Discovery of carbazole carboxamides as novel RORγt inverse agonists

Article abstract of DOI:10.1016/j.ejmech.2018.02.050

A novel series of carbazole carboxamides was discovered as potent RORγt inverse agonists using a scaffold hybridization strategy. Structure-activity relationship exploration on the amide linker, carbazole ring and arylsulfone moiety of the hybrid amide 3a led to identification of potent RORγt inverse agonists. Compound 6c was found to have a good RORγt activity with an IC₅₀ of 58.5 nM in FRET assay, and reasonable inhibitory activity in mouse Th17 cell differentiation assay (58.8% inhibition at 0.3 μM). The binding mode of carbazole carboxamides in RORγt ligand binding domain was discussed.

Full text of DOI:10.1016/j.ejmech.2018.02.050

European Journal of Medicinal Chemistry 148 (2018) 465e476
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of carbazole carboxamides as novel RORgt inverse agonists
Yafei Huang, Mingcheng Yu, Nannan Sun, Ting Tang, Fazhi Yu, Xiaoxia Song, Qiong Xie*,
Wei Fu, Liming Shao, Yonghui Wang**
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of carbazole carboxamides was discovered as potent RORgt inverse agonists using a
Received 21 December 2017
Received in revised form
29 January 2018
Accepted 14 February 2018
Available online 16 February 2018
scaffold hybridization strategy. Structure-activity relationship exploration on the amide linker, carbazole
ring and arylsulfone moiety of the hybrid amide 3a led to identification of potent ROR t inverse agonists.
Compound 6c was found to have a good ROR t activity with an IC50 of 58.5 nM in FRET assay, and
reasonable inhibitory activity in mouse Th17 cell differentiation assay (58.8% inhibition at 0.3 M). The
binding mode of carbazole carboxamides in ROR t ligand binding domain was discussed.
© 2018 Elsevier Masson SAS. All rights reserved.
g
g
m
g
Keywords:
Carbazole carboxamides
RORgt inverse agonists
Th17 cells
Autoimmune diseases
Binding mode
1. Introduction
and was therefore employed as a unique tool compound for both
in vitro and in vivo biological and immunological studies [10,27].
However, compound 1 showed some developability issues which
prevented it to be further progressed as a clinical candidate. The
developability issues could be associated with the compound 1's
high molecular weight (MW ¼ 532) and high hydrophobicity
(cLogP ¼ 5.23), mainly attributed to its biaryl moiety [28]. Thus, one
of our lead optimization goals for the N-aryl amide series is to
identify novel structural moieties to improve compounds' devel-
opability profiles.
Th17 cells, a lineage of CD4þ effector T cells characterized by the
production of IL-17A and IL-17F, are pathogenic in autoimmune
diseases such as multiple sclerosis and psoriasis [1e4]. The retinoic
acid receptor-related orphan receptor-gamma-t (ROR
nuclear receptor of the ROR family that has been implicated in the
differentiation and function of Th17 cells [5,6]. Therefore, ROR
inhibitors have potential utility in reducing the activity of Th17 cells
and can be developed as therapeutic agents for the treatment of
Th17-mediated autoimmune diseases [7e14].
gt) is a key
gt
Recently, Takeda Pharmaceutical Company disclosed in a PCT
Started with digoxin [15], SR1001 [16] and ursolic acid [17], a
few small molecule RORgt inhibitors have been reported in the
literature [18e21]. Previously, we reported the discovery of several
N-aryl amides such as thiazole/thiophene ketone amides [22],
thiazole ether amides [23], indole amides [24] and biaryl amides
patent a series of carbazole-containing compounds as ROR
hibitors [29]. The representative compound 2 (example 14 in
Takeda's patent) showed good ROR
FRET assay). However, the compound 2 has much higher cLogP
(6.44) although MW is reasonable (473). Structural superimposi-
gt in-
g
t activity (IC50 ¼ 50.6 nM in our
[25,26] as ROR
GSK805) exhibited excellent ROR
FRET assay), strong inhibition of Th17 cell differentiation, good oral
bioavailability and CNS penetration, and remarkable efficacy in
mouse experimental autoimmune encephalomyelitis (EAE) model,
g
t inverse agonists (Fig. 1). Biaryl amide 1 (or
tion of compounds 1 with 2 in ROR
indicated that both carbazole and biaryl moieties occupied the
same hydrophobic pocket of the ROR t LBD (Fig. 2a) [24]. Stimu-
gt ligand binding domain (LBD)
g
t activity (IC50 ¼ 34.0 nM in our
g
lated by the docking results along with relatively lower MW and
cLogP of carbazole structural moiety, we had a hypothesis that
replacing the biaryl moiety in 1 with the carbazole moiety in 2,
resulting in hybrid compounds such as 3a, could reduce MW and
cLogP of the amides (3a: MW ¼ 421, cLogP ¼ 4.16) while possibly
* Corresponding author.
** Corresponding author.
maintaining the RORgt activity based on overlay results of 3a with 1
E-mail addresses: qxie@fudan.edu.cn (Q. Xie), yonghuiwang@fudan.edu.cn
(Y. Wang).
(Figs. 2b and 3). To test this hypothesis, we prepared the hybrid
https://doi.org/10.1016/j.ejmech.2018.02.050
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

466
Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
Fig. 1. N-Aryl amide-based RORgt inverse agonists.
Fig. 2. (a) Structural superimposition of the docked poses of 1 (salmon stick) with 2 (yellow stick) in RORgt LBD; (b) Structural superimposition of the docked poses of 1 (salmon
stick) with 3a (cyan stick) in ROR
g
t LBD (PDB: 4NIE). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
c
c
c
Fig. 3. Hybridization strategy for the design of novel ROR
g
t inhibitors.

Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
467
compound 3a and tested it in the ROR
the hybrid carbazole amide compound 3a showed a decent ROR
activity with an IC50 of 170 nM and could be employed as new
chemical starting point for further optimization. In this paper, we
reported the design, synthesis, structure-activity relationship (SAR)
and binding mode studies of a series of carbazole-containing
g
FRET assay. To our delight,
of 12 with amines 14 obtained from reduction of cyanides 13
afforded the desired carbazolyl amides (3b, 5a-5h, 6a-6d, 7a-7d).
Reagents and conditions: (a1) Pd(OAc)2, rac-BINAP, K2CO3,
toluene; (a2) Pd(OAc)2, rac-BINAP, Cs2CO3, toluene; (b1) Pd(OAc)2,
HOAc, 130 ꢀC; (b2) PdCl2(PPh3)2, NaOAc$3H2O, DMF, microwave; (c)
NaH; (d) KOH, EtOH/H2O, 100 ꢀC; (e) Raney Ni, H2, MeOH, rt; (f)
HATU, DIEA, DCM, rt.
gt
compounds as novel RORgt inhibitors.
For the non-carbazolyl carboxamides analogues (4a-4d)
(Scheme 2), the synthesis started with Fischer indole synthesis
from 4-hydrazinylbenzoic acid and ketones to form indole-5-
carboxylic acids 15, which were protected as esters 16, then con-
verted to 17 upon ethylation with ethyl iodide. Condensation of 18,
the hydrolysis product of 17, with amine 14a afforded compounds
4a-4d.
Reagents and conditions: (a) HCl, 1,4-dioxane, reflux; (b) MeOH,
H2SO4; (c) NaH, EtI, DMF, 0ꢀC-rt; (d) LiOH, EtOH/H2O, rt; (e) HATU,
DIEA, DCM, rt.
2. Result and discussion
2.1. Compound design
Since Takeda's compounds such as 2 have “longer” linker be-
tween the left-hand side (LHS) carbazole moiety and the right-
hand side (RHS) aryl moiety than that in the biaryl amides such
as 1, we first designed a few compounds (3a-3e) with different
linkers, trying to identify a suitable one to connect the LHS carba-
zole moiety and RHS arylsulfone moiety. Then, with the best linker
(-CONHCH2-) identified, we designed some new compounds (4a-
4d, 5a-5h, 6a-6d, 7a-7d) with the LHS carbazole moiety and RHS
arylsulfone moiety modified, respectively, trying to optimize the
Synthetic procedures of 3a-3e were described in the supporting
information.
2.3. Structure-activity relationship
carboxamides for better RORgt activity (Fig. 4).
Firstly,
carbazole and arylsulfone moieties were designed, synthesized and
evaluated in the ROR FRET assay (Table 1). Compound 3b with a
reversed amide linker relative to 3a showed essentially the same
ROR t activity. Overlay of 3a and 3b in ROR t LBD revealed that the
two compounds aligned to each other very well, maintaining the
same key hydrogen bonding interactions with the receptor (Fig. 5).
Replacing the amide linker in 3a with a urea linker (3c) dropped the
a few compounds with different linkers between
2.2. Chemistry
g
A versatile synthesis of the general structures of carbazole car-
boxamides was developed and outlined in Scheme 1. The synthesis
started with Buchwald-Hartwig cross coupling of amines and ha-
lides to form diphenylamines 8 or 9, which could be converted to
carbazoles 10 through cyclization. To introduce different N-sub-
stituents at the carbazolyl moiety, various bromides or iodides were
reacted with 10 under alkaline condition to yield 11, which pro-
duced carbazolyl carboxylic acids 12 upon hydrolysis. Condensation
g
g
RORgt activity dramatically. Extending the length of the linker by
adding polar amide groups further lowered the activity (3d) or
completely aborted the activity (3e) although cLogPs of 3d and 3e
Fig. 4. Target compounds with amide linker (3a-e), carbazole ring (4a-d, 5a-h, 6a-d) and arylsulfone moiety (7a-d) modified.

468
Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
Scheme 1. General synthetic procedures for carbazolyl carboxamides analogues.
Scheme 2. General synthetic procedures for non-carbazolyl carboxamides analogues.
were reduced. In view of the ROR
g
t activity and the potential
maximum percentage of RORgt inhibition and increased MW and
genotoxicity of 3a associated with its aniline moiety, we selected
the reversed amide linker in 3b as the preferred linker for further
LHS and RHS optimization.
cLogP, we turned our next attention to the modification of carba-
zole ring with an ethyl group capped on the nitrogen. Replacing the
left phenyl ring of the carbazole with a pyridine ring (6a) dropped
We subsequently explored the LHS SAR of the carbazole car-
boxamide 3b. Results were summarized in Table 2. Changing
the ROR
g
t activity dramatically, indicating that polar moiety in LHS
t activity.
of the carbazole carboxamides disfavored the ROR
g
carbazole ring (3b) to tetrahydrocarbazole ring (4a) lowered ROR
activity. Reducing the size of the tetrahydrocarbazole ring to indole
ring and its derivatives (4b-4d) further lowered the ROR t activity
g
t
Introduction of a small substituent on the 6-position of the carba-
zole ring such as fluorine (6b), chlorine (6c) or methyl (6d)
g
improved RORgt potency. For example, compound 6c exhibited a
although the MW and cLogPs of these indole derivatives were
reduced, indicating the importance of occupying a hydrophobic
pocket by the larger carbazole moiety of 3b. Not surprisingly, the
size of substituents on nitrogen of the carbazole ring modulated the
decent ROR t potency with an IC50 of 58.5 nM and a maximum
inhibition of 161% and had reasonable MW (455) and cLogP (5.03)
comparing to GSK805 (1) or Takeda's compound (2). The effect of
g
small substituents such as Cl and Me on ROR
explained by the binding mode of the carbazole carboxamides in
ROR t LBD (see section 2.4). Either Cl (6c) or Me (6d) on 6-position
of the carbazole moiety was found to fit well into the hydrophobic
gt activity could be
RORgt activity (3b, 5a-5h). The RORgt inhibitory potency (IC50) and
ligand lipophilicity (cLogP) were generally increased with the size
of substituents, with an order of H ≪ Me < i-Pr < Et < Pr < c-
PrCH2 < i-Bu < c-BuCH2 < c-HexCH2. However, the percentage of
g
pocket of the RORgt LBD and therefore exhibited better potency
RORgt maximum inhibition (% max. inh.) started to decrease when
than 3b.
the size of N-substituents was larger than propyl, e.g., Pr > c-
PrCH2 > i-Bu > c-BuCH2 > c-HexCH2. This observation could be
explained by the binding mode of the carbazole carboxamides in
Fixing the LHS moiety as N-ethyl carbazole, SAR of the RHS was
briefly explored based on the SAR knowledge learnt from the other
chemical series with the same RHS moiety [23,25], and summa-
rized in Table 3. Changing the ethyl sulfone in 3b with either a
RORgt LBD (see Section 2.4). Since larger N-substituents lowered

Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
469
Table 1
SAR exploration of the linkers.
Compd
linker
MW
421
cLogP
4.16
RORg
FRET IC50 (nM)a (% max. inh.)b
3a
170 (156)
167 (153)
16200 (61)
7670 (61)
3b
3c
3d
421
436
478
4.30
4.37
3.38
3e
506
3.76
N/Ac
1
2
GSK805
Example 14
in Takeda's patent
532
472
5.23
6.44
34.0 (127)
54.0 (176)
a
IC50 value is the average of at least two independent determinations.
Percent of maximum inhibition measured against activation by the surrogate agonist.
N/A represents no activity.
b
c
Fig. 5. Structural superimposition of the docked poses of 3a (cyan stick) with 3b (magenta stick) in ROR
figure legend, the reader is referred to the Web version of this article.)
gt LBD (PDB: 4NIE). (For interpretation of the references to colour in this
methyl sulfone (7a) or a propyl sulfone (7b) resulted in a drop of
ROR t potency no matter cLogP is lowered or increased, indicating
2.4. Binding mode study
g
that the ethyl group in ethylsulfone could adopt an optimal
conformation to facilitate H-bonding interactions between the
sulfone oxygen and Arg367. Switching ethyl sulfone to a secondary
Docking of N-ethyl-carbazole carboxamide (3b) into RORgt LBD
revealed the binding mode of a typical carbazole carboxamide-
based ROR t inverse agonist (Fig. 6). In the binding mode, carba-
g
sulfonamide (7c) led to a lower ROR
moiety was replaced by pyridylsulfone (7d) with reduced cLogP, the
ROR t activity was greatly reduced.
We further evaluated the representative carbazole carbox-
amides (3b, 6b-6d) in the mouse Th17 cell differentiation assay and
metabolic stability assay (Table 4). All the four compounds tested
showed good inhibitory activity in the Th17 cell differentiation
assay and exhibited reasonable metabolic stability in mouse liver
microsomes, among which 6c stands out with an inhibition of
g
t activity. When phenylsulfone
zole moiety in the LHS of the amide, functioning as the biaryl
moiety in 1, provided preferred intermolecular interactions with
surrounding hydrophobic residues in the hydrophobic site near
Tyr502 and His479, which was believed to be important for the
RORgt binding affinity. In the RHS, the sulfone moiety formed two
hydrogen bonds: one with Arg367 and the other with the backbone
of Leu287. The linker amide formed hydrogen bond interaction
g
with the backbone of Phe377. In addition, two
p-p stacking in-
teractions were observed: one between the sulfone-substituted
phenyl ring and Phe377, and the other between the carbazole
moiety and Phe378.
58.8% at 0.3
respectively.
mM and intrinsic clearance of 0.197 mL/min/g (liver),

470
Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
Table 2 (continued)
Table 2
SAR exploration on the LHS of the carboxamides.
Compd
LHS
MW
455
cLogP
5.03
RORg
FRET IC50 (nM)a
(% max. inh.)b
6c
58.5 (161)
Compd
LHS
MW
421
cLogP
4.30
RORg
FRET IC50 (nM)a
6d
435
4.80
47.8 (112)
(% max. inh.)b
3b
167 (153)
1
2
GSK805
Example 14
in Takeda's patent
532
472
5.23
6.44
34.0 (127)
54.0 (176)
4a
4b
4c
425
399
411
4.48
3.86
3.93
426 (151)
914 (152)
1320 (141)
a
IC50 value is the average of at least two independent determinations.
Percent of maximum inhibition measured against activation by the surrogate
b
agonist.
Table 3
SAR exploration on the RHS of the carboxamides.
4d
370
2.91
5160 (134)
Compd
RHS
MW
421
cLogP
4.30
RORg
FRET IC50 (nM)a
(% max. inh.)b
3b
167 (153)
5a
5b
5c
392
407
435
3.28
3.77
4.83
5400 (93)
566 (140)
138 (150)
7a
7b
7c
7d
407
435
422
422
3.76
4.83
4.18
3.41
1100 (167)
1450 (169)
313 (161)
1220 (158)
5d
5e
435
447
4.61
4.74
358 (126)
81.9 (115)
1
2
GSK805
Example 14
in Takeda's patent
532
472
5.23
6.44
34.0 (127)
54.0 (176)
a
IC50 value is the average of at least two independent determinations.
Percent of maximum inhibition measured against activation by the surrogate
b
agonist.
5f
449
461
489
5.22
5.30
6.42
69.6 (91)
54.9 (82)
33.5 (32)
Table 4
Results in Th17 cell differentiation assay and metabolic stability assay.
5g
5h
Compd
mTh17, % inh.@0.3
m
M
CLint, mL/min/g (liver)
3b
6b
6c
6d
40.0
45.9
58.8
43.6
0.305
0.348
0.197
0.368
In the SAR exploration, we found that when the N-substituent at
carbazole ring is larger, the maximum percentage of inhibition of a
carbazole carboxamide is lower. For example, N-cyclohexylmethyl
6a
6b
422
439
2.98
4.46
3000 (103)
156 (149)
carbazole carboxamide (5h) only had a max. inh. of 32% in ROR
g
FRET assay, compared to that of 153% for the N-ethyl counterpart
(3b). Fig. 7a showed the predicted binding modes of 5h compared
with 3b. The cyclohexylmethyl substituent of 5h was found to make
the carbazole ring naturally rotated 180ꢀ compared to 3b, more
extended toward helix 12 (H12) and closer to the side chain of

Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
471
Fig. 6. (a) Zoomed-in view of 3b in the binding pocket of RORgt LBD (PDB: 4NIE); (b) A two-dimensional plot showing the interactions between 3b and the surrounding amino
acids.
Fig. 7. (a) Structural superimposition of 3b (magenta stick) and 5h (slate stick); (b) Structural superimposition of 5h (slate) and the RORgt agonist (grey) in co-crystal structure of
4NIE. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
His479 and Tyr502 on H12 in a low energy conformation. Overlay of
5h with the previously discovered agonist (N-(4-((benzyl(propyl)
amino)methyl)phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide [24])
modes of the carbazole carboxamides in ROR
understanding of agonist/inverse agonist conversion in the aryl
amide-based RORgt modulators [23,24,30].
gt LBD accord with our
in RORgt co-crystal structure (PDB: 4NIE) (Fig. 7b) indicated that
the N-cyclohexylmethyl moiety occupied almost the same place of
the agonist's benzyl group, being able to partially stabilize the
activation function 2 (AF2) domain (H12) toward recruitment of
steroid receptor co-activator (SRC) and possibly make 5h a partial
agonist. To confirm, we tested 5h in a dual FRET assay (without
adding a surrogate agonist) and found it indeed a partial agonist
with a EC50 of 18 nM and maximum activation of 54%. Binding
3. Conclusions
In summary, we have discovered a series of carbazole carbox-
amides as novel ROR t inverse agonists by a scaffold hybridization
strategy. Structure-activity relationship exploration on the amide
linker, the LHS carbazole ring and the RHS arylsulfone moiety of the
g
hybrid amide 3a led to the identification of potent RORgt inverse

472
Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
agonists. Compound 6c was found to have good ROR
g
t activity in
4.2.1.1. 9-Ethyl-N-(4-(ethylsulfonyl)benzyl)-9H-carbazole-3-
both FRET assay and mouse Th17 cell differentiation assays as well
as reasonable metabolic stability in mouse liver microsomes.
Further optimization of the carbazole carboxamide lead series with
the aid of the binding mode study is undergoing.
carboxamide (3b). Off white solid (71%); mp 161.3e162.3 ꢀC. 1H
NMR (400 MHz, CD3OD)
d
8.70 (s, 1H), 8.15 (d, J ¼ 7.7 Hz, 1H), 8.03
(d, J ¼ 8.7 Hz, 1H), 7.89 (d, J ¼ 8.3 Hz, 2H), 7.66 (d, J ¼ 8.2 Hz, 2H),
7.61e7.54 (m, 2H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.26 (t, J ¼ 7.3 Hz, 1H), 4.75
(s, 2H), 4.47 (q, J ¼ 7.1 Hz, 2H), 3.19 (q, J ¼ 7.4 Hz, 2H), 1.42 (t,
J ¼ 7.2 Hz, 3H), 1.20 (t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz, DMSO-d6)
4. Experimental
d
167.41, 146.88, 141.73, 140.67, 137.34, 128.45, 128.41, 126.74,
4.1. Materials and methods
125.70, 125.15, 122.81, 122.23, 120.87, 120.55, 119.94, 110.02, 109.12,
49.74, 42.94, 37.66, 14.16, 7.64. MS (ESI) m/z: 421.1 [MþH]þ. HRMS
(ESIþ) m/z calcd for C24H24N2O3S [MþH]þ: 421.1580; found:
421.1575.
All the reagents used were commercially available and were
used without further purification unless otherwise indicated. Mi-
crowave reaction was conducted with a Biotage Initiator™ micro-
wave synthesizer. Melting point was recorded by WRS-1B digital
instrument. Analytical thin-layer chromatography and preparative
thin-layer chromatography were performed with silica gel plates
using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd.,
Qingdao, Shandong Province, China). 1H NMR spectra were recor-
ded in DMSO-d6, CD3OD or CDCl3 solution on a Bruker 400 MHz
spectrometer, with chemical shifts expressed in parts per million
using tetramethylsilane (TMS) as an internal standard. Mass spec-
troscopy was carried out on Electrospray ionization (ESI) in-
struments or MALDI-TOF (Bruker).
4.2.1.2. N-(4-(Ethylsulfonyl)benzyl)-9H-carbazole-3-carboxamide
(5a). Off white solid (38.1%); mp 185.0e187.0 ꢀC. 1H NMR
(400 MHz, CD3OD)
d
8.68 (d, J ¼ 1.2 Hz, 1H), 8.12 (d, J ¼ 7.8 Hz, 1H),
7.96 (d, J ¼ 8.5, 1.7 Hz, 1H), 7.89 (d, J ¼ 8.3 Hz, 2H), 7.67 (d, J ¼ 8.3 Hz,
2H), 7.49 (t, J ¼ 8.0 Hz, 2H), 7.42 (t, J ¼ 7.7 Hz, 1H), 7.22 (t, J ¼ 7.4 Hz,
1H), 4.74 (s, 2H), 3.19 (q, J ¼ 7.4 Hz, 2H), 1.20 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (151 MHz, CD3OD)
d 169.76, 146.13, 142.26, 140.77, 137.06,
128.11, 127.85, 125.97, 124.68, 124.01, 122.89, 122.76, 119.76, 119.49,
119.17, 110.75, 110.10, 49.87, 42.77, 6.18. MS (ESI) m/z: 393.0[MþH]þ.
HRMS (ESIþ) m/z calcd for C22H20N2O3S [MþH]þ: 393.1267; found:
393.1259.
4.2. Synthesis
4.2.1.3. N-(4-(Ethylsulfonyl)benzyl)-9-methyl-9H-carbazole-3-
4.2.1. General procedure A for the synthesis of carbazole
carboxamide (5b). Off white solid(10.2%); mp 178.6e180.3 ꢀC. 1H
carboxamides analogues (3b, 5a-5h, 6b-6d, 7a-7d)
NMR (400 MHz, CD3OD)
d
8.70 (s, 1H), 8.15 (d, J ¼ 7.7 Hz, 1H), 8.04
Step 1: To a sealed vial was added aniline (1.2 eq), methyl 4-
(phenylamino)benzoate (1 eq), Pd(OAc)2 (0.05 mmol), rac-BINAP
(0.05 eq), K2CO3 (3 eq) and toluene. Then the sealed vial was irra-
diated in the microwave at 130 ꢀC for 2 h. After the reaction
completed, ethyl acetate was added to dilute the reaction mixture,
and then filtered by diatomite. The filtrate was concentrated under
reduced pressure and the residue was purified by column chro-
matography on silica gel to afford diphenylamine intermediate (8).
Step 2: To a solution of diphenylamine (1 eq) in acetic acid was
added Pd(OAc)2 (1.1 eq). The mixture was stirred under N2 at 120 ꢀC
for 1 h. After the reaction was complete, acetic acid was removed
under reduced pressure. The residue was purified by column
chromatography on silica gel to afford methyl 9H-carbazole-3-
carboxylate intermediate (10).
(dd, J ¼ 8.6, 1.7 Hz, 1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.67 (d, J ¼ 8.4 Hz,
2H), 7.57 (m, 2H), 7.51 (t, J ¼ 7.9 Hz, 1H), 7.27 (t, J ¼ 6.7 Hz, 1H), 4.75
(s, 2H), 3.92 (s, 3H), 3.20 (q, J ¼ 7.4 Hz, 2H),1.21 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (151 MHz, DMSO-d6)
d 166.77 (s), 146.23 (s), 142.12 (s), 141.11
(s), 136.67 (s), 127.80 (d, J ¼ 7.2 Hz), 126.10 (s), 125.06 (s), 124.47 (s),
121.96 (s), 121.37 (s), 120.09 (s), 119.80 (s), 119.34 (s), 109.44 (s),
108.54 (s), 49.08 (s), 42.30 (s), 29.04 (s), 7.01 (s). MS (ESI) m/z: 407.0
[MþH]þ. HRMS (ESIþ) m/z calcd for C23H22N2O3S [MþH]þ:
407.1424; found: 407.1412.
4.2.1.4. N-(4-(Ethylsulfonyl)benzyl)-9-propyl-9H-carbazole-3-
carboxamide (5c). Off white solid (12.7%); mp 149.0e151.0 ꢀC. 1H
NMR (400 MHz, CD3OD)
d
8.70 (d, J ¼ 1.4 Hz, 1H), 8.15 (d, J ¼ 7.6 Hz,
1H), 8.03 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.67 (d,
J ¼ 8.5 Hz, 2H), 7.60e7.55 (m, 2H), 7.50 (t, J ¼ 8.0 Hz, 1H), 7.26 (t,
J ¼ 8.0 Hz, 1H), 4.75 (s, 2H), 4.39 (t, J ¼ 7.1 Hz, 2H), 3.19 (q, J ¼ 7.4 Hz,
2H), 1.96e1.87 (m, 2H), 1.20 (t, J ¼ 7.1 Hz, 3H), 0.95 (t, J ¼ 8 Hz, 3H).
Step 3: To
a stirred solution of methyl 9H-carbazole-3-
carboxylate (1 eq) in DMF was added NaH (3 eq) at 0 ꢀC. The re-
action mixture was stirred at 0 ꢀC for 30 min, and then bromides or
iodides (3 eq) were added. The mixture was reacted at room tem-
perature until the reaction completed. Water was added to quench
the reaction and the mixture was extracted with ethyl acetate
several times. Then the organic layer was combined and the solvent
was removed under reduced pressure to afford substituted carba-
zole carboxylate intermediate (11).
13C NMR (151 MHz, CD3OD)
d 169.60, 146.09, 142.54, 141.21, 137.07,
128.11, 127.87, 126.09, 124.81, 124.08, 122.68, 122.46, 119.92, 119.55,
119.34,109.11,108.44, 49.87, 44.05, 42.79, 21.90,10.43, 6.18. MS (ESI)
m/z: 435.0[MþH]þ. HRMS (ESIþ) m/z calcd for C25H26N2O3S
[MþH]þ: 435.1737; found: 435.1730.
Step 4: To a stirred solution of substituted carbazole carboxylate
(1 eq) in ethanol/H2O (5:1) was added LiOH or KOH (3 eq). The
mixture was stirred at 90 ꢀC. After the reaction completed, ethanol
was removed under reduced pressure. Then pH of the mixture was
adjusted to 3 with 2 N hydrochloric acid solution. The mixture was
filtered to afford the carbazole carboxylic acid intermediate (12) as
solid.
Step 5: To a stirred solution of carbazole carboxylic acid (1 eq) in
DCM was added HATU (1.2 eq), N,N-diisopropylethylamine (3 eq)
and benzylamine (1.2 eq). The mixture was stirred at room tem-
perature. After the reaction completed, solvent was removed under
reduced pressure, and the residue was purified by column chro-
matography on silica gel to afford the target compounds (3b, 5a-5h,
6b-6d, 7a-7d).
4.2.1.5. N-(4-(Ethylsulfonyl)benzyl)-9-isopropyl-9H-carbazole-3-
carboxamide (5d). Off white solid (35.4%); mp 138.8e140.8 ꢀC.1H
NMR (400 MHz, CD3OD)
d
8.70 (d, J ¼ 1.6 Hz, 1H), 8.15 (d, J ¼ 7.8 Hz,
1H), 8.00 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.89 (d, J ¼ 8.3 Hz, 2H), 7.70e7.65
(m, 4H), 7.47 (t, J ¼ 7.3 Hz, 1H), 7.24 (t, J ¼ 7.5 Hz, 1H), 5.18e5.08 (m,
1H), 4.74 (s, 2H), 3.19 (q, J ¼ 7.4 Hz, 2H), 1.71 (d, J ¼ 7.0 Hz, 6H), 1.20
(t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz, CD3OD)
d 169.50, 146.06,
141.45, 140.15, 137.04, 128.09, 127.88, 125.90, 124.59, 123.84, 123.18,
122.97, 119.95, 119.51, 119.20, 110.37, 109.59, 49.85, 46.89, 42.78,
19.52, 6.18. MS (ESI) m/z: 435.0 [MþH]þ. HRMS (ESIþ) m/z calcd for
C
25H26N2O3S [MþH]þ: 435.1737; found: 435.1731.
4.2.1.6. 9-(Cyclopropylmethyl)-N-(4-(ethylsulfonyl)benzyl)-9H-
carbazole-3-carboxamide (5e). Off white solid (38.3%); mp

Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
473
177.6e179.6 ꢀC.1H NMR (400 MHz, CD3OD)
d
8.70 (d, J ¼ 1.3 Hz, 1H),
4.2.1.11. 6-Chloro-9-ethyl-N-(4-(ethylsulfonyl)benzyl)-9H-carbazole-
8.14 (d, J ¼ 7.7 Hz, 1H), 8.02 (d, J ¼ 8.6, 1.7 Hz, 1H), 7.88 (d, J ¼ 8.3 Hz,
2H), 7.66 (d, J ¼ 8.3 Hz, 2H), 7.61e7.57 (m, 2H), 7.49 (t, J ¼ 7.7 Hz,
1H), 7.25 (t, J ¼ 7.5 Hz, 1H), 4.74 (s, 2H), 4.33 (d, J ¼ 6.6 Hz, 2H), 3.18
(q, J ¼ 7.4 Hz, 2H), 1.38e1.32 (m, 1H), 1.20 (t, J ¼ 7.4 Hz, 3H),
0.56e0.48 (m, 2H), 0.48e0.41 (m, 2H). 13C NMR (151 MHz, DMSO-
3-carboxamide (6c). Off white solid (77.8%); mp 155.8e157.9 ꢀC. 1H
NMR (400 MHz, DMSO-d6)
d
9.11 (t, J ¼ 5.9 Hz,1H), 8.80 (s,1H), 8.26
(d, J ¼ 1.9 Hz, 1H), 8.09e8.02 (m, 1H), 7.84 (d, J ¼ 8.3 Hz, 2H), 7.71 (d,
J ¼ 3.3 Hz, 2H), 7.69 (d, J ¼ 3.3 Hz, 2H), 7.61 (d, J ¼ 8.3 Hz, 2H), 7.50
(dd, J ¼ 8.7, 2.0 Hz, 1H), 4.63 (d, J ¼ 5.8 Hz, 2H), 4.47 (q, J ¼ 6.9 Hz,
2H), 3.24 (q, J ¼ 7.4 Hz, 2H), 1.29 (t, J ¼ 7.1 Hz, 3H), 1.07 (t, J ¼ 7.4 Hz,
d6)
d 167.40, 146.88, 142.32, 141.25, 137.33, 128.44, 128.40, 126.73,
125.67, 125.14, 122.73, 122.15, 120.76, 120.46, 119.94, 110.43, 109.53,
49.73, 46.77, 42.93, 11.20, 7.64, 4.06. MS (ESI) m/z: 447.0 [MþH]þ.
HRMS (ESIþ) m/z calcd for C26H26N2O3S [MþH]þ:447.1737; found:
447.1752.
3H). 13C NMR (151 MHz, DMSO-d6)
d 167.30, 146.78, 142.21, 139.14,
137.36, 128.46, 128.41, 126.49, 126.32, 125.66, 124.34, 124.12, 121.36,
121.25, 120.53, 111.64, 109.51, 49.73, 42.94, 37.89, 14.15, 7.64. MS
(ESI) m/z: 454.9[MþH]þ. HRMS (ESIþ) m/z calcd for C24H23ClN2O3S
[MþH]þ: 455.1191; found: 455.1207.
4.2.1.7. N-(4-(Ethylsulfonyl)benzyl)-9-isobutyl-9H-carbazole-3-
carboxamide (5f). Off white solid (20.2%); mp 166.4e168.4 ꢀC. 1H
4.2.1.12. 9-Ethyl-N-(4-(ethylsulfonyl)benzyl)-6-methyl-9H-carba-
NMR (400 MHz, CD3OD)
d
8.71 (d, J ¼ 1.4 Hz, 1H), 8.16 (d, J ¼ 7.8 Hz,
zole-3-carboxamide
(6d). Off
white
solid
(69.4%);
mp
1H), 8.02 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.90 (d, J ¼ 8.4 Hz, 2H), 7.67 (d,
J ¼ 8.4 Hz, 2H), 7.59e7.54 (m, 2H), 7.49 (t, J ¼ 7.6 Hz, 1H), 7.26 (t,
J ¼ 7.4 Hz,1H), 4.75 (s, 2H), 4.22 (d, J ¼ 7.5 Hz, 2H), 3.20 (q, J ¼ 7.4 Hz,
2H), 2.46e2.31 (m, 1H), 1.21 (t, J ¼ 7.4 Hz, 3H), 0.97 (d, J ¼ 6.7 Hz,
160.4e161.5 ꢀC.1H NMR (400 MHz, CDCl3)
d 8.62 (s, 1H), 7.96 (dd,
J ¼ 8.6, 1.3 Hz, 1H), 7.87 (s, 1H), 7.74 (d, J ¼ 8.2 Hz, 2H), 7.48 (d,
J ¼ 8.2 Hz, 2H), 7.35 (d, J ¼ 8.6 Hz, 1H), 7.32e7.27 (m, 3H), 4.74 (d,
J ¼ 5.9 Hz, 2H), 4.32 (q, J ¼ 7.2 Hz, 2H), 3.05 (q, J ¼ 7.4 Hz, 2H), 2.49
(s, 3H), 1.39 (t, J ¼ 7.2 Hz, 3H), 1.24 (d, J ¼ 7.4 Hz, 3H). 13C NMR
6H). 13C NMR (151 MHz, DMSO-d6)
d 167.41, 146.88, 142.61, 141.50,
137.33, 128.43, 128.40, 126.66, 125.61, 125.11, 122.61, 122.00, 120.74,
120.43, 119.91, 110.57, 109.69, 50.14, 49.74, 42.93, 28.84, 20.50, 7.64.
MS (ESI) m/z: 448.9[MþH]þ. HRMS (ESIþ) m/z calcd for
(151 MHz, DMSO-d6) d 167.41, 146.89, 141.91, 139.00, 137.34, 128.75,
128.44, 128.40, 128.01, 125.53, 124.86, 122.95, 122.03, 120.68,
120.44,109.77,109.02, 49.74, 42.92, 37.66, 21.51,14.16, 7.64. MS (ESI)
m/z: 435.2[MþH]þ. HRMS (ESIþ) m/z calcd for C25H26N2O3S
[MþH]þ: 435.1737; found: 435.1744.
C
26H28N2O3S [MþH]þ: 449.1893; found: 449.1906.
4.2.1.8. 9-(Cyclobutylmethyl)-N-(4-(ethylsulfonyl)benzyl)-9H-carba-
zole-3-carboxamide (5g). Off white solid (58.3%). mp
203.5e205.5 ꢀC .1H NMR (400 MHz, CD3OD)
8.70 (s, 1H), 8.14 (d,
4.2.1.13. 9-Ethyl-N-(4-(methylsulfonyl)benzyl)-9H-carbazole-3-
d
carboxamide (7a). Off white solid (48.5%); mp 206.4e208.1 ꢀC.1H
J ¼ 7.8 Hz, 1H), 8.02 (d, J ¼ 8.6 Hz, 1H), 7.89 (d, J ¼ 8.2 Hz, 2H), 7.67
(d, J ¼ 8.2 Hz, 2H), 7.63e7.57 (m, 2H), 7.49 (t, J ¼ 7.6 Hz, 1H), 7.25 (t,
J ¼ 7.4 Hz,1H), 4.75 (d, J ¼ 4.5 Hz, 2H), 4.43 (s, 2H), 3.19 (q, J ¼ 7.4 Hz,
2H), 2.98e2.95 (m, 1H), 2.01e1.96 (m, 2H), 1.93e1.89 (m, 4H), 1.21
NMR (400 MHz, DMSO-d6)
d
9.15 (t, J ¼ 5.8 Hz, 1H), 8.75 (s, 1H), 8.17
(d, J ¼ 7.7 Hz, 1H), 8.07e8.00 (m, 1H), 7.88 (d, J ¼ 8.3 Hz, 2H),
7.70e7.62 (m, 2H), 7.60 (d, J ¼ 8.3 Hz, 2H), 7.49 (t, J ¼ 7.7 Hz, 1H),
7.24 (t, J ¼ 7.4 Hz, 1H), 4.62 (d, J ¼ 5.8 Hz, 2H), 4.47 (q, J ¼ 7.0 Hz, 2H),
3.17 (s, 3H), 1.31 (t, J ¼ 7.1 Hz, 3H). 13C NMR (151 MHz, DMSO-d6)
(t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz, DMSO-d6)
d 167.40, 146.88,
142.52, 141.43, 137.33, 128.44, 128.40, 126.67, 125.63, 125.11, 122.62,
122.03, 120.73, 120.43, 119.91, 110.38, 109.49, 49.74, 47.56, 42.93,
38.72, 35.62, 26.31, 18.40, 7.64. MS (ESI) m/z: 461.0 [MþH]þ. HRMS
(ESIþ) m/z calcd for C27H28N2O3S [MþH]þ: 461.1893; found:
461.1908.
d 167.39, 146.72, 141.72, 140.66, 139.70, 128.46, 127.55, 126.74,
125.69, 125.16, 122.80, 122.22, 120.87, 120.53, 119.94, 110.02, 109.12,
44.13, 42.96, 37.66, 14.16. MS (ESI) m/z: 407.0 [MþH]þ. HRMS (ESIþ)
m/z calcd for C23H22N2O3S [MþH]þ: 407.1424; found: 407.1434.
4.2.1.14. 9-Ethyl-N-(4-(propylsulfonyl)benzyl)-9H-carbazole-3-
4.2.1.9. 9-(Cyclohexylmethyl)-N-(4-(ethylsulfonyl)benzyl)-9H-carba-
carboxamide (7b). Off white solid (40.2%); mp 150.8e152.5 ꢀC .1H
zole-3-carboxamide
(5h). Off
white
solid
(8.0%);
mp
NMR (400 MHz, CDCl3)
d
8.63 (s, 1H), 8.12 (d, J ¼ 7.8 Hz, 1H), 7.97 (d,
190.2e192.0 ꢀC. 1H NMR (400 MHz, CDCl3)
d
8.61 (s, 1H), 8.12 (d,
J ¼ 8.6 Hz, 1H), 7.82 (d, J ¼ 8.1 Hz, 2H), 7.55 (d, J ¼ 8.2 Hz, 2H), 7.51
(d, J ¼ 7.9 Hz, 1H), 7.47e7.40 (m, 2H), 7.28 (d, J ¼ 7.3 Hz, 1H), 6.93 (s,
1H), 4.80 (d, J ¼ 5.6 Hz, 2H), 4.39 (q, J ¼ 7.2 Hz, 2H), 3.08e3.00 (m,
2H), 1.75e1.69 (m, 2H), 1.45 (t, J ¼ 7.2 Hz, 3H), 0.97 (t, J ¼ 7.4 Hz, 3H).
J ¼ 7.7 Hz,1H), 7.94 (d, J ¼ 8.6 Hz,1H), 7.87 (d, J ¼ 8.1 Hz, 2H), 7.59 (d,
J ¼ 8.2 Hz, 2H), 7.51 (t, J ¼ 7.5 Hz, 1H), 7.43 (d, J ¼ 10.7 Hz, 2H), 7.29
(d, J ¼ 7.6 Hz, 1H), 6.76 (t, J ¼ 5.3 Hz,1H), 4.82 (d, J ¼ 5.7 Hz, 2H), 4.14
(d, J ¼ 7.3 Hz, 2H), 3.10 (q, J ¼ 7.4 Hz, 2H), 2.00 (m, 1H), 1.66 (m, 10H),
13C NMR (151 MHz, DMSO-d6)
d 167.41, 146.84, 141.73, 140.67,
1.28 (t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz, CD3OD)
d
169.51, 147.04,
137.94, 128.44, 128.28, 126.74, 125.70, 125.15, 122.81, 122.23, 120.87,
120.55, 119.94, 110.02, 109.12, 56.81, 42.94, 37.66, 16.69, 14.16, 12.98.
MS (ESI) m/z: 432.9 [MeH]e, MS (ESI) m/z: 435.2 [MþH]þ. HRMS
(ESIþ) m/z calcd for C25H26N2O3S [MþH]þ: 435.1737; found:
435.1749.
143.54, 142.31, 138.00, 128.97, 128.74, 127.03, 125.85, 125.20, 123.39,
122.91, 120.89, 120.50, 120.27, 110.60, 109.86, 50.56, 49.68, 43.49,
31.59, 26.79, 26.32, 7.32. MS (ESI) m/z: 489.3[MþH]þ. HRMS (ESIþ)
m/z calcd for C29H32N2O3S [MþH]þ: 489.2206; found: 489.2217.
4.2.1.10. 9-Ethyl-N-(4-(ethylsulfonyl)benzyl)-6-fluoro-9H-carbazole-
3-carboxamide (6b). Off white solid (69.4%); mp 166.8e170.9 ꢀC. 1H
4.2.1.15. 9-Ethyl-N-(4-(N-methylsulfamoyl)benzyl)-9H-carbazole-3-
carboxamide (7c). Off white solid (31.2%); mp 226.0e228.0 ꢀC.1H
NMR (400 MHz, CDCl3)
d
8.56 (s, 1H), 7.97 (d, J ¼ 8.7 Hz, 1H), 7.87 (d,
NMR (400 MHz, DMSO-d6)
d
9.12 (t, J ¼ 6.0 Hz, 1H), 8.76 (s, 1H), 8.17
J ¼ 8.2 Hz, 2H), 7.77 (dd, J ¼ 8.6, 2.4 Hz, 1H), 7.58 (d, J ¼ 8.2 Hz, 2H),
7.43 (d, J ¼ 8.5 Hz, 1H), 7.36 (dd, J ¼ 8.8, 4.1 Hz, 1H), 7.25e7.22 (m,
1H), 6.79 (s, 1H), 4.82 (d, J ¼ 5.7 Hz, 2H), 4.37 (t, J ¼ 7.2 Hz, 2H), 3.10
(d, J ¼ 7.7 Hz, 1H), 8.04 (d, J ¼ 8.6 Hz, 1H), 7.73 (d, J ¼ 8.2 Hz, 2H),
7.70e7.62 (m, 2H), 7.55 (d, J ¼ 8.1 Hz, 2H), 7.49 (t, J ¼ 7.7 Hz, 1H),
7.40 (q, J ¼ 4.8 Hz, 1H), 7.24 (t, J ¼ 7.5 Hz, 1H), 4.60 (d, J ¼ 5.8 Hz, 2H),
4.47 (q, J ¼ 6.9 Hz, 2H), 2.37 (d, J ¼ 5.0 Hz, 3H), 1.31 (t, J ¼ 7.0 Hz, 3H).
(q, J ¼ 7.4 Hz, 2H), 1.44 (t, J ¼ 7.2 Hz, 3H), 1.28 (d, J ¼ 7.4 Hz, 3H).13
C
NMR (151 MHz, DMSO-d6)
d
166.71, 157.49, 155.94, 146.18, 141.90),
13C NMR (151 MHz, DMSO-d6)
d 167.37, 145.34, 141.71, 140.66,
136.68, 136.51, 127.80), 127.78,125.46, 124.55, 122.72, 122.66, 121.21,
120.68, 113.80, 113.64, 110.51, 110.45, 108.80, 106.11, 105.96, 49.08,
42.29, 37.23, 13.54), 7.01. MS (ESI) m/z: 435.2 [MþH]þ. HRMS (ESIþ)
m/z calcd for C24H23FN2O3S [MþH]þ: 439.1486; found: 439.1500.
138.13, 128.27, 127.26, 126.73, 125.70, 125.19, 122.81, 122.21, 120.87,
120.54, 119.93, 110.02, 109.11, 42.99, 37.56, 29.20, 14.16. MS (ESI) m/
z: 422.1[MþH]þ. HRMS (ESIþ) m/z calcd for C23H23N3O3S [MþH]þ:
422.1533; found: 422.1549.

474
Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
4.2.1.16. 9-Ethyl-N-((5-(ethylsulfonyl)pyridin-2-yl)methyl)-9H-
carbazole-3-carboxamide (7d). Off white solid (17.0%); mp
silica gel to afford the target compound 5-ethyl-N-(4-(ethyl-
sulfonyl)benzyl)-5H-pyrido[3,2-b]indole-8-carboxamide (6a) as a
white solid (22.8%); mp 164.4e166.5 ꢀC. 1H NMR (400 MHz, CDCl3)
167.3e169.3 ꢀC. 1H NMR (400 MHz, CD3OD)
d
8.99 (d, J ¼ 1.7 Hz,1H),
8.73 (d, J ¼ 1.1 Hz, 1H), 8.26 (dd, J ¼ 8.3, 2.2 Hz, 1H), 8.15 (d,
J ¼ 7.8 Hz, 1H), 8.05 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.68 (d, J ¼ 8.3 Hz, 1H),
7.61e7.53 (m, 2H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.25 (t, J ¼ 7.4 Hz, 1H), 4.85
(s, 2H), 4.46 (q, J ¼ 7.1 Hz, 2H), 3.26 (q, J ¼ 7.4 Hz, 2H), 1.41 (t,
J ¼ 7.2 Hz, 3H), 1.24 (t, J ¼ 7.4 Hz, 3H).13C NMR (151 MHz, DMSO-d6)
d
8.79 (s, 1H), 8.54 (d, J ¼ 4.6 Hz, 1H), 8.23 (dd, J ¼ 8.7, 1.7 Hz, 1H),
7.85 (d, J ¼ 8.3 Hz, 2H), 7.76 (d, J ¼ 8.3 Hz, 1H), 7.56 (d, J ¼ 8.2 Hz,
2H), 7.53 (d, J ¼ 8.7 Hz,1H), 7.43e7.40 (m,1H), 7.15 (t, J ¼ 5.5 Hz,1H),
4.78 (d, J ¼ 5.9 Hz, 2H), 4.40 (q, J ¼ 7.3 Hz, 2H), 3.10 (q, J ¼ 7.4 Hz,
2H), 1.46 (t, J ¼ 7.2 Hz, 3H), 1.28 (d, J ¼ 7.4 Hz, 3H). 13C NMR
d
166.92 (s), 164.84 (s), 147.86 (s), 141.13 (s), 140.03 (s), 136.58 (s),
(151 MHz, CD3OD) d 169.10, 146.01, 142.49, 141.12, 140.68, 137.11,
133.02 (s), 126.13 (s), 125.08 (s), 124.31 (s), 122.16 (s), 121.59 (s),
121.16 (s), 120.24 (s), 120.00 (s), 119.34 (s), 109.41 (s), 108.52 (s),
49.27 (s), 44.77(s), 37.03 (s), 13.54 (s), 6.82 (s). MS (ESI) m/z: 423.1
[MþH]þ. HRMS (ESIþ) m/z calcd for C23H23N3O3S [MþNa]þ:
444.1352; found: 444.1363.
134.54, 128.72, 128.13, 127.91, 127.32, 125.61, 120.68, 119.83, 117.38,
109.20, 49.87, 42.82, 37.39, 12.72, 6.20. MS (ESI) m/z: 422.2 [MþH]þ.
HRMS (ESIþ) m/z calcd for C23H23N3O3S [MþH]þ: 422.1533; found:
422.1550.
4.2.3. General procedure C for the synthesis of non-carbazolyl
carboxamides analogues (4a-4d)
4.2.2. General procedure B for the synthesis of 5-ethyl-N-(4-
(ethylsulfonyl)benzyl)-5H-pyrido [3,2-b]indole-8-carboxamide (6a)
Step1: To a sealed vial was added 2-chloro-3-iodopyridine (1
eq), ethyl 4-aminobenzoate (1.2 eq), Pd(OAc)2 (0.06 eq), Cs2CO3 (3
eq), rac-BINAP (0.05 eq) and toluene. Then the sealed vial was
irradiated in the microwave 140 ꢀC for 1.5 h. After the reaction
completed, ethyl acetate was added to dilute the reaction mixture,
and then filtered by diatomite. The filtrate was concentrated under
reduced pressure, and the residue was purified by column chro-
matography on silica gel to afford ethyl 4-((2-chloropyridin-3-yl)
Step 1: To a stirred solution of 4-hydrazinobenzoic acid (1 eq) in
1,4-dioxane was added ketone (1.1 eq) and concentrated hydro-
chloric acid. The reaction mixture was stirred at 120 ꢀC overnight. A
large amount of solid appeared and was filtered to afford the solid
as indole acid (15).
Step 2: To a stirred solution of indole acid (1 eq) in methanol
was added sulfuric acid. The mixture was stirred at 85 ꢀC for 2 h.
After cooled to room temperature, the mixture was neutralized by
adding saturated Na2CO3 and then extracted with ethyl acetate.
Organic layer was combined and solvent was removed under
reduced pressure. The residue was purified by column chroma-
tography on silica gel to afford indole carboxylate (16).
Step 3: To a stirred solution of indole carboxylate (1 eq) in DMF
was added NaH (3 eq) at 0 ꢀC. The reaction mixture was stirred at
0 ꢀC for 30 min, and then bromides or iodides (3 eq) were added.
The mixture was reacted at room temperature until the reaction
completed. Water was added to quench the reaction and extracted
with ethyl acetate several times. And then the organic layer was
combined and the solvent was removed under reduced pressure to
afford substituted indole carboxylate (17).
Step 4: To a stirred solution of substituted indole carboxylate (1
eq) in ethanol/H2O (5:1) was added LiOH or KOH (3 eq). The
mixture was stirred at 90 ꢀC. After the reaction completed, ethanol
was removed under reduced pressure. And then pH of the mixture
was adjusted to 3 with 2 N hydrochloric acid solution. The mixture
was filtered to afford the indole acid (18) as solid.
amino)benzoate (9). 1H NMR (400 MHz, CDCl3)
d 8.05e7.93 (m, 3H),
7.69 (d, J ¼ 7.9 Hz, 1H), 7.18 (dd, J ¼ 8.0, 4.6 Hz, 1H), 7.12 (d,
J ¼ 8.7 Hz, 2H), 6.35 (s, 1H), 4.35 (t, J ¼ 7.1 Hz, 2H), 1.38 (t, J ¼ 7.1 Hz,
3H).
Step 2: To a sealed vial was added ethyl 4-((2-chloropyridin-3-
yl)amino)-benzoate (1 eq), NaOAc$3H2O (2.5 eq), PdCl2(PPh3)2
(0.1 eq) and DMF. Then the sealed vial was irradiated in the mi-
crowave 180 ꢀC for 2 h. After the reaction completed, H2O was
added and the mixture was extracted with ethyl acetate three
times. Organic layer was combined, washed with H2O for 5 times,
and dried with Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by column chromatography
on silica gel to afford ethyl 5H-pyrido[3,2-b]indole-8-carboxylate
(10, X ¼ N). 1H NMR (400 MHz, CDCl3)
d 9.12 (s, 1H), 8.98 (s, 1H),
8.62 (dd, J ¼ 4.7, 1.1 Hz, 1H), 8.23 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.78 (dd,
J ¼ 8.2, 1.1 Hz, 1H), 7.48 (d, J ¼ 8.6 Hz, 1H), 7.37 (dd, J ¼ 8.2, 4.7 Hz,
1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 1.43e1.36 (m, 3H).
Step 3: To a stirred solution of ethyl 5H-pyrido[3,2-b]indole-8-
carboxylate (1 eq) in DMF was added NaH (3 eq) at 0 ꢀC. The re-
action mixture was stirred at 0 ꢀC for 30 min, and then bromides or
iodides (3 eq) were added. The mixture was reacted at room tem-
perature until the reaction completed. Water was added to quench
the reaction and the mixture was extracted with ethyl acetate
several times. Then the organic layer was combined and the solvent
was removed under reduced pressure to afford ethyl 5-ethyl-5H-
pyrido[3,2-b]indole-8-carboxylate (11, X ¼ N). MS (ESI) m/z: 241.1
[MþH]þ.
Step 5: To a stirred solution of indole acid (1 eq) in DCM was
added HATU (1.2 eq), N,N-diisopropylethylamine (3 eq) and ben-
zylamine (1.2 eq). The mixture was stirred at room temperature.
After the reaction completed, solvent was removed under reduced
pressure. The residue was purified by column chromatography on
silica gel to afford the target compound (4a-4d).
4.2.3.1. 9-Ethyl-N-(4-(ethylsulfonyl)benzyl)-2,3,4,9-tetrahydro-1H-
carbazole-6-carboxamide (4a). Off yellow solid (74.7%); mp
193.3e195.3 ꢀC.1H NMR (400 MHz, CD3OD)
d 8.02 (s, 1H), 7.85 (d,
Step 4: To a stirred solution of ethyl 5-ethyl-5H-pyrido[3,2-b]
indole-8-carboxylate (1 eq) in ethanol/H2O (5:1) was added LiOH (3
eq). The mixture was stirred at 90 ꢀC for 2 h. After the reaction was
complete, ethanol was removed under reduced pressure. Then pH
of the mixture was adjusted to 3 with 2 N hydrochloric acid solu-
tion. The mixture was filtered to afford 5-ethyl-5H-pyrido[3,2-b]
indole-8-carboxylic acid (12, X ¼ N) as solid.
J ¼ 8.1 Hz, 2H), 7.65 (d, J ¼ 8.6 Hz,1H), 7.61 (d, J ¼ 8.1 Hz, 2H), 7.35 (d,
J ¼ 8.6 Hz, 1H), 4.68 (s, 2H), 4.16e4.10 (q, J ¼ 7.1, 2H), 3.17 (q,
J ¼ 7.4 Hz, 2H), 2.73 (d, J ¼ 6.1 Hz, 4H),1.95 (d, J ¼ 5.5 Hz, 2H),1.87 (d,
J ¼ 5.2 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz, 3H), 1.19 (t, J ¼ 7.4 Hz, 3H). 13C NMR
(151 MHz, CDCl3)
d 168.95, 145.63, 137.62, 137.19, 136.78, 128.51,
128.18, 127.21, 124.05, 119.52, 117.65, 110.71, 108.39, 50.67, 43.34,
37.67, 23.11, 23.02, 22.03, 20.96, 15.43, 7.42. MS (ESI) m/z: 425.0
[MþH]þ. HRMS (ESIþ) m/z calcd for C24H28N2O3S [MþH]þ:
425.1893; found: 425.1889.
Step 5: To a stirred solution of 5-ethyl-5H-pyrido[3,2-b]indole-
8-carboxylic acid (1 eq) in DCM was added HATU (1.2 eq), N,N-
diisopropylethylamine (3 eq) and (4-(ethylsulfonyl)phenyl)meth-
anamine (1.2 eq). The mixture was stirred at room temperature.
After the reaction completed, solvent was removed under reduced
pressure. The residue was purified by column chromatography on
4.2.3.2. 1-Ethyl-N-(4-(ethylsulfonyl)benzyl)-2,3-dimethyl-1H-indole-
5-carboxamide (4b). Off yellow solid (39.5%); mp 155.9e157.9 ꢀC.1H
NMR (400 MHz, CD3OD)
d
8.05 (s, 1H), 7.85 (d, J ¼ 8.3 Hz, 2H), 7.65

Y. Huang et al. / European Journal of Medicinal Chemistry 148 (2018) 465e476
475
(d, J ¼ 8.5 Hz, 1H), 7.61 (d, J ¼ 8.2 Hz, 2H), 7.33 (d, J ¼ 8.6 Hz, 1H),
4.68 (s, 2H), 4.16 (q, J ¼ 7.2 Hz, 2H), 3.16 (q, J ¼ 7.4 Hz, 2H), 2.36 (s,
3H), 2.25 (s, 3H), 1.27 (t, J ¼ 7.1 Hz, 3H), 1.18 (t, J ¼ 7.4 Hz, 3H). 13C
mL, Peprotech) and IL-6 (20 ng/mL, Peprotech) for 4 days before
analysis. Compounds or DMSO control were added to the culture on
day 0 of Th17 differentiation at indicated concentrations. Percent-
age of IL-17 production from CD4þ T cells were analyzed by intra-
cellular staining followed by flow cytometry. Dose-response curves
were plotted to determine half-maximal inhibitory concentrations
(IC50) for the compounds using the GraphPad Prism 5 (GraphPad
Software, San Diego CA, USA).
NMR (151 MHz, CDCl3)
d 168.99, 145.70, 137.34, 137.06, 133.61,
128.42, 128.39, 128.15, 124.09, 119.51, 117.93, 108.27, 107.85, 50.66,
43.29, 38.01, 15.35, 10.03, 8.74, 7.40. MS (ESI) m/z: 399.0[MþH]þ.
HRMS (ESIþ) m/z calcd for C22H26N2O3S [MþH]þ: 399.1737; found:
399.1733.
4. 2. 3. 3. 4-Ethyl-N -(4-(ethylsul fo nyl )ben zyl)-1, 2, 3, 4-
4.3.3. Molecular docking studies
tetrahydrocyclopenta[b]indole-7- carboxamide (4c). Off white solid
Molecular docking was carried out using Schrodinger 3.5 soft-
ware package. The co-crystal structure of RORgt LBD (PDB: 4NIE)
(40.2%); mp 162.0e164.0 ꢀC.1H NMR (400 MHz, CD3OD)
d 7.96 (s,
1H), 7.87 (d, J ¼ 8.3 Hz, 2H), 7.62 (d, J ¼ 8.4 Hz, 3H), 7.36 (d,
J ¼ 8.7 Hz, 1H), 4.69 (s, 2H), 4.15 (q, J ¼ 7.3 Hz, 2H), 3.18 (q, J ¼ 7.4 Hz,
2H), 2.90 (t, J ¼ 7.1 Hz, 2H), 2.85 (t, J ¼ 7.0 Hz, 2H), 2.61e2.52 (m,
2H),1.35 (t, J ¼ 7.2 Hz, 3H),1.19 (t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz,
was selected and processed using the Protein Preparation Wizard
including water deletion, addition of missing hydrogen atoms as
well as adjustment of the tautomerization and protonation states of
histidine. The compound 3D structures were subjected to energy
minimization with force field (OPLS_2005) before submitting to the
docking procedure. The docking grid was centered according to the
ligand position, and the bounding box was set to 15 Å. This docking
was performed with Glide-docking using Extra Precision (GlideXP)
algorithm. The final ranking from the docking was based on the
docking score, which combines the Epik state penalty with the
Glide Score. High-scoring complexes were inspected visually to
select the most reasonable solution.
CDCl3) d 168.95, 147.51, 145.64, 142.25, 137.14, 128.47, 128.17, 124.41,
124.05, 119.03, 118.25, 118.25, 109.26, 50.66, 43.32, 39.68, 28.37,
25.07, 24.48, 15.57, 7.42. MS (ESI) m/z: 411.0[MþH]þ. HRMS (ESIþ)
m/z calcd for C23H26N2O3S [MþH]þ: 411.1737; found: 411.1731.
4.2.3.4. 1-Ethyl-N-(4-(ethylsulfonyl)benzyl)-1H-indole-5-
carboxamide (4d). Off white solid (89.7%); mp 111.0e113.0 ꢀC.1H
NMR (400 MHz, CDCl3)
d
8.17 (s, 1H), 7.72 (d, J ¼ 8.6 Hz, 1H), 7.66 (d,
J ¼ 8.1 Hz, 2H), 7.56e7.45 (m, 1H), 7.40 (d, J ¼ 8.0 Hz, 2H), 7.30 (d,
J ¼ 8.6 Hz, 1H), 7.14 (d, J ¼ 2.9 Hz, 1H), 6.48 (d, J ¼ 2.4 Hz, 1H), 4.65
(d, J ¼ 5.8 Hz, 2H), 4.14 (q, J ¼ 7.2 Hz, 2H), 3.01 (q, J ¼ 7.4 Hz, 2H),1.42
(t, J ¼ 7.2 Hz, 3H), 1.18 (t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz, DMSO-
Acknowledgements
This work was supported by National Science Foundation of
China (Grant Number: 81573276) and Shanghai Bio-
pharmaceutical Science and Technology Supporting Plan (Grant
Numbers: 15431900300 and 17431902100).
d6)
d 167.82, 146.97, 137.41, 137.28, 129.91, 128.37, 128.04, 125.53,
120.95, 120.92, 109.78, 102.22, 49.74, 42.86, 40.95, 15.91, 7.63. MS
(ESI) m/z: 371.0[MþH]þ. HRMS (ESIþ) m/z calcd for C20H22N2O3S
[MþH]þ: 371.1424; found: 371.1423.
Appendix A. Supplementary data
4.3. Biological assays
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.02.050.
4.3.1. RORg FRET assay
The assays were performed in an assay buffer consisting of
50 mM NaF, 50 mM 3-(N-morpholino)propanesulfonic acid, pH 7.4,
0.05 mM 3-[(3-cholamidopropyl) dimethylammonio]propane-
sulfonate, 0.1 mg/mL bovine serum albumin, and 10 mM dithio-
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