Chalcone based azacarboline analogues as novel antitubulin agents: Design, synthesis, biological evaluation and molecular modelling studies

Article abstract of DOI:10.1016/j.ejmech.2014.08.005

The present study involves the design of a series of 3-aryl-9-acetyl- pyridazino[3,4-b]indoles as constrained chalcone analogues. A retrosynthetic route was proposed for the synthesis of target compounds. All the synthesized compounds were evaluated for in-vitro cytotoxicity against THP-1, COLO-205, HCT-116 and A-549 human cancer cell lines. The results indicated that 2a, 3a, 5a and 6a possessed significant cytotoxic potential with an IC₅₀ value ranging from 1.13 to 5.76 μM. Structure activity relationship revealed that the nature of both Ring A and Ring B influences the activity. Substitution of methoxy groups on the phenyl ring (Ring A) and unsubstituted phenyl ring (Ring B) were found to be the preferred structural features. The most potent compound 2a was further tested for tubulin inhibition. Compound 2a was found to significantly inhibit the tubulin polymerization (IC₅₀ value - 2.41 μM against THP-1). Compound 2a also caused disruption of microtubule assembly as evidenced by Immunoflourescence technique. The significant cytotoxicity and tubulin inhibition by 2a was rationalized by molecular modelling studies. The most potent structure was docked at colchicine binding site (PDB ID-1SA0) and was found to be stabilized in the cavity via various hydrophobic and hydrogen bonding interactions.

Full text of DOI:10.1016/j.ejmech.2014.08.005

Accepted Manuscript
Chalcone based azacarboline analogues as novel antitubulin agents: Design,
synthesis, biological evaluation and molecular modelling studies
Sahil Sharma, Charanjeet Kaur, Abhishek Budhiraja, Dr. Kunal Nepali, Assistant
Professor Manish K. Gupta, A.K. Saxena, P.M.S. Bedi
PII:
S0223-5234(14)00732-6
10.1016/j.ejmech.2014.08.005
EJMECH 7238
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2014
Revised Date: 27 July 2014
Accepted Date: 4 August 2014
Please cite this article as: S. Sharma, C. Kaur, A. Budhiraja, K. Nepali, M.K Gupta, A.K Saxena, P.
Bedi, Chalcone based azacarboline analogues as novel antitubulin agents: Design, synthesis, biological
evaluation and molecular modelling studies, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.08.005.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Chalcone based azacarboline analogues as novel antitubulin agents: Design, synthesis, biological evaluation and molecular modelling
studies.
Sahil Sharma , Charanjeet Kaur , Abhishek Budhiraja Kunal Nepalia*, Manish K Guptac, A.K Saxena , PMS Bedi
a
a
b,
d
a
R'
SITES FOR PLACING CONSTRAINTS
N
N
R'
N
N
N
1
N
O
1
MOLECULAR
HYBRIDIZATION
AZACARBOLINE
R
3
R
A
3
B
2
R
A
2
B
R
3
-CARBON
3
-CARBON
BRIDGE
O
BRIDGE
R' = -COCH3
R
A
B
CHALCONE FRAMEWORK
R
AZACARBOLINE- CHALCONE
HYBRIDS AS CONSTRAINED CHALCONE
ANALOGUES.
CHALCONE
FRAMEWORK

ACCEPTED MANUSCRIPT
Highlights
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis of chalcone based Azacarboline analogues
In-vitro cytotoxicity study against a panel of human cancer cell line
In-vitro tubulin polymerization assay
Microtubule assembly disruption by immunoflorescence technique
Molecular modelling studies

ACCEPTED MANUSCRIPT
PRELIMINARY COMMUNICATION
Chalcone based azacarboline analogues as novel antitubulin agents: Design, synthesis, biological
evaluation and molecular modelling studies.
a
a
b
a*
c
Sahil Sharma , Charanjeet Kaur , Abhishek Budhiraja , Kunal Nepali , Manish K Gupta , A.K
d
a
Saxena , PMS Bedi
a
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005, Punjab
Faculty of Pharmaceutical Sciences, Shoolini University, Solan, Himachal Pradesh, India
Molecular Modeling and Pharmacoinformatics Lab, Department of Pharmaceutical chemistry,
ISF College of Pharmacy, Moga, Punjab-143005, India
b
c
d
Indian Insitute of Integrative Medicine, Jammu, India
Corresponding author: Dr. Kunal Nepali
Assistant Professor, Department of Pharmaceutical Sciences
Guru Nanak Dev University, Amritsar, Punjab-143005, India
Email: kunal_8855@gmail.com
1

ACCEPTED MANUSCRIPT
Abstract: The present study involves the design of a series of 3-aryl-9-acetyl-pyridazino[3,4-
b]indoles as constrained chalcone analogs. A retrosynthetic route was proposed for the synthesis
of target compounds. All the synthesised compounds were evaluated for in-vitro cytotoxicity
against THP-1, COLO-205, HCT-116 and A-549 human cancer cell lines . The results indicated
2
5
a, 3a, 5a and 6a possesed significant cytotoxic potential with an IC value ranging from 1.13 -
50
.76 µM. Structure activity relationship revealed that the nature of both Ring A and Ring B
influences the activity. Substitution of methoxy groups on the phenyl ring (Ring A) and
unsubstituted phenyl ring (Ring B) were found to be the preferred structural features. The most
potent compound 2a was further tested for tubulin inhibition. Compound 2a was found to
significantly inhibit the tubulin polymerization (IC50 value – 2.41 µM against THP-1).
Compound 2a also caused disruption of microtubule assembly as evidenced by
Immunoflourescence technique. The significant cytotoxicity and tubulin inhibtion by 2a was
rationalized by molecular modelling studies. The most potent structure was docked at colchicine
binding site (PDB ID-1SA0) and was found to be stabilized in the cavity via various
hydrophobic and hydrogen bonding interactions.
Keywords: Carboline, Chalcone, Cytotoxic, Cell line, Tubulin, Constrained, Molecular
modelling studies
2

ACCEPTED MANUSCRIPT
1
. Introduction
Microtubules, key cytoskeletal filaments, are extremely important in the process of mitosis and
cell division which makes them an important target for anticancer drugs. Inhibiting tubulin
polymerization or interfering with microtubule disassembly ultimately leads to cell cycle arrest
or cell apoptosis induction. [1-4].
Chalcones are well known to inhibit microtubule polymerization and have been most extensively
explored among the various classes of tubulin inhibitors. Chalcones are precursors of flavonoids
and isoflavones and possess diverse array of biological activities, such as anti-cancer [5–7] anti-
inflammatory [8, 9], anti-tuberculosis [10] and anti-fungal activities [11]. Chemically, chalcones
(1,3-diaryl-2-propen-1-ones) possess an enone bridge between two aromatic rings [12]. The
extensive investigation on chalcones can be attributed to the ease of synthesis and their relatively
simpler chemical architecture. Recently the tubulin inhibitory potential of boronic acid chalcone
analogs [13], cinnamic acyl sulfonamide derivatives [14], guanidine derivatives of chalcones [4]
and antiproliferative effects of flavonoids based novel tetrahydropyran conjugates, chalcone
based sulfones and bisulfones [15] has been reported. Thus the recent literature clearly indicates
that chalcones are an area of focus at present. The well established structure activity relationship
for chalcones reveals that the rigid arrangement of the two aryl rings remarkably enhances the
anticancer potential which tempted us to focus on constrained analogues [16, 17]. The rigid
arrangement of chalcones is usually obtained by the incorporation of heterocyclic rings as
constraints in place of the enone bridge linking the two aromatic rings.
3

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Carboline skeleton belongs to the group of indole alkaloids and consist of pyridine ring fused to
an indole skeleton. Both α and β carboline skeleton have been extensively reported to exert
anticancer potential via varied mechanisms [18-24]. Recent reports about the
cytotoxic/anticancer effects of ruthenium(II)-β-carboline complex [18], β-carboline amino acid
ester conjugates [19], 3-Benzylamino-β-carboline derivatives [20], 1,6,8,9-substituted α-
carboline derivatives [21] and β-Carboline-3-(4-benzylidene)-4H-oxazol-5-one [22] highlights
the cell killing potential of this alkaloid.
With this background, 3-aryl-9-acetyl-pyridazino[3,4-b]indoles as Chalcone based Azacarboline
analogues were designed in the present study. The incorporation of azacarboline skeleton within
the chalcone framework provides a rigid arrangement of the two aryl rings (Fig. 1). The chemical
architecture of the designed analogues provides a similar arrangement of two aromatic rings (
two aryl rings separated by a three carbon linker) as in chalcones. The design strategy for the
synthesis of the target tubulin inhibitors is shown in Fig. 1. The reason for the inclusion of
azacarboline rather than α or β-carboline was primarily due to its bioisosterism to both the
carbolines. The retro synthetic path for the designed hybrids is shown in Fig. 2.
2
. Results
2
.1. Chemistry
Target compounds were synthesised via a sequence of reactions (Scheme 1) starting from base
catalysed condensation of isatin and differently substituted acetophenones. The attack of
generated carbanions from acetophenones at C-3 of isatins yielded 3-hydroxy-3-(2-oxo-2-
aryl/heteroaryl-ethyl)-1,3-dihydro-indol-2-ones (1). The condensed product of isatin and
acetophenonoes (1) were characterized by the appearance of two doublets for the non equivalent
geminal protons at 3.65 and 3.95 with a J value of 17.5 Hz. The reaction worked smoothly with
electronically and sterically diverse acetophenones i.e acetophenones substituted with electron
withdrawing and electron donating groups, acetyl naphthalenes as well as heteroaryl ketones,
however only halogenated isatins were tolerated by the reaction as the condensation of
acetophenones with methoxy substituted isatin did not proceed. The reason for this is being dealt
seperately. The condensed product (1) was then refluxed with a drop of sulphuric acid in glacial
acetic acid to yield the dehydrated product (2). The dehydrated product was then refluxed with
4

ACCEPTED MANUSCRIPT
hydrazine hydrate in methanol to obtain the target compound. However, only the undesired
hydrazone was afforded. The hydrazone was charaterized by the appearance of a doublet band
-1
for NH stretch at 3350-3340 cm in Infra red spectrum. This could be attributed to weak
2
nd
reactivity of the carbonyl of the isatin ring (2 position). Thus a different route was followed and
acetylation of the dehydrated product (2) was carried out to yield 3. The acetylated product was
characterized by the appearance of the singlet with an integration of three methyl protons at 2.7
ppm. Acetylation of the NH of isatin ring was done to increase the reactivity of the carbonyl
nd
group (2 position) of isatin ring. Grinding of the acetylated product with hydrazine hydrate
ultimately yielded the cyclized hybrid (1a). The proton resonances of the charaterstic compound
i.e 1a are represented in the Fig. 3.
2
.2. Biological Evaluation
All the synthetics were assayed for in vitro cytotoxicity against THP-1, COLO-205, HCT-116
and A-549 human cancer cell lines using sulforhodamine B19 [25]. The cells were allowed to
proliferate in the presence of test material for 48 h. All the synthetics were screened against the
cell lines at 50 µM. The molecules displaying percentage inhibition (Table 1) of greater than 60
%
at least against one cell line were only evaluated at different concentrations and IC values
50
(Table 2) were calculated. From Table 1 and Table 2, it is clear that the THP-1 cell lines were the
most sensitive towards the designed compounds whereas A-549 cell line were the most resistant.
Compound 2a, 3a, 5a and 6a displayed significant cytotoxicity with IC50 value ranging from
1
.13 – 4.15 µM, 2.33 – 5.69 µM, 2.41 – 5.76 µM and 2.39 – 5.74 µM against THP-1, COLO-205
and HCT-116. The most active hybrid 2a with trimethoxy phenyl ring as Ring A (IC = 1.13
5
0
µM) was almost two folds more active than 3a (dimethoxy phenyl ring as Ring A, IC50 = 2.33
µM), the second most potent hybrid against THP-1 cell line. Careful observation of Table 1 and
2
revealed that the substitution of methoxy groups on the phenyl ring (Ring A) remarkably
enhances the cytotoxic potential (compare 1a with 2a, 3a and 4a) whereas the placement of halo
and nitro groups diminishes the effect (compare 1a with 11a, 12a, 13a and 14a). Placement of a
heteroaryl ring in place of unsubstituted phenyl ring (Ring A) such as pyrrole and thiophene
improved the activity profile (compare 1a with 7a and 8a). Both 7a and 8a displayed a similar
cytotoxic profile. A positive effect was observed with the increased number of methoxy
5

ACCEPTED MANUSCRIPT
substituents on Ring A such as trimethoxy > dimethoxy > methoxy (compare 2a and 3a with 4a).
However placement of naphthyl ring as Ring A also proved to be a good surrogate for methoxy
substituted phenyl rings as their IC values were similar to the compounds bearing dimethoxy
5
0
phenyl rings (compare 3a with 5a and 6a). No competition was observed between cytotoxic
effects of 5a (1-naphthyl) and 6a (2-naphthyl). Overall the preference order of Ring A is as
follows: trimethoxy phenyl ring > dimethoxy phenyl ring = 1-naphthyl = 2-naphthyl > methoxy
phenyl > heteroaryl > phenyl > phenyl with halo and nitro groups. Any substitution on Ring B
significantly decreased the cytotoxic profile of the compounds (compare 2a with 2b, 2c, 2d and
2
e). The influence of the size of the halogens on ring B was also observed. The preference order
for substituents at Ring B is as follows H > chloro > bromo > Iodo = dibromo. The structure
activity relationship has been depicted in Fig. 4.
The most active compounds (2a, 3a, 5a and 6a) were evaluated for their inhibitory effects on
tubulin polymerization as per the reoprted assay using a cytoskeleton tubulin polymerization
assay kit [26, 27]. Compound 2a (most potent hybrid) with a trimethoxy ring (Ring A) also
displayed the most potent antitubulin activity with IC50 value of 2.41 µM. 3a also exhibited
significant inhibition of tubulin polymerization with an IC value of 4.92 µM. 5a and 6a with
5
0
naphthyl ring (Ring A) which displayed similar cytotoxic profile as that to 3a were found to be
endowed with weak inhibitory potential for tubulin polymerization. The results of the in-vitro
tubulin polymerization assay clearly indicate that both 2a and 3a exert their cytotoxic effects
through tubulin inhibition.
The immunofluorenscence technique was used to evaluate the effect of 2a on microtubules. The
effect on microtubule structure was determined using confocal microscopy. In this study,
untreated THP-1 cell lines showed the normal distribution of microtubules. However, cells
treated with compound 2a showed the disrupted microtubule organization at 5µM concentration.
The standard compound paclitaxel disrupted the microtubule assembly at 1µM (Fig. 5).
2
.3 Molecular modelling studies
In order to investigate the recognition process of azacarboline based constrained chalcone
analogues at the colchicine-binding site of tubulin, a flexible docking study was performed with
6

ACCEPTED MANUSCRIPT
most potent tubulin inhibitor (2a) using GOLD software [28]. The natural tubulin inhibitor
‘colchicine’ binds at the interface of α and β subunits of tubulin (PDB entry: 1SA0) [29].
Compound 2a was docked at the colchicine binding site of tubulin and the best fit conformation
was selected on the basis of Gold score and visual inspection. The Fig. 6 shows the binding
conformation of 2a at the colchicine-binding site of tubulin. The docking study showed that the
2
a gets stabilized in tubulin by various hydrogen bonding and hydrophobic interactions. Ring A
of 2a is situated in a polar cavity formed by Asn101A, Thr179A, Lys254B and Asn258B. The
oxygen atom of para-methoxy group of ring A acts as hydrogen bond acceptor and involved in
2
H-bond interactions with side chain NH of Asn101A (d=2.28Å). An additional but weak H-
bond was also observed between methoxy function at meta-position (CH: H-bond donor) and
3
main chain carbonyl function of Thr179A (C=O: H-bond acceptor; d= 2.23 Å). The ring B, C
and D of 2a are extended towards the β subunits of tubulinand positioned in a hydrophobic
cavity formed by Leu242B, Leu248B, Leu255B, Ala316B, Ala317B, Val318B and Ile378B. The
ring B is sandwiched between Leu248B and Leu255B. This may be suggested that the aliphatic
site chain of Leu248B is involved in alkyl-aryl interactions with ring B. [29] The similar
interaction was also observed between ring A and aliphatic side chain Lys254B. Rind D is
situated in a hydrophobic cleft formed by Ala316B, Ala317B, Val318B and Ile378B (Fig. 6)
[30].
3
. Conclusions
Chalcone based Azacarboline analogues were designed, synthesised and evaluated for
cytotoxicty against a panel of human cancer cell lines in the present study. The nature of both
Ring A and Ring B was found to have significant influence on the cytotoxic potential. Tubulin
inhibitory potential of the most potent compound 2a (with a trimethoxy phenyl ring as Ring A
and unsubstituted Ring B) was confirmed by in vitro tubulin assay and Immunoflourescence
technique The significant inhibition of tubulin by compound 2a was also rationalized by
molecular modelling studies. Detailed investigation of compound 2a is under progress.
Experimental section
7

ACCEPTED MANUSCRIPT
4
.1. Chemistry
The reagents were purchased from Sigma–aldrich, Loba and CDH, India and used without
further purification. All yields refer to isolated products after purification. Products were
1
13
characterized by comparison with authentic samples and by spectroscopic data ( H and
C
1
13
NMR). H-NMR and C-NMR Spectra were recorded on a 300 MHz FT-NMR and 75 or 125
MHz respectively. The spectra were measured in CDCl and DMSO-d relative to TMS (0.00
3
6
1
ppm). In H-NMR chemical shifts were reported in δ values using tetramethylsilane as internal
standard with number of protons, multiplicities (s-singlet, d-doublet, t-triplet, q-quartet, m-
multiplet, dd-double doublet) and coupling constants (J) in Hz (Hertz) in the solvent indicated.
Melting points were determined in open capillaries and were uncorrected.
4
1
.2. Experimental procedure for the synthesis of substituted 2-(3-hydroxy-2-oxoindolin-3-yl)-
-arylethanone (1):
An aqueous solution of sodium hydroxide (5%, 10 mL) was added slowly to the stirring solution
of isatin (1 mmol) and appropriate aryl acetophenone (1 mmol) in ethanol (20 mL) in 100 mL
conical flask. The stirring was continued for 2 hours and the completion of reaction was
monitored by TLC. The reaction on completion was poured onto ice, solid obtained after
filtration was crystallized from ethanol. The physical data for the characteristic compound is
shown below:
◦
4
.2.1. 2-(3-hydroxy-2-oxoindolin-3-yl)-1-phenylethanone (1) White solid, yield 92%; mp: 132 -
◦
1
1
3
33 C; H NMR (300 MHz, CDCl ): δ = 10.21 (s, 1H), 7.86 (d, J = 7.8 Hz, 2H), 7.56 (t, J = 7.2
Hz, 1H), 7.42-7.46 (2H, m), 7.24 (d, J = 7.2 Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H), 6.87 (m, 2H), 6.08
s, 1H), 3.95 (d, J = 17.5 Hz, 1H), 3.65 (d, J = 17.5 Hz, 1H). Anal. Calcd. For C16 : C,
(
H13NO
3
7
1.90; H, 4.90; N, 5.24. Found: C, 72.28; H, 5.13; N, 5.01.
4
.3. Experimental procedure for the synthesis of (Z)-3-(2-oxo-2-arylethylidene)indolin-2-one
(2):
8

ACCEPTED MANUSCRIPT
A mixture of 2-(3-hydroxy-2-oxoindolin-3-yl)-1-phenylethanone (1 mmol) and 10 mL of acetic
acid and a drop of sulfuric acid was refluxed for 1 hour. The completion of reaction was
monitored by TLC. The reaction mixture on completion was poured onto ice and the precipitates
were collected and crystallized from ethanol. The physical data for the characteristic compound
is shown below:
◦
4
1
7
2
.3.1. (Z)-3-(2-oxo-2-phenylethylidene)indolin-2-one (2) Orange red solid; yield 90%; mp:187 -
◦
1
3 2
88 C; H NMR (300 MHz, CDCl ); δ = 10.15 (s, 1H, D O exchangeable proton); 8.26 (d, J =
.8 Hz, 1H); 8.10 (d, J = 7.5 Hz, 2H); 7.80 (s, 1H); 7.62 (t, J = 7.5 Hz, 1H); 7.52 (t, J = 7.5 Hz,
H); 7.26 (t, J = 7.5 Hz, 1H); 6.93 (t, J = 7.8 Hz, 1H); 6.85 (d, J = 7.8 Hz, 1H). Anal. Calcd. For
C H NO : C, 77.10; H, 4.45; N, 5.62. Found: C, 77.32; H, 4.18; N, 5.73.
16
11
2
4
.4. Experimental procedure for the synthesis of (Z)-1-acetyl-3-(2-oxo-2-arylethylidene)
indolin-2-one (3):
A mixture of (Z)-3-(2-oxo-2-arylethylidene)indolin-2-one (1 mmol) and acetic anhydride was
stirred in the presence of DMAP (0.1 mmol) for 2 hours under anhydrous condition. The
completion of reaction was monitored by TLC. The reaction mixture on completion was poured
onto ice and the precipitates were collected and crystallized from ethanol. The physical data for
the characteristic compound is shown below:
4
1
7
.4.1. (Z)-1-acetyl-3-(2-oxo-2-phenylethylidene)indolin-2-one (3) Yellow solid; yield 85%; mp:
◦
◦
1
55 -156 C; H NMR (300 MHz, CDCl ); δ = 8.32 (d, J = 8.4 Hz, 1H), 8.10 (s, J = 7.5 Hz, 2H),
3
.88 (s, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7. 56 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 8.4 Hz, 1H), 7.19 (t, J
=
7.2 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 2.77 (s, 3H). Anal. Calcd. For C18
3
H13NO : C, 74.22; H,
4
.50; N, 4.81. Found: C, 74.05; H, 4.64; N, 4.77.
4
.5. Experimental procedure for the synthesis of 3-Aryl-9-acetyl-pyridazino[3,4-b]indoles:
A mixture of (Z)-1-acetyl-3-(2-oxo-2-phenylethylidene)indolin-2-one (1 mmol) and hydrazine
monohydrate 80% (1 mmol) was ground in china dish for 15 mins. The completion of reaction
was monitored by TLC. The reaction mixture on completion was poured onto ice and the
precipitates were collected and crystallized from ethanol. The physical data for the characteristic
compound is shown below:
9

ACCEPTED MANUSCRIPT
4
1
2
.5.1. 3-Phenyl-9-acetyl-pyridazino[3,4-b]indole (1a) Pale yellow fluffy powder; yield 90%; Mp
◦
◦
1
25 -126 C; H NMR (300 MHz, DMSO-d ): δ = 8.40 (d, J = 8.4 Hz, 1H); 8.24 (d, J = 6.9 Hz,
6
H); 8.13 (d, J = 8.4 Hz, 1H); 8.10 (s, 1H); 7.73-7.78 (m, 2H); 7.45-7.61 (m, 3H); 2.08 (s, 3H).
C NMR (DMSO-d6, 75 MHz, δ, TMS = 0): 20.48, 116.78, 123.33, 125.31, 127.18, 128.88,
1
3
-
1
1
3
29.42, 129.86, 130.32, 138.00, 141.16, 147.77, 155.62, 165.67, 167.87, 168.46. IR (KBr, cm )
.
+
13 3
030, 2998, 1665, 1565, 1552, 1130 MS: M at m/z 287. Anal. Calcd. For C18H N O: C, 75.25;
H, 4.56; N, 14.63. Found: C, 75.19; H, 4.73; N, 14.81.
4
.5.2. 3-(3,4,5-Trimethoxyphenyl)-9-acetyl-pyridazino[3,4-b]indole (2a) Pale yellow fluffy
◦
◦
1
powder; yield 90%; Mp 197 -198 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.46 (bs, 1H), 8.21-
8
6
1
.34 (m, 2H), 7.96 (d, J = 8.7 Hz, 1H), 7.83 (dd, J = 1.8 and 9.0 Hz, 1H), 7.55 (s, 2H), 3.91 (s,
1
3
H), 3.76 (s, 3H), 2.05 (s, 3H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 19.58, 56.59, 60.63,
6
,
05.31, 118.34, 124.82, 131.33, 131.98, 133.71, 139.94, 146.61, 153.77, 156.38, 166.00, 169.13.
-1
.
+
+
IR (KBr, cm ) 3045, 2943, 1667, 1561, 1545, 1211, 1137 MS: M at m/z 287. MS: M at m/z
3
1
77. Anal. Calcd. For C21
1.45.
19 3 4
H N O : C, 66.83; H, 5.07; N, 11.13. Found: C, 66.84; H, 5.17; N,
4
7
8
8
5
1
.5.3. 3-(3,4-Dimethoxyphenyl)-9-acetyl-pyridazino[3,4-b]indole (3a) Light yellow solid; yield
◦
◦
1
5%; Mp 180 -181 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.38 (d, J = 7.8 Hz, 1H); 8.12 (d, J =
.1 Hz, 1H); 8.07 (s, 1H); 7.93 (s, 1H); 7.72-7.79 (m, 2H); 7.58 (t, J = 7.2 Hz, 1H); 7.03 (d, J =
1
3
.4 Hz, 1H); 3.92 (s, 6H); 2.01 (s, 3H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 19.53,
6
,
6.02, 110.60, 112.12, 116.98, 120.81, 123.54, 125.78, 127.30, 129.71, 130.78, 131.09, 141.46,
+
48.19, 149.48, 151.07, 155.84, 166.36, 168.20. MS: M at m/z 347. Anal. Calcd. For
C
20
H
17
N
3
O
3
: C, 69.15; H, 4.93; N, 12.10. Found: C, 69.45; H, 5.28; N, 12.43.
4
7
8
1
=
.5.4. 3-(4-Methoxyphenyl)-9-acetyl-pyridazino[3,4-b]indole (4a) Cream colored solid; yield
◦
◦
1
8%; Mp 157 -158 C; H NMR (300 MHz, DMSO-d ): δ = 8.33 (d, J = 8.4 Hz,1H); 8.27 (d, J =
6
.8 Hz, 2H); 8.10 (d, J = 8.4 Hz, 1H); 8.09 (s, 1H); 7.82 (t, J = 7.2 Hz, 1H); 7.62 (t, J = 7.6 Hz,
13
H); 7.13 (d, J = 8.8 Hz, 2H); 3.86 (s, 3H); 1.99 (s, 3H). C NMR (DMSO-d 75 MHz, δ, TMS
6
,
0): 20.89, 55.79, 114.80, 116.85, 123.56, 125.87, 127.27, 128.45, 129.22, 129.74, 130.76,
+
1
30.97, 141.62, 148.32, 155.79, 161.35, 166.29, 168.54, 169.00. MS: M at m/z 317. Anal.
Calcd. For C H N O : C, 71.91; H, 4.76; N, 13.24. Found: C, 71.83; H, 4.47; N, 13.44.
1
9
15
3
2
10

ACCEPTED MANUSCRIPT
4
.5.5. 3-Naphth-2-yl-9-acetyl-pyridazino[3,4-b]indole (5a) Creamish white solid; yield 70%;
◦
1
Mp 251-252 C; H NMR (300 MHz, DMSO-d ): δ = 8.88 (s, 1H); 8.51 (dd, J = 1.2 and 8.4 Hz,
6
1
H); 8.37 (d, J = 8.8 Hz, 1H); 8.33 (s, 1H) ; 8.20 (d, J = 8.8 Hz, 1H); 8.11-8.16 (m, 2H); 8.01
(dd, J = 3.2 and 6.04 Hz, 1H); 7.88 (t, J = 8.8 Hz, 1H); 7.69 (t, J = 7.6 Hz, 1H); 7.60-7.62 (m,
1
3
2
1
1
H); 2.05 (s, 3H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 21.08, 117.48, 123.95, 125.00,
6,
25.93, 127.17, 127.53, 127.71, 127.84, 128.10, 129.00, 129.31, 130.00, 130.97, 133.51, 134.09,
+
35.91, 141.87, 148.38, 155.98, 166.27, 169.05. MS: M at m/z 337. Anal. Calcd. For
C H N O: C, 78.32; H, 4.48; N, 12.46. Found: C, 78.22; H, 4.19; N, 12.74.
22
15
3
4
.5.6. 3-Naphth-1-yl-9-acetyl-pyridazino[3,4-b]indole (6a) Creamish white solid; yield 70%;
◦
◦
1
Mp 245 -246 C; H NMR (300 MHz, CDCl ): δ = 8.44 (dd, J = 1.2 and 8.4 Hz, 1H); 8.38 (d, J =
3
8
.8 Hz, 1H); 8.35 (s, 1H) ; 8.22 (d, J = 8.8 Hz, 1H); 8.11-8.16 (m, 2H); 8.01 (dd, J = 3.2 and 6.04
13
Hz, 1H); 7.88 (t, J = 8.8 Hz, 1H); 7.69 (t, J = 7.6 Hz, 1H); 7.60-7.62 (m, 3H); 2.05 (s, 3H). C
NMR (DMSO-d 75 MHz, δ, TMS = 0): 20.98, 122.44, 124.31, 124.71, 125.67, 125.94, 126.72,
6
,
1
1
1
27.47, 128.75, 128.96, 130.07, 130.82, 131.52, 132.14, 132.71, 133.95. 137.41, 140.33, 146.66,
+
59.21, 165.73, 169.18. MS: M at m/z 337. Anal. Calcd. For C H N O: C, 78.32; H, 4.48; N,
2
2
15
3
2.46. Found: C, 78.58; H, 4.66; N, 12.15.
4
.5.7. 3-(1H)-Pyrrol-2-yl-9-acetyl-pyridazino[3,4-b]indole (7a) Pale brown solid; yield 56%;
◦
◦
1
Mp 232 -233 C; H NMR (300 MHz, CDCl ): δ = 6.18 (s, 1H, D O exchangeable proton); 8.48
3
2
(d, J = 8.4 Hz, 1H); 7.99 (d, J = 7.8 Hz, 1H) ; 7.84 (s, 1H); 7.48-7.53 (m, 1H), 7.12-7.27 (m, 2H),
1
3
6
1
1
6
.90 (t, J = 7.2 Hz, 2H), 2.06 (s, 3H). C NMR (DMSO-d6, 75 MHz, δ, TMS = 0): 20.90, 110.35,
11.14, 116.20, 123.04, 125.97, 126.26, 128.80, 130.69, 131.33, 141.43, 148.19, 150.41, 166.36,
+
69.02. MS: M at m/z 276. Anal. Calcd. For C16
12 4
H N O: C, 69.55; H, 4.38; N, 20.28. Found: C,
6.89; H, 4.08; N, 19.89.
4
2
7
.5.8. 3-Thiophen-2-yl-9-acetyl-pyridazino[3,4-b]indole (8a) Pale brown solid; yield 56%; Mp
◦
◦
1
02 -203 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.29 (d, J = 8.4 Hz, 1H); 8.00-8.10 (m, 3H),
1
3
.76-7.82 (m, 2H), 7.61 (m, 1H), 7.23 (m, 1H), 1.98 (s, 3H). C NMR (DMSO-d 75 MHz, δ,
6
,
TMS = 0): 20.90, 116.11, 123.85, 126.00, 127.44, 128.19, 129.17, 130.66, 131.11, 141.97,
+
1
44.52, 148.01, 151.93, 166.05, 168.48, 169.04. MS: M at m/z 293. Anal. Calcd. For
C H N OS: C, 65.51; H, 3.78; N, 14.32; S, 10.93. Found: C, 65.82; H, 4.05; N, 14.66; S, 10.58.
16
11
3
11

ACCEPTED MANUSCRIPT
4
.5.9. 3-(2-Aminoacetylphenyl)-9-acetyl-pyridazino[3,4-b]indole (9a) Yellow coloured solid;
◦
◦
1
yield 85%; Mp 185 -186 C; H NMR (300 MHz, DMSO-d ): δ = 10.44 (s, 1H, D O
6
2
exchangeable proton); 8.40 (d, J = 8.4 Hz, 1H); 8.20 (d, J = 8.0 Hz, 1H); 8.00-8.03 (m, 2H); 7.92
13
(
t, J = 7.6 Hz, 1H); 7.75 (t, J = 7.6 Hz, 1H); 7.48-7.52 (m, 3H); 2.35 (s, 3H); 2.01 (s, 3H). C
NMR (DMSO-d 75 MHz, δ, TMS = 0): 19.55, 24.48, 114.24, 117.29, 117.96, 121.00, 122.44,
6
,
1
1
24.00, 125.55, 126.92, 130.06, 131.00, 139.11, 141.22, 141.92, 148.66, 150.88, 155.99,
+
66.232, 169.87. Anal. Calcd. For C H N O : 69.76; H, 4.68; N, 16.27. MS: M at m/z 344.
2
0
16
4
2
Anal. Calcd. For C H N O : C, 69.76; H, 4.68; N, 16.27. Found: C, 70.03; H, 4.38; N, 16.26.
2
0
16
4
2
4
.5.10. 3-(3-Aminoacetylphenyl)-9-acetyl-pyridazino[3,4-b]indole (10a) Yellow colored solid;
◦
◦
1
yield 70%; Mp 130 -131 C; H NMR (300 MHz, DMSO-d ): δ = 10.20 (s, 1H, D O
6
2
exchangeable proton); 8.44 (s, 1H); 8.33 (d, J = 8.0 Hz, 1H); 8.13 (d, J = 8.4 Hz, 1H); 8.09 (s,
1
1
1
1
H); 7.96 (d, J = 7.8 Hz, 1H); 7.83-7.88 (m, 2H); 7.68 (t, J = 7.2 Hz, 1H); 7.55 (t, J = 8.0 Hz,
13
H); 2.35 (s, 3H); 2.02 (s, 3H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 19.56, 24.47,
6
,
14.20, 117.23, 118.16, 121.04, 122.49, 123.90, 125.85, 127.12, 129.86, 131.00, 139.01, 140.42,
+
41.84, 148.33, 150.83, 155.95, 166.26, 169.07. MS: M at m/z 344. Anal. Calcd. For
C H N O : 69.76; H, 4.68; N, 16.27. Found: C, 69.43; H, 4.83; N, 16.65.
20
16
4
2
4
.5.11. 3-(4-Fluorophenyl)-9-acetyl-pyridazino[3,4-b]indole (11a) Yellow powder; yield 92% ;
◦
◦
1
Mp 235 -236 C; ; H NMR (300 MHz, DMSO-d ); δ = 8.41 (d, J = 8.1 Hz, 1H); 8.29-8.32 (m,
6
2
7
1
1
1
H); 8.12 (d, J = 8.4 Hz, 1H); 8.09 (s, 1H); 7.77 (t, J = 7.8 Hz, 1H); 7.59 (t, J = 7.8 Hz, 1H);
1
3
.26 (m, 2H); 2.08 (s, 3H). C NMR (DMSO-d6, 75 MHz, δ, TMS = 0): 20.90, 116. 22, 116.50,
17.18, 123.78, 125.78, 127.82, 129.91, 130.01, 130.12, 130.98, 135.03, 141.79, 148.23, 155.11,
+
62.24, 166.18, 169.07. MS: M at m/z 305. Anal. Calcd. For C18
3.76. Found: C, 71.00; H, 3.65; N, 13.77.
3
H12FN O: C, 70.81; H, 3.96; N,
4
2
8
3
1
.5.12. 3-(4-Chlorophenyl)-9-acetyl-pyridazino[3,4-b]indole (12a) White solid; yield 85%; Mp
◦
1
60-261 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.33-8.37 (m, 2H); 8.15 (d, J = 8.8 Hz, 2H);
.16 (s, 1H); 7.87 (t, J = 7.2 Hz, 1H); 7.69 (t, J = 7.6 Hz, 1H); 7.66 (d, J = 8.8 Hz, 2H); 1.99 (s,
1
3
H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 20.90, 117.17, 123.95, 125.89, 128.00, 129.50,
6
,
+
29.97, 131.05, 135.38, 137.30, 141.86, 148.23, 154.89, 166.12, 168.48, 169.04. MS: M at m/z
12

ACCEPTED MANUSCRIPT
3
1
21. Anal. Calcd. For C18
1.09.
3
H12ClN O: C, 67.19; H, 3.76; N, 13.06. Found: C, 67.42; H, 3.57; N,
4
.5.13. 3-(4-Bromophenyl)-9-acetyl-pyridazino[3,4-b]indole (13a) White coloured powder;
◦
1
yield 89%; Mp 269-270 C; H NMR (300 MHZ, DMSO-d
6
): δ = 8.33-8.37 (m, 2H); 8.25 (d, J =
8
.8 Hz, 2H); 8.16 (s,1H); 7.87 (t, J = 7.2 Hz, 1H); 7.69 (t, J = 7.6 Hz, 1H); 7.81 (d, J = 8.8 Hz,
1
3
2
H); 1.99 (s, 3H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 20.41, 116.63, 123.51, 123.73,
6,
1
25.41, 127.50, 129.28, 129.48, 130.54, 131.92, 137.20, 141.41, 147.79, 154.52, 165.60, 167.94,
+
1
68.51. MS: M at m/z 365. Anal. Calcd. For C H BrN O: C, 59.03; H, 3.30; N, 11.47. Found:
1
8
12
3
C, 59.14; H, 3.68; N, 11.69.
4
.5.14. 3-(4-Nitrophenyl)-9-acetyl-pyridazino[3,4-b]indole (14a) Yellowish powder; yield 85%;
◦
1
Mp 255-256 C; H NMR (300 MHz, DMSO-d ); δ = 8.56 (d, J = 9.0 Hz, 2H); 8.38-8.45 (m, 3H);
6
8
3
1
.26 (s, 1H); 8.18 (d, J = 8.1 Hz, 1H); 7.83 (t, J = 7.0 Hz, 1H); 7.67 (d, J = 7.5 Hz, 1H); 1.99 (s,
1
3
H). C NMR (DMSO-d 75 MHz, δ, TMS = 0): 20.90, 117.75, 124.32, 124.59, 125.96, 128.68,
6
,
+
29.01, 130.22, 131.33, 142.18, 144.38, 148.28, 148.69, 153.94, 165.93, 168.44, 169.02. MS: M
at m/z 332. Anal. Calcd. For C18
N, 16.51.
12 4 3
H N O : C, 65.06; H, 3.64; N, 16.86. Found: C, 65.43; H, 3.86;
4
.5.15. 3-Phenyl-5-chloro-9-acetyl-pyridazino[3,4-b]indole (1b) Pale yellow fluffy powder;
◦
1
6
yield 90%; Mp 255-256 C; H NMR (300 MHz, DMSO-d ): δ = 8.45 (s, 1H), 8.29 (m, 2H), 8.14-
1
3
8
1
1
.21 (m, 2H), 7.86 (d, J = 9.00 Hz, 1H), 7.56-7.58 (m, 3H), 2.05 (s, 3H). C NMR (DMSO-d
6,
25 MHz, δ, TMS = 0): 25.71, 123.13, 123.24, 129.35, 129.46, 129.55, 132.36, 132.47, 133.89,
+
35.78, 137.26, 143.07, 144.85, 151.69, 161.35, 170.47, 173.73. MS: M at m/z 321. Anal.
Calcd. For C H ClN O: C, 67.19; H, 3.76; N, 13.06. Found: C, 66.94; H, 3.81; N, 12.99.
1
8
12
3
4
.5.16. 3-(3,4,5-Trimethoxyphenyl)-5-chloro-9-acetyl-pyridazino[3,4-b]indole (2b) Pale yellow
◦
1
fluffy powder; yield 90%; Mp 280-281 C; H NMR (300 MHz, DMSO-d ): δ = 8.47 (s, 1H),
6
8
.10-8.15 (m, 2H), 7.65 (d, J = 9.0 Hz, 1H), 7.52 (s, 2H), 3.94 (s, 6H), 3.77 (s, 3H), 2.04 (s, 3H).
C NMR (DMSO-d6, 125 MHz, δ, TMS = 0): 20.36, 55.71, 59.78, 109.05, 118.14, 126.24,
1
3
+
1
31.83, 131.98, 133.64, 139.89, 146.79, 153.28, 156.97, 165.34, 169.71. MS: M at m/z 411.
3 4
Anal. Calcd. For C21H18ClN O : C, 61.24; H, 4.41; N, 10.20. Found: C, 61.48; H, 4.49; N, 10.01.
13

ACCEPTED MANUSCRIPT
4
.5.17. 3-(3,4-Dimethoxyphenyl)-5-chloro-9-acetyl-pyridazino[3,4-b]indole (3b) Light yellow
◦
1
solid; yield 75%; Mp 268-269 C; H NMR (300 MHz, DMSO-d ): δ = 8.44 (d, J = 2.1 Hz, 1H),
6
8
.08-8.15 (m, 2H), 8.00 (s, 1H), 7.69-7.92 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.92
13
(
s, 3H), 2.07 (s, 3H). C NMR (DMSO-d6, 125 MHz, δ, TMS = 0): 20.93, 56.10, 110.53, 111.42,
1
1
18.15, 120.71, 124.26, 124.66, 130.73, 131.15, 131.37, 132.20, 139.50, 146.88, 149.41, 150.96,
+
56.71, 165.71, 169.12. MS: M at m/z 381. Anal. Calcd. For C20
H16ClN O
3 3
: C, 62.91; H, 4.22;
N, 11.01. Found: C, 63.06; H, 4.35; N, 10.88.
4
.5.18. 3-(4-Methoxyphenyl)-5-chloro-9-acetyl-pyridazino[3,4-b]indole (4b) Cream colored
◦
1
solid; yield 78%; Mp 275-276 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.41 (s, 1H), 8.22-8.28 (d,
J = 8.7 Hz, 2H), 8.08-8.15 (m, 2H), 7.77 (d, J = 9.00 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 3.83 (s,
1
3
3
1
H), 2.02 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.93, 55.51, 114.43, 117.99,
6,
24.21, 124.67, 129.06, 130.79, 131.44, 132.04, 139.73, 146.93, 156.22, 161.36, 165.77, 169.19.
+
MS: M at m/z 351. Anal. Calcd. For C H ClN O : C, 64.87; H, 4.01; N, 11.94.. Found: C,
1
9
14
3
2
6
4.76; H, 3.89; N, 12.08.
4
.5.19. 3-Naphth-2-yl-5-chloro-9-acetyl-pyridazino[3,4-b]indole (5b) Creamish white solid;
◦
1
yield 70%; Mp 288-289 C; H NMR (300 MHz, DMSO-d ): δ = 8.86 (s, 1H), 8.47 (s, 1H), 8.39
6
(s, 1H), 8.18-8.24 (m, 2H), 8.06-8.09 (m, 2H), 7.85 (m, 1H), 7.82 (m, 1H), 7.60 (m, 2H), 2.01 (s,
1
3
3
1
1
7
H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 21.38, 118.88, 123.97, 125.98, 126.83,
6,
27.13, 127.81, 127.07, 127.55, 128.99, 129.50, 129.23, 130.39, 131.05, 133.15, 134.44, 135.75,
+
41.21, 148.66, 156.77, 165.28, 169.17. MS: M at m/z 371. Anal. Calcd. For C22
1.07; H, 3.80; N, 11.30. Found: C, 70.85; H, 4.02; N, 11.44.
3
H14ClN O: C,
4
.5.20. 3-Naphth-1-yl-5-chloro-9-acetyl-pyridazino[3,4-b]indole (6b) Creamish white solid;
◦
1
yield 70%; Mp 285-286 C; H NMR (300 MHz, CDCl
3
): δ = 8.48 (s, 1H), 8.09-8.28 (m, 5H),
1
3
7
0
1
.69-7.91 (m, 2H), 7.43-7.58 (m, 3H), 1.98 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS =
6
,
): 20.99, 122.24, 124.32, 124.72, 125.68, 125.94, 126.72, 127.47, 128.75, 128.96, 130.07,
+
30.83, 131.52, 132.15, 132.71, 133.95, 137.42, 140.34, 146.67, 159.21, 165.71, 169.20. MS: M
at m/z 371. Anal. Calcd. For C H ClN O: C, 71.07; H, 3.80; N, 11.30. Found: C, 71.12; H,
2
2
14
3
3
.99; N, 11.03.
14

ACCEPTED MANUSCRIPT
4
.5.21. 3-(1H)-Pyrrol-2-yl-5-chloro-9-acetyl-pyridazino[3,4-b]indole (7b) Pale brown solid;
◦
1
yield 56%; Mp 272-273 C; H NMR (300 MHz, CDCl ): δ = 6.24 (s, 1H, D O exchangeable
3
2
proton); 8.32 (d, J = 2.1 Hz, 1H), 7.94-7.99 (m, 2H), 7.77 (dd, J = 1.8 and 8.7 Hz, 1H), 6.93-7.40
1
3
(
m, 2H), 6.24 (d, J = 2.1 Hz, 1H), 1.99 (s, 3H). C NMR (DMSO-d6, 125 MHz, δ, TMS = 0):
2
1
1.01, 110.50, 111.75, 117.17, 120.51, 122.10, 123.48, 123.82, 124.85, 130.64, 130.83, 140.50,
+
46.75, 150.86, 165.86, 169.13. MS: M at m/z 310. Anal. Calcd. For C16
H11ClN
4
O: C, 61.84;
H, 3.57; N, 18.03. Found: C, 62.01; H, 3.63; N, 17.95.
4
8
8
3
1
3
1
.5.22. 3-(4-Chlorophenyl)-5-chloro-9-acetyl-pyridazino[3,4-b]indole (8b) White solid; yield
◦
1
5%; Mp 276-277 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.46 (s, 1H), 8.34 (d, J = 7.5 Hz, 2H),
.25 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.5 Hz, 2H), 2.01 (s,
1
3
H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 21.00, 118.43, 123.59, 126.87, 128.12,
6
,
+
29.45, 129.95, 131.33, 135.47, 137.56, 141.22, 147.32, 156.11, 165.28, 169.16. MS: M at m/z
55. Anal. Calcd. For C H Cl N O: C, 60.69; H, 3.11; N, 11.80. Found: C, 60.91; H, 2.93; N,
1
8
11
2
3
2.04.
4
.5.23. 3-(4-Bromophenyl)-5-chloro-9-acetyl-pyridazino[3,4-b]indole (9b) White coloured
◦
1
powder; yield 89%; Mp 280-281 C; H NMR (300 MHZ, DMSO-d ): δ = 8.48 (s, 1H), 8.20 (d, J
6
1
3
=
9.0 Hz, 2H), 8.11 (d, J = 9.0 Hz, 1H), 7.94 (bs, 2H), 7.69 (d, J = 9.0 Hz, 2H), 2.08 (s, 3H). C
NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.98, 118.11, 124.55, 124.68, 124.76, 129.84,
6
,
+
1
3
1
31.63, 132.11, 132.47, 132.63, 137.27, 141.00, 146.74, 155.58, 165.68, 169.27. MS: M at m/z
99. Anal. Calcd. For C18
0.54.
3
H11ClBrN O: C, 53.96; H, 2.77; N, 10.49. Found: C, 54.16; H, 2.56; N,
4
.5.24. 3-(4-Nitrophenyl)-5-chloro-9-acetyl-pyridazino[3,4-b]indole (10b) Yellowish powder;
◦
1
yield 85%; Mp 268-269 C; H NMR (300 MHz, DMSO-d
6
); δ = 8.51-8.57 (m, 3H), 8.34-8.41
13
(
m, 3H), 8.14 (m, 1H), 7.82 (d, J = 9.0 Hz, 1H), 2.05 (s, 3H). C NMR (DMSO-d 125 MHz, δ,
6
,
TMS = 0): 20.93, 118.74, 124.18, 124.82, 125.01, 128.78, 131.42, 132.01, 133.90, 140.11,
+
1
44.15, 146.89, 148.63, 154.20, 165.33, 169.19. MS: M at m/z 366. Anal. Calcd. For
C H ClN O : C, 58.95; H, 3.02; N, 15.28. Found: C, 59.11; H, 2.87; N, 15.33.
18
11
4
3
15

ACCEPTED MANUSCRIPT
4
.5.25. 3-Phenyl-5-bromo-9-acetyl-pyridazino[3,4-b]indole (1c) Pale yellow fluffy powder;
◦
1
yield 90%; Mp 258-259 C; H NMR (300 MHz, DMSO-d ): δ = 8.42-8.64 (m, 2H), 8.39-8.42
6
1
3
(
m, 2H), 8.09 (d, J = , 8.7 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.45-7.61 (m, 3H), 2.00 (s, 3H). C
NMR (DMSO-d6, 125 MHz, δ, TMS = 0): 21.01, 118.25, 121.05, 125.08, 127.83, 127.99,
1
29.49, 129.99, 130.72, 132.16, 134.00, 136.55, 138.15, 140.71, 146.97, 156.76, 165.74, 169.11.
+
MS: M at m/z 365. Anal. Calcd. For C18
H12BrN
3
O: C, 59.03; H, 3.30; N, 11.47. Found: C,
5
8.84; H, 3.49; N, 11.29.
4
.5.26. 3-(3,4,5-Trimethoxyphenyl)-5-bromo-9-acetyl-pyridazino[3,4-b]indole (2c) Pale yellow
◦
1
fluffy powder; yield 90%; Mp 290-291 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.42 (s, 1H),
8
.09-8.15 (m, 2H), 7.66 (d, J = 9.0 Hz, 1H), 7.50 (s, 2H), 3.99 (s, 6H), 3.77 (s, 3H), 2.01 (s, 3H).
1
3
C NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.78, 55.98, 59.88, 109.93, 119.31, 124.97,
6
,
+
1
4
8
31.39, 131.48, 133.92, 139.83, 146.67, 153.61, 156.88, 161.82, 166.55, 169.32. MS: M at m/z
55. Anal. Calcd. For C H BrN O : C, 55.28; H, 3.98; N, 9.21. Found: C, 55.43; H, 4.25; N,
2
1
18
3
4
.91.
4
.5.27. 3-(3,4-Dimethoxyphenyl)-5-bromo-9-acetyl-pyridazino[3,4-b]indole (3c) Light yellow
◦
1
solid; yield 75%; Mp 285-286 C; H NMR (300 MHz, DMSO-d ): δ = 8.72 (s, 1H), 8.14 (s, 1H),
6
8
1
1
1
.05 (d, J = 8.4 Hz, 1H), 7.85-7.88 (m, 3H), 7.12 (d, J = 8.1 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 3H),
1
3
.98 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.47, 56.88, 58.01, 111.36, 113.82,
6
,
18.98, 121.43, 123.48, 125.36, 127.38, 129.72, 130.71, 131.95, 141.98, 148.99, 149.66, 155.61,
+
51.07, 161.41, 165.47, 169.74. MS: M at m/z 425. Anal. Calcd. For C20
H16BrN
3
O
3
: C, 56.35;
H, 3.78; N, 9.86. Found: C, 56.71; H, 3.92; N, 9.99.
4
.5.28. 3-(4-Methoxyphenyl)-5-bromo-9-acetyl-pyridazino[3,4-b]indole (4c) Cream colored
◦
1
solid; yield 78%; Mp 278-279 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.58 (s, 1H), 8.25-8.37
1
3
(
m, 5H), 7.13-7.16 (m, 2H), 3.89 (s, 3H), 1.99 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS =
6
,
0
1
): 20.97, 55.89, 114.99, 118.34, 119.85, 125.61, 126.32, 128.05, 129.60, 130.09, 136.37,
+
41.53, 143.78, 156.82, 161.93, 165.41, 169.19. MS: M at m/z 395. Anal. Calcd. For
C H BrN O : C, 57.59; H, 3.56; N, 10.60;. Found: C, 57.41; H, 3.72; N, 10.42;.
19
14
3
2
16

ACCEPTED MANUSCRIPT
4
.5.29. 3-Naphth-2-yl-5-bromo-9-acetyl-pyridazino[3,4-b]indole (5c) Creamish white solid;
◦
1
yield 70%; Mp 295-296 C; H NMR (300 MHz, DMSO-d ): δ = 8.89 (s, 1H), 8.61 (s, 1H), 8.37-
6
1
3
8
1
1
1
.50 (m, 2H), 8.09-8.14 (m, 3H), 7.79 (m, 2H), 7.59 (m, 2H), 2.01 (s, 3H). C NMR (DMSO-d
6,
25 MHz, δ, TMS = 0): 21.04, 118.40, 121.10, 124.90, 125.14, 127.22, 127.80, 127.85, 128.06,
28.12, 129.05, 129.36, 132.16, 133.48, 134.05, 134.20, 135.49, 140.86, 147.03, 156.60, 165.79,
+
69.16. MS: M at m/z 415. Anal. Calcd. For C22
H14BrN
3
O: C, 63.48; H, 3.39; N, 10.09. Found:
C, 63.56; H, 3.42; N, 9.89.
4
.5.30. 3-Naphth-1-yl-5-bromo-9-acetyl-pyridazino[3,4-b]indole (6c) Creamish white solid;
◦
1
3
yield 70%; Mp 288-289 C; H NMR (300 MHz, CDCl ): δ = 8.42 (s, 1H), 8.03-8.18 (m, 5H),
1
3
7
0
1
.78-7.92 (m, 2H), 7.56-7.69 (m, 3H), 1.96 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS =
6,
): 20.67, 117.27, 122.86, 124.24, 124.69, 125.34, 125.47, 126.82, 127.75, 128.66, 130.38,
+
30.84, 131.25, 132.36, 132.84, 133.89, 137.52, 141.87, 146.53, 159.44, 165.31, 169.79. MS: M
at m/z 415. Anal. Calcd. For C H BrN O: C, 63.48; H, 3.39; N, 10.09. Found: C, 63.65; H,
2
2
14
3
3
.48; N, 9.81.
4
8
8
2
1
1
.5.31. 3-(4-Chlorophenyl)-5-bromo-9-acetyl-pyridazino[3,4-b]indole (7c) White solid; yield
◦
1
5%; Mp 282-283 C; H NMR (300 MHz, DMSO-d ): δ = 8.64 (d, J = 2.4 Hz, 1H), 8.42 (d, J =
6
.7 Hz, 2H), 8.24 (s, 1H), 8.08 (d, J = 9.00 Hz, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.64 (d, J = 8.4 Hz,
1
3
H), 2.00 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 21.02, 116.87, 118.98, 123.76,
6
,
25.68, 128.42, 129.82, 130.64, 131.45, 135.78, 137.21, 141.86, 147.12, 155.33, 165.21, 168.11,
+
69.22. MS: M at m/z 399. Anal. Calcd. For C18
H
11ClBrN
3
O: C, 53.96; H, 2.77; N, 10.49.
Found: C, 53.77; H, 2.94; N, 10.58.
4
.5.32. 3-(4-Bromophenyl)-5-bromo-9-acetyl-pyridazino[3,4-b]indole (8c) White coloured
◦
1
powder; yield 89%; Mp 285-286 C; H NMR (300 MHZ, DMSO-d
m, 3H), 8.06-8.09 (m, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 1.85 (s, 3H). C
NMR (DMSO-d6, 125 MHz, δ, TMS = 0): 19.99, 116.77, 118.23, 123.81, 124.62, 125.97,
6
): δ = 8.36 (s, 1H), 8.15-8.18
1
3
(
1
27.63, 128.72, 129.84, 131.42, 131.97, 137.78, 141.94, 147.27, 155.55, 165.76, 167.12, 168.22.
+
MS: M at m/z 443. Anal. Calcd. For C H Br N O: C, 48.57; H, 2.49; N, 9.44. Found: C,
1
8
11
2
3
4
8.76; H, 2.59; N, 9.22.
17

ACCEPTED MANUSCRIPT
4
.5.33. 3-(4-Nitrophenyl)-5-bromo-9-acetyl-pyridazino[3,4-b]indole (9c) Yellowish powder;
◦
1
yield 85%; Mp 268-269 C; H NMR (300 MHz, DMSO-d ); δ = 8.62-8.68 (d, J = 8.4 Hz, 2H),
6
1
3
8
1
1
.42-8.48 (m, 2H), 8.32 (m, 2H), 8.15 (d, J = 8.4 Hz, 2H), 2.03 (s, 3H). C NMR (DMSO-d
6,
25 MHz, δ, TMS = 0): 21.01, 117.72, 119.48, 123.43, 124.79, 125.76, 128.17, 129.51, 130.43,
+
31.82, 142.57, 144.85, 148.71, 148.96, 153.59, 165.69, 168.79, 169.03. MS: M at m/z 410.
Anal. Calcd. For C18
H11BrN
4
O
3
: C, 52.57; H, 2.70; N, 13.62. Found: C, 52.81; H, 2.89; N, 13.39.
4
.5.34. 3-Phenyl-5-iodo-9-acetyl-pyridazino[3,4-b]indole (1d) Pale yellow fluffy powder; yield
◦
1
9
0%; Mp 264-265 C; H NMR (300 MHz, DMSO-d ): δ = 8.29 (d, J = 8.4 Hz, 1H), 8.00-8.10
6
1
3
(
m, 3H), 7.76-7.89 (m, 3H), 7.59 (m, 1H), 7.23 (m, 1H), 2.05 (s, 3H). C NMR (DMSO-d6, 125
MHz, δ, TMS = 0): 20.78, 93.33, 118.33, 125.50, 127.58, 128.99, 130.12, 131.59, 134.43,
+
1
38.37, 138.70, 139.40, 147.44, 156.81, 165.59, 168.46. MS: M at m/z 413. Anal. Calcd. For
C
18
H
12IN
3
O: C, 52.32; H, 2.93; N, 10.17. Found: C, 52.43; H, 3.03; N, 10.02.
4
.5.35. 3-(3,4,5-Trimethoxyphenyl)-5-iodo-9-acetyl-pyridazino[3,4-b]indole (2d) Pale yellow
◦
1
fluffy powder; yield 90%; Mp > 300 C; H NMR (300 MHz, DMSO-d ): δ = 8.48 (s, 1H), 8.26-
6
1
3
8
.38 (m, 2H), 7.84 (d, J = 9.3 Hz, 1H), 7.57 (s, 2H), 3.93 (s, 6H), 3.72 (s, 3H), 2.04 (s, 3H). C
NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.26, 55.84, 59.28, 93.47, 109.12, 119.41, 124.42,
6
,
+
1
31.16, 131.83, 133.25, 139.91, 146.78, 153.65, 155.81, 165.93, 169.39. MS: M at m/z 503.
Anal. Calcd. For C H IN O : C, 50.12; H, 3.60; N, 8.35. Found: C, 49.98; H, 3.78; N, 8.44.
2
1
18
3
4
4
.5.36. 3-(3,4-Dimethoxyphenyl)-5-iodo-9-acetyl-pyridazino[3,4-b]indole (3d) Light yellow
◦
1
solid; yield 75%; Mp 288-289 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.74 (s, 1H), 8.15 (s, 1H),
8
2
1
1
8
.07 (d, J = 9.00 Hz, 1H), 7.87-7.90 (m, 3H), 7.14 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 3.85 (s, 3H),
1
3
.04 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.21, 55.83, 93.78, 110.42, 114.56,
6
,
18.48, 121.20, 123.73, 125.69, 127.43, 129.10, 130.97, 131.44, 141.64, 147.67, 149.85, 151.13,
+
55.45, 165.63, 169.28. MS: M at m/z 473. Anal. Calcd. For C H IN O : C, 50.76; H, 3.41;,
2
0
16
3
3
.88. Found: C, 50.67; H, 3.55; N, 8.99.
4
.5.37. 3-(4-Methoxyphenyl)-5-iodo-9-acetyl-pyridazino[3,4-b]indole (4d) Cream colored solid;
◦
1
yield 78%; Mp 281-282 C; H NMR (300 MHz, DMSO-d ): δ = 8.58 (s, 1H), 8.35-8.38 (m, 4H),
6
1
3
8
.25 (s, 1H), 7.12 (d, J = 7.8 Hz, 2H), 3.85 (s, 3H), 1.99 (s, 3H). C NMR (DMSO-d6, 125 MHz,
18

ACCEPTED MANUSCRIPT
δ, TMS = 0): 21.03, 56.76, 93.38, 115.85, 118.23, 123.67, 125.83, 127.21, 128.56, 129.48,
+
1
4
9
29.89, 130.46, 130.88, 141.93, 147.31, 155.57, 161.33, 165.80, 167.66, 169.82. MS: M at m/z
43. Anal. Calcd. For C H IN O : C, 51.49; H, 3.18; N, 9.48. Found: C, 51.66; H, 3.04; N,
1
9
14
3
2
.58.
4
7
7
2
1
.5.38. 3-Naphth-2-yl-5-iodo-9-acetyl-pyridazino[3,4-b]indole (5d) Creamish white solid; yield
◦
1
0%; Mp > 300 C; H NMR (300 MHz, DMSO-d ): δ = 8.67 (s, 1H), 8.01-8.12 (m, 5H), 7.74-
6
1
3
.75 (m, 2H), 7.41-7.64 (m, 3H), 2.01 (s, 3H). C NMR (DMSO-d6, 125 MHz, δ, TMS = 0):
0.68, 93.12, 118.18, 123.21, 125.46, 125.97, 127.93, 127.36, 127.57, 127.86, 128.51, 129.92,
+
29.66, 130.53, 130.99, 133.59, 134.39, 135.41, 141.97, 147.31, 155.75, 165.53, 169.70. MS: M
at m/z 463. Anal. Calcd. For C H IN O: C, 57.04; H, 3.05; N, 9.07. Found: C, 56.77; H, 2.83;
2
2
14
3
N, 9.41.
4
7
7
2
1
.5.39. 3-Naphth-1-yl-5-iodo-9-acetyl-pyridazino[3,4-b]indole (6d) Creamish white solid; yield
◦
1
0%; Mp 295-296 C; H NMR (300 MHz, CDCl ): δ = 8.53 (s, 1H), 8.06-8.49 (m, 4H), 7.82-
3
1
3
.99 (m, 2H), 7.43-7.82 (4H, m), 2.01 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0):
6
,
1.03, 93.83, 122.14, 124.43, 124.79, 125.66, 125.95, 126.24, 127.93, 128.31, 128.85, 130.42,
+
30.65, 131.26, 132.74, 132.98, 133.96, 137.34, 141.11, 147.86, 161.22, 165.64, 169.31. MS: M
at m/z 463. Anal. Calcd. For C22
N, 8.79.
3
H14IN O: C, 57.04; H, 3.05; N, 9.07. Found: C, 57.32; H, 3.31;
4
8
2
1
1
.5.40. 3-(4-Chlorophenyl)-5-iodo-9-acetyl-pyridazino[3,4-b]indole (7d) White solid; yield
◦
1
5%; Mp 287-288 C; H NMR (300 MHz, DMSO-d
6
): δ = 8.59 (s, 1H), 8.32 (d, J = 6.9 Hz,
H), 8.22 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 7.62 (d, J = 6.9 Hz, 2H),
1
3
.99 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.88, 93.78, 118.91, 123.46, 125.83,
6
,
28.92, 129.05, 129.67, 131.75, 135.86, 137.23, 141.42, 147.34, 155.66, 165.76, 167.41, 169.19.
+
MS: M at m/z 447. Anal. Calcd. For C H ClIN O: C, 48.29; H, 2.48; N, 9.39. Found: C,
1
8
11
3
4
8.38; H, 2.37; N, 9.39.
4
.5.41. 3-(4-Bromophenyl)-5-iodo-9-acetyl-pyridazino[3,4-b]indole (8d) White coloured
◦
1
powder; yield 89%; Mp > 300 C; H NMR (300 MHZ, DMSO-d ): δ = 8.78 (s, 1H), 8.26 (d, J =
6
8
.1 Hz, 2H), 8.19 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 9.3 Hz, 1H), 7.78 (d, J = 8.0 Hz,
19

ACCEPTED MANUSCRIPT
1
3
2
H), 2.00 (s, 3H). C NMR (DMSO-d6, 125 MHz, δ, TMS = 0): 20.83, 93.47, 118.35, 123.46,
1
23.76, 125.48, 127.64, 129.30, 129.72, 130.32, 131.44, 137.94, 141.15, 147.77, 155.11, 165.17,
+
1
67.49, 169.85. MS: M at m/z 491. Anal. Calcd. For C H BrIN O: C, 43.93; H, 2.25; N, 8.54.
1
8
11
3
Found: C, 44.08; H, 2.43; N, 8.67.
4
8
8
9
1
1
1
.5.42. 3-(4-Nitrophenyl)-5-iodo-9-acetyl-pyridazino[3,4-b]indole (9d) Yellowish powder; yield
◦
1
5%; Mp 283-284 C; H NMR (300 MHz, DMSO-d ); δ = 8.82 (d, J = 1.8 Hz, 1H), 8.57 (d, J =
6
.7 Hz, 2H), 8.40 (d, J = 8.7 Hz, 2H), 8.31 (s, 1H), 8.15 (dd, J = 1.8 and 9.0 Hz, 1H), 7.95 (d, J =
1
3
.0 Hz, 1H), 2.08 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0): 20.21, 93.66, 118.22,
6
,
24.32, 124.86, 125.69, 128.21, 129.92, 130.82, 131.13, 142.88, 144.22, 147.23, 149.61, 155.12,
+
65.60, 167.91, 169.90. MS: M at m/z 458. Anal. Calcd. For C H IN O : C, 47.18; H, 2.42; N,
1
8
11
4
3
2.23. Found: C, 47.28; H, 2.49; N, 12.02.
4
.5.43. 3-Phenyl-5,7-dibromo-9-acetyl-pyridazino[3,4-b]indole (1e) Pale yellow fluffy powder;
◦
1
yield 90%; Mp 284-285 C; H NMR (300 MHz, DMSO-d ): δ = 8.61 (d, J = 1.5 Hz, 1H), 8.39
6
1
3
(
d, J = 7.5 Hz, 2H), 8.34 (d, J = 1.8 Hz, 1H), 8.31 (s, 1H), 7.58 (bs, 3H), 2.03 (s, 3H). C NMR
(DMSO-d6, 125 MHz, δ, TMS = 0): 21.01, 115.96, 117.23, 118.87, 120.13, 127.71, 128.89,
+
1
29.47, 129.96, 130.48, 138.91, 141.82, 147.92, 156.71, 165.76, 169.74. MS: M at m/z 443.
Anal. Calcd. For C18
H11Br
2
N
3
O: C, 48.57; H, 2.49; N, 9.44. Found: C, 48.39; H, 2.62; N, 9.28.
4
.5.44. 3-Naphth-2-yl-5,7-dibromo-9-acetyl-pyridazino[3,4-b]indole (2e) Creamish white solid;
◦
1
yield 70%; Mp > 300 C; H NMR (300 MHz, DMSO-d ): δ = 8.96 (s, 1H), 8.58-8.65 (m, 2H),
6
1
3
8
.50 (s, 1H), 8.43 (d, J = 2.1 Hz, 1H), 7.99-8.14 (m, 3H), 7.63 (m, 2H), 2.01 (s, 3H). C NMR
DMSO-d 125 MHz, δ, TMS = 0): 20.88, 115.68, 117.56, 119.63, 125.37, 127.83, 127.57,
(
6
,
1
1
27.78, 127.94, 128.18, 129.81, 129.99, 130.13, 130.79, 133.15, 134.91, 135.90, 141.97, 147.48,
+
56.64, 165.68, 169.25. MS: M at m/z 493. Anal. Calcd. For C22
H13Br
2
N
3
O: C, 53.36; H, 2.65;
N, 8.49. Found: C, 53.36; H, 2.65; N, 8.49.
4
.5.45. 3-Naphth-1-yl-5,7-dibromo-9-acetyl-pyridazino[3,4-b]indole (3e) Creamish white solid;
◦
1
yield 70%; Mp > 300 C; H NMR (300 MHz, CDCl
.49 (m, 2H), 8.11 (d, J = 7.8 Hz, 2H), 7.99 (d, J = 7.2 Hz, 1H), 7.61 (m, 2H), 2.00 (s, 3H).
NMR (DMSO-d6, 125 MHz, δ, TMS = 0): 19.99, 115.44, 117.13, 119.74, 124.89, 125.63,
3
): δ = 8.96 (s, 1H), 8.58-8.64 (m, 2H), 8.43-
1
3
8
C
20

ACCEPTED MANUSCRIPT
1
26.81, 127.41, 128.78, 128.31, 130.86, 130.98, 131.39, 132.40, 132.48, 133.79, 137.67, 141.85,
+
1
47.16, 156.61, 165.59, 169.96. MS: M at m/z 493. Anal. Calcd. For C H Br N O: C, 53.36;
2
2
13
2
3
H, 2.65; N, 8.49. Found: C, 53.36; H, 2.65; N, 8.49.
4
.5.46. 3-(4-Fluorophenyl)-5,7-dibromo-9-acetyl-pyridazino[3,4-b]indole (4e) Yellow powder;
◦
1
yield 92% ; Mp 298-299 C; H NMR (300 MHz, DMSO-d ); δ = 8.62 (d, J = 1.8 Hz, 1H), 8.31-
6
1
3
8
2
1
.48 (m, 4H), 7.40-7.46 (m, 2H), 2.02 (s, 3H). C NMR (DMSO-d 125 MHz, δ, TMS = 0):
6,
0.92, 116.47, 116.64, 118.72, 120.52, 125.89, 126.51, 128.10, 130.34, 130.41, 134.17, 136.58,
+
41.81, 143.71, 156.09, 163.32, 165.32, 169.16. MS: M at m/z 461. Anal. Calcd. For
C
18
H
10Br
2
3
FN O: C, 46.68; H, 2.18; N, 9.07. Found: C, 46.68; H, 2.18; N, 9.07.
4
.5.47. 3-(4-Bromophenyl)-5,7-dibromo-9-acetyl-pyridazino[3,4-b]indole (5e) White coloured
◦
1
powder; yield 89%; Mp > 300 C; H NMR (300 MHZ, DMSO-d ): δ = 8.64 (d, J = 1.5 Hz, 1H),
6
13
8
.43 (s, 1H), 8.34-8.36 (s, 3H), 7.81 (d, J = 8.4 Hz, 2H), 2.00 (s, 3H). C NMR (DMSO-d6, 125
MHz, δ, TMS = 0): 20.85, 115.79, 119.71, 123.93, 126.58, 127.39, 129.82, 129.44, 131.04,
+
1
31.92, 136.21, 142.45, 148.28, 155.66, 164.99, 169.15. MS: M at m/z 521. Anal. Calcd. For
C
18
H
10Br
3
N
3
O: C, 41.26; H, 1.92; N, 8.02. Found: C, 41.26; H, 1.92; N, 8.02.
4
.6. In-vitro cytotoxic assay
In vitro cytotoxicity against four human cancer cell lines was determined using 96-well tissue
culture plate. The cells were allowed to grow in carbon dioxide incubator (37°C) for 24 h. Test
materials in complete growth medium (100ꢀl) were added after 24 h of incubation to the wells
containing cell suspension. The plates were further incubated for 48 h in a carbon dioxide
incubator. The cell growth was stopped by gentle layering trichloroacetic acid (50%, 50 ꢀl) on
top of the medium in all the wells. The plates were incubated at 4°C for 1 h to fix the cells
attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and
discarded. The plates were washed five times with distilled water to remove trichloroacetic acid,
growth medium low-molecular weight metabolites, serum on a mechanical stirrer. The optical
density (OD) was recorded on ELISA reader at 540 nm. The cell growth was determined by
subtracting the mean OD value of respective blank from the mean OD value of the experimental
set. Percent growth in presence of test material was calculated considering the growth in the
21

ACCEPTED MANUSCRIPT
absence of any test material as 100%, and in turn, percent growth inhibition in presence of test
material was calculated proteins, etc. and air-dried. The plates were stained with sulforhodamine
B dye (0.4% in 1% acetic acid, 100 ꢀl) for 30 min. The plates were washed five times with 1%
trichloroacetic acid and then air-dried . The adsorbed dye was dissolved in Tris–HCl buffer
(100ꢀL, 0.01 M, pH 10.4), and the plates were gently stirred for 10 min [25].
4
.7. In-vitro tubulin polymerization assay
A Cytoskeleton tubulin polymerization assay kit was purchased from Labex India. For assessing
the tubulin polymerization inhibition potential, 100µM stock solution of each compound was
made in molecular biology grade DMSO. Further dilutions (50 µM, 25 µM, 10 µM) of each
compound were made by diluting the stock solution. General tubulin buffer was prepared by
reconstituting the constituents (available in kit) in 10 mL distilled water. General Tubulin Buffer
(1 mL) was supplemented with 10 µL of 100mM GTP solution and 10 mg Tubulin was
suspended in it and mixture was stored at 4˚C until used. Before starting the experiment 96 well
plate and Elisa plate reader were warmed at 37˚C as this temperature is required to achieve the
proper polymerization of tubulin. Tubulin polymerization buffer was prepared by mixing
General tubulin buffer (750 µL), Tubulin glycerol buffer (250 µL) and 100mM GTP (10 µL). 10
µL General tubulin buffer was added in the wells, where first two wells were kept as control
(without any test compound), and different concentrations of compounds were subsequently
added in previously marked wells and plate was kept in plate reader for 2 minutes at 37˚C.
Meanwhile tubulin suspension was diluted with Tubulin polymerization buffer and 100 µL of
diluted tubulin was added in each well. Plate was again kept in plate reader at 37˚C for 1 hr. and
absorbance was measured at 340 nm (Lee and Timasheff, 1977). Percentage inhibition and IC50
values were calculated according to the absorbance values obtained after 1 hr. [26, 27]
4
.8 Molecular modelling study
The 3D coordinates of tubulin was obtained from protein data bank (PDB entry: 1sa0) [29]. The
structure of 2a was drawn in ChemDraw and subjected to energy minimization in the MOPAC
module, using the AM1 procedure for closed shell systems, implemented in the CS Chem3D
Ultra [31]. The ligands were docked at the colchicine-binding site of tubulin using the GOLD
5
.0.1. [28]. Gold performs genetic algorithm based ligand docking to optimize the conformation
22

ACCEPTED MANUSCRIPT
of ligand at the receptor binding site. It utilizes Gold Score fitness function to evaluate the
various conformations of ligand at the binding site and comprises of four components: protein–
ligand hydrogen bond energy, protein–ligand van der Waals (vdw) energy, ligand internal vdw
energy, and ligand torsional strain energy [28]. 2a was docked 10 times and each pose was
ranked according to its GoldScore fitness function. The conformation with highest score was
selected for discussion.
Acknowledgement
The authors are grateful to the DST for providing the financial support under DST Purse scheme
necessary for the present study.
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Captions
Fig. 1 Design strategy
Fig. 2 Retrosynthetic route
2 3
Scheme 1: a) 5% NaOH, ethanol, stirring, rt, 2 hr b) a drop of H SO4, CH COOH, reflux, 1 hr; c)
NH NH .H O, methanol, reflux, 1 hr d) (CH CO) O, DMAP, stirring, 2 hr e) NH .NH .H O,
2
2
2
3
2
2
2
2
grinding, 15 min.
Fig. 3 Proton resonances of 1a
Fig. 4 Structure activity relationship
Fig. 5 Effect of compound on the microtubule network of THP-1 cells untreated (control), 1µM
Paclitaxel and 5 µM 2a for 48 h. Microtubules and unassembled tubulin are shown in green.
DNA, stained with 4,6-diamidino-2-phenylindole (DAPI) shown in blue.
Fig. 6 a) Importantbinding site residues around 2a (pink: polar; blue:hydrophobic); b) binding
conformation of 2a (green) in tubulin
Table 1 represents the structures and % age inhibition of the designed analogues
Table 2 represents IC values of selected analogues
5
0
Table 3 represents tubulin polymerization inhibitory activity of selected compounds
27