Triazolopyridazine derivatives: Synthesis, cytotoxic evaluation, c-Met kinase activity and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2019.103272

Novel series of some triazolo[4,3-b]pyridazine derivatives were designed and synthesized. All the newly synthesized compounds were evaluated for their cytotoxic activity at 10^?5 M concentration towards 60 cancer cell lines according to USA NCI protocol. Most of the synthesized compounds showed good activity against SR (leukemia) cell panel. The most active compounds, 2f and 4a were subjected for further evaluation at a five dose level screening and their efficacy for c-Met kinase inhibition was determined in vitro. Binding mode of these derivatives was explored via molecular docking.

Full text of DOI:10.1016/j.bioorg.2019.103272

BioorganicChemistry92(2019)103272
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Triazolopyridazine derivatives: Synthesis, cytotoxic evaluation, c-Met kinase
activity and molecular docking
⁎
Eman M. Ahmeda, Nadia A. Khalila, Azza T. Tahera,b, Rana H. Refaeyc, , Yassin M. Nissanc,d
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, October 6 University, Giza, Egypt
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza, Egypt
d Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini St., Cairo 11562, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel series of some triazolo[4,3-b]pyridazine derivatives were designed and synthesized. All the newly syn-
thesized compounds were evaluated for their cytotoxic activity at 10−5 M concentration towards 60 cancer cell
lines according to USA NCI protocol. Most of the synthesized compounds showed good activity against SR
(leukemia) cell panel. The most active compounds, 2f and 4a were subjected for further evaluation at a five dose
level screening and their efficacy for c-Met kinase inhibition was determined in vitro. Binding mode of these
derivatives was explored via molecular docking.
Triazolopyridazines
Pyridazines
Cytotoxic activity
c-Met kinase
1. Introduction
evaluated as c-Met kinase inhibitors in vitro. Compound I showed IC50
value of 9.3 nM while that for compound II was less than 100 nM and
C-Met kinase is a tyrosine kinase receptor that is activated by
binding of hepatocyte growth factor (HGF), resulting in receptor di-
merization followed by downstream signalling [1]. In normal cells, this
pathway plays a key role in normal embryonic development, as well as
damage repair and wound healing in adults [2–6]. It has been reported
that the c-Met kinase pathway is deregulated in various human cancers,
such as colorectal, lung and gastric and other carcinomas, through the
overexpression of the c-Met kinase receptor. This deregulation is asso-
ciated with tumour proliferation, angiogenesis and metastasis, as well
as poor prognosis [7]. In addition, c-Met kinase has also been im-
plicated in tumour resistance to therapy, including EGFR and VEGFR
inhibitors such as gefitinib, cetuximab and panitumumab [8]. There-
fore, the inhibition of c-Met kinase using small molecules, particularly
through the targeting of the ATP-binding site as demonstrated for other
kinases, is an attractive strategy for pursuing novel anticancer agents
[1,9]. Crizotinib and cabozantinib are c-Met kinase inhibitors that have
been recently approved by FDA for treatment of various cancers, along
with several other agents, such as tivantinib and fortinib, reported to be
undergoing clinical trials [10–14]. Among the compounds reported to
show high c-Met kinase inhibitory potency, as well as high selectivity
towards c-Met kinase in comparison to other kinases, are triazolopyr-
idazine derivatives, Fig. 1. Hence, this research was centered on the
synthesis and investigation of the cytotoxic potential of novel triazo-
lopyridazines. Compounds exhibiting promising results were further
finally compound III 3 nM [15–20]. These results encouraged further
developments of new pyrazolopyridazines targeting c-Met kinase
hoping to discover better inhibitors for this key enzyme.
2. Results and discussion
2.1. Chemistry
The synthetic route for the target compounds 2a-i, 4a,b and 6a,b is
outlined in Schemes 1 and 2. The intermediates 1a-i and 3b were
prepared from the commercially available 3,6-dichloropyridazine and
the appropriate amines or amides viz; anthranilamide or salicylamide as
a
starting materials, according to previously reported procedure
[21,22]. The target triazolopyridazine derivatives 2a-i and 4a,b were
obtained in 65–84% yield by heating the precursors 1a-i or 3a,b with
isonicotinic acid hydrazide in n-butanol for 6 h.
Supporting evidence for the structure of the two new key com-
pounds 1f, 1i and the final targets 2a-i was achieved on the basis of
elemental analyses and various spectroscopic methods. Spectral data IR,
1H NMR, 13C NMR and MS were found to be in full agreement with the
proposed structure. IR spectra of 1f, 1i and 2a-i showed prominent
bands of NH str. at 3476–3267 cm−1. In 1H NMR spectra, the aromatic
protons were observed at the expected region. The signals due to NH
protons (controlled with D2O) were resonated at δ 9.86–10.39 ppm. In
⁎ Corresponding author.
E-mail address: rhosny@msa.eun.eg (R.H. Refaey).
https://doi.org/10.1016/j.bioorg.2019.103272
Received 22 May 2019; Received in revised form 15 August 2019; Accepted 9 September 2019
Availableonline10September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
Fig. 1. Structures of lead compounds (I, II, IIII) with known c-Met kinase inhibitory activity.
Cl
Cl
Cl
N
N
N
N
a
N
N
b
N
N
ArNH2
N
NHAr
NHAr
2a-i
1a-i
CONH2
2a-i
Ar
a
2a
2b
2-BrC6H4
2-ClC6H4
XH
2c
2d
4-ClC6H4
2,6-Cl2C6H3
N
Cl
N
2-ClC5H3N
2e
2f
N
N
N
2-COOHC6H4
3-COOHC6H4
4-COOHC6H4
N
N
b
2g
CONH2
CONH2
2h
2i
X
X
2-COOH-4-ClC6H3
,
3a,b
4a,b
3a, 4a:
3b, 4b:
X=NH
X= O
Scheme 1. Regents and condition: (a) isopropanol/k2CO3/reflux 4 h. (b) INH/butanol/reflux 6 h.
Cl
Cl
NH2
N
Cl
N
N
N
N
N
N
N
a
b
N
HN
SO2NHR
NH
SO2NHR
SO2NHR
6a,b
5a,b
,
5a, 6a: R= H
5b, 6b: R=
C NH2
NH
Scheme 2. Reagents and condition: (a) isopropanol/k2CO3/reflux 4 h, (b) INH/butanol/reflux 6 h.
2

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
Table 1
Percentage growth inhibition (GI%) of in vitro sensitive tumour cell lines for compounds 2f & 4a at 10−5 M.
Compound
Mean growth inhibition
Range of growth
Most sensitive cell panel/cell line growth %
%
inhibition %
2f
42.18
45.87
20–75
18–89
L/CCRF-CEM 69, L/MOLT-4 46, L/RPMI- 8226 32, L/SR 75, NSCLc/A549 31, NSCLc/NCI-H460 49, NSCLc/
NCI-H522 64, Cc/SW-620 43, Cc/HCT-116 68, Cc/HCT-15 71, Cc/HT29 33, M/LOX IMVI 75, M/SK-MEL-5
43,Oc IGROV1 51, Rc/ACHN 32, Bc/MCF-7 63, Bc/MDA-MB 468 70
4a
L/CCRF-CEM 77, L/K-560 35, L/MOLT-4 58, L/RPMI-8226 36, L/SR 78, NSCLc/NCI-H460 60, NSCLc/NCI-
H522 81, Cc/HCT-116 71, Cc/HCT-15 72, Cc/HT-29 30, Cc/SW620 50, CNSc/SF-268 41, M/LOX IMV1 89,
M/SK-MEL-5 52, Oc/IGROV1 36, Oc/OVCAR-8 30, Rc/ACHN 41, Rc/SN 12C 39, Bc/MCF-7 68, Bc/BT-549
45, Bc/MDA-MB-468 77
L leukemia, NSCLc non-small cell lung cancer, Cc colon cancer, CNSc CNS cancer, M melanoma, Oc ovarian cancer, Rc renal cancer and Bc breast cancer.
Scheme 2, using methodology previously described [22], the inter-
mediates 5a, b were synthesized from a commercially available 3,6-
dichloropyridazine and either sulphanilamide or sulphadiazine.
Thereafter, reaction of 5a, b with isonicotinic acid hydrazide afforded
6a, b in 76–72% yield.
in an attempt to explain their cytotoxic mechanism of action. The
profile of 2f and 4a against c-Met kinase was evaluated by employing
the standardized assay methodology [24,25]. The results were reported
as % activity change compared to control (Table 5). Furthermore, the
data was used to plot dose response graph which was used to determine
the IC50 value for both compounds (Fig. 2).
IR spectra of compounds 6a, b showed two bands for SO2 str. in the
range of 1315–1323 cm−1 and 1134–1153 cm−1. The structures were
further established by 1H NMR spectral data. The additional D2O ex-
changeable signals due to SO2NH2 protons in 6a and SO2NH in 6b were
observed at δ 5.75 and δ 6.65 ppm respectively.
Upon examination of the results, it was found that both compounds,
2f and 4a showed increasing inhibition of c-Met kinase activity with
increasing concentrations, with compound 2f showing higher inhibitory
activity than compound 4a. Compound 2f, at 100 μM inhibited the c-
Met kinase activity by 51% and exhibited an IC50 value of 106.9 μM. On
the other hand, compound 4a, at 100 μM concentration inhibited the c-
Met kinase activity by 35%, with an IC50 value of 242.2 μM (Fig. 2).
2.2. Biological studies
2.2.1. Cytotoxic activity
All the synthesized compounds underwent preliminary cytotoxic
assay against the full panel of 60 cell lines, at a single dose of 10−5 M.
The preliminary screening results showed compounds 2e, 2h and 6b
were completely devoid of cytotoxic activity. It was also reported that
compounds 2g, 4b and 6a showed moderate activity only against renal
cancer cell lines while compound 2d only showed cytotoxic activity
against leukemia cell lines. On the other hand, compounds 2a, 2b, 2c,
2f, 2i and 4a exhibited significant cytotoxic activity against a wide
variety of cell lines. However, only compounds 2f and 4a showed
considerable cytotoxic activity with a broad spectrum against several
cancer cell lines (Table 1).
2.3. Molecular docking
In order to investigate the molecular basis of the mechanism of
action of the synthesized compounds, the interactions formed between
the synthesized compounds and the c-Met kinase receptor was explored
by carrying out docking studies. The docking studies revealed that the
synthesized compounds adopted different orientations into the ATP-
binding pocket. The compounds showing high to moderate cytotoxic
activity (2a, 2b, 2c, 2f, 2i & 4a), adopted “Bent U-shaped” con-
formations, with different degrees of bending, within the ATP binding
site of the c-Met kinase receptor (Fig. 3). This binding mode is reported
to be characteristic for compounds type I inhibitors selective to c-Met
Kinase [26].
Accordingly, compounds 2f and 4a were selected for further eva-
luation at a five-dose assay with concentrations ranging from 0.01 to
100 µM, the response parameters GI50, TGI50 and LC50 against each cell
line were calculated. GI50 represents molar concentration of the com-
pound that produce 50% decrease in the net cell growth and is viewed
as a growth inhibitory level of effect; TGI (cytostatic activity) is the
molar concentration of the compound that results in total growth in-
hibition; LC50 is the cytotoxicity parameter that reflects the molar
concentration needed to cause 50% net cell death (Table 2).
Additionally, the mean graph midpoints (MG_MID), representing
the average of GI50 and TGI over all the cell lines (full panel), as well as
subpanel averages for each of the tested molecules were also calculated.
Furthermore, a selectivity index was calculated by dividing the full
panel MID by their individual subpanel MID and was used to determine
the criterion for selectivity of a compound [23] (Tables 3 and 4).
Compounds 2f and 4a showed effective growth inhibition with MG-
MID values of GI50 9.51 and 8.69 µM, respectively and MG-MID values
of TGI (MG-MID) of 30.13 and 24.53 µM, respectively. The calculated
selectivity index ranged from 0.57 to 1.68 and from 0.30 to 1.65, for
compounds 2f and 4a respectively. Selectivity index values lower than
3 indicate lack of selectivity [23]. These values suggest that compounds
2f and 4a have non-selective, broad spectrum antitumor activity
against all tumor subpanels tested.
In addition to the distinctive conformation, Type I inhibitors are
also reported to form distinctive interactions with the ATP binding site,
a hydrogen bonds with Met1160 in the hinge region, as well as π
stacking with Tyr1230 residue [10,27]. Upon examination of the se-
lected docked poses of the most active compounds, it was found that the
previously mentioned compounds were able to form the π stacking with
Tyr1230 residue, whereas only compound 4a was able to form a hy-
drogen bond with Met1160 residue backbone NH and the triazole ring
(Fig. 4). The difference in the ability to form the hydrogen bond is due
to the different orientation the molecules adopt in the binding pocket.
The triazolopyridazine ring of compound 4a is oriented towards the
hinge ring thus able to form the hydrogen bond with Met1160 residue,
and the phenyl ring is oriented towards the Tyr1230 (Fig. 4, top). For
compounds 2f, the pyridine ring is oriented towards Tyr1230 residue
forming the π stacking, therefore the molecule is unable to interact with
the Met1160 residue (Fig. 4, middle). The remaining compounds, 2a,
2b, 2c & 2i, adopted a different conformation, where the triazolopyr-
idazine is oriented towards the Tyr1230 residue, leaving the phenyl
ring oriented towards the Met1160 residue, and the compound unable
to form hydrogen bond. This orientation is in agreement with the or-
ientation reported by Albrecht et al. and Buchanan et al., where the
crystal structures of c-Met kinase co-crystallized with triazolopyr-
idazine derivatives (Fig. 1, compounds I and II) where the triazolo-
pyridazine moiety is responsible for the π stacking [15,20]. Compounds
I and II were able to form Hydrogen bonds with Met1160 through
2.2.2. In vitro c-Met kinase inhibition
In light of the promising cytotoxic activity exhibited by compounds
2f and 4a, their activity as c-Met kinase inhibitors in vitro was evaluated
3

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
Table 2
Table 2 (continued)
In vitro anticancer activity of compounds 2f and 4a against 60 human cancer
Panel cell
line
Compound
2f
cell lines.
Panel cell
Compound
2f
4a
line
4a
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
MCF7
2.85
5.27
24.60
24.20
> 100 3.10
16.10 54.20
25.60 67.10
MDA-MB-231/
95.40 9.39
Leukaemia
ATCC
CCRF-CEM
HL60(TB)
K-562
4.28
32.5
15.1
6.99
–
> 100 > 100 3.12
17.40 > 100
HS 578T
BT-549
18.30 53.90
> 100 18.10 58.30 > 100
> 100 > 100 14.30 39.60 > 100
> 100 > 100 11.10 60.40 > 100
2.59
4.30
1.96
8.33
22.30
4.59
98.20
98.20
25.20
1.90
4.69
1.98
3.83 7.73
20.30 68.60
7.32 76.80
T-47D
MOLT-4
RPMI-8226
SR
> 100 > 100 5.18
30.40 > 100
17.40 > 100
MDA-MB-468
–
> 100 3.43
5.23
> 100 > 100 2.83
14.6
> 100
Non-Small Cell Lung Cancer
A549/ATCC
HOP-62
Table 3
Median growth inhibitory concentrationsa (GI50, µM) of in vitro subpanel tu-
13.30 62.00
15.40 33.50
16.90 35.80
> 100
73.10
76.00
–
–
–
–
9.93
–
22.40 50.30
mour cell lines.
Subpanel cell line
HOP-92
–
–
Compound
NCI-H226
–
–
15.00 44.40 > 100
NCI-H23
14.10 54.20
10.60 23.60
> 100 15.00 40.40 > 100
2f
4a
NCI-H322M
NCI-H460
52.60
3.63
14.90 38.60
16.90 49.00
18.00 42.90
4.27
24.00
> 100 4.44
MG-MID Selectivity
index
MG-MID Selectivity
index
NCI-H522
10.40 22.80
50.10
4.46
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
13.00 30.80
23.50 51.90
73.40
11.60 26.70 61.30
Leukemia
12.82
12.14
0.74
0.78
6.66
8.74
1.31
0.99
> 100 16.60 34.50 71.50
Non-small cell lung
cancer
4.09
6.88
5.66
7.34
6.48
21.00
66.30
24.20
23.90
36.00
> 100 3.77
> 100 5.48
> 100 6.39
14.30 37.80
22.40 71.10
19.20 47.00
21.20 46.10
19.10 52.80
Colon cancer
CNS cancer
9.56
0.99
0.78
1.51
0.70
1.68
0.76
1.62
8.39
1.04
0.77
1.09
0.72
1.32
0.57
1.33
HT29
12.20
6.28
11.25
7.95
KM12
67.90
9.34
Melanoma
SW-620
> 100 5.60
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Full panel MG-MIDb
13.55
5.66
12.09
6.57
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3 M
M14
12.45
5.88
15.25
6.53
5.60
29.20
> 100 5.56
21.00 58.70
13.30 40.10
14.60 55.90
14.20 40.80
12.20 25.00
13.30 28.00
> 100
–
–
–
9.51
8.69
> 100 15.30 28.90 54.40
> 100 14.40 31.80 70.20
a
Median value calculated according to the data obtained from NCI's in vitro
51.60
59.00
13.60 26.50 51.50
7.40
20.60 46.00
disease-oriented human tumour cell screen.
b
GI50 (µM) full panel mean-graph mid point(MG-MID) = the average sen-
sitivity of all cell lines towards the test agents.
1.97
7.97
7.03
4.02
3.15
4.10
8.54
1.87
8.38
3.75
7.55
26.30
40.90
23.10
9.08
78.50
22.50 53.60
> 100 11.10 26.50 63.40
Table 4
Median total growth inhibitory concentrationsa (TGI, µM) of in vitro subpanel
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
> 100 7.10
26.90 84.50
59.20
68.70
40.50
–
–
–
tumor cell lines.
Subpanel cell line
12.70 29.60
15.30 29.80 57.90
3.92
10.6
5.18
14.80
33.60
19.50
4.74
16.90 43.10
22.20 55.30
Compound
> 100 7.15
49.90
–
–
–
2f
4a
Ovarian Cancer
MG-MID Selectivity
index
MG-MID Selectivity
index
IGROV1
5.29
9.70
–
22.90
24.80
–
99.20
62.40
–
2.23
5.41
22.10
OVCAR-3
13.70 26.60 51.60
15.10 30.50 61.60
OVCAR-4
Leukemia
> 100
36.56
0.30
0.82
29.97
26.17
0.82
0.94
OVCAR-5
17.40 79.30
> 100 15.40 29.80 58.00
Non-small cell lung
cancer
OVCAR-8
8.10
18.80 77.40
22.00 > 100 > 100 15.40 44.30 > 100
41.70
> 100 8.07
31.50 > 100
NCI/ADR-RES
SK-OV-3
> 100 14.70 44.70 > 100
Colon cancer
CNS cancer
36.30
36.50
22.33
57.68
18.24
28.75
22.99
30.13
0.83
0.83
1.35
0.52
1.65
1.05
1.30
22.49
25.76
21.22
30.40
19.00
28.80
21.91
24.53
1.09
0.95
1.16
0.81
1.29
0.85
1.12
Renal Cancer
Melanoma
786-0
4.64
19.70
73.00
56.20
63.30
44.40
39.90
83.00
77.10
44.80
10.80 23.10 49.60
14.20 27.20 52.20
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Full panel MG-MIDb
A498
15.50 29.50
ACHN
4.24
3.22
3.18
3.56
7.13
3.81
17.40
11.70
9.98
6.32
2.45
3.77
3.69
6.81
4.49
20.80 53.90
CAKI-1
RXF 393
SN12C
TK-10
6.42
24.20
18.10 72.70
18.10 70.30
20.70 50.50
17.60 43.60
17.10
25.00
15.50
a
Median value calculated according to the data obtained from NCI's in vitro
UO-31
disease-oriented human tumor cell screen.
b
TGI (µM) full panel mean-graph mid point(MG-MID) = the average sensi-
Prostate Cancer
tivity of all cell lines toward the test agents.
PC-3
13.40 31.90
11.50 25.60
76.20
56.70
16.20 30.30 56.70
14.30 27.30 52.30
DU-145
quinolone moieties that is connected to the triazolopyridazine through
a spacer, which is absent in our compounds (Fig. 4, bottom). The
docking score of the compounds showing the highest activity are listed
in Table 6.
Breast Cancer
4

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
Table 5
substitution at position 2 of the phenyl ring showed the highest cyto-
Inhibition activity of 2f and 4a towards c-Met kinase receptor.
toxic activity (4a).
Compound
% c-Met kinase activity
IC50 (µM)
Concentration (µM)
3. Experimental
0.001
0.01
0.1
1
10
50
100
3.1. Chemical synthesis
2f
−3
−1
−3
1
−5
0
−9
−5
−17
−8
−38
−22
−51
−35
106.9
242.2
4a
All chemicals and reagents were obtained from Aldrich
(Sigma–Aldrich, St. Louis, MO, USA), and were used without further
purification. Reactions were monitored by TLC, performed on silica gel
glass plates containing 60 GF-254, and visualization on TLC was
achieved by UV light or iodine indicator. IR spectra were determined on
Shimadzu IR 435 spectrophotometer (KBr, cm−1). 1H NMR spectra
were carried out using a Mercury 300-BB 300 MHz using TMS as an
internal standard. Chemical shifts (δ) were recorded in ppm on δ scale,
Micro analytical Centre, Cairo University, Egypt. 13C NMR spectra were
carried out using a Mercury 300-BB 75 MHz using TMS as an internal
standard. Chemical shifts (δ) were recorded in ppm on δ scale, Micro
analytical Centre, Cairo University, Egypt. Mass spectra were recorded
on Shimadzu Qp-2010 plus Spectrometer, Micro analytical Centre,
Cairo University, Egypt. Elemental analyses were carried out at the
Micro analytical Centre, Cairo University, Egypt. Their results corre-
sponded to the calculated values within 0.4% experimental error.
Melting points were determined with Stuart apparatus and are un-
corrected.
The intra-assay variability is less than 10%.
Only values of more than 25% change in activity compared to the control are
considered to be significant.
The starting material, 3,6-dichloropyridazine was commercially
available. The key intermediates; 6-chloro-3-substituted-aminopyr-
idazines [21,22] 1a-e and 1 g,h 2-(6-chloropyridazine-3-yloxy)benza-
mide [21] (3b) 4-(6-chloropyridazin-3-ylamino)benzene sulphonamide
[22] (5a) and N-carbamimidoyl-4-[(6-chloropyridazin-3-yl)amino]
benzene sulphonamide [22] (5b) were prepared according to the re-
ported procedures.
Fig. 2. Dose response curve for compounds 2f and 4a (log inhibitor con-
centration (μM) vs. % normalized c-Met kinase activity).
2.4. Structure activity relationship
Upon examination of both the biological screening results, as well as
the docking results it was found that the use of an oxygen spacer, in-
stead of carbon between the pyradizine ring and substituted aromatic
3.1.1. General procedure for 1f, 1i and 3a
ring resulted in
a compound that showed poor activity (4b).
A mixture of 3,6-dichloropyridazine (1.48 g, 0.01 mol), anhydrous
K2CO3 (0.02 mol) and the appropriate amino compound (0.01 mol) in
isopropanol (30 mL) was heated under reflux for 4–5 h. The reaction
mixture was concentrated under reduced pressure to half its volume
and cooled. The separated solid was filtered, washed with water
(20 mL), dried and crystallized from isopropanol.
Additionally, the substituted of the aromatic ring attached to the pyr-
idazine with a heterocyclic ring produced a compound with no cycto-
toxic activity (6a & 6b). Similarly, the substitution of the aromatic ring
with a sulphonamide or a carbamimidoylsulphonamide group yielded
compounds with little or no cytotoxic activity (2e). The use of halogen
substituents yielded mixed results, monohalogenated compounds pro-
duced compounds with moderate activity (2a, 2b, 2c & 2i), while the
dihalogenated derivative showed poor activity (2d). Similarly, the use
of carboxylic acid substituent also yielded mixed results, the addition of
the carboxylic acid substituent in position 3 or 4 produced compounds
that showed little or no activity (2g & 2h), while the addition of the
carboxylic acid group position 2 produced a compound that showed
excellent cytotoxic activity (2f). The compound bearing an amide
3.1.2. 2-(6-Chloropyridazin-3-ylamino)benzoic acid (1f)
Yield 72%; mp 220–221 °C; IR (KBr) (cm−1): 3400–2318 (br., OeH
and NeH), 1747 (C]O), 1620 (C]N); 1H NMR (300 MHz, DMSO‑d6) δ:
7.60–7.65 (m, 2H, Ar-H), 7.69 (d, 1H, pyridazine, J = 9.9 Hz), 7.80 (d,
1H, Ar-H, J = 8.7 Hz), 7.94 (d, 1H, pyridazine, J = 9.9 Hz), 8.32 (d, 1H,
Ar-H, J = 8.7 Hz); MS (EI, 70 ev) m/z (%): 249 (M+· , 0.1), 205 (3.2), 137
(0.8), 128 (1.3), 121 (0.9), 113 (5.3). Anal. Calcd for C11H8ClN3O2
Fig. 3. Orientation of compounds 2a (green), 2b (blue), 2c (grey), 2f (pink), 2i (orange) & 4a (yellow) in the ATP binding site of c-Met kinase. Hydrogen atoms not
shown for clarity.
5

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
Fig. 4. Binding modes of compounds 2f, shown in purple (top), 4a, shown in yellow (middle), compound 2c, shown in grey, superimposed with the crystal structure
of ligand I, shown in blue (bottom), in the c-Met kinase binding pocket. Images showing key interacting residues, as well as key interactions formed. Hydrogen atoms
not shown for clarity.
(249.65): C, 52.92; H, 3.23; N, 16.83, Found, C, 52.66; H, 2.94; N,
16.95.
and NeH), 1728 (C]O), 1616 (C]N); 1H NMR (300 MHz, DMSO‑d6) δ:
7.62–7.65 (m, 2H, Ar-H), 7.70 (d, 1H, pyridazine, J = 9.9 Hz), 7.87 (s,
1H, NH, D2O exchangeable), 7.95 (d, 1H, pyridazine, J = 9.9 Hz), 8.30
(s, 1H, Ar-H); MS (EI, 70 ev) m/z (%): 283 (M+· , 0.05), 239 (5.6), 171
(0.2), 128 (0.4), 113 (1.7). Anal. Calcd for C11H7Cl2N3O2 (284.10): C,
3.1.3. 5-Chloro-2-(6-chloropyridazin-3-ylamino)benzoic acid (1i)
Yield 70%; mp 204–205 °C; IR (KBr) (cm−1): 3433–2364 (br., OeH
Table 6
Docking score and interactions of the compounds showing the highest activity (2f & 4a).
Compound
Energy score (Kcal/mol)
Amino acid
Interacting group
Length (Å)
2f
−7.53
−6.88
Tyr 1230 (π-π stacking)
Tyr 1230 (π-π stacking)
Met 1160 (H-bond)
Pyrimidine ring
Phenyl ring
3.75
3.65
3.20
4a
N of triazole ring
6

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
46.50; H, 2.48; N, 14.79, Found, C, 46.30; H, 2.65; N, 14.98.
0.4), 211 (1.9), 196 (2.0), 161 (3.2), 145 (2.9). Anal. Calcd for
C16H10Cl2N6 (357.20): C, 53.80; H, 2.82; N, 23.50, Found, C, 53.70; H,
2.45; N, 23.23.
3.1.4. 2-(6-Chloropyridazin-3-ylamino)benzamide (3a)
Yield 68%; mp 150–151 °C; IR (KBr) (cm−1): 3401, 3392 (NH2),
3350 (NeH), 1710 (C]O), 1619 (C]N); 1H NMR (300 MHz, DMSO‑d6)
δ: 7.36 (s, 2H, NH2, D2O exchangeable), 7.61 (d, 1H, pyridazine,
J = 9.6 Hz), 7.77–7.92 (m, 3H, Ar-H), 7.95 (d, 1H, pyridazine,
J = 9.6 Hz), 8.30 (d, 1H, Ar-H, J = 9.3 Hz); MS (EI, 70 ev) m/z (%): 250
(M + 2, 14.87), 248 (M+· , 15.6), 231 (100.0), 135 (0.7), 113 (24.5).
Anal. Calcd for C11H9ClN4O (248.67): C, 53.13; H, 3.65; N, 22.53,
Found, C, 52.95; H, 3.74; N, 22.48.
3.1.10. N-(2-Chloropyridin-3-yl)-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-6-amine (2e)
Yield 70%; mp 269–270 °C; IR (KBr) (cm−1): 3394 (NeH), 1631
(C]N); 1H NMR (300 MHz, DMSO‑d6) δ: 7.68 (d, 1H, pyridazine,
J = 9.6 Hz), 7.92–8.51 (m, 3H, pyridine), 8.64 (d, 1H, pyridazine,
J = 9.6 Hz), 8.84 (d, 2H, pyridine, J = 6.3 Hz), 8.94 (d, 2H, pyridine,
J = 6.3 Hz), 10.19 (s, 1H, NH, D2O exchangeable); MS (EI, 70 ev) m/z
(%): 325 (M + 2, 1.4), 323 (M+· , 0.1), 211 (0.6), 196 (100.0), 146 (1.9),
127 (1.1), 92 (9.1). Anal. Calcd for C15H10ClN7 (323.74): C, 55.65; H,
3.11; N, 30.29, Found, C, 55.76; H, 2.95; N, 30.45.
3.1.5. General procedure 2a-i, 4a,b and 6a,b
A solution of an appropriate either of 1a-i or 3a,b or 5a,b
(0.001 mol) and isonicotinic acid hydrazide (0.14 g, 0.001 mol) in n-
butanol (20 mL) was heated under reflux for 6–8 h. The solution was
filtered while hot, the filtrate was concentrated under reduced pressure
to half its volume and cooled. The separated solid was filtered, washed
by sodium carbonate solution (10%, 20 mL), then crystallized from
benzene.
3.1.11. 2-[3-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-ylamino]
benzoic acid (2f)
Yield 72%; mp 200–201 °C; IR (KBr) (cm−1): 3390–2476 (br.,
OeH), 3244 (NeH), 1708 (C]O), 1624 (C]N); 1H NMR (300 MHz,
DMSO‑d6) δ: 7.58–7.79 (m, 3H, Ar-H), 7.91 (d, 1H, pyridazine,
J = 9.3 Hz), 7.94–7.97 (m, 1H, Ar-H), 8.32 (d, 1H, pyridazine,
J = 9.3 Hz), 8.64 (d, 2H, pyridine, J = 6 Hz), 8.80 (d, 2H, pyridine,
J = 6 Hz), 10.21 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6
ppm) δ: 119.0, 119.1, 126.7, 126.8, 126.9, 127.1, 127.2, 128.7, 135.1,
135.2, 137.5, 144.2, 146.6, 146.7 (aromatic C's), 156.7 (C]O); MS (EI,
70 ev) m/z (%): 332 (M+· , 2.3), 211 (0.9), 121 (6.5), 136 (1.7), 93
(11.1). Anal. Calcd for C17H12N6O2 (332.32): C, 61.44; H, 3.64; N,
25.29, Found, C, 61.76; H, 3.23; N, 25.43.
3.1.6. N-(2-Bromophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-6-amine (2a)
Yield 76%; mp 220–221 °C; IR (KBr) (cm−1): 3383 (NeH), 1631
(C]N); 1H NMR (300 MHz, DMSO‑d6) δ: 7.63 (d, 1H, pyridazine,
J = 9.9 Hz), 7.84–7.92 (m, 2H, Ar-H), 8.40–8.45 (m, 2H, Ar-H), 8.52 (d,
1H, pyridazine, J = 9.9 Hz), 8.80 (d, 2H, pyridine, J = 6.3 Hz), 8.88 (d,
2H, pyridine, J = 6.3 Hz), 10.02 (s, 1H, NH, D2O exchangeable); MS
(EI, 70 ev) m/z (%): 366 (M+· , 30.2), 288 (15.4), 197 (6.0), 92 (28.8), 91
(6.0). Anal. Calcd for C16H11BrN6 (367.20): C, 52.33; H, 3.02; N, 22.89,
Found, C, 52.56; H, 2.96; N, 22.95.
3.1.12. 3-[3-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-ylamino]
benzoic acid (2g)
Yield 76%; mp > 300 °C; IR (KBr) (cm−1): 3400–2538 (br., OeH),
3290 (NeH), 1685 (C]O), 1631 (C]N); 1H NMR (300 MHz, DMSO‑d6)
δ: 7.25 (d, 1H, pyridazine, J = 9.9 Hz), 7.50–7.73 (m, 3H, Ar-H), 8.24
(d, 1H, pyridazine, J = 9.9 Hz), 8.50 (d, 2H, pyridine, J = 6 Hz), 8.63
(s, 1H, Ar-H), 8.84 (d, 2H, pyridine, J = 6 Hz), 10.21 (s, 1H, NH, D2O
exchangeable), 13.00 (br, 1H, OH, D2O exchangeable); MS (EI, 70 ev)
m/z (%): 332 (M+· , 6.9), 288 (99.7), 196 (15.0), 136 (6.1), 93 (9.0).
Anal. Calcd for C17H12N6O2 (332.32): C, 61.44; H, 3.64; N, 25.29,
Found, C, 61.28; H, 3.44; N, 25.05.
3.1.7. N-(2-Chlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-6-amine (2b)
Yield 65%; mp 209–210 °C; IR (KBr) (cm−1): 3417 (NeH), 1631
(C]N); 1H NMR (300 MHz, DMSO‑d6) δ: 7.51–7.61 (m, 2H, Ar-H), 7.77
(d, 1H, pyridazine, J = 9.6 Hz), 8.14–8.17 (m, 2H, Ar-H), 8.38 (d, 1H,
pyridazine, J = 9.6 Hz), 8.95 (d, 2H, pyridine, J = 6 Hz), 8.99 (d, 2H,
pyridine, J = 6 Hz), 9.86 (s, 1H, NH, D2O exchangeable); MS (EI, 70 ev)
m/z (%): 324 (M + 2, 33.9), 322 (M+· , 100.0), 212 (4.4), 196 (31.5),
128 (16.2), 126 (4.4), 111 (22.7). Anal. Calcd for C16H11ClN6 (322.07):
C, 59.54; H, 3.44; N, 26.04, Found, C, 59.88; H, 3.56; N, 26.14.
3.1.13. 4-[3-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-ylamino]
benzoic acid (2h)
Yield 70%; mp 240–241 °C; IR (KBr) (cm−1): 3440–2546 (br.,
OeH), 3294 (NeH), 1666 (C]O), 1612 (C]N); 1H NMR (300 MHz,
DMSO‑d6) δ: 7.24 (d, 1H, pyridazine, J = 9.9 Hz), 7.30 (d, 2H, Ar-H,
J = 9.3 Hz), 7.63 (d, 2H, Ar-H, J = 9.3 Hz), 8.27 (d, 1H, pyridazine,
J = 9.9 Hz), 8.40 (d, 2H, pyridine, J = 6 Hz), 8.91 (d, 2H, pyridine,
J = 6 Hz), 10.25 (s, 1H, NH, D2O exchangeable), 12.55 (br, 1H, OH,
D2O exchangeable); MS (EI, 70 ev) m/z (%): 332 (M+· , 0.3), 288 (0.2),
212 (0.6), 196 (2.0), 136 (6.3), 93 (11.4). Anal. Calcd for C17H12N6O2
(332.32): C, 61.44; H, 3.64; N, 25.29, Found, C, 61.58; H, 3.55; N,
25.22.
3.1.8. N-(4-Chlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-6-amine (2c)
Yield 68%; mp 275–276 °C; IR (KBr) (cm−1): 3267 (NeH), 1635
(C]N); 1H NMR (300 MHz, DMSO‑d6) δ: 7.27 (d, 1H, pyridazine,
J = 9 Hz), 7.48 (d, 2H, Ar-H, J = 8.4 Hz), 7.70 (d, 2H, Ar-H,
J = 8.4 Hz), 8.20 (d, 1H, pyridazine, J = 9 Hz), 8.52 (d, 2H, pyridine,
J = 6.3 Hz), 8.98 (d, 2H, pyridine, J = 6.3 Hz), 10.39 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6 ppm) δ: 118.6, 120.7, 121.3, 121.6,
126.4, 128.7, 137.3, 138.0, 143.5, 144.5, 146.3, 1+49.7 (aromatic C’s);
MS (EI, 70 ev) m/z (%): 324 (M + 2, 33.6), 322 (M· , 100.0), 212 (7.9),
196 (48.5), 126 (8.3), 111 (43.7), 91 (8.4). Anal. Calcd for C16H11ClN6
(322.07): C, 59.54; H, 3.44; N, 26.04, Found, C, 59.46; H, 3.48; N,
25.88.
3.1.14. 5-Chloro-2-[3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-
ylamino]benzoic acid (2i)
Yield 62%; mp 250–251 °C; IR (KBr) (cm−1): 3410–2623 (br., OeH),
3272 (NeH), 1693 (C]O), 1620 (C]N); 1H NMR (300 MHz, DMSO‑d6) δ:
7.44 (d, 1H, pyridazine, J = 9.9 Hz), 7.51–7.80 (m, 2H, Ar-H), 8.01 (s, 1H,
Ar-H), 8.16 (d, 1H, pyridazine, J = 9.9 Hz), 8.85 (d, 2H, pyridine,
J = 6.3 Hz), 8.90 (d, 2H, pyridine, J = 6.3 Hz), 9.80 (s, 1H, NH, D2O ex-
changeable), 11.13 (s, 1H, OH, D2O exchangeable); MS (EI, 70 ev) m/z
(%): 368 (M + 2, 2.9), 366 (M+· , 7.8), 321 (0.3), 212 (2.6), 196 (1.9), 169
(1.9), 125 (1.8). Anal. Calcd for C17H11ClN6O2 (366.76): C, 55.67; H, 3.02;
N, 22.91, Found, C, 55.86; H, 2.96; N, 22.82.
3.1.9. N-(2,6-Dichlorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-6-amine (2d)
Yield 84%; mp 245–246 °C; IR (KBr) (cm−1): 3394 (NeH), 1631
(C]N); 1H NMR (300 MHz, DMSO‑d6) δ: 7.64 (d, 1H, pyridazine,
J = 9.6 Hz), 7.84–7.93 (m, 1H, Ar-H), 8.35 (d, 1H, pyridazine,
J = 9.6 Hz), 8.60 (d, 2H, Ar-H, J = 9.6 Hz), 8.80 (d, 2H, pyridine,
J = 6 Hz), 8.88 (d, 2H, pyridine, J = 6 Hz), 10.02 (s, 1H, NH, D2O ex-
changeable); MS (EI, 70 ev) m/z (%): 360 (M + 4, 0.4), 358 (M + 2,
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3.1.15. 2-[3-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-ylamino]
benzamide (4a)
and growth; a 48 h drug exposure protocol and sulforhodamine B (SRB)
protein assay were employed [28–31]. The results were reported as the
percentage growth of the treated cells in comparison to the untreated
control cells. Additionally, a dose response curve was plotted and the
following parameters, GI50, representing the molar concentration of the
tested compound that results in a 50% decrease in cell growth, TGI,
representing the molar concentration of the tested compound that re-
sults in total growth inhibition and LC50, reflecting the molar con-
centration of the tested compound that results in 50% net cell death
were calculated.
Yield 75%; mp 205–206 °C; IR (KBr) (cm−1): 3437, 3417 (NH2),
3387 (NeH), 1665 (C]O), 1624 (C]N); 1H NMR (300 MHz, DMSO‑d6)
δ: 4.62 (br, 2H, NH2, D2O exchangeable), 6.85–6.88 (m, 3H, Ar-H), 6.98
(d, 1H, pyridazine, J = 9.9 Hz), 7.51 (d, 1H, pyridazine, J = 9.9 Hz),
7.73 (d, 1H, Ar-H, J = 6 Hz), 7.82 (d, 2H, pyridine, J = 6.3 Hz), 8.65 (s,
1H, NH, D2O exchangeable), 8.70 (d, 2H, pyridine, J = 6.3 Hz); MS (EI,
70 ev) m/z (%): 331 (M+· , 6.2), 315 (1.9), 287 (4.1), 212 (1.3), 196
(4.6), 136 (10.8), 121 (10.8). Anal. Calcd for C17H13N7O (331.33): C,
61.62; H, 3.95; N, 29.59, Found, C, 61.58; H, 4.10; N, 29.65.
3.2.2. In vitro c-Met kinase inhibition
Using proprietary methods; c-Met kinase was cloned, expressed and
purified to be employed in the compound profiling process. Moreover,
quality control testing was regularly done to ensure compliance to ac-
ceptable standards. 33P-ATP was purchased from PerkinElmer, while all
other materials were of standard laboratory grade. Stock solution of the
synthesized compounds was prepared in DMSO, which was then diluted
to be used for profiling against c-Met protein kinase.
3.1.16. 2-[3-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yloxy]
benzamide (4b)
Yield 80%; mp 79–80 °C; IR (KBr) (cm−1): 3410, 3390 (NH2), 1666
(C]O), 1631 (C]N); 1H NMR (300 MHz, DMSO‑d6) δ: 4.98 (br, 2H,
NH2, D2O exchangeable), 7.60 (d, 1H, pyridazine, J = 9.9 Hz),
7.65–7.95 (m, 4H, Ar-H), 7.94 (d, 2H, pyridine, J = 6.6 Hz), 7.96 (d,
1H, pyridazine, J = 9.9 Hz), 8.35 (d, 2H, pyridine, J = 6.6 Hz); MS (EI,
70 ev) m/z (%): 332 (M+· , 0.1), 288 (0.2), 212 (0.2), 196 (0.4), 136
(0.9), 78 (87.2). Anal. Calcd for C17H12N6O2 (332.32): C, 61.44; H,
3.64; N, 25.29, Found, C, 61.50; H, 3.74; N, 25.43.
To evaluate the profiling of the protein kinase target; a radioisotope
assay format was employed and all assays were performed in a desig-
nated radioactive working area. Protein kinase assays were performed
in singlicates at room temperature for 20–30 min in a final volume of
25 μl according to the following assay reaction recipe: Component 1.
5 μl of diluted active c-Met (∼10–50 nM final concentration in the
assay), component 2.5 μl of stock solution of substrate, component
3.5 μl of kinase assay buffer, component 4.5 µl of compound (various
concentration) or 10% DMSO and component 5.5 µl of 33P-ATP (250 μM
stock solution, 0.8 μCi).
3.1.17. 4-[3-(Pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-ylamino]
benzene- sulfonamide (6a)
Yield 76%; mp 100–101 °C; IR (KBr) (cm−1): 3464, 3356 (NH2),
3271 (NeH), 1666 (C]O), 1631 (C]N), 1315, 1153 (SO2); 1H NMR
(300 MHz, DMSO‑d6) δ: 5.75 (br, 2H, NH2, D2O exchangeable), 6.57 (d,
1H, pyridazine, J = 9.6 Hz), 6.59–7.43 (m, 4H, Ar-H), 6.85 (s, 1H, NH,
D2O exchangeable), 7.45 (d, 1H, pyridazine, J = 9.6 Hz), 7.73 (d, 2H,
pyridine, J = 6 Hz), 8.70 (d, 2H, p+yridine, J = 6 Hz); MS (EI, 70 ev) m/z
(%): 368 (M + H, 13.6), 367 (M· , 15.5), 352 (18.6), 287 (13.6), 78
(85.0). Anal. Calcd for C16H13N7O2S (367.39): C, 52.31; H, 3.57; N,
26.69, Found, C, 52.16; H, 3.66; N, 26.73.
33P-ATP was added to initiate the assay and the reaction mixture
was incubated at room temperature for 20–30 min. To terminate the
assay after the incubation period; 10 μl of the reaction mixture were
spotted onto multiscreen phosphocellulose P81 plate, which was then
washed 3 times in 1% phosphoric acid solution for approximately
15 min. The radioactivity on the P81 plate was counted using a Trilux
scintillation counter in the presence of scintillation fluid. A blank
control was set up in the same manner, however, the appropriate
substrate was replaced with equal volume of assay dilution buffer.
Finally, to determine the corrected activity for protein kinase target; the
blank control values were removed [24,25].
3.1.18. N-Carbamimidoyl-4-[3-(pyridin-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-6-ylamino] benzenesulfonamide (6b)
Yield 72%; mp 129–130 °C; IR (KBr) (cm−1): 3417, 3325 (NH2),
3201 (NeH), 1631 (C]N), 1323, 1134 (SO2); 1H NMR (300 MHz,
DMSO‑d6) δ: 4.41 (br, 2H, NH2, D2O exchangeable), 6.63 (d, 2H, Ar-H,
J = 8.7 Hz), 6.75 (s, 1H, NH, D2O exchangeable), 7.43 (d, 2H, Ar-H,
J = 8.7 Hz), 7.68 (d, 1H, pyridazine, J = 9.3 Hz), 8.43 (d, 2H, pyridine,
J = 6.3 Hz), 8.64 (d, 1H, pyridazine, J = 9.3 Hz), 8.91 (d, 2H, pyridine,
J = 6.3 Hz), 10.00 (s, 1H, NH, D2O exchangeable), 11.02 (s, 1H, NH,
D2O exchangeable); MS (EI, 70 ev) m/z (%): 409 (M+· , 0.1), 393 (0.1),
351 (0.2), 287 (0.3), 213 (4.9), 196 (8.9), 123 (13.2), 78 (4.4) 57
(100.0). Anal. Calcd for C17H15N9O2S (409.43): C, 49.87; H, 3.69; N,
30.79; Found, C, 49.96; H, 3.45; N, 30.88.
3.3. Molecular docking
Molecular docking in the ATP binding site of c-Met was carried out
for all the synthesized compounds using Autodock 4.2 [32]. The crystal
structure of c-Met kinase (PDB code: 3CD8) [15] was downloaded from
the protein databank (https://www.rcsb.org/pdb) [33] and was pre-
pared using Autodock tools [32]. All water molecules were deleted from
the crystal structure before docking. This was then followed by as-
signing partial charges using Gasteiger charges to both the protein
structure and ligands. The binding pocket was defined using a grid box
cantered on the native ligand with dimensions 60 × 60 × 60 points
with a spacing of 0.375 Å. The Lamarckian genetic algorithm was used
to carry out a 100 docking runs for each compound using the default
Autodock parameters [32]. UCSF Chimera [34] was then used to vi-
sualize and analyse the results.
3.2. Biological studies
3.2.1. Cytotoxic activity
The cytotoxic activity of the synthesized target compounds (2a-i,
4a, b and 6a, b) was evaluated using the NCI’s disease-oriented human
cell lines screening assay. The cytotoxic assays were performed in ac-
cordance with the protocol of the Drug Evaluation Branch, NCI,
Bethesda, MD, USA. Initially, all the synthesized compounds were first
evaluated at a single concentration of 10−5 M towards a panel of ap-
proximately 60 cancer lines. The human tumor cell lines used were
derived from nine different cancer types: leukemia, melanoma, lung,
colon, CNS, ovarian, renal, prostate and breast cancers. The cultures
were incubated for 48 h, and the sulforhodamine B (SRB) protein assay
was employed as previously reported [28–31]. Compounds showing
significant cytotoxic activity, were then reevaluated using 5 different
concentrations ranging for 10−4–10−8 M. To estimate the cell viability
4. Conclusion
This study reports the synthesis and anticancer activity assessment
of 3-(pyridin-4-yl)-[1,2,4] triazolo[4,3-b]pyridazine derivatives 2a-i,
4a,b and 6a,b according to the protocol of the Drug Evaluation Branch,
NCI, USA. Interestingly, the majority of the tested compounds demon-
strated promising activity against various leukaemia cell panels. Two
compounds, namely 2f and 4a, bearing carboxylic acid and carbox-
amide substituents respectively at the 2- position of the phenyl ring
8

E.M. Ahmed, et al.
BioorganicChemistry92(2019)103272
scaffold, emerged as promising cytotoxic agents against the entire panel
of tumor cell lines. The MG-MID GI50 (µM) values of the two most
potent analogs 2f and 4a recorded 9.51 and 8.69 respectively.
Additionally, these compounds also showed significant activity as c-Met
kinase inhibitors, with IC50 values of 106.9 μM and 242.2 μM, respec-
tively.
[14] T.C.C. Yau, R. Lencioni, W. Sukeepaisarnjaroen, Y. Chao, C.-J. Yen,
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K.E. Raffensperger, D.A. Kulkarni, H. Kallender, L.H. Ottesen, R.T.P. Poon,
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Declaration of Competing Interest
[19] T.L. Underiner, T. Herbertz, S.J. Miknyoczki, Discovery of small molecule c-Met
inhibitors: evolution and profiles of clinical candidates, anti-cancer agents in
medicinal, Chem.- Anti-Cancer Age. 10 (1) (2010) 7–27.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[20] S.G. Buchanan, J. Hendle, P.S. Lee, C.R. Smith, P.-Y. Bounaud, K.A. Jessen,
C.M. Tang, N.H. Huser, J.D. Felce, K.J. Froning, M.C. Peterman, B.E. Aubol,
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Acknowledgment
Thanks are due to the NCI, Bethesda, MD, U.S.A. for performing the
antitumor testing of the synthesized compounds and for Kinexus,
Canada for performing the c-Met inhibition activity.
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