Synthesis and anti-proliferative activity studies of 2-(2-(trifluoromethyl)-6-(substituted)imidazo[1,2-b]pyridazin-3-yl)-N-(substituted)acetamide derivatives

Article abstract of DOI:10.1002/jhet.3920

A series of novel imidazo[1,2-b]pyridazin-3-yl acetamide derivatives (9a-9j) were synthesized from a 3,6-dichloropyridazine. We have developed a simple strategy for the synthesis of functionally diverse imidazole, and pyridiazine derivatives were reported via a series of steps. The work involves bicyclic imidazo-pyridazine ring formation, halogenation, cynation, hydrolysis, peptide coupling, and Buchwald reaction. The structure of the synthesized compounds was confirmed by IR, ¹H NMR, ¹³C NMR,¹⁹F NMR, mass spectra, and elemental analysis, and purity is checked by HPLC. All synthesized compounds were screened for anticancer activity against A-549 and Du-145 cancer cell lines by MTT assay. The preliminary bioassay suggests that most of the compounds show anti-proliferation with different degrees; doxorubicin was used as positive control. The synthesized compound shows IC₅₀ values in the range of 1.74μM to 16.17μM in both cell lines. The compounds 9e, 9g, and 9h were active compared with doxorubicin in both the cell lines. The compounds having cyclopentyl ring are active compared with higher and lower carbon analogues.

Full text of DOI:10.1002/jhet.3920

Received: 27 August 2019
DOI: 10.1002/jhet.3920
Revised: 27 December 2019
Accepted: 22 January 2020
A R T I C L E
Synthesis and anti-proliferative activity studies
of 2-(2-(trifluoromethyl)-6-(substituted)imidazo[1,2-b]
pyridazin-3-yl)-N-(substituted)acetamide derivatives
Dattatraya D. Gaikwad1 | Umakant D. Pawar2 | Sadhana L. Chavan1
|
Chandrakant D. Pawar3 | Dattatraya N. Pansare1
Santosh L. Chavan4 | Ashok M. Zine5
| Rohini N. Shelke1
|
1Department of Chemistry, Deogiri
Abstract
College, Aurangabad, Maharashtra, India
2Department of Chemistry, Regional
Forensic Science Laboratories,
Aurangabad, Maharashtra, India
3Department of Chemical Technology,
Dr. Babasahaeb Ambedkar Marathwada
University, Aurangabad, Maharashtra,
India
4Department of Chemistry, Maharashtra
Pollution Control Board, Aurangabad,
Maharashtra, India
5Department of Chemistry, Vinayakrao
Patil College, Vaijapur, Maharashtra,
India
A series of novel imidazo[1,2-b]pyridazin-3-yl acetamide derivatives (9a-9j)
were synthesized from a 3,6-dichloropyridazine. We have developed a simple
strategy for the synthesis of functionally diverse imidazole, and pyridiazine
derivatives were reported via a series of steps. The work involves bicyclic
imidazo-pyridazine ring formation, halogenation, cynation, hydrolysis, peptide
coupling, and Buchwald reaction. The structure of the synthesized compounds
was confirmed by IR, 1H NMR, 13C NMR,19F NMR, mass spectra, and elemen-
tal analysis, and purity is checked by HPLC. All synthesized compounds were
screened for anticancer activity against A-549 and Du-145 cancer cell lines by
MTT assay. The preliminary bioassay suggests that most of the compounds
show anti-proliferation with different degrees; doxorubicin was used as posi-
tive control. The synthesized compound shows IC50 values in the range of
1.74μM to 16.17μM in both cell lines. The compounds 9e, 9g, and 9h were
active compared with doxorubicin in both the cell lines. The compounds hav-
ing cyclopentyl ring are active compared with higher and lower carbon
analogues.
Correspondence
Dattatraya N. Pansare, Department of
Chemistry, Deogiri College, Aurangabad
431005, Maharashtra, India.
Email: dnpansare74@gmail.com
1 | INTRODUCTION
new chemotherapeutic targets with selective action has
to be identified, as many classes of hetterocycle scaffolds
Cancer is considered as one of the major causes of
human health concerns with increasing number of
patients with the time all over the world. Although
many types of chemotherapeutic drugs were used for
the treatment, although still, there is a challenge to
identify safe and effective drug for the cancers. Drug
resistance occurred during treatment is a major concern
of present time. The design and development of new
anti-proliferative agents with increased efficiency, less
side effective, cost effective, and time concern for the
treatment were the major challenges for present
researchers. Considering these facts, the development of
were used for the different types of cancers. For normal
functioning of cells in the human kinome, there are
518 kinases that are involved in different phases of life
and all are associated with each other.[1] Different
kinases were responsible for different functioning of
cells; some kinases are TOR signaling, which are
responsible for cell growth,[2,3] and some are protein
tyrosine kinase inhibitors.[4] The imbalance in the
kinases occurs in several diseases like cancer, neurode-
generative disorders, and inflammation. By considering
the importance of kinases, we need to develop new
kinase inhibitors with diversified activity. In present
J Heterocyclic Chem. 2020;1–11.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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GAIKWAD ET AL.
work, we have chosen substituted imidazole and
pyridazine nuclei and its derivatives for cell line and
kinases study. Several heterocycles were intensively
studied to discover new anticancer agents. Imidazole
scaffolds were identified as one of the important classes
of heterocyclic compounds because of their significant
and diversified pharmacological and biological proper-
ties. Imidazole derivatives acts as anti-proliferative,[5]
TGR5 receptor agonists,[6] calcitonin gene-related pep-
tide (CGRP) antagonists,[7] and antimicrobial.[8] The
imidazole scaffold plays an important role in drug dis-
covery. The imidazole scaffold shows varied biological
activities such as antifungal,[9] antibacterial,[10] anti-
inflammatory, analgestic,[11] antitubercular,[12] antide-
pressant, antiviral,[13] anticancer,[14] anti-lishmanial.[15]
The substituted imidazole shows multiple biological
activities.[16–18] Recent literature review reveals that
reactions involving hero atoms such as pyridazine were
very advantageous in design of anti-proliferative agents
as they resemble many biomolecules. Piridazine and
derivatives mainly act as potent and selective factor Xia
inhibitors,[19] inhibitors of TNF-α production,[20] potent
PDE10a inhibitors,[21] CNS penetrant pan-muscarinic
antagonists,[22] Janus kinase (JAK) inhibitors.[23] Piridazine
in combination of other groups shows diversified activity
like FXIa inhibitors.[24–26]
The imidazo[1,2-b]pyridazine was identified as one of
the promising building blocks in drug discovery as it
shows diversified biological activities like TNF-α
inhibitors,[27] treatment of rheumatoid arthritis, treat-
ment of dermal allergic inflammation,[28] and treatment
of inflammatory diseases caused by dysfunctions of
kinases. Several derivatives of imidazo-pyridazine series
act as inhibitors of Plasmodium falciparum calcium-
dependent protein kinase inhibitors,[29] some as protein
kinase inhibitors.[30] By considering the diversified bio-
logical activities of imidazole and pipridazine, we have
tried to incorporate both scaffolds in one framework as
imidazo[1,2-b]pyridazin, and in continuation of our
research,[31–35] for the search of anti-proliferateve target
and kinase inhibitors, we have synthesized a series of
compounds having imidazo[1,2-b]pyridazin nuclei.
2 | RESULTS AND DISCUSSION
In present work, we have done synthesis of
2-(2-(trifluoromethyl)-6-(substituted)imidazo[1,2-b]
pyridazin-3-yl)-N-(substituted)acetamide (9a-9j) by
using 3,6-dichloropyridazine (1) in below Scheme 1. We
have optimized the synthesis of compound 9a-9j in step-
wise manner to get good yield, neat reaction profile, and
FIGURE 1 General structure of biologically active molecules having imidazo[1,2-b]pyridazin nuclei: Figure 1 compromising the
biological active molecules reported with imidazo-pyridazine nuclei in combination. Ponatinib is an oral drug used for the treatment of
chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia; in short, it is used as multitargeted tyrosine
kinase inhibitor. Neflamapimod is used for treatment of Alzheimer and other neurodegenerative disorders. Copanlisib: drug used for the
treatment of follicular lymphoma. Pim-1 inhibitor: imidazo[1,2-b]pyridazine 1 inhibitor; it acts as inhibitor for PIM-1 dependent cancer
types. IKKβ inhibitor: It is known for inhibitor of nuclear factor kappa-B kinases. SYK inhibitors: These are known as spleen tyrosine kinase
inhibitors as these are used for the treatment of autoimmune diseases as asthma and rheumatoid arthritis. PfCDK1 inhibitors: These are
known as Plasmodium falciparum calcium-dependent protein kinase

GAIKWAD ET AL.
3
SCHEME 1 2-(2-(Trifluoromethyl)-6-(substituted)imidazo[1,2-b]pyridazin-3-yl)-N-(substituted)acetamide (9a-9j): Reagents and
conditions: (a): liq. Ammonia, −10ꢀC to room temperature, 150ꢀC, 8 hours; (b): 3-bromo-1,1,1-trifluorobutan-2-one, DME, 150ꢀC, 4 hours;
(c): NBS, AIBN, CAN, 100ꢀC for 1 hour; (d) NaCN, DMF, H2O, room temperature, 12 hours; (e) NaOH, 100ꢀC, 2 hours; (f) 3-flouroaniline,
EDCI, DIPEA, DCM, room temperature, 12 hours; (g) pyrrolidin-2-one, Cu(OAc)2, K3PO4, 1,2-dimethylethylenediamine, DMF, 150ꢀC,
12 hours
less harsh condition by keeping these things in mind, and
the optimized steps are depicted in below Scheme 1.
obtain yield >90%. We have used heating condition for
this bromination as to decrease the time of reaction. At
room temperature for getting good yields, we have to run
the reaction for 12 hours. The new non polar spot was
1
seen on TLC, afterisolation the H NMR shows singlet of
Sr. No.
8a = 9a
8b = 9b
8c = 9c
8d = 9d
8e = 9e
R1
Sr. No.
8f = 9f
8g = 9g
8h = 9h
8i = 9i
8j = 9j
R1
methylene peak for 2 protons confirmingformation of
compound 3.
In step d, we have done cynation of comp-4 by using
sodium cyanide in DMF: H2O. The optimization reaction
for step d was done by using different solvents obtaining
better yields.
methylamine
ethylamine
propylamine
butylamine
pyrrolidine
piperidine
4-methylpiperidine
pyrrolidin-2-one
piperidin-2-one
1-methylpiperazine
We have done reaction by using MeOH, toluene
and DMF to get 30%, 34% and 50% yields respectively.
Further we have used DMF assolvent along with com-
bination of water for increasing the yield of compound
5. We have used 1:1 (DMF-H2O) mixture; we got 55%
yield; further, we have decreased the water content to
(2:1), (3:1), (4:1), and (5:1); in all these combinations,
we got yield of 80% in (4:1) combination condition. The
yields in higher combinations and lower combinations
of H2O ware less as compared with 4 DMF and 1 H2O
combination. We used the same condition for scale up
of compound 5.
The compound 5 was treated with aq. NaOH solu-
tion at room temperature for 16 h there is 50% forma-
tion of compound 6. We have heated compound
5 along with NaOH at 100ꢀC for 1 hour; there is 60%
formation of product, and after 2 hours, there is >90%
formation of compound 6; we used the same condition
for scale up of compound 6.
In step a, we have done the synthesis of
6-chloropyridazine-3-amine [cas no. 5469-69-2; 2] from
commercially available 3,6-dichloropyridazine (1). We
have used liquid ammonia for the reaction; when the
reaction was performed in sealed tube, the starting mate-
rial was not consuming as product formation was very
less as there is possibility of ammonia leakage at higher
temperature. There are also strong chances of sealed tube
breakage at higher temperature. We have used steel
bomb vessel for performing this reaction, and it worked
well getting good yields of product. The compound 2 was
characterized by melting point as it matches with
reported literature.
In step b, the compound 2 reacted with 3-bromo-1,1,
1-trifluorobutan-2-one in DME at higher temperature in
sealed tube, as this cyclization required high tempera-
ture. The compound 3 was confirmed from 13C NMR, as
two aliphatic carbons are vanished, and only one signal
was there for aromatic ring attached methyl group, also
observed different spot on TLC.
In step f, we have used peptide coupling condition for
the reaction of compound 6 and 3-flouro aniline to obtain
88% of compound 7. [36,37]
The compound 3 on bromination by using crystalized
NBS in acetonitrile and AIBN was added as catalyst to
In step g, we have used Buchwald coupling reaction
condition to obtain different compounds as 9a-9j with

4
GAIKWAD ET AL.
TABLE 1 Screening of catalyst,
ligand, base, and solvent for synthesis of
Entry
1
Catalyst
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
Ligand
Base
Solvent
Dioxane
DMF
Temp (ꢀC)
110
Yielda (%)
i
Cs2CO3
K3PO4
KOtBu
Cs2CO3
K2CO3
K3PO4
Cs2CO3
K3PO4
NaOtBu
Cs2CO3
Cs2CO3
K3PO4
KOtBu
Cs2CO3
K3PO4
Cs2CO3
K3PO4
K3PO4
K2CO3
20
32
18
24
20
75
45
36
30
40
20
15
-
compound (9h)
2
i
150
3
i
Toluene
Dioxane
Toluene
DMF
110
4
ii
ii
ii
iii
iii
iii
iv
i
110
5
110
6
150
7
Dioxane
Dioxane
Toluene
DMF
110
8
110
9
110
10
11
12
13
14
15
16
17
18
19
150
Dioxane
Toluene
Toluene
Dioxane
Dioxane
Dioxane
Dioxane
DMF
110
i
110
i
110
ii
ii
iii
iii
iv
iv
110
20
30
30
28
40
25
110
110
110
150
DMF
150
aIsolated yield, Comp-7 (1 eq.), pyrrolidin-2-one (8h), (1.5 eq.), catalyst (10 mol%), ligand (15 mol%), base
(2 eq.), solvent (10 Vol) time 12 hours.
SCHEME 2
2-(2-(Trifluoromethyl)-
6-(2-oxopyrrolidin-1-yl)imidazo[1,2-b]
pyridazin-3-yl)-N-(3-fluorophenyl)
acetamide (9h)
>70% yields.[38] We have optimized Buchwald coupling
reaction by modifying catalyst, ligand, base, solvent, and
temperature to get suitable condition.
By using Cu(OAc)2 as catalysts, we have used four
ligands, different bases, and different solvents as in
entries 1 to 10; the yields are in the range of 18% to 75%.
We got 75% yield by using DMEDA as ligand K3PO4 as
base and DMF as solvent in entry 6; we have heated the
reaction at high temperature 150ꢀC remaining reactions
we have done at 110ꢀC. By using CS2CO3 as base, we got
yields of 30%, 24%, 45%, and 30% in dioxane and DMF as
solvents in entries 1, 4, 7, and 10. By using K3PO4 in dif-
ferent solvents like toluene and dioxane, we got <40%
yields in entries 2 and 8. For toluene and dioxane, we
have done all the reactions at 110ꢀC, and for DMF, we
have used 150ꢀC temperature. The reaction at higher
temperature works well. In entry 10, we have used
xanthophos as ligand CS2CO3 as base DMF as solvent,
and we heated the reaction at 150 C to obtain 40% of
desired product. In entries 11 to 19, we have used
For optimization purpose, we have taken pyrrolidin-
2-one as model amine reacted with comp-7; we have used
catalyst Cu(OAc)2 and Pd(OAc)2, ligands as BINAP (i),
1,2-Dimethylethylenediamine (DMEDA) (ii), trans-N,N0-
dimethylcyclohexane-1,2-diamine (DMCDA) (iii), and
Xanthophos (iv). We have used bases like CS2CO3,
K2CO3, K3PO4, NaOtBu, and KOtBu and solvents like
dioxane, toluene, and DMF. For all the optimization reac-
tions, we have used catalyst 10 mol%, ligands 15 mol%, base
2 equivalents, and solvents 10 vol for all the reactions. The
results of the optimization reaction were given in Table 1.
Synthesis of 2-(2-(trifluoromethyl)-6-(2-oxopyrrolidin-1-yl)
imidazo[1,2-b]pyridazin-3-yl)-N-(3-fluorophenyl)acetamide
(9h) shown in Scheme 2.

GAIKWAD ET AL.
5
Pd(OAc)2 as catalyst, BINAP, DMEDA, DMCDA,
xanthophos as ligands, CS2CO3, K3PO4, K2CO3, KOtBu, and
NaOtBu as bases. We have used dioxane, toluene, and DMF
as solvents; the reaction temperature and isolated yields of
the product were given in Table 1. From all those optimiza-
tions, it is clear that when we use Cu(OAc)2 as catalyst,
DMEDA as ligand, K3PO4 as base, and DMF as solvent at
150ꢀC, we obtain highest yield. The same reaction condition
works well for the synthesis of remaining compounds from
the series; in most of the cases, we got yields in the range of
65% to 75%. The detailed experimental procedure, workup,
and yield details are given in experimental section.
The crude obtained was used further for next reaction
without purification.
3.1.2 | Step b—synthesis of 6-chloro-
2-(trifluoromethyl)-3-methylimidazo[1,2-b]
pyridazine (3)
To a stirred solution of comp-2 (1.29 g, 1.0 mmol) and
DME (13 mL) in a sealed tube was added 3-bromo-1,1,
1-trifluorobutan-2-one (2.25 g, 1.1 mmol) at room tem-
perature. Heat the reaction mixture at 150ꢀC for 4 hours.
Progress of reaction was monitored by TLC and LCMS
for the consumption of starting material. After comple-
tion, reaction mixture was poured in cold H2O (10 mL),
precipitation formed in reaction mixture, which was
filtered in vacuo washed with water (25 mL), brine
(20 mL), cold diethyl ether (25 mL), and cold pentane
(25 mL) to afford 6-chloro-2-(trifluoromethyl)-3-methyl-
imidazo[1,2-b]pyridazine (3, 2 g, 85.3%) as brown solid.
The crude obtained was used further for next reaction
without purification.
3 | EXPERIMENTAL SECTION
All chemicals, unless otherwise specified, were purchased
from commercial sources and were used without further
purification. The major chemicals were purchased from
Sigma Aldrich and Avra labs. The development of reactions
was monitored by thin layer chromatography (TLC) analysis
on Merck precoated silica gel 60 F254 aluminum sheets, visu-
alized by UV light. Melting points were recorded on SRS
Optimelt, melting point apparatus, and are uncorrected. The
1H NMR spectra were recorded on a 400-MHz Varian NMR
spectrometer. The 13C were recorded on a 100-MHz Varian
NMR spectrometer. The chemical shifts are reported as NMR
spectra δppm units. The following abbreviations are used:
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m),
and broad (br s). LCMS mass spectra were taken with
Micromass-QUATTRO-II of WATER mass spectrometer.
3.1.3 | Step c—synthesis of
3-(bromomethyl)-6-chloro-
2-(trifluoromethyl)imidazo[1,2-b]
pyridazine (4)
To a stirred solution of comp-3 (2.35 g, 1.0 mmol) and
ACN (25 mL) in a sealed tube was added freshly
crystalized NBS (1.95 g, 1.1 mmol) and AIBN (25 mg) at
room temperature. Heat the reaction mixture at 100ꢀC
for 1 hour. Progress of reaction was monitored by TLC
and LCMS for the consumption of starting material. After
completion, the reaction mixture was evaporated in
vacuo to obtain crude, which was poured in cold H2O
(20 mL). Reaction mixture was extracted with DCM
(2 × 20 mL). The organic layer was separated, washed
with H2O (15 mL) and brine (10 mL), dried over anhy-
drous Na2SO4 and concentrated in vacuo to afford
3-(bromomethyl)-6-chloro-2-(trifluoromethyl)imidazo
[1,2-b]pyridazine (4, 2.9 g, 92%) as yellow solid.
3.1 | Experimental procedure for
synthesis of 2-(2-(trifluoromethyl)-
6-(substituted)imidazo[1,2-b]pyridazin-
3-yl)-N-(substituted)acetamide derivatives
(9a-9j)
3.1.1 | Step a—synthesis of
6-chloropyridazin-3-amine (2)
To a stirred solution of comp-1 (14.9 g, 1.0 mmol) in a steel
bottomed flask was added Liq. ammonia (150 mL) at −10ꢀC.
Allowed reaction mixture to come to room temperature and
stirred at 150ꢀC for 8 hours. Progress of reaction was moni-
tored by TLC and LCMS for the consumption of starting
material. After completion, reaction mixture was cooled to
−10ꢀC, solid precipitates out in steel bottomed flask which is
filtered. The obtained solid was washed with hexane
(250 mL), cold diethyl ether (200 mL), and cold pentane
(200 mL) and dried in vacuo to afford 6-chloropyridazin-
3-amine (2, 10 g, 77.2%) [CAS - 5469-69-2] as yellow solid.
The crude obtained was used further for next reaction
without purification.
3.1.4 | Step d—synthesis of 2-(6-chloro-
2-(trifluoromethyl)imidazo[1,2-b]pyridazin-
3-yl)acetonitrile (5)
To a stirred solution of comp-4 (2.35 g, 1.0 mmol) and
DMF: H2O (19 mL: 5 mL) in a round bottom flask was

6
GAIKWAD ET AL.
carefully added NaCN (0.98 g, 2.0 mmol) at room temper-
ature. Stirred the reaction mixture at room temperature
for 12 hours. Progress of reaction was monitored by TLC
and LCMS for the consumption of starting material. After
completion, the reaction mixture was poured in cold H2O
(30 mL). Reaction mixture was extracted with DCM
(2 × 15 mL). The organic layer was separated, washed
with H2O (3 × 15 mL) and brine (10 mL), dried over
anhydrous Na2SO4, and concentrated in vacuo to afford
2-(6-chloro-2-(trifluoromethyl)imidazo[1,2-b]pyridazin-
3-yl)acetonitrile (5, 2.08 g, 80%) as off white solid.
The crude obtained was used further for next reaction
without purification.
was extracted with DCM (2 × 10 mL). The organic layer was
separated, washed with 1 N aq. cold HCl (5 mL), brine
(5 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo. The crude obtained was purified by washing
with 15:85% of DCM: hexane (20 mL), cold diethyl ether
(20 mL), and cold pentane (20 mL) to afford2-(6-chloro-
2-(trifluoromethyl)imidazo[1,2-b]pyridazin-3-yl)-N-
(3-fluorophenyl)acetamide (7, 0.33 g, 88%) as a white solid.
IR (KBr) (νmax, cm−1): 1625 (C═O), 1588 and 1520
(Ar). LC-MS m/z (%): 372.7 (M + H); 1H NMR (400 MHz,
DMSO-d6) δ 8.21 (s, 1H, NH), 7.42 (d, J = 7.2 Hz, 1H,
ArH), 7.36-7.25 (m, 2H, ArH), 7.22 (d, J = 7.2 Hz, 1H,
ArH), 6.80 (d, J = 7.6 Hz, 1H, ArH), 6.70 (d, J = 6.8 Hz,
1H, ArH), 3.40 (s, 2H, CO-CH2).
3.1.5 | Step e—synthesis of 2-(6-chloro-
2-(trifluoromethyl)imidazo[1,2-b]pyridazin-
3-yl)acetic acid (6)
3.1.7 | Step g—synthesis of
2-(2-(trifluoromethyl)-6-(2-oxopyrrolidin-
1-yl)imidazo[1,2-b]pyridazin-3-yl)-N-
(3-fluorophenyl)acetamide (9h)
To a stirred solution of comp-5 (2.60 g, 1.0 mmol) in a
round bottom flask was added aq. 20% NaOH solution
(25 mL) at room temperature. Heat the reaction mixture
at 100ꢀC for 2 hours. Progress of reaction was monitored
by TLC and LCMS for the consumption of starting
material.
After completion, reaction mixture was poured in
cold H2O (30 mL) and extracted with DCM (25 mL). Col-
lected the aqueous layer acidified it by using 6 N aq. HCl
solution up to pH 3 solid precipitated in reaction mixture.
The formed precipitation was filtered in vacuo washed
with water (25 mL), brine (20 mL), and diethyl ether
(25 mL) to afford 2-(6-chloro-2-(trifluoromethyl)imidazo
[1,2-b]pyridazin-3-yl)acetic acid (6, 2.52 g, 90%) as a
white solid.
To a stirred solution of Comp-7 (0.19 g, 0.5 mmol) in DMF
(5 mL) was added pyrrolidin-2-one (0.065 g, 0.75 mmol),
Cu(OAc)2 (0.019 g, 10 mol%), K3PO4 (0.216 g, 1.0 mmol),
and N,N-dimethylethylenediamine (0.028 g, 15 mol%).
Degas reaction mixture using argon and heat reaction mix-
ture to 150ꢀC for 12 hours. Progress of reaction was moni-
tored by TLC and LCMS for the consumption of starting
material. After completion, reaction mixture was poured in
cold H2O (10 mL) and stirred for 10 minutes. Reaction mix-
ture was extracted with EtOAc (2 × 10 mL), collected
organic layer washed with H2O (5 mL) and Brine (5 mL).
Separated organic layer dried over anhydrous Na2CO3 and
evaporated in vacuo to obtain crude material. Crude was
purified by silica gel (100-200 mesh) column chromatogra-
phy by using (ethyl acetate-hexane; 30:60%) to obtain
2-(2-(trifluoromethyl)-6-(2-oxopyrrolidin-1-yl)imidazo[1,2-b]
pyridazin-3-yl)-N-(3-fluorophenyl)acetamide (9h, 160 mg,
75%) as a white solid.
The crude obtained was used further for next reaction
without purification.
1
LC-MS m/z (%): 280.7 (M + H); H NMR (400 MHz,
DMSO-d6, ppm) δ 11.8 (brs, 1H, COOH), 7.70 (d, J = 8.4 Hz,
1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, Ar-H), 3.388 (s, 2H,
CO-CH2).
3.1.8 | 2-(2-(Trifluoromethyl)-
6-(methylamino)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (9a)
3.1.6 | Step f—synthesis of 2-(6-chloro-
2-(trifluoromethyl)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (7)
Yellow Solid; m.p. 163-164ꢀC; Yield - 68%; IR (KBr) (νmax
,
To a stirred solution of Comp-6 (0.28 g, 0.01 mmol) in DCM
(5 mL) was added EDCI (0.28 g, 0.015 mmol) and DIPEA
(0.52 mL, 0.03 mmol) at 0ꢀC. Added 3-flouroaniline (0.13 g,
0.012 mmol) at 0ꢀC and stirred reaction mixture at room tem-
perature for 12 hours. Progress of the reaction was monitored
by TLC and LCMS. After completion, the reaction mixture
was diluted with cold water (10 mL). The reaction mixture
cm−1): 1627 (C═O), 1582 and 1520 (Ar). Anal. calc. for
C16H13F4N5O: C, 52.32; H, 3.57; N, 19.07; Found: C,
52.36; H, 3.61; N, 19.04. LC-MS m/z (%): 367.3 (M + H);
HPLC-98.9% RT-8.22 minutes; 1H NMR (400 MHz,
DMSO-d6) δ 8.22 (s, 1H, NH), 7.43 (d, J = 7.2 Hz, 1H,
ArH), 7.38-7.28 (m, 2H, ArH), 7.24 (d, J = 7.2 Hz,
1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH), 6.72 (d, J = 6.8 Hz,

GAIKWAD ET AL.
7
1H, ArH), 5.16 (q, 1H, CH3-NH), 3.42 (s, 2H, CO-CH2), 2.46
(d, J = 6.6 Hz, 3H, NH-CH3). 13C NMR (CDCl3, 100 MHZ,
ppm) = δ 24.3, 28.7, 110.1, 112.2, 117.2, 118.2, 120.2, 120.4,
124.6, 130.4, 135.6, 136.3, 136.8, 162.3, 163.4, 168.6.
2H, ArH), 7.24 (d, J = 7.2 Hz, 1H, ArH), 6.88 (d,
J = 7.6 Hz, 1H, ArH), 6.72 (d, J = 6.8 Hz, 1H, ArH), 5.16
(q, 1H, CH3-NH), 3.42 (s, 2H, CO-CH2), 3.16-3.09 (d,
J = 12.6, 6.4 Hz, 2H, CH2-CH2), 1.58-142 (q, J = 14.8,
7.4 Hz, 4H, CH2-CH2), 0.96 (t, J = 7.4 Hz, 3H, CH2-CH3).
13C NMR (CDCl3, 100 MHZ, ppm) = δ 12.8, 20.3, 28.7,
33.5, 47.2, 110.2, 112.2, 117.2, 118.2, 120.2, 120.4, 124.8,
130.4, 135.6, 136.3, 137.4, 162.4, 163.4, 168.6.
3.1.9 | 2-(6-(Ethylamino)-
2-(trifluoromethyl)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (9b)
Gray Solid; m.p. 167-168ꢀC; Yield - 70%; IR (KBr) (νmax
,
3.1.12 | 2-(2-(Trifluoromethyl)-
6-(pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (9e)
cm−1): 1624 (C═O), 1580 and 1524 (Ar). Anal. calc. for
C17H15F4N5O: C, 53.55; H, 3.96; N, 18.37; Found: C,
53.51; H, 3.99; N, 18.34; Mol. Wt.: 381.33; HPLC-97.2% RT-
1
8.16 minutes; H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H,
Off white Solid; m.p. 136-137ꢀC; Yield - 68%; IR (KBr) (νmax
,
NH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.38-7.28 (m, 2H, ArH),
7.24 (d, J = 7.2 Hz, 1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH),
6.72 (d, J = 6.8 Hz, 1H, ArH), 5.16 (q, 1H, CH3-NH), 3.42
(s, 2H, CO-CH2), 3.16-3.08 (q, J = 14.4, 7.2 Hz, 2H, CH3-
CH2), 1.16 (s, 3H, CH2-CH3).13C NMR (CDCl3, 100 MHZ,
ppm) = δ14.8, 28.7, 40.4, 110.2, 112.2, 117.4, 118.2, 120.2,
120.4, 124.6, 130.4, 135.6, 136.3, 136.8, 162.2, 163.3, 168.6.
cm−1): 1628 (C═O), 1580 and 1520 (Ar). Anal. calc. for
C19H17F4N5O: C, 56.02; H, 4.21; N, 17.19; Found: C,
55.95; H, 4.18; N, 17.22; Mol. Wt.: 407.36; HPLC-99.1% RT-
1
7.87 minutes; H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H,
NH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.38-7.28 (m, 2H, ArH),
7.24 (d, J = 7.2 Hz, 1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH),
6.72 (d, J = 6.8 Hz, 1H, ArH), 3.42 (s, 2H, CO-CH2), 2.78
(t, J = 8.4, 4.4 Hz, 4H, N-(CH2)2), 1.58 (q, J = 7.6, 3.8 Hz,
4H, CH2-(CH2)2). 13C NMR (CDCl3, 100 MHZ, ppm) = δ
25.6, 28.7, 52.8, 110.1, 112.1, 117.7, 118.7, 120.2, 120.4, 124.6,
130.4, 135.6, 136.3, 136.8, 156.3, 163.4, 168.4.
3.1.10 | 2-(2-(Trifluoromethyl)-
6-(propylamino)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (9c)
Gray Solid; m.p. 181-182ꢀC; Yield - 69%; IR (KBr) (νmax
,
3.1.13 | 2-(2-(Trifluoromethyl)-
6-(piperidin-1-yl)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (9f)
cm−1): 1620 (C═O), 1576 and 1524 (Ar). Anal. calc. for
C18H17F4N5O: C, 54.68; H, 4.33; N, 17.71; Found: C,
54.69; H, 4.38; N, 17.75; Mol. Wt.: 395.35; HPLC-98.3% RT-
1
7.89 minutes; H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H,
Off white Solid; m.p. 139-140ꢀC; Yield - 64%; IR (KBr) (νmax
,
NH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.38-7.28 (m, 2H, ArH),
7.24 (d, J = 7.2 Hz, 1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH),
6.72 (d, J = 6.8 Hz, 1H, ArH), 5.16 (q, 1H, CH3-NH), 3.42
(s, 2H, CO-CH2), 3.16-3.14 (d, J = 12.6, 6.4 Hz, 2H, CH2-CH2),
1.54 (q, J = 14.8, 7.4 Hz, 2H, CH3-CH2), 0.94 (t, J = 7.4 Hz, 3H,
CH2-CH3). 13C NMR (CDCl3, 100 MHZ, ppm) = δ 11.2, 24.3,
28.7, 47.4, 110.4, 112.2, 117.1, 118.5, 120.2, 120.4, 124.6, 130.2,
135.6, 136.3, 136.6, 162.3, 163.6, 168.8.
cm−1): 1632 (C═O), 1584 and 1528 (Ar). Anal. calc. for
C20H19F4N5O: C, 57.00; H, 4.54; N, 16.62; Found: C,
57.06; H, 4.59; N, 16.67; Mol. Wt.: 421.39; HPLC-98.2% RT-
1
7.44 minutes; H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H,
NH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.38-7.28 (m, 2H, ArH),
7.24 (d, J = 7.2 Hz, 1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH),
6.72 (d, J = 6.8 Hz, 1H, ArH), 3.42 (s, 2H, CO-CH2), 2.82
(t, J = 8.4, 4.4 Hz, 4H, N-(CH2)2), 1.48 (q, J = 7.6, 3.8 Hz,
4H, CH2-(CH2)3). 13C NMR (CDCl3, 100 MHZ, ppm) = δ
24.6, 26.1, 28.7, 52.4, 110.1, 112.2, 117.2, 118.2, 120.2, 120.4,
124.6, 130.4, 135.6, 136.3, 136.8, 157.3, 163.8, 168.2.
3.1.11 | 2-(6-(Butylamino)-
2-(trifluoromethyl)imidazo[1,2-b]pyridazin-
3-yl)-N-(3-fluorophenyl)acetamide (9d)
3.1.14 | 2-(2-(Trifluoromethyl)-
6-(4-methylpiperidin-1-yl)imidazo[1,2-b]
pyridazin-3-yl)-N-(3-fluorophenyl)
acetamide (9g)
Brown Solid; m.p. 162-163ꢀC; Yield- 75%; IR (KBr) (νmax
,
cm−1): 1628 (C═O), 1586 and 1516 (Ar). Anal. calc. for
C19H19F4N5O: C, 55.74; H, 4.68; N, 17.11; Found: C,
55.70; H, 4.62; N, 17.08; Mol. Wt.: 409.38; HPLC-99.3%
1
RT-8.44 minutes; H NMR (400 MHz, DMSO-d6) δ 8.22
Yellow Solid; m.p. 149-150ꢀC; Yield - 52%; IR (KBr)
(s, 1H, NH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.38-7.28 (m,
(νmax, cm−1): 1634 (C═O), 1588 and 1528 (Ar). Anal.

8
GAIKWAD ET AL.
calc. for C21H21F4N5O: C, 57.93; H, 4.86; N, 16.08;
Found: C, 57.90; H, 4.89; N, 16.02; Mol. Wt.: 435.42;
HPLC-99.8% RT-7.38 minutes; 1H NMR (400 MHz,
DMSO-d6) δ 8.22 (s, 1H, NH), 7.43 (d, J = 7.2 Hz, 1H,
ArH), 7.38-7.28 (m, 2H, ArH), 7.24 (d, J = 7.2 Hz, 1H,
ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH), 6.72 (d, J = 6.8 Hz,
1H, ArH), 3.42 (s, 2H, CO-CH2), 2.82 (t, J = 8.4, 4.4 Hz,
4H, N-(CH2)2), 1.54 (q, J = 8.0, 4.0 Hz, 1H, CH3-CH),
1.48 (q, J = 7.6, 3.8 Hz, 4H, CH2-(CH2)2), 1.02
(d, J = 6.2 Hz, 3H, CH-CH3). 13C NMR (CDCl3,
100 MHZ, ppm) = δ 20.3, 28.7, 31.9, 32.2, 48.6, 110.4,
112.6, 117.2, 118.2, 120.2, 120.4, 124.6, 130.4, 135.6,
136.3, 136.4, 157.3, 163.1, 168.3.
32.4, 48.8, 110.8, 113.2, 117.6, 118.2, 120.2, 121.4, 124.6,
130.4, 135.6, 136.3, 136.8, 153.3, 162.4, 167.3, 169.8.
3.1.17 | 2-(2-(Trifluoromethyl)-
6-(4-methylpiperazin-1-yl)imidazo[1,2-b]
pyridazin-3-yl)-N-(3-fluorophenyl)
acetamide (9j)
Yellow Solid; m.p. 188-189ꢀC; Yield - 80%; IR (KBr)
(νmax, cm−1): 1622 (C═O), 1578 and 1518 (Ar). Anal.
calc. for C20H20F4N6O: C, 55.04; H, 4.62; N, 19.26;
Found: C, 55.01; H, 4.67; N, 19.29; Mol. Wt.: 436.41;
HPLC-98.9% RT-8.22 minutes; 1H NMR (400 MHz,
DMSO-d6) δ 8.22 (s, 1H, NH), 7.43 (d, J = 7.2 Hz, 1H,
ArH), 7.38-7.28 (m, 2H, ArH), 7.24 (d, J = 7.2 Hz,
1H, ArH), 6.88 (d, J = 7.6 Hz, 1H, ArH), 6.72
(d, J = 6.8 Hz, 1H, ArH), 3.42 (s, 2H, CO-CH2), 3.16
(t, J = 8.4, 4.4 Hz, 4H, N-(CH2)2), 2.48 (t, J = 7.6,
3.8 Hz, 4H, N-(CH2)2), 2.27 (s, 3H, N-CH3). 13C NMR
(CDCl3, 100 MHZ, ppm) = δ 26.7, 42.1, 46.7, 56.6,
110.2, 114.2, 116.2, 118.2, 120.2, 122.4, 124.6, 130.4,
135.6, 136.3, 136.8, 153.3, 164.4, 167.6.
3.1.15 | 2-(2-(Trifluoromethyl)-
6-(2-oxopyrrolidin-1-yl)imidazo[1,2-b]
pyridazin-3-yl)-N-(3-fluorophenyl)
acetamide (9h)
White Solid; m.p. 183-184ꢀC; Yield - 75%; IR (KBr)
(νmax, cm−1): 1636 (C═O), 1576 and 1524 (Ar). Anal.
calc. for C19H15F4N5O2: C, 54.16; H, 3.59; N, 16.62;
Found: C, 54.10; H, 3.63; N, 16.64; Mol. Wt.: 421.7;
HPLC-98.2% RT-7.67 minutes; 1H NMR (400 MHz,
DMSO-d6) δ 8.22 (s, 1H, NH), 7.97 (d, J = 7.6 Hz, 1H,
ArH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.38-7.28 (m, 2H,
ArH), 7.24 (d, J = 7.2 Hz, 1H, ArH), 6.72 (d, J = 6.8 Hz,
1H, ArH), 3.42 (s, 2H, CO-CH2), 3.02 (t, J = 6.4 Hz, 2H,
N-CH2), 2.56 (d, J = 6.2 Hz, 2H, CO-CH2), 2.16
(q, J = 6.8, 2.2 Hz, 2H, CH2-CH2). 13C NMR (CDCl3,
100 MHZ, ppm) = δ 18.6, 28.7, 33.2, 49.2, 110.8, 112.8,
117.2, 118.2, 120.2, 120.4, 124.7, 130.4, 135.4, 136.3,
136.8, 154.3, 163.6, 168.8, 174.2.
3.2 | Biological evaluation
All the synthesized compounds were tested for their
in vitro anticancer activity against human lung cancer
cell line and human prostate cancer cell lines. The
anticancer activity test is performed according to the
procedure developed by the National Cancer Institute
(NCI, USA) in the “In vitro Anticancer Drug Discovery
Screen” that uses the 3-(4,5-dimethythiazol-2yl)-
2,5-diphenyl tetrazolium bromide by mitochondrial
succinate dehydrogenase.[39,40] All the newly synthe-
sized compounds 9a-9j are evaluated for their anti-
proliferative activities against two human cancer cell
lines. The results are summarized in Table 2. These
values represent the concentration required to inhibit
50% cell population compared with the control cells
treated with DMSO and positive control Doxorubicin
under similar conditions. The synthesized compounds
were evaluated for their in vitro anticancer activity
against human lung cancer cell line (A549) and pros-
tate cell line (DU-145).
3.1.16 | 2-(2-(Trifluoromethyl)-
6-(2-oxopiperidin-1-yl)imidazo[1,2-b]
pyridazin-3-yl)-N-(3-fluorophenyl)
acetamide (9i)
Off white Solid; m.p. 180-190ꢀC; Yield - 75%; IR (KBr)
(νmax, cm−1): 1628 (C═O), 1580 and 1530 (Ar). Anal. calc.
for C20H17F4N5O2: C, 55.17; H, 3.94; N, 16.09; Found: C,
55.16; H, 3.97; N, 16.04; Mol. Wt.: 435.37; HPLC-99,1%
RT-8.38 minutes; 1H NMR (400 MHz,DMSO-d6)δ 8.21
(s, 1H, NH), 7.97 (d, J = 7.6 Hz, 1H, ArH), 7.44
(d, J = 7.2 Hz, 1H, ArH), 7.36-7.28 (m, 2H, ArH), 7.24
(d, J = 7.2 Hz, 1H, ArH), 6.74 (d, J = 6.8 Hz, 1H, ArH),
3.44 (s, 2H, CO-CH2), 3.03 (t, J = 6.4 Hz, 2H, N-CH2),
2.57 (d, J = 6.2 Hz, 2H, CO-CH2), 1.98 (q, J = 6.8, 2.2 Hz,
2H, CH2-CH2), 1.81 (q, J = 6.8, 2.2 Hz, 2H, CH2-CH2).
13C NMR (CDCl3, 100 MHZ, ppm) = δ 22.3, 26.6, 28.7,
3.3 | Cell culture
Human cancer cell lines A549 (lungs) and DU-145 (pros-
tate) were grown in DMEM + GlutaMax (Invitrogen, Carls-
bad, CA, USA), supplemented with 10% heat-inactivated

GAIKWAD ET AL.
9
TABLE 2 In vitro anticancer
screening of the synthesized
Sr. No.
A-549a
Sib
6.85
DU-145c
Sib
4.87
HUVECd
9a
12.27 0.10
8.12 0.06
11.10 0.07
18.08 0.14
2.12 0.04
4.18 0.11
3.10 0.24
1.73 0.18
9.77 0.03
14.13 0.12
1.82 0.06
17.26 0.08
7.11 0.11
9.17 0.16
13.48 0.10
2.06 0.07
5.10 0.04
4.10 0.24
1.76 0.06
16.42 0.11
8.36 0.04
1.72 0.12
84.06 0.03
82.03 0.05
87.11 0.08
86.16 0.05
81.11 0.07
89.11 0.08
87.06 0.05
81.06 0.06
89.04 0.08
87.11 0.04
87.04 0.06
compounds against two cell lines; data
are expressed as IC50 (μM) SD (n = 3)
9b
10.10
7.84
11.53
9.49
9c
9d
4.76
6.39
9e
38.26
21.31
28.08
46.85
9.11
33.79
17.47
21.23
46.05
5.42
9f
9g
9h
9i
9j
6.16
10.42
50.81
Doxorubicin
48.02
aA-549: Human lung cancer cell line.
bSelectivity Index (SI): IC50 of pure compound in normal cell line/IC50 of same compound.
in cancer cell line. IC50, the concentration required to inhibit 50% of cell population.
cDU-145: Human prostate cancer cell line.
dHUVEC: Human Umbilical vein endothelial cell line.
bovine serum (Gibco) and penicillin-streptomycin (Gibco,
Gaithersburg, MD, USA) at 37ꢀC in a humified chamber
with 5% CO2 supply.
For lung cancer cell line (A549), the compounds
9e, 9g, and 9h are most active with 38.26%, 28.08%,
and 46.85% inhibitions with IC50 value of 2.12μM,
3.10μM, and 1.73μM, respectively. The compounds 9f
is moderately active with 21.31% inhibition and with
IC50 value of 4.18μM; the remaining compounds were
less active with IC50 values in the range of 8.12μM to
18.08μM. For Du-145 cell line, the compounds 9e and
9h are most active with IC50 value of 2.06μM and
1.76μM and inhibitions of 33.79% and 46.05%, respec-
tively. For compounds 9f and 9g are moderately active
with IC50 value of 5.10μM to 4.10μM and inhibitions of
17.47% and 21.23%, respectively; remaining com-
pounds are very less active or inactive with IC50 value
in the range of 7.11 to 17.26. Compound 9h is most
active for A-549 and Du-145 cell line with IC50 value of
1.73μM and 1.76μM, respectively. Similar results were
obtained for compound 9e for cell lines A-549 and Du-
145 with IC50 value of 2.12μM and 2.06μM, respec-
tively. The remaining compounds were mostly inactive
for both the cancer cell lines.
The SAR can be drawn like compounds having methyl
acetamide, ethyl acetamide, propyl acetamide and butyl acet-
amide wareless active for both the cancer cell lines. These
derivatives don’t involve anyring structure and electron
donating or electron with drawing substituent. These com-
pounds showed very less activity among the synthesized com-
pounds. Instead of straight chain, compounds were replaced
by chain compounds like pyrrolidine, piperidine,
4-methylpiperidine, and pyrrolidin-2-one; the activity of final
targets is increasing for both the cell lines. In those targets, the
ring strain on the molecule plays a key role in the activity. We
3.4 | Cytotoxicity assay
Cells were seeded (105 cells/well) in 96-well flat-bottom
plates (Becton-Dickinson Labware, Franklin Lakes, NJ,
USA) a day before treatment and grown overnight. Com-
pounds were dissolved in dimethyl sulfoxide (DMSO;
Sigma) and finally prepared as 1.0 mg/mL stocks, respec-
tively, in the culture media. The final concentration of
DMSO never exceeded 0.1% in the treatment doses. Six
different doses of compounds (400μM, 200μM, 100μM,
50μM, 25μM, and 10μM) were further prepared by dilut-
ing the stocks in culture media, and cells were treated
(in triplicate/dose). Doxorubicin was included as stan-
dard reference drug (positive control), and untreated cul-
ture was considered as negative control. The cultures
were further incubated for 48 hours. At 48-hour post-
treatment, cell viability test was performed using TACS
MTT Cell Proliferation and Viability Assay Kit (TACS) as
per manufacturer's instructions. The optical density
(OD) was recorded at 570 nm in a microplate reader
(ELx800, BioTek, Winooski, VT, USA), and cell survival
fraction was determined.[39,40] The cell survival fraction
was calculated as [(A-B)/A], where A and B are the OD
of untreated and of treated cells, respectively. The con-
centration required for 50% inhibition of cell viability
(IC50) was calculated and compared with the reference
drug Doxorubicin, and the results are given in Table 2.

10
GAIKWAD ET AL.
TABLE 3 Inhibitory activity of selected compounds against
panel of five human kinases
a series of reactions including imidazo-pyridazine ring
formation, halogenation, cynation, peptide coupling,
and Buchwald reaction. We have tried to report simple
reaction condition, easy workup, short reaction time, and
good to high yields. The synthesized compounds were
screened for anticancer activity against A-549 and Du-145
cancer cell lines. The compounds 9e, 9g, and 9h are most
active with IC50 values in the range of 1.73μM to 3.10μM.
The compounds 9e, 9g, and 9h show promising inhibi-
tions (>54%) for EGFR and m-TOR human kinases. The
compounds having electron-donating substituents along
with aliphatic ring and without electron deficient atom
show promising activity and greater inhibitions for protein
kinases.
Kinase
9e (% Inh.)
9g (% Inh.)
9h (% Inh.)
CDK2/cyclinA
CDK5/P25
EGFR
16
36
72
54
23
42
28
66
48
28
51
44
63
66
39
mTOR
PDK1
tried to introduce a hetero atom in the ring system like
pyrrolidin-2-one, piperidin-2-one, and 1-methylpiperazine;
the activity was decreased to greater extent. The compound
with pyrrolidine-2-one is more active than standard for both
the cell lines. As the ring size increases to piperidin-2-one,
there is decrease in activity because of larger ring size. The less
activity was seen in the case of 1-methylpiperazine. The rings
having oxygen as a heteroatom and larger rings are less active.
The compounds having electron donating substituent’s ware
less active than compounds having ring structures attached
with them. The compounds having electron deficient hetero
atom shows decrease in activity due to ring delocalization.
The active compounds from cell line studies were fur-
ther tested for their kinase inhibitors as these targets
show good percentage of inhibitions for some kinases,
which is key aspect in target identification in cancer, by
following the earlier protocols.[41–45]
The compounds 9e, 9g, and 9h are most active in cell
line studies, so further, we have tested them for their
activity against a panel of five human kinase at 10μM
concentration. The inhibition results are summarized in
Table 3. Protein kinase plays a key role in cell prolifera-
tion, differentiation of cell, migration of cell, survival of
cell, and angiogenesis of cells.
ACKNOWLEDGMENT
The authors are thankful to the Head, Department of
Chemistry, Deogiri College, Aurangabad, Dr. Babasaheb
Ambedkar Marathwada University, Aurangabad 431004,
MS, India for providing the laboratory facility.
ORCID
Dattatraya N. Pansare https://orcid.org/0000-0002-
0419-3538
Rohini N. Shelke https://orcid.org/0000-0002-2053-4785
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Among CMGC kinase family for CDK2/cyclinA and
CDK5/P25 kinases, the inhibition is in the range of 16% to
44% for all the compounds. The compounds 9e, 9g, and 9h
show 72%, 66%, and 63% inhibitions, respectively, (Table 3)
for EGFR kinase. For mTOR kinase, the inhibitions are in
the range of 48% to 66% for all the compounds. For PDK1
kinase, the inhibitor for all the compounds has very less
inhibitions in the range of 23% to 39%. The active com-
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4 | CONCLUSION
We have synthesized 2-(2-(trifluoromethyl)-6-(substituted)
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derivatives (9a-9j) from 3,6-dichloropyridazine through

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SUPPORTING INFORMATION
Additional supporting information may be found online
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How to cite this article: Gaikwad DD,
Pawar UD, Chavan SL, et al. Synthesis and anti-
proliferative activity studies of 2-(2-
(trifluoromethyl)-6-(substituted)imidazo[1,2-b]
pyridazin-3-yl)-N-(substituted)acetamide
derivatives. J Heterocyclic Chem. 2020;1–11. https://
doi.org/10.1002/jhet.3920