Click chemistry based multicomponent approach in the synthesis of spirochromenocarbazole tethered 1,2,3-triazoles as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.01.070

A series of spirochromenocarbazole tethered 1,2,3-triazoles were synthesized via click chemistry based one-pot, five component reaction between N-propargyl isatins, malononitrile, 4-hydroxycarbazole, aralkyl halides and sodium azide using cellulose suppor

Full text of DOI:10.1016/j.bioorg.2019.01.070

Accepted Manuscript
Click chemistry based multicomponent approach in the synthesis of spirochro-
menocarbazole tethered 1,2,3-triazoles as potential anticancer agents
Pramod V. Chavan, Uday V. Desai, Prakash P. Wadgaonkar, Savita R Tapase,
Kisan M Kodam, Amit Choudhari, Dhiman Sarkar
PII:
S0045-2068(18)30321-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.070
YBIOO 2774
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
8 April 2018
25 January 2019
30 January 2019
Please cite this article as: P.V. Chavan, U.V. Desai, P.P. Wadgaonkar, S.R. Tapase, K.M. Kodam, A. Choudhari,
D. Sarkar, Click chemistry based multicomponent approach in the synthesis of spirochromenocarbazole tethered
1,2,3-triazoles as potential anticancer agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.01.070
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Click chemistry based multicomponent approach in the synthesis of
spirochromenocarbazole tethered 1,2,3-triazoles as potential anticancer agents
Pramod V. Chavan,1 Uday V. Desai,*1 Prakash P. Wadgaonkar,2 Savita R Tapase,3 Kisan M
Kodam,3 Amit Choudhari,4 Dhiman Sarkar*4
1: Department of Chemistry, Shivaji University, Kolhapur – 416 004, India.
2: Polymer Science and Engineering Division, CSIR, National Chemical Laboratory, Pune - 411 008, India.
3. Biochemistry Division, Department of Chemistry, Savitribai Phule Pune University, Pune – 411007,
India
4: Combi - Chem. Bio-Resource Centre, CSIR National Chemical Laboratory, Pune - 411 008, India.
E-mail: uvdchem2011@gmail.com;d.sarkar@ncl.res.in
Phone: 0231-2609161; 2609354; Fax: 091-0231-2692333
_____________________________________________________________________________
Abstract: A series of spirochromenocarbazole tethered 1,2,3–triazoles were synthesized via
click chemistry based one-pot, five component reaction between N-propargyl isatins,
malononitrile, 4-hydroxycarbazole, aralkyl halides and sodium azide using cellulose supported
CuI nanoparticles (Cell-CuI NPs) as the heterogeneous catalyst. Antiproliferative activity of all
the synthesized compounds was investigated against panel of cancer cell lines such as MCF-7,
MDA-MB-231, HeLa, PANC-1, A-549, and THP-1. Many of the synthesized compounds
exhibited good anti-proliferative activity against breast (MCF-7 and MDA-MB-231) and cervical
(HeLa) cancer cells with IC50 values less than 10 μM. In case of MCF-7 cells, among the nine
compounds that showed good anti-proliferative activity, compounds 6f and 6j were found to be
highly potent (IC50 = 2.13 μM and 4.80 μM, respectively). In case of MDA-MB-231, three
compounds (6k, 6j and 6s) showed antiproliferative activity amongst which 6k was the most
potent one (IC50 = 3.78 μM). On the other hand, in cervical cancer HeLa cells, compounds 6b,
6g, 6s and 6u showed excellent antiproliferative activity (IC50 = 4.05, 3.54, 3.83, 3.35 μM,
respectively). All the compounds were found to be nontoxic to the human umbilical vein
endothelial cells (HUVECs). AO and EtBr staining and fluorescence microscopy studies of the
active compounds (IC50 < 5 μM) suggested that these compounds induce cell death by
apoptosis.
Keywords: Click chemistry, 1,2,3-triazolylspirochromenocarbazole, anticancer, cytotoxicity,
apoptotic assay, heterogeneous catalysis, multicomponent synthesis
____________________________________________________________________________
Introduction
Cancer is a multifaceted disease caused due to uncontrolled growth of abnormal cells and
after cardiovascular disease it is the most common cause of death worldwide [1,2]. While great
strides have been made in the treatment of cancer, it still continues to be a major health concern

[3]. In the treatment of almost all types of cancers, after surgery the use of chemotherapeutics
that inhibit hyper proliferation of cancer cells is routinely practisized [4,5]. However, this therapy
is plagued with the side effects that include off-target toxicity and drug resistance [6].
Consequently, the discovery of new drugs that can inhibit proliferation or induce death of cancer
cells without disturbing normal cells is highly desirable.
Many heterocyclic compounds containing isatin, thiazolidinone, benzimidazole, oxindole,
β-carboline, etc. as the structural motif serve as potential anticancer agents [7-10]. In the recent
years based upon the hypothesis that, the molecules synthesized by the combination of
heterocyclic pharmacophores can have enhanced biological potential [11,12] the most attractive
strategy in the design of new anticancer agents involves combination of pharmacophoric
templates through molecular hybridization [13-16]. With all these concepts in mind and our
continued interest in multicomponent synthesis as well as in the synthesis of hybrid molecules
[17-20], we planned to undertake the synthesis of novel hybrid molecules as potential anti-
cancer agents.
Carbazoles are probably the most widely spread nitrogen heterocycles in nature [21, 22].
Especially aryl and hetero annulated carbazoles like pyridocarbazoles, indolocarbazoles,
pyrrolocarbazoles, pyranocarbazoles, etc. are distributed in numerous natural products with
diverse biological activities [23-26]. The hetero annulated carbazoles of synthetic origin have
attracted growing attention these days due to their antidiabetic, antimicrobial, antioxidant,
anticancer, antitubercular as well as anticonvulsant activities [27-32].
Alike carbazole, 1,2,3-triazole is another medicinally privileged scaffold serving as a key
structural feature in many bioactive molecules as well as pharmaceuticals [33]. Many molecules
containing 1,2,3-triazole as the structural motif are known to possess anti-tubercular, anti-fungal,
anti-allergic, anti-viral, anti-tumor, anti-malarial as well as anti-HIV properties [34-42] while many
1,2,3-triazole conjugates have already been reported to exhibit potent anticancer activity [43-49].
For instance, carboxyamidotriazole is a commercially available anticancer drug that contains
1,2,3-triazole as the structural unit [50]. On the other hand, isatin is a medicinally privileged
scaffold that has led to the generation of large number of structurally related compounds that
exhibit anticancer agents [51]. Representative biologically potent compounds containing
carbazole, triazole and isatin are shown in Fig.1. Considering the biological potential of
compounds containing carbazole, isatin as well as 1,2,3-triazole as the structural units, we
surmised that hybrid molecules containing all these structural units may possess enhanced
anticancer activity.

Cl
O
O
N
Cl
Cl
N
O
H2N
H2N
N
NH
N
HO
O
H
Isomahanine
Carboxyamidotriazole
Girinimbine
R1
N
O
N
O
R1
N
O
N
H
R2
N
S
O
N
R2
O
N
H
N
N
N
N
R3
Ph
R1 = H,Cl, Br
R1 = Cl, OH
R2, R3 = Ph piperazinyl, etc.
R2 = Ph, subst.Ph, etc.
Isatin-benzimidazole
Napthalimide -1,2.3- triazole
Spirothiazolidinone-isatins
Fig. 1 Biologically active compounds containing 1,2,3 triazole, isatin and carbazole moieties
We have earlier reported cellulose supported CuI nanoparticles (Cell-CuI NPs) as a
heterogeneous catalyst in the synthesis of 1,2 3- triazoles [52] and as an extension to that work,
we have also reported the synthesis of spirochromene tethered 1,2,3–triazoles, 8 (Scheme 1A)
by the reaction between N-propargyl isatin, malononitrile, dimedone, aralkyl halide and sodium
azide. The synthesized compounds were found to exhibit potential anti-tubercular activity [20].
Based on these results and the focused literature survey on biological potential of compounds
containing pyranocarbazole, 1, 2, 3 - triazole as well as isatin as the structural units (Fig. 1) we
speculated that, replacement of dimedone component from the aforementioned multicomponent
reaction by 4-hydroxycarbazole would furnish spirochromenocarbazole tethered 1,2,3- triazoles
with anti-cancer activity. As an outcome of this philosophy, herein we report the synthesis of
spirochromenocarbazole tethered 1,2,3-triazoles by one-pot, five component reaction between
N-propargyl isatins, malononitrile, 4-hydroxycarbazole, aralkyl halides and sodium azide using
cellulose supported CuI nanoparticles (Cell-CuI NPs) as the catalyst. The studies on their
anticancer activity have also been described (Scheme 1B).

HO
NH
O
H2N
NC
N
3
H
O
R1
N
N
O
N
N
6
Br
CN
R1
Cell - CuI NPs
R2
O
+
+
+
NaN3
5
N
DMF - H2O
70 oC
O
R2
4
CN
2
O
NH2
1
CN
O
O
7
O
R1
N
N
N
8
N
R2
(Ref. 20)
Scheme1. One-pot, five component synthesis of spirochromenocarbazole tethered 1,2,3- triazoles
Result and discussion:
Chemistry
The synthetic route was initiated with the preparation of N-propargyl isatins. Various
substituted N-propargylated isatins were prepared by potassium carbonate catalyzed
propargylation of isatins with propargyl bromide using DMF as solvent and TBAB as the phase
transfer catalyst. We then turned our attention to examine the first model reaction for the
synthesis of 6a. In this regard, we decided to employ the reaction conditions described earlier by
us for the synthesis of spirochromene tethered 1, 2, 3- triazoles [20]. Accordingly, to a well
stirred solution of N-propargyl isatin, malononitrile, 4-hydroxycarbazole, benzyl bromide (1 mmol,
each) and sodium azide (1.1 mmol) in DMF: H2O (1: 2, v / v) was added Cell- CuI NPs (200 mg,
o
7.4 mol % CuI) as the catalyst. The reaction mixture was heated with stirring at 70 C and the
progress of the reaction was monitored by TLC. Upon completion of the reaction followed by
work-up, it was truly gratifying to notice the formation of expected spirochromenocarbazole
tethered 1,2,3 -triazole, 6a, in excellent yield (92 %). So as to examine the scope of the
developed protocol, few reactions were then performed by replacement of benzyl bromide
component from the model reaction with various aralkyl as well as alkyl bromides. In each case,
respective spirochromenocarbazole tethered 1,2,3-triazole was obtained in acceptable yield
(Table1, entry 6b to 6k). So as to explore generality of the reaction conditions, keeping
malononitrile, 4-hydroxycarbazole and sodium azide components from the model reaction same,
various N-propargylated isatins (1b – 1e) were allowed to react with various aralkyl bromides
(4a-4d). Once again, the reactions furnished expected spirochromenocarbazole tethered 1,2,3-
triazoles in decent yields (Table1, entry 6l to 6aa).

Table 1 : Cell-CuI NPs catalyzed synthesis of spiro [indoline-3,4’-pyrano[3,2-c] carbazole tethered
1,2,3-triazolesa
NH
H2N
NC
O
Br
HO
O
CN
CN
Cell - CuI NPs
R1
NaN3
5
+
+
+
O
+
DMF : H2O
70 oC
O
N
R1
N
H
N
R2
4
N
N
N
R2
1
2
6
3
O
O
O
O
O
Br
Me
O
Cl
F
H
Br
Br
Br
O
O
N
O
O
N
F
N
N
Me
N
1b
4b
4a
1a
1d
1e
4c
1c
MeO
MeO
4h
Br
O
Br
Br
Br
Br
O2N
Br
Et
Br
Br
O
Cl
F3C
NC
OMe
4g
4j
4d
4e
4i
Yieldb
(%)
4f
4k
Product
(6)
Time
(h)
M.P. (oC)
Entry
N–Propargyl isatin(1)
Aralkyl or alkyl
bromide(4)
1
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1c
1c
1c
1c
1d
1d
1d
1d
1e
1e
1e
1e
4a
4b
4c
4d
4e
4f
6a
6b
6c
6d
6e
6f
3
3
3
4
4
4
4
5
3
5
5
3
4
3
3
4
3
4
5
4
4
5
4
3
4
4
4
92
90
87
89
84
87
85
83
91
86
84
92
87
89
90
85
87
88
86
87
85
83
81
88
86
89
84
160 - 162
185 - 187
190 - 192
195 - 197
168 - 170
178 - 180
160 - 162
176 - 178
165 - 167
158 - 160
156 - 157
197 - 199
160 - 165
190 - 192
187 - 189
182 - 184
202 - 204
190 - 192
185 - 187
168 - 170
175 - 177
180 - 182
170 - 172
201 - 203
218 - 220
205 - 208
190-192
2
3
4
5
6
7
4g
4h
4i
6g
6h
6i
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4j
6j
4k
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
6aa
a: Reaction conditions: N-propargyl isatin, malononitrile, 4 - hydroxyl carbazole, aralkyl bromide(1
mmol, each) and NaN3 (1.1 mmol), Cell-CuI (7 mol %), DMF - water– (1: 2:, v/v, 6 mL), 70 oC.

After the library synthesis of spirochromenocarbazole tethered 1,2,3- triazoles, 6a – 6aa,
it was planned to undertake screening of the synthesized compounds for their anti-cancer
activity. In this regard it was essential that the synthesized compounds are free from copper
content and the same in the synthesized compounds is possible if the catalyst undergoes
leaching under the employed reaction conditions. Hence, it was decided to examine the leaching
behavior of the catalyst. Thus, upon completion of the model reaction, the reaction mixture was
diluted with water and resultant solid was filtered. Resultant filtrate was analyzed for it’s copper
content by AAS. Results being negative it was inferred that the synthesized compounds are
suitable for the biological assay.
Biological evaluation
All the synthesized compounds were evaluated for their antiproliferative activity against a
panel of six human cancer cells namely MCF-7 and MDA-MB-231 (Breast Carcinoma), HeLa
(Cervical Carcinoma), PANC-1 (Pancreas Carcinoma), A-549 (Lung Carcinoma) and THP-1
(acute monocytic leukemia). In addition, non-cancerous HUVEC cell line was chosen to compare
the effect of synthesized compounds between cancerous and non-cancerous cells. Paclitaxel
and Doxorubicin were used as a positive control.
Cells were treated with different concentration (0-100 μg/ml) of all the synthesized
compounds and incubated for 48 h. Screening studies revealed that, most of the compounds
were active against breast cancer cells MCF-7 and MDA-MB-231 (ER, PR positive) and cervical
cancer cells, HeLa (HPV-positive) with IC50 values in low micro-molar range (> 10 μM). However,
they did not exhibit any cytotoxicity against PANC-1, A-549 and THP-1 cancer cell lines (SI,
Table 1). IC50 values of all the synthesized compounds with respect to MCF-7, MDA-MB-231
and HeLa cancer cell lines and one, normal HUVEC cell line, are summarized in Table 2.
Among the synthesized compounds, 6a, 6f, 6g, 6h, 6j, 6p, 6q, and 6r were notably
active against MCF-7 cells with IC50 values in the range 2.1 – 7.06 μM. Especially compounds 6f
and 6j were highly active (IC50 = 2.13 and 4.80 μM, respectively). Similarly, compounds 6j, 6k
and 6s showed very good activity against MDA-MB-231 cells with IC50 values in the range 3.78 -
9.35 μM and among these 6k showed excellent activity (IC50 = 3.78 μM). On the other hand,
compounds 6b, 6g, 6j, 6s, 6u and 6z showed very good activity against HeLa cells with IC50
values in the range 3.35 - 9.2 μM and among these compound 6u was noticed to be the most
active one (IC50 = 3.35 μM). During the screening studies it was noticed that, compound 6j,
shows pronounced activity against all the aforementioned cell lines with IC50 in the range 4.8 -
9.2 μM. In order to check the biocompatibility of the synthesized compounds towards normal
cell, MTT assay was also performed with HUVEC cell and it was truly delighting that all the
synthesized compounds exhibited biocompatibility above 100 μM concentrations (Table 2).
These results reveal that, synthesized compounds are nontoxic to noncancerous cell line.

Table 2: Anticancer activity data of spirochromenocarbazole tethered 1,2,3-triazoles on various
human cancer cell lines.
Sr.
No.
Compound
Codes
MCF-7 Cells
MDA-MB-231
HeLa Cell
HUVEC
IC50 (μM) ± SD
IC50 (μM) ± SD
IC50 (μM) ± SD
IC 50 (μM) ± SD
1
6a
7.02 ± 0.93
25.56 ± 2.12
14.37 ± 0.95
>100
17.25 ± 0.08
12.74 ± 2.32
13.33 ± 0.48
>100
12.35 ± 0.17
4.05 ± 0.69
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>10
2
6b
3
6c
4
6d
>100
5
6e
25.51 ± 0.32
2.13 ± 0.16
6.31 ± 0.63
6.97 ± 0.83
18.00 ± 0.32
4.80 ± 0.25
51.16 ± 0.53
>100
37.66 ± 0.55
24.89 ± 0.74
14.86 ± 0.12
23.66 ± 1.02
41.64 ± 0.04
8.39 ± 1.84
3.78 ± 0.74
>100
>100
6
6f
>100 ± 1.6
3.54 ± 1.24
15.88 ± 1.12
10.83 ± 0.6
9.21 ± 0.34
61.89 ± 2.53
>100
7
6g
8
6h
9
6i
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
6j
6k
6l
6m
>100
>100
>100
6n
62.74 ± 0.17
42.50 ± 0.67
9.40 ± 0.36
7.06 ± 0.35
6.57 ± 0.26
14.81 ± 0.61
9.72 ± 0.89
>100
71.26 ± 1.18
60.82 ± 0.38
42.37 ± 0.55
30.33 ± 3.8
25.38 ± 0.69
9.35 ± 1.10
99.59 ± 0.55
75.01 ± 0.28
63.08 ± 2.14
>100
75.81 ± 0.61
13.25 ± 0.37
32.49 ± 0.15
61.29 ± 1.67
42.77 ± 0.22
3.83 ± 0.51
>100
6o
6p
6q
6r
6s
6t
6u
3.35 ± 0.17
37.87 ± 4.11
>100
6v
>100
6w
>100
6x
88.85 ± 0.06
>100
>100
59.12 ± 0.32
>100
6y
>100
6z
16.54 ± 0.51
23.69 ± 1.45
0.0026±0.00
4.63±0.41
27.88 ± 1.67
50.32 ± 0.59
0.1 ± 0.07
0. 55 ± 0. 01
7.80 ± 0.58
94.77 ± 0.20
0.0061±0.00
2.66±0.32
6aa
Paclitaxel
Doxorubicin
>10

Structure activity relationship:
So as to arrive at the relationship between the structures of the synthesized compounds
and their anticancer activity it would be rational to categorize these compounds in to two groups:
i) Those prepared using N-propargyl isatin (6a-6k) and, ii) prepared using substituted N-
propargyl isatins (6l-6aa). Among the twenty seven compounds screened for their anti-cancer
activity, nine compounds (6a, 6f, 6g, 6h, 6j, 6p, 6q, 6r and 6t) were prominently active against
MCF- 7 cells, six compounds (6b, 6g, 6j, 6u, 6s and 6z) showed excellent activity against HeLa
cells and only three compounds (6j, 6k and 6s) showed good activity against MDA-MB-231
cells. It is worthy to note that among these eighteen compounds, ten active compounds [6a, 6b,
6f, 6h, 6k, 6g (two cell lines) and 6j (three cell lines)] were prepared using N-propargyl isatin
as the starting compound. On the other hand, remaining eight compounds (6p, 6q, 6r, 6s, 6t,
6u, and 6z) were prepared using 5-chloro, 5-bromo, 5-fluoro and 5-methyl-N-propargyl isatin as
the starting materials. Furthermore out of these eight compounds, five compounds [6p, 6q, 6r,
6s (two cell lines)] were prepared using 5-bromo-N-propargyl isatin as the starting material, the
remaining three compounds 6t and 6u were prepared using 5-fluoro-N-propargyl isatin while 6z
was prepared using 5-methyl-N-propargyl isatin. On analysis of this data it could broadly be
concluded that, most active compounds are derived from N-propargyl isatin and 5-bromo-N-
propargyl isatin whereas substitution of chloro, fluro as well as methyl group on N-propargyl
isatin does not assist in improvement of anticancer activity. As regards the effect of aralkyl or
alkyl bromide component on anticancer activity is concerned it was noticed that, compounds
prepared using benzyl bromides bearing electron-withdrawing groups (6f and 6g) were more
potent than those prepared using benzyl bromide (6a) or the one bearing electron-donating
group (6h) and the one prepared using aliphatic long chain bromide, 6k, was active against
Breast as well as cervical cancer cells.
In summary, in the multicomponent synthesis of spirochromenocarbazole tethered 1,2,3-
triazoles as anticancer agents, the use of unsubstituted n-propargyl isatin, 1a, in combination
with benzyl bromides bearing electron-withdrawing group, 4f and 4g, or aliphatic bromides, 4j
and 4k has furnished the compounds with excellent anticancer activity against MCF-7 cells. In
case HeLa cells, excellent anticancer activity was noticed by four compounds (6b, 6g, 6s and
6u) and it was interesting to note that, out of these four compounds three compounds (6g, 6s
and 6u) that showed excellent and comparable activity were containing fluorine or bromine
substituent in them. Thus, presence of halogen substituent may be responsible for the
improvement of anticancer activity against HeLa cells.
Based upon these studies we propose that, there certainly exists scope for the synthesis
novel spirochromenocarbazole tethered 1,2,3-triazoles with improved anticancer activity by
using difluoro or dibromo isatin in combination with 4-triflorobenzyl bromide, 4-nitrobenzyl
bromide and aliphatic long chain bromides.

Apoptosis assays:
To gain insight of the mechanisms by which the tested compounds reduced proliferation
of breast cancer cells, the compounds showing significant anti-proliferative activity were
assessed for cell death. Thus, MCF-7, MDA-MB-231 and HeLa cells were treated at the
concentrations in the range of IC50 for 48 h and after staining with acridine orange and ethidium
bromide (AO/EB) were assessed by ArrayScan™ 4.0 High content screening system.
Depending upon the overall morphological characteristics and the cell membrane integrity, the
cells were distinguished from one another as necrotic, apoptotic or live cells. Apoptotic cells
containing apoptotic bodies (condensed nucleus) were observed as yellow / orange colored cells
while the necrotic cells were observed as red colored cells. Interestingly, compared to
compounds 6a,6g, 6h,6p, 6q, 6r and 6t (SI Fig 1) the number of apoptotic and necrotic events
were significantly high in case of 6f and 6j treated MCF-7 cells (Fig. 2A). Similarly, compared to
compounds 6j and 6s (SI, Fig. 2), 6k treated MDA MB-231 cells showed higher apoptotic and
necrotic population (Fig. 2B). On the other hand, in case of HeLa cells, compared to 6b, 6g, 6j,
6u and 6z (SI Fig.3), 6s treated cells (Fig. 2C) showed significant increase in apoptotic events.
However, the morphological assessment of 6b, 6g, 6j, 6u, 6z treated Hela Cells revealed a
decrease in cell number by cell cycle arrest. The results of the most active compounds in the
respective cell line are summarized in Table 3 and the same of all the compounds are
summarized in SI, Table 3. The results obtained are in agreement with the results of MTT assay.
In summary, we could assume that under the applied conditions apoptotic cell death was the
major cause of reduced proliferation of the breast cancer cells but not in the cervical cancer
cells.

Figure 2. Fluorescent images showing apoptosis induced by selected chromenocarbazole tethered 1,2,3-
triazoles derivatives in (A) MCF-7, (B) MDA MB 231 and (C) HeLa cells. Control and treated cells were
stained with Acridine orange (AO) / Ethidium bromide (EtBr); and the staining pattern was monitored by
The ArrayScan™ 4.0 system. For detection of AO (green) (Panel I) and EtBr (red) (Panel II), mercury-
xenon lamp was excited at 488 nm and 590 nm, respectively. Panel III represents the merge images
Table 3. Percent of viable, apoptotic and necrotic cell population
as measured by AO/EB fluorescent staining studies
Viable
Apoptotic
Necrotic
MCF-7
Control
89.5 ± 2.3
42.0 ± 2.3
49.6 ± 10.2
5.4± 1.4
14.3± 1.4
43.2 ±2.4
5.1 ± 1.8
38.6 ± 1.8
7.2 ± 4.1
6f
6j
MDA-MB-231
92.8 ± 1.8
42.8 ± 2.2
5.6 ±1.3
1.6 ± 0.01
21.1 ± 2.1
Control
36.1 ±1.8
6k
HeLa
Control
97.8 ± 1.2
41.4 ± 2.8
1.4 ±0.01
46.2 ±2.3
0.8 ± 0.2
12.4 ± 7.2
6s

Conclusions:
Taking clues from the structures of the compounds with proven biological activities and
our experiences in click chemistry as well as multicomponent reactions, studies were undertaken
on the synthesis of novel anticancer agents. During the studies, we have designed and
synthesized for the first time a series of spirochromenocarbazole tethered 1,2,3- triazoles by
one-pot, five component condensation reaction between N-propargyl isatins, malononitrile, 4-
hydroxycarbazole, alkyl / arylalkyl halides and sodium azide using cellulose supported CuI
nanoparticles (Cell-CuI NPs) as the heterogeneous catalyst. Simple experimental procedure,
wide scope and reusability of the catalyst are the main merits of the developed protocol. The
synthesized compounds were found to be selectively cytotoxic against breast (MCF-7, MDA-MB-
231) and cervical (HeLa) cancer cells. Among all screened compounds, 6f, 6k, 6g, 6s and 6u
showed excellent activity towards MCF-7, MDA-MB-231 and HeLa cancer cell lines. Especially
compound 6j showed good activity for all three cancer cell lines. The synthesized compounds
being nontoxic towards normal cells, they may be useful towards specific cancer cell lines.
Furthermore, AO/EB staining suggested that under the applied conditions apoptotic cell death
was the major cause of reduced proliferation of the breast cancer cells but not in the cervical
cancer cells.
Acknowledgments
P. V. Chavan wishes to thank University Grants Commission (UGC), New Delhi, India for
the award of teacher fellowship. Dr. A. S. Choudhari is grateful to Science and Engineering
Research Board (SERB), Government of India, New Delhi for the award of National Post-
Doctoral Fellowship (N-PDF, File no: PDF/2016/003615). We gratefully acknowledge Dr. K. G.
Kanade, Principal, Yashavantrao Chavan Institute of Science, Satara, for encouragement.
Experimental
General
Isatins (Aldrich), benzyl halides (Aldrich), sodium azide (S. D. Fine-Chem. Limited,
Mumbai), 4-hydroxycarbazole, malononitrile, copper (I) iodide (Spectrochem, Mumbai) and
microcrystalline cellulose (SRL, Mumbai) were used, as received. The catalyst Cell-CuI NPs was
prepared according to the procedure developed and reported by us earlier [20]. Melting points
were recorded using Kumar melting point apparatus. IR spectra were recorded as neat using
1
13
Thermo Scientific Nicolet S10 FT-IR Spectrometer. H-NMR (300 MHz) and C-NMR (75 MHz)
spectra were recorded using Bruker Avance – II spectrometer. High resolution mass spectra
(HRMS) were recorded using Thermo Scientific Q-Exactive, Accela 1250 pump, instrument.
Representative procedure for the preparation of N - propargylation of isatin:
Isatin (1mmol), potassium carbonate (1 mmol), propargyl bromide (1.3 mmol) and TBAB
(0.05 mmol) were placed in a round bottom flask. DMF (3 mL) was added and the reaction

mixture was stirred at room temperature for 24 h. After completion of reaction (TLC), crushed ice
was added and stirring was continued for 15 min more. Resultant solid was filtered and washed
with water (4 x 5 mL). It was dried in air and recrystallized from ethanol.
Representative procedure for one-pot synthesis of spirochromenocarbazole tethered
1,2,3-triazoles [1,4-disubstituted-1,2,3–triazolyl spiroindoline pyrano [3,2-c] carbazoles,
6a-6aa]:
N-Propargyl isatin (1 mmol), malononitrile (1 mmol), 4-hydroxycarbazole (1 mmol),
benzyl bromide (1 mmol), and sodium azide (1.1 mmol) were placed in a round bottom flask.
DMF-Water (1: 2, 6 mL, v / v) and cellulose-CuI NPs catalyst (0.2 g) were added and the
reaction mixture was heated at 70 °C. After completion of the reaction (TLC), the reaction
mixture was filtered and the filter was washed with ethyl acetate (4 x 10 mL). The organic extract
was washed with water, brine and dried over Na2SO4. Removal of solvent under vacuum
furnished corresponding 1,4-disubstituted-1,2,3-triazolyl spiroindoline pyrano [3, 2-c] carbazole
as a solid product. The resultant solid was successively washed using a mixture of hexane -
chloroform (80: 20 v/v), and dried. The resultant product did not require any further purification.
The recovered catalyst was washed with acetone, dried in air. Reusability studies showed that
the catalyst can be reused for five consecutive runs without significant loss in its activity.
Biological methods
Cytotoxicity assay
Six human cancer cell lines, MCF-7 and MDA-MB-231, HeLa, PANC-1, A-549, THP-1
were used to check the cytotoxicity of the synthesized compounds. The cell lines were obtained
from National Center for Cell Science (NCCS), Pune and maintained in T25 flasks with 10 %
(v/v) fetal bovine serum (FBS) containing Dulbecco’s Modified Eagle Medium (DMEM). HUVECs
cells were purchased from Life Technologies and maintained in basal medium M200 (Life
Technologies) containing 10 % FBS and growth factors (LSGS, Life Technologies). Cell line
containing T25 flasks were maintained at 37°C under 5 % CO2 and 95 % air in a humidified
atmosphere. Medium were replaced twice a week.
The cytotoxicity of the synthesized compounds was tested against the mentioned cell
lines by using modified MTT assay [53]. Briefly, cells were seeded at the density of 1×104
cells/mL in a 96 well plate. The plates were incubated overnight into CO2 incubator (37 ºC under
5 % CO2 and 95% air in a humidified atmosphere) to adhere the cells. Next day, cells were
treated with different concentration of test compounds (100-0 µg/ml) and incubated for additional
48 h. Post incubation, cell medium was replaced with MTT (0.5 mg/mL) - PBS medium and
incubated for 2-4 h to form Formazan crystals. The crystals were solubilized by addition of
acidified iso-propanol. The optical density was read on a micro plate reader at 570 nm filter
against a blank prepared from cell-free wells. Absorbance given by cells treated with the vehicle
alone was taken as 100% cell growth. IC50 and MIC values were calculated by using Origin Pro

software. The viability and growth in the presence of test material is calculated by using the
following formula: Percent cytotoxicity = [(average absorbance of control − absorbance of
compound) / (absorbance of control – absorbance of blank)] × 100, where control is the culture
medium with cells and DMSO while blank is the culture medium without cells. Paclitaxel and
Doxorubicin were used as a positive control.
Morphological assessment by acridine orange and ethidium bromide double staining
The dual-fluorescence staining using acridine orange (AO) and ethidium bromide (EtBr)
is a nuclear staining method used to assess apoptotic cell morphology. Stain AO is able to
permeate into both live and dead cells to stain all the nucleated cells and excite green
fluorescence, while stain EtBr enters only into dead cells with damaged membranes and
generates red fluorescence. Thus, late apoptotic and necrotic cells take up both stains. Briefly,
MCF-7, MDA-MB-231 and HeLa cells were seeded at the density of 1×104 cells/mL in a 96 well
plate. The plates were incubated overnight into CO2 incubator. Next day, the cells were treated
with IC50 concentration of test compounds for additional 48 h. Post incubation, cell medium was
replaced with AO and EtBr solution mixture (1 part each of 10 μg mL-1 of AO and EtBr in PBS)
and examined under Array Scan VTI HCS 600‑type high content live cell imaging system. The
Fluorescence microphotographs in the green, and red channels of at least 20 fields were
acquired in each well with a 20X objective, corresponding to at least 2000 cells counted. An
analysis of the fluorescence intensity was performed for AO and EtBr stained samples by the
HCS Assay Scan software.
Spectral data
All the synthesized compounds are new and were fully characterized by spectral
methods.
2-Amino-4,7-dihydro-1’((1-(benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-3’,4-
chromene] pyrano [3,2-c]carbazole-3-carbonitrile, 6a: Off-white solid; M.P.: 160 -162 °C; IR
1
(KBr):3310, 3187, 2927, 2190, 1708, 1652, 1607, 1398, 1338, 1098, 748 cm-1; H NMR (300
MHz, DMSO - d6): δ 4.97 (m, 2H,indolinyl-CH2), 5.58 (s, 2H, benzylic-CH2), 6.35 (br s, 1H, Ar-
H), 7.04 (s, 1H, Ar-H), 7.14 (d, 2H, J = 8.4 Hz, Ar-Hs), 7.21 (d, 1H, J = 7.8 Hz, Ar-Hs), 7.29 (m,
3H, Ar-Hs), 7.34 - 7.42 (m, 4H, Ar- Hs), 7.45 (d, 2H, J = 7.2 Hz, Ar-Hs), 7.51 (d, 1H, J = 8.1
Hz, Ar-H), 7.59 (s, 2H, NH2), 8.08 (s, 1H, triazolyl-H), 8.58 (d, 1H, J = 7.8 Hz, Ar-H), 11.55 (s,
1H, NH); 13C NMR (75.4MHz, DMSO-d6): δ 35.70, 50.37, 53.31, 54.55, 108.50, 109.51,
110.04, 110.59, 111.42, 119.25, 119.74, 120.65, 123.68, 128.31, 128.59, 129.21, 129.39,
130.20, 136.41, 140.08, 140.77, 142.47, 142.76, 144.44, 161.86, 177.87 ppm; HRMS: mass
calculated for [C33H23N7O2]: 550.1991 [M + H] + and 572.1811 [M + Na] +; obs. mass: 550.1986
[M + H] + and 572.1805 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-
3’,4- chromene] pyrano[3,2-c] carbazole-3-carbonitrile, 6b: cream colored solid; M.P.: 185 -187

1
°C; IR (KBr): 3308, 3190, 2928, 2195, 1710, 16525, 1609, 1390, 1335, 1094, 745 cm-1; H
NMR (300 MHz, DMSO-d6): δ 2.26 (s, 3H, Ar-CH3), 5.10 (m, 2H, indolinyl-CH2), 5.18 - 5.56 (m,
2H, benzylic-CH2), 6.37 (d, 1H, J = 8.4 Hz, Ar-H), 7.06 (d, 2H, J = 8.4 Hz, Ar-Hs), 7.13 (m, 4H,
Ar-Hs), 7.19 (d, 2H, J = 8.1 Hz, Ar-Hs), 7.24 (d, 1H, J = 7.5 Hz, Ar-H), 7.34 (d, 1H, J = 7.8 Hz,
Ar-H), 7.42 (d, 1H, J = 7.8 Hz, Ar-H), 7.47 – 7.51 (m, 1H, Ar-H), 7.54 (br s, 2H, NH2), 8.02 (s,
1H, triazolyl-H), 8.57 (d, 1H, J = 7.8 Hz, Ar-H), 11.55 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-
d6): δ 21.14, 35.70, 50.37, 53.13, 54.50, 108.53, 109.51, 110.03, 110.60, 111.43, 119.25,
119.77, 120.64, 123.64, 123.93, 125.18, 126.30, 128.34, 129.41, 129.74, 133.34, 135.17,
137.96, 140.08, 140.78, 142.46, 142.71, 144.44, 161.87, 177.88 ppm; HRMS: mass calculated
for [C34H25N7O2]: 564. 2148 [M + H] + and 586.1968 [M + Na] +; obs. mass: 564.2142 [M + H] +
and 586.1962 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-
3’,4- chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6c: yellow solid; M.P.: 190 - 192 °C; IR
1
(KBr): 3317, 3190, 2915, 2198, 1714, 1625, 1610, 1398, 1331, 1092, 745cm-1; H NMR (300
MHz, DMSO -d6): δ 4.98 (m, 2H, indolinyl-CH2), 5.57 (br s, 2H, benzylic-CH2), 6.38 (d, 1H, J =
8.4 Hz, Ar-H), 7.07 (d, 2H, J = 8.4 Hz, Ar-Hs), 7.13 (d, 1H, J = 6 Hz, Ar-H), 7.18 (d, 2H, J = 8.7
Hz, Ar-Hs), 7.23 - 7.26 (m, 2H, Ar-Hs), 7.31 - 7.37 (m, 3H, Ar-Hs), 7.42 - 7.47 (m, 1H, Ar-H),
7.51 (d, 1H, J = 8.1 Hz, Ar-H), 7.57 (br s, 2H, NH2), 8.17 (s, 1H, triazolyl-H), 8.57 (d, 1H, J = 7.8
Hz, Ar-H), 11.54 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 35.70, 50.36, 52.50, 54.57,
108.51, 109.50, 110.03, 110.59, 111.42, 115.88, 116.16, 119.62, 119.73, 120.64, 123.67,
123.76, 123.84, 125.19, 126.27, 129. 40, 130.59, 130.70, 132.63, 135.17, 140. 08, 140.78,
142.48, 142.79, 144.45, 160.70, 161.85, 177.87 ppm; HRMS: mass calculated for
[C33H22FN7O2]: 568.1897 [M + H] + and 590.1717 [M + Na] +; obs. mass: 568.1892 [M + H] + and
590.1711 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-
3’,4- chromene] pyrano[3,2-c]carbazole-3 carbonitrile, 6d: brown solid; M.P.: 195 - 197 °C; IR
1
KBr): 3317, 3190, 2915, 2198, 1714, 1625, 1610, 1398, 1331, 1092, 745 cm-1; H NMR (300
MHz, DMSO-d6): δ 5.03 - 5.05 (m, 2H, indolinyl-CH2), 5.59 (s, 2H, benzylic-CH2), 6.38 (d, 1H, J
= 8.4 Hz, Ar-H), 7.07 (d, 2H, J = 8.1 Hz, Ar-Hs), 7.14 (d, 1H, J = 6.9 Hz, Ar-Hs), 7.21 (d, 2H, J
= 7.5 Hz, Ar-Hs),7.31- 7.37 (m, 3H, Ar-Hs), 7.40 - 7.47 (m, 3H, Ar-Hs), 7.51 (d, 2H, J = 7.8 Hz,
Ar-Hs), 7. 58 (s, 2H, NH2), 8.10 (s, 1H, triazolyl-H), 8.57 (br s, 1H, Ar-H), 11.55 (s, 1H, NH); 13C
NMR (75.4 MHz, DMSO - d6): δ 35.69, 50.36, 52.47, 54.58, 108.51, 109.50, 110.02, 110.59,
111.41, 119.20, 119.71, 120.65, 123.67, 123.87, 125.20, 126.25, 129. 17, 129.38, 130.23,
133.28, 135.18, 135.42, 138.25, 140. 08, 140.78, 142.49, 142.80, 144.45, 147.88, 161.84,
177.85 ppm; HRMS: mass calculated for [C33H22ClN7O2]: 584.1602 [M+ H] + and 606.1422 [M +
Na]+; obs. mass: 584.1596 [M+H] + and 606.1416 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(4-cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-

3’,4 chromene] pyrano [3,2-c]carbazole-3-carbonitrile, 6e: brown solid; M. P.: 168 - 170 °C; IR
(KBr): 3313, 3183, 2968, 2189, 1987, 1714, 1653, 1608, 1365, 1340, 1214, 1051, 749 cm-1; 1H
NMR (300 MHz, DMSO- d6): δ 5.05 - 5.06 (br s, 2H, indolinyl-CH2), 5.72 (s, 2H, benzylic-CH2,),
6.39 (d, 1H, J = 8.4 Hz, Ar-H), 7.07 (d, 1H, J = 7.8 Hz, Ar-H), 7.14 (d, 1H, J = 7.9 Hz, Ar-H), 7.2
(m, 2H, Ar-Hs), 7.32 (d, 1H, J = 7.2 Hz, Ar-H), 7.39 - 7.47 (m, 4H, Ar-Hs), 7.51 (d, 1H, J = 8.1
Hz, Ar-H), 7.59 (s, 2H, NH2), 7.81 (d, 2H, J = 7.8 Hz, Ar-Hs), 8.17 (s, 1H, triazolyl-H), 8.57 (d,
1H, J = 7.8 Hz, Ar-H), 11.55 (s, 1H, NH); 13C NMR (75.4MHz, DMSO-d6): δ 35.66, 50.36,
52.65, 54.54, 108.53, 109.47, 110.05, 110.58, 111.34, 111.43, 118.99, 119.21, 119.76, 120.63,
123.66, 123.89, 123.92, 124.36, 125.21, 126. 28, 129.01, 129.42, 133.15, 135.17, 140. 07,
140.78, 141.91, 142.47, 142.89, 144.45, 161.84, 177.89 ppm; HRMS: mass calculated for
[C34H22N8O2]: 575.1944 [M + H] + and 597.1764 [M + Na] +; obs. mass: 575.1938 [M + H] + and
597.1758 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-3’,4-
chromene] pyrano [3,2-c]carbazole-3-carbonitrile, 6f: brown solid; M.P.: 178 - 180 °C; IR (KBr):
1
3315, 3173, 2978, 2179,1985, 1714, 1650, 1610, 1360, 1338, 1212, 1055, 750 cm-1; H NMR
(300 MHz, DMSO-d6): δ 5.06 (s, 2H, indolinyl-CH2), 5.78 (s, 2H, benzylic-CH2), 6.41 (d, 1H, J =
8.4 Hz, Ar-H), 7.08 - 7.14 (m, 3H, Ar-Hs), 7.24 (d, 2H, J = 7.0 Hz, Ar-Hs), 7.32 (d, 1H, J = 7.5
Hz, Ar-H), 7.44 - 7.53 (m, 4H, Ar-Hs), 7.58 (s, 2H, NH2), 8.19 (s, 1H, triazolyl-H), 8.24 (br s, 2H,
Ar-Hs), 8.57 (d, 1H, J = 7.8 Hz, Ar-H), 11.54 (s, 1H, NH); 13C NMR (75.4MHz, DMSO-d6): δ
35.65, 50.36, 52.65, 54.54, 108.54, 109.47, 110.05, 110.58, 111.34, 111.43, 118.99, 119.74,
120.63, 123.66, 123.92, 124.32, 124.42, 125.22, 126.29, 129. 33, 129.42, 133.15, 135.17,
140.07, 140.78, 141.91, 142.47, 142.89, 144.45, 161.84, 177.89 ppm; HRMS: mass calculated
for [C33H22N8O4]: 595.1842 [M+H]+ and 617.1662 [M+Na]+; obs. mass: 595.1837 [M + H] + and
617.1662 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(4-trifluoromethylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-
spiro[indoline-3’,4 chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6g: brown solid; M. P.: 160
- 162 °C; IR (KBr): 3315, 3173, 2978, 2179,1985, 1714, 1650, 1610, 1360, 1338, 1212, 1055,
1
750 cm-1; H NMR (300 MHz, DMSO-d6): δ 5.06 (s, 2H, indolinyl-CH2), 5.72 (s, 2H, benzylic-
CH2 ), 6.41 (d, 1H, J = 8.1 Hz, Ar-H) , 7.08 (d, 2H, J = 7.8 Hz, Ar-Hs), 7.14 (br s, 1H, Ar-H),
7.24 (br s, 2H, Ar-Hs), 7.32 (d, 1H, J = 7.2 Hz, Ar-H), 7.46 - 7.50 (m, 3H, Ar-Hs), 7.51 (d, 1H, J
= 7.8 Hz, Ar-H), 7.58 (s, 2H, NH2), 7.70 (d, 2H, J = 7.2 Hz, Ar-Hs), 8.17 (s, 1H, triazolyl-H), 8.57
(d, 1H, J = 7.5 Hz, Ar-H), 11.55 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 35.68, 50.38,
52.62, 54.59, 108.54, 109.50, 110.05, 110.60, 111.43, 119.23, 119.74, 120.64, 122.73, 123.67,
123.91, 124.29, 125.21, 126. 07, 126.11, 126.28, 128.95, 129.31, 129.41, 135. 19, 140.10,
140.79, 141.10, 142.49, 142.87, 144.46, 161.86, 177.90 ppm; HRMS: mass calculated for
+
[C34H22F3N7O2]: 618.1865 [M+H]+ and 640.1685 [M+Na]+; obs. mass: 618.1860 [M+H] and
640.1679 [M + Na] +.

2-Amino-4,7-dihydro-1’((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-
spiro[indoline-3’,4- chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6h: brown solid; M.P.: 176
- 178 °C; IR (KBr): 3315, 3173, 2978, 2179, 1985, 1714, 1650, 1610, 1360, 1338, 1055, 750
cm-1; 1H NMR (300 MHz, DMSO-d6): δ 3.57 (s, 3H, Ar-OCH3), 3.66 (s, 6H, 2xAr-OCH3), 5.03 -
5.06 (br s, 2H, indolinyl-CH2), 5.49 (s, 2H, benzylic-CH2), 6.39 (d, 1H, J = 8.4 Hz, Ar-H), 6.55 -
6.68 (m, 3H, Ar-Hs), 7.04 (d, 1H, J = 8.4 Hz, Ar-H), 7.13 (d, 1H, J = 6.0 Hz, Ar-H), 7.17 (d, 1H,
J = 7.8 Hz, Ar-H), 7.24 (d, 1H, J = 7.5 Hz, Ar-H), 7.28 (d, 1H, J = 7.5 Hz, Ar-H), 7.42 (d, 1H, J =
7.8 Hz, Ar-H), 7.51 (d, 1H, J = 8.4 Hz, Ar-H), 7.57 (s, 2H, NH2), 8.10 (s, 1H, triazolyl-H), 8.53
(d, 1H, J = 7.2 Hz, Ar-H), 11.70 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 35.65, 50.38,
53.51, 56.26, 60.44, 105.70, 105.98, 108.48, 109.48, 110.00, 111.45, 119.27, 120.62, 123. 67,
123.83, 123.87, 123.96, 125.79, 126. 28, 129.33, 131.76, 131.84, 135.22, 137.62, 140.08,
140.76, 142.48, 142.69, 144.46, 153.03, 153.44, 161.81, 178.11 ppm; HRMS: mass calculated
for [C36H29N7O5]: 640.2308 [M + H] + and 662.2128 [M + Na]+; obs. mass: 640.2303 [M + H] +
and 662.2122 [M + Na] +.
2-Amino-4,7-dihydro-1’((1-(carboethoxy)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-3’,4-
chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6i: brown solid; M.P.: 165 - 167 °C; IR
(KBr):3313, 3186, 2926, 2190,1985, 1739, 1650, 1608, 1518, 1459, 1367, 1214, 1098, 745
1
cm-1; H NMR (300 MHz, DMSO-d6): δ 1.19 (t, 3H, J = 7.2 Hz, CH3), 4.17 (q, 2H,J = 7.2 Hz,
CH3CH2), 5.07 (q, 2H, J = 39.3 Hz, indolinyl-CH2), 5.39 (s, 2H, COCH2), 6.37 (d, 1H, J = 8.4
Hz, Ar-H), 7.05 – 7. 53 (m, 8H, Ar-Hs), 7.60 (s, 2H, NH2), 8.07 (s, 1H, triazolyl-H), 8.57 (d, 1H,
J = 7.8 Hz, Ar-H), 11.56 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 14.39, 35.61, 50.37,
50.89, 54.52, 61.95, 108.59, 109.57, 110.06, 110.56, 111.41, 119.21, 119.73, 120.64, 123.67,
123.90, 125.17, 126.26, 129.37, 135. 20, 140.06, 140.77, 142.47, 144.40, 161.87, 167.58,
177.89 ppm; HRMS: mass calculated for [C30H23N7O4]: 546.1890 [M + H] + and 568.1710 [M +
Na]+; obs. mass: 546.1884 [M + H] + and 568.1704 [M + Na].+
2-Amino-4,7-dihydro-1’((1-(pentyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-3’,4-
chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6j: brown solid; M.P.: 158 - 160 °C; IR (KBr):
3313, 3186, 2926, 2190, 1985, 1739, 1650, 1608, 1518, 1459, 1367, 1214, 1098, 745 cm-1; 1H
NMR (300 MHz, DMSO-d6): δ 0.80 (t, 3H,J = 7.2 Hz, CH3), 1.09 - 1.26 (m, 4H, CH2), 1.74 -
1.78 (m, 2H, CH2), 4.31 (t, 2H, J = 6.9 Hz CH2), 5.04 (m, 2H, indolinyl-CH2), 6.39 (d, 1H, J =
8.4 Hz, Ar-H), 7.08 (m, 2H, Ar-Hs), 7.12 – 7.20 (m, 2H, Ar-Hs), 7.24 (d, 1H, J = 7.2 Hz, Ar-H),
7.30 (d, 1H, J = 7.8 Hz, Ar-H), 7.42 (d, 1H, J = 7.2 Hz, Ar-H), 7.50 (d, 1H, J = 8.1 Hz, Ar-H),
7.56 (s, 2H, NH2), 7.98 (s, 1H, triazolyl-H), 8.56 (d, 1H, J = 7.8 Hz, Ar-H), 11.53 (s, 1H, NH);
13C NMR (75.4 MHz, DMSO-d6): δ 14.19, 21.90, 28.34, 29.70, 35.86, 49.84, 50.38, 108.51,
109.46, 110.05, 110.57, 111.42, 119.34, 119.76, 120.62, 123.41, 123.65, 123.88, 123.90,
125.16, 126.29, 129.37, 135.21, 140.06, 140.77, 142.39, 144.43, 161.82, 177.88 ppm; HRMS:
+
+
mass calculated for [C31H27N7O2]: 530.2304 [M + H] and 552.2124 [M + Na] ; obs. mass:

530.2299 [M + H] + and 552.2118[M + Na].+
2-Amino-4,7-dihydro-1’((1-(secondarybutyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[indoline-
3’,4-chromene] pyrano[3,2-c] carbazole-3-carbonitrile, 6k: brown solid; M.P.: 156-158 °C; IR
(KBr): 3307, 3184, 2967, 2189, 1709, 1652, 1608, 1458, 1397, 1305, 1051, 746 cm-1; 1H NMR
(300 MHz, DMSO-d6): δ 0.63 - 0.70 (m, 3H, CH3), 1.43 (dd, 3H, J = 6.6 and 6.1 Hz, CH3), 1.74
- 1.80 (m, 2H, CH2), 4.56 (sextet, 1H, J = 6.6 Hz, CH), 5.04 (br s, 2H, indolinyl-CH2), 6.38 (d,
1H, J = 8.4 Hz, Ar-H), 7.07 (m, 2H, Ar-H), 7.15 (d, 1H, J = 8.4 Hz, Ar-H), 7.19 (d, 2H, J = 7.2
Hz, Ar-H), 7.31 - 7.36 (m, 1H, Ar-H), 7.44 (d, 1H, J = 7.2 Hz, Ar-H), 7.50 (d, 1H, J = 7.8 Hz, Ar-
H), 7.59 (s, 2H, NH2), 7.99 (s, 1H, triazolyl-H), 8.57 (d, 1H, J = 7.8 Hz, Ar-H), 11.54 (s, 1H,
NH); 13C NMR (75.4 MHz, DMSO-d6): δ 10.56, 20.99, 29.92, 35.85, 50.39, 54.59, 58.50,
108.51, 109.43, 110.05, 110.58, 111.42, 119.37, 119.75, 120.64, 121.56, 123.66, 123.89,
123.91, 125.16, 126.28, 129.38, 135.27, 140.06, 140.78, 142.37, 142.37, 142.42, 144.45,
161.78, 177. 89 ppm; HRMS: mass calculated for [C30H25N7O2]: 516.2148 [M + H]+ and
538.1968 [M + Na]+; obs. mass: 516.2142 [M + H] + and 538.1962[M + Na] +.
2-Amino-4,7-dihydro-1’((1-(benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-chloroindoline-
3’,4- chromene] pyrano[3,2-c] carbazole-3-carbonitrile, 6l: brown solid; M.P.: 197-199 °C; IR
(KBr): 3307, 3184, 2967, 2189, 1709, 1652, 1608, 1458, 1397, 1305, 1051, 746 cm-1; 1H NMR
(300 MHz, DMSO-d6): δ 5.00 (s, 2H, indolinyl-CH2), 5.49 (br s, 2H, benzylic-CH2), 6.34 (d, 1H,
J = 8.4 Hz, Ar-H), 7.00 (d, 2H, J = 8.4 Hz, Ar-Hs), 7.11 (d, 2H, J = 8.4 Hz, Ar-Hs), 7.17 (d, 2H, J
= 8.4 Hz, Ar-Hs), 7.23 - 7.33 (m, 4H, Ar-Hs), 7.36 - 7.38 (br s, 2H, NH2), 7.43 (d, 1H, J = 8.1
Hz, Ar-H), 7.90 (br s, 1H, Ar-H), 8.00 (s, 1H, triazolyl-H), 8.50 (d, 1H, J = 7.8 Hz, Ar-H), 11.30
(s, 1H, NH); 13C NMR (75.4MHz, DMSO-d6): δ 35.85, 50.57, 53.62, 54.00, 108.33, 108.49,
110.80, 111.15, 111.25, 119.17, 119.57, 120.70, 123.43,123.51, 123.63, 125.18, 125.96,
128.12, 128.34, 128.50, 129.03, 129.11, 135.71, 136.97, 140.03, 140.92, 140.99, 142.40,
144.44, 161. 93, 177.59 ppm; HRMS: mass calculated for [C33H22ClN7O2]: 584.1602 [M + H]+;
obs. mass: 584.1596 [M + H] +.
2-Amino-4,7-dihydro-1’((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
chloroindoline-3’,4-chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6m: brown solid; M.P.: 160
- 165 °C; IR (KBr): 3307, 3184, 2923, 2190, 1777, 1705, 1650, 1608, 1487, 1454, 1335, 1216,
1
1107, 749 cm-1; H NMR (300 MHz, DMSO-d6): δ 2.26 (s, 3H, Ar-CH3), 4.99 (s, 2H, indolinyl-
CH2), 5.44 (s, 2H, benzylic-CH2), 6.36 (d, 1H, J = 8.4 Hz, Ar- H), 7.03 (d, 2H, J = 8.7 Hz, Ar-
Hs), 7.11 - 7.15 (m, 4H, Ar-Hs), 7.18 (d, 1H, J = 7.5 Hz, Ar- H), 7.26 (d, 1H, J = 8.4 Hz, Ar- H),
7.35 (d, 2H, J = 7.2 Hz, Ar- Hs), 7.44 (br s, 2H, NH2), 7.88 (d, 1H, J = 9.0 Hz), 8.07 (s, 1H,
triazolyl-H), 8.52 (d, 1H, J = 7.8 Hz, Ar-H), 11.36 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6):
δ 21.18, 35.91, 50.56, 53.44, 54.12, 108.33, 108.45, 110.80, 111.13, 111.20, 119.16, 119.53,
120.71, 123.29, 123.45, 123.63, 125.17, 125.93, 128.14, 128.26, 128.32, 129.07, 129.60, 132.
64, 136.95, 137.99, 140.02, 140.92, 140.99, 144.45, 161.91, 177.55 ppm; HRMS: mass

calculated for [C34H24ClN7O2]: 598.1758[M+ H]+; obs. mass: 598.1753 [M + H] +.
2-Amino-4,7-dihydro-1’((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
chloroindoline-3’,4 -chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6n: brown solid; M.P.: 190
- 192 °C; IR (KBr): 3307, 3184, 2923, 2190, 1777,1705, 1650, 1608, 1487, 1454, 1335, 1216,
1
1107, 749 cm-1; H NMR (300 MHz, DMSO-d6): δ 5.04 (br s, 2H, indolinyl-CH2), 5.58 (s, 2H,
benzylic-CH2), 6.41 (d, 1H, J = 8.4 Hz, Ar-H), 7.09 - 7.12 (d, 1H, J = 8.7 Hz, Ar-H), 7.15 - 7.28
(m, 2H, Ar-Hs), 7.21 - 7.25 (m, 3H, Ar-Hs), 7.33 (d, 2H, J = 6.3 Hz, Ar-Hs), 7.35 (d, 1H, J = 6.0
Hz, Ar-H), 7.43 (d,1H, J = 9.0 Hz, Ar-H), 7.52 (d, 1H, J = 7.8 Hz, Ar-H), 7.64 (s, 2H, NH2 ), 8.10
(s, 1H, triazolyl-H), 8.57 (d, 1H, J = 7.8 Hz, Ar-H), 11.57 (s, 1H, NH); 13C NMR (75.4 MHz,
DMSO-d6): δ 35.80, 50.62, 52.51, 53.91, 108.68, 110.64, 110.45, 111.74, 115.89, 116.17,
119.13, 119.79, 120.63, 123.71, 123.82, 125.32, 126.34, 128.01, 129.42, 130.59, 130.70,
132.61, 132.65, 137.15, 140.08, 140.90, 141.45, 142.50, 144.43, 160.71, 161.86, 163.95,
+
177.66 ppm; HRMS: mass calculated for [C33H21ClFN7O2]:602. 1507 [M + H] ; obs. mass:
602.1502 [M + H].+
2-Amino-4,7-dihydro-1’((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
chloroindoline-3’,4 chromene]pyrano[3,2-c]carbazole-3-carbonitrile, 6o: brown solid; M. P.: 187
- 189 °C; IR (KBr): 3307, 3184, 2923, 2190, 1777,1705, 1650, 1608, 1487, 1454, 1335, 1216,
1
1107, 749 cm-1; H NMR (300 MHz, DMSO-d6): δ 5.05 (br s, 2H, indolinyl-CH2), 5.58 (s, 2H,
benzylic-CH2), 6.42 (d, 1H, J = 8.4 Hz, Ar- H), 7.10 (d, 1H, J = 8.4 Hz, Ar- H), 7.23 - 7.29 (m,
5H, Ar-Hs), 7.39 - 7.45 (m, 4H, Ar-Hs), 7.47 - 7.54 (m, 1H, Ar-H), 7.65 (s, 2H, NH2), 8.10 (s,
1H, triazolyl-H), 8.56 (d, 1H, J = 7.8 Hz, Ar-H), 11.57 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-
d6): δ 35.78, 50.61, 52.52, 53.91, 108.65, 110.64, 111.47, 111.75, 119.13, 119.84 120.60,
123.67, 123.73, 124.00, 125.29, 126.38, 128.03, 129.18, 129.44, 130.21, 130.34, 133.32,
135.32, 137.13, 140.07, 140.90, 142.51, 142.60, 144.42, 161.87, 177.62 ppm; HRMS: mass
calculated for [C33H21Cl2N7O2]: 618. 1212 [M + H]; + obs. mass: 618.1207 [M + H].+
2-Amino-4,7-dihydro-1’((1-(benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-bromoindoline-
3’,4- chromene] pyrano[3,2-c] carbazole-3-carbonitrile, 6p: brown solid; M.P.: 182 - 184 °C; IR
(KBr): 3308, 3174, 2933, 2180, 1767, 1715, 1640, 1618, 1485, 1451, 1335, 1216, 1109, 749
1
cm-1; H NMR (300 MHz, DMSO-d6): δ 5.04 (s, 2H, indolinyl-CH2), 5.59 (s, 2H, benzylic-CH2),
6.40 (d, 1H, J = 8.0 Hz, Ar-H), 6.99 (br s, 1H, Ar-H), 7.25 - 7.35 (m, 9H, Ar-Hs), 7.54 (d, 2H, J =
4.0 Hz, Ar- Hs), 7.66 (s, 2H, NH2), 8.10 (s, 1H, triazolyl-H), 8.59 (d, 1H, J = 8.0 Hz, Ar-H),
11.58 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 35.25, 50.04, 52.79, 53.37, 108.14,
110.11, 110.92, 111.73, 115.19, 118.59 118.81, 119.26, 120.10, 123.14, 123.39, 125.82,
127.44, 127.76, 127. 82, 128.07, 128.68, 131.72, 135.83, 137.00, 139.55, 140.36, 141.30,
143.85, 143.88, 161.32, 176.94 ppm; HRMS: mass calculated for [C33H22BrN7O2]: 628.1096 [M
+ H]; + obs. mass: 628.1091 [M + H].+
2-Amino-4,7-dihydro-1’((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-

bromoindoline-3’,4-chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6q: brown solid; M. P.:
202 - 204 °C; IR (KBr): 3310, 3170, 2938, 2154, 1777,1710, 1645, 1608, 1495, 1471, 1365,
1
1226, 1106, 753 cm-1; H NMR (300 MHz, DMSO-d6): δ 2.26 (s, 3H, Ar-CH3), 4.99 (s, 2H,
indolinyl-CH2), 5.36 (dd, 2H, J = 19.5 and 18.9 Hz benzylic-CH2), 6.37 (d, 1H, J = 8.1 Hz, Ar-H),
7.02 (d, 2H, J = 8.4 Hz, Ar-H), 7.05 - 7.09 ( m, 4H, Ar-Hs), 7.18 (s, 2H, NH2), 7.33 - 7.38 (m,
3H, Ar-Hs), 7.42 (d, 2H, J = 8.1 Hz, Ar-H), 7.83 (s, 1H, triazolyl-H), 8.51 (d, 1H, J = 7.8 Hz, Ar-
H), 11.26 (s, 1H, NH); 13C NMR (75.4MHz, DMSO-d6): δ 21.19, 35.91, 50.52, 53.64, 54.17,
108.37, 108.45, 110.83, 111.10, 111.69, 115.85, 119.14, 119.53, 120.72, 123.27, 123.46,
123.64, 125.93, 127.90, 128.14, 129.61, 131.92, 132.60, 137.33, 138.00, 140.03, 140.93,
141.44, 144.46, 161.91, 177.43 ppm; HRMS: mass calculated for [C34H24BrN7O2]: 642.1253 [M
+ H]; + obs. mass: 642.1248 [M + H].+
2-Amino-4,7-dihydro-1’((1-(4-flourobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
bromoindoline-3’,4-chromene] pyrano[3,2-c] carbazole-3-carbonitrile, 6r: brown solid; M.P.: 190
- 192 °C; IR (KBr): 3320, 3174, 2959, 2191, 1710,1715, 1652, 1625, 1451, 1335, 1216, 1021,
1
750 cm-1; H NMR (300 MHz, DMSO -d6): δ 5.01 (s, 2H, indolinyl-CH2), 5.50 (s, 2H, benzylic-
CH2), 6.40 (d, 1H, J = 8.4 Hz, Ar-H), 7.04 - 7.51 (m, 13H, 11-Ar-Hs, NH2), 7.98 (s, 1H,
triazolyl-H), 8.55 (d, 1H, J = 7.2 Hz, Ar-H), 11.38 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6):
δ 35.86, 50.52, 50.80, 54.10, 108.32, 108.47, 110.81, 111.14, 111.75, 115.70, 115.85, 115.99,
119.15, 119.56, 120.71, 123.48, 123.65, 125.97, 127.90, 130.29, 130.40, 131.85, 131.96,
137.35, 140.03, 140.93, 141.48, 142.43, 144.46, 160.77, 161.91, 164.03, 177.45 ppm; HRMS:
mass calculated for [C33H21BrFN7O2]: 646.1002 [M + H];+ obs. mass: 646.0997 [M + H].+
2-Amino-4,7-dihydro-1’((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
bromoindoline-3’,4-chromene] pyrano[3,2-c] carbazole-3-carbonitrile, 6s: brown solid; M. P.:
185 - 187 °C; IR (KBr): 3325, 3138, 2959, 2191, 1710, 1652, 1602, 1457, 1398, 1333, 1220,
1115, 1047, 749 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 5.03 (s, 2H, indolinyl-CH2), 5.56 (s, 2H,
benzylic-CH2), 6.42 (d, 1H, J = 8.4 Hz, Ar-H), 7.07 (d, 1H, J = 8.4 Hz, Ar-H), 7.16 - 7.50 (m,
10H, Ar-Hs), 7.58 (s, 2H, NH2), 8.06 (s, 1H, triazolyl-H), 8.55 (d, 1H, J = 7.8 Hz, Ar-H), 11.47 (s,
1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 35.80, 50.54, 52.61, 53.94, 108.49, 108.54,
110.73, 111.26, 112.02, 115.74, 119.12, 119.61, 120.69, 123.62, 123.71, 123.84, 126.08,
127.90, 129.07, 130.05, 132.08, 133.48, 135.05, 137.45, 140.06, 140.92, 141.71, 142.43,
144.45, 161.87, 177.43 ppm; HRMS: mass calculated for [C33H21BrClN7O2]: 662.0707 [M + H];+
obs. mass: 662.0701 [M + H].+
2-Amino-4,7-dihydro-1’((1-(benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-fluoroindoline-
3’,4- chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6t: brown solid; M. P.: 168-170 °C; IR
(KBr): 3328, 3148, 2951, 2198, 1717, 1657, 1612, 1458, 1398, 1338, 1227, 1105, 1049, 759
1
cm-1; H NMR (300 MHZ, DMSO-d6): δ 5.03 (dd, 2H, J = 24.6 Hz, indolinyl-CH2), 5.57 (s, 2H,
benzylic-CH2), 6.37 (d, 1H, J = 8.7 Hz, Ar-H), 7.06 - 7.54 (m, 12H, Ar-Hs), 7.63 (s, 2H, NH2),

8.07 (s, 1H, triazolyl-H), 8.56 (d, 1H, J = 7.8 Hz, Ar-H), 11.56 (s, 1H, NH); 13C NMR (75.4 MHz,
DMSO-d6): δ 35.78, 50.34, 53.34, 54.17, 108.63, 108.85, 110.63, 111.13, 111.24, 111.46,
112.81, 113.14, 115.74, 116.05, 119.15, 119.83, 120.60, 123.65, 123.87, 126.36, 128.29,
128.62, 129.22, 136.32, 136.92, 138.68, 140.07, 140.87, 142.58, 144.40, 158.04, 161.88,
177.84 ppm; HRMS: mass calculated for [C33H22FN7O2]: 568.1897 [M + H] + and 590.1717 [M +
Na];+ obs. mass: 568.1892 [M + H]+ and 590.1711 [M + Na].+
2-Amino-4,7-dihydro-1’((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
fluoroindoline-3’,4 chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6u: brown solid; M. P.: 175
- 178 °C; IR (KBr): 3321, 3138, 2942, 2182, 1727, 1656, 1617, 1448, 1395, 1339, 1228, 1145,
1
1039, 755 cm-1; H NMR (300 MHz, DMSO-d6): δ 2.29 (s, 3H, Ar-CH3), 4.95 (dd, 2H, J = 23.4
Hz, indolinyl-CH2), 5.50 (s, 2H, benzylic-CH2), 6.38 (d, 1H, J = 8.4 Hz, Ar-H), 7.07 – 7.54 (m,
11H, Ar-Hs), 7.64 (s, 2H, NH2), 8.02 (s, 1H, triazolyl-H), 8.55 (d, 1H, J = 7.5 Hz, Ar-H), 11.57 (s,
1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 21.14, 35.86, 50.79, 53.36, 54.17, 108.51, 110.75,
110.90, 111.22, 112.58, 112.91, 115.51, 115.83, 119.14, 119.65, 120.67, 123.49, 123.61,
126.05, 128.20, 129.64, 132.80, 136.72, 136.82, 137.98, 138. 38, 140.04, 140.91, 142.46,
144.44, 158.01, 161.20, 161.95, 177.84 ppm; HRMS: mass calculated for [C34H24FN7O2]: 582.
2054[M + H] + and 604.1874[M + Na]; + obs. mass: 582.2048 [M + H] + and 604.1868[M + Na]. +
2-Amino-4,7-dihydro-1’((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
fluoroindoline-3’,4 chromene ] pyrano[3,2-c]carbazole-3-carbonitrile, 6v: brown solid; M. P.: 180
- 182 °C; IR (KBr): 3317, 2924, 2853, 2191, 1707, 1652, 1600, 1488, 1451, 1399, 1220, 1167,
1020, 786 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 5.01 (dd, 2H, J = 20.1 and 21.0 Hz, indolinyl
CH2) , 5.55 (s, 2H, benzylic CH2), 6.39 (d, 1H, J = 8.4 Hz, Ar-H), 7.07 - 7.54 (m, 11H, Ar-Hs),
7.63 (s, 2H, NH2), 8.07 (s, 1H, triazolyl-H), 8.55 (d, 1H, J = 7.8 Hz, Ar-H), 11.56 (s, 1H, NH);
13C NMR (75.4 MHz, DMSO-d6): δ 35.32, 50.33, 52.02, 53.59, 108.12, 108.40, 110.16, 110.95,
112.45, 115.29, 115.46, 115.63, 118.65, 119.26, 120.17, 123.21, 123.33, 125.82, 130.13,
130.20, 132.16, 136.43, 138.26, 139.60, 140.41, 142.15, 143.96, 158.20, 160.10, 160.88,
161.38, 162.82, 177.32 ppm; HRMS: mass calculated for [C33H21F2N7O2]: 586.1803 [M + H] +
and 608.1623 [M + Na]; + obs. mass: 586.1798 [M + H]+ and 608.1617 [M + Na]. +
2-Amino-4,7-dihydro-1’((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
fluoroindoline-3’,4-chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6w: brown solid; M.P.: 170-
172 °C; IR (KBr): 3310, 3164, 2930, 2185, 1757, 1705, 1645, 1608, 1485, 1459, 1385, 1216,
1
1129, 749 cm-1; H NMR (300 MHz, DMSO-d6): δ 5.03 (br s, 2H, indolinyl CH2) , 5.59 (s, 2H,
benzylic-CH2), 6.43 (d, 1H, J = 7.8 Hz, Ar-H), 7.09 - 7.54 (m, 11H, Ar-Hs), 7.64 (s, 2H, NH2),
8.11 (s, 1H, triazolyl-H), 8.56 (d, 1H, J = 7.8 Hz, Ar-H), 11.57 (s, 1H, NH); 13C NMR (75.4MHz,
DMSO-d6): δ 35.79, 50.84, 52.65, 54.16, 108.63, 108.85, 110.63, 111.45, 115.74, 119.12,
119.79, 120.63, 123.67, 123.72, 124.00, 126.34, 129.18, 129.40, 130.22, 133.31, 135. 36 136.
82, 136.90, 138.72, 140.08, 140.87, 142.64, 144.43, 158.16, 161.86, 177.81 ppm; HRMS:

mass calculated for [C33H21ClFN7O2]: 602.1507 [M + H]+ and 624.1327 [M + Na]+; obs. mass:
602.1502 [M + H]+ and 624.1322 [M + Na].+
2-Amino-4,7-dihydro-1’((1-(4-benzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
methylindoline-3’,4 chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6x: brown solid; M. P.:
201 - 203 °C; IR (KBr): 3318, 2933, 2180, 1767, 1715, 1640, 1618, 1485, 1451, 1335, 1216,
1109, 755 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 2.21 (s, 3H, Ar-CH3), 4.99 (dd, 2H, J = 27.0
and 27.9 Hz, indolinyl-CH2) , 5.58 (s, 2H, benzylic-CH2), 6.35 (d, 1H, J = 8.3 Hz, Ar-H), 6.96 (s,
1H, Ar-H), 7.03 – 7.51 (m, 11H, Ar-Hs), 7.56 (s, 2H, NH2), 8.06 (s, 1H, triazolyl-H), 8.57 (d, 1H,
J = 8.1 Hz, Ar-H), 11.55 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 20.99, 35.73, 50.41,
53.29, 54.67, 108.54, 109.58, 109.82, 110.54, 111.41, 119.32, 119.73, 120.64, 123.66, 123.76,
123.89, 125.63, 126.25, 128.32, 128.59, 129.21, 129.64, 132.99, 135.42, 136.42, 139.99,
140.06, 140.72, 142.83, 144.36, 161.73, 177.79 ppm; HRMS: mass calculated for
[C34H25N7O2]: 564.2148 [M + H] + and 586.1968 [M + Na]; + obs. mass: 564.2142 [M + H] + and
586.1962[M + Na]. +
2-Amino-4,7-dihydro-1’((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
methylindoline-3’,4-chromene ] pyrano[3,2-c]carbazole- 3 carbonitrile, 6y : brown solid; M. P.:
268 - 270 °C; IR (KBr): 3318, 3171, 2937, 2185, 1757,1705, 1630, 1628, 1488, 1441, 1338,
1220, 1169, 752 cm-1; 1H NMR (300 MHz, DMSO-d6): δ 2.12 (s, 3H, Ar-CH3), 2.26 (s, 3H, Ar-
CH3), 4.98 (br s, 2H, indolinyl-CH2), 5.51 (s, 2H, benzylic-CH2), 6.36 (d, 1H, J = 8.4 Hz, Ar-Hs),
6.95 (br s, 1H, Ar-H), 7.06 – 7.44 (m, 10H, Ar-Hs), 7.55 (s, 2H, NH2), 8.01 (s, 1H, triazolyl-H),
8.56 (d, 1H, J = 8.1 Hz, Ar-H), 11.54 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 20.99,
21.15, 35.74, 50.41, 53.11, 54.67, 108.54, 109.58, 109.81, 110.55, 111.41, 119.33, 119.73,
120.64, 123.57, 123.66, 123.92, 125.63, 126.26, 128.37, 129. 65, 129.73, 132.99, 133.38,
135.41, 137.43, 140.00, 140.07, 140.73, 142.79, 144.37, 161.74, 177.86 ppm; HRMS: mass
calculated for [C35H27N7O2]: 578.2304 [M + H]+ and 600.2124 [M + Na]+ obs. mass: 578.2299
;
[M + H] + and 600.2118 [M + Na]. +
2-Amino-4,7-dihydro-1’((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
methylindoline-3’,4-chromene ] pyrano[3,2-c]carbazole- 3 carbonitrile, 6z: brown solid; M. P.:
205 - 207 °C; IR (KBr): 3317, 2923, 2854, 2362, 2190, 1703, 1652, 1625, 1496, 1398, 1336,
1
1220, 1180, 1098, 809, 785, 750 cm-1; H NMR (300 MHz, DMSO-d6): δ 2.21 (s, 3H, Ar-CH3),
4.98 (dd, 2H, J = 24.0 Hz, indolinyl-CH2), 5.57 (s, 2H, benzylic-CH2), 6.38 (d, 1H, J = 8.4 Hz,
Ar-H), 7.06 – 7.51 (m, 11H, Ar-Hs), 7.55 (s, 2H, NH2), 8.07 (s, 1H, triazolyl-H), 8.58 (d, 1H, J =
7.8 Hz, Ar-H), 11.54 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO - d6): δ 20.99, 35.74, 50.42,
52.49, 54.69, ,108.54, 109.58, 109.81, 110.56, 111.42, 115.88, 116.17, 119.32, 119.73,
120.65, 123.71, 123.94, 125.65, 126. 26, 129.65, 130. 61, 130.72, 132.64, 133.00, 135.41,
140.01, 140.07, 140.75, 142.87, 144.39, 160.70, 161.74, 163.94, 177.81 ppm; HRMS: mass
calculated for [C34H24FN7O2]: 582.2054 [M + H] + and 604.1874[M + Na]; + obs. mass: 582.2048

[M + H] + and 604.1868[M + Na]. +
2-Amino-4,7-dihydro-1’((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2’oxo-spiro[5’-
methylindoline-3’,4-chromene] pyrano[3,2-c]carbazole-3-carbonitrile, 6aa: brown solid; M. P.:
190 - 192 °C; IR (KBr): 3308, 3174, 2933, 2180, 1767,1715, 1640, 1618, 1485, 1451, 1335,
1
1216, 1109, 749 cm-1; H NMR (300 MHz, DMSO-d6): δ 2.21 (s, 3H, Ar-CH3), 5.00 (br s, 2H,
indolinyl-CH2), 5.59 (s, 2H, benzylic-CH2 ), 6.39 (d, 1H, J = 8.4 Hz, Ar-H), 6.96 (s, 1H, Ar-H),
7.11 – 7.53 (m, 10H, Ar-Hs), 7.56 (s, 2H, NH2), 8.08 (s, 1H, triazolyl-H), 8.58 (d, 1H, J = 7.8 Hz,
Ar-H), 11.54 (s, 1H, NH); 13C NMR (75.4 MHz, DMSO-d6): δ 21.00, 35.72, 50.41, 52.47, 54.70,
,108.54, 109.57, 109.81, 110.53, 111.41, 119.30, 119.72, 120.64, 123.66, 123.88, 123. 95,
125.64, 126.25, 129.17, 129.18, 129. 64, 130.24, 132.99, 133.29, 135.41, 140.00, 140.07,
140.74, 142.87, 144.38, 161.73, 177.80 ppm; HRMS: mass calculated for [C34H24FN7O2]:
598.1758 [M + H] + and 620.1578[M + Na];+ obs. mass: 598.1753 [M + H] + and 620.1572[M +
Na].+
1
Supporting information: Original copies of H, 13C-NMR and HRMS spectra of all the
synthesized compounds and morphological phase contrast microscopy results of selected
compounds are provided in ESI.

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