Design, synthesis and evaluation of hybrid of tetrahydrocarbazole with 2,4-diaminopyrimidine scaffold as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2018.11.016

Several 6-substituted tetrahydrocarbazole derivatives were designed, synthesized and evaluated for the antibacterial activities against Staphylococcus aureus Newman strain. Subsequently, 2,4-diaminopyrimidine scaffold was merged with the tetrahydrocarbazole unit to generate a series of novel hybrid derivatives and the antibacterial activities were also investigated. Among these novel hybrids, compound 12c showed the most potent activity with a MIC of 0.39–0.78 μg/mL against S. aureus Newman and Escherichia coli AB1157 strain. In addition, compound 12c exhibited low MIC values against a panel of multidrug-resistant strains of S. aureus.

Full text of DOI:10.1016/j.ejmech.2018.11.016

Accepted Manuscript
Design, synthesis and evaluation of hybrid of tetrahydrocarbazole with 2,4-
diaminopyrimidine scaffold as antibacterial agents
Liqiang Su, Jiahui Li, Zhen Zhou, Dongxia Huang, Yuanjin Zhang, Haixiang Pei,
Weikai Guo, Haigang Wu, Xin Wang, Mingyao Liu, Cai-Guang Yang, Yihua Chen
PII:
S0223-5234(18)30970-X
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.016
EJMECH 10873
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 December 2017
Revised Date: 25 October 2018
Accepted Date: 6 November 2018
Please cite this article as: L. Su, J. Li, Z. Zhou, D. Huang, Y. Zhang, H. Pei, W. Guo, H. Wu, X. Wang,
M. Liu, C.-G. Yang, Y. Chen, Design, synthesis and evaluation of hybrid of tetrahydrocarbazole with 2,4-
diaminopyrimidine scaffold as antibacterial agents, European Journal of Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.ejmech.2018.11.016.
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Graphical abstract
HN
N
n R2
H
HN
8a-c
Br
Hybrids
N
N
HN
N
3
N
H
H
H
H
12c
N
N
N
N
diaminopyrimidine
SAR analysis resulted in the discovery of hybrids of tetrahydrocarbazole with
2,4-diaminopyrimidine scaffold with potent antibacterial activities not only against S. aureus
strain Newman and Escherichia coli AB1157 strain but also multidrug-resistant S. aureus strains.
1

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Design, synthesis and evaluation of hybrid of
tetrahydrocarbazole with
2,4-diaminopyrimidine scaffold as
antibacterial agents
Liqiang Sua,#, Jiahui Lib, #, Zhen Zhoua , Dongxia Huanga, Yuanjin Zhanga, Haixiang
Peia, Weikai Guoa, Haigang Wua, Xin Wanga, Mingyao Liua, Cai-Guang Yangb,**,
Yihua Chena,*
a
Shanghai Key Laboratory of Regulatory Biology, The Institute of Biomedical
Sciences and School of Life Sciences, East China Normal University, Shanghai,
200241, China.
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
* To whom correspondence should be addressed:
Dr. Yihua Chen or Dr. Cai-Guang Yang
*
Phone:
+86-21-24206647.
Fax:
+86-21-54344922.
E-mail:
yhchen@bio.ecnu.edu.cn
** Phone: +86-21-50806029. Fax: +86-21-50807088. E-mail: yangcg@simm.ac.cn
#
These authors contributed equally to this work.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if
required according to the journal that you are submitting your paper to)
Abstract: Several 6-substituted tetrahydrocarbazole derivatives were designed,
synthesized and evaluated for the antibacterial activities against Staphylococcus
aureus Newman strain. Subsequently, 2,4-diaminopyrimidine scaffold was merged
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with the tetrahydrocarbazole unit to generate a series of novel hybrid derivatives and
the antibacterial activities were also investigated. Among these novel hybrids,
compound 12c showed the most potent activity with a MIC of 0.39-0.78 µg/mL
against S. aureus Newman and Escherichia coli AB1157 strain. In addition,
compound 12c exhibited low MIC values against a panel of multidrug-resistant strains
of S. aureus.
1. Introduction
The discovery of antibiotics has been vital to human survival, and has added an
average of 20 years to the human lifespan [1, 2]. Unfortunately, the overuse of
antibiotics has put pressure on bacteria to evolve resistance. Nowadays, bacteria are
resistant to almost all antibiotics [3, 4]. To make matters worse, the drug-discovery
pipeline of new antibacterial drugs is almost empty [3]. According to the World
Health Organization, infections caused by resistant bacteria have become increasingly
widespread, weakening the efficacy of antibiotics which have been regarded as the
most successful discovery in medical history [5]. This poses a significant future risk
as common infections become untreatable. Therefore, discovery of novel classes of
antibiotics that are effective against a broad range of bacteria, including those which
have developed resistance to known drugs, has become an urgent challenge [6].
Tetrahydrocarbazole is a well-known privileged skeleton, which possesses many
different biological functions such as anti-tumor [5, 7, 8], antibiotics [6, 9], analgesia
[10], anti-depression [11, 12], and anti-virus [13, 14] (2, Fig. 1). Beyond that,
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compound 1 (GSK983), as a novel tetrahydrocarbazole (Fig. 1), has shown broad
spectrum antiviral activity [14], and recently was reported to take effect through
inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase
(DHODH) [15]. Interestingly, the 2,4-diaminopyrimidine scaffold was generally
existed in bioactive small-molecule agents, including the antibiotic trimethoprim
(TMP, 3), and Bacillus anthracis inhibitor 4 (Fig. 1) [16, 17]. As for its antibiotic
effect, the diaminopyrimidine can interfere with the folic acid metabolism by binding
dihydrofolate reductase, which eventually leads to the prevention of bacterial
reproduction [16-18].
R1
Cl
N
HN
N
N
H
HN
H
O
1 (GSK983)
NH2
2
NH2
N
O
R
O
N
N
N
N
O
N
NH2 H2N
OMe
O
OMe
4
3
Fig. 1. Several molecules of tetrahydrocarbazoles and diaminopyrimidines with
antibacterial and antiviral activities.
In order to identify novel antibacterial drug candidates, we synthesized a series of
tetrahydrocarbazole derivatives and evaluated their activities. Compound 5 was
identified as a potential hit with the antibacterial activity against S. aureus Newman
with MIC values of 5.0 - 6.6 µg/mL (Fig. 2, Table 1). In an extension of our previous
study, several novel tetrahydrocarbazole derivatives (8a-c, Table 2) of compound 5
were designed and optimized with a goal of exploring the active site, necessary
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groups and suitable length of linker motifs. The in vitro antibacterial activities against
S. aureus Newman were evaluated. Subsequently, since many substituted
2,4-diaminopyrimidine derivatives have also shown to have potent antibacterial
efficacy, the structural combination of tetrahydrocarbazole and diaminopyrimidine
scaffolds might lead to several novel antibacterial compounds (Fig. 3, 12a-p, Table 2,
3). These hybrids not only could retain the excellent antibacterial activities as
observed in our previous work, but also might overcome the deficiency of
trimethoprim antibiotics which are susceptible to resistance development [19]. Five
compounds with the most potent in vitro antibacterial activities against S. aureus
Newman and E. coli AB1157 were evaluated against a panel of multidrug-resistant S.
aureus, respectively.
HN
N
H
8a-c
N
( )
4
HN
R2
H
5
Fig. 2. Simple modification of based on tetrahydrocarbazole 5.
F3CO
4
5
3
6
H
2
7
1
N
N
HN
N
N
8
H
HN
12a
or
H
R2
N
8a-c
3
4
H
H
HN
5
N
N
N
R1
6
2
1
N
N
N
HN
12b-p
N
7
n
N
H
8
H
diaminopyrimidine
Fig. 3. Hybridation and optimization based on the tetrahydrocarbazole and
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diaminopyrimidine scaffolds.
Table 1 MICs of compound 5 against Newman and multidrug-resistant S. aureus.
MIC (µg/mL)
Compound
Newman NRS-1 NRS-70 NRS-100 NRS-108
NRS-271
5.0-6.6
< 18.70
< 3.75
< 2.50
2.9-4.2
>75
2.9-4.2
> 75
2.9-4.2
< 9.38
< 1.25
< 1.56
4.2-5.8
> 75
4.2-5.8
< 9.38
< 1.25
< 1.56
5
Kanamycin
Vancomycin
Tetracycline
1.25-2.5
> 12.5
< 1.25
< 1.56
< 1.25
< 1.56
2. Chemistry
With the purpose of discovering novel antibacterial agents with higher activities, we
first synthesized tetrahydrocarbazole derivatives 8a-c as demonstrated in Scheme 1.
Intermediate 7 was constructed from p-tolylhydrazine hydrochloride and
1,2-cyclohexadione in the presence of hydrochloric acid and acetic acid using the
Fischer indole synthesis [20]. Subsequently, treatment of 7 with different primary
amines generated compounds 8a-c [21].
HCl
b, c
a
NH2
N
N
H
O
N
H
H
HN
8a-c
n=1, 2, 3
n
6
7
Scheme 1. Synthesis of compounds 8a-c. Reagents and conditions: (a)
1,2-cyclohexanedione, conc. HCl, AcOH, MeOH, 60 oC; (b) p-TsOH, amines, toluene,
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140 oC, 16 h; (c) NaBH4, MeOH, 80 oC, 4 h.
Compound 12a was prepared as described in Scheme 2. The starting material 7a was
synthesized using procedures similar to those in Scheme 1. Compound 9a was
prepared from 6-trifluoromethoxy-2,3,4,9-tetrahydro-1H-1-carbazolone by a similar
method. Debenzylation of 9a with Pd/C under H2 then afforded the key intermediate
10a, which was subsequently coupled with 2,4-dichloropyrimidine to provide 11a.
Finally, the target compound 12a was obtained by nucleophilic aromatic substitution
of chloro by isobutylamine (Scheme 2).
F3CO
a, b
F3CO
F3CO
c
NH2
N
H
O
N
N
H
HN
H
Bn
7a
9a
10a
d
F3CO
F3CO
e
N
N
HN
HN
N
H
N
H
N
N
HN
Cl
11a
12a
Scheme 2. Synthesis of compound 12a. Reagents and conditions: (a) p-TsOH,
o
benzylamine, toluene, 140 C, 16 h; (b) NaBH4, MeOH, 80 oC, 4 h; (c) Pd/C (10%),
H2, MeOH, r.t.; (d) 2,4-dichloropyrimidine, DIEA, EtOH, r.t.; (e) isobutylamine,
o
n-BuOH, 120 C.
In the process of preparing the target compounds 12b-k and 12m-p (Scheme 3), there
were just two steps different from the synthesis of compound 12a. The free amino
groups of starting compounds 7b-k were reacted with various aliphatic diamines
protected by a Boc group that was then readily cleaved under acidic conditions to give
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compounds 14b-k and 14m-p. The remaining steps paralleled to those used to prepare
12a. Compound 12l was prepared by hydrolyzed 12k with the sodium hydroxide
solution.
a
b
R
R
R
HN
N
HN
13b-k, 13m-p
O
N
N
NH HCl
n
2
NHBoc
n
H
H
H
14b-k, 14m-p
7b-k
c
Cl
N
HN
N
N
N
d
e
R
R
HN
N
H
HN
N
H
N
H
n
N
n
H
15b-k, 15m-p
12b-k, 12m-p
HN
HN
MeO2C
HO2C
N
N
N
N
HN
N
HN
N
N
N
3
3
H
H
H
H
12k
12l
Scheme 3. Synthesis of compounds 12b-p. Reagents and conditions: (a) p-TsOH,
o
o
corresponding amines, toluene, 140 C, 16 h; NaBH4, MeOH, 80 C, 4 h; (b)
o
anhydrous DCM, 1,4-dioxane, 0 C to r.t.; (c) 2,4-dichloropyrimidine, DIEA, EtOH,
r.t.; (d) isobutylamine, n-BuOH, 120 oC; (e) NaOH, EtOH.
3. Antibacterial studies
To evaluate the antibacterial activities of the synthesized tetrahydrocarbazole
derivatives, we chose the lab-cultured S. aureus Newman and Escherichia coli
AB1157 as the experimental strains, which are representatives of Gram-positive and
Gram-negative bacteria, respectively. We also investigated the activities of the
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synthetic compounds against a panel of multidrug-resistant strains of S. aureus,
including NRS-1 (resistant to aminoglycosides and tetracycline), NRS-70 (resistant to
erythromycin), NRS-100 (resistant to oxacillin and tetracycline), NRS-108 (resistant
to gentamicin), and NRS-271 (resistant to linezolid) [22]. The MIC values of these
compounds were in the range of 0.39 - 50 µg/mL.
4. Results and discussion
In this study, novel antibiotics were designed and synthesized for the treatment of
multiple S. aureus strains and E. coli AB1157. In order to demonstrate which groups
or moieties were beneficial or essential for enhancing the antibacterial activities of the
compounds, several modifications to the linker moiety of compound 5 were
performed to afford compounds 8a-c (Table 1). The results showed that the linker
with three or four methylenes may be more suitable for maintaining antibacterial
activities. In addition, the aromaticity of the group at the end of the linker is essential
for the antibacterial activities of the compounds based on our previous results.
Table 2 MICs of compounds dating from explorative modification for compound 5
against S. aureus Newman.
R1
HN
N
H
R2
Compound
R1
n
R2
MIC
µg/mL
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-CH3
-CH3
-CH3
-CH3
4
3
2
1
Ph
Ph
Ph
Ph
5.0-6.6
4.0-8.0
5
8a
8b
8c
15.02-30.2
14.5-20.9
-OCF3
0
0.78-1.56
12a
-
-
-
-
-
-
-
-
-
0.78-1.56
1.95-3.90
<0.20
Vancomycin
Kanamycin
Tetracycline
By referring to the literature, we noticed that the 2,4-diaminopyrimidine scaffold is
one of the dominant pharmacophores in antibacterial agents. Therefore, compound
12a was designed innovatively by hybridizing tetrahydrocarbazole scaffold with the
2,4-diaminopyrimidine moiety directly. As we expected, the inclusion of these rings
in 12a showed an improved antibacterial activity against S. aureus strain Newman
with a MIC of 0.78-1.56 µg/mL. A series of novel derivatives 12b-p were further
synthesized to investigate the influence of linker-length and tetrahydrocarbazole
substituents on the antibacterial activities.
Compounds 12b-g were obtained by modifying the length of the linker between
tetrahydrocarbazole skeleton and the terminal pyrimidine ring to identify the optimum
linker length. As shown in Table 3, the antibacterial effect of compounds 12b-d and
12e-g indicated a parabolic trend for two-carbon, three-carbon and four-carbon linkers,
with 12c, having a three-carbon linker exhibiting the best activity against S. aureus
Newman and E. coli AB1157 among the investigated compounds. The above studies
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illustrated that the antibacterial activity was enhanced by maintaining three methylene
group in the linker moiety of the hybrid. Therefore, in the subsequent modifications,
the linker containing three methylene groups was retained.
Table 3 MICs of synthetic hybrid derivatives against S. aureus Newman and E. coli
AB1157
HN
R1
N
N
HN
N
N
n
H
H
R1
Compound
MIC (µg/mL)
n
Newman
1.56-3.13
0.39-0.78
0.78-1.56
3.13-6.25
1.56-3.13
1.56-3.13
12.5-25.0
1.56-3.13
1.56-3.13
25.0-50.0
> 50.0
AB1157
1.56-3.13
0.39-0.78
1.56-3.13
3.13-6.25
1.56-3.13
3.13-6.25
12.5-25.0
1.56-3.13
1.56-3.13
> 50.0
6-Br
6-Br
2
3
4
2
3
4
3
3
3
3
3
3
3
3
3
-
12b
12c
6-Br
12d
6-OCF3
6-OCF3
6-OCF3
6-F
12e
12f
12g
12h
6-Cl
12i
6-CF3
6-CO2Me
6-COOH
6-OMe
6-H
12j
12k
> 50.0
12l
25.0-50.0
12.5-25.0
3.13-6.25
12.5-25.0
0.78-1.56
3.13-6.25
> 50.0
12m
12.5-25.0
6.25-12.5
12.5-25.0
> 50.0
12n
8-Br
12o
8-OMe
-
12p
Vancomycin
Kanamycin
-
6.25-12.5
-
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Compounds 12h-p were prepared by altering the substituents on the
tetrahydrocarbazole ring of the hybrid as presented in Table 3. The phenyl rings with
electron-withdrawing substituents, such as chlorine (12i), bromine (12c and 12o),
trifluoromethyl (12j) and trifluoromethoxy (12f) exhibited more potent activity with
MICs of 0.39-12.5 µg/mL, while compounds with electron-donating substituents such
as methoxy (12m and 12p) and unsubstituted compound 12n with MICs of 12.5-25.0
µg/mL. However, the fluorine substituent (12h) was an exception, showing an MIC of
12.5-25.0 µg/mL. Surprisingly, analogues with carboxyl (12l) or methyl ester (12k)
substituents showed little or no inhibition (>50 µg/mL). In addition, it seems that
tetrahydrocarbazole substitution at the 6-position with electron-withdrawing
substituent increased the inhibitory effects obviously compared to the corresponding
compounds substituted at C8 (see 12c and 12o). The SARs of these compounds
revealed that the antibacterial activity was enhanced by the introduction of small
electron-withdrawing substituents into the tetrahydrocarbazole ring at the 6-position.
The five most potent compounds against S. aureus Newman and E. coli AB1157 were
further evaluated against multidrug-resistant strains of S. aureus NRS-1, NRS-70,
NRS-100, NRS-108 and NRS-271 (Table 4). The derivatives incorporating
tetrahydro-carbazole and diaminopyrimidine moieties exhibited good antibacterial
activity in vitro, with MICs ranging from 0.39-6.25 µg/mL. Among them, compounds
12c, 12f, 12i and 12j were more effective with MICs of 0.39 -3.13 µg/mL, because all
compounds have electron-withdrawing substituents at C6, compound 12c was the
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most potent one against Newman strain and multidrug-resistant strains of S. aureus,
and its effectiveness was comparable to vancomycin. The mechanism and target of
compound 12c is unclear, however. It appears likely that the compound binds to the
bacterial DNA clamp in the previously described “Subsite I”.[9] We also suspect that
the 2,4-diaminopyrimidine moieties may be able to reach around to “Subsite II”.
Table 4 MICs of the hybrid derivatives against five multidrug-resistant strains of S.
aureus: NRS-1, NRS-70, NRS-100, NRS-108, NRS-271.
Compound
MIC (µg/mL)
NRS-100
0.78-1.56
1.56-3.13
3.13-6.25
1.56-3.13
6.25-12.5
0.78-1.56
0.39-0.78
NRS-1
0.39-0.78
1.56-3.13
1.56-3.13
1.56-3.13
3.13-6.25
1.56-3.13
> 50.0
NRS-70
0.78-1.56
1.56-3.13
3.13-6.25
1.56-3.13
6.25-12.5
0.78-1.56
> 50.0
NRS-108
0.78-1.56
1.56-3.13
1.56-3.13
1.56-3.13
6.25-12.5
0.78-1.56
> 50.0
NRS-271
0.78-1.56
1.56-3.13
1.56-3.13
1.56-3.13
6.25-12.5
0.78-1.56
6.25-12.5
12c
12f
12i
12j
12o
Vancomycin
Kanamycin
Preliminary pharmacokinetic studies of compound 12c in vivo
In order to further evaluate the potential of this series of compounds as an
antibacterial lead, compound 12c was selected to explore the preclinical
pharmacokinetic properties with oral administration, the plasma concentration in SD
rats after a single oral gavage administration at a dose of 10 mg/kg was measured, and
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pharmacokinetic parameters were calculated, as shown in Table 5. The concentration
of compound 12c reached the peak value (Cmax = 86.32 ± 25.57 ng/mL) at 3.60 ±
0.89 hr (Tmax), and the area under the curve (AUC0-t) was 1196.68 ± 238.10. The
relatively low concentration in rats may be due to the poor dissolution and absorption
rate of compound 12c, which indicate more hydrophilic groups should be introduced
to the scaffold. In addition, it possessed of a long half-life in plasma (t½ = 10.80 ±
0.89 hr). Also, the plasma clearance (CL) was 6.60 ± 1.20 L/min/kg. Which means
that the compound was not easily metabolized in the body, reminding us to pay
attention to dosing intervals in subsequent multiple dosing trials. The mean values of
the apparent volume of distribution (Vd) were quite high for oral administration,
indicating that compound 12c was widely distributed in tissues.
Table 5. Pharmacokinetic parameters of 12c after oral administration in rats (n = 5,
mean ± SD)
Dose (mg/kg)
Cmax (ng/mL)
Tmax (hr)
10
86.32 ± 25.57
3.60 ± 0.89
t
½
(hr)
10.80 ± 0.57
1196.68 ± 238.10
1557.25 ± 298.17
103.15 ± 21.46
6.60 ± 1.20
AUC0-t (hr*ng/mL)
AUC0-∞ (hr*ng/mL)
Vd (L/kg)
CL (L/min/kg)
MRT (min)
16.19 ± 0.82
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Conclusion
In the initial screening of a small molecule library, a tetrahydrocarbazole compound 5
displayed antibacterial activity and classified as a hit compound. A series of new
analogues and hybrids bearing the 2,4-diaminopyridimine scaffold were designed and
synthesized based on hit compound 5 and investigated on the antibacterial activity in
vitro against S. aureus Newman and E. coli AB1157. Several potent compounds were
further evaluated against five multidrug-resistant S. aureus strains (NRS-1, NRS-70,
NRS-100, NRS-108 and NRS-271). The structure-activity relationship analysis
indicated that the linker and the electric properties of substituents on the terminal
aromatic ring had a significant influence on the antibacterial activities. Compound 12c
exhibited excellent activity. For Gram-positive bacterium S. aureus Newman and
Gram-negative bacterium E. coli AB1157, 12c showed highest activity with a MIC of
0.39-0.78 µg/mL; for multidrug-resistant bacteria of S. aureus, 12c displayed the most
potent activity with a MIC of 0.39-1.56 µg/mL. Here we offered a series of promising
novel hybrid of tetrahydrocarbazole and diaminopyrimidine derivatives for the
discovery of new chemical entities against multidrug-resistant bacteria. Further
in-depth SAR studies of the active scaffolds and the mechanism (of action) are also
required.
6. Experiment section
6.1 General methods for chemistry
All reagents and chemicals used in experiments were purchased from Adamas-beta
and Aladdin Reagents Inc., Sigma-Aldrich Inc., Bide Pharmatech Ltd., and J&K
Scientific Ltd.. Unless otherwise indicated, all reagents were used as received. All
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reactions were carried out with standard techniques under an inert atmosphere (Ar or
N2). Column chromatography was performed on silica gel (Qingdao, 200-300 mesh)
using the indicated eluents. Preparative thin layer chromatography was carried out on
200 mm × 200 mm plate coated with 0.4-0.5 mm of silica gel. NMR spectra were
collected on a Bruker 300 or 500 MHz instrument using CDCl3 or DMSO-d6;
High-resolution mass data were obtained on an Agilent 6550 iFunnel Q-TOF LC/MS
System. HPLC was employed for purity detection with an Agilent Technologies 1200
Series, using the following method: Eclipse XDB C18 column, 5 µm, 4.6 mm × 150
mm, column temperature 40 °C; solvent A: ultrapure water (with 0.1% triethylamine),
solvent B: MeOH (with 0.1% trimethylamine); flow rate = 1.5 mL/min. The
biological activities of compounds were tested on the assumption that compound
purity was more than 95%.
6.2. Synthesis of 6-methyl-1-(4-phenylbutylamino)-1H-2,3,4,9-tetrahydrocarbazole
(5)
To a solution of 1,2-cyclohexanedione (2243 mg, 20 mmol) and concentrated
hydrochloric acid (13 mL) in acetic acid (40 mL), p-tolylhydrazine hydrochloride
(1586 mg, 10 mmol) in methanol (25 mL) was added dropwise slowly over 10 mins.
After the addition, the resulting mixture was heated to 60 °C, and stirred overnight.
The solvent was evaporated, and the residue was pH adjusted to weak alkaline with
saturated NaHCO3. The mixture was extracted with AcOEt (3 × 20 mL). The
combined organic extract was washed with brine, dried over anhydrous Na2SO4, and
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concentrated. The residue was purified by silica gel chromatography (petroleum
ether/AcOEt, 12/1 v/v) to give intermediate 7 (1235 mg, 62%) as a brown powder.
The mixture of intermediate 7 (102 mg, 0.5 mmol), 4-phenylbutylamine (0.12 mL, 0.8
mmol) and catalytic p-TsOH in toluene (10 mL) was refluxed at 140 oC for 16 h with
a Dean-Stark trap in place. The solvent was evaporated and the residue was dissolved
in methanol. NaBH4 (177 mg) was then added at 0 oC. The solution was heated to 80
oC until TLC indicated the reaction was complete. The reaction was quenched with
water and concentrated. Subsequently, the mixture was extracted with AcOEt twice
and the combined organic layers were dried over anhydrous Na2SO4. After
evaporation of the solvent, the resulting residue was purified by column
chromatography on silica gel (petroleum ether/AcOEt, 4/1 v/v) to give compound 5
1
(123 mg, Yield: 72%). H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 7.33 – 7.27 (m,
2H), 7.26 – 7.23 (m, 1H), 7.23 – 7.11 (m, 4H), 6.99 – 6.92 (m, 1H), 3.97 – 3.85 (m,
1H), 2.89 – 2.77 (m, 1H), 2.71 – 2.63 (m, 4H), 2.63 – 2.59 (m, 1H), 2.44 (s, 3H), 2.28
13
– 2.14 (m, 1H), 2.08 – 1.95 (m, 1H), 1.83 –1.65 (m, 4H), 1.63–1.48 (m, 4H). C
NMR (125 MHz, DMSO-d6) δ 142.28, 136.74, 134.21, 128.29, 128.18, 127.10,
126.27, 125.57, 121.96, 117.38, 110.68, 108.76, 51.61, 46.07, 35.22, 29.72, 29.34,
28.92, 21.27, 20.89, 20.76. HR MS (ESI): calcd for C23H29N2 [M + H]+; 333.2331;
found 333.2328.
6.3. Syntheses of compounds 8a-c
6.3.1. 6-Methyl-1-(3-phenylpropylamino)-1H-2,3,4,9-tetrahydrocarbazole (8a)
16

ACCEPTED MANUSCRIPT
The title compound 8a was obtained according to the procedure for compound 5
1
except utilizing 3-phenylpropylamine instead of 4-phenylbutylamine. Yield: 71%. H
NMR (300 MHz, CDCl3) δ 8.15 (s, 1H), 7.38 – 7.27 (m, 2H), 7.25 – 7.10 (m, 4H),
6.97 (d, J = 8.2 Hz, 1H), 3.96 – 3.87 (m, 1H), 2.92 – 2.78 (m, 1H), 2.76 – 2.57 (m,
5H), 2.45 (s, 3H), 2.29 – 1.95 (m, 2H), 1.92 – 1.68 (m, 3H), 1.66 – 1.57 (m, 1H), 1.27
13
(s, 1H). C NMR (125 MHz, DMSO-d6) δ 142.33, 136.89, 134.20, 128.31, 128.19,
127.11, 126.25, 125.55, 121.94, 117.37, 110.67, 108.69, 51.57, 45.67, 33.06, 32.04,
29.42, 21.27, 20.89, 20.71. HR MS (ESI): calcd for C22H27N2 [M + H]+; 319.2174;
found 319.2172.
6.3.2. 6-Methyl-1-(2-phenylethylamino)-1H-2,3,4,9-tetrahydrocarbazole (8b)
The title compound 8b was obtained according to the procedure for compound 5
except utilizing 2-phenylethylamine instead of 4-phenylbutylamine. Yield: 51%. 1H
NMR (300 MHz, CDCl3) δ 8.00 (s, 1H), 7.38 – 7.19 (m, 6H), 7.14 (d, J = 8.2 Hz, 1H),
6.95 (dd, J = 8.6, 0.8 Hz, 1H), 4.01 – 3.90 (m, 1H), 3.06 – 2.91 (m, 2H), 2.68 – 2.56
(m, 2H), 2.43 (s, 3H), 2.17 – 2.07 (m, 1H), 2.06 – 1.91 (m, 1H), 1.82 – 1.64 (m, 2H),
1.63 – 1.57 (m, 3H). 13C NMR (125 MHz, DMSO-d6) δ 140.41, 136.52, 134.19,
128.57, 128.25, 127.03, 126.30, 125.85, 122.03, 117.40, 110.71, 108.80, 51.36, 48.20,
36.50, 29.37, 21.25, 20.81, 20.50. HR MS (ESI): calcd for C21H25N2 [M + H]+;
305.2018; found 305.2011.
6.3.3. 6-Methyl-1-benzylamino-1H-2,3,4,9-tetrahydrocarbazole (8c)
The title compound 8c was obtained according to the procedure for compound 5
1
except utilizing benzylamine instead of 4-phenylbutylamine. Yield: 50%. H NMR
17

ACCEPTED MANUSCRIPT
(300 MHz, CDCl3) δ 8.12 (s, 1H), 7.45 – 7.13 (m, 7H), 6.95 (d, J = 8.2 Hz, 1H), 4.00
(d, J = 12.9 Hz,1H), 3.98 (s, 1H), 3.86 (d, J = 12.9 Hz, 1H), 2.73 – 2.59 (m, 2H), 2.43
(s, 3H), 2.30 (dd, J = 12.0, 5.1 Hz, 1H), 2.14 – 1.98 (m, 1H), 1.86 – 1.60 (m, 3H). 13C
NMR (125 MHz, DMSO-d6) δ 141.29, 136.82, 134.29, 128.10, 128.02, 127.17,
126.52, 126.39, 122.06, 117.44, 110.77, 108.97, 51.28, 49.95, 29.41, 21.31, 20.97,
20.92. HR MS (ESI): calcd for C20H23N2 [M + H]+; 291.1861; found 291.1856.
6.4. Synthesis of compounds 12a-p
6.4.1.
6-Trifluoromethoxy-1-((2-isobutylaminopyrimidine)-4-amino)-1H-2,3,4,9-tetrahydro
carbazole (12a)
To a solution of intermediate 10a (132 mg, 0.5 mmol) in EtOH (5 mL) at r.t. was
added 2,4-dichloropyrimidine (112 mg, 0.75 mmol) and DIEA (0.25 mL) and the
reaction was stirred for 8 h. Upon completion, the mixture was quenched with water,
concentrated, and extracted with AcOEt (3 × 20 mL). The combined organic extracts
were washed with brine, dried over Na2SO4, and concentrated to give crude
intermediate 11a (48 mg). A solution of intermediate 11a (48 mg, 0.13 mmol),
o
isobutylamine (0.13 mL), DIEA (67 µL) in n-butanol (3 mL) was heated at 120 C
overnight. Upon completion, the title compound 12a was obtained by the
post-processing steps which were the same as above. Yield: 67%. 1H NMR (500 MHz,
DMSO-d6) δ 12.26 (s, 1H), 7.77 (d, J = 6.0 Hz, 1H), 7.47 – 7.32 (m, 2H), 7.04 (d, J =
8.5 Hz, 1H), 6.09 (d, J = 6.0 Hz, 1H), 5.43 (d, J = 5.5 Hz, 1H), 3.30 – 3.10 (m, 2H),
18

ACCEPTED MANUSCRIPT
3.10 – 3.01 (m, 1H), 2.78 – 2.59 (m, 2H), 2.09 (s, 1H), 1.94 (s, 2H), 1.90 – 1.78 (m,
2H), 1.25 – 1.14 (m, 1H), 0.90 (d, J = 5.5 Hz, 6H). 13C NMR (125 MHz, DMSO-d6):
δ 161.62, 154.15, 141.46, 135.13, 134.57, 126.51, 121.49, 119.47, 114.81, 112.15,
111.54, 110.42, 97.18, 47.64, 45.30, 44.47, 29.36, 27.76, 20.34, 19.95. HR MS (ESI):
calcd for C21H25F3N5O [M + H]+; 420.2011; found 420.2004.
6.4.2.
6-Bromo-1-((2-isobutylaminopyrimidine)-4-aminoethyl)amino-1H-2,3,4,9-tetrahydro
carbazole (12b)
Intermediate 7a was synthesized according to the procedure for compound 5 except
utilizing
4-bromophenylhydrazine
instead
of
p-tolylhydrazine,
N-Boc-ethylene-diamine instead of 4-phenylbutylamine. Compound 14b was
synthesized after removal of Boc group with 4.6 N hydrogen chloride dissolved in
1,4-dioxane. The remaining steps are essentially the same as for the conversion of 12a
1
to 12b. Yield: 11%. H NMR (500 MHz, DMSO-d6) δ 10.87 (s, 1H), 7.61 (s, 1H),
7.51 (d, J = 1.5 Hz, 1H), 7.26 (d, J = 8.5 Hz, 1H), 7.11 (dd, J = 8.5, 1.8 Hz, 1H), 6.78
(s, 1H), 6.35 (s, 1H), 5.70 (d, J = 5.7 Hz, 1H), 3.89 (s, 1H), 3.45 – 3.35 (m, 2H), 3.00
(t, J = 6.4 Hz, 2H), 2.85 – 2.71 (m, 2H), 2.55 (m, 2H), 2.07 – 1.92 (m, 2H), 1.85 –
1.74 (m, 1H), 1.73 – 1.64 (m, 2H), 1.25 (m, 1H), 0.83 (d, J = 6.7 Hz, 6H). 13C NMR
(125 MHz, DMSO-d6): δ 162.61, 162.16, 154.46, 138.49, 134.47, 128.70, 122.84,
119.98, 112.89, 110.62, 109.23, 95.08, 62.80, 51.21, 48.28, 45.45, 29.33, 27.89, 21.32,
20.56, 20.29. HR MS (ESI): calcd for C22H30BrN6 [M + H]+; 457.1715; found
457.1759.
19

ACCEPTED MANUSCRIPT
6.4.3.
6-Bromo-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahydr
ocarbazole (12c)
The title compound 12c was synthesized according to the procedure for compound
12b except utilizing N-Boc-propylene-1,3-diamine instead of N-Boc-ethylenediamine.
Yield: 47%. 1H NMR (500 MHz, DMSO-d6) δ 10.85 (s, 1H), 7.60 (s, 1H), 7.52 (d, J =
1.5 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 7.12 (dd, J = 8.5, 2.0 Hz, 1H), 6.85 (s, 1H),
6.35 (s, 1H), 5.68 (d, J = 5.5 Hz, 1H), 3.91 (s, 1H), 2.98 (t, J = 6.5 Hz, 2H), 2.79 –
2.62 (m, 2H), 2.57 (s, 2H), 2.04 – 1.89 (m, 3H), 1.82 – 1.63 (m, 5H), 1.24 (s, 2H),
0.83 (d, J = 7.0 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 162.36, 149.93, 134.59,
130.42, 130.26, 127.87, 124.57, 120.83, 113.41, 112.93, 111.33, 96.48, 54.95, 50.75,
47.97, 42.58, 27.78, 25.89, 25.65, 20.33, 20.11, 19.66. HR MS (ESI): calcd for
C23H32BrN6 [M + H]+; 471.1872; found 471.1867.
6.4.4.
6-Bromo-1-((2-isobutylaminopyrimidine)-4-aminobutyl)amino-1H-2,3,4,9-tetrahydro
carbazole (12d)
The title compound 12d was synthesized according to the procedure for compound
12b except utilizing N-Boc-butylene-1,4-diamine instead of N-Boc-ethylenediamine.
Yield: 14%. 1H NMR (500 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.63 (m, 2H), 7.34 (d, J
= 8.6 Hz, 1H), 7.21 (dd, J = 8.6, 1.8 Hz, 1H), 5.77 (d, J = 6.0 Hz, 1H), 4.39 (s, 1H),
3.39 (s, 1H), 3.28 (s, 2H), 3.12 – 2.88 (m, 4H), 2.73 – 2.56 (m, 3H), 2.16 – 2.06 (m,
1H), 2.04 – 1.95 (m, 2H), 1.85 – 1.74 (m, 2H), 1.67 (m, 2H), 1.59 (d, J = 6.4 Hz, 2H),
20

ACCEPTED MANUSCRIPT
13
1.27 – 1.21 (m, 1H), 0.85 (d, J = 6.7 Hz, 6H). C NMR (125 MHz, DMSO-d6): δ
162.45, 152.79, 134.56, 131.65, 128.01, 124.32, 120.72, 115.72, 113.35, 112.30,
111.23, 95.90, 62.81, 50.81, 48.19, 44.73, 27.85, 26.28, 26.19, 24.30, 20.25, 20.17,
19.67. HR MS (ESI): calcd for C24H34BrN6 [M + H]+; 485.2028; found 485.2023.
6.4.5.
6-Trifluoromethoxy-1-((2-isobutylaminopyrimidine)-4-aminoethyl)amino-1H-2,3,4,9-
tetra-hydrocarbazole (12e)
The title compound 12e was synthesized according to the procedure for compound
1
12b. Yield: 23%. H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.64 (s, 1H), 7.39
(d, J = 8.8 Hz, 1H), 7.34 (s, 1H), 7.01 (d, J = 8.6 Hz, 1H), 5.80 (d, J = 5.9 Hz, 1H),
4.09 (s, 1H), 3.50 – 3.35 (m, 2H), 3.02 (t, J = 5.8 Hz, 2H), 2.95 – 2.79 (m, 2H), 2.61
(s, 2H), 2.03 (m, 1H), 1.91 (s, 1H), 1.82 – 1.69 (m, 3H), 1.37 – 1.19 (m, 3H), 0.84 (d,
J = 6.5 Hz, 6H). 13C NMR (125 MHz, DMSO-d6): δ 162.57, 154.23, 141.65, 134.36,
130.34, 126.10, 121.45, 119.42, 115.88, 114.17, 112.50, 110.99, 97.11, 50.78, 47.46,
45.62, 42.68, 27.82, 25.42, 20.06, 19.86 ,19.75. HR MS (ESI): calcd for C23H30F3N6O
[M + H]+; 463.2433; found 463.2423.
6.4.6.
6-Trifluoromethoxy-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,
9-tetrahydrocarbazole (12f)
The title compound 12f was synthesized according to the procedure for compound
12c
except
utilizing
4-trifluoromethoxyphenylhydrazine
instead
of
4-bromophenyl-hydrazine. Yield: 49%. 1H NMR (500 MHz, DMSO-d6) δ 11.02 (s,
21

ACCEPTED MANUSCRIPT
1H), 7.61 (s, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.33 (s, 1H), 7.00 (d, J = 9.0 Hz, 1H), 5.70
(d, J = 5.5 Hz, 1H), 4.03 (s, 2H), 2.98 (t, J = 6.5 Hz, 2H), 2.76 (m, 2H), 2.65 – 2.56
(m, 3H), 1.91 (s, 3H), 1.83 – 1.64 (m, 4H), 1.25 (m, 4H),0.82 (d, J = 6.5 Hz, 6H). 13C
NMR (125 MHz, DMSO-d6): δ 162.40, 154.61, 141.64, 134.35, 130.46, 126.08,
121.44, 119.42, 115.84, 114.03, 112.52, 110.95, 96.88, 54.92, 50.68, 47.69, 42.40,
27.72, 26.36, 25.41, 20.09, 19.99, 19.58. HR MS (ESI): calcd for C24H32F3N6O [M +
H]+; 477.2590; found 477.2586.
6.4.7.
6-Trifluoromethoxy-1-((2-isobutylaminopyrimidine)-4-aminobutyl)amino-1H-2,3,4,9
-tetrahydrocarbazole (12g)
The title compound 12g was synthesized according to the procedure for compound
12d
except
utilizing
4-trifluoromethoxyphenylhydrazine
instead
of
1
4-bromophenyl-hydrazine. Yield: 19%. H NMR (500 MHz, DMSO-d6) δ 11.74 (s,
1H), 9.03 (s, 1H), 8.24 (s, 1H), 7.68 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 9.1
Hz, 1H), 6.08 (d, J = 6.3 Hz, 1H), 4.61 (t, J = 5.3 Hz, 1H), 3.39 (s, 2H), 3.17 (s, 2H),
3.09 – 3.00 (m, 2H), 2.74 – 2.61 (m, 2H), 2.23 – 2.00 (m, 3H), 1.89 – 1.75 (m, 4H),
13
1.65 (s, 2H), 1.33 – 1.21 (m, 1H), 0.90 (d, J = 6.7 Hz, 6H). C NMR (125 MHz,
DMSO-d6): δ 162.19, 154.21, 141.66, 134.34, 130.41, 126.10, 121.46, 119.44, 115.86,
114.07, 112.53, 110.97, 96.94, 62.82, 50.64, 47.63, 44.00, 27.75, 25.45, 25.31, 23.26,
20.09, 19.99, 19.59. HR MS (ESI): calcd for C25H34F3N6O [M + H]+; 491.2746; found
491.2734.
6.4.8.
22

ACCEPTED MANUSCRIPT
6-Fluoro-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahydr
ocarbazole (12h)
The title compound 12h was synthesized according to the procedure for compound
12c except utilizing 4-fluorophenylhydrazine instead of 4-bromophenylhydrazine.
1
Yield: 37%. H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 7.75 – 7.54 (m, 2H),
7.38 (dd, J = 8.7, 4.3 Hz, 1H), 7.28 – 7.22 (m, 1H), 7.02 – 6.94 (m, 1H), 5.98 (s, 1H),
4.22 (t, J = 6.5 Hz, 1H), 3.14 – 2.96 (m, 4H), 2.71 – 2.56 (m, 2H), 2.23 – 2.10 (m, 2H),
2.05 – 1.92 (m, 4H), 1.87 – 1.72 (m, 3H), 1.67 – 1.60 (m, 1H), 1.41 – 1.33 (m, 1H),
0.87 (d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, DMSO-d6): δ δ 162.30, 157.74, 155.89,
154.62, 132.60, 130.05, 126.22, 113.69, 112.48, 110.44, 103.36, 96.90, 61.83, 50.80,
47.75, 42.45, 29.05, 27.77, 25.46, 20.23, 20.05, 19.63. HR MS (ESI): calcd for
C23H32FN6 [M + H]+; 411.2672; found 411.2661.
6.4.9.
6-Chloro-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahydr
ocarbazole (12i)
The title compound 12i was synthesized according to the procedure for compound 12c
except utilizing 4-chlorophenylhydrazine instead of 4-bromophenylhydrazine. Yield:
1
53%. H NMR (500 MHz, DMSO-d6) δ 10.85 (s, 1H), 7.59 (s, 1H), 7.37 (d, J = 1.7
Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.00 (dd, J = 8.5, 1.9 Hz, 1H), 6.86 (s, 1H), 6.37 (s,
1H), 5.67 (s, 1H), 3.86 (s, 1H), 2.97 (t, J = 6.2 Hz, 2H), 2.79 – 2.59 (m, 3H), 2.56 (s,
2H), 2.05 – 1.88 (m, 3H), 1.81 – 1.73 (m, 1H), 1.73 – 1.61 (m, 4H), 1.23 (s, 1H), 0.81
(d, J = 6.2 Hz, 6H). 13C NMR (125 MHz, DMSO-d6): δ 162.59, 162.24, 154.89,
23

ACCEPTED MANUSCRIPT
138.61, 134.25, 127.96, 122.65, 120.26, 116.92, 112.41, 109.27, 99.50, 59.74, 51.45,
48.30, 43.87, 29.93, 29.09, 27.85, 20.59, 20.51, 20.26. HR MS (ESI): calcd for
C23H32ClN6 [M + H]+; 427.2377; found 427.2364.
6.4.10.
6-Trifluoromethyl-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-
tetrahydrocarbazole (12j)
The title compound 12j was synthesized according to the procedure for compound
12c
except
utilizing
4-trifluoromethylphenylhydrazine
instead
of
1
4-bromophenylhydrazine. Yield: 44%. H NMR (500 MHz, DMSO-d6) δ 11.76 (s,
1H), 8.53 (s, 1H), 7.88 (s, 1H), 7.66 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.6
Hz, 1H), 5.94 (s, 1H), 4.56 (s, 1H), 3.41 (s, 2H), 3.07 (s, 4H), 2.72 (m, 2H), 2.20 –
2.02 (m, 3H), 1.99 (m, 2H), 1.87 – 1.75 (m, 2H), 1.24 (s, 2H), 0.86 (d, J = 6.7 Hz, 6H).
13C NMR (125 MHz, DMSO-d6): δ 162.39, 154.13, 137.38, 126.63, 125.42, 124.47,
119.74, 119.50, 118.42, 116.16, 114.15, 112.11, 96.37, 54.92, 50.71, 45.62, 42.64,
27.77, 26.37, 25.80, 20.10, 19.75, 19.65. HR MS (ESI): calcd for C24H32F3N6 [M +
H]+; 461.2641; found 461.2621.
6.4.11.
6-Methoxylcarbonyl-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,
9-tetrahydrocarbazole (12k)
The title compound 12k was synthesized according to the procedure for compound
12c except utilizing methyl 4-hydrazinylbenzoate instead of 4-bromophenylhydrazine.
1
Yield: 70%. H NMR (500 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.10 – 8.07 (m, 1H ),
24

ACCEPTED MANUSCRIPT
7.84 (s, 1H), 7.73 – 7.63 (m, 1H), 7.60 (d, J = 7.3 Hz, 1H), 7.45 – 7.35 (m, 1H), 5.70
(s, 1H), 4.04 (s, 1H), 3.89 – 3.78 (m, 3H), 2.97 (s, 2H), 2.83 (s, 2H), 2.64 (s, 2H), 1.98
– 1.84 (m, 4H), 1.81 – 1.66 (m, 4H), 1.32 – 1.18 (m, 3H), 0.81 (d, J = 6.1 Hz, 6H).
13C NMR (125 MHz, DMSO-d6) δ 167.08, 166.60, 162.23, 138.46, 129.93, 125.62,
123.20, 121.06, 120.62, 120.32, 114.93, 111.41, 96.89, 60.16, 57.65, 51.69, 50.65,
42.52, 28.96, 27.72, 25.31, 19.98, 19.60, 19.56. HR MS (ESI): calcd for C25H35N6O2
[M + H]+; 451.2821; found 451.2824.
6.4.12.
6-Carboxy-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahy
drocarbazole (12l)
To a solution of compound 12k (301 mg, 0.67 mmol) in 12 mL of ethanol was added
the aqueous sodium hydroxide solution (134 mg in 4 mL of H2O) and the reaction
mixture was stirred overnight. The reaction was then acidized with 1.0 N of
hydrochloric acid solution. The mixture was extracted with AcOEt twice and the
combined organic layers were dried over anhydrous Na2SO4. After evaporation of the
solvent, the resulting residue was purified by column chromatography on silica gel to
1
give the title compound 12l (64 mg, yield: 37%). H NMR (500 MHz, DMSO-d6) δ
12.37 (s, 1H), 11.76 (s, 1H), 8.81 (s, 1H), 8.16 (s, 1H), 8.11 – 7.91 (m, 1H), 7.76 (dd,
J = 8.6, 1.6 Hz, 1H), 7.68 (d, J = 4.1 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 6.02 (d, J =
2.0 Hz, 1H), 4.59 (s, 1H), 3.59 – 3.41 (m, 2H), 3.23 – 2.95 (m, 4H), 2.81 – 2.61 (m,
2H), 2.26 – 2.08 (m, 2H), 2.07 – 1.95 (m, 2H), 1.89 – 1.69 (m, 2H), 1.25 – 1.21 (m,
13
2H), 0.88 (d, J = 6.7 Hz, 6H). C NMR (125 MHz, DMSO-d6) δ 172.06, 168.37,
25

ACCEPTED MANUSCRIPT
162.34, 155.70, 138.35, 129.91, 125.62, 123.49, 121.59, 121.17, 114.75, 111.14,
96.69, 62.82, 50.73, 47.84, 42.51, 29.02, 27.78, 25.47, 21.16, 20.08, 19.66. HR MS
(ESI): calcd for C24H33N6O2 [M + H]+; 437.2665; found 437.2645.
6.4.13.
6-Methoxy-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahy
drocarbazole (12m)
The title compound 12m was synthesized according to the procedure for compound
12c except utilizing 4-methoxyphenylhydrazine instead of 4-bromophenylhydrazine.
1
Yield: 46%. H NMR (500 MHz, CDCl3) δ 10.31 (s, 1H), 8.73 (s, 1H), 7.64 (s, 1H),
6.95 – 6.61 (m, 2H), 6.14 (s, 1H), 5.05 – 4.81 (m, 1H), 4.67 (s, 1H), 3.81 (s, 2H), 3.71
– 3.60 (m, 1H), 3.49 (s, 2H), 3.14 (s, 2H), 3.05 – 2.88 (m, 2H), 2.68 (s, 2H), 2.36 –
13
2.19 (m, 3H), 1.83 (m, 2H), 1.61 (m, 1H), 1.28 (s, 3H), 0.91 (d, J = 6.1 Hz, 6H). C
NMR (125 MHz, DMSO-d6): δ 162.28, 153.24, 142.30, 131.01, 129.63, 128.47,
126.28, 113.18, 112.39, 112.11, 100.25, 96.74, 55.33, 54.93, 50.90, 47.74, 42.27,
27.74, 25.47, 22.09, 20.33, 20.03, 19.78. HR MS (ESI): calcd for C24H35N6O [M +
H]+; 423.2872; found 423.2862.
6.4.14.
1-((2-Isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahydro-carbazo
le (12n)
The title compound 12n was synthesized according to the procedure for compound
12c except utilizing phenylhydrazine instead of 4-bromophenylhydrazine. Yield: 8%.
1H NMR (500 MHz, DMSO-d6) δ 11.29 (s, 1H), 8.39 (s, 1H), 7.66 (s, 1H), 7.48 (d, J
26

ACCEPTED MANUSCRIPT
= 7.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.17 – 7.11 (m, 1H), 7.05 – 6.99 (m, 1H),
5.91 (s, 1H), 4.56 (s, 1H), 3.41 (s, 2H), 3.14 – 3.01 (m, 4H), 2.75 – 2.58 (m, 2H), 2.22
– 1.93 (m, 5H), 1.87 – 1.74 (m, 2H), 1.27 – 1.21 (m, 1H), 0.87 (d, J = 6.7 Hz, 6H).
13C NMR (125 MHz, DMSO-d6) δ 162.41, 152.41, 145.77, 135.93, 128.29, 126.02,
122.23, 118.83, 118.49, 113.26, 111.38, 96.10, 57.49, 50.87, 47.99, 42.42, 27.79,
25.85, 25.63, 20.27, 20.14, 19.77. HR MS (ESI): calcd for C23H33N6 [M + H]+;
393.2767; found 393.2763.
6.4.15.
8-Bromo-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahydr
ocarbazole (12o)
The title compound 12o was synthesized according to the procedure for compound
12c except utilizing 2-bromophenylhydrazine instead of 4-bromophenylhydrazine.
Yield: 25%. 1H NMR (500 MHz, CDCl3) δ 10.67 (s, 1H), 8.74 – 8.54 (m, 1H), 7.77 –
7.60 (m, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 6.94 – 6.87 (m, 1H),
6.06 (br s, 1H), 4.63 (s, 1H), 3.56 – 3.40 (m, 2H), 3.23 – 3.08 (m, 2H), 3.07 – 2.94 (m,
2H), 2.75 – 2.61 (m, 2H), 2.33 – 2.21 (m, 3H), 2.15 – 2.08 (m, 2H), 2.03 – 1.96 (m,
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1H), 1.91 – 1.71 (m, 3H), 1.65 – 1.56 (m, 1H), 0.91 (d, J = 6.0 Hz, 6H). C NMR
(125 MHz, DMSO-d6): δ 162.32, 154.89, 134.60, 129.40, 127.56, 124.70, 120.38,
118.07, 114.94, 108.34, 104.17, 96.67, 54.93, 50.56, 47.74, 41.82, 27.75, 25.58, 24.92,
22.08, 20.35, 20.05. HR MS (ESI): calcd for C23H32BrN6 [M + H]+; 471.1872; found
471.1866.
6.4.16.
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ACCEPTED MANUSCRIPT
8-Methoxy-1-((2-isobutylaminopyrimidine)-4-aminopropyl)amino-1H-2,3,4,9-tetrahy
drocarbazole (12p)
The title compound 12p was synthesized according to the procedure for compound
12c except utilizing 2-methoxyphenylhydrazine instead of 4-bromophenylhydrazine.
Yield: 18%. 1H NMR (500 MHz, DMSO-d6) δ 11.07 (s, 1H), 7.67 (m, 1H), 7.07 (d, J
= 7.9 Hz, 1H), 6.95 (dd, J = 7.9, 7.7 Hz, 1H), 6.72 (d, J = 7.7 Hz, 1H), 6.03 (d, J = 6.3
Hz, 1H), 3.91 (s, 2H), 3.46 (m, 2H), 3.16 (m, 2H), 3.08 (m, 2H), 2.71 – 2.61 (m, 2H),
2.20 – 2.11 (m, 2H), 2.04 (m, 4H), 1.82 (m, 2H), 1.41 – 1.32 (m, 1H), 0.89 (d, J = 6.7
13
Hz, 6H). C NMR (125 MHz, DMSO-d6) δ 162.19, 153.91, 145.93, 140.61, 127.30,
126.14, 119.49, 114.04, 111.20, 102.82, 96.98, 55.24, 54.95, 50.71, 47.54, 41.80,
37.70, 27.68, 25.07, 20.39, 20.13, 19.94. HR MS (ESI): calcd for C24H35N6O [M +
H]+; 423.2872; found 423.2868.
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation
of China (81673304 to Y.C., and 81661138004 and 21725801 to C.Y.), the
Innovation
Program
of
Shanghai
Municipal
Education
Commission
(2017-01-07-00-05-E00011 to M.L.) and the Science and Technology Commission of
Shanghai Municipality (15431902200 to Y.C. and 17XD1404400 to C.Y.).
References
[1] M.E. Hulscher, J.W. van der Meer, R.P. Grol, Antibiotic use: how to improve it?,
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