Synthesis, crystal structures and biological evaluation of new pyridazine derivatives

Article abstract of DOI:10.1016/j.molstruc.2019.127084

A series of functionalized pyridazine derivatives (1–10) 4-phenyl-3,6-di(pyridine-4-yl)pyridazine (1), 3-(3,6-di(pyridin-4-yl)pyridazin-4-yl)aniline (2), 4-(pyridin-3-yl)-3,6-di(pyridin-4-yl)pyridazine (3), 3,6-di(pyridin-4-yl)-4-(thiophen-2-yl)pyridazine

Full text of DOI:10.1016/j.molstruc.2019.127084

Journal of Molecular Structure 1200 (2020) 127084
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, crystal structures and biological evaluation of new
pyridazine derivatives
, b, *
Bholey Singh a, Rohit Bhatia c, Balaram Pani a, Deepak Gupta a
a Department of Chemistry, Bhaskaracharya College of Applied Sciences (University of Delhi), Sector-2, Dwarka, New Delhi, 110075, India
b Present Address: Institut de La Matie're Condense et des Nanosciences (IMCN), Universite’ Catholique de Louvain (UCL), Place Louis Pasteur 1, 1348,
Louvain-la-Neuve, Belgium
c Rudraksh Proudhyogiki Sangathan, Delhi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of functionalized pyridazine derivatives (1e10) 4-phenyl-3,6-di(pyridine-4-yl)pyridazine (1), 3-
(3,6-di(pyridin-4-yl)pyridazin-4-yl)aniline (2), 4-(pyridin-3-yl)-3,6-di(pyridin-4-yl)pyridazine (3), 3,6-
Received 13 May 2019
Received in revised form
12 September 2019
di(pyridin-4-yl)-4-(thiophen-2-yl)pyridazine
(4),
6-methyl-1,4-di(pyridin-4-yl)-5H-pyrrolo[3,4-d]pyr-
idazine-5,7(6H)-dione (5), 4-phenyl-3,6-di(pyridine-3-yl)pyridazine (6), 3-(3,6-di(pyridin-4-yl)pyridazin-3-
yl)aniline (7), 3,4,6-tri(pyridin-3-yl)pyridazine (8), 3,6-di(pyridin-3-yl)-4-(thiophen-2-yl)pyridazine (9), 6-
methyl-1,4-di(pyridin-3-yl)-5H-pyrrolo[3,4-d]pyridazine-5,7(6H)-dione (10) were synthesized by one-
step methodologies which also include Inverse Electron Demand Diels-Alder reaction. The compounds
were isolated in high yields without any tedious purification procedures and characterized by NMR, Mass
spectrometry, Elemental Analysis and X-ray diffraction techniques. The crystal structures of five com-
pounds were studied. The pyridazines were subjected to anti-microbial evaluations where few of the
compounds showed moderate to high activity against most of the bacteria and fungi presented in this
study.
Accepted 15 September 2019
Available online 18 September 2019
Keywords:
Anti-biotic
Crystal
Diels-alder
Pyridazine
Tetrazine
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
[9,10]. In these systems, the tetrazine unit acts as a functional
building block and play diverse roles such as site for multiple-
Nitrogeneous heterocyclic compounds such as tetrazines are
known as efficient photoꢀ and redoxꢀ active compounds, which
have found applications in variety of areas such as dyes, bio-
activity, non-linear optics and light emission [1e3]. Considered as
highly electron-poor aromatic system, tetrazine-based compounds
are also being developed for electron-storage and photo-oxidation
[4,5]. Aiming for significant utilization of tetrazines in material
science and intrigued by such properties, polytetrazines and
tetrazine-containing polymers and covalent organic frameworks
are also coming-up with promising applications [6e8]. Moreover,
tetrazine moiety has also been elegantly deployed as a part of
ligand system in coordination chemistry in the form of supramo-
lecular coordination complexes and metal-organic framework
electron uptake and appending various functional units (applica-
tion oriented functionalization). Tetrazine platform have also been
employed as synthetic intermediates as well as starting material in
several natural product synthesis via tetrazine to pyridazine con-
version [11]. Pyridazines belong to the family of diazine compounds
containing two sp2-hybridized adjacent nitrogen atoms. The pyr-
idazine derivatives have been widely studied as biologically-active
compounds particularly for their antimicrobial activities [12e15].
Functionalized pyridazines unit containing compounds have been
identified as highly electron deficient compounds which prompted
their utilization as electrochromic materials and in metal-organic
frameworks [16,17]. In order to improve the existing properties
and to generate new functions of nitrogen-based materials, modi-
fication of the tetrazine unit to functionalized pyridazines is
extremely useful.
In the last few decades, a number of synthetic strategies such as
Cu-catalyzed azide-alkyne based “click reaction” and Diels-Alder
reactions have been developed as versatile tools for the conjuga-
tion of molecules via carbon-carbon bond formation. Among these,
iEDDA (inverse Electron Demand DielsꢀAlder) reaction has been
* Corresponding author. Department of Chemistry Institution, Bhaskaracharya
College of Applied Sciences (University of Delhi), Sector-2, Dwarka, New Delhi,
110075, India.
E-mail addresses: deepakg2003@gmail.com, deepak.gupta@uclouvain.be
(D. Gupta).
https://doi.org/10.1016/j.molstruc.2019.127084
0022-2860/© 2019 Elsevier B.V. All rights reserved.

2
B. Singh et al. / Journal of Molecular Structure 1200 (2020) 127084
introduced and gained importance as potential click reaction
scheme involving a 1,2,4,5ꢀtetrazine and olefins [18]. It has
emerged as a metal-free route for bio-orthogonal substrate label-
ling which offers fast reactivity (even at RT), clean products and
compatibility [19e21]. Very recently, this reaction platform has
been applied to append pyridazines on DNA [22]. Herein, we report
on a series of functionalized pyridazines derived from the reaction
of tetrazine compounds with various functional units via the single
step iEDDA approach. All the pyridazines have been thoroughly
characterized and studied for their anti-biotic properties such as
anti-bacterial and anti-fungal.
2. Results and discussion
The inverse-electron demand Diels-Alder (iEDDA) reaction has
been widely employed to append organic unit/s on a variety of
tetrazines by converting the tetrazine into a substituted pyridazine.
The reaction requires normal reflux conditions to react a tetrazine
moiety with the organic unit to be appended without using any
external reagent. The water-based work-up of these reactions
provide clean products in high yields. Owing to the fast reaction
kinetics, this reaction has been widely utilized to append biological
as well as luminescent molecules on tetrazine moiety containing
building blocks [23]. Therefore, as compared to the Copper-based
“click reactions”, the implementation of iEDDA reaction of tetra-
zines is one of the excellent methodologies to achieve pyridazine-
based compounds where a number of photo-, redox- and biologi-
cally active units can be appended on the pyridazine ring in one
step.
Scheme 1. One-step synthesis of pyridyl-functionalized pyridazines 1e10.
2.1. Synthesis of functionalized pyridazines
The pyridazine derivatives 1, 2, 5, 6, 7 and 10 were synthesized
by single step procedure using the iEDDA reaction between tetra-
zine ring of the di-3,6-(4-pyridyl)-1,2,4,5-tetrazine/di-3,6-(3-
pyridyl)-1,2,4,5-tetrazine precursor with acetylene-based dien-
ophiles (phenylacetylene for 1 and 6; 3-aminophenylacetylene for
2 and 7) and N-methylmaleimide for 5 and 10 in DMF followed by
extraction in DCM resulting in off-white to light yellow colored
compounds (Scheme 1). All the compounds were obtained in more
than 80% yield. The compounds were found to be soluble in most of
the polar organic solvents. The X-ray quality single crystals of the
pyridazines were grown by slow evaporation of DCM. The com-
pounds were analyzed with the help 1H NMR, 13C NMR, 2D-COSY,
Mass spectrometry, single crystal X-ray diffraction and elemental
analysis.
The pyridyl functionalized pyridazines 3, 4, 8 and 9 were ob-
tained by a slightly different procedure where 2-acetylpyridine for
3 and 8, 2-acetylthiophene for 4 and 9 were reacted with dipyr-
idyltetrazine precursor in the presence of 2.5% methanolic solution
of KOH at RT (Scheme 1). Refluxing these overnight resulted in
brown color solutions which on solvent removal produced light
yellow to light brown color powder/semi-solid. The products were
suspended in water and extracted with DCM. Fine crystals of the
products were obtained by slow evaporation of the DCM solution.
All the products are soluble in most of the polar organic solvents
resulting in clear colorless solutions.
were identified and assigned according to the theoretical mass
values. The elemental analysis data further justified the high purity
of all the compounds. The 1H NMR spectra of the 4-pyridyl-func-
tionalized pyridazines 1e5 were recorded in CDCl3. The peak po-
sition and integration ratios established the formation of functional
group appended pyridazines in pure form with the formation of a
single isomer in all the cases (Fig. 1). The four distinct doublets in
the aromatic region corresponds to the four pyridyl ring protons
which appears in the region (7.3e8.9 ppm) due to asymmetrical
structure.
The signal for the only pyridazine ring proton H5 can be seen as
singlet at different positions between 7.8 and 8.3 ppm for different
compounds which justifies the indirect effect of the pendant group
on the pyridazine ring whereas the signals for protons of the
functional group (phenyl, aniline, thiophenyl and pyridyl) appeared
in their usual positions without any major shift in the peak posi-
tions in all the compounds. The assignment of the peaks was aided
by HeH CoSy data. The correlations were observed for H2eH3 and
H20ꢀH3’. The NH proton in 2 can be seen at 5.25 ppm as broad
singlet. The 13C NMR data also supported the products formation.
The 1H NMR spectra of 3-pyridyl functionalized pyridazines
6e10 were also recorded in CDCl3 which showed entirely different
pattern as compared to NMR spectra of 4-pyridyl functionalized
pyridazines (Fig. 2). The peaks were assigned on the basis of CoSy
NMR spectra of all the compounds. In the 1H NMR spectra, the
signals for the H2 protons of the two pyridyl rings being nearest to
the pyridyl nitrogen and the pyridazine unit are most down-fielded
and appear at around 9.3 and 8.8 ppm for different compounds. The
next appearing peaks could be assigned to the H6 and H6’ protons
2.2. Characterization
The HRMS data for all the pyridazine compounds (1e10) was
found to be in accordance with the calculated values (m/
z ¼ 311.1282 for 1; 326.1399 for 2; 312.1247 for 3; 317.0848 for 4;
318.0865 for 5; 311.1305 for 6; 326.1405 for 7; 312.1248 for 8;
317.0871 for 9; 318.0995 for 10) (see SI). The molecular ion peaks
of the two pyridyl rings. The lone pyridazine ring proton (Ha) signal
appeared at around similar position (7.9 ppm) in all the NMR
spectra. The subsequent peaks in the high field aromatic region

B. Singh et al. / Journal of Molecular Structure 1200 (2020) 127084
3
Fig. 1. 1H NMR spectra of functionalized pyridazines 1e5 (CDCl3, RT, 400 MHz).
Fig. 2. 1H NMR spectra of functionalized pyridazines 6e10 (CDCl3, RT, 400 MHz).
could be easily assigned to the other pendant group on the pyr-
idazine ring. Notably, the signal for the H proton in 10 was found
absent unlike other pyridazines (6e9) further establishing the
be suitable to X-ray diffraction studies and collected by slow
evaporation of a saturated DCM solution at RT (Table 2). Fine quality
colorless crystals were decanted and exposed to X-rays on a
diffractometer. The crystal structures of the pyridazine compounds
resemble; featuring the central pyridazine ring containing the
pendant functional group at the fourth position (aniline for 2,
phenyl for 6, pyridyl for 3 & 8, thiophenyl 9) and two 4-pyridyl
units (for 2, 3) and 3-pyridyl units (for 6, 8 and 9) at the third
and sixth positions respectively (Fig. 3). The bond length data
a
structure of the compound.
2.3. Crystal structure
The X-ray diffraction quality single crystals of five out of the ten
functionalized pyridazine compounds 2, 3, 6, 8 and 9 were found to

4
B. Singh et al. / Journal of Molecular Structure 1200 (2020) 127084
Table 1
confirms the electron density delocalization in the ring. The three
substituents on the pyridazine ring namely, two pyridyl and the
functional group are not co-planar and exist in different orienta-
tions (Table 1). One common feature in the crystal structures is the
slight tilt of one of the pyridyl rings (ring 1) which is close to the
pendant functional group (ring 3) where the angle between the C3
carbon of the pyridyl ring and the nearest pyridazine nitrogen is in
the range ca. 112ꢁꢀ114ꢁ which is less than the normal bond angle
found in the parent tetrazine compounds. The crystal structure of 6
is isostructural with the structures presented previously, though
the temperature of collections of data sets are different [24,25].
Non-covalent interactions play pivotal role in numerous bio-
logical phenomenon where molecules interact in the form of loose
aggregates to generate supramolecules. These weaker supramo-
lecular interactions form the core of crystal engineering and are
therefore essential [26]. Interestingly, all the crystal structures
shown in the present study display a variety of non-covalent in-
The dihedral angles between various rings (shown here) and the pyridazine ring in
all the crystal structures.
.
Code
Ring 1
Ring 2
Ring 3
2
3
6
8
9
49.48
45.10
49.67
54.42
74.76
35.78
21.63
1.57
2.40
14.75
53.09
39.36
53.51
55.12
9.17
The values are in degree (o).
teractions such as H-bonding and
pꢀp stacking in the crystal
structures (Figs. 4 and 5). To mention a few, compound 2 shows a
rather strong network of inter-molecular H-bonding interactions
which extend along the b-axis where one of the eNH2 hydrogens of
pendant aniline ring is interacting with the pyridazine nitrogen
(~2.3 Å) of the adjacent molecule in the same direction while the
other one is involved with pyridyl nitrogen (~2.4 Å) of different
molecule in the other direction. The molecules of compound 3
showed that the pyridazine rings in all the compounds contain the
normal N]N bond (1.31e1.34 Å). Unlike the pyridine substituents,
the bond length data of various bonds in the pyridazine ring in all
the five crystal structures do not reflect uniformity but lie within
the range of the normal pyridazine bond lengths and therefore
Table 2
Crystallographic data for compounds 2, 3, 6, 8 and 9.
Sample Code
2
3
6
8
9
Chemical Formula
Formula Mass
Crystal System
a/Å
C20H15N5
325.37
C19H13N5
311.34
C20H14N4
310.35
C19H13N5
311.34
C18H12N4S
316.38
Monoclinic
19.2721(16)
8.1066(7)
10.3200(7)
90.0000
99.887(7)
90.0000
1588.4(2)
293
Orthorhombic
7.3813(3)
10.6853(4)
38.6185(16)
90.0000
90.0000
90.0000
3045.9(2)
293
Monoclinic
14.3445(10)
7.1214(4)
15.8417(10)
90.0000
105.483(7)
90.0000
1559.55(18)
293
Monoclinic
14.3445(6)
7.0963(3)
15.4880(7)
90.0000
104.798(5)
90.0000
1524.28(12)
293
Orthorhombic
5.3569(3)
16.5036(8)
16.8191(8)
90.0000
90.0000
90.0000
1486.95(13)
293
b/Å
c/Å
ꢁ
ꢁ
ꢁ
/
a
b
g
/
/
Unit Cell Volume/Å
Temperature/K
Space Group
P 21/c
4
1.361
680
Pbca
8
1.358
1296.0
P 21/n
4
1.322
648.0
P 21/n
4
1.357
648.0
P 21 21 21
4
1.413
No. of formula unit per unit cell, Z
Density (g cmꢀ3
F(000)
)
656.0
No. of reflections measured
22050
24386
22268
22304
21688
No. of independent reflections
3968
3938
3957
3910
3766
Observed [I > 2
s
(I)] reflections
4279
0.0646, 0.1738
1.041
23257
0.0612, 0.1684
1.066
23257
0.0555, 0.0917
1.030
5679
0.0489, 0.1340
1.015
4911
0.0467, 0.1175
1.056
R [F2 > 2
s
(F2)], wR(all data)
S
Fig. 3. Crystal structures of functionalized pyridazines 2, 3, 6, 8 & 9. Grey: C, Blue: N, White: H, Yellow: S.

B. Singh et al. / Journal of Molecular Structure 1200 (2020) 127084
5
Fig. 4. Network of hydrogen-bonding and CHꢀ
p
interaction formed molecules of 2.
Fig. 6. Absorption spectra of functionalized pyridazine compounds (1e10).
9 were most active in comparison to the standard respectively. For
S. aureus, functionalized-pyridazine 7 was most active whereas 1
and 4 were least active. Similar trend was observed in case of
K. pneumonia where again 4 displayed least activity and 9 was most
active. In case of B. subtilis, 1 was seen to be least active while 7
showed highest active in comparison to standard among all. Similar
to the anti-bacterial studies, all pyridazines showed dose response
behavior and possess moderate activity in comparison to the
standard against tested Fungi. Compound 3 and 8 were found to be
most active against in case of R. solani whereas the rest of com-
pounds showed average to moderate activity. In case of S. rolfsii, 2
were least active and 7 was most active whereas 8 displayed
highest activity against F. oxysporum, In case of A. niger, most of the
pyridazines showed poor activity but 3 showed moderate activity
(see Table 4).
Fig. 5. Inter-molecular
of 3.
p p stacked arrangement of pyridyl rings in crystal structure
ꢀ
undergo intermolecular p-p stacking interactions.
In the crystal structure of 3, the 3-pyridyl ring is stacked over the
electron deficient pyridazine ring of the adjacent molecule leading
to an offset packing forming a linear chain in a hand-shake manner
along the a-axis. These linear chains are inter-connected through
H-bonds and thus stabilize the crystal packing structure. Thus the
ability to make intermolecular networks and linear chains induces
an additional feature to these compounds to become suitable for
making supramolecular coordination structures when bonded with
metal-ions.
The absorption properties of all the pyridazines (1e10) were
studied by using UVeVis absorption spectroscopy in dichloro-
methane solution at room temperature. All the compounds show
intense bands in UV region which can be assigned to the aromatic
ring based transitions. The molar extinction coefficients were in the
range of 15000e90000 Mꢀ1cmꢀ1 in UV region (see supporting in-
formation). No significant absorption was found in the visible re-
gion (see Fig. 6).
2.4.1. Materials and methods
All the starting materials and solvents were purchased from
local vendors and utilized without any purification. All the solvents
were dried and distilled using general procedures prior to re-
actions. The reactions were carried out in air-atmosphere. The NMR
spectra were collected on Bruker Avance-400 spectrometer using
TMS as internal standard. The absorption spectra were recorded on
an Analytikjena Spectrophotometer (specord 250). The crystals
were mounted on Oxford Xcalibur Nova X-ray diffractometer with a
four circle
MoK . Single crystal X-ray data was recorded on Oxford callibur
Sapphire3 diffractometer employing a graphite monochromatized
MoK at room temperature. The data of all the crystals was reduced
k Goniometer employing a graphite monochromatized
a
a
using CrysAlis pro software available with the diffractometer and
thereby reduced the Rint. Further least square refinement after
introduction of anisotropic displacement parameters yielded the R
values mentioned in Table 2. Elemental Analysis were carried out
on Flash EA series 1112 CHNS analyser. The ESI MS data was
collected from a Agilent Technologies 6530 Accurate-Mass Q-TOF
LC/MS. Fungal culture of R. solani (ITCC 4502), S. rolfsii (ITCC 6263) F.
oxysporum (ITCC 4884) and A. niger (ITCC 1624) were obtained
from the Indian type culture collection, Division of Mycology and
Plant Pathology, Indian Agricultural Research Institute (IARI), New
Delhi. Bacterial culture E. coli (MTCC 443), Staphylococcus aureus
(MTCC 3160), Pseudomonas aeruginosa (MTCC 2581), B. subtilis
(MTCC 441) and Klebsiella pneumoniae (MTCC 7028) were procured
2.4. Antimicrobial activity
The in-vitro antimicrobial activity for the synthesized com-
pounds was determined against various bacteria e.g. E. coli,
S. aureus, P. aeruginosa, K. pneumonia and B. subtilis as average
diameter of the inhibition zone in mm whereas the antifungal ac-
tivity was estimated against R. solani, S. rolfsii, F. oxysporum and
A. niger. These data are shown in Tables 3 and 4. All the compounds
showed dose response behavior and possessed moderate to good
activity against both gram negative and gram positive bacteria in
comparison to the standard against tested bacteria. In case of E. coli
and P. aeruginosa, 4 was found to be least active whereas 2, 3, 7 and

6
B. Singh et al. / Journal of Molecular Structure 1200 (2020) 127084
Table 3
Antimicrobial screening results of few of the synthesized compounds (1e9).
Code
E. coli Zone of inhibition
S. aureus Zone of inhibition
P. aeruginosa Zone of
K. pneumonia Zone of
B. subtilis Zone of inhibition
(mm)
(mm)
inhibition (mm)
inhibition (mm)
(mm)
200 100 50 25 12.5 200 100 50 25 12.5 200 100 50 25 12.5 200 100 50 25 12.5 200 100 50 25 12.5
1
2
3
4
6
7
8
9
20
27
33
21
31
34
30
29
19
24
29
17
29
28
25
23
35
16 13
19 15 08
24 19 11
e
23
30
31
23
32
35
28
29
38
18
27
26
19
28
30
20
26
33
15 09
21 19 14
21 16 09
14 07
22 14 08
24 21 19
14 08
21 18 12
30 28 26
e
29
31
29
19
27
30
28
32
39
24
26
24
15
24
26
23
28
34
19 16 11
19 14 09
17 11 07
21
25
17
14
23
28
21
27
32
15
21
11
09
16
23
19
23
30
12 06
16 13 08
e
18
22
24
20
19
26
23
24
28
16
18
16
16
15
21
18
19
26
13 10 05
14 07
12 06
12 08
e
e
e
08
e
e
e
e
e
13
e
e
e
07
e
e
23 19 15
21 17 14
19 16 12
17 14 10
32 30 26
16 09
e
11 09 05
19 14 08
16 14 09
18 15 11
29 27 25
11 09 06
16 11 08
15 12 08
23 19 15
17 13 09
21 18 14
32 30 28
e
16 07
e
Control 37
25 23 19
Table 4
Anti-fungal screening results of few of the synthesized compounds (1e9).
Code
Fungal inhibition (%)
R. solani
S. rolfsii
F. oxysporum
A. niger
100
50
25
12.5
100
50
25
12.5
100
50
25
12.5
100
50
25
12.5
1
2
3
4
6
7
8
9
51
38
83
77
81
67
84
45
92
48
29
79
68
74
59
71
39
84
41
14
73
52
67
47
62
33
77
22
5
53
33
71
65
76
79
77
52
85
42
30
69
55
66
64
71
43
83
21
28
62
51
47
56
63
36
75
14
15
57
47
31
42
51
27
72
42
40
87
82
83
55
88
61
93
39
36
78
74
77
47
78
53
86
30
25
69
69
68
41
67
45
79
25
19
57
59
42
39
52
34
71
51
43
84
79
68
77
80
58
94
41
36
71
66
44
63
76
45
85
31
26
69
57
36
54
64
39
76
29
15
49
44
19
43
48
21
69
61
41
56
36
53
25
73
Standard
from the microbial type culture collection and gene bank, Institute
of Microbial Technology, Chandigarh.
from the concentration (mgLꢀ1) and corresponding IC data of each
compound with the help of statistical package (GW BASIC) [24].
2.4.3. Disk diffusion assay
2.4.2. Poison food assay
The antimicrobial potential of synthesized metal complexes was
tested using a disk diffusion assay. Briefly the nutrient agar medium
(25 mL) was poured into petri dishes (90 mm in diameter) under
aseptic conditions in a laminar flow hood. The plates were kept in
the laminar flow chamber for solidification of the media. After so-
lidification 100 mL of fresh culture (log phase) was spread on the
surface of the solidified medium with the help of a spreader. The
plates were then kept in laminar flow for drying. Once dried, five
Antifungal assay was carried out by a poisoned food technique
using potato-dextrose-agar (4% PDA) medium against four phyto-
pathogenic fungi Rhizoctonia solani, Sclerotium rolfsii, Fusarium
oxysporum and A. niger at different concentrations (200e12.5 ppm)
[27]. 1 mL of stock solution was added to 50 mL of PDA medium in
to obtain desired concentrations. The medium was then poured
into two Petri plates under aseptic conditions in a laminar flow
chamber. Similarly other concentrations were prepared by serial
dilution process. 1 mL of methanol was used as a control. A 5 mm
thick disc of fungus was put at the center of the medium in the test
petri plate and the plates were kept in BOD incubator at 28 1 ꢁC
till the fungal growth in the control dishes was completed (6e10
days). The mycelial growth (cm) in both treated (T) and control (C)
petri-plates use measured diametrically in three different di-
rections. From the mean growth of above readings, percentage in-
hibition of growth (I) was calculated by using the following
equation:
plain sterile disks were placed in the plate and 5
of different concentration (200e12.5 ppm) was loaded on each
disk. In control plate, commercially procured ampicillin (10 g/
mL of test solution
m
disk) was used. Plates were then kept at 37 ꢁC for 24 h in the
incubator and zone of inhibition (in mm) was recorded for all the
compounds tested and commercial antibiotic. All experiments
were performed in triplicate for each treatment against each
bacteria.
2.4.4. Experimental section
2.4.4.1. General procedure for the preparation of functionalized pyr-
idazines (1, 2, 5, 6, 7, 10). In a round bottom flask, di-3,6-(4-
pyridyl)-1,2,4,5ꢀtetrazine (1 mmol) for 1, 2 & 5 or di-3,6-(3-
pyridyl)-1,2,4,5ꢀtetrazine (1 mmol) for 6, 7 & 10 was suspended
in DMF at 100 ꢁC with stirring. After half an hour, phenylacetylene
for 1, 6; 3-ethynylaniline for 2, 7; N-methylmaleimide for 5, 10
(1 mmol) was added to the reaction mixture. This was left for
stirring. The formation of product was monitored by TLC. The light
brown color reaction mixture was poured into ice-cold water which
was washed with 40 ꢂ 3 mL DCM. Solvent layer was evaporated on
a rotary evaporator to obtain the desired product as light yellow
solids.
Percent growth inhibition I(%) ¼ [(Tꢀ C)/C] ꢂ 100
EC50 (effective concentration for 50% inhibition of mycelial
growth) was calculated from the percent inhibition (IC) as per the
following equations:
IC ¼ [(% Ie C$F.) / (100ꢀ C$F.)] ꢂ 100
C.F. (Correction Factor) ¼ [(90ꢀ C)/C] ꢂ 100
where 90 is the diameter of the petri dishes (mm) and C is the
growth of the fungus (mm) in control. EC50 (mgLꢀ1) was calculated

B. Singh et al. / Journal of Molecular Structure 1200 (2020) 127084
7
2.4.4.2. Synthesis of 4-phenyl-3,6-di(pyridine-4-yl)pyridazine (1).
desired product as light yellow/off-white solids.
Prepared from phenylacetylene, light yellow crystal, yield ¼ 88.6%,
mp ¼ 190.8 ꢁC; MS: (m/z) 311.1282 (MHþ); 1H NMR (CDCl3,
2.4.4.9. Synthesis of 4-(pyridin-3-yl)-3,6-di(pyridin-4-yl)pyridazine
(3). Prepared from 3-acetylpyridine, light yellow product,
yield ¼ 89.6%, mp ¼ 218.3 ꢁC. MS: (m/z) 312.1247 (MHþ); 1H NMR
400 MHz,
d
): 8.74 (dd, 2H, J ¼ 4Hz, H2), 8.51 (dd, 2H, J ¼ 4Hz, H2’),
8.014 (dd, 2H, J ¼ 4 Hz, H3), 7.91 (s, 1H, H
a), 7.39e7.32 (m, 5H,
H30,300,400), 7.21e7.19 (m, 2H, H200). Anal. Calcd. for C20H14N4: C,
(CDCl3, 400 MHz, d
): 8.81 (d, 2H, H2), 8.68 (d, 1H, H600), 8.60 (d, 3H,
H2’, H200), 8.06 (d, 2H, H3), 7.96 (s, 1H, H ), 7.51 (d, 1H, H400), 7.36 (d,
a
77.40; H, 4.55; N, 18.05. Found: C, 77.52; H, 4.34; N, 18.14.
2H, H3’), 7.33e7.30 (dd, J ¼ 4Hz, 1H, H500). Anal. Calcd. for C19H13N5:
C, 73.30; H, 4.21; N, 22.49. Found: C, 73.46; H, 4.16; N, 22.38.
2.4.4.3. Synthesis of 3-(3,6-di(pyridin-4-yl)pyridazin-4-yl)aniline
(2). Prepared from 3-ethynylaniline, light yellow crystal,
yield ¼ 92.3%, mp ¼ 212 ꢁC; MS: (m/z) 326.1399 (MHþ); 1H NMR
2.4.4.10. Synthesis of 3,6-di(pyridin-4-yl)-4-(thiophen-2-yl)pyr-
idazine (4). Prepared from 2-acetylthiophene, off-white product,
yield ¼ 85.8%, mp ¼ 225 ꢁC. MS: (m/z) 317.0848 (MHþ); 1H NMR
(CDCl3, 400 MHz,
H2’), 8.31 (s, 1H, H
H3’), 6.99 (t, 1H, H500), 6.58 (d, 1H, J ¼ 4 Hz, H600), 6.52 (m, 1H, H200),
6.37 (d, 1H, J ¼ 4 Hz, H400), 5.23 (broad singlet, 2H, NH). Anal. Calcd.
for C20H15N5: C, 73.83; H, 4.65; N, 21.52. Found: C, 73.98; H, 4.48; N,
21.54.
d
a
): 8.74 (d, 2H, J ¼ 4 Hz, H2), 8.55 (d, 2H, J ¼ 4 Hz,
), 8.22 (d, 2H, J ¼ 4 Hz, H3), 7.40 (d, 2H, J ¼ 4 Hz,
(CDCl3, 400 MHz,
d
): 8.76e8.74 (dd, J ¼ 4Hz, 2H, H2), 8.63e8.61 (dd,
J ¼ 4Hz, 2H, H2’), 8.00 (dd, J ¼ 4Hz, 2H, H3), 7.94 (s, 1H, H
a), 7.44
(dd, J ¼ 4Hz, 2H, H3’), 7.43 (d, 1H, H500), 6.98 (dd, J ¼ 4Hz, 2H, H400),
6.93 (dd, 1H, J ¼ 4Hz, 2H, H300). Anal. Calcd. for C18H12N4S: C, 68.33;
H, 3.82; N, 17.71; S, 10.13. Found: C, 68.20; H, 4.06; N, 17.60; S, 10.14.
2.4.4.4. Synthesis of 6-methyl-1,4-di(pyridin-4-yl)-5H-pyrrolo[3,4-d]
pyridazine-5,7(6H)-dione (5). Prepared from N-methylmaleimide,
off-white crystal, yield ¼ 78.5%, mp ¼ 205.6 ꢁC; MS: (m/z) 318.0995
2.4.4.11. Synthesis
of
3,4,6-tri(pyridin-3-yl)pyridazine
(8).
Prepared from 3-acetylpyridine, light yellowþ product,
yield ¼ 85.8%, mp ¼ 248.2 ꢁC. MS: (m/z) 312.1248 (MH ); 1H NMR
(MHþ). 1H NMR (CDCl3, 400 MHz,
d): 8.78 (d, 4H, J ¼ 4 Hz, H2), 8.01
(CDCl3, 400 MHz,
d
): 9.26 (d, 1H, H2), 8.67 (dd, J ¼ 4Hz, 1H, H6), 8.57
(d, 2H, J ¼ 4 Hz, H3), 7.57 (d, 2H, H5), 8.22 (d, 2H, J ¼ 4 Hz, H3’), 2.50
(s, 3H, N-methyl). Anal. Calcd. for C17H11N5O2: C, 64.35; H, 3.49; N,
22.07. Found: C, 64.42; H, 3.42; N, 21.94.
(m, 2H, H2’, H6’), 8.53e8.51 (m, 2H, H200, H600), 8.49e8.46 (ddd, 1H,
H4), 7.9 (s, 1H, Ha), 7.78e7.75 (ddd, 1H, H4’), 7.52e7.49 (ddd, 1H,
H400), 744e7.41 (dd, J ¼ 4Hz, 1H, H5), 7.27e7.21 (ddd, 2H, H5’, H500).
Anal. Calcd. for C19H13N5: C, 73.30; H, 4.21; N, 22.49. Found: C,
73.42; H, 4.14; N, 22.44.
2.4.4.5. Synthesis of 4-phenyl-3,6-di(pyridine-3-yl)pyridazine (6).
Prepared from phenylacetylene, off-white crystal, yield ¼ 84.4%,
mp ¼ 183.8 ꢁC; MS: (m/z) 311.1305 (MHþ); 1H NMR (CDCl3,
2.4.4.12. Synthesis of 3,6-di(pyridin-3-yl)-4-(thiophen-2-yl)pyr-
idazine (9). Prepared from 2-acetylthiophene, off-whþite product,
yield ¼ 87.6%, mp ¼ 251.6 ꢁC. MS: (m/z) 317.0871 (MH ); 1H NMR
400 MHz, d): 9.33 (s, 1H, H2), 8.76 (d, 1H, H6), 8.69 (s, 1H, H2’),
8.60e8.56 (m, 2H, H6’, H4), 7.92 (s, 1H, Ha), 7.87 (dd, 1H, H4’),
7.52e7.49 (dd, 1H, H5), 7.42e7.37 (m, 3H, H300, H400), 7.30e7.25 (m,
3H, H5’, H200). Anal. Calcd. for C20H14N4: C, 77.40; H, 4.55; N, 18.05.
Found: C, 77.49; H, 4.53; N, 17.98.
(CDCl3, 400 MHz, d): 9.25 (s, 1H, H2), 8.71 (s, 1H, H2’), 8.67 (d, 1H,
H2’), 8.59 (d, J ¼ 4Hz, 1H, H6), 8.45 (d, 1H, H6’), 7.92 (s, 1H, H
a), 7.86
(d, 1H, H4’), 7.43e7.40 (dd, J ¼ 4Hz, 1H, H5), 7.37 (d, 1H, H500),
7.31e7.28 (dd, J ¼ 4Hz, 1H, H5’), 6.97e6.93 (m, 2H, H300, H400). Anal.
Calcd. for C18H12N4S: C, 68.33; H, 3.82; N, 17.71; S, 10.13. Found: C,
68.26; H, 3.98; N, 17.58; S, 10.18.
2.4.4.6. Synthesis of 3-(3,6-di(pyridin-4-yl)pyridazin-3-yl)aniline
(7). Prepared
from
3-ethynylaniline,
off-white
crystal,
yield ¼ 91.2%, mp ¼ 204.4 ꢁC; MS: (m/z) 326.1405 (MHþ); 1H NMR
(CDCl3, 400 MHz,
d): 9.29 (s, 2H, J ¼ 4 Hz, H2), 8.74 (2, 2H, J ¼ 4 Hz,
Acknowledgement
H2’), 8.71 (d, 1H, H6), 8.54 (d,1H, H6’), 8.50 (d, J ¼ 4 Hz,1H, H4), 7.87
(s, 1H, H
a
), 7.85 (d, J ¼ 4 Hz, 1H, H4’), 7.47e7.44 (dd, J ¼ 4 Hz, 1H,
DG is indebted to Science and Engineering Research Board (In-
dia) for a generous funding (YSS/2015/001820) and Bhaskaracharya
College of Applied Sciences (University of Delhi) for research fa-
cilities. BS thanks UGC for Junior Research Fellowship (162000320).
Authors acknowledge scientific support from University Science
Instrumentation Center and Department of Chemistry (University
of Delhi). Authors thank Dr. Koen Robeyns (Institut de la Matie're
Condense et des Nanosciences (IMCN), Universite' Catholique de
Louvain, Belgium) for frutiful discussions on improving the crys-
tallography studies.
1878879 (for 2), 1878881 (for 3), 1878877 (for 6), 1881615 (for 8)
and 1882828 (for 9) contains the supplementary crystallographic
data for the respective compounds. These data can be obtained free
of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
H5), 7.25e7.22 (m, 1H, H5’), 7.10 (t, 1H, H500), 6.66 (d, 1H, H400), 6.54
(s, 1H, H200), 6.52 (d, 1H, H600). Anal. Calcd. for C20H15N5: C, 73.83; H,
4.65; N, 21.52. Found: C, 73.94; H, 4.55; N, 21.51.
2.4.4.7. Synthesis of 6-methyl-1,4-di(pyridin-3-yl)-5H-pyrrolo[3,4-d]
pyridazine-5,7(6H)-dione (10). Prepared from N-methylmaleimide,
off-white crystal, yield ¼ 80.4%, mp ¼ 183.2 ꢁC; MS: (m/z) 318.0995
(MHþ). 1H NMR (CDCl3, 400 MHz,
d): 9.28 (s, 2H, H2), 8.79 (m, 2H,
H6), 8.43e8.39 (m, 2H, H4), 7.52 (t, 2H, H5). Anal. Calcd. for
17H11N5O2: C, 64.35; H, 3.49; N, 22.07. Found: C, 64.46; H, 3.41; N,
C
21.98.
2.4.4.8. General procedure for the preparation of functionalized
pyridazines (3, 4, 8, 9). In a round bottom flask, di-3,6-(4-pyridyl)-
1,2,4,5ꢀtetrazine (for 3 & 4)/di-3,6-(3-pyridyl)-1,2,4,5ꢀtetrazine
(for
8 & 9) (1 mmol) and 2-acetylpyridine (for 3 & 8)/2-
Appendix A. Supplementary data
acetylthiophene (for 4 & 9) (1 mmol) were suspended in meth-
anol at 65 ꢁC with stirring. To this, 1 mL of 2.5% methanolic solution
of KOH was added to the reaction mixture. This was left for stirring.
The formation of product was monitored by TLC. The solvent was
reduced under vacuum. The brown color reaction mixture was
poured into ice-cold water which was washed with 40 ꢂ 3 mL DCM.
Solvent layer was evaporated on a rotary evaporator to obtain the
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127084.
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