482.2556; found 482.2569.
5.2.2. 9-Ethyl-6,6-dimethyl-8-(4-(morpholine-3-carbonyl)piperazin-1-yl)-11-oxo-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (8b)
1
White solid (64% yield in two steps). H NMR (300 MHz, CDCl3) δ 10.92 (s,
1H), 8.52 (d, J = 8.1 Hz, 1H), 8.30 (s, 1H), 7.75 (s, 1H), 7.53 (dd, J = 8.2, 1.4 Hz, 1H),
7.19 (s, 1H), 4.03 – 3.68 (m, 7H), 3.53 – 3.40 (m, 2H), 3.11 – 2.95 (m, 6H), 2.74 (q, J
= 7.5 Hz, 2H), 1.85 (s, 6H), 1.26 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
180.7, 169.0, 159.3, 154.5, 146.4, 137.8, 135.7, 128.4, 127.7, 127.5, 125.5, 122.7,
120.2, 116.3, 116.0, 111.0, 105.8, 69.6, 68.1, 55.2, 52.9, 52.0, 45.9, 44.6, 42.4, 36.7,
30.8, 23.2, 14.4. MS (ESI) m/z 512 [M + H]+. HRMS: calcd for C30H34N5O3 [M + H]+,
16

ACCEPTED MANUSCRIPT
512.2662; found 512.2668.
5.3. Synthesis of 9-ethyl-6,6-dimethyl-11-oxo-8-(1,2,3,6-tetrahydropyridin-4-yl)-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (7b)
To a solution of 6b (300 mg, 0.68 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (422 mg, 1.36 mmol) and
Pd(PPh3)4 (158 mg, 0.136 mmol) in dioxane (4.5 mL) was added Na2CO3 (2 M in
water, 1.5 mL). The mixture was degassed with nitrogen for 5 min and then heated at
110 °C under microwave for 1.5 h. After the completion of the reaction, the mixture
was diluted with EtOAc (20 mL) and filtered. The filtrate was evaporated in vacuum
to afford a crude product, which was purified by chromatograph (DCM/MeOH)
followed by treatment with TFA (100 equiv) in DCM at room temperature overnight.
The mixture was concentrated in vacuum and the residue was basified with sodium
bicarbonate until pH > 7. After extraction with DCM, the combined organic phase
was washed with brine, dried over sodium sulfate, filtered, and evaporated to afford
1
the title product 7b as a pale yellow solid (203 mg, 75% yield): H NMR (300 MHz,
CDCl3 + CD3OD) δ 8.37 (d, J = 8.1 Hz, 1H), 8.14 (s, 1H), 7.70 (s, 1H), 7.44 (dd, J =
8.2, 0.8 Hz, 1H, 1H), 7.24 (s, 1H), 5.63 – 5.56 (m, 1H), 3.48 – 3.42 (m, 2H), 3.09 –
3.01 (m, 2H), 2.64 (q, J = 7.6 Hz, 2H), 2.33 – 2.24 (m, 2H), 1.68 (s, 6H), 1.19 (t, J =
7.5 Hz, 3H).
5.4. Synthesis of 9-ethyl-6,6-dimethyl-8-(1-(morpholine-3-carbonyl)-1,2,3,6-tetra
hydropyridin-4-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (9a)
This compound was prepared as a white solid in 76% overall yield from 7b and
17

ACCEPTED MANUSCRIPT
4-(tert-butoxycarbonyl)morpholine-3-carboxylic acid by following a procedure
similar to that of preparation of 8b. 1H NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 8.52
(d, J = 8.2 Hz, 1H), 8.27 (s, 1H), 7.78 (s, 1H), 7.55 (dd, J = 8.2, 1.3 Hz, 1H), 7.29 (s,
1H), 5.71 – 5.62 (m, 1H), 4.41 – 4.29 (m, 1H), 4.23 – 4.12 (m, 1H), 4.06 – 3.96 (m,
2H), 3.89 – 3.68 (m, 3H), 3.51 – 3.42 (m, 2H), 3.11 – 2.99 (m, 2H), 2.66 (q, J = 7.6
13
Hz, 2H), 2.59 – 2.38 (m, 2H), 1.84 (s, 6H), 1.22 (t, J = 7.5 Hz, 3H). C NMR (126
MHz, CDCl3) δ 180.7, 169.3, 159.4, 145.9, 145.0, 140.4 and 140.3 (1C), 138.6, 136.7
and 135.7 (1C), 130.9, 128.4, 126.7 and 126.6 (1C), 126.0 and 125.8 (1C), 125.6,
123.1 and 121.6 (1C), 122.8, 120.2, 116.0, 111.1, 106.0, 69.6 and 69.4 (1C), 68.1,
55.9 and 55.5 (1C), 44.6, 42.5 and 42.2 (1C), 39.2, 36.5, 31.2 and 30.2 (1C), 30.7,
25.7, 15.5 and 15.4 (1C). MS (ESI) m/z 509 [M + H]+. HRMS: calcd for C31H33N4O3
[M + H]+, 509.2553; found 509.2567.
5.5. Synthesis of 9-ethyl-6,6-dimethyl-11-oxo-8-(1-(tetrahydro-2H-pyran-4-carbonyl)
-1,2,3,6-tetrahydropyridin-4-yl)-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile
(9b)
This compound was prepared as a white solid in 84% yield from 7b and
tetrahydro-2H-pyran-4-carboxylic acid by following a procedure similar to that of
1
preparation of 8a (without the last deprotection procedure). H NMR (300 MHz,
CDCl3 + CD3OD) δ 8.45 (d, J = 8.1 Hz, 1H), 8.22 (s, 1H), 7.72 (s, 1H), 7.48 (d, J =
8.1 Hz, 1H), 7.31 – 7.23 (m, 1H), 5.75 – 5.59 (m, 1H), 4.31 – 4.14 (m, 2H), 4.08 –
3.95 (m, 2H), 3.88 – 3.70 (m, 2H), 3.56 – 3.41 (m, 2H), 2.89 – 2.77 (m, 1H), 2.65 (q,
J = 7.2 Hz, 2H), 2.50 – 2.36 (m, 2H), 2.01 – 1.87 (m, 2H), 1.75 (s, 6H), 1.70 – 1.58
18

ACCEPTED MANUSCRIPT
13
(m, 2H), 1.23 (t, J = 7.3 Hz, 3H). C NMR (126 MHz, CDCl3 + CD3OD) δ 180.7,
173.5, 159.8, 145.8, 145.2, 140.2 and 140.1 (1C), 138.7, 136.6 and 135.9 (1C), 130.9
and 130.8 (1C), 128.30, 126.6 and 126.5 (1C), 126.0 and 125.7 (1C), 125.2, 123.4 and
121.7 (1C), 122.5, 120.4, 116.0, 110.6, 105.5, 67.3, 44.7, 42.5 and 42.3 (1C), 39.1,
38.4 and 37.9 (1C), 36.4, 31.3, 30.3, 29.0 and 28.8 (1C), 25.7, 15.5 and 15.4 (1C). MS
(ESI) m/z 506 [M – H]-. HRMS: calcd for C32H32N3O3 [M – H]-, 506.2444; found
506.2430.
5.6. Synthesis of 8-(1-(azetidine-3-carbonyl)-1,2,3,6-tetrahydropyridin-4-yl)-9-ethyl-
6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (9c)
This compound was prepared as a white solid in 62% overall yield from 7b and
1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid by following a procedure similar
to that of preparation of 8a. 1H NMR (400 MHz, CDCl3 + CD3OD) δ 8.41 (d, J = 8.0
Hz, 1H), 8.18 (s, 1H), 7.72 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.24 – 7.19 (m, 1H), 5.67
– 5.52 (m, 1H), 4.23 – 4.15 (m, 1H), 4.07 – 3.88 (m, 3H), 3.86 – 3.74 (m, 2H), 3.66 (t,
J = 7.6 Hz, 2H), 3.52 – 3.46 (m, 1H), 2.62 (t, J = 7.5 Hz, 2H), 2.41 – 2.32 (m, 2H),
1.71 (s, 6H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3+ CD3OD) δ 180.7,
170.9, 160.0, 145.7, 145.2 , 140.1 and 140.0 (1C), 138.5, 136.6 and 136.0 (1C), 130.9,
128.3, 126.5, 125.9 and 125.7 (1C), 125.1, 123.0 and 121.4 (1C), 122.5, 120.3, 116.0,
110.6, 105.4, 48.9 and 48.8 (1C), 44.3, 42.2, 39.03, 36.6 and 36.4 (1C), 36.4, 31.0 and
30.1 (1C), 30.2, 25.7, 15.4. MS (ESI) m/z 479 [M + H]+. HRMS: calcd for
C30H31N4O2 [M + H]+, 479.2447; found 479.2455.
5.7. General procedure for the synthesis of compounds 10a, 10b and 12
19

ACCEPTED MANUSCRIPT
A mixture of 7a (0.2 mmol), an appropriate bromide or methanesulfonate (0.4
mmol), K2CO3 (0.6 mmol), DIEA (0.4 mmol) and KI (0.04 mmol) in 4 mL DMF was
heated at 55 °C to 80 °C for 2 days until the completion of the reaction. The mixture
was treated with EtOAc (100 mL), washed with brine and then dried over with
Na2SO4, filtered, and evaporated. The crude product was purified by chromatograph
(CHCl3/MeOH) to give the corresponding products.
5.7.1. (S)-9-Ethyl-6,6-dimethyl-11-oxo-8-(4-((5-oxopyrrolidin-2-yl)methyl)piperazin-1
-yl)-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (10a)
This compound was obtained as pale yellow solid in 60% yield from 7a and
1
(S)-5-(bromomethyl)pyrrolidin-2-one. H NMR (400 MHz, CDCl3 + CD3OD) δ 8.46
(d, J = 8.4 Hz, 1H), 8.23 (s, 1H), 7.73 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.15 (s, 1H),
3.93 – 3.81 (m, 1H), 3.05 – 2.93 (m, 4H), 2.82 – 2.72 (m, 2H), 2.69 (q, J = 7.5 Hz,
2H), 2.62 – 2.51 (m, 2H), 2.49 – 2.42 (m, 2H), 2.40 – 2.35 (m, 2H), 2.27 – 2.19 (m,
1H), 1.76 (s, 6H), 1.72 – 1.65 (m, 1H), 1.28 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz,
CDCl3 + CD3OD) δ 180.9, 178.3, 159.8, 155.0, 146.6, 137.4, 135.9, 128.3, 127.2,
127.1, 125.1, 122.5, 120.4, 116.0, 115.9, 110.6, 105.3, 64.3, 52.0, 51.3, 36.6, 30.4,
30.1, 25.0, 23.2, 14.4. MS (ESI) m/z 496 [M + H]+. HRMS: calcd for C30H34N5O2 [M
+ H]+, 496.2713; found 496.2723.
5.7.2.
9-Ethyl-8-(4-((3-(hydroxymethyl)oxetan-3-yl)methyl)piperazin-1-yl)-6,6-di
methyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (10b)
This compound was obtained as ivory white solid in 45% yield from 7a and
(3-(bromomethyl)oxetan-3-yl)methanol. 1H NMR (300 MHz, CDCl3) δ 10.78 (s, 1H),
20

ACCEPTED MANUSCRIPT
8.52 (d, J = 7.8 Hz, 1H), 8.28 (s, 1H), 7.72 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.16 (s,
1H), 4.71 – 4.52 (m, 2H), 4.52 – 4.38 (m, 2H), 4.22 (s, 2H), 3.13 – 2.88 (m, 6H), 2.82
– 2.54 (m, 6H), 1.83 (s, 6H), 1.26 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
180.7, 159.3, 154.8, 146.4, 137.6, 135.7, 128.4, 127.4, 127.3, 125.4, 122.7, 120.2,
115.9, 115.8, 111.0, 105.9, 78.3, 70.0, 65.6, 54.4, 52.1, 42.3, 36.7, 30.8, 23.2, 14.3.
MS (ESI) m/z 499 [M + H]+. HRMS: calcd for C30H35N4O3 [M + H]+, 499.2709;
found 496.2714.
5.7.3. 9-Ethyl-6,6-dimethyl-11-oxo-8-(4-((tetrahydro-2H-pyran-4-yl)methyl)piperazin-
1-yl)-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (12)
This compound was obtained as white solid in 58% yield from 7a and
1
4-(bromomethyl)tetrahydro-2H-pyran. H NMR (300 MHz, CDCl3) δ 10.12 (s, 1H),
8.53 (d, J = 8.1 Hz, 1H), 8.28 (s, 1H), 7.73 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.22 (s,
1H), 4.05 – 3.96 (dd, J = 11.1, 3.3 Hz, 2H), 3.42 (t, J = 11.4 Hz, 2H), 3.11 – 2.99 (m,
4H), 2.72 (d, J = 7.6 Hz, 2H), 2.69 – 2.53 (m, 4H), 2.31 (d, J = 6.6 Hz, 2H), 1.86 –
13
1.70 (m, 9H), 1.38 – 1.23 (m, 5H). C NMR (126 MHz, CDCl3 + CD3OD) δ 180.9,
159.5, 155.4, 146.4, 137.4, 135.7, 128.4, 127.2, 126.9, 125.2, 122.6, 120.4, 115.9 (2),
110.8, 105.5, 67.9, 64.9, 54.0, 52.1, 36.6, 32.5, 31.7, 30.6, 23.2, 14.4. MS (ESI) m/z
497 [M + H]+. HRMS: calcd for C31H37N4O2 [M + H]+, 497.2917; found 497.2930.
5.8. Synthesis of 9-ethyl-6,6-dimethyl-11-oxo-8-(1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,2,3,6-tetrahydropyridin-4-yl)-6,11-dihydro-5H-benzo[b]carbazole-3-carbon
itrile (13)
This compound was prepared as a white solid in 67% yield from 7b and
21

ACCEPTED MANUSCRIPT
4-(bromomethyl)tetrahydro-2H-pyran by following a procedure similar to that of
1
preparation of 12. H NMR (300 MHz, CDCl3) δ 10.77 – 10.49 (m, 1H), 8.52 (d, J =
8.1 Hz, 1H), 8.26 (s, 1H), 7.74 (s, 1H), 7.54 (dd, J = 8.0, 1.2 Hz, 1H), 7.38 (s, 1H),
5.63 (s, 1H), 4.08 – 3.93 (m, 2H), 3.44 (t, J = 11.7 Hz, 2H), 3.20 – 3.17 (m, 2H), 2.77
– 2.54 (m, 4H), 2.48 – 2.39 (m, 2H), 2.37 (d, J = 6.9 Hz, 2H), 1.93 – 1.77 (m, 9H),
13
1.41 – 1.29 (m, 2H), 1.22 (t, J = 7.5 Hz, 3H). C NMR (126 MHz, CDCl3) δ 181.1,
159.9, 147.2, 145.0, 140.6, 136.6, 135.8, 130.4, 128.5, 126.4, 126.2, 125.5, 122.7,
120.3, 116.1, 111.0, 105.8, 68.0, 64.9, 53.5, 50.5, 36.6, 32.7, 31.8, 31.4, 30.6, 25.6,
15.5. MS (ESI) m/z 494 [M + H]+. HRMS: calcd for C32H36N3O2 [M + H]+, 494.2808;
found 494.2810.
5.9. Synthesis of 8-(2,5-diazabicyclo[2.2.1]heptan-2-yl)-9-ethyl-6,6-dimethyl-11-oxo-
6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (7c)
To a solution of 6b (150 mg, 0.34 mmol), tert-butyl 2,5-diazabicyclo[2.2.1]
heptane-2-carboxylate (109 mg, 0.51 mmol), Pd2(dba)3 (16 mg, 0.017 mmol) and
S-Phos (28 mg, 0.068 mmol) in dry 1,4-dioxane (8.5 mL) was added NaHMDS (2 M
in THF, 0.6 mL) under a nitrogen atmosphere. The mixture was purged with nitrogen
for 5 min, and then heated at 60 oC until the completion of the reaction. After cooled
to room temperature, the mixture was diluted with DCM (20 mL) and filtered. The
filtrate was concentrated in vacuum and purified by chromatograph (CHCl3/MeOH) to
1
give the coupling intermediate as pale yellow solid (103 mg, 59% yield): H NMR
(400 MHz, CDCl3) δ 11.60 – 11.40 (m, 1H), 8.52 (d, J = 7.6 Hz, 1H), 8.19 (s, 1H),
7.80 – 7.64 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 6.83 (s, 1H), 4.72 – 4.54 (m, 1H), 4.53
22

ACCEPTED MANUSCRIPT
– 4.37 (m, 1H), 3.88 – 3.75 (m, 1H), 3.74 – 3.21 (m, 3H), 2.85 – 2.53 (m, 2H), 2.09 –
1.97 (m, 2H), 1.86 (s, 6H), 1.59 – 1.40 (m, 9H), 1.23 – 1.08 (m, 3H). Subsequent
deprotection of the intermediate with TFA (100 equiv) in DCM at room temperature
afforded compound 7c as a yellow solid (69 mg, 49% yield in two steps).
5.10. Synthesis of 9-ethyl-6,6-dimethyl-11-oxo-8-(5-((tetrahydro-2H-pyran-4-yl)
methyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-6,11-dihydro-5H-benzo[b]carbazole-3-c
arbonitrile (11)
This compound was prepared as a white solid in 54% yield from 7c and
4-(bromomethyl)tetrahydro-2H-pyran by following a procedure similar to that of
1
preparation of 12. H NMR (400 MHz, CDCl3) δ 11.16 (s, 1H), 8.53 (d, J = 8.0 Hz,
1H), 8.18 (s, 1H), 7.70 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 6.82 (s, 1H), 4.25 (s, 1H),
4.01 – 3.89 (m, 2H), 3.62 – 3.50 (m, 2H), 3.45 – 3.32 (m, 3H), 3.09 (d, J = 8.4 Hz,
1H), 2.89 (d, J = 9.6 Hz, 1H), 2.67 (q, J = 7.4 Hz, 2H), 2.52 – 2.37 (m, 2H), 2.02 –
1.96 (m, 1H), 1.93 – 1.84 (m, 7H), 1.73 – 1.57 (m, 3H), 1.31 – 1.22 (m, 2H), 1.15 (t, J
= 7.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 181.1, 159.6, 151.2, 147.0, 135.8,
131.0, 128.8, 128.5, 125.1, 122.8, 122.5, 120.4, 116.1, 111.8, 110.8, 105.3, 68.0, 67.9,
62.6, 60.8, 60.6, 58.2, 55.2, 36.7, 35.7, 35.0, 31.6, 31.5, 31.0, 30.9, 26.4, 13.7. MS
(ESI) m/z 509 [M + H]+. HRMS: calcd for C32H37N4O2 [M + H]+, 509.2917; found
509.2924.
5.11. Synthesis of 9-ethyl-6,6-dimethyl-8-(4-(methyl(oxetan-3-yl)amino)piperidin-1-yl)
-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (15f)
Compound 7d (130 mg, 0.3 mmol) was dissolved in dry THF/CH3COOH (6
23

ACCEPTED MANUSCRIPT
mL/0.2 mL), and oxetan-3-one (140 ꢁL, 2.4 mmol) was added at room temperature.
The mixture was stirred for 1 h, then NaBH(OAc)3 (318 mg, 1.5 mmol) was added by
o
portions. The solution was heated at 40 C until the completion of the reaction. After
cooled to room temperature, the mixture was evaporated and then water was added.
The water layer was extracted with DCM and the combined mixture was washed with
brine, dried over sodium sulfate, filtered, and concentrated. The crude product was
purified by chromatograph (CHCl3/MeOH) to give the title product as white solid (68
1
mg, 46% yield): H NMR (300 MHz, CDCl3 + CD3OD) δ 8.43 (d, J = 8.4 Hz, 1H),
8.20 (s, 1H), 7.69 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.12 (s, 1H), 4.73 – 4.59 (m, 4H),
4.00 (q, J = 6.9 Hz, 1H), 3.24 (d, J = 11.4 Hz, 2H), 2.76 – 2.62 (m, 4H), 2.48 – 2.38
13
(m, 1H), 2.24 (s, 3H), 1.80 – 1.60 (m, 10H), 1.26 (t, J = 7.5 Hz, 3H). C NMR (126
MHz, CDCl3 + CD3OD) δ 181.0, 159.8, 155.6, 146.5, 137.3, 135.9, 128.3, 127.0,
126.8, 125.0, 122.4, 120.4, 116.0, 115.8, 110.6, 105.2, 76.3, 59.7, 56.1, 52.3, 36.6,
32.9, 30.4, 28.4, 23.1, 14.3. MS (ESI) m/z 483 [M + H]+. HRMS: calcd for
C30H35N4O2 [M + H]+, 483.2760; found 483.2756.
5.12. Synthesis of 8-(4-(azetidine-3-carbonyl)piperazin-1-yl)-6,6-dimethyl-11-oxo-
6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (14)
This compound was prepared as a white solid in 74% yield in two steps from 7f
and 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid by following a procedure
similar to that of preparation of 8a. 1H NMR (300 MHz, CDCl3+ CD3OD) δ 8.32 (dd,
J = 8.3, 0.8 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 7.67 (dd, J = 1.4, 0.8 Hz, 1H), 7.39 (dd,
J = 8.1, 1.5 Hz, 1H), 6.93 – 6.83 (m, 2H), 3.92 – 3.86 (m, 2H), 3.78 – 3.66 (m, 3H),
24

ACCEPTED MANUSCRIPT
13
3.59 (t, J = 8.0 Hz, 2H), 3.45 – 3.35 (m, 2H), 3.28 – 3.22 (m, 4H), 1.66 (s, 6H). C
NMR (126 MHz, CDCl3+ CD3OD) δ 180.4, 170.4, 159.3, 153.3, 149.7, 135.9, 128.4,
128.2, 125.0, 124.0, 122.4, 120.4, 116.0, 114.3, 111.4, 110.4, 105.2, 48.8, 48.2, 47.8,
44.8, 41.6, 36.9, 35.8, 30.5. MS (ESI) m/z 454 [M + H]+. HRMS: calcd for
C27H28N5O2 [M + H]+, 454.2243; found 454.2251.
5.13. General procedure for the synthesis of compounds 15b, 15d and 15e
These compounds were prepared by treating compound 7e with an appropriate
bromide or p-toluenesulfonate following a procedure similar to that of preparation of
12. The obtained intermediate was submitted to the hydride reduction with 10% Pd/C
(60% w/w) under hydrogen gas at room temperature by following a standard
hydrogenation procedure. The crude product was purified by chromatograph
(CHCl3/MeOH) to give the corresponding product.
5.13.1. 6,6-Dimethyl-11-oxo-8-(1-((tetrahydro-2H-pyran-4-yl)methyl)piperidin-4-yl)-
6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (15b)
This compound was obtained as white solid in 47% overall yield from 7e and
4-(bromomethyl)tetrahydro -2H-pyran. 1H NMR (500 MHz, CDCl3 + CD3OD) δ 8.47
(d, J = 8.5 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 7.72 (s, 1H), 7.50 (dd, J = 8, 1 Hz, 1H),
7.47 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 3.98 (dd, J = 11.3, 3.3 Hz, 2H), 3.41 (t, J = 11.0
Hz, 2H), 3.07 – 2.98 (m, 2H), 2.66 – 2.58 (m, 1H), 2.24 (d, J = 7.0 Hz, 2H), 2.11 –
2.02 (m, 2H), 1.90 – 1.80 (m, 5H), 1.78 (s, 6H), 1.70 (d, J = 13.0 Hz, 2H), 1.30 (qd, J
13
= 12.5, 4.0 Hz, 2H). C NMR (126 MHz, CDCl3 + CD3OD) δ 180.8, 159.7, 151.4,
147.9, 135.8, 130.0, 128.3, 126.9, 126.1, 125.3, 124.2, 122.6, 120.3, 116.0, 110.7,
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ACCEPTED MANUSCRIPT
105.6, 68.0, 65.4, 54.8, 43.1, 36.8, 33.3, 32.7, 31.8, 30.5. MS (ESI) m/z 468 [M + H]+.
HRMS: calcd for C30H34N3O2 [M + H]+, 468.2651; found 468.2639.
5.13.2. 6,6-Dimethyl-8-(1-(oxetan-3-ylmethyl)piperidin-4-yl)-11-oxo-6,11-dihydro-5H
-benzo[b]carbazole-3-carbonitrile (15d)
This compound was obtained as white solid (in 30% overall yield) from 7e and
oxetan-3-ylmethyl 4-methylbenzenesulfonate. 1H NMR (400 MHz, CDCl3 + CD3OD)
δ 8.46 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.71 (s, 1H), 7.50 (d, J = 8.0 Hz,
1H), 7.46 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 4.84 (dd, J = 7.6, 6.0 Hz, 2H), 4.46 (t, J =
6.2 Hz, 2H), 3.36 – 3.22 (m, 1H), 2.98 – 2.86 (m, 2H), 2.77 (d, J = 7.2 Hz, 2H), 2.66
– 2.57 (m, 1H), 2.15 – 2.08 (m, 2H), 1.91 – 1.80 (m, 4H), 1.78 (s, 6H). 13C NMR (126
MHz, CDCl3 + CD3OD) δ 180.8, 160.1, 149.7, 148.3, 136.0, 130.3, 128.3, 126.9,
125.9, 125.1, 124.2, 122.4, 120.3, 116.2, 110.4, 105.4, 76.3, 61.5, 53.8, 41.8, 36.8,
32.4, 31.8, 30.2. MS (ESI) m/z 440 [M + H]+. HRMS: calcd for C28H30N3O2 [M + H]+,
440.2338; found 440.2344.
5.13.3. 6,6-Dimethyl-11-oxo-8-(1-((tetrahydrofuran-3-yl)methyl)piperidin-4-yl)-6,11-
dihydro-5H-benzo[b]carbazole-3-carbonitrile (15e)
This compound was obtained as white solid in 32% overall yield from 7e and
(tetrahydrofuran-3-yl)methyl 4-methylbenzenesulfonate. 1H NMR (400 MHz, CDCl3)
δ 11.13 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 7.58 –
7.46 (m, 2H), 7.34 (d, J = 8.4 Hz, 1H), 3.99 – 3.87 (m, 2H), 3.79 (q, J = 7.7 Hz, 1H),
3.59 (dd, J = 8.4, 6.0 Hz, 1H), 3.22 – 3.04 (m, 2H), 2.71 – 2.62 (m, 1H), 2.60 – 2.44
(m, 3H), 2.27 – 2.06 (m, 3H), 2.01 – 1.88 (m, 4H), 1.86 (s, 6H), 1.72 – 1.64 (m, 1H).
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ACCEPTED MANUSCRIPT
13C NMR (126 MHz, CDCl3) δ 181.0, 160.1, 148.1, 136.0, 130.1, 128.4, 126.9, 126.2,
125.4, 124.4, 122.6, 120.3, 116.3, 110.8, 105.7, 72.5, 67.8, 621, 54.7, 54.2, 42.8, 37.0,
36.8, 33.0, 31.1, 30.6. MS (ESI) m/z 454 [M + H]+. HRMS: calcd for C29H32N3O2 [M
+ H]+, 454.2495; found 454.2485.
5.14. Synthesis of 8-(1-((4-hydroxytetrahydro-2H-pyran-4-yl)methyl)piperidin-4-yl)-
6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (15c)
A mixture of 7e (130 mg, 0.35 mmol), 1,6-dioxaspiro[2.5]octane (80 mg, 0.7
o
mmol) and DIEA (122 ꢁL, 0.7 mmol) in EtOH (4 mL) was heated at 70 C until the
completion of the reaction. After being cooled to room temperature, the solvent was
removed in vacuum. The residue was purified by chromatograph (CHCl3/MeOH) to
afford an intermediate, which was then hydrogenated with H2 to yield the title product
as white solid (63 mg, 37% yield in two steps): 1H NMR (400 MHz, CDCl3 + CD3OD)
δ 8.46 (d, J = 8.0 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.50 (d, J = 8.0 Hz,
1H), 7.44 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 3.84 – 3.73 (m, 4H), 3.07 – 2.91 (m, 2H),
2.66 – 2.57 (m, 1H), 2.56 – 2.45 (m, 2H), 2.42 – 2.34 (m, 2H), 1.89 – 1.80 (m, 4H),
1.77 (s, 6H) 1.66 – 1.58 (m, 2H), 1.54 – 1.45 (m, 2H). 13C NMR (126 MHz, CDCl3 +
CD3OD) δ 180.8, 159.7, 150.8, 148.0, 135.9, 130.2, 128.3, 126.9, 125.9, 125.2, 124.1,
122.5, 120.4, 116.0, 110.6, 105.5, 68.3, 67.6, 63.9, 56.9, 42.4, 36.7, 36.7, 33.7, 30.3.
MS (ESI) m/z 484 [M + H]+. HRMS: calcd for C30H34N3O3 [M + H]+, 484.2600;
found 484.2592.
5.15. Synthesis of 6,6-dimethyl-8-(1-(morpholine-3-carbonyl)piperidin-4-yl)-11-oxo-
6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (15a)
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This compound was prepared as white solid in 45% overall yield from 7e and
N-Boc-morpholine-3-carboxylic acid by following a procedure similar to that of
1
preparation of 8b and then hydrogenated. H NMR (300 MHz, CDCl3 + CD3OD) δ
8.45 (d, J = 8.1 Hz, 1H), 8.31 (d, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.48 (d, J = 8.1 Hz,
1H), 7.40 (s, 1H), 7.31 – 7.23 (m, 1H), 4.83 – 4.67 (m, 1H), 4.18 – 4.04 (m, 1H), 4.02
– 3.88 (m, 2H), 3.84 – 3.75 (m, 1H), 3.50 – 3.32 (m, 2H), 3.28 – 3.13 (m, 1H), 3.08 –
2.83 (m, 3H), 2.79 – 2.60 (m, 1H), 2.08 – 1.88 (m, 2H), 1.77 (s, 6H), 1.74 – 1.50 (m,
2H). 13C NMR (126 MHz, CDCl3+ CD3OD) δ 180.5, 168.6 and 168.5 (1C), 159.6 and
159.5 (1C), 149.4 and 149.3 (1C), 148.10, 135.9, 130.5, 128.2, 127.1, 126.1 and 125.7
(1C), 125.2, 124.1 and 123.9 (1C), 122.6, 120.3, 116.0, 110.6, 105,6, 69.6 and 69.3
(1C), 67.8, 54.9 and 54.8 (1C), 46.1 and 45.9 (1C), 44.2 and 44.1 (1C), 43.1, 42.8 and
42.5 (1C), 36.7, 34.0 and 33.6 (1C), 32.8 and 32.6 (1C), 30.4 and 30.3 (1C). MS (ESI)
m/z 483 [M + H]+. HRMS: calcd for C29H31N4O3 [M + H]+, 483.2396; found
483.2408.
5.16. Synthesis of 6,6-dimethyl-11-oxo-8-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-
4-yl)-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile (17)
This compound was prepared as a white solid in 65% yield from 6c and 16a by
following a Suzuki coupling reaction condition similar to that of preparation of 7b. 1H
NMR (400 MHz, CDCl3 + CD3OD) δ 8.39 (d, J = 8.0 Hz, 1H), 8.29 (d, J = 8.4 Hz,
1H), 7.84 (s, 1H), 7.82 (s, 1H), 7.75 – 7.70 (m, 1H), 7.65 (d, J = 1.2 Hz, 1H), 7.51 (dd,
J = 8.0, 1.6 Hz, 1H), 7.46 (dd, J = 8.2, 1.4 Hz, 1H), 4.42 – 4.28 (m, 1H), 4.11 – 4.04
(m, 2H), 3.53 (td, J = 11.2, 2.8 Hz, 2H), 2.15 – 2.02 (m, 4H), 1.76 (s, 6H). 13C NMR
28