Design and synthesis of dithiocarbamate linked β-carboline derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing ability

Article abstract of DOI:10.1016/j.bmc.2015.07.037

A series of new β-carboline-dithiocarbamate derivatives bearing phenyl, dithiocarbamate and H/methyl substitutions at position-1, 3 and 9, respectively, were designed and synthesized. These derivatives 8a-l and 13a-l and their starting precursors (7a-d and 12a-d) have been evaluated for their in vitro cytotoxic activity on selected human cancer cell lines. Among the derivatives tested, 7c, 12c, 8a, 8d, 8i, 8j, 8k, 8l and 13d-l exhibited considerable cytotoxicity against most of the tested cancer cell lines (IC₅₀ <10 μM). Interestingly, most of the derivatives (8a-l and 13a-l) exhibited enhanced activity than their precursors (7a-d and 12a-d), which indicates that the combination of dithiocarbamate with β-carboline enhances the cytotoxicity of 8a-l and 13a-l. Moreover, the derivatives 8j and 13g exhibited significant cytotoxic activity with IC₅₀ values of 1.34 μM and 0.79 μM on DU-145 cancer cells, respectively. Further, the induction of apoptosis by these derivatives was confirmed by Annexin V-FITC and Hoechst staining assays. However, both biophysical as well as molecular docking studies suggested a combilexin-type of interaction between these derivatives and DNA, unlike simple β-carbolines. With a view to understand their mechanism of action, DNA topoisomerase II (topo II) inhibition assay was also performed. Overall, the present study emphasizes the importance of linking a dithiocarbamate moiety to the β-carboline scaffold for exhibiting profound activity.

Full text of DOI:10.1016/j.bmc.2015.07.037

Accepted Manuscript
Design and synthesis of dithiocarbamate linked β-carboline derivatives: DNA
topoisomerase II inhibition with DNA binding and apoptosis inducing ability
Ahmed Kamal, Manda Sathish, V. Lakshma Nayak, Vunnam Srinivasulu, Botla
Kavitha, Yellaiah Tangella, Dinesh Thummuri, Chandrakant Bagul, Nagula
Shankaraiah, Narayana Nagesh
PII:
S0968-0896(15)00621-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.037
BMC 12466
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 April 2015
17 July 2015
19 July 2015
Please cite this article as: Kamal, A., Sathish, M., Lakshma Nayak, V., Srinivasulu, V., Kavitha, B., Tangella, Y.,
Thummuri, D., Bagul, C., Shankaraiah, N., Nagesh, N., Design and synthesis of dithiocarbamate linked β-carboline
derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing ability, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.037
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Design and synthesis of dithiocarbamate linked β-carboline derivatives:
DNA topoisomerase II inhibition with DNA binding and apoptosis inducing
ability
Ahmed Kamal,*a,b Manda Sathish,a V. Lakshma Nayak,a Vunnam Srinivasulu,a Botla
Kavitha,b Yellaiah Tangella,a Dinesh Thummuri,b Chandrakant Bagul,a,b Nagula
Shankaraiah,b and Narayana Nagesh*c
aMedicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad-500 007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500 037, India
cCentre for Cellular and Molecular Biology, Hyderabad-500 007, India
Abstract: A series of new β-carboline-dithiocarbamate derivatives bearing phenyl,
dithiocarbamate and H/methyl substitutions at position-1, 3 and 9 respectively were designed
and synthesized. These derivatives 8a–l and 13a–l and their starting precursors (7a–d and
12a d) have been evaluated for their in vitro cytotoxic activity on selected human cancer
cell lines. Among the derivatives tested, 7c, 12c, 8a, 8d, 8i, 8j, 8k, 8l and 13d–l exhibited
considerable cytotoxicity against most of the tested cancer cell lines (IC50 <10 µM).
Interestingly, most of the derivatives (8a–l and 13a–l) exhibited enhanced activity than their
precursors (7a–d and 12a–d), which indicates that the combination of dithiocarbamate with
β-carboline enhances the cytotoxicity of 8a–l and 13a–l. Moreover, the derivatives 8j and
13g exhibited significant cytotoxic activity with IC50 values of 1.34 µM and 0.79 µM on DU-
145 cancer cells respectively. Further, the induction of apoptosis by these derivatives was
confirmed by Annexin V-FITC and Hoechst staining assays. However, both biophysical as
well as molecular docking studies suggested a combilexin-type of interaction between these
derivatives and DNA, unlike simple β-carbolines. With a view to understand their mechanism
of action, DNA topoisomerase II (topo II) inhibition assay was also performed. Overall, the
present study emphasizes the importance of linking a dithiocarbamate moiety to the β-
carboline scaffold for exhibiting profound activity.
Keywords: β-Carboline; dithiocarbamate; topoisomerase II; anticancer activity; DNA-
binding affinity; pharmacophore.
*Corresponding author. Phone: (+)91-40-27193157; Fax: (+)91-40-27193189; E-mail:
ahmedkamal@iict.res.in
1

1. Introduction
DNA is one of the most essential pharmacological targets of many drugs presently in
clinical usage. Small molecules that bind to DNA have proven to be effective antiviral,
antibacterial and anticancer therapeutic agents.1 Many, small molecules could interact with
DNA through (i) intercalation; (ii) surface binding; (iii) minor groove, and (iv) major groove
binding.2 An important class of drugs in anticancer therapy is DNA intercalators and the
study of the interaction of such agents with DNA, in particular their structural basis could
offer newer insights into the design of new DNA targeting anticancer drugs.3 In addition, the
combilexin class of molecules, with mixed DNA intercalation and DNA groove-binding
properties are capable of enhancing DNA-binding affinity due to their double mode of DNA
interaction, for example NetAmsa (F, Figure 1).4
Presently, a large number of anticancer drugs that are in clinical use are derived from
natural products, it is the rationale to design improved DNA-binding agents based on natural
products (Figure 1).5 The β-carboline alkaloids are a large group of natural and synthetic
indole alkaloids with a broad spectrum of biological and pharmaceutical profiles.6 Recent
reports have revealed that β-carbolines are potential anticancer agents, which exert their
anticancer effects through different mechanisms such as DNA intercalation,7,8 inhibition of
DNA topoisomerase I and II,9 CDK,10 MK-2,11 PLK1,12 kinesin Eg513 and IKK.14 Among
these, DNA intercalation is one of the important mode of action in clinical oncology and
some of such drugs are currently used for the treatment of various cancers.15 β-Carbolines
have been characterized as DNA intercalators due to the presence of planar polycyclic
aromatic pharmacophore, which is capable of stacking between DNA base pairs.16 Moreover,
some of the β-carbolines act as DNA topoisomerase II (topo II) inhibitors, which damage the
DNA.9 Earlier reports have shown that β-carboline derivatives containing a phenyl group at
position-1, some biologically active heterocyclic scaffolds, such as 1,3,4-oxadiazole,17 1,2,4-
triazole,17 4-benzylidene-4H-oxazol-5-one,18 or a substituted carbohydrazide19 moiety at
position-3 and some alkyl or aryl substitutions at position-9 demonstrated significant
anticancer properties.20 In addition, Chen and co-workers have reported that 3-chlorobenzyl
and 3-phenylpropyl substituents at position-9 of β-carbolines showed significant anticancer
activity.20 Ikeda and co-workers have also reported 3-benzylamino-β-carboline derivatives as
potential anticancer agents21 and their SAR analysis revealed that (i) the common β-carboline
skeleton is very important for the cytotoxic activity; (ii) the introduction of appropriate
2

substitution at positions-1, 3 and 9 of the β-carboline nucleus enhanced the cytotoxic
activity.22 Recently, we have reported various β-carboline derivatives bearing different
substituents at positions-1 and 3 of the β-carboline nucleus as promising cytotoxic agents.23,24
Figure 1. Pharmaceutically important β-carboline derivatives (A-E), combilexin molecule (F), dithiocarbamate
derivatives (G and H) and synthesized derivatives (8a l and 13a l).
On the other hand, dithiocarbamates are a common class of organic molecules that
display a wide range of biological activities such as antibacterial, antifungal and anticancer
activities.25 Moreover, brassinin (G, Fig. 1),26,27 an indole based dithiocarbamate isolated
from cruciferous vegetables such as Chinese cabbage, was found to be a significant
chemopreventive agent.27 Similarly, sulforamate (H, Fig 1) is another potent phase II enzyme
inducer that could be used as a cancer chemopreventive agent.28 However, the structure
activity relationship (SAR) of brassinin revealed that chemopreventive nature was due to the
dithiocarbamate motif and not the indole ring.27
In continuation of our earlier attempts toward the discovery of new molecules, we have
developed a number of conjugate/derivative based scaffolds as potent anticancer agents.29 In
3

this context, a series of new β-carboline-3-methyl amines (7a–d and 12a–d) and β-carboline-
dithiocarbamate derivatives (8a–l and 13a–l) respectively have been designed, synthesized
and evaluated for their cytotoxic potential as well as DNA-binding ability. Furthermore, the
in silico study of the absorption, distribution, metabolism and excretion (ADME) properties
of these derivatives (8a–l and 13a–l) were carried out by investigating Lipinski’s parameters,
topological polar surface area (TPSA) and percentage of absorption (% ABS).
2. Results and discussion
2.1. Chemistry
These β-carboline-dithiocarbamate derivatives 8a l and 13a l were synthesized as
shown in Scheme 1. The L tryptophan methyl ester (2) was obtained by esterification of L-
tryptophan (1) by using SOCl2. Pictet–Spengler condensation of 2 with various substituted
benzaldehydes by employing p-TSA on refluxing yielded the corresponding methyl
tetrahydro-β-carboline-3-carboxylates (3a d) which were directly used for further
aromatization with trichloroisocyanuric acid (TCC) to afford the fully aromatized methyl-β-
carboline-3-carboxylates (4a–d). The N-methylation of 4a–d by methyl iodide and sodium
hydride afforded the 9-methyl-β-carboline-3-carboxylates (9a d) in good yields. Next, the
reduction of 4a–d and 9a d with LiBH4 in dry THF afforded the corresponding alcohols
5a d and 10a d respectively, which were taken up for further azidation with
diphenylphosphoryl azide and DBU to provide the corresponding azides 6a d and 11a d
respectively. Then, Staudinger reduction of azide 6a d and 11a d to the corresponding
amines was achieved by triphenylphosphene in acetonitrile and water to afford both 9-H and
9-methyl-β-carboline-3-methyl amine (7a d and 12a d). Finally, the desired
dithiocarbamate linked β-carboline derivatives (8a l and 13a l) were obtained in good
yields by the reaction of 7a d and 12a d with carbon disulfide, alkyl halides (methyl
iodide, allyl bromide and benzyl bromide) and TEA in pyridine.
4

O
O
O
O
NH
OH
O
N
H
NH2
NH2
CHO
N
H
N
H
R1
R1
3a-d
2
1
a
: R1 = 3,4,5-trimethoxy
b
: R1 = 4-methoxy
c
: R1 = 4-triflouromethyl
d
: R1 = 4-flouro
O
O
N3
OH
N
N
N
N
H
N
H
N
H
R1
R1
R1
6a-d
4a-d
5a-d
8a:
8b:
8c:
8d:
8e:
8f:
8g:
8h:
8i:
:13a
:13b
:13c
:13d
:13e
:13f
:13g
:13h
:13i
R1 = 3,4,5-trimethoxy, R2 = methyl
R1 = 3,4,5-trimethoxy, R2 = allyl
R1 = 3,4,5-trimethoxy, R2 = benzyl
R1 = 4-methoxy, R2 = methyl
R1 = 4-methoxy, R2 = allyl
R1 = 4-methoxy, R2 = benzyl
R1 = 4-triflouromethyl, R2 = methyl
R1 = 4-triflouromethyl, R2 = allyl
R1 = 4-triflouromethyl, R2 = benzyl
R1 = 4-flouro, R2 = methyl
O
O
NH2
N
N
N
N
H
R1
R1
7a-d
9a-d
8j:
:13j
d 10a-d)
i)
(
e (11a-d)
f (12a-d)
:13k
:13l
ii)
iii)
8k:
8l:
R1 = 4-flouro, R2 = allyl
R1 = 4-flouro, R2 = benzyl
S
S
R2
R2
N
H
S
N
H
S
N
N
N
H
N
R1
R1
13a-l
8a-l
o
Scheme 1. Reagents and conditions: a) SOCl2, MeOH, 0 C rt, 12 h, 90%; b) substituted benzaldehyde, p-
o
o
TSA, EtOH, reflux, 12 h; c) TCC, TEA, DMF, 0 C-rt, 2 h, 70-85%; d) LiBH4, THF, 0 C rt, 4 h; e) DPPA,
DBU, dry THF, rt,16 h, 60–80%; f) TPP, acetonitrile : water (1:1), rt, 12 h, 55 70%; g) alkyl halide, CS2, TEA,
pyridine, 0 oC, 30 min, 80 95 %; h) MeI, NaH, dry DMF, 0 oC, 4 h, 70 90%.
2.2. Biology
2.2.1. Cytotoxic activity
To evaluate the cytotoxicity of β-carboline-3-methyl amines (7a–d and 12a–d) and
the dithiocarbate linked β-carboline derivatives (8a–l and 13a–l) MTT assay30 was carried out
on the selected human cancer cell lines, such as lung cancer (A549), breast cancer (MCF-7),
prostate cancer (DU-145), and cervical cancer (HeLa). The cytotoxicity results are expressed
5

Table 1
Cytotoxicity (IC50 values in µM)a of β-carboline-3-methylamines (7a d and 12a d) and β-
carboline-dithiocarbamate derivatives 8a l and 13a l on selected human cancer cell lines
Compound
A549b
MCF-7c
DU-145d
Helae
7a
15.13
17.84
15.84
21.10
18.15
20.09
19.19
19.49
8.12
12.8
27.5
7.94
51.4
17.7
16.9
18.6
9.33
4.36
16.7
39.8
12.5
18.1
14.1
5.12
10.0
9.12
3.54
4.26
5.12
5.62
6.02
6.76
1.58
18.62
18.91
12.02
17.95
15.13
16.62
11.74
16.59
8.91
15.8
25.8
8.51
38.8
10.0
15.8
10.9
12.3
2.45
6.60
7.41
15.2
20.6
13.9
1.51
3.20
3.36
1.09
4.78
4.31
1.86
3.01
2.75
0.96
18.26
17.39
9.12
13.77
14.77
12.78
7.76
10.00
5.62
13.8
17.3
1.14
46.7
13.8
9.54
7.76
5.88
1.34
3.98
3.71
6.16
19.4
10.6
1.41
2.50
6.91
0.79
1.99
2.08
1.45
2.88
4.78
1.38
21.66
18.59
15.76
22.50
19.95
14.79
15.07
20.51
11.3
23.2
14.5
7.78
59.1
24.1
12.6
10.6
3.98
3.46
4.46
3.81
16.9
19.9
12.3
2.57
8.30
13.8
1.47
4.67
4.78
2.39
2.57
3.16
2.63
7b
7c
7d
12a
12b
12c
12d
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
Doxorubicinf
a
50% Inhibitory concentration after 48 h of drug treatment and the values are average of three individual
experiments.
b Human lung cancer (A549).
c Human breast cancer (MCF-7).
d Human prostate cancer (DU-145).
e Human cervical cancer (HeLa) cells.
f Doxorubicin was included as reference agent.
6

in IC50 values that are tabulated in Table 1 and compared with the positive control
doxorubicin. It is evident that most of the derivatives exhibited moderate to good cytotoxicity
against the cancer cell lines tested, with IC50 values ranging from <1.0 µM to 60 µM. Among
the derivatives, 8a, 8d, 8i–l and 13d–l exhibited considerable cytotoxicity against all the
cancer cell lines (<10.0 µM). The most active derivatives in this series are 8d, 8j, 13d, 13j,
13g and 13h with IC50 values ranging from <1 µM to 2.0 µM. However, most of these
derivatives showed higher cytotoxicity on DU-145 (prostate cancer) cell line in comparison
to other cell lines. Derivatives 8j and 13g were found to be the most effective derivatives
amongst the series exhibiting IC50 value of 1.34 and 0.79 µM respectively against DU-145
cell line and have also shown considerable activity against the other cell lines tested.
Based on the cytotoxicity assay, it is observed that the activity depends upon the
substitution at positions 1, 3 and 9 of the β-carboline as well as on the nature of the
substituents present on the phenyl ring (R1) at position-1, substituents on the dithiocarbamate
(R2) and also H/Me at position-9 of β-carboline. Overall, the cytotoxicity of β-carboline-3-
methyl amines (7a–d and 12a–d) was comparatively lower than the dithiocarbamate linked
β-carboline derivatives (8a–l and 13a–l), indicating that the enhanced cytotoxicity of 8a–l
and 13a–l is due to the attachment of dithiocarbamate side chain. Moreover, the new
derivatives with a fluoro and trifluoromethyl (8g–l and 13g–l) substituent on the phenyl ring
at position-1 have shown significant cytotoxicity, probably due to the lipophilic nature of
fluorine. Methyl dithiocarbamate linkage at position-3 of the β-carboline (incase of 8a, 8d,
8g, 8j, 13a, 13d, 13g and 13j) enhanced the cytotoxic activity in comparison to the other
derivatives, thus indicats the importance of dithiocarbamate group. Furthermore, methyl
substitution on position-9 also plays a crucial role for activity as all the 9-methyl β-carboline
derivatives (13a–l) synthesized are most active with IC50 values <20 µM than the 9-H β-
carboline derivatives (8a–l) with IC50 values <60 µM. Other derivatives possessing 3,4,5-
trimethoxy and 4-methoxy at R1, as well as allyl and benzyl at R2 (8a, 8d, 13a and 8d and 8f)
were also considerably active against most of the cell lines tested. However, derivative with a
4-methoxy substituent at R1 and allyl substituent at R2 (8e) was less active with IC50 value of
>30 μM in this series. These results imply that the methyl group at R2 represents the most
appropriate structure for this class of derivatives, which displays remarkable cytotoxic
activity. From the SAR studies, some interesting observations were made, such as
trifluoromethyl and fluoro substitutions on the phenyl ring (R1) at position-1 and a
methyldithiocarbamate group at position-3 of the β-carboline is crucial for cytotoxicity,
7

nevertheless some derivatives with methyl group at position-9 also display potent
cytotoxicity.
2.2.2. Cell cycle analysis
To understand the effect of the active derivatives on cell cycle progression, cell cycle
analysis was performed on DU-145 cell line. On treatment of DU-145 cells with 0.5 µM
concentrations of 8j and 13g, a small increment in population of cells at sub-G1 stage (7.3 %
and 10.2 %) was observed compared to control cells (6.5 %). Whereas, on doubling the 8j
and 13g concentrations (1.0 µM), the population of cells in sub-G1 further increased to
15.6% and 18.6% respectively. These results imply that the cell cycle arrests at the sub-G1
phase and thereby induces cellular apoptosis.31 The cell cycle histograms obtained for
treatment of 13g and 8j at 0.5 µM and 1µM concentrations are shown in Fig. 2 and the results
are displayed in Table 2.
Figure 2. Flow cytometric analysis in DU-145 prostate cancer cell lines after treatment with derivatives 8j and
13g and 0.5 and 1 µM concentrations for 48 h; A. control cells (DU-145); B. 8j (0.5 µM); C. 8j (1.0 µM); D.
13g (0.5 µM) and E. 13g (1.0 µM).
8

Table 2
Cell cycle results obtained when DU-145 cells were treated with 0.5 µM and 1.0 µM
concentration of 8j and 13g.
Sample
Sub G1 %
G0/G1 %
85.94
84.83
75.52
78.90
S %
4.02
3.36
3.34
5.18
5.31
G2/M %
1.97
1.98
2.33
3.73
A: Control
B: 8j (0.5 µM)
C: 8j (1 µM)
D: 13g (0.5 µM)
E: 13g (1 µM)
6.52
7.34
15.61
10.12
18.60
70.81
5.36
2.2.3. Apoptosis induction studies
2.2.3.1. Annexin V-FITC
To further confirm the effect of 8j and 13g in inducing apoptosis, Annexin V-FITC/PI
(AV/PI) dual staining assay was performed. This study is also useful to examine the
occurrence of phosphatidylserine externalization as well as to find out whether it is due to
physiological apoptosis or nonspecific necrosis.32 Phosphatidylserine is used as a marker for
cell death, moving from the inner cell membrane to the outer cell membrane during the
cascade of apoptosis.33 Annexin V is a cellular protein of the annexin group which selectively
binds to phosphatidylserine.
Table 3
Percentage of apoptotic cells observed when DU-145 cells were treated with 0.5 and 1.0 µM
concentrations of 8j and 13g.
Sample
UL %
UR %
LL%
LR %
A: Control
B: 8j (0.5 µM)
C: 8j (1 µM)
D: 13g (0.5 µM)
E: 13g (1 µM)
0.10
0.14
0.96
0.93
1.65
1.52
4.17
14.68
13.70
21.19
97.95
94.63
79.95
81.55
74.99
0.43
1.07
4.40
3.82
2.18
In this study, DU-145 cells were treated with 8j and 13g for 48 h at 0.5 and 1.0 µM
concentrations, and we found that these derivatives demonstrated significant apoptosis, as
tabulated in Table 3 and dot plots are shown in Fig. 3. The results indicated that 8j and 13g
showed 19.0% and 23.3% apoptosis at a concentration of 1.0 µM, whereas 1.9% of apoptosis
was observed in the control, this shows that derivative 13g is a better inducer of apoptosis
than 8j.
9

Figure 3. a) Annexin V-FITC/PI (AV/PI) dual staining assay. b) Hoechst (H33258) staining assay.
2.2.3.2. Hoechst staining
Apoptosis is one of the main pathways that lead to cell death in which the chromatin
condensation and fragmented nuclei are known as the classic characteristics.34 Flow
cytometry experiments demonstrated that 8j and 13g were effective in inducing apoptosis on
DU 145 cells. Further, to visualize the apoptosis-inducing effect of such derivatives,
Hoechst (H33258) staining was performed in DU 145 cells. Manual field quantification of
apoptotic cells based on the presence of apoptotic bodies, cellular condensation, and nuclear
fragmentation under the microscope in the presence of tested derivatives 8j and 13g showed a
significant increase in the percentage of apoptotic cells in treated cells compared to the
control. However, this study shows that 13g is more effective than 8j in causing apoptosis as
more number of wrinkled and DNA fragmented cells were observed in cells treated with 13g
(0.5 µM) than in 8j (0.5 µM) as shown in Fig. 3.
2.2.4. DNA-topoisomerase II inhibition study
DNA-topoisomerase II (topo II) is a nuclear enzyme that controls the DNA structure
by catalysing the DNA cleavage and relegating the phosphodiester bonds. In recent years,
DNA topo II inhibitors have gained importance as clinically useful chemotherapeutic agents.
Topo II inhibition studies were performed by using topo II Drug Screening Kit (TG 1009,
Topogen, USA), which is suitable for screening the enzyme inhibition by both catalytic
inhibitory compounds (CICs) and interfacial poisons (IFPs). CICs inhibit the activity of the
10

Figure 4. Effect of 8j and 13g on topo II inhibition.
enzyme, whereas IFP compounds stimulate the formation of the cleavage complexes and
block the relegation which leads to form linear DNA. Catenated DNA in the presence of topo
II enzyme incubated with etoposide at concentration of 1, 10 and 100 µM (lane E to G in
control, Fig. 4) showed clear linear DNA formation indicating that it acts as IFP which blocks
the resealing of the dsDNA. Catenated DNA in the presence of topo II enzyme incubated
with compound 8j (lane E to G in 8j, Fig. 4) at concentration of 1-100 µM did not show the
formation of linear DNA, but most of the catenated DNA was highly super coiled and
remained in the wells documenting the formation of only nicked circular and relaxed DNA,
indicated that the compounds interference in the catalytic activity of the topo II enzyme and
suggesting that the role of 8j as CIC. Further, catenated DNA in the presence of topo II
incubated with compound 13g showed the formation of linear DNA at concentrations of 1-
100 µM in a similar pattern to etoposide indicating that it acts as IFP (lane E to G in 13g, Fig.
4). This assay indicates that both derivatives 8j and 13g affect the topo II enzyme activity,
however derivative 8j acts as CIC while derivative 13g acts as IFP.
2.2.5. DNA binding studies
To further substantiate the biological activities of these active derivatives (8j and 13g)
and to understand the nature of interaction with DNA, spectroscopic studies were performed.
2.2.5.1. UV-visible spectroscopy
In order to investigate the nature of binding of these derivative molecules to DNA, the
UV absorption spectra of active derivatives 8j and 13g in the presence and absence of
increasing concentrations of CT DNA were examined (Fig. 5). On addition of DNA to
derivative solution (8j or 13g) the absorption band at 210 nm intensity gradually increases
11

Figure 5. UV–visible spectra (left), fluorescence spectra (middle) and CD spectra (right) of derivatives 8j and
13g.
without exhibiting any shift, whereas the absorption band at 340 nm showed gradual decrease
for derivative 8j and slight decrease for derivative 13g in its intensity. Since the absorption
band at 210 nm shows hyperchromicity upon addition of DNA, it is evident that these
derivatives could bind to the surface of DNA,35 however hypochromicity of absorption band
at 340 nm indicates partial intercalation to DNA.36 Interestingly, this data indicates that 8j
and 13g exhibit combilexin-type of interaction with the DNA. However, the extent of
variation in absorption band intensity at 210 nm and 340 nm is more with 8j than 13g,
emphasizing higher ability of 8j to interact with the DNA.
2.2.5.2. Fluorescence spectroscopy
Fluorescence titration is another valuable technique to understand the binding mode
of small molecules to DNA at comparatively lower concentrations.37 As these derivative
molecules show fluorescence properties, their interaction with CT-DNA could provide
certain observations regarding the nature of their interactions with DNA. In this study, when
CT-DNA was added to the derivative, the fluorescence emission peak of 8j and 13g,
exhibited hypochromicity. Hypochromicity of the fluorescence emission peak was observed
at 617 and 567 nm for the 8j and 13g respectively, indicating that the fluorescence of each
derivative molecule getting quenched due to its interaction with CT-DNA. The quenching
may be due to energy transfer from excited derivative molecules to the DNA bases.
Fluorescence titration results indicate that these derivative molecules may intercalate between
the DNA bases.38 The fluorescence spectra recorded on the interaction of 8j and 13g with
CT-DNA was depicted in Fig. 5. Among 8j and 13g, the extent of quenching of emission
12

band intensities is more with 8j, indicating its higher level of intercalation with CT-DNA.
Most of the catalytic inhibitory compounds (CIC) are strong intercalators and probably 8j
acts as a CIC due to its higher intercalation with DNA (as demonstrated by fluorescence
studies).
2.2.5.3. Circular dichroism spectroscopy
The circular dichroism (CD) is a potential technique to study the nucleic acid
conformational change due to interaction of DNA with molecules or changes in
environmental conditions.39 The CD spectrum of the calf thymus DNA (CT DNA) exhibits a
positive band at 275 nm and a negative band at 245 nm due to π–π base stacking and right-
hand helicity. On the addition of 8j at a concentration of 10 µM to the same concentration of
CT DNA solution (DNA: 8j ligand, 1:1), positive band at 275 nm exhibits slight
hyperchromicity, which is an indication of stabilization of the DNA–ligand complex.40,41
Interestingly, on further increasing the concentration of 8j (DNA:8j ligand, 1:2) the positive
band at 275 nm also increases its intensity, indicating further stabilization of the DNA on
ligand binding. Interestingly, a similar interaction was observed when 13g was added to the
CT-DNA solution, the positive CD band at 275 nm exhibited continuous hyperchromicity at
both lower and higher concentrations, demonstrating complex stabilization. The negative
band intensity at 245 nm reduced to a greater extent with 8j compared to 13g, indicating its
highest ability to minimize DNA staking. From Fig. 5, it is evident that hyperchromicity is
more in case of 8j, indicating its higher potential to stabilize CT-DNA by intercalation,
compared to 13g , which may be due to its planarity.
2.3. In silico computational studies
An in silico computational study of the representative substituted dithiocarbamate
linked β-carboline derivatives 8a l and 13a l was performed for determining the Lipinski’s
parameters, topological polar surface area (TPSA) and percentage of absorption (% ABS).42
Calculations were performed by using the Molinspiration Online Property Calculation
Toolkit (http://www.molinspiration.com).43 The percentage of absorption was estimated by
using the equation: % ABS = 109 − 0.345 × TPSA and the data generated are shown in Table
4. In vivo absorption of the newly synthesized β-carboline-dithiocarbamate derivatives was
tentatively assessed by means of theoretical calculations following Lipinski’s rule of five,
which implies that the absorption or permeation of an orally administered compound is more
13

Table 4
Lipinski’s parameters for derivatives 8a l and 13a l
Lipinsk’s parameters
Compound
TPSA
nHBA
(NO)
nHBD
(OHNH)
No.
Violations
logP
MW
8a
8b
68.412
68.412
68.412
49.944
49.944
49.944
40.71
6
6
6
4
4
4
3
3
3
3
3
3
6
6
6
4
4
4
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
4.623
453.589
0
1
2
1
1
1
1
1
2
0
1
1
0
1
2
1
1
1
1
1
2
1
1
1
5.267
6.218
5.048
5.693
6.643
5.887
6.532
7.482
5.155
5.80
479.627
529.687
393.537
419.575
469.635
431.508
457.546
507.606
381.501
407.537
457.599
467.616
493.654
543.714
407.564
433.602
483.662
445.535
471.573
521.633
395.528
421.566
471.626
8c
8d
8e
8f
8g
8h
40.71
8i
40.71
8j
40.71
8k
40.71
8l
40.71
6.750
4.691
5.335
6.285
5.116
5.761
6.711
5.955
6.599
7.55
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
57.555
57.555
57.555
39.087
39.087
39.087
29.853
29.853
29.853
29.853
29.853
29.853
5.223
5.868
6.818
14

likely to be better if the molecule satisfies the following rules: (i) hydrogen bond donors ≤5
(OH and NH groups); (ii) hydrogen bond acceptors ≤10 (N and O atoms); (iii) molecular
weight <500; (iv) calculated log P <5.42 Compounds that violate more than one of these rules
could have problems related to bioavailability.
Most of these new derivatives violated only one of the Lipinski’s rules, except 8c, 8i, 13c and
13i. Gratifyingly, the derivatives such as 8a, 8j, and 13a in this series satisfied all the
Lipinski’s rules. Furthermore, derivatives that violated Lipinski’s rules either have an
octanol–water partition coefficients (log P) >5.0 or molecular weight larger than 500 Da only.
All these derivatives have a number of hydrogen bond acceptors between 3 and 6 and 1–2
hydrogen bond donors respectively and these values are in agreement with the Lipinski’s
rules. Moreover, the calculated percentage of absorption of all derivatives ranged between
29.8% and 68.4%, demonstrating that these new derivatives may have the required cell
membrane permeability.
2.4. Molecular docking studies
Most of the β-carboline derivatives that bind to topoisomerase enzyme, results in
significant anticancer activity.9 In the present study, the derivatives 8j and 13g have shown
better topo II inhibition and cytotoxicity on selected human cancer cell lines. These results
are encouraged us to perform molecular docking studies to understand the binding mode of
these new derivatives to the different targets. Herein, all the co-ordinates of the crystal
structures were obtained from Protein Data Bank. Necessary corrections to the protein and
DNA structures were carried out by using Protein Preparation Wizard from Schrödinger
package. Geometry optimization of these derivatives was carried out by Gaussian 09 package
using PM3 semiempirical method.44 All the docking studies were performed using AutoDock
4.2 docking software45 and results were visualized through PyMOL.46 These studies were
performed on the new derivatives (8a l and 13a l)) and their amine intermediates (7a d
and 12a d)) at the ATP binding site of the Topo-II (PDB ID 1ZXN).47 Docking studies
showed that these molecules fit well in the ATP binding site of the Topo-II (Fig. 6).
Superimposed pose of the 13g and co-crystallize ligand shown phosphate groups of the ADP
and methyldithiocarbamate group was at the same position and β-carboline ring and
nucleoside group of co-crystallized ligand was at the same position. However, β-carboline
ring forms
a
T-type π-π stacking interaction with Phe142. Besides this,
methyldithiocarbamate forms a hydrophobic contact with the Ile141, Thr147, Ser148,
15

Gly161, Arg162, Asn163, Gly164, Tyr165, Gly166, and Ala167 amino acids and two
hydrogen-bonding interactions with
Figure 6. Docking poses for 13g; A) Binding pose in topo II; B) superimposed pose of 13g and co-crystal
ligand in topo II; C) binding pose of 13g in CT-DNA showing intercalation as binding mode.
Gly166 and Ser148. Further, β-carbolin ring forms a hydrophobic interaction with Tyr34,
Ile88, Asn91, Ala92, Asn95, Ilu118, Asn120, Ile125, Tyr215 and Ile217 amino acids.
However, trifluoromethylphenyl ring forms a hydrophobic interaction with Asp94,
Arg98, Ser149, Asn150, Lys157 and Thr159. In view of the biological activity, it is observed
that the derivatives with 9-methyl substitution (13a l) having the higher activity than the 9-
H substitution (8a l). To understand the binding poses of these derivatives, the both series
(8a l and 13a-l) were examined. Superimposed poses of 8g and 13g (with 9-methyl
substituent) shows that both are having different binding pose where only
methyldithiocarbamate groups are overlapped. Derivative 8g is below that of 13g and which
is persistent to all the remaining compounds in both the series. Also it is observed that the
series with 9-H substitution having distinct binding pose than the co-crystallized ligand.
Hence, 9-H substituted derivatives having different binding pose from 9-methyl substituted
derivatives and co-crystallized ligand, therefore methyl group assist to keep this binding pose
like co-crystal ligand. Deviation of this binding pose may be accountable for overall decrease
in the biological activity of 9-H substituted derivatives. Moreover, the most active derivative
13g having trifluoromethyl group and that fluorine is having orthogonal multipolar
interactions with the carbonyl oxygen of Asn150 which may be responsible for the highest
activity of 13g. Additionally, the docking studies were also performed for the amine
intermediates 7a d and 12a d and these results are showing different binding poses in
compare to the final molecules. However, these intermediates (7a d and 12a d) were
lacking the two additional hydrogen bonding interactions which were fulfilled by
dithiocarbamate group in final derivatives and this could be the reason for lesser activity of
these intermediates.
16

To know the binding mode of action for these new molecules, DNA docking studies
were performed on two different DNA structures, first with already intercalated co-crystal
ligand (PDB ID 1NAB)48 and second with co-crystallized ligand that binds in the minor
groove of DNA (PDB ID 195D).49 Docking studies on the 1NAB showed that these
molecules bind properly at the intercalation site, whereas trifluoromethylphenyl and
methyldithiocarbamate were extended out to the minor groove site and β-carboline ring
stacks with the nucleotide base pairs, and NH of methyldithiocarbamate group shows strong
hydrogen-bonding interactions with the carbonyl oxygen of cytosine. While docking studies
on 195D showed that these molecules have potential to bind in the minor groove where β-
carboline ring and methyldithiocarbamate groups are towards the minor groove and 4-
trifluoromethylphenyl group extends out from the minor groove. Whereas in NH group of the
methyldithiocarbamate forms additional hydrogen bonding interactions with sugar moiety of
the nucleotide. Docking studies have shown that these molecules having potential for
multiple modes of binding.
3. Conclusions
In conclusion, we have designed and synthesized a new class of dithiocarbamate
linked β-carboline derivatives by introducing a substitution of the phenyl at the position-1,
dithiocarbamate at position-3 and a H/methyl group at the position-9. These derivatives that
contain two pharmacphoric groups such as β-carboline ring, dithiocarbamate and the
derivatives 8j and 13g displayed promising cytotoxic activity and induced apoptosis. In
addition, these derivatives also inhibit topo II enzymatic activity. However, the binding mode
of both these derivatives with DNA is combilexin-type, which is different from the usual β-
carboline alkaloids. Although both the derivatives exhibit good interaction with DNA,
however the interaction of 8j is better with DNA due to its planar structure. Perhaps, due to
the influence of pharmacophores on the β-carboline ring, 13g scored higher potential in cell
biology studies. Nevertheless, these studies are useful for understanding the role of different
pharmacophores that are connected to β-carboline nucleus in enhancing cytotoxicity,
apoptosis, DNA binding ability and inhibition of DNA topo II.
4. Experimental Section
4.1. Materials and methods
Chemistry: Chemical reagents were purchased from Sigma–Aldrich and were used
without further purification. Anhydrous THF, CH2Cl2, CH3CN, toluene, DMF and ether used
17

in reactions were prepared by distillation under nitrogen over Na/benzophenone, CaH2,
Na/P2O5, and CaH2/molecular sieves, respectively. Solvents for extraction and column
chromatography were distilled prior to use. TLC analysis were performed with silica gel
plates (0.25 mm, E. Merck, 60 F254) using ninhydrine, p-anisaldehyde, KMnO4, iodine, and
UV lamp for visualization. 1H and 13C NMR experiments were performed on 300 or 500 and
75 or 125 MHz respectively, on a Bruker Avance. Chemical shifts are reported in parts per
million (ppm) downstream from the internal tetramethylsilane standard. Spin multiplicities
are described as s (singlet), bs (broad singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet) and m (multiplet). Coupling constants are reported in Hertz (Hz). Melting points
were determined on an Electrothermal melting point apparatus and are uncorrected. FT-IR
spectra for all compounds were recorded using thin film as well as a KBr pellet. Mass spectra
were recorded by electrospray ionization mass spectrometry (ESI MS). High-resolution mass
spectra (HRMS) were recorded on a QSTAR XL Derivative MS-MS mass spectrometer.
4.2. (S)-Methyl 2-amino-3-(1H-indol-3-yl)propanoate (2)19
To the stirred solution of L-tryptophan (0.1 mol) in methanol (50 ml), thionyl chloride
o
8.02 ml (0.11mol) was added drop wise at 0 C and continued stirring for 12 h at room
temperature. Then, the excess amount of solvent was removed under vacuum and the crude
product was azeotroped with toluene (2×10 ml) to obtain dry solid. Then, the resulting solid
was dissolved in CH2Cl2, basified with saturated NaHCO3 solution and extracted with excess
amount of CH2Cl2. Then, the organic layer was dried over anhydrous sodium sulphate and
concentrated under reduced pressure to obtain pure product. White solid; 19.62 g; 90% yield;
Mp: 102 103 oC; 1H NMR (300 MHz, CDCl3): δ 2.00 (s, 2H), 3.00 (dd, J = 4.12, 13.8 Hz,
1H), 3.25 (dd, J = 4.45, 13.8 Hz, 1H), 3.73 (s, 3H), 3.84 (t, J = 4.90, 5.42 Hz, 1H), 6.90 (s,
1H), 7.08 (t, J = 6.90, 7.14 Hz, 1H), 7.14 (t, J = 6.90, 7.14 Hz, 1H), 7.25 (d, J = 7.90 Hz,
1H), 7.58 (d, J = 7.90 Hz, 1H), 8.81 (bs, 1H); MS (ESI): m/z 219 [M + H] +.
4.3. General reaction procedure for the preparation of compounds (4a d).
To the mixture of L tryptophan ester (2, 1 mmol) and substituted benzaldeyde (1.1
mmol) in ethanol, catalytic amount of p-TSA was added and the mixture was refluxed for 12
h. After completion of the reaction, ethanol was removed under vacuum and the resulting
crude product 3a d (1.0 mmol) was taken in DMF and added triethylamine (3.0 mmol), a
solution of trichloroisocyanuric acid (1.1 mmol) in DMF was added dropwise at -20 oC. The
reaction mixture was slowly warmed up to 0 oC and stirred at this temperature for 2 h. After
18

the completion of the reaction, added ice water, the resulted precipitate was filtered, washed
with water, and dried under vacuum to give the compound (4a d).
4.3.1. Methyl 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4a)23
Yellow solid: 76% yield; mp: 229–230 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.38
(bs, 1H), 8.75 (s, 1H), 8.21(d, J = 7.5 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.56–7.61 (m, 1H),
7.32–7.38 (m, 1H), 6.94 (s, 2H), 4.03 (s, 3H), 3.84 (s, 3H), 3.77 (s, 6H); 13C NMR (75 MHz,
DMSO d6 + CDCl3) δ (ppm): 165.8, 152.7, 142.1, 141.2, 137.8, 136.1, 134.4, 132.8, 128.7,
128.0, 121.1, 120.9, 119.9, 116.1, 112.4, 105.5, 59.8, 55.5, 51.6; MS (ESI): m/z 393 [M +
H]+; HRMS calculated for C22H21O5N2 m/z 393.14450 [M + H]+, found m/z 393.14320.
4.3.2. Methyl 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4b)23
White solid; 85% yield; mp: 226–230 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.18
(bs, 1H), 8.80 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.57–7.55 (m, 2H),
13
7.36–7.33 (m, 1H), 6.88 (d, J = 8.6 Hz, 2H), 4.03 (s, 3H), 3.77 (s, 3H); C NMR (75 MHz,
DMSO d6 + CDCl3) δ (ppm): 165.9, 159.5, 141.9, 141.2, 136.2, 134.3, 129.9, 129.6, 128.6,
127.8, 121.0, 120.9, 119.8, 115.6, 113.5, 112.4, 54.8, 51.6; MS (ESI): m/z 333 [M + H]+;
HRMS calculated for C20H17O3N2 m/z 333.12337 [M + H]+, found m/z 333.12194.
4.3.3. Methyl 1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4c)23
1
Pale yellow solid; 75% yield; mp: 250–252 °C; H NMR (300 MHz, DMSO–d6) δ
(ppm): 11.99 (bs, 1H), 8.93 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 8.23 (d, J = 7.7 Hz, 2H), 7.92
(d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H),
3.94 (s, 3H); 13C NMR (75 MHz, DMSO d6) δ(ppm): 165.8, 141.4, 141.2, 140.2, 136.6,
134.6, 129.6, 129.5, 129.4, 129.2, 128.8, 125.5 (q, J = 3.3 Hz), 122.0, 120.9, 120.5, 117.2,
112.6, 52.0; MS (ESI): m/z 371 [M + H]+; HRMS calculated for C20H14F3O2N2 m/z
371.33257 [M + H]+, found m/z 371.09941.
4.3.4. Methyl 1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4d)23
White solid: 70% yield; mp: 195–198 °C; 1H NMR (500 MHz, CDCl3) δ (ppm): 8.87
(s, 1H), 8.83 (bs, 1H), 8.22 (d, J = 7.8 Hz, 1H), 7.92 (q, J = 5.3, 8.7 Hz, 2H), 7.63–7.55 (m,
2H), 7.39 (t, J = 7.9 Hz, 1H), 7.22 (t, J = 8.7 Hz, 2H), 4.06 (s, 3H); 13C NMR (75 MHz,
19

CDCl3) δ (ppm): 165.7, 160.7 and 164.0 (d, J = 247.0 Hz), 141.3, 140.8, 136.3, 134.3, 133.7,
130.4 (d, J = 8.2 Hz), 129.0, 128.2, 121.3, 120.9, 120.0, 116.2, 115.2 (d, J = 21.4 Hz), 112.4,
51.6; MS (ESI): m/z 321 [M + H]+; HRMS calculated for C19H14O2N2F m/z 321.10338 [M +
H]+, found m/z 321.10298.
4.4. General rection procedure for the preparation of compounds (9a d)
To a stirred solution of 4a d (1 mmol) in DMF was added 60% NaH in paraffin oil
o
(4 mmol) at 0 C and stirred for 15 min. Then, added methyl iodide (1.2 mmol) and the
reaction mixture was stirred for 4 hr and added ice cold water slowly, extracted with ethyl
acetate, washed with water, dried over sodium sulfate and concentrated under reduced
pressure to get N methyl compounds 9a d. These products were directly used for next step
without any further purification.
4.4.1. Methyl 9-methyl-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-
carboxylate (9a)
1
Off white solid: 72% yield; mp: 195–198 °C; H NMR (300 MHz, CDCl3) δ (ppm):
8.93 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.40
(t, J = 7.6 Hz, 1H), 6.85 (s, 2H), 4.04 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.55 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ (ppm): 166.7, 153.1, 143.6, 143.0, 138.4, 136.6, 136.2, 134.5,
129.8, 128.9, 121.6, 121.2, 120.8, 116.8, 110.0, 107.1, 60.7, 56.2, 52.6, 32.7; MS (ESI): m/z
407 [M + H]+, HRMS calculated for C23H23N2O5 m/z 407.16015 [M + H]+, found m/z
407.16019.
4.4.2. Methyl 1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (9b)
1
Cream white solid: 80% yield; mp: 210–215 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.88 (s, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.66–7.62 (m, 1H), 7.58 (d, J = 8.7 Hz, 2H),
7.46 (d, J = 8.4 Hz, 1H), 7.40–7.36 (m, 1H), 7.05 (d, J = 8.7 Hz, 2H), 4.03 (s, 3H), 3.90 (s,
3H), 3.51 (s, 3H); 13C NMR (125 MHz, CDCl3) δ (ppm): 166.8, 160.0, 143.8, 143.1, 136.8,
136.5, 131.5, 131.0, 129.8, 128.7, 121.6, 121.3, 120.7, 116.4, 113.6, 110.0, 55.3, 52.6, 32.9;
MS (ESI): m/z 347 [M + H]+, HRMS calculated for C21H19N2O3 m/z 347.13902 [M + H]+,
found m/z 347.13894.
4.4.3. Methyl 9-methyl-1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-3-
carboxylate (9c)
20

White solid: 73% yield; mp: 220–225 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.94
(s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.80 (s, 4H), 7.68 (t, J = 7.9 Hz, 1H), 7.49 (d, J = 8.2 Hz,
1H), 7.41 (t, J = 7.9 Hz, 1H), 4.04 (s, 3H), 3.50 (s, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm):
166.5, 143.1, 142.7, 142.0, 137.1, 136.3, 130.4, 130.3, 129.2, 125.8, 125.2 (q, J = 3.3 Hz),
122.2, 121.7, 121.2, 121.0, 117.1, 110.1, 52.7, 33.2; MS (ESI): m/z 385 [M + H]+, HRMS
calculated for C21H16O2N2F3 m/z 385.11584 [M + H]+, found m/z 385.11554.
4.4.4. Methyl 1-(4-fluorophenyl)-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (9d)
1
Cream white solid: 76% yield; mp: 223–225 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.91 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.67–7.63 (m, 3H), 7.48 (d, J = 8.4 Hz, 1H),
7.40 (t, J = 7.2 Hz, 1H), 7.23 (t, J = 8.7 Hz, 2H), 4.04 (s, 3H), 3.50 (s, 3H); 13C NMR (75
MHz, CDCl3) δ (ppm): 166.6, 164.7 and 161.4 (d, J = 248.1 Hz), 143.1, 142.7, 136.9, 136.4,
135.2, 131.6 (d, J = 8.2 Hz), 130.1, 129.0, 121.7, 121.3, 120.9, 116.8, 115.3 (d, J = 22.0 Hz),
110.0, 52.7, 32.9; MS (ESI): m/z 335 [M + H]+, HRMS calculated for C20H16O2N2F m/z
335.11903 [M + H]+, found m/z 335.11872.
4.5. General reaction procedure for the preparation of compounds (5a d and 10a d)
A stirred suspension of compounds 4a d/9a d (20 mmol) in dry THF (60 mL) was
o
added LiBH4 (22 mmol) at 0 C and stirred for 4 h at room temperature. Then, the reaction
mixture was quenched with saturated NH4Cl solution and extracted with ethyl acetate and
washed with water. The organic layer was dried over anhydrous sodium sulphate and
evaporated under reduced pressure. Then, the crude residue was used directly for the next
step without any further purification.
4.6. General reaction procedure for the preparation of compounds (6a d and 11a d).
To a stirred solution of 5a d/10a d (10 mmol) in dry THF was added DBU (15
o
mmol) and DPPA (12 mmol) at 0 C. The reaction mixture was stirred for 16 h at rt and the
reaction mixture was quenched with water, extracted with ethyl acetate and washed with
water. The combined organic phases were dried over sodium sulphate and concentrated under
reduced pressure to get crude products, which were purified by column chromatography
using silica gel to get pure compounds 6a d/11a d.
4.6.1. 3-(Azidomethyl)-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole (6a)
Light yellow solid: 75% yield mp: 130–135 °C; 1H NMR (300 MHz, CDCl3) δ (ppm):
8.75 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.95 (s, 1H), 7.60–7.54 (m, 2H), 7.33 (t, J = 7.9 Hz,
21

1H), 7.18 (s, 2H), 4.66 (s, 2H), 3.93 (s, 6H), 3.92 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
(ppm): 153.9, 145.4, 142.6, 140.7, 138.8, 133.7, 132.8, 130.7, 128.7, 121.8, 120.5, 112.3,
111.7, 105.6, 60.9, 56.4, 55.8; MS (ESI): m/z 390 [M + H]+, HRMS calculated for
C21H20O3N5 m/z 390.15607 [M + H]+, found m/z 390.15609.
4.6.2. 3-(Azidomethyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole (6b)
Yellow solid: 80% yield; mp: 134–136 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.50
(s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.95–7.92 (m, 3H), 7.58–7.54 (m, 1H), 7.51 (d, J = 8.0 Hz,
13
1H), 7.32 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 8.7 Hz, 2H), 4.68 (s, 2H), 3.90 (s, 3H); C NMR
(125 MHz, CDCl3) δ (ppm): 160.2, 145.2, 142.5, 140.6, 132.6, 130.6, 130.5, 129.4, 128.5,
121.8, 121.7, 120.3, 114.6, 111.8, 111.5, 56.1, 55.4; MS (ESI): m/z 330 [M + H]+, HRMS
calculated for C19H16ON5 m/z 330.13494 [M + H]+, found m/z 330.13530.
4.6.3. 3-(Azidomethyl)-1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole (6c)
Yellow solid: 68% yield; mp: 138–140 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.49
(s, 1H), 8.17 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 7.9 Hz, 2H), 8.01 (s, 1H), 7.84 (d, J = 8.0 Hz,
2H), 7.61–7.57 (m, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.37–7.33 (m, 1H), 4.69 (s, 2H); 13C NMR
(125 MHz, CDCl3) δ (ppm): 145.6, 141.5, 140.7, 132.8, 131.2, 130.9, 130.6, 129.0, 128.4,
126.1 (q, J = 3.6 Hz), 125.0, 121.8, 121.6, 120.7, 113.0, 111.6, 55.9; MS (ESI): m/z 368 [M +
H]+, HRMS calculated for C19H13N5F3 m/z 368.11176 [M + H]+, found m/z 368.11170.
4.6.4. 3-(Azidomethyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole (6d)
1
Cream white solid: 86% yield; mp: 140–143 °C; H NMR (300 MHz, CDCl3) δ (ppm): 8.46
(s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.99–7.93 (m, 3H), 7.61–7.48 (m, 2H), 7.36–7.23 (m, 3H),
4.67 (s, 2H); 13C NMR (125 MHz, CDCl3) δ (ppm): 164.1 and 162.1 (d, J = 248.8 Hz), 145.3,
141.5, 140.6, 134.2, 132.7, 130.8, 129.9 (d, J = 8.2 Hz), 128.7, 121.8, 121.7, 120.5, 116.2 (d,
J = 21.8 Hz), 112.4, 111.6, 56.0; MS (ESI): m/z 318 [M + H]+, HRMS calculated for
C18H13N5F m/z 318.11495 [M + H]+, found m/z 318.11458.
4.6.5. 3-(Azidomethyl)-9-methyl-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole
(11a)
1
Light yellow solid: 85% yield; mp: 128–132 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.19 (d, J = 7.8 Hz, 1H), 8.03 (s, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 8.4 Hz,
1H), 7.34 (t, J = 7.5 Hz, 1H), 6.8 (s, 2H), 4.69 (s, 2H), 3.93 (s, 3H), 3.91 (s, 6H), 3.55 (s, 3H);
13C NMR (125 MHz, CDCl3) δ (ppm): 153.1, 143.9, 143.6, 143.2, 138.2, 134.8, 134.3, 130.9,
22

128.7, 121.5, 120.9, 120.0, 112.2, 109.8, 106.8, 60.9, 56.1, 55.8, 32.8; MS (ESI): m/z 404 [M
+ H]+, HRMS calculated for C22H22O3N5 m/z 404.17172 [M + H]+, found m/z 404.17199.
4.6.6. 3-(Azidomethyl)-1-(4-methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indole (11b)
1
Light yellow solid: 78% yield; mp: 120–125 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.18 (d, J = 7.9 Hz, 1H), 7.99 (s, 1H), 7.63–7.60 (m, 1H), 7.59 (d, J = 8.7 Hz, 2H),
7.43 (d, J = 8.2 Hz, 1H), 7.34–7.30 (m, 1H), 7.06 (d, J = 8.7 Hz, 2H), 4.68 (s, 2H), 3.90 (s,
3H), 3.50 (s, 3H); 13C NMR (125 MHz, CDCl3) δ (ppm): 159.9, 144.0, 143.7, 143.3, 134.6,
131.9, 130.8, 128.5, 121.5, 121.0, 119.9, 113.7, 111.8, 109.8, 56.0, 55.4, 32.9; MS (ESI): m/z
344 [M + H]+, HRMS calculated for C20H18ON5 m/z 344.15059 [M + H]+, found m/z
344.15067.
4.6.7. 3-(Azidomethyl)-9-methyl-1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole
(11c)
Yellow solid: 67% yield; mp: 120–123 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.20
(d, J = 7.8 Hz, 1H), 8.06 (s, 1H), 7.80 (s, 4H), 7.66–7.62 (m, 1H), 7.45 (d, J = 8.4 Hz, 1H),
13
7.37–7.33 (m, 1H), 4.69 (s, 2H), 3.48 (s, 3H); C NMR (125 MHz, CDCl3) δ (ppm): 144.3,
143.3, 143.0, 141.9, 134.4, 131.4, 130.7, 130.4, 130.0, 129.0, 125.2 (q, J = 3.6 Hz), 121.6,
120.9, 120.3, 112.7, 109.9, 55.8, 33.2; MS (ESI): m/z 382 [M + H]+, HRMS calculated for
C20H15N5F3 m/z 382.12741 [M + H]+, found m/z 382.12788.
4.6.8. 3-(Azidomethyl)-1-(4-fluorophenyl)-9-methyl-9H-pyrido[3,4-b]indole (11d)
1
Light yellow solid: 83% yield; mp: 140–143 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.19 (d, J = 7.7 Hz, 1H), 8.03 (s, 1H), 7.67–7.59 (m, 3H), 7.44 (d, J = 8.3 Hz, 1H),
7.33 (t, J = 7.5 Hz, 1H), 7.25–7.19 (m, 2H), 4.68 (s, 2H), 3.48 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ (ppm): 163.9 and 162.0 (d, J = 247.9 Hz), 144.1, 143.3, 142.6, 135.5, 134.5, 131.4
(d, J = 8.2 Hz), 131.1, 128.7, 121.5, 120.9, 120.1, 115.3 (d, J = 21.8 Hz), 112.2, 109.8, 55.9,
32.9; MS (ESI): m/z 332 [M + H]+, HRMS calculated for C19H15N5F m/z 332.13060 [M +
H]+, found m/z 332.13056.
4.7. General reaction procedure for the preparation of compounds (7a d and 12a d)
To a stirred solution of azide compounds 6a d/11a d (1 mmol) in acetonitrile and
water (1:1) was added tripthenylphosphene (1.1 mmol) and stirred for 12 h at rt. The solvent
was removed in vacuo, to this residue was added 6N HCl solution and washed with ethyl
23

acetate to remove non basic impurities. Then separated acidic medium and basified with 20%
NaOH solution (up to pH=10), extracted with chloroform, washed with water, dried over
sodium sulphate and concentrated under reduced pressure to get crude amine products which
were triturated in 10% ethyl acetate/ hexane, decanted and dried to get pure amine products
(7a d /12a d).
4.7.1. (1-(3,4,5-Trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methanamine (7a)
1
Cream white solid: 68% yield; mp: 140–145 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.65 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H), 7.57–7.50 (m, 2H), 7.30 (t, J = 7.8
Hz, 1H), 7.13 (s, 2H), 4.19 (s, 2H), 3.94 (s, 6H), 3.92 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
(ppm): 153.7, 148.6, 142.3, 140.8, 138.4, 134.0, 132.5, 130.6, 128.4, 121.7, 120.0, 112.3,
111.6, 111.1, 105.5, 60.8, 56.3, 47.3; MS (ESI): m/z 364 [M + H]+, HRMS calculated for
C21H22O3N3 m/z 364.16557 [M + H]+, found m/z 364.16557.
4.7.2. (1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methanamine (7b)
1
Cream white solid: 70% yield; mp: 170–173 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.49 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.86 (s, 1H), 7.55–
7.51 (m, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.31–7.27 (m, 1H), 7.12 (d, J = 8.7 Hz, 2H), 4.18 (s,
2H), 3.90 (s, 3H); 13C NMR (75 MHz, CDCl3+DMSO d6) δ (ppm): 159.4, 150.4, 141.6,
141.1, 132.1, 130.9, 130.0, 129.3, 127.5, 121.1, 120.9, 119.0, 113.8, 111.6, 109.8, 54.9, 47.5;
MS (ESI): m/z 304 [M + H]+, HRMS calculated for C19H18ON3 m/z 304.14444 [M + H]+,
found m/z 304.14414.
4.7.3. (1-(4-(Trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-yl)methanamine (7c)
1
Cream white solid: 78% yield; mp: 210–213 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.77 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 8.1 Hz, 2H), 7.90 (s, 1H), 7.81 (d, J
= 8.1 Hz, 2H), 7.57–7.53 (m, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.32–7.28 (m, 1H), 4.17 (s, 2H);
13C NMR (75 MHz, CDCl3+DMSO d6) δ (ppm): 150.9, 142.1, 141.4, 139.9, 132.5, 130.8,
127.8, 128.0, 125.7, 125.3 (q, J = 3.3 Hz), 122.1, 121.2, 121.1, 119.4, 111.8, 111.2, 47.7; MS
(ESI): m/z 342 [M + H]+, HRMS calculated for C19H15N3F3 m/z 342.12126 [M + H]+, found
m/z 342.12124.
4.7.4. (1-(4-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)methanamine (7d)
24

1
Cream white solid: 66% yield; mp: 190–195 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.54 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.96 (q, J = 5.3, 8.5 Hz, 2H), 7.90 (s, 1H), 7.54
13
(t, J = 8.1 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.32–7.25 (m, 3H), 4.18 (s, 2H); C NMR (75
MHz, CDCl3 + DMSO d6) δ (ppm): 162.8 and 159.6 (d, J = 247.0 Hz), 149.6, 140.4, 139.1,
133.6, 130.9, 129.2 (d, J = 7.1 Hz), 129.1, 126.6, 119.9, 119.8, 118.0, 114.2 (d, J = 21.4 Hz),
110.9, 109.2, 46.5; MS (ESI): m/z 292 [M + H]+, HRMS calculated for C18H15N3F m/z
292.12445 [M + H]+, found m/z 292.12400.
4.7.5. (9-Methyl-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methanamine
(12a)
1
Light yellow solid: 80% yield; mp: 125–130 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.17 (d, J = 7.7 Hz, 1H), 8.00 (s, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 8.3 Hz,
1H), 7.31 (t, J = 7.4 Hz, 1H), 6.85 (s, 2H), 4.20 (s, 2H), 3.93 (s, 3H), 3.91 (s, 6H), 3.50 (s,
13
3H); C NMR (125 MHz, CDCl3) δ (ppm): 153.0, 143.3, 142.2, 138.2, 131.0, 128.6, 128.3,
121.5, 119.8, 119.6, 112.1, 111.2, 109.7, 106.9, 106.8, 66.6, 60.9, 56.2, 32.6; MS (ESI): m/z
378 [M + H]+, HRMS calculated for C22H24O3N3 m/z 378.18122 [M + H]+, found m/z
378.18221.
4.7.6. (1-(4-Methoxyphenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)methanamine (12b)
1
Cream white solid: 75% yield; mp: 120–125 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.16 (d, J = 7.8 Hz, 1H), 7.94 (s, 1H), 7.61–7.56 (m, 3H), 7.41 (d, J = 8.2 Hz, 1H),
7.29 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 4.17 (s, 2H), 3.90 (s, 3H), 3.47 (s, 3H); 13C
NMR (125 MHz, CDCl3) δ (ppm): 159.7, 150.6, 143.3, 143.2, 134.1, 132.3, 132.0, 130.8,
128.2, 121.4, 121.1, 119.5, 113.5, 110.4, 109.6, 55.3, 48.0, 32.7; MS (ESI): m/z 318 [M +
H]+, HRMS calculated for C20H20ON3 m/z 318.16009 [M + H]+, found m/z 318.16033.
4.7.7. (9-Methyl-1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-yl)methanamine
(12c)
1
Cream white solid: 68% yield; mp: 125–130 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.18 (d, J = 7.7 Hz, 1H), 8.02 (s, 1H), 7.80 (s, 4H), 7.65–7.59 (m, 1H), 7.42 (d, J = 7.9
13
Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 4.20 (s, 2H), 3.46 (s, 3H); C NMR (125 MHz, CDCl3) δ
(ppm): 150.8, 143.5, 143.3, 141.5, 134.1, 131.3, 130.5, 130.0, 128.6, 125.1 (q, J = 3.7),
25

123.0, 121.5, 121.0, 119.9, 111.3, 109.7, 47.8, 33.1; MS (ESI): m/z 356 [M + H]+, HRMS
calculated for C20H17N3F3 m/z 356.13691 [M + H]+, found m/z 356.13739.
4.7.8. (1-(4-Fluorophenyl)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)methanamine (12d)
Cream white solid mp: 127–130 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.1 (d, J =
7.8 Hz, 1H), 7.97 (s, 1H), 7.64–7.59 (m, 3H), 7.41 (d, J = 8.2 Hz, 1H), 7.30 (t, J = 7.9 Hz,
1H), 7.22 (t, J = 8.7 Hz, 2H), 4.18 (s, 2H), 3.44 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
(ppm): 163.8 and 161.8 (d, J = 247.9 Hz), 150.5, 143.3, 142.2, 135.9, 134.1, 132.0, 131.4 (d,
J = 8.2 Hz), 128.4, 121.4, 121.0, 119.7, 115.2 (d, J = 20.8 Hz), 110.9, 109.6, 47.8, 32.8; MS
(ESI): m/z 306 [M + H]+, HRMS calculated for C19H17N3F m/z 306.14010 [M + H]+, found
m/z 306.14041.
4.8. General reaction procedure for the preparation of compounds (8a l and 13a l)
To a stirred solution of amine compounds 7a–d/12a–d (1 mmol) in pyridine (1 ml)
o
was added TEA (1.3 mmol) and carbon disulfide (1.5 mmol) at 0 C and stirred for 15 min.
Then, to this reaction mixture was added alkyl bromide (1.1 mmol) and reaction mixture was
o
stirred at 0 C for 30 min. The reaction mixture was quenched with water, extracted with
chloroform, washed with water, dried over sodium sulphate and concentrated under reduced
pressure to get crude product. The crude product was purified by silica gel column
chromatography by using ethyl acetate/hexane.
4.8.1. Methyl ((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8a)
1
Light green solid: 86% yield; mp: 197–198 °C; H NMR (300 MHz, CD6CO) δ
(ppm): 10.77 (s, 1H), 9.59 (bs, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.10 (s, 0.9H), 7.68–7.52 (m,
2H), 7.30–7.25 (m, 3H), 5.20 (s, 1.8H), 3.94 (s, 6H), 3.83 (s, 3H), 2.65 (s, 3H) and peaks at
13
8.02 and 5.00 are due to 10% minor rotamer; C NMR (75 MHz, CD6CO) δ (ppm): 199.7,
155.5, 146.3, 143.7, 143.6, 135.7, 134.6, 132.5, 130.2, 123.4, 123.3, 121.6, 114.1, 113.6,
113.3, 107.7, 61.6, 57.4, 53.7, 18.9; FT-IR: 3299.85, 3147.58, 2961.84, 2910.37, 1624.14,
1
1585.03, 1501.36, 1389.10, 1233.81 cm ; MS (ESI): m/z 454 [M + H]+, HRMS calculated
for C23H24O3N3S2 m/z 454.12536 [M + H]+, found m/z 454.12516.
26

4.8.2. Allyl ((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8b)
1
Off white solid: 78% yield; mp: 195–197 °C; H NMR (300 MHz, DMSo-d6) δ
(ppm): 11.53 (s, 1H), 10.53 (bt, J = 5.1 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 8.00 (s, 1H), 7.65–
7.51 (m, 2H), 7.30–7.22 (m, 3H), 5.97–5.82 (m, 1H), 5.30 (d, J = 16.9 Hz, 1H), 5.15–5.08
(m, 3H), 3.95 (d, J = 8.1 Hz, 2H), 3.92 (s, 6H), 3.77 (s, 3H) and peaks at 10.63, 4.88 and 4.02
are due to 10% minor rotamer; 13C NMR (75 MHz, CD6CO) δ (ppm): 195.5, 153.0, 144.5,
141.5, 141.4, 137.9, 133.4, 132.1, 129.9, 128.3, 121.5, 120.7, 119.6, 118.1, 112.5, 111.7,
105.8, 60.1, 55.8, 51.8, 37.0; FT-IR: 3297.48, 3134.44, 2971.05, 2920.87, 1623.77, 1504.51,
1
1470.65, 1387.38, 1231.35 cm ; MS (ESI): m/z 480 [M + H]+, HRMS calculated for
C25H26O3N3S2 m/z 480.14101 [M + H]+, found m/z 480.13983.
4.8.3. Benzyl ((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8c)
1
Cream colour solid: 80% yield; mp: 155–160 °C; H NMR (300 MHz, DMSO-d6) δ
(ppm): 11.53 (s, 1H), 10.57 (bt, J = 5.3 Hz, 0.9H), 8.21 (d, J = 7.7 Hz, 1H), 8.00 (s, 1H),
7.68–7.49 (m, 2H), 7.43–7.36 (m, 2H), 7.34–7.21 (m, 6H), 5.13 (d, J = 5.3 Hz, 1.8H), 4.56 (s,
2H), 3.90 (s, 6H), 3.77 (s, 3H) and peaks at 10.66 and 4.88 are due to 10% minor rotamer;
13C NMR (125 MHz, CDCl3) δ (ppm): 196.4, 153.7, 143.4, 141.7, 141.1, 138.7, 136.5, 133.4,
132.8, 131.1, 129.0, 128.9, 128.5, 127.4, 121.9, 121.5, 120.5, 112.1, 111.8, 105.4, 60.9, 56.3,
51.7, 39.8; FT-IR: 3321.70, 3129.08, 2932.96, 1625.07, 1584.30, 1496.18, 1453.10, 1389.25,
1
1234.75, 1127.46 cm ; MS (ESI): m/z 530 [M + H]+, HRMS calculated for C29H28O3N3S2
m/z 530.15666 [M + H]+, found m/z 530.15701.
4.8.4. Methyl ((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8d)
1
Off white solid: 85% yield; mp: 197–200 °C; H NMR (300 MHz, DMSO-d6) δ
(ppm): 11.04 (s, 1H), 9.95 (s, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.83 (s,
1H), 7.55 (d, J = 8.1 Hz, 1H), 7.43 (t, J = 7.4 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.04 (d, J =
8.5 Hz, 2H), 5.10 (d, J = 4.9 Hz, 1.8H), 3.84 (s, 3H), 2.57 (s, 3H) and peak at 4.86 is due to
13
10% minor rotamer; C NMR (125 MHz, CDCl3+DMSO–d6) δ (ppm): 197.6, 159.5, 144.3,
141.4, 141.2, 131.9, 130.4, 129.8, 129.6, 127.7, 121.0, 120.6, 119.1, 113.8, 112.2, 110.8,
55.0, 51.8, 17.4; FT-IR: 3295.41, 3236.34, 2923.86, 1739.36, 1575.92, 1624.78, 1489.43,
27

1
1437.74, 1247.16, 1232.27, 1175.75 cm ; MS (ESI): m/z 394 [M + H]+, HRMS calculated
for C21H20ON3S2 m/z 394.10423 [M + H]+, found m/z 394.10425.
4.8.5. Allyl ((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)carbamodithioate
(8e)
1
Cream colour solid: 78% yield; mp: 170–175 °C; H NMR (300 MHz, CDCl3) δ
(ppm): 8.88 (bs, 0.9H), 8.50 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.85
(s, 0.9H), 7.60–7.48 (m, 2H), 7.31 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H), 6.05–5.85 (m,
1H), 5.30 (d, J = 17.2 Hz, 1H), 5.16 (d, J = 4.2 Hz, 1.8H), 5.12 (d, J = 9.6 Hz, 1H), 3.96 (d, J
= 6.8 Hz, 1.8H), 3.91 (s, 3H) and peaks at 8.98, 7.79, 4.92 and 4.10 is due to 10% minor
rotamer; 13C NMR (75 MHz, CDCl3+DMSO–d6) δ (ppm): 195.4, 159.2, 142.7, 141.0, 140.8,
132.1, 131.9, 129.9, 129.8, 129.1, 127.5, 120.6, 120.4, 118.8, 117.4, 113.4, 111.6, 110.6,
54.6, 51.2, 37.2; FT-IR: 3244.40, 3058.12, 2951.63, 1739.00, 1625.31, 1607.56, 1490.25,
1
1439.96, 1320.20, 1234.54, 1182.23 cm ; MS (ESI): m/z 420 [M + H]+, HRMS calculated
for C23H22ON3S2 m/z 420.11988 [M + H]+, found m/z 420.11971.
4.8.6. Benzyl ((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8f)
1
Light yellow solid: 82% yield; mp: 172–176 °C; H NMR (500 MHz, CDCl3) δ
(ppm): 8.98 (bs, 1H), 8.64 (bs, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.88–7.83 (m, 3H), 7.58–7.49
(m, 2H), 7.38 (d, J=7.3 Hz, 1.7H), 7.34–7.22 (m, 4H), 7.07 (d, J = 8.4 Hz, 2H), 5.17 (d, J =
3.9 Hz, 1.7H), 4.58 (s, 1.7H), 3.87 (s, 3H) and peak at 7.44, 4.88 and 4.68 are due to 15%
minor rotamer; 13C NMR (75 MHz, CDCl3) δ (ppm): 196.2, 160.2, 143.1, 141.3, 140.9,
136.4, 132.5, 130.9, 129.3, 129.0, 128.8, 128.5, 127.3, 121.8, 121.7, 121.4, 120.4, 114.5,
111.6, 111.1, 55.4, 51.5, 39.8; FT-IR: 3300.56, 3030.98, 2970.36, 1738.88, 1625.65, 1606.60,
1
1490.34, 1441.79, 1352.69, 1230.64, 1181.26 cm ; MS (ESI): m/z 470 [M + H]+, HRMS
calculated for C27H24ON3S2 m/z 470.13553 [M + H]+, found m/z 470.13538.
4.8.7. Methyl ((1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8g)
White solid: 80% yield; mp: 218–222 °C; 1H NMR (500 MHz, CDCl3) δ (ppm): 8.69
(bs, 0.9H), 8.45 (bs, 1H), 8.17 (d, J = 7.9 Hz, 1H), 8.10 (d, J = 8.1 Hz, 2H), 7.98 (s, 0.8H),
7.89 (d, J = 8.2 Hz, 2H), 7.60 (t, J = 8.1 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.35 (t, J = 7.8 Hz,
28

1H), 5.22 (d, J = 4.4 Hz, 1.7H), 2.69 (s, 2.5H) and peak at 8.90, 7.94, 4.97 and 2.76 are due to
13
15% minor rotamer; C NMR (75 MHz, CDCl3 + DMSO–d6) δ (ppm): 197.4, 143.5, 141.2,
139.0, 132.3, 130.4, 129.4, 128.9, 128.5, 128.2, 127.8, 124.7, 120.9, 120.6, 120.2, 119.1,
111.6, 51.2, 17.5; FT-IR: 3389.50, 3294.16, 1738.88, 1625.66, 1491.81, 1438.23, 1491.81,
1
1332.54, 1322.30, 1229.78, 1110.42, 851.03, 747.76 cm ; MS (ESI): m/z 432 [M + H]+,
HRMS calculated for C21H17N3F3S2 m/z 432.08105 [M + H]+, found m/z 432.08042.
4.8.8. Allyl ((1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8h)
White solid: 88% yield; mp: 188–192 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.71
(bs, 0.8H), 8.52 (bs, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.06 (d, J = 7.9 Hz, 2H), 7.91 (s, 1H), 7.85
(d, J = 8.1 Hz, 1.7H), 7.59 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.34 (t, J = 7.5 Hz,
1H), 5.98–5.88 (m, 1H), 5.3 (d, J = 17.1 Hz, 1H), 5.18 (d, J = 4.3 Hz, 1.7H), 5.13 (d, J = 9.9
Hz, 1H), 3.96 (s, 1.7H) and peaks at 8.90, 7.80, 4.92 and 4.10 are due to 15% minor rotamer;
13C NMR (75 MHz, CDCl3 + DMSO–d6) δ (ppm): 195.8, 143.3, 141.3, 141.2, 139.1, 132.3,
130.4, 129.0, 128.6, 128.2, 127.9, 124.8, 121.6, 121.0, 120.6, 120.2, 119.0, 117.5, 111.6,
51.3, 37.3; FT-IR: 3398.05, 3259.43, 1738.89, 1623.97, 1493.40, 1396.98, 1320.80, 1233.66,
1
1154.66, 1068.51, 1108.71, 850.92, 739.03 cm ; MS (ESI): m/z 458 [M + H]+, HRMS
calculated for C23H19N3F3S2 m/z 458.09670 [M + H]+, found m/z 458.09634.
4.8.9. Benzyl ((1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)carbamodithioate (8i)
1
Off white solid: 82% yield; mp: 196–200 °C; H NMR (500 MHz, CDCl3) δ (ppm):
8.70 (bs, 0.8H), 8.51 (bs, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 7.6 Hz, 2H), 7.97 (s, 1H),
7.86 (d, J = 7.8 Hz, 2H), 7.62–7.51 (m, 2H), 7.40–7.33 (m, 3H), 7.30–7.21 (m, 3H), 5.22 (d, J
= 4.3 Hz, 1.7H), 4.59 (s, 1.7H) and peak at 8.90, 4.95 and 4.68 are due to 15% minor
rotamer; 13C NMR (75 MHz, CDCl3 + DMSO–d6) δ (ppm): 196.3, 156.5, 143.2, 141.5, 141.4,
136.2, 135.7, 132.7, 130.9, 130.0, 129.1, 128.6, 128.3, 128.1, 126.9, 125.2, 121.2, 120.6,
119.5, 112.3, 111.9, 51.5, 39.9; FT-IR: 3415.74, 3290.78, 1627.48, 1568.88, 1495.42,
1
1397.88, 1319.00, 1241.30, 1175.09, 1110.59, 1065.20, 851.13, 748.53 cm ; MS (ESI): m/z
508 [M + H]+, HRMS calculated for C27H21N3F3S2 m/z 508.11235 [M+H]+, found m/z
508.11199.
29