Aminopropyl carbazole analogues as potent enhancers of neurogenesis

Article abstract of DOI:10.1016/j.bmc.2013.08.066

Neural stem cells are multipotent and self-renewing cells that can differentiate into new neurons and hold great promise for treating various neurological disorders including multiple sclerosis, Parkinson's disease, and Alzheimer's disease. Small molecules that can trigger neurogenesis and neuroprotection are particularly useful not only because of their therapeutic implications but also because they can provide an invaluable tool to study the mechanisms of neurogenesis. In this report, we have developed and screened 25 aminopropyl carbazole derivatives that can enhance neurogenesis of cultured neural stem cells. Among these analogues, compound 9 demonstrated an excellent proneurogenic and neuroprotective activity with no apparent toxicity. We believe that compound 9 can serve as an excellent lead to develop various analogues and to study the underlying mechanisms of neurogenesis.

Full text of DOI:10.1016/j.bmc.2013.08.066

Accepted Manuscript
Aminopropyl Carbazole Analogues as Potent Enhancers of Neurogenesis
Hye Jin Yoon, Sun-Young Kong, Min-Hye Park, Yongsung Cho, Sung-Eun
Kim, Jae Yeon Shin, Sung Hye Jung, Jiyoun Lee, Farhanullah, Hyun-Jung Kim,
Jeewoo Lee
PII:
S0968-0896(13)00769-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.066
BMC 11082
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 August 2013
28 August 2013
29 August 2013
Please cite this article as: Yoon, H.J., Kong, S-Y., Park, M-H., Cho, Y., Kim, S-E., Shin, J.Y., Jung, S.H., Lee, J.,
Farhanullah, Kim, H-J., Lee, J., Aminopropyl Carbazole Analogues as Potent Enhancers of Neurogenesis,
Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.066
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Aminopropyl Carbazole Analogues as Potent
Enhancers of Neurogenesis
b
a
a
Hye Jin Yoon a,#, Sun-Young Kong b,#, Min-Hye Park , Yongsung Cho , Sung-Eun Kim , Jae
Yeon Shin b, Sung Hye Jung b, Jiyoun Lee c, Farhanullah d, Hyun-Jung Kim b,*, and Jeewoo Lee
a,
*
a
Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 151-742, Korea
b
Laboratory of Molecular and Stem Cell Pharmacology, College of Pharmacy, Chung-Ang
University, Seoul 156-756, Korea
c Department of Global Medical Science, Sungshin Women’s University, Seoul 142-732, Korea
d Medicinal Chemistry Division, GVK Biosciences Pvt Ltd, Hyderabad 500076, India
# These authors contributed equally
* To whom correspondence should be addressed.
Prof. Jeewoo Lee
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
Prof. Hyun-Jung Kim
Phone: 82-2-820-5619
Fax: 82-2-816-7338
E-mail: hyunjungkim@cau.ac.kr
1

Keywords: Neurogenesis, neuroprotective agents, neurodegenerative diseases, aminopropyl
carbazole, neural stem cells
Abstract
Neural stem cells are multipotent and self-renewing cells that can differentiate into new
neurons and hold great promise for treating various neurological disorders including multiple
sclerosis, Parkinson’s disease, and Alzheimer’s disease. Small molecules that can trigger
neurogenesis and neuroprotection are particularly useful not only because of their therapeutic
implications but also because they can provide an invaluable tool to study the mechanisms of
neurogenesis. In this report, we have developed and screened 25 aminopropyl carbazole
derivatives that can enhance neurogenesis of cultured neural stem cells. Among these analogues,
compound 9 demonstrated an excellent proneurogenic and neuroprotective activity with no
apparent toxicity. We believe that compound 9 can serve as an excellent lead to develop various
analogues and to study the underlying mechanisms of neurogenesis.
2

1. Introduction
Neural stem cells (NSCs) are multipotent and self-renewing cells located in the
developing and adult nervous system of mammalian species.1, 2 NSCs have the capacity to
differentiate into multiple cell types and replace lost or damaged neurons.3 More specifically,
NSCs in the adult brain are found in two regions of the brain, first, in the subgranular zone of the
hippocampal dentate gyrus, in which NSCs produce the neurons supporting hippocampal
structure and function, and second, in the subventricular zone where NSCs can migrate along the
rostral migratory stream and eventually repair or replace damaged neural cells.4, 5
Since the process of generating and differentiating NSCs into new neurons, also called
neurogenesis, occurs continually throughout an adult’s life, NSCs hold great promise for treating
neurological disorders including multiple sclerosis, Parkinson’s disease, and Alzheimer’s
disease.6, 7 To date, clinical applications of NSCs are mostly focused on cell replacement therapy
via intracerebral transplantation of cultured NSCs, however, they have not been successful due to
inefficient survival and possible immune responses. An alternative approach to the cell
replacement therapy is the activation of endogenous neurogenesis and neuroprotection via
chemical or genetic modification.8-11 Small molecules that can trigger neurogenesis and
neuroprotection are particularly intriguing not only because of their therapeutic implications, but
also because they can serve as an invaluable tool to study the mechanisms of neurogenesis.12
Recently, McKnight and coworkers identified an aminopropyl carbazole, termed P7C3, as
a proneurogenic and neuroprotective compound.8 Based on computational methods, they selected
1,000 small molecules from a library of 200,000 drug-like chemicals, and screened in vivo using
mice lacking the ability of hippocampal neurogenesis. P7C3 appeared to suppress apoptosis of
newborn hippocampal neurons, and enhance neurogenesis in aged rats (Fig. 1). Furthermore,
3

several more derivatives with better pharmacokinetic properties were developed in their follow-
up study, however, the underlying mechanisms of these compounds are still unknown.13
Inspired by this approach, we have developed a series of aminopropyl carbazole (Fig. 1)
and investigated their biological activity in cultured NSCs derived from rat cortex at embryonic
day (E) 14. Here, we describe the synthesis of aminopropyl carbazole derivatives, the detailed
analysis of structure-activity relationship (SAR), and the mechanistic investigation of the selected
compounds.
Figure 1. (a) P7C3, previously reported proneurogenic, neuroprotective chemical; (b) Newly
designed derivatives based on the aminopropyl carbazole scaffold
4

2. Result and discussion
2.1. Chemistry
A series of aminopropyl carbazole derivatives were synthesized to analyze SAR on
proneurogenic and neuroprotective effects. These previously reported aminopropyl carbazole
derivatives can be divided into three structural elements, a dibromocarbazole connected to
substituted aniline through a 2-hydroxypropyl linker. Since it was reported that the
dibromocarbazole group appeared to be essential for the neuroprotective effect, we decided to
focus on modifying the 2-hydroxypropyl linker region and the substituted aniline ring to study
how the structural modification would affect biological activity.
First, the substituted aniline derivatives were synthesized by introducing an epoxide into
various aniline fragments and then subsequent coupling of 3,6-dibromo-9H-carbazole to obtain
compounds 1-6 (Scheme 1). Modification of the 2-hydroxypropyl linker region was carried out
by using compound 1 as starting material (Scheme 2). The N-alkylated derivatives 7-11 were
prepared by simple substitution reactions with corresponding alkyl halides. The O-alkylated and
acylated derivatives, compounds 12-18, were also synthesized. Among these compounds, two
derivatives, compounds 9 and 11 were further subjected to an oxidation reaction by using PCC to
obtain the analogues with a 2-ketopropyl linker (compounds 19-20) (Scheme 3). Subsequently,
the ketone group of compound 20 was converted to an oxime group to yield compound 21.
The analogues with a 2-fluoropropyl linker were synthesized from compound 1 via a
fluorine displacement reaction using (dimethylamino)sulfur trifluoride (methyl DAST) as the
fluorine source (Scheme 4). Finally, the analogues containing a 3-methoxyphenol ring that
replaces the aniline were synthesized by using 3-methoxyphenol as a starting material (Scheme
5).
5

Scheme 1. Reagents and Conditions : (a) epichlorohydrin, ZnOTf, DMF, rt, overnight; (b) KOH,
dioxane, rt, overnight (c) 3,6-dibromo-9H-carbazole, NaH, THF, 0 °C to rt, overnight
Scheme 2. Reagents and Conditions : (a) CH3I, K2CO3, DMF, 50 °C, 4 h for 7; acetyl chloride,
NaH, CH2Cl2, 0 °C to rt, 2 h for 8;RCl, NEt3, CH2Cl2, 0 °C to rt, overnight for 9-10 ; Ag2O,
BnBr, DMF, rt, overnight for 11; (b) CH3I, KOH, DMF, 0 °C to rt, 3 h for R1=OCH3; acetyl
chloride, pyridine, CH2Cl2, 0 °C to rt, 4 h for for R1=OAc; (c) Pd/C, Ph2S, THF:EtOH(1:1), rt, 17
h; (d) Boc2O, DMAP, NEt3, CH2Cl2, 0 °C to rt, 2 h
6

Scheme 3. Reagents and Conditions : (a) PCC, H5IO6, CH3CN, 0 °C to rt, 5 h or DMP, CH2Cl2, rt;
(b) 5% Pd/C, Ph2S, THF:EtOH(1:1), rt, 17 h; (c) hydroxylamine, HCl, pyridine, rt, 3 h
Scheme 4. Reagents and Conditions : (a) Ag2O, BnBr, DMF, rt, overnight; (b) methylDAST,
CH2Cl2, -78 °C, 8 h; (c) Pd/C, Ph2S, THF:EtOH(1:1), rt, 17h
Scheme 5. Reagents and Conditions : (a) 2-(chloromethyl)oxirane, K2CO3, DMF, rt to 50 °C,
overnight; (b) 3,6-dibromocarbazole, NaH, THF, 0 °C to rt, 4 h; (c) methylDAST, CH2Cl2, -
78 °C, 8 h
7

2.2. Biological Activity
In order to identify small molecules that induce neurogenesis, all the synthesized
derivatives were tested in NSCs at 5.0 μM. Briefly, 1 week after expansion, NSCs derived from
rat cortex were treated with 5.0 μM of each compound (P7C3, P7C3A20, and compounds 1-26)
and differentiated for 4 days in the absence of epithelial growth factor (EGF) and fibroblast
growth factor 2 (FGF2), neurons were immunostained with TUJ1 antibody, and the number of
TUJ1 positive neurons was counted and normalized (Table 1). Among these derivatives,
compounds 1, 2, 3, 5, 7, 8, 9, 18 and 21 significantly increased the number of TUJ1 positive
neurons compared to dimethyl sulfoxide (DMSO), indicating that these compounds enhance
neuronal differentiation. Among the effective compounds, 3, 5, and 9 exhibited comparable
activity to P7C3 that was used as a positive control (Fig. 2).
It is interesting to note that various substituents in the aniline region (R2) do not appear to
affect overall activity unless the ring is constrained as compound 6. This result suggests that the
aniline ring is likely to accommodate a possible modification site to attach a linker for affinity
purification. Several analogues with N-substituted anilines (7-9) seemed to exhibit slightly
improved activity compared to their amine counterpart 1, whereas the N-benzyl analogues, 11
and 22, demonstrated substantially low number of TUJ1 positive cells indicating that these
particular derivatives might act as inhibitors of neuronal differentiation. The analogues with the
2-hydroxyl-modified derivatives (12-21) mostly demonstrated lower number of TUJ1 positive
neurons compared to the 2-hydroxyl analogues indicating that the 2-hydroxyl group appears to be
essential for proneurogenic activity. Interestingly, however, replacing the 2-hydroxyl group with
a fluorine atom did not seem to affect activity of each parent compound (Fig. 2).
8

Table 1. Structures and TUJ1 immunoreactivity of the aminocarbazole derivatives
X
R1
Y
R2
Fold
1
DMSO
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
H, H
OH
OH
OH
OH
OH
OH
OH
OH
OH
NH
NH
NH
NH
NH
H
1.47
1.16
1.29
1.88
1.17
1.67
0.88
1.42
1.25
1.52
0.57
1.12
0.73
0.92
0.96
0.65
0.63
0.39
1.25
0.52
1.02
1.13
1.16
0.58
0.86
1.07
0.87
P7C3
3-OCH3
4-Cl
4-CH3
1
2
3
4
5
6
7
8
9
11
25
12
13
15
14
16
17
4-OCH3
3,4-(OCH3)2
3,4-OCH2O
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
NH
NH
N-CH3
N-Ac
N-Ts
N-Bn
O
N-Ts
N-Ns
NH
N-Ts
NH
NH
NH
N-Ts
NH
OH
OH
OH
OCH3
OCH3
OCH3
OAc
OAc
OAc
OBoc
(C)=O
(C)=O
(C)=N-OH
F
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
Br, Br
H, Br
18
19
20
21
NH
NH
N-Bn
NH
NH
P7C3A20
22
F
F
F
F
23
24
26
H, H
Br, Br
O
9

Figure 2. Effects of aminopropyl carbozole derivatives in neurogenesis. After one week of
expansion, NSCs were dissociated and plated onto PDL/laminin-coated coverslips. NSCs were
treated with vehicle (0.1% DMSO) or 5.0 μM chemicals (P7C3 and its derivatives, 1-26) and
differentiated for additional 4 days in the absence of EGF and FGF2. Cells were immunostained
with TUJ1 antibody and nuclei were identified by DAPI staining. NSCs labelled by TUJ1
antibody were counted, and the ratio of labelled cells to total cells (DAPI positive) was calculated
and divided with that of control (DMSO) to yield fold changes. The values are presented as mean
± standard deviation (SD) (n=3, *p < 0.05 and **P < 0.01, vs. control). Statistical analysis was
performed using Student’s t-test.
To confirm the effect on neurogenesis of those compounds, we examined the mRNA
expression levels of neuronal gene βIII tubulin by reverse-transcription polymerase chain reaction
(RT PCR) followed by real-time PCR. As shown in the real-time PCR data (Fig. 3A), 4 days of
treatment of 5.0 μM of 5 and 9 significantly increased the mRNA expression levels of βIII
tubulin by 1.47 and 1.43 fold, respectively, compared to DMSO treated control. A recent study
revealed that P7C3 protected newborn neurons from apoptosis and enhanced neurogenesis in the
hippocampal dentate gyrus in mice and rats.8, 13 Thus to test whether compound 3, 5, and 9 also
induce neurogenesis by improving neuronal survival, we observed the morphology of NSCs
treated with each compound at 10.0 μM (Fig. 3B). However, the phase contrast morphology and
10

4',6-diamidino-2-phenylindole (DAPI)-stained nuclei of NSCs showed that compound 3 and 5
were toxic to NSCs. In contrast, compound 9 did not induce any toxicity to NSCs. These data
suggest that 9 is the safe and effective compound to induce neurogenesis in NSCs. Therefore we
chose this compound to perform further experiments to identify mechanisms of how neurogenesis
is enhanced by aminopropyl carbazole derivatives.
Figure 3. Compound 9 is safe and effective in neurogenesis in NSCs. (A) Induction of βIII
tubulin by compound 5 and 9. Total RNA was prepared from the NSCs treated with DMSO, 5.0
µM of P7C3, 3, 5, or 9 for 4 days, cDNA was synthesized, and subjected to real-time PCR using
specific primer for βIII tubulin. Gapdh was used as an internal control. Values are the mean ±
standard error of mean (SEM) (n = 2, **P < 0.01, vs. control). Statistical analysis was performed
using Student’s t-test. (B) Representative digital images (upper) and fluorescence images of
DAPI-stained nuclei (lower) of NSCs treated with DMSO, 10.0 µM of 3, 5, or 9. The scale bar
represents 50 μm.
11

As shown in Figure 4, different concentrations (5.0, 10.0, and 20.0 μM) of 9 enhanced
neurogenesis in NSCs. Treatment of 5.0 and 10.0 μM of compound 9 significantly increased the
mRNA expression levels of neuronal genes, neuroD and βIII tubulin, while repressing the mRNA
expression of astroglial gene gfap in a dose-dependent manner (Figs. 5A-5C). These data indicate
that 9 induces neuronal differentiation and inhibits astrocytogenesis in NSCs.
Figure 4. Dose-dependent increase of neurogenesis by compound 9. (A) Representative
immunofluorescence images of neurons (TUJ1 positive, green) and nuclei (DAPI positive, blue)
in NSCs treated with DMSO, 10.0 µM of P7C3A20 (positive control), or 5.0 and 10.0 µM of 9.
Scale bar, 50 μm. (B) Quantification of neurons in NSCs treated with DMSO, 10.0 µM of
P7C3A20, or various concentrations (1.0 – 20.0 μM) of 9. The values are presented as the mean ±
SEM (n = 2, **P < 0.01, vs. control). Statistical analysis was performed using Student’s t-test.
12

Figure 5. Compound 9 induces neurogenesis while inhibiting astrocytogenesis and
proliferation of NSCs. (A-E) The relative mRNA expression levels of neuroD (A), βIII tubulin
(B), gfap (C), notch1 (D), and hes1 (E) in NSCs treated with DMSO, 10.0 µM of P7C3A20, or
5.0 and 10.0 µM of 9. Gapdh was used as an internal control. Values are the mean ± SEM (n = 2,
**P < 0.01, vs. control). (F) The volume of neurospheres was calculated by measuring the
diameter of individual neurospheres treated with DMSO (black circle) or 5.0 μM (white circle) or
10.0 μM (black triangle) of 9 for 5 days in the presence of EGF and FGF2. Values are presented
as mean ± SD (n ≥ 4, for each treatment, *p < 0.05, vs. control). Statistical analysis was
performed using Student’s t-test.
Furthermore, the mRNA expression levels of notch1 and hairy and enhancer of split 1
(hes1), which inhibit neurogenesis while promoting proliferation and astrocytogenesis of NSCs,
were down-regulated in NSCs treated with 9 in a dose dependent manner (Figs. 5D and 5E).14, 15
To test the effects of 9 on the proliferation of NSCs, neurosphere growth assays were performed.
13

The sizes of neurospheres treated with DMSO or 9 (5.0 and 10.0 μM) were measured in the
presence of EGF and FGF2 for 5 days and each sphere volume was calculated. Compound 9
treated NSCs grew significantly less than DMSO treated control (Fig. 5F). Taken together, these
data suggest that 9 increases neurogenesis, reduces astrocytogenesis, and inhibits proliferation of
NSCs.
To explore the effects of 9 on the survival of NSCs, cell viability was assessed by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in the absence of EGF and
FGF2. As shown in Figure 6A, both positive control P7C3A20 and 9 significantly increased the
cell viability compared to DMSO treated control. However, 9 increased viability of NSCs
significantly better than P7C3A20. As shown in Figure 6B, NCSs treated with 20.0 and 50.0 μM
of P7C3A20 shrunken and rounded up, suggesting P7C3A20 may be toxic to NSCs at the
concentrations. On the other hand, NSCs treated with 9 increased the cell number and appeared
healthy at the same concentrations. Furthermore, the mRNA ratio of anti-apoptotic gene bcl2 to
pro-apoptotic gene bax in P7C3A20 (10.0 μM) and 9 treated NSCs decreased significantly when
compared with that of control. However, the mRNA ratio of bcl2 to bax in 9 (5.0 and 10.0 μM)
treated NSCs was significantly increased from that of P7C3A20 control (Fig. 6C). P7C3A20
reduced bcl2 expression while maintaining bax expression comparable to DMSO treated control
(data not shown). Interestingly, compound 9 not only lowered the expression levels of bcl2 but
also bax. Thus, by reducing pro-apoptotic bax expression, compound 9 may protect NSCs better
than P7C3A20. From these results, 9 is a better neuroprotective agent than P7C3A20.
14

Figure 6. Compound 9 increases survival of NSCs. (A) Cell viability was assessed using the
MTT assay 2 days after treatment with DMSO or various concentrations (1.0 – 50.0 μM) of
P7C3A20 or 9 in the absence of EGF and FGF2. The values are presented as the mean ± SEM (n
= 3, *P < 0.05 and **P < 0.01, vs. control; #P < 0.001, vs. P7C3A20). (B) Representative digital
images of NSCs treated with various concentrations (0.0 – 50.0 μM) of P7C3A20 or 9 in the
presence of EGF and FGF2 at 48 h. The scale bars represents 50 μm. (C) The mRNA ratio of
bcl2 to bax in NSCs treated with DMSO, 10.0 µM of P7C3A20, or 5.0 and 10.0 µM of 9. Gapdh
was used as an internal control. Values are the mean ± SEM (n = 2, *P < 0.05 and **P < 0.01, vs.
control; #P < 0.001, vs. P7C3A20). Statistical analysis was performed using Student’s t-test.
3. Conclusion
We have developed and screened 25 aminopropyl carbazole derivatives for proneurogenic
and neuroprotective activity in NSCs. Among these analogues, compound 9 demonstrated an
excellent proneurogenic and neuroprotective activity with no apparent toxicity. It was also
observed that compound 9 not only promoted neurogenesis, but also suppressed astrocytogenesis
and proliferation of NSCs, thereby boosting overall survival of NSCs. These results provide some
basic insights into the underlying mechanisms of neuroprotective effect of the aminopropyl
carbazole derivatives, and also can help us design affinity probes to identify the molecular targets.
15

4. Experimental
4.1. Chemistry
4.1.1. General
All chemical reagents were commercially available. Melting points were determined on a melting
point Buchi B-540 apparatus and are uncorrected. Silica gel column chromatography was
performed on Silica Gel 60, 230–400 mesh, Merck. Proton NMR spectra were recorded on a
JEOL JNM-LA 300 at 300 MHz and Bruker Analytik, DE/AVANCE Digital 400 at 400 MHz.
Chemical shifts were reported in ppm units with Me4Si as a reference standard. Mass spectra
were recorded on a VG Trio-2 GC–MS.
4.1.2. General Procedure for the synthesis of various aromatic amine compounds (1-6)
Epichlorohydrin (0.19 mL, 2.44 mmol, 1 eq) was dissolved in DMF (8 mL). The reaction mixture
was added ZnOTf (17.7 mg, 0.05 mmol, 0.02 eq), aromatic amine compounds (2.44 mmol, 1 eq)
and stirred at room temperature for overnight. The reaction mixture was diluted with water and
extracted with EA. The organic part was washed with water and brine. The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The crude was purified by column
chromatography on silica gel to give pure compounds. To a solution of pure compounds (0.70
mmol, 1 eq) in 1.4-dioxane (2.5 mL) was added KOH (47.5 mg, 0.85 mmol, 1.2 eq) and stirred at
room temperature for overnight. The reaction mixture was diluted with water and extracted with
EA. The organic part was washed with water and brine. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was purified by column chromatography
on silica gel to give oxirane compounds. To a solution of 3,6-dibromo-9H-carbazole (69.5 mg,
0.21 mmol, 1 eq) in THF (1 mL) was added NaH (9.6 mg, 0.24 mmol, 1.1 eq) and stirred at 0 °C.
16

The oxirane compound (0.26 mmol, 1.2 eq) was added and stirred at room temperature for
overnight. TLC showed complete consumption of starting material. The reaction mixture was
diluted with water and extracted with EA. The organic part was washed with water and brine.
The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude
was purified by column chromatography on silica gel to afford the desired product.
4.1.2.1. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((3-methoxyphenyl)amino)propan-2-ol (1).
1
65% yield; white solid; mp = 140-141 °C; H NMR (300MHz, CDCl3) δ 8.15 (d, 2H, J = 1.83
Hz), 7.56 (dd, 2H, J = 8.61, 1.83 Hz), 7.34 (d, 2H, J = 8.58 Hz), 7.08 (t, 1H, J = 8.25 Hz), 6.32 (d,
1H, J = 8.07 Hz), 6.23 (d, 1H, J = 7.35 Hz), 6.15 (t, 1H, J = 2.19 Hz), 4.41 (m, 3H), 3.73 (s, 3H),
3.32 (m, 1H), 3.20 (m, 1H); MS (ESI) m/z 504.90 [M+H]+.
4.1.2.2. 1-((4-Chlorophenyl)amino)-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-ol (2).
1
44% yield; yellow solid; mp = 162-163 °C; H NMR (300MHz, CDCl3) δ 8.14 (d, 2H, J = 2.01
Hz), 7.54 (dd, 2H, J = 8.61, 1.83 Hz), 7.32 (d, 2H, J = 8.79 Hz), 7.01-7.15 (m, 2H), 6.50-6.55 (m,
2H), 4.35-4.44 (m, 3H), 4.01 (brs, 1H), 3.27-3.32 (m, 1H), 3.15 (dd, 1H, J = 13.20, 6.24 Hz),
2.15 (brs, 1H); MS (ESI) m/z 508.9 [M+H]+.
4.1.2.3. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-(p-tolylamino)propan-2-ol (3).
37% yield; yellow solid; mp = 170-173 °C; 1H NMR (300MHz, CD3OD) δ 8.20 (d, 2H, J = 1.65
Hz), 7.48 (dd, 2H, J = 8.79, 1.86 Hz), 7.43 (d, 2H, J = 8.79 Hz), 6.93 (d, 2H, J = 8.04 Hz), 6.55
(d, 2H, J = 8.43 Hz), 4.43 (m, 2H), 4.24 (m, 1H), 3.10-3.25 (m, 2H), 2.19 (s, 3H); MS (ESI) m/z
488.9 [M+H]+.
17

4.1.2.4. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((4-methoxyphenyl)amino)propan-2-ol (4).
32% yield; dark yellow solid; mp = 96-99 °C; 1H NMR (300MHz, CDCl3) δ 8.15 (d, 2H, J = 1.83
Hz), 8.55 (dd, 2H, J = 8.78, 2.04 Hz), 7.36 (d, 2H, J = 8.58 Hz), 6.75-6.80 (m, 2H), 6.57-6.61 (m,
2H), 4.35-4.43 (m, 3H), 3.75 (s, 3H), 3.29 (dd, 1H, J = 12.81, 3.66 Hz), 3.13 (dd, 1H, J = 12.81,
6.96 Hz); MS (ESI) m/z 504.9 [M+H]+.
4.1.2.5. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((3,4-dimethoxyphenyl)amino)propan-2-ol (5).
52% yield; brown solid; mp = 93-95 °C; 1H NMR (300MHz, CDCl3) δ 8.15 (s, 2H), 7.55 (d, 2H,
J = 8.61 Hz), 7.36 (d, 2H, J = 9.15 Hz), 6.72 (d, 1H, J = 8.40 Hz), 6.19 (s, 1H), 6.13 (d, 1H, J =
8.61 Hz), 4.41 (m, 2H), 4.36 (brs, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.28 (d, 1H, J = 12.24 Hz), 3.12
(dd, 1H, J = 12.81, 6.78 Hz); MS (ESI) m/z 534.9 [M+H]+.
4.1.2.6. 1-(Benzo[d][1,3]dioxol-5-ylamino)-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-ol (6).
1
20% yield; brown solid; H NMR (400MHz, CDCl3) δ 8.13 (s, 2H), 7.53 (dd, 2H, J = 6.92, 1.32
Hz), 7.33 (d, 2H, J = 6.96 Hz), 6.62 (d, 1H, J = 6.6 Hz), 6.24 (d, 1H, J = 1.72 Hz), 6.03 (dd, 1H,
J = 6.64, 1.76 Hz), 5.85 (s, 2H), 4.39 (t, 2H, J = 4.2 Hz), 4.33-4.35 (m, 1H), 3.25 (dd, 1H, J =
10.48, 3.00 Hz), 3.10 (dd, 1H, J = 10.28, 5.56 Hz).
4.1.3. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((3-methoxyphenyl)(methyl)amino)propan-2-ol
(7).
To a solution of compound 1 (50 mg, 0.10 mmol, 1 eq) in anhydrous DMF (0.3 mL) was added
K2CO3 (21 mg, 0.15 mmol, 1.5 eq) and CH3I (0.01 mL, 0.11 mmol, 1.1 eq) at room temperature
18

to 50 °C for 4 h. TLC showed complete consumption of starting material. The reaction mixture
was diluted with water and extracted with EA. The organic part was washed with water and brine.
The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude
was purified by column chromatography to give pure compound 7 (25 mg, 48%) as a white solid;
mp = 76-78 °C; 1H NMR (300MHz, CDCl3) δ 8.14 (d, 2H, J = 1.83 Hz), 7.54 (dd, 2H, J = 8.79,
2.04 Hz), 7.32 (d, 2H, J = 8.79 Hz), 7.12 (t, 1H, J = 8.25 Hz), 6.32-6.37 (m, 2H), 6.24 (t, 1H, J =
2.37 Hz), 4.34-4.42 (m, 3H), 3.69 (s, 3H), 3.38 (d, 2H, J = 6.03 Hz), 2.95 (s, 3H), 2.26 (brs, 1H);
MS (ESI) m/z 518.90 [M+H]+.
4.1.4.
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-(3-methoxyphenyl)
acetamide (8).
To a solution of compound 1 (50 mg, 0.10 mmol, 1 eq) in anhydrous MC (0.3 mL) was added
NaH (4.4 mg, 0.11 mmol, 1.1 eq) and acetylchloride (0.01 mL, 0.11 mmol, 1.1 eq) at 0 °C to
room temperature for 2 h. TLC showed complete consumption of starting material. The reaction
mixture was diluted with water and extracted with EA. The organic part was washed with water
and brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure.
The crude was purified by column chromatography to give pure compound 8 (24 mg, 44%) as a
white solid; mp = 167-170 °C; 1H NMR (300MHz, CDCl3) δ 8.09 (d, 2H, J = 1.83 Hz), 7.51 (dd,
2H, J = 8.79, 2.01 Hz), 7.22-7.29 (m, 2H), 6.85 (dd, 1H, J = 8.25, 1.47 Hz), 6.66 (d, 1H, J = 7.89
Hz), 6.60 (t, 1H, J = 2.19 Hz), 4.30 (m, 3H), 3.87 (brs, 1H), 3.75 (s, 3H), 3.48 (d, 1H, J = 11.88
Hz), 1.89 (s, 3H); MS (ESI) m/z 546.90 [M+H]+.
19

4.1.5.
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-(3-methoxyphe-nyl)-4-
methylbenzenesulfonamide (9).
To a solution of compound 1 (100 mg, 0.20 mmol, 1 eq) in MC was added TEA (0.06 mL, 0.44
mmol, 2.2 eq), TsCl (83.2 mg, 0.44 mmol, 2.2 eq) at 0 °C to room temperature for overnight.
TLC showed complete consumption of starting material. The reaction mixture was diluted with
water and extracted with EA. The organic part was washed with water and brine. The organic
layer was dried over MgSO4 and concentrated under reduced pressure. The crude was purified by
column chromatography to give pure compound 15 (132 mg, 99%) as a white solid; mp = 108-
110 °C; 1H NMR (300MHz, CDCl3) δ 8.07 (d, 2H, J = 1.83 Hz), 7.48 (dd, 2H, J = 8.79, 2.01 Hz),
7.42 (d, 2H, J = 8.25 Hz), 7.21 (m, 5H), 6.85 (dd, 1H, J = 8.25, 2.37 Hz), 6.64 (t, 1H, J = 2.01 Hz)
6.58 (dd, 1H, J = 7.5, 1.83 Hz), 4.42 (dd, 1H, J = 14.82, 3.3 Hz), 4.21-4.29 (m, 1H), 4.17 (brs,
1H), 3.73 (s, 3H), 3.63 (d, 2H, J = 6.03 Hz), 2.43 (s, 3H); MS (ESI) m/z 657.00 [M+H]+.
4.1.6.
(11).
1-(Benzyl(3-methoxyphenyl)amino)-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-ol
To a solution of compound 1 (762 mg, 1.51 mmol, 1 eq) in DMF (5 mL) was added Ag2O (420
mg, 1.81 mmol, 1.2 eq) and benzylbromide (0.22 mL, 1.81 mmol, 1.2 eq) at 0 °C to room
temperature for overnight. TLC showed complete consumption of starting material. The reaction
mixture was diluted with water and extracted with EA. The organic part was washed with water
and brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure.
The crude was purified by column chromatography to give pure compound 9 (310 mg, 35%) as a
white solid; mp = 167-168 °C; 1H NMR (300MHz, CDCl3) δ 8.10 (d, 2H, J = 1.83 Hz), 7.50 (dd,
2H, J = 8.79, 2.01 Hz), 7.21-7.36 (m, 5H), 7.14-7.16 (m, 2H), 7.07 (t, 1H, J = 8.25 Hz), 6.30-6.35
20

(m, 2H), 6.24 (t, 1H, J = 2.37 Hz), 4.51-4.63 (m, 2H), 4.36-4.41 (m, 1H), 4.19-4.32 (m, 2H), 3.62
(s, 3H), 3.40-3.57 (m, 2H), 2.12 (d, 1H, J = 3.09 Hz); MS (ESI) m/z 594.90 [M+H]+.
4.1.7.
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-methoxypropyl)-N-(3-methoxyphenyl)-4-
methylbenzenesulfonamide (12).
To a solution of compound 9 (200 mg, 0.33 mmol, 1 eq) in DMF was added KOH (28 mg, 0.50
mmol, 1.5 eq), CH3I (0.02 mL, 0.36 mmol, 1.1 eq) at 0 °C to room temperature for 5 h. TLC
showed complete consumption of starting material. The reaction mixture was diluted with water
and extracted with EA. The organic part was washed with water and brine. The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The crude was purified by column
chromatography to give pure compound 16 (117 mg, 53%) as a white solid; mp = 176-177 °C; 1H
NMR (300MHz, CDCl3) δ 8.11 (d, 2H, J = 1.86 Hz), 7.51 (dd, 2H, J = 8.79, 2.01 Hz), 7.44 (d,
2H, J = 8.25 Hz), 7.28 (m, 3H), 7.21 (t, 2H, J = 7.86 Hz), 6.86 (dd, 1H, J = 7.68, 1.83 Hz), 6.71 (t,
1H, J = 2.22 Hz), 6.64 (dd, 1H, J = 7.14, 1.08 Hz), 4.55 (dd, 1H, J = 15.39, 2.4 Hz), 4.25 (q, 1H),
3.75 (s, 3H), 3.59-3.68 (m, 3H), 2.88 (s, 3H); MS (ESI) m/z 671.02 [M+H]+.
4.1.8.
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-methoxypropyl)-N-(3-methoxyphenyl)-4-
nitrobenzenesulfonamide (13).
To a solution of compound 1 (100 mg, 0.56 mmol, 1 eq) in MC (2 mL) was added TEA (0.17 mL,
1.23 mmol, 2.2 eq) and 4-nitrobenzenesulfonyl chloride (273 mg, 1.23 mmol, 2.2 eq) at -78 °C to
room temperature for overnight. TLC showed complete consumption of starting material. The
reaction mixture was diluted with water and extracted with EA. The organic part was washed
with water and brine. The organic layer was dried over MgSO4 and concentrated under reduced
21

pressure. The crude was purified by column chromatography to obtain compound 10. This
compound (10) was reacted with KOH and CH3I at 0 °C to room temperature for 3 h following
the procedure for the synthesis of 7 to give 13 as a white solid; 41% yield; mp = 173-174 °C; 1H
NMR (300MHz, CDCl3) δ 8.13 (d, 2H, J = 1.8 Hz), 7.93-7.97 (m, 2H), 7.52 (dd, 2H, J = 8.7, 2.1
Hz), 7.27 (t, 1H, J = 8.1 Hz), 7.21 (d, 2H, J = 8.7 Hz), 6.78 (dd, 1H, J = 8.7, 2.4 Hz), 6.70–6.74
(m, 1H), 6.67 (t, 1H, J = 2.4 Hz), 6.61–6.65 (m, 2H), 4.35 (dd, 1H, J = 15, 6.6 Hz), 4.24 (dd, 1H,
J = 15, 4.5 Hz), 3.89–3.93 (m, 2H), 3.78-3.87 (m, 2H), 3.74 (s, 3H), 3.14 (s, 3H); MS (ESI) m/z
654.98 [M+H]+.
4.1.9.
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-methoxypropyl)-N-(3-methoxyphenyl)-4-
methylbenzenesulfonamide (14).
To a solution of compound 9 (70 mg, 0.11 mmol, 1 eq) in MC (1 mL) was added pyridine (0.07
mL, 0.81 mmol, 7 eq), acetyl chloride (0.06 mL, 0.81 mmol, 7 eq) and stirred at 0 °C to room
temperature for 4 h. TLC showed complete consumption of starting material. The reaction
mixture was diluted with water and extracted with EA. The organic part was washed with water
and brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure.
The crude was purified by column chromatography to give pure compound 14 (58 mg, 75%) as a
yellow solid; mp = 88-89 °C; 1H NMR (300MHz, CDCl3) δ 8.09 (d, 2H, J = 1.83 Hz), 7.51 (dd,
2H, J = 8.61, 1.86 Hz), 7.41 (d, 2H, J = 8.22 Hz), 7.22 (m, 5H), 6.87 (dd, 1H, J = 8.43, 2.58 Hz),
6.63-6.68 (m, 2H), 5.17-5.21 (m, 1H), 4.48-4.61 (m, 2H), 3.79-3.88 (m, 2H), 3.75 (s, 3H), 2.42 (s,
3H), 1.55 (s, 3H); MS (ESI) m/z 699.01 [M+H]+.
4.1.10. General Procedure for the debenzylation (15-17).
22

N-benzyl compound (0.14 mmol, 1 eq) was dissolved in THF : EtOH (1 : 1). Ph2S (0.26 mg,
0.0014 mmol, 0.01 eq) was added to it. And Pd/C (5 wt% Pd on carbon, cat. amount) was added
at room temperature under H2 for 17 h. TLC showed complete consumption of starting material.
The mixture was filtered through celite bed and the filterate was concentrated under reduced
pressure. The reaction mixture was diluted with water and extracted with EA. The organic part
was washed with water and brine. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The crude was purified by column chromatography on silica gel to afford
the desired product.
4.1.10.1. N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-methoxypropyl)-3-methoxyaniline (15).
Compound 11 was reacted with K2CO3 and CH3I at room temperature to 50 °C for 4 h following
the procedure for the synthesis of 7 and debenzylation was followed by the general procedure to
give 15 as a white solid in 26% yield.; mp = 142-144 °C; 1H NMR (300MHz, CDCl3) δ 8.13 (d,
2H, J = 1.65 Hz), 7.51 (dd, 2H, J = 8.61, 1.83 Hz), 7.32 (d, 2H, J = 8.79 Hz), 7.08 (t, 1H, J =
8.07 Hz), 6.33 (dd, 1H, J = 8.07, 2.19 Hz), 6.21 (dd, 1H, J = 8.04, 1.44 Hz), 6.12 (t, 1H, J = 2.37
Hz), 4.35-4.50 (m, 2H), 4.03 (brs, 1H), 3.82-3.89 (m, 1H), 3.74 (s, 3H), 3.29 (s, 3H), 3,27 (m,
1H), 3.11 (m, 1H); MS (ESI) m/z 518.9 [M+H]+.
4.1.10.2. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((3-methoxyphenyl)amino)propan-2-yl acetate
(16).
Compound 11 was reacted with NaH and acetylchloride at 0 °C to room temperature for 2 h
following the procedure for the synthesis of 8 and debenzylation was followed by the general
procedure to give 16 as a brown solid in 27% yield.; mp = 86-89 °C; 1H NMR (300MHz, CDCl3)
23

δ 8.12 (d, 2H, J = 1.83 Hz), 7.52 (dd, 1H, J = 8.61, 1.83 Hz), 7.37 (d, 2H, J = 8.79 Hz), 7.08 (t,
1H, J = 8.04 Hz), 6.33 (dd, 1H, J = 8.25, 2.37 Hz), 6.19 (dd, 1H, J = 7.71, 2.22 Hz), 6.13 (t, 1H, J
= 2.37 Hz), 5.38-5.42 (m, 1H), 4.42-4.58 (m, 2H), 3.93 (brs, 1H), 3.71 (s, 3H), 3.26-3.30 (m, 2H),
1.98 (s, 3H); MS (ESI) m/z 546.9 [M+H]+.
4.1.10.3. 1-(9H-Carbazol-9-yl)-3-((3-methoxy phenyl)amino)propan-2-yl acetate (17).
1
The procedure is same as the procedure for the synthesis of 16; 67% yield; H NMR (300MHz,
CDCl3) δ 8.07 (d, 2H, J = 7.68 Hz), 7.50 (d, 2H, J = 8.04 Hz), 7.40-7.45 (m, 2H), 7.21-7.25 (m,
2H), 7.04 (t, 1H, J = 8.04 Hz), 6.29 (dd, 1H, J = 8.22, 2.37 Hz), 6.18 (dd, 1H, J = 8.04, 2.01 Hz),
6.11 (t, 1H, J = 2.19 Hz), 5.43-5.51 (m, 1H), 4.26-4.60 (m, 2H), 3.91 (brs, 1H), 3.66 (s, 3H),
3.25-3.42 (m, 2H), 1.98 (s, 3H); MS (ESI) m/z 389.1 [M+H]+.
4.1.11. tert-Butyl (1-(3,6-dibromo-9H-carbazol-9-yl)-3-((3-methoxyphenyl)amino) propan-2-
yl) carbonate (18).
To a solution of compound 1 (500 mg, 0.99 mmol, 1 eq) in MC was added DMAP (12 mg, 0.1
mmol, 0.1 equip), TEA (0.28 mL, 1.98 mmol, 2 eq) and Boc2O (476 mg, 2.18 mmol, 2.2 eq) at
0 °C to room temperature for 2 h. TLC showed complete consumption of starting material. The
reaction mixture was diluted with water and extracted with EA. The organic part was washed
with water and brine. The organic layer was dried over MgSO4 and concentrated under reduced
pressure. The crude was purified by column chromatography to give pure compound 18 (513 mg,
1
86%) as a white solid; mp = 85-88 °C; H NMR (300MHz, CDCl3) δ 8.11 (d, 2H, J = 2.01 Hz),
7.51 (dd, 2H, J = 6.03, 1.83 Hz), 7.38 (d, 2H, J = 8.76 Hz), 7.05 (t, 1H, J = 8.04 Hz), 6.32 (dd,
1H, J = 8.07, 2.22 Hz), 6.16 (dd, 1H, J = 7.89, 1.47 Hz), 6.07 (t, 1H, J = 2.22 Hz), 5.19-5.26 (m,
24

1H), 4.44-4.59 (m, 2H), 3.98 (brs, 1H), 3.70 (s, 3H), 3.29 (d, 2H, J = 4.56 Hz), 1.35 (s, 9H); MS
(ESI) m/z 604.90 [M+H]+.
4.1.12.
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-oxopropyl)-N-(3-methoxyphenyl)-4-
methylbenzenesulfonamide (19).
i) Periodic acid (0.18 mmol, 1.05 eq) was dissolved in CH3CN (1 mL) and stirred at room
temperature for 15 min. Compound 9 (0.17 mmol, 1 eq) was added in an ice bath and PCC
(0.0034 mmol, 2 mol%) in CH3CN was added via syringe at 0 °C to room temperature for 5 h.
TLC showed complete consumption of starting material. The reaction mixture was diluted with
water and extracted with EA. The organic part was washed with water and brine. The organic
layer was dried over MgSO4 and concentrated under reduced pressure. The crude was purified by
column chromatography to give pure compound 19 (30 mg, 27%) as a white solid; ii) To a
solution of compound 9 (0.50 mmol, 1 eq) in anhydrous MC (3 mL) was added Dess-martin
periodinane (0.61 mmol, 1.2 eq) at room temperature for overnight under N2. TLC showed
complete consumption of starting material. The reaction mixture was diluted with sodium
thiosulfate solution (in H2O) and sodium bicarbonate solution and extracted with EA. The
organic part was washed with water and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The crude was purified by column chromatography to give
pure compound 19; 27% yield; white solid; mp = 114-115 °C; 1H NMR (300MHz, CDCl3) δ 8.13
(d, 2H, J = 1.83 Hz), 7.50 (dd, 2H, J = 8.61, 1.83 Hz), 7.42 (d, 2H, J = 8.43 Hz), 7.14-7.24 (m,
2H), 7.07-7.13 (m, 1H), 7.04 (s, 1H), 7.01 (s, 1H), 6.88 (dd, 1H, J = 7.68, 1.65 Hz), 6.69 (t, 1H, J
= 2.22 Hz), 6.58 (d, 1H, J = 7.35 Hz), 5.38 (s, 2H), 4.19 (s, 2H), 3.75 (s, 3H), 2.42 (s, 3H); MS
(ESI) m/z 654.98 [M+H]+.
25

4.1.13. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((3-methoxyphenyl)amino)propan-2-one (20).
To a solution of compound 11 (300 mg, 0.50 mmol, 1 eq) in anhydrous MC (3 mL) was added
Dess-martin periodinane (257 mg, 0.61 mmol, 1.2 eq) at room temperature for overnight under
N2. TLC showed complete consumption of starting material. The reaction mixture was diluted
with sodium thiosulfate solution (in H2O) and sodium bicarbonate solution and extracted with EA.
The organic part was washed with water and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The crude was purified by column chromatography to give
pure compound and then debenzylation was followed by the general procedure to give 20 as a
1
yellow solid in 32% yield; mp = 92-95 °C; H NMR (300MHz, CDCl3) δ 8.17 (d, 2H, J = 1.83
Hz), 7.56 (dd, 2H, J = 8.61, 1.83 Hz), 7.10 (d, 2H, J = 8.79 Hz), 7.06 (t, 1H, J = 8.04 Hz), 6.31
(dd, 1H, J = 7.71, 1.65 Hz), 6.08 (dd, 1H, J = 7.35, 1.65 Hz), 5.99 (t, 1H, J = 2.19 Hz), 5.07 (s,
2H), 3.89 (s, 2H), 3.73 (s, 3H); MS (ESI) m/z 502.90 [M+H]+.
4.1.14. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((3-methoxyphenyl)amino)propan-2-one oxime
(21).
The reaction of compound 20 with hydroxylamine hydrochloride in pyridine at room temperature
1
for 3 h gave to pure compound 21 as a brown solid in 60% yield; mp = 103-105 °C; H NMR
(300MHz, CDCl3) δ 8.10 (d, 2H, J = 7.71 Hz), 7.43-7.46 (m, 4H), 7.13 (t, 1H, J = 8.07 Hz), 6.36
(dd, 1H, J = 8.04, 1.83 Hz), 6.22 (m, 1H), 6.15 (t, 1H, J = 2.19 Hz), 4.98 (s, 2H), 3.91 (s, 2H),
3.78 (s, 3H)
26

4.1.15.
(22).
N-Benzyl-N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-fluoropropyl)-3-methoxyaniline
Compound 1 was reacted with Ag2O and benzylbromide at 0 °C to room temperature for
overnight following the procedure for the synthesis of 9 to give pure compound. To a solution of
pure compound (300 mg, 0.50 mmol, 1 eq) in anhydrous MC (10 mL) was added methyl DAST
(0.06 mL, 0.61 mmol, 1.2 eq) at -78 °C for 8 h under N2. TLC showed complete consumption of
starting material. The reaction mixture was diluted with water and extracted with EA. The
organic part was washed with water and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The crude was purified by column chromatography to give
pure compound 22 (167 mg, 56%) as a white solid; mp = 158-160 °C; 1H NMR (300MHz, CDCl3)
δ 8.11 (d, 2H, J = 1.83 Hz), 7.51 (dd, 2H, J = 8.61, 2.01 Hz), 7.26-7.31 (m, 2H), 7.25-7.16 (m,
5H), 7.07 (t, 1H, J = 8.25 Hz), 6.24-6.32 (m, 2H), 6.17 (t, 1H, J = 2.19 Hz), 5.05-5.24 (m, 1H),
4.58 (q, 2H), 4.34-4.47 (m, 2H), 3.61-3.72 (m, 2H), 3.63 (s, 3H); MS (ESI) m/z 595.03 [M+H]+.
4.1.16. N-(3-(3-Bromo-9H-carbazol-9-yl)-2-fluoropropyl)-3-methoxyaniline (23).
Compound 22 (83 mg, 0.14 mmol, 1 eq) was dissolved in THF : EtOH (1 : 1). Ph2S (0.26 mg,
0.0014 mmol, 0.01 eq) was added to it. And Pd/C (8.3 mg, 0.1 eq) was added at room
temperature under H2 for 17 h. TLC showed complete consumption of starting material. The
mixture was filtered through celite bed and the filterate was concentrated under reduced pressure.
The reaction mixture was diluted with water and extracted with EA. The organic part was washed
with water and brine. The organic layer was dried over MgSO4 and concentrated under reduced
pressure. The crude was purified by column chromatography to give pure compound 23 (14 mg,
23%) as a brown solid; mp = 90-93 °C; 1H NMR (400MHz, CDCl3) δ 8.18 (d, 1H, J = 1.36 Hz),
27

8.02 (d, 1H, J = 7.76 Hz), 7.45-7.52 (m, 2H), 7.39 (d, 1H, J = 8.16 Hz), 7.25-7.30 (m, 2H), 7.06
(t, 1H, J = 8.12 Hz), 6.31 (d, 1H, J = 8.08 Hz), 6.18 (d, 1H, J = 8.04 Hz), 6.11 (s, 1H), 5.02-5.17
(m, 1H), 4.54-4.62 (m, 2H), 3.94 (brs, 1H), 3.71 (s, 3H), 3.26-3.46 (m, 2H); MS (ESI) m/z 427.00
[M+H]+.
4.1.17. N-(3-(9H-Carbazol-9-yl)-2-fluoropropyl) -3-methoxyaniline (24).
The procedure is same as the procedure for the synthesis of 23; 31% yield; brown solid; mp = 98-
100 °C; 1H NMR (400MHz, CDCl3) δ 8.08 (d, 2H, J = 7.72 Hz), 7.40-7.46 (m, 4H), 7.20-7.26 (m,
2H), 7.05 (t, 1H, J = 8.12 Hz), 6.29 (d, 1H, J = 7.96 Hz), 6.18 (d, 1H, J = 8 Hz), 6.10 (s, 1H),
5.05-5.20 (m, 1H), 4.56-4.68 (m, 2H), 3.94 (brs, 1H), 3.68 (s, 3H), 3.28-3.47 (m, 2H); MS (ESI)
m/z 349.10 [M+H]+.
4.1.18. 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-(3-methoxyphenoxy)propan-2-ol (25).
To a solution of 3-methoxyphenol (100 mg, 0.81 mmol, 1 eq) in anhydrous DMF (3 mL) was
added KOH (54 mg, 0.97 mmol, 1.2 eq) at room temperature. And epichlorohydrin (0.16 mL,
2.03 mmol, 2.5 eq) was added via syringe and stirred at 50 °C for overnight. The reaction mixture
was diluted with water and extracted with EA. The organic part was washed with water and brine.
The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude
was purified by column chromatography to give pure compound (115 mg, 79%). To a solution of
3,6-dibromocarbazole (114 mg, 0.35 mmol, 1 eq) in THF (2 mL) was added NaH (9.3 mg, 0.39
mmol, 1.1 eq) and stirred at 0 °C. The synthesized pure compound (76 mg, 0.42 mmol, 1.2 eq)
was added and stirred at room temperature for 4 h. The reaction mixture was diluted with water
and extracted with EA. The organic part was washed with water and brine. The organic layer was
28

dried over MgSO4 and concentrated under reduced pressure. The crude was purified by column
chromatography to give pure compound 25 (62 mg, 35%) as a white solid; mp = 130-132 °C; 1H
NMR (300MHz, CDCl3) δ 8.14 (d, 2H, J = 1.65 Hz), 7.52 (dd, 2H, J = 8.79, 2.01 Hz), 7.36 (d,
2H, J = 8.79 Hz), 7.19 (t, 1H, J = 8.25 Hz), 6.56 (dd, 1H, J = 8.4, 2.37 Hz), 6.42-6.48 (m, 2H),
4.57 (dd, 1H, J = 16.47, 7.89 Hz), 4.43-4.48 (m, 2H), 3.99 (dd, 1H, J = 9.69, 4.2 Hz), 3.88 (dd,
1H, J = 9.54, 4.41 Hz), 3.79 (s, 3H); MS (ESI) m/z 503.98 [M+H]+.
4.1.19. 3,6-Dibromo-9-(2-fluoro-3-(3-methoxyphenoxy)propyl)-9H-carbazole (26).
Compound 25 was reacted with methyl DAST at -78 °C for 8 h under N2 following the procedure
1
for the synthesis of 22 to give pure compound; 45% yield; white solid; mp = 134-135 °C; H
NMR (300MHz, CDCl3) δ 8.13 (d, 2H, J = 2.01 Hz), 7.52 (dd, 2H, J = 8.61, 1.83 Hz), 7.33 (d,
2H, J = 8.79 Hz), 7.20 (t, 1H, J = 8.4 Hz), 6.57 (dd, 1H, J = 8.22, 2.37 Hz), 6.46–6.51 (m, 2H),
5.06-5.25 (m, 1H), 4.58-4.80 (m, 2H), 4.01-4.16 (m, 2H), 3.80 (s, 3H); MS (FAB) m/z 507
[M+H]+.
4.2. Biology
4.2.1. Cell Culture
NSCs were cultured as previously described.2 We cultured NSCs from the cortex of Sprague–
Dawley rat embryos at E14. The NSCs (200,000 cells/mL) differentiated into neurospheres for 7
days in Dulbecco’s modified Eagle’s medium/F12 media (Gibco, CA, USA) supplemented with 2%
(v/v) B27 (Invitrogen, NY, USA), 20 ng/mL EGF (Chemicon, CA, USA), and 20 ng/mL FGF2
(Chemicon). The media was replaced every 2 days. To induce differentiation, neurospheres were
dispersed into a single-cell suspension with accutase (Chemicon) for 10 min at 37 °C and plated
29