Synthesis and bioevaluation of 6-chloropyridazin-3-yl hydrazones and 6-chloro-3-substituted-[1,2,4]triazolo[4,3-b]pyridazines as cytotoxic agents

Article abstract of DOI:10.1016/j.bioorg.2019.01.049

An efficient synthesis of a series of 6-chloro-3-substituted-[1,2,4]triazolo[4,3-b]pyridazines is described via intramolecular oxidative cyclization of various 6-chloropyridazin-3-yl hydrazones with iodobenzene diacetate. The structures of the newly synth

Full text of DOI:10.1016/j.bioorg.2019.01.049

BioorganicChemistry86(2019)288–295
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and bioevaluation of 6-chloropyridazin-3-yl hydrazones and 6-
chloro-3-substituted-[1,2,4]triazolo[4,3-b]pyridazines as cytotoxic agents
⁎
Mamtaa, Ranjana Aggarwala, , Rachna Sadanab, Jeziel Ilagb, Garima Sumranc
a Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
b Department of Natural Sciences, University of Houston, Downtown, Houston 77002, USA
c Department of Chemistry, D. A. V. College (Lahore), Ambala City 134 002, Haryana, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
An efficient synthesis of a series of 6-chloro-3-substituted-[1,2,4]triazolo[4,3-b]pyridazines is described via in-
tramolecular oxidative cyclization of various 6-chloropyridazin-3-yl hydrazones with iodobenzene diacetate.
The structures of the newly synthesized compounds were assigned on the basis of elemental analysis, IR, NMR
(1H and 13C) and mass spectral data. All the thirty three compounds 3a-q and 4b-q synthesized in the present
study were evaluated for their in vitro cytotoxic activities against two Acute Lymphoblastic Leukemia (ALL) cell
lines named, SB-ALL and NALM-6, and a human breast adenocarcinoma cell lines (MCF-7). The results revealed
that triazoles 4 exhibit better cytotoxicity than their hydrazone precursors 3. Among triazoles, compounds 4f, 4j
and 4q exhibited potent cytotoxic activity against SB-ALL and NALM-6 with IC50 values in the range of
∼1.64–5.66 μM and ∼1.14–3.7 μM, respectively, compared with doxorubicin (IC50 = 0.167 μM, SB-ALL).
Compounds 4f, 4j and 4q were subjected to apoptosis assay after 48 h treatment and these compounds induced
apoptosis of NALM-6 cells via caspase 3/7 activation. Results revealed that compound 4q represents potential
promising lead.
6-Chloro-3-substituted-[1,2,4]triazolo[4,3-b]
pyridazines
Iodobenzene diacetate
6-Chloropyridazin-3-yl hydrazones
6-Chloro-3-hydrazinopyridazine
Cytotoxicity
1. Introduction
besides being used as various enzyme inhibitors such as Leucine rich
repeat kinase 2 (LRRK2) [10], phosphodiesterase (PDE4) [11]. Meti-
The 1,2,4-triazole nucleus, an important five-membered hetero-
cyclic scaffold, is found in large number of marketed drugs such as
Vorozole, Letrozole and Anastrozole which are potent and selective
non-steroidal inhibitors of cytochrome P450 aromatase and used in
treatment of hormone receptor-positive breast cancer (Fig. 1) [1,2].
Recently our group reported a series of 1,2,4-triazolo[4,3-a]quinox-
alines exhibiting promising DNA photocleaving activity [3]. Pyridazine
nucleus play a key role in drug discovery as it can improve the physi-
cochemical profile of drug candidates by increasing their water solu-
bility. Pyridazine ring has been known to be present in several natural
products (antifungal antibiotic Pyridazomycin and meroterpenoid
Azamerone) and drugs (nonsteroidal anti-inflammatory drug such as
Emorfazone, phosphodiesterase inhibitors Amipizone, Pimobendan,
Zardaverine, Milrinone, Imazodan, and cardiotonic drug Indolidan) [4].
1,2,4-Triazolo[4,3-b]pyridazine derivatives, in particular, possess ex-
tensive therapeutic properties like anxiolytic [5], anticonvulsant [6],
antimicrobial properties [7,8], antituberculostatic [8], simultaneous
inhibitor of α5 subunit-containing GABAA receptors (GABAARs) and
enhances α7 neuronal nicotinic-acetylcholine receptors (nAChRs) [9]
culously, (S)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]
pyridazin-3-yl)ethyl)-quinoline (I) has been reported as potent and
highly selective c-MET inhibitors (Fig. 1) having an important role in
tumor invasive growth and metastasis [12]. 4-(2-(6-Methyl-[1,2,4]
triazolo[4,3-b]pyridazin-8-ylamino)ethyl)-phenol (II) and (R)-3-(2,5-
dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-
[1,2,4]triazolo[4,3-b]pyridazine (III) have been established as highly
potent tankyrases (TNKSs) [13] and PDE4A inhibitors [11], respec-
tively. 3,6-Diaryl-[1,2,4]triazolo[4,3-b]pyridazines (IV) displayed the
highly active antiproliferative activity against SGC-7901, A549 and HT-
1080 cell lines with IC50 values of in the range 0.008–0.014 M, re-
spectively and effectively inhibited tubulin polymerization [14].
A number of synthetic methods have been developed for the
synthesis of [1,2,4]triazolo[4,3-b]pyridazine derivatives which involve
the oxidation of hydrazones with various reagents such as lead tetra-
acetate [15], bromine [15], nitrobenzene [16], copper dichloride [17],
mixture of Me4NBr and oxone® [7] etc, reaction of hydrazinopyridazine
with aroyl chlorides [8]/ethyl orthoformate or ethyl orthoacetate
[5,16,18], reaction of 3-chloropyridazines with acylhydrazines or
⁎ Corresponding author.
E-mail address: ranjanaaggarwal67@gmail.com (R. Aggarwal).
https://doi.org/10.1016/j.bioorg.2019.01.049
Received 22 August 2018; Received in revised form 24 December 2018; Accepted 17 January 2019
Availableonline02February2019
0045-2068/©2019PublishedbyElsevierInc.

Mamta et al.
BioorganicChemistry86(2019)288–295
N
C N
N
C
N
N
N
N
N
H
N
N
N
Cl
C
N
N
C
N
N
N
Anastrozole
Letrozole
N
Vorozole
N
N
N
N
H
N
N
N
I
N
N
N
OH
CH3
II
N
Tankyase inhibitor
c-MET inhibitor
N
N
H3CO
O
O
H3CO
N
O
N
H3C
H3CO
OCH3
N
N
OCH3
IV
N
N
NH2
OCH3
III
Antitubulin agent
PDE4 inhibitor
Fig. 1. Commercial anticancer drugs containing 1,2,4-triazole moiety and biologically active [1,2,4]triazolo[4,3-b]pyridazines.
formic hydrazide in n-butanol under reflux for 10–20 h [5,6]. Un-
fortunately, most of these methods suffer from various disadvantages
such as hazardous materials, poor yields, longer reaction time, elevated
temperatures and tedious work-up procedures. Therefore, a more effi-
cient and mild synthesis of [1,2,4]triazolo[4,3-b]pyridazines is desir-
able. Utility of iodobenzene diacetate (IBD) in oxidative transformation
is a valuable strategy for greener synthesis because of its easy avail-
ability, mild reaction condition and ease of handling.
triazolo[4,3-b]pyridazine 4a in good yield (80%). The structure and
purity of 4a was established by TLC and its spectral (IR, 1H NMR, 13C
NMR, Mass) and elemental analytical data. Encouraged by the success
of the reaction, different aldehydes having electron donating group,
electron withdrawing group, disubtituted aryl and heteroaryl groups
were condensed with 2 and subsequent oxidation with IBD under
identical conditions led to the formation of desired triazolo[4,3-b]pyr-
idazines 4b-q in 81–91% yields. The reaction has a broad scope in terms
of substitution diversity and is scalable to gram level.
Keeping in view the significance, and in continuation of our ongoing
research work on hypervalent iodine reagents in organic transforma-
tions [19–22], we herein report the greener synthesis of a series of 6-
chloro-3-substituted-[1,2,4]triazolo[4,3-b]pyridazines from hydrazone
precursors using IBD and evaluate in vitro anticancer activity of hy-
drazones and triazoles against three different human cancer cell lines.
Additionally, we have attempted cyclization of 3-benzylidenehy-
drazino-6-chloropyridazine (3c) with chloramine-T, another eco-
friendly and cheap oxidising reagent known for such type of oxidative
cyclization. Contrary to our expectations, the reaction did not proceed
to furnish the desired product 6-chloro-3-phenyl-[1,2,4]triazolo-[4,3-
b]-pyridazine (4c) even after refluxing 3c in ethanol for 15 h which was
confirmed by running co-TLC with synthesized compound. Compound
4c exhibits a fluorescent spot under long UV (365 nm) using TLC vi-
sualizer which was absent when the oxidative reaction was conducted
with chloramine-T.
2. Results and discussion
2.1. Chemistry
IR spectra of compounds 3 exhibited one characteristic absorption
band in the range of 3024–3217 cm−1 for NeH stretching. 1H NMR
spectra of 3 displayed two characteristic singlets at δ 7.52–8.67 and
9.82–11.83 ppm assignable to methine proton (N]CH) and NeH pro-
tons, respectively, a pair of doublets of one proton intensity each for H-
4 and H-5 of pyridazine ring at δ 7.00–7.82 and 7.48–7.92 ppm, re-
spectively, having the coupling constant 3J = ∼9.36 Hz. The char-
acterization of products 4 were based upon comparison of their spectral
Synthetic pathway to [1,2,4]triazolo-[4,3-b]-pyridazines is depicted
in Scheme 1. Initially, 6-chloro-3-hydrazinopyridazine 2 was obtained
by refluxing of 3,6-dichloropyridazine 1 with hydrazine hydrate in tert-
BuOH. Compound 2 on reaction with equimolar amount of acet-
aldehyde in refluxing ethanol afforded the key intermediate, ethylide-
nehydrazino-6-chloropyridazine 3a. The intramolecular oxidative cy-
clization of 3a with 1.1 equivalents IBD in dichloromethane at room
temperature provided the desired compound 6-chloro-3-methyl-[1,2,4]
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Mamta et al.
BioorganicChemistry86(2019)288–295
Scheme 1. Synthesis of 6-chloropyridazin-3-yl hydrazones 3a-q and their oxidation to [1,2,4]triazolo[4,3-b]pyridazines 4a-q.
data (IR and 1H NMR) with those of intermediate hydrazones 3. IR
spectra of 4 were found to be transparent in the region of NH stretch
and bend, thus confirming the oxidation of 3 into 4. Further con-
firmation was established by 1H NMR spectra of 4 where the dis-
appearance of signals was observed at δ 7.52–8.67 and 9.82–11.83 ppm
for aldehydic H and NH, respectively. Further 1H NMR spectra of 4
exhibited a pair of doublets for H-4 and H-5 of pyridazine ring at δ
7.55–8.53 and 6.88–7.53 ppm, respectively having the coupling con-
stant 3J = ∼9.6 Hz. It is noteworthy that in compounds 4 the downfield
shift of proton at position-4 of pyridazine ring in comparison with
compound 3 may be attributed to the lone pair effect of the nitrogen of
the triazole ring on the H-4. Known products were identified by com-
parison of their mp with those reported in literature [15,17].
cytotoxicity against different human cancer cell lines (Table 2). It is also
evident that triazole derivatives 4f, 4j and 4q, having p-chlorophenyl,
p-bromophenyl and 3-indolyl group, respectively, at position-3 of tria-
zole ring are the most potent amongst the tested compounds. Interest-
ingly, except for MCF-7 cell line, it was noticed that substitution pattern
on phenyl residue at position-3 of triazole ring has little influence on
cytotoxicity of these triazoles. For triazole derivative 4f which is sub-
stituted at para-position of phenyl ring with chloro group leads to an
increase in cytotoxicity against SB-ALL and NALM-6 as compared to
ortho and meta-substituted phenyl derivatives 4d and 4e, respectively.
Similarly incorporation of para-substituent on the phenyl ring viz. Br
(4h) and F (4j) increases the cytotoxicity as compared to ortho deri-
vative 4 g and meta derivative 4i. No cytotoxicity was observed for
compounds 4m and 4n having disubstitution of phenyl ring. It is also
interesting to note that substitution of position-3 of triazole by a het-
eroaryl groups have led to the different effects on the cytotoxicity, as
the sharp increase of cell viability against three cancer lines in case of
compound 4o (furan-2′-yl) and 4p (thiophen-2′-yl). In contrast, com-
pound 4q with indol-3′-yl substituent in the triazole core was the most
cytotoxic against all human cancer cell lines therefore indicating that
indole group at position-3 of triazole ring plays a crucial role in im-
parting cytotoxicity to the molecule. For instance, at 10 µM con-
centration, compound 4q shows only 14.8% cell survival against SB-
ALL, 14.2% against NALM-6 and 31.0% against MCF-7 cell lines, re-
spectively comparable to commercial drug doxorubicin with percentage
cell survival 11.2%, 16.8% and 19.0%.
2.2. In vitro cytotoxic evaluation and structure–activity relationship
Cytotoxic activity of all the synthesized compounds (3a-q and 4b-q)
were evaluated in vitro employing three human cancer cell lines namely
SB-ALL, NALM-6 and MCF-7 by using the well-established MTT [3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] cell prolifera-
tion assay [23,24]. The three cell lines are efficient in vitro models for
their cytotoxic evaluation of different types of chemotherapeutic agents
such as DNA minor groove binders [25], protein kinase inhibitors [26]
and caspases inhibitors [27,28] among others. Doxorubicin was used as
standard drug.
When all the synthesized compounds were initially screened at
10 µM concentration against these three cancer cell lines, compounds
4f, 4j and 4q showed significant cytotoxic effects comparable to com-
mercial drug doxorubicin against the tested cancer cell lines (Tables 1
and 2).
Compounds 4f, 4j and 4q with less than 50% cell survival were
chosen as lead compounds and further investigated for their IC50 values
(Table 3). Compounds 4f and 4j were moderately active against MCF-7
cancer cell line with an IC50 value of 21.2 and 11.6 µM, respectively,
but 4f and 4j were highly active against SB-ALL and NALM-6 with IC50
values in the range of 1.23–5.66 μM. Compound 4q exhibited excellent
As evident from results summarized in Table 1, hydrazone deriva-
tives 3 showed poor cytotoxicity against these cell lines. Only two hy-
drazones 3m and 3n exhibited less than 50% cell survival against
NALM-6 cell line. In contrast, most triazoles 4 show moderate to high
cytotoxicity
against
SB-ALL
(IC50 = 1.64 µM),
NALM-6
(IC50 = 1.14 µM), and MCF-7 cancer (IC50 = 3.55 µM) cell lines.
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BioorganicChemistry86(2019)288–295
Table 1
Percentage cell survival of compounds 3a-q at 10 µM concentration using MTT assay against three cancer cell lines.
Tested Compd.
R
% Cell Survival
S.D.a
SB-ALL
NALM-6
MCF-7
Cells
100
7.6
100
10.6
100
14.5
16.3
18.7
20.8
17.4
17.0
40.7
16.8
23.5
7.6
3a
eCH3
80.3
76.5
93.2
64.8
89.8
71.9
70.5
72.2
74.3
79.0
73.3
61.7
63.4
61.1
97.0
80.7
76.0
11.2
23.4
5.6
93.0
92.5
111.2
51.9
117.7
62.9
68.6
58.4
63.2
72.1
55.3
53.0
46.5
45.2
101.8
82.7
104.4
16.8
9.0
8.0
87.4
81.2
83.2
75.3
76.4
83.6
66.7
74.7
65.9
62.9
61.7
69.8
74.5
75.5
86.3
81.8
71.4
19.0
3b
eCH2CH3
3c
eC6H5
14.4
23.1
21.9
5.9
10.6
3d
-o-ClC6H4
4.9
3e
-m-ClC6H4
-p-ClC6H4
16.1
3f
11.6
22.9
7.9
3 g
-o-FC6H4
12.1
15.5
10.2
13.5
11.0
12.7
8.2
3h
-p-FC6H4
3i
-m-BrC6H4
-p-BrC6H4
11.4
15.5
15.5
8.6
3j
21.0
10.6
14.1
12.9
19.8
24.9
11.4
17.9
3.0
3k
-p-CH3C6H4
-p-OCH3C6H4
−2,5-di(OCH3)C6H3
−3,4-di(OCH3)C6H3
−2′-furyl
3l
3m
3.9
3n
7.2
3.6
3o
21.4
21.8
9.2
14.8
3p
−2′-thienyl
−3′-indolyl
12.3
13.4
7.6
3q
Doxorubicin
3.8
a
The activity data represents mean values
SD of experiments conducted in triplicates at three independent times.
Compounds 4f, 4j and 4q were 2–9 fold more selective for SB-ALL and
NALM-6 cell lines compared to MCF-7 cells. Results of cytotoxicity
shows that compound 4q could serve as potent cytotoxic agent by
further derivatization.
Table 3
IC50 values of compounds 4f, 4j and 4q against three cancer cell lines.
a
Compound
IC50 value (in µM) against cancer cell line
SB-ALL
Nalm-6
MCF-7
To gain into deeper insight to mode of action for cytotoxic activity,
compounds 4f, 4j and 4q were analyzed for their ability to cause
apoptosis (Fig. 2). Caspase 3/7 activation is one of the biochemical
hallmarks of apoptotic cell death. Camptothecin was used a positive
control.
4f
5.66
1.87
1.64
0.167
2.6
3.7
1.6
21.2
11.6
3.55
0.48
3.6
4j
0.56
0.74
1.23
1.14
0.32
0.26
2.32
0.54
0.12
4q
0.24
0.08
Doxorubicin
0.032
As shown in Fig. 2, compounds 4f, 4j and 4q caused ∼ 3 to 3.5 fold
increase in fluorescence respectively, compared to control indicative of
caspase 3/7 activation. This data suggests that these compounds kill
cancer cells via apoptosis induction.
a
IC50 = half maximal concentration represents the concentration of drug
able to inhibit by 50% the in vitro growth. Each value represents mean
SD of
three experiments.
diacetate as an oxidant. Compounds 4f, 4j and 4q were found to have
significant cytotoxic potency against three cancer cell lines (SB-ALL,
NALM-6 and MCF-7) and induced apoptosis of NALM-6 cells via caspase
3/7 activation. Preliminary results indicate that the varying substitu-
tion patterns at 3-position of triazole ring could have an effect on the
3. Conclusion
In summary, a series of triazolo[4,3-b]pyridazines 4a-q have been
synthesized by an efficient and ecofriendly route utilizing iodobenzene
Table 2
Percentage cell survival of compounds 4b-q at 10 µM concentration using MTT assay against three cancer cell lines.
Tested Compd.
R
% Cell Survival
S.D.a
SB-ALL
NALM-6
MCF-7
Cells
100
7.6
100
10.6
13.7
8.7
13.5
13.1
24.1
100
14.5
20.2
15.6
14.6
12.5
17.2
16.1
11.1
20.1
14.9
33.2
14.1
21.5
32.9
25.0
16.5
25.8
3.0
4b
eCH2CH3
77.3
68.4
72.8
72.5
37.8
82.2
60.2
68.3
23.8
48.4
87.8
82.3
65.7
74.9
42.1
14.8
11.2
12.5
11.9
10.9
13.6
15.3
11.9
15.3
8.3
101.8
96.0
87.8
103.7
32.4
104.6
41.7
73.0
22.0
96.9
100.3
103.4
94.4
97.1
74.8
14.2
16.8
70.7
74.7
81.4
78.4
91.2
75.9
79.3
65.7
77.3
89.2
79.4
78.7
70.7
63.8
75.8
31.0
19.0
4c
eC6H5
4d
-o-ClC6H4
4e
-m-ClC6H4
4f
-p-ClC6H4
4g
-o-FC6H4
14.0
4h
-p-FC6H4
7.1
4i
-m-BrC6H4
-p-BrC6H4
12.9
6.7
4j
17.7
22.7
24.8
23.3
17.1
5.0
4k
-p-CH3C6H4
-p-OCH3C6H4
−2,5-di(OCH3)C6H3
−3,4-di(OCH3)C6H3
−2′-furyl
10.5
6.8
4l
4m
10.6
4n
10.9
11.9
21.6
6.4
4o
4p
−2′-thienyl
−3′-indolyl
15.9
4.4
4q
Doxorubicin
3.8
7.6
a
The activity data represents mean values
SD of experiments conducted in triplicates at three independent times.
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4.2.2. 3-Benzylidenehydrazino-6-chloropyridazine (3c)
Yield 77%; mp 258.5 °C; Lit. mp [15] 260.5 °C. 13C NMR (100 MHz,
DMSO): 115.45, 126.10, 128.36, 128.78, 129.41, 134.61, 141.66,
147.19, 158.72.
4.2.3. 3-(2′-Chlorobenzylidenehydrazino)-6-chloropyridazine (3d)
Yield 74%; mp 225.5 °C; IR (KBr, cm−1): 3194 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 7.32–7.35 (m, 2H, Ph-4′, 5′-H), 7.40–7.42 (m, 1H,
Ph-3′-H), 7.53–7.56 (d, 1H, J = 9.36 Hz, pyridazine-4H), 7.65–7.67 (d,
1H, J = 9.36 Hz, pyridazine-5H), 8.01–8.04 (m, 1H, Ph-6′-H), 8.51 (s,
1H, methine-H), 11.83 (bs, 1H, eNH). 13C NMR (100 MHz, DMSO): δ
116.04, 126.48, 127.45, 128.16, 129.82, 130.11, 131.87, 132.16,
137.64, 147.81, 158.69. Ms: m/z = 266.01 [M]+, 268.10 [M+2]+
,
270.03 [M+4]+, (9:6:1). Anal. Calcd for C11H8Cl2N4: C, 49.46; H, 3.02;
N, 20.98. Found: C, 49.24; H, 3.41, N, 20.84.
Fig. 2. Caspase activation in NALM-6 cells treated with 4f, 4j and 4q (Data
represents mean values
independent times).
SD of experiments conducted in duplicates at two
4.2.4. 3-(3′-Chlorobenzylidenehydrazino)-6-chloropyridazine (3e)
Yield 76%; mp 224 °C. IR (KBr, cm−1): 3194 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 7.48–7.55 (m, 3H, Ph-4′, 5′, 6′-H), 7.60 (m, 1H,
Ph-2′-H), 7.80–7.82 (d, 1H, J = 9.6 Hz, pyridazine-4H), 7.90–7.92 (d,
1H, J = 9.6 Hz, pyridazine-5H), 8.67 (s, 1H, methine-H), 11.83 (bs, 1H,
eNH). 13C NMR (100 MHz, DMSO) δ: 116.50, 127.41, 128.52, 130.30,
130.85, 131.20, 134.41, 135.61, 143.20, 145.50, 160.21. Ms: m/
z = 266.01 [M]+, 268.11 [M+2]+, 270.01 [M+4]+, (9:6:1). Anal.
Calcd for C11H8Cl2N4: C, 49.46; H, 3.02; N, 20.98. Found: C, 49.83; H,
3.33, N, 20.58.
cytotoxic activity and compound 4q has the potential to be promising
lead.
4. Experimental section
4.1. General chemistry
Melting points were determined in digital melting point apparatus
MEPA. IR spectra were recorded on a Buck Scientific IR M−500
spectrophotometer in KBr pellets (νmax in cm−1). Analytical TLC was
performed using Merck Kieselgel 60 F254 silica gel plates. Visualisation
was performed under UV light using Ultra Violet Flourescence
Inspection Cabinet (Perfit, India). 1H and 13C NMR spectra for analy-
tical purpose were recorded in CDCl3 and DMSO‑d6 on a Bruker in-
strument; chemical shifts are expressed in δ-scale downfield from TMS
as an internal standard. Elemental analyses were performed at
Sophisticated Analytical Instrument Facility, Panjab University,
Chandigarh, India.
4.2.5. 3-(4′-Chlorobenzylidenehydrazino)-6-chloropyridazine (3f)
Yield 70%; mp 260 °C; Lit. mp [15] 295–296 °C.
4.2.6. 6-Chloro-3-(2′-fluorobenzylidenehydrazino)pyridazine (3 g)
Yield 76%; mp 240 °C. IR (KBr, cm−1): 3027 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 7.14–7.21 (m, 2H, Ph-3′, 5′-H), 7.34–7.39 (m, 1H,
Ph-4′-H), 7.55–7.57 (d, 1H, J = 9.36 Hz, pyridazine-4H), 7.64–7.66 (d,
1H, J = 9.36 Hz, pyridazine-5H), 7.93–7.97 (t, 1H, J = 7.44 Hz, Ph-6′-
H), 8.33 (s, 1H, methine-H), 11.78 (bs, 1H, eNH). 13C NMR (100 MHz,
DMSO) δ: 115.78, 122.17, 124.76, 125.99, 126.02, 130.87, 130.95,
134.39, 147.70, 158.73, 161.32. Ms: m/z = 250.00 [M]+, 252.00 [M
+2]+, (3:1). Anal. Calcd for C11H8ClFN4: C, 52.71; H, 3.22, N, 22.35.
Found: C, 52.31; H, 3.50; N, 22.58.
6-Chloro-3-hydrazinopyridazine (2) was synthesized according to
the literature procedure [29,30].
4.2. Synthesis of 6-chloro-3-ethylidenehydrazinopyridazine (3a)
To an ethanolic solution (15 ml) of 3-chloro-6-hydrazinopyridazine
(2) (0.36 g, 2.5 mmol) was added acetaldehyde (0.11 g, 2.5 mmol), and
the reaction mixture was refluxed for 30 min. During refluxing a solid
separated out. The solvent was evaporated in vacuo and the reaction
mixture cooled to room temperature. The obtained solid was filtered
off, washed with cold ethanol and recrystallized with ethanol to afford
3a.
4.2.7. 6-Chloro-3-(4′-fluorobenzylidenehydrazino)pyridazine (3h)
Yield 72%; mp 256 °C. IR (KBr, cm−1): 3024 (eNH str). 1H NMR
(400 MHz, CDCl3) δ: 7.17–7.21 (t, 2H, J = 8.8 Hz, Ph-3′, 5′-H),
7.57–7.59 (d, 1H, J = 9.4 Hz, pyridazine-4H), 7.63–7.66 (d, 1H,
J = 9.32 Hz, pyridazine-5H), 7.71–7.75 (m, 2H, Ph-2′, 6′-H), 8.11 (s,
1H, methine-H), 11.68 (bs, 1H, eNH). 13C NMR (100 MHz, DMSO) δ:
115.64, 116.16, 128.38, 130.00, 131.28, 132.59, 147.38, 158.87,
159.63, 160.46. Ms: m/z = 250.01 [M]+, 252.00 [M+2]+, (3:1). Anal.
Calcd for C11H8ClFN4: C, 52.71; H, 3.22, N, 22.35. Found: C, 52.63; H,
3.61; N, 22.02.
Yield 78%; mp > 315 °C. IR (KBr, cm−1): 3202 (eNH str). 1H NMR
(400 MHz, CDCl3) δ: 2.01 (s, 3H, eCH3), 7.28–7.30 (d, 1H, J = 9.36 Hz,
pyridazine-4H), 7.50–7.52 (d, 1H, J = 9.36 Hz, pyridazine-5H), 7.54 (s,
1H, methine-H), 10.51 (bs, 1H, eNH). 13C NMR (100 MHz, CDCl3): δ
13.18, 116.29, 140.89, 143.65, 147.22, 159.16. Ms: m/z = 170.04
[M]+, 172.04 [M+2]+, (3:1). Anal. Calcd for C6H7ClN4: C, 42.24; H,
4.14, N, 32.84; Found: C, 42.44; H, 4.19, N, 32.94.
4.2.8. 3-(3′-Bromobenzylidenehydrazino)-6-chloropyridazine (3i)
Yield 83%; mp 195 °C. IR (KBr, cm−1): 3024 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 7.32–7.36 (t, 1H, J = 7.84 Hz, Ph-5′-H), 7.48–7.51
(m, 1H, Ph-4′-H), 7.57–7.60 (d, 1H, J = 9.4 Hz, pyridazine-4H),
7.62–7.64 (d, 1H, J = 7.76 Hz, Ph-6′-H), 7.68–7.70 (d, 1H, J = 9.4 Hz,
pyridazine-5H), 7.88–7.89 (t, 1H, J = 1.68 Hz, Ph-2′-H), 8.07 (s, 1H,
methine-H), 11.83 (bs, 1H, eNH). 13C NMR (100 MHz, DMSO) δ:
116.12, 122.22, 125.62, 128.26, 130.04, 130.84, 131.61, 137.17,
140.03, 147.67, 158.78. Anal. Calcd for C11H8BrClN4: C, 42.41; H, 2.59;
N, 17.98. Found: C, 42.33; H, 2.77, N, 17.81.
Similarly, other compounds (3b-q) are synthesized by following the
above procedure using appropriate aldehydes.
4.2.1. 6-Chloro-3-propylidenehydrazinopyridazine (3b)
Yield 72%; mp 144.5 °C. IR (KBr, cm−1): 3209 (eNH str). 1H NMR
(400 MHz, CDCl3) δ: 1.13–1.17 (t, 3H, J = 7.52 Hz, eCH3), 2.33–2.37
(q, 2H, J = 7.52 Hz, eCH2), 7.29–7.31 (d, 1H, J = 9.32 Hz, pyridazine-
4H), 7.48–7.51 (d, 1H, J = 9.32 Hz, pyridazine-5H), 7.52 (s, 1H, me-
thine-H), 9.82 (bs, 1H, eNH). 13C NMR (100 MHz, CDCl3): δ 10.53,
25.85, 116.18, 129.89, 147.14, 148.27, 159.00. Ms: m/z = 184.05
[M]+, 186.05 [M+2]+, (3:1). Anal. Calcd for C7H9ClN4: C, 45.54; H,
4.91, N, 30.35; Found: C, 45.50; H, 4.90; N, 30.33.
4.2.9. 3-(4′-Bromobenzylidenehydrazino)-6-chloropyridazine (3j)
Yield 82%; mp 215 °C. IR (KBr, cm−1): 3217 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 7.56–7.58 (d, 2H, J = 8.52 Hz, Ph-2′, 6′-H),
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BioorganicChemistry86(2019)288–295
7.60–7.62 (d, 1H, J = 9.4 Hz, pyridazine-4H), 7.65–7.67 (d, 1H,
J = 9.36 Hz, pyridazine-5H), 7.68–7.70 (d, 2H, J = 8.52 Hz, Ph-3′, 5′-
H), 8.08 (s, 1H, methine-H), 11.75 (bs, 1H, eNH). 13C NMR (100 MHz,
DMSO) δ: 115.93, 122.20, 128.22, 130.05, 131.68, 133.98, 140.58,
147.57, 158.79. Anal. Calcd for C11H8BrClN4: C, 42.41; H, 2.59; N,
17.98. Found: C, 42.27; H, 2.36, N, 18.19.
(3:1). Anal. Calcd for C9H7ClN4S: C, 45.29; H, 2.96; N, 23.47. Found: C,
45.73; H, 2.85; N, 23.13.
4.2.16. 6-Chloro-3-(indole-3′-ylmethylenehydrazino)pyridazine (3q)
Yield 75%; mp 235 °C. IR (KBr, cm−1): 3094 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 7.12–7.20 (m, 2H, indole-5′, 6′-H), 7.41–7.43 (d,
1H, J = 7.4 Hz, indole-7′-H), 7.53–7.55 (d, 1H, J = 9.4 Hz, pyridazine-
4H), 7.57–7.59 (d, 1H, J = 9.4 Hz, pyridazine-5H), 7.62–7.63 (d, 1H,
J = 2.68 Hz, indole-2′-H), 8.17–8.19 (t, 1H, J = 7.16 Hz, indole-4′-H),
4.2.10. 6-Chloro-3-(4′-methylbenzylidenehydrazino)pyridazine (3k)
Yield 78%; mp 212 °C. IR (KBr, cm−1): 3209 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 2.34 (s, 3H, Ph-4′-CH3), 7.18–7.20 (d, 2H,
J = 7.48 Hz, Ph-3′, 5′-H), 7.51–7.53 (d, 1H, J = 9.2 Hz, pyridazine-4H),
7.54–7.56 (d, 2H, J = 7.48 Hz, Ph-2′, 6′-H), 7.60–7.63 (d, 1H,
J = 9.2 Hz, pyridazine-5H), 8.08 (s, 1H, methine-H), 11.53 (bs, 1H,
eNH). 13C NMR (100 MHz, DMSO) δ: 20.95, 115.77, 126.33, 129.34,
129.95, 131.95, 138.86, 142.01, 147.18, 158.88. Ms: m/z = 246.01
[M]+, 248.01 [M+2]+, (3:1). Anal. Calcd for C12H11ClN4: C, 58.42; H,
4.49; N, 22.71. Found: C, 58.74; H, 4.86, N, 22.63.
8.34 (s, 1H, methine-H), 11.23 (bs, 1H, eNH), 11.38 (bs, 1H, eNH). 13
C
NMR (100 MHz, DMSO) δ: 111.63, 111.94, 114.90, 120.18, 121.50,
122.31, 123.97, 128.54, 129.37, 136.98, 139.81, 145.99, 158.81. Anal.
Calcd for C13H10ClN5: C, 57.47; H, 3.71, N, 25.78. Found: C, 57.19; H,
3.99, N, 25.41.
4.3. Synthesis of 6-chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine (4a)
To a solution of 6-chloropyridazin-3-yl hydrazone (3a) (0.34 g,
2 mmol) in dichloromethane (25 ml), IBD (0.70 g, 2.2 mmol) was added
in small portions and the reaction mixture was stirred for 2–3 h or until
the completion of reaction as monitored by TLC. Excess solvent was
distilled off in vacuo, and the residual mass was triturated with petro-
leum ether to remove the excess of iodobenzene and a solid product
separated out, which was recrystallized from aqueous ethanol to afford
4a.
4.2.11. 6-Chloro-3-(4′-methoxybenzylidenehydrazino)pyridazine (3l)
Yield 74%; mp 207 °C. IR (KBr, cm−1): 3204 (eNH str). 1H NMR
(400 MHz, DMSO) δ: 3.83 (s, 3H, Ph-4′-OCH3), 6.94–6.96 (d, 2H,
J = 8.72 Hz, Ph-3′, 5′-H), 7.00–7.02 (d, 1H, J = 9.36 Hz, pyridazine-
4H), 7.59–7.60 (d, 1H, J = 9.36 Hz, pyridazine-5H), 7.78–7.80 (d, 2H,
J = 8.72 Hz, Ph-2′, 6′-H), 8.59 (s, 1H, methine-H), 11.81 (bs, 1H, eNH).
13C NMR (100 MHz, DMSO) δ: 55.21, 114.22, 115.65, 127.28, 127.88,
141.91, 146.97, 158.90, 160.14. Ms: m/z = 262.01 [M]+, 264.01 [M
+2]+, (3:1). Anal. Calcd for C12H11ClN4O: C, 54.87; H, 4.22, N, 21.33.
Found: C, 54.36; H, 4.56; N, 21.69.
Yield 80%; mp 75.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 2.74 (s, 3H, eCH3), 7.01–7.03
(d, 1H, J = 9.64 Hz, pyridazine-5H), 7.97–7.99 (d, 1H, J = 9.6 Hz,
pyridazine-4H). 13C NMR (100 MHz, DMSO) δ: 9.70, 121.77, 126.30,
4.2.12. 6-Chloro-3-(2′,5′-dimethoxybenzylidenehydrazino)pyridazine
(3m)
142.65, 147.28, 149.16. Ms: m/z = 168.02 [M]+, 170.01 [M+2]+
,
(3:1). Anal. Calcd for C6H5ClN4: C, 42.75; H, 2.99, N, 33.23 Found: C,
42.33; H, 3.19, N, 33.38.
Yield 74%; mp 229 °C. IR (KBr, cm−1): 3016 (eNH str). 1H NMR
(400 MHz, CDCl3) δ: 3.76 (s, 3H, Ph-5′-OCH3), 3.81 (s, 3H, Ph-2′-
OCH3), 6.78–6.85 (m, 2H, Ph-3′, 4′-H), 7.28–7.31 (d, 1H, J = 9.32 Hz,
pyridazine-4H), 7.39–7.40 (d, 1H, J = 2.76 Hz, Ph-6′-H), 7.57–7.59 (d,
1H, J = 9.28 Hz, pyridazine-5H), 8.39 (s, 1H, methine-H), 10.11 (bs,
1H, eNH). 13C NMR (100 MHz, CDCl3) δ: 55.00, 55.10, 115.22, 116.30,
116.85, 117.81, 120.31, 132.00, 142.33, 146.32, 152.30, 158.36.
158.58; Ms: m/z = 292.01 [M]+, 294.11 [M+2]+, (3:1). Anal. Calcd
for C13H13ClN4O2: C, 53.34; H, 4.48; N, 19.14. Found: C, 53.87; H, 4.20,
N, 18.93.
Similarly, other compounds (4b-q) are synthesized by using the
above procedure.
4.3.1. 6-Chloro-3-ethyl-[1,2,4]triazolo[4,3-b]pyridazine (4b)
Yield 82%; mp 93 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (300 MHz, CDCl3) δ: 1.48–1.53 (t, 3H, J = 7.5 Hz,
eCH3), 3.17–3.24 (q, 2H, J = 7.5 Hz, eCH2), 7.08–7.11 (d, 2H,
J = 9.6 Hz, pyridazine-5H), 8.04–8.07 (d, 1H, J = 9.6 Hz, pyridazine-
4H). 13C NMR (100 MHz, DMSO) δ: 10.44, 17.16, 121.85, 126.57,
142.44, 148.49, 150.34. Ms: m/z = 182.00 [M]+, 184.01 [M+2]+
,
4.2.13. 6-Chloro-3-(3′,4′-dimethoxybenzylidenehydrazino)pyridazine
(3:1); Anal. Calcd for C7H7ClN4: C, 46.04; H, 3.86, N, 30.68. Found: C,
46.37; H, 3.51, N, 30.88.
(3n)
Yield 78%; mp 234 °C; Lit. mp [17] 235–237 °C.
4.3.2. 6-Chloro-3-phenyl-[1,2,4]triazolo[4,3-b]pyridazine (4c)
4.2.14. 6-Chloro-3-(furan-2′-ylmethylenehydrazino)pyridazine (3o)
Yield 81%; mp 196.5 °C. IR (KBr, cm−1) 3132 (eNH str). 1H NMR
(400 MHz, CDCl3) δ: 6.49–6.50 (dd, 1H, J3′, 4′ = 3.4 Hz, J4′, 5′ = 1.8 Hz,
furan-4′-H), 6.69–6.70 (d, 1H, J3′, 4′ = 3.4 Hz, furan-3′-H), 7.37–7.39
(d, 1H, J = 9.36 Hz, pyridazine-4H), 7.51–7.52 (d, 1H, J4′, 5′ = 1.6 Hz,
furan-5′-H), 7.65–7.68 (d, 1H, J = 9.36 Hz, pyridazine-5H), 8.11 (s, 1H,
methine-H), 11.02 (bs, 1H, eNH). 13C NMR (100 MHz, DMSO) δ:
110.71, 111.52, 115.51, 129.34, 132.16, 143.24; 147.17, 149.74,
158.27. Ms: m/z = 222.06 [M]+, 224.00 [M+2]+, (3:1). Anal. Calcd
for C9H7ClN4O: C, 48.55; H, 3.17, N, 25.17. Found: C, 48.18; H, 3.63, N,
25.55.
Yield 82%; mp 199.5 °C; Lit. mp [15] 200–201 °C.
4.3.3. 6-Chloro-3-(2′-chlorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4d)
Yield 84%; mp 155 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 7.17–7.20 (d, 1H, J = 9.64 Hz,
pyridazine-5H), 7.44–7.48 (dt, 1H, J = 7.46 Hz, J = 1.2 Hz, Ph-4′-H),
7.52–7.56 (dt, 1H, J = 7.42 Hz, J = 1.68 Hz, Ph-5′-H), 7.59–7.61 (d,
1H, J = 8.0 Hz, Ph-3′-H), 7.67–7.69 (dd, 1H, J = 7.58 Hz, J = 1.6 Hz,
Ph-6′-H), 8.16–8.18 (d, 1H, J = 9.54 Hz, pyridazine-4H). 13C NMR
(100 MHz, DMSO) δ: 122.65, 124.76, 126.48, 126.96, 130.26, 132.16,
132.60, 134.81, 149.57. Ms: m/z = 264.08 [M]+, 266.01 [M+2]+
,
268.00 [M+4]+, (9:6:1). Anal. Calcd for C11H6Cl2N4: C, 49.84; H, 2.28,
N, 21.13. Found: C, 49.55; H, 2.67, N, 21.01.
4.2.15. 6-Chloro-3-(thiophen-2′-ylmethylenehydrazino)pyridazine (3p)
Yield 78%; mp > 315 °C. IR (KBr, cm−1): 3024 (eNH str). 1H NMR
(400 MHz, CDCl3) δ: 6.82–6.84 (m, 1H, thiophene-4′-H), 7.20–7.22 (d,
1H, J = 9.8 Hz, pyridazine-4H), 7.34–7.35 (m, 1H, thiophene-3′-H),
7.41–7.42 (m, 1H, thiophene-5′-H), 7.52–7.54 (d, 1H, J = 9.8 Hz, pyr-
idazine-5H), 8.14 (s, 1H, methine-H), 11.10 (bs, 1H, eNH). 13C NMR
(100 MHz, DMSO) δ: 115.12, 115.84, 121.00, 124.52, 130.65, 132.56,
4.3.4. 6-Chloro-3-(3′-chlorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4e)
Yield 85%; mp 201.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 7.17–7.20 (d, 1H, J = 9.6 Hz,
pyridazine-5H), 7.06–7.36 (m, 3H, Ph-4′,5′,6′-H), 7.48–7.50 (m, 1H,
Ph-2′H), 7.55–7.59 (d, 1H, J = 9.6 Hz, pyridazine-4H). 13C NMR
(100 MHz, DMSO) δ: 121.00, 123.24, 125.36, 126.52, 132.10, 132.19,
140.12, 146.23, 150.12. Ms: m/z = 238.00 [M]+, 240.02 [M+2]+
,
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133.26, 134.52, 148.56. Ms: m/z = 264.00 [M]+, 266.01 [M+2]+
,
(m, 3H, Ph-3′, 5′-H, pyridazine-5H), 8.13–8.16 (d, 1H, J = 9.6 Hz,
pyridazine-4H), 8.42–8.45 (d, 2H, J = 9.0 Hz, Ph-2′, 6′-H). 13C NMR
(100 MHz, CDCl3) δ: 55.42, 114.24, 117.97, 121.53, 126.63, 129.28,
143.37, 147.09, 149.18, 161.39. Ms: m/z = 260.01 [M]+, 262.01 [M
+2]+, (3:1). Anal. Calcd for C12H9ClN4O: C, 55.29; H, 3.48, N, 21.49.
Found: C, 55.46; H, 3.75, N, 21.66.
268.02 [M+4]+ (9:6:1). Anal. Calcd for C11H6Cl2N4: C, 49.84; H, 2.28,
N, 21.13. Found: C, 49.49; H, 2.12, N, 21.36.
4.3.5. 6-Chloro-3-(4′-chlorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4f)
Yield 81%; mp 191.5 °C; Lit. mp [15] 193–194 °C. IR (KBr, cm−1):
transparent in the region of eNH str. 1H NMR (400 MHz, DMSO) δ:
7.50–7.53 (d, 1H, J = 9.64 Hz, pyridazine-5H), 7.63–7.67 (d, 2H,
J = 8.6 Hz, Ph-3′, 5′-H), 8.35–8.38 (d, 2H, J = 8.5 Hz, Ph-2′, 6′-H),
8.51–8.53 (d, 1H, J = 9.68 Hz, pyridazine-4H).
4.3.12. 6-Chloro-3-(2′,5′-dimethoxyphenyl)-[1,2,4]triazolo[4,3-b]
pyridazine (4m)
Yield 84%; mp 78 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 3.81 (s, 3H, Ph-5′-OCH3), 3.82
(s, 3H, Ph-2′-OCH3), 7.03–7.05 (m, 1H, Ph-3′-H) 7.10–7.12 (d, 1H,
J = 9.72 Hz, pyridazine-5H), 7.14 (m, 1H, Ph-4′-H), 7.21–7.22 (m, 1H,
J = 2.72 Hz, Ph-6′-H), 8.11–8.13 (d, 1H, J = 9.52 Hz, pyridazine-4H).
13C NMR (100 MHz, DMSO) δ: 56.00, 56.50, 113.41, 114.86, 116.47,
118.43, 122.19, 126.36, 143.37, 147.91, 148.98, 152.59, 153.57. Ms:
4.3.6. 6-Chloro-3-(2′-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4 g)
Yield 83%; mp 160.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (300 MHz, CDCl3) δ: 7.18–7.21 (d, 1H, J = 9.6 Hz,
pyridazine-5H), 7.28–7.37 (m, 2H, Ph-3′, 5′-H), 7.60 (m, 1H, Ph-4′-H),
7.89 (m, 1H, Ph-6′-H), 8.16–8.19 (d, 1H, J = 9.3 Hz, pyridazine-4H).
13C NMR (100 MHz, DMSO) δ: 113.46, 116.43, 124.50, 126.46, 131.50,
132.83, 143.46, 145.56, 149.64, 159.22, 161.75. Ms: m/z = 248.00
[M]+, 250.01 [M+2]+, (3:1). Anal. Calcd for C11H6ClFN4: C, 53.14; H,
2.43, N, 22.53. Found: C, 53.43; H, 2.79, N, 22.84.
m/z = 290.00 [M]+
,
292.04 [M+2]+
, (3:1). Anal. Calcd for
C13H11ClN4O2: C, 53.71; H, 3.81; N, 19.27. Found: C, 53.28; H, 4.09, N,
19.39.
4.3.13. 6-Chloro-3-(3′,4′-dimethoxyphenyl)-[1,2,4]triazolo[4,3-b]
pyridazine (4n)
4.3.7. 6-Chloro-3-(4′-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4h)
Yield 86%; mp 192.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 7.16–7.18 (d, 1H, J = 9.48 Hz,
pyridazine-5H), 7.24–7.29 (m, 2H, Ph-3′, 5′-H), 8.15–8.17 (d, 1H,
J = 9.56 Hz, pyridazine-4H), 8.46–8.51 (m, 2H, Ph-2′, 6′-H). 13C NMR
(100 MHz, CDCl3) δ: 115.98, 116.20, 121.90, 126.73, 129.89, 147.36,
Yield 84%; mp 230.5 °C; Lit. m.pt. [17] 235–237 °C. IR (KBr, cm−1):
transparent in the region of eNH str. 1H NMR (300 MHz, CDCl3) δ: 3.98
(s, 3H, Ph-4′-OCH3), 4.02 (s, 3H, Ph-3′-OCH3), 7.04–7.07 (d, 1H,
J = 8.4 Hz, Ph-5′-H), 7.11–7.15 (d, 1H, J = 9.6 Hz, pyridazine-5H),
8.03 (s, 1H, Ph-2′-H), 8.12–8.17 (m, 2H, Ph-6′-H, pyridazine-4H).
149.53, 162.91, 165.34. Ms: m/z = 248.06 [M]+, 250.00 [M+2]+
,
(3:1). Anal. Calcd for C11H6ClFN4: C, 53.14; H, 2.43, N, 22.53. Found:
C, 52.14; H, 2.49, N, 22.91.
4.3.14. 6-Chloro-3-(furan-2′-yl)-[1,2,4]triazolo[4,3-b]pyridazine (4o)
Yield 83%; mp 212 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 6.61–6.62 (dd, 1H, J3′,
4′ = 3.4 Hz, J4′, 5′ = 1.8 Hz, furan-4′-H), 6.88–6.91 (d, 1H,
J = 10.08 Hz, pyridazine-5H), 7.40–7.41 (dd, 1H, J3′, 4′ = 3.32 Hz, J3′,
5′ = 0.64 Hz, furan-3′-H), 7.65–7.66 (dd, 1H, J4′, 5′ = 1.72 Hz, J3′,
5′ = 0.68 Hz, furan-5′-H), 7.86–7.88 (d, 1H, J = 10.08 Hz, pyridazine-
4H). 13C NMR (100 MHz, DMSO) δ: 111.88, 113.24, 122.26, 126.54,
4.3.8. 6-Chloro-3-(3′-bromophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4i)
Yield 90%; mp 179.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (300 MHz, CDCl3) δ: 7.17–7.20 (d, 1H, J = 9.6 Hz,
pyridazine-5H), 7.42–7.47 (t, 1H, J = 7.8 Hz, Ph-5′-H), 7.65–7.68 (d,
1H, J = 8.1 Hz, Ph-4′-H), 8.16–8.19 (d, 1H, J = 9.6 Hz, pyridazine-4H);
8.43–8.45 (d, 1H, J = 7.8 Hz, Ph-6′-H), 8.65 (m, 1H, Ph-2′-H). 13C NMR
(100 MHz, CDCl3) δ: 122.19, 122.91, 126.06, 126.73, 127.38, 130.39,
133.64, 139.11, 149.74. Anal. Calcd for C11H6BrClN4: C, 42.68; H, 1.95,
N, 18.10. Found: C, 42.87; H, 2.09, N, 18.33.
140.09, 141.95, 142.86, 144.86, 150.00. Ms: m/z = 220.03 [M]+
,
222.02 [M+2]+, (3:1). Anal. Calcd for C9H5ClN4O: C, 49.00; H, 2.28,
N, 25.40. Found: C, 49.00; H, 2.28, N, 25.40.
4.3.15. 6-Chloro-3-(thiophen-2′-yl)-[1,2,4]triazolo[4,3-b]pyridazine (4p)
Yield 86%; mp 179.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (300 MHz, DMSO): 7.25–7.32 (m, 1H, thiophene-4′-
H), 7.50–7.54 (d, 1H, J = 9.6 Hz, pyridazine-5H), 7.68–7.76 (m, 1H,
thiophene-3′-H), 8.10–8.20 (m, 1H, thiophene-5′-H), 8.47–8.50 (d, 1H,
J = 9.6 Hz, pyridazine-4H). 13C NMR (100 MHz, DMSO) δ: 122.16,
126.00, 126.84, 127.71, 127.83, 128.59, 142.99, 143.76, 149.43. Ms:
4.3.9. 6-Chloro-3-(4′-bromophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (4j)
Yield 91%; mp 171 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 7.16–7.18 (d, 1H, J = 9.6 Hz,
pyridazine-5H), 7.69–7.72 (d, 2H, J = 8.64 Hz, Ph-2′, 6′-H), 8.15–8.17
(d, 1H, J = 9.6 Hz, pyridazine-4H), 8.35–8.38 (d, 2H, J = 8.6 Hz, Ph-3′,
5′-H). 13C NMR (100 MHz, DMSO) δ: 122.05, 124.41, 125.23, 125.86,
126.73, 129.03, 129.95, 132.14, 149.65. Anal. Calcd for C11H6BrClN4:
C, 42.68; H, 1.95, N, 18.10. Found: C, 42.42; H, 1.63, N, 18.21.
m/z = 236.02 [M]+
,
238.01 [M+2]+
, (3:1). Anal. Calcd for
C9H5ClN4S: C, 45.67; H, 2.13, N, 23.67. Found: C, 45.24; H, 2.61, N,
23.89.
4.3.10. 6-Chloro-3-(4′-methylphenyl)-[1,2,4]triazolo[4,3-b]pyridazine
(4k)
4.3.16. 6-Chloro-3-(1H-indole-3′-yl)-[1,2,4]triazolo[4,3-b]pyridazine
(4q)
Yield 83%; mp 253.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, CDCl3) δ: 2.45 (s, 3H, Ph-4′-CH3),
7.12–7.14 (d, 1H, J = 9.6 Hz, pyridazine-5H), 7.37–7.39 (d, 2H,
J = 8.08 Hz, Ph-3′, 5′-H), 8.12–8.14 (d, 1H, J = 9.6 Hz, pyridazine-4H),
8.33–8.35 (d, 2H, J = 8.28 Hz, Ph-2′, 6′-H). 13C NMR (100 MHz, DMSO)
δ: 22.68, 122.13, 122.67, 125.71, 127.00, 129.21, 133.43, 140.12,
143.14, 149.12. Ms: m/z = 244.01 [M]+, 246.01 [M+2]+, (3:1). Anal.
Calcd for C12H9ClN4: C, 58.90; H, 3.71, N, 22.90. Found: C, 58.72; H,
3.66, N, 23.12.
Yield 82%; mp 173.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (400 MHz, DMSO) δ: 7.21–7.27 (m, 2H, indole-5′, 6′-
H), 7.34–7.37 (d, 1H, J = 9.52 Hz, pyridazine-5H), 7.52–7.55 (m, 1H,
indole-7′-H), 8.38–8.41 (d, 1H, J = 9.52 Hz, pyridazine-4H), 8.44–8.45
(d, 1H, J = 2.72 Hz, indole-2′-H), 8.51–8.53 (d, 1H, J = 7.24 Hz, in-
dole-4′-H), 11.73 (s, 1H, eNH). Ms: m/z = 269.02 [M]+, 271.01 [M
+2]+, (3:1). Anal. Calcd for C13H8ClN5: C, 57.90; H, 2.99; N, 25.97.
Found: C, 57.57; H, 3.12; N, 25.69.
4.3.11. 6-Chloro-3-(4′-methoxyphenyl)-[1,2,4]triazolo[4,3-b]pyridazine
(4l)
4.4. Biological assays
Yield 88%; mp 215.5 °C. IR (KBr, cm−1): transparent in the region of
eNH str. 1H NMR (300 MHz, CDCl3) δ: 3.92 (s, 3H, –OCH3), 7.09–7.15
4.4.1. Cytotoxic activity
4.4.1.1. Cell culture. NALM-6 and SB-ALL cell lines were maintained in
294

Mamta et al.
BioorganicChemistry86(2019)288–295
Roswell Park Memorial Institute medium (RPMI-1640) supplemented
with 10% fetal serum albumin (FBS) and 50 µg/ml of penicillin and
streptomycin (pen/strep) at 37 °C with 5% of CO2. MCF-7 cell line was
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS and 50 µg/ml of pen/strep at 37 °C with 5% of CO2.
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(#30–1010 K). After adding 10 µL MTT reagent to each well, cells
were placed back in incubator for 4 hr at 37 °C. 100 µL of detergent
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% Cell survival = (100/At As)
where At and As are the absorbance of wells treated with test
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4.4.1.3. Caspase 3/7 assay. 100 000 JURKAT cells were plated in a 24
well plate and treated with 1 µM of 4f, 4j and 4q for 48 h, 100 µL
sample was mixed with equal amount of substrate reagent and
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Acknowledgments
[19] R. Aggarwal, G. Sumran, S.P. Singh, A. Saini, Hypervalent iodine oxidation of
benzil-α-arylimino oximes: An efficient synthesis of 2,3-diphenylquinoxaline-1-
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We are grateful to the Haryana State Council for Science and
Technology (HSCST), Haryana for providing financial assistance to
Mamta. We also thank the Sophisticated Analytical Instrumentation
Facility (SAIF), Panjab University, Chandigarh for providing mass
spectra and elemental analyses. We would like to thank University of
Houston-Downtown for providing organized research and creative ac-
tivities (ORCA) funds to Dr. Rachna Sadana.
[20] R. Aggarwal, C. Rani, G. Sumran, Efficient synthesis of some new 2,3-di-
methylquinoxaline-1-oxides using bis(acetoxy)phenyl-λ 3-iodane as an oxidant,
Synth. Commun. 44 (2014) 923–928.
[21] G. Sumran, R. Aggarwal, A convenient [hydroxy(tosyloxy)iodo]benzene-mediated
one-pot synthesis of 2-arylimidazo[2,1-b]benzothiazoles, J. Sulfur Chem. 36 (2015)
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[22] G. Sumran, R. Aggarwal, M. Hooda, D. Sanz, R.M. Claramunt, An unusual synthesis
of azines and their oxidative degradation to carboxylic acid using iodobenzene
diacetate, Synth. Commun. 48 (2018) 439–446.
Appendix A. Supplementary material
[23] S. Kumar, R. Aggarwal, V. Kumar, R. Sadana, B. Patel, P. Kaushik, D. Kaushik,
Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-in-
flammatory agents, Eur. J. Med. Chem. 123 (2016) 718–726.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.01.049.
[24] Mamta, R. Aggarwal, J. Smith, R. Sadana, Synthesis and cytotoxic evaluation of 1-
(6-chloropyridazin-3-yl)-3-H/alkyl-4-phenyl-1H-pyrazole-5-amines and their deri-
vatives, Indian J. Het. Chem. 26 (2016) 59–68.
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