Synthesis and structural studies of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3′,4′-f]pyridazines

Article abstract of DOI:10.1016/j.molstruc.2019.02.082

Synthesis of a series of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3′,4′-f]pyridazines (4) was accomplished by the oxidative intramolecular cyclization of 6-arylidenehydrazino-3-aryl-1,2,4-triazolo[4,3-b]pyridazine (3) using iodobenzene diacetate (IBD), as a green oxidant, in dichloromethane at room temperature. The compounds 3 and 4 were characterized by IR, NMR (¹H and¹³C), mass spectral data and elemental analyses. X-ray crystal analysis of sterically strained 3,6-di-(2′-fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3′,4′-f]pyridazine 4f and 3,6-di-(4′-fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3′,4′-f]pyridazine 4g indicated that pyridazine ring has twisted conformation leading to nonplanar tricyclic core. Both compounds crystallized in orthorhombic P2₁2₁2₁ space group, containing one single molecule per asymmetric unit. The studies reveal that the compound 4f is associated with weak, centrosymmetric F?F interactions (distance of 2.882(3) ?), with cis geometry, between adjacent molecules which are responsible for the formation of chains along a axis. Additionally, compounds 4 were screened for their cytotoxic activity against the human cervical carcinoma (HeLa) cell line using MTT assay, however, with not much significant activity.

Full text of DOI:10.1016/j.molstruc.2019.02.082

Accepted Manuscript
Synthesis and structural studies of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b]
[3′,4′-f]pyridazines
Ranjana Aggarwal, Mamta, Garima Sumran, Mari Carmen Torralba
PII:
S0022-2860(19)30218-2
DOI:
https://doi.org/10.1016/j.molstruc.2019.02.082
MOLSTR 26239
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 September 2018
Revised Date: 7 February 2019
Accepted Date: 21 February 2019
Please cite this article as: R. Aggarwal, Mamta, G. Sumran, M.C. Torralba, Synthesis and structural
studies of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3′,4′-f]pyridazines, Journal of Molecular Structure
(2019), doi: https://doi.org/10.1016/j.molstruc.2019.02.082.
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Graphical abstract

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Synthesis and structural studies of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-
b][3',4'-f]pyridazines
Ranjana Aggarwala*, Mamtaa, Garima Sumranb, Mari Carmen Torralbac
aDepartment of Chemistry, Kurukshetra University, Kurukshetra, 136119, India
bDepartment of Chemistry, D. A. V. College (Lahore), Ambala City 134 002, Haryana, India
cDepartamento de Química Inorgánica, Facultad de Ciencias Químicas, Universidad
Complutense de Madrid (UCM), E-28040 Madrid, Spain
*Corresponding Author: Phone: +91(1744)238734, +919896740740; Fax: +91(1744)238277
Email: ranjanaaggarwal67@gmail.com
Abstract: Synthesis of a series of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazines
(4) was accomplished by the oxidative intramolecular cyclization of 6-arylidenehydrazino-3-
aryl-1,2,4-triazolo[4,3-b]pyridazine (3) using iodobenzene diacetate (IBD), as a green oxidant, in
dichloromethane at room temperature. The compounds 3 and 4 were characterized by IR, NMR
(1H and13C), mass spectral data and elemental analyses. X-ray crystal analysis of sterically
strained 3,6-di-(2'-fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine 4f and 3,6-di-(4'-
fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine 4g indicated that pyridazine ring has
twisted conformation leading to nonplanar tricyclic core. Both compounds crystallized in
orthorhombic P212121 space group, containing one single molecule per asymmetric unit. The
studies reveal that the compound 4f is associated with weak, centrosymmetric F···F interactions
(distance of 2.882(3) Å), with cis geometry, between adjacent molecules which are responsible
for the formation of chains along a axis. Additionally, compounds 4 were screened for their
cytotoxic activity against the human cervical carcinoma (HeLa) cell line using MTT assay,
however, with not much significant activity.
Keywords: 6-Hydrazino-1,2,4-triazolo[4,3-b]pyridazine; 6-Arylidenehydrazino-3-aryl-1,2,4-
triazolo[4,3-b]pyridazine; Bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazines; Iodobenzene diacetate;
X-ray crystallography.
1. Introduction
1,2-Diazine, commonly referred as pyridazine, is heteroaromatic compound containing
two nitrogen atoms with free electron pairs that permit the pyridazine ring to act like as an
effective and stable complexing agent for transition metal ions and to be used for the
development of new π-conjugated organic compounds [1]. The chemistry of 1,2,4-triazoles fused
with pyridazine ring has received considerable attention owing to their versatile pharmacological
properties such as anticonvulsant [2], antitubercular [3], antitublin [4], antiproliferative [5],

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antimicrobial [6, 7], anxiolytic [8], and cognition enhancing activity [9]. These compounds also
act as c-Met kinase inhibitors [10], tankyrase inhibitors [11], pan-phosphodiesterase (PDE)
inhibitors [12, 13] besides being highly selective antagonists of canbinoid receptors [14].
Meticulously, 3,6-diaryl-[1,2,4]triazolo[4,3-b]pyridazine (I) (Figure 1) exhibited significant
antiproliferative effects (IC50 = 0.008-0.014 ꢀM) against SGC-7901, A549 and HT-1080 cell
lines and cell cycle studies revealed that compound I arrested cell cycle progression at G2/M
phase in A549 cells [4]. (S)-6-(1-(6-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-
3-yl)ethyl)quinoline (II) was reported as selective c-Met inhibitor which inhibited tumor growth
in GTL-16 xenograft tumor model besides being pan-PDE family inhibitor [10]. Very recently
our group reported anticancer potential of [1,2,4]triazolo[4,3-b]pyridazines against three human
cancer cell lines namely SB-ALL, NALM-6 and MCF-7. Among them 6-chloro-3-(1H-indole-3'-
yl)-[1,2,4]triazolo[4,3-b]pyridazine (III) displayed potent cytotoxic activity with IC50 values
ranging from 1.14 to 3.55 ꢀM and induced apoptosis of NALM-6 cells via caspase 3/7 activation
[15]. Additionally, pyridazine derivatives play an imperative role in chemistry because of their
unique property of forming and strengthening of supramolecular assembly [16-19].
Figure 1. Representative [1,2,4]triazolo[4,3-b]pyridazine derivatives with anticancer activity.

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The DNA photocleavage and antifungal activities of a series of 1-aryl-4-methyl-1,2,4-
triazolo[4,3-b]quinoxalines [20, 21] has been reported by our group and the study further
extended to synthesis of 3,10-disubtituted-bis-1,2,4-triazolo[4,3-a][3',4'-c]-quinoxalines [22].
Inspired by biological importance of 1,2,4-triazoles fused with pyridazine ring it was envisioned
to extend our work to the synthesis of bis-1,2,4-triazolopyridazines. The most widely used
method for the synthesis of bis-1,2,4-triazolopyridazines involves the cyclization of 6-
arylidenehydrazino-3-aryl-1,2,4-triazolo[4,3-b]pyridazine into bis-1,2,4-triazolo-[4,3-b][3',4'-
f]pyridazine upon treatment with Br2/AcOH or lead tetracetate [23], or Me4NBr/oxone® [24],
thereby constructing the second triazole ring. Whereas simultaneous formation of bis-triazole has
been reported by oxidative cyclization of 3,6-bis-(arylidenehydrazino)pyridazines using
Br2/AcOH or lead tetraacetate [23] and by the reaction of 3,6-dichloropyridazine with two moles
of acid hydrazides [25]. However, these methods suffer from one or more disadvantages such as
harsh reaction conditions, use of toxic and expensive reagents, higher reaction temperature,
tedious workup reaction condition and longer reaction time. Recently, iodobenzene diacetate
(IBD) emerged as a powerful oxidizing reagent for various synthetic transformations replacing
toxic metal based oxidants, such as Tl(III), Pb(IV) and Hg(II), because of its similarity in
oxidation reactions, comparative less toxic nature, ready availability, ease of handling and easy
reaction workup condition.
Keeping in view the significance of 1,2,4-triazolopyridazines and in continuation of our
ongoing research work on hypervalent iodine reagents in organic transformations [26-30], we
herein report the facile synthesis of a series of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3',4'-
f]pyridazines from hydrazone precursors using iodobenzene diacetate, structural studies by X-ray
crystallography and in vitro cytotoxicity study.

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2. Experimental
2.1. Materials and methods
Melting points were determined in digital melting point apparatus MEPA and are
uncorrected. IR spectra were recorded on a Buck Scientific IR M-500 spectrophotometer in KBr
1
pellets (νmax in cm-1). H (400 MHz) and 13C NMR (100 MHz) spectra for analytical purpose
were recorded on a Bruker Avance II 400 NMR spectrometer using DMSO-d6 or CDCl3 as a
solvent; chemical shifts are expressed in δ units (parts per million) downfield from TMS as an
internal reference. The coupling constants (J) are given in Hz. Mass spectra were measured in EI
mode on a Kratos MS-50 spectrometer at MS Facilities at Sophisticated Analytical Instrument
Facility (SAIF), Panjab University, Chandigarh, India. Elemental analyses were also performed
at SAIF, Panjab University, Chandigarh, India.
2.2. Synthesis of 6-hydrazino-3-aryl/heteroaryl-1,2,4-triazolo[4,3-b]pyridazine (2a-l)
6-Chloro-3-aryl/heteroaryl-1,2,4-triazolo[4,3-b]pyridazine (1) (2.0 mmol) was dissolved
in 10 mL of ethanol and then hydrazine hydrate (0.1 g, 2.0 mmol) was added. The resulting
mixture was refluxed for 3 hours. After cooling to room temperature, a solid residue separated
out which was filtered off, washed with water and dried to afford hydrazine derivative 2.
2.2.1. 6-Hydrazino-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine (2a)
1
Yield 70%; m.p. 245 ºC; IR (KBr, cm-1): 3233 ( br NH str.), 1602 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.32 (bs, 2H, -NH2), 6.85-6.88 (d, 1H, J=9.88 Hz, pyridazine-5H), 7.46-7.48
(m, 1H, Ph-4'-H), 7.51-7.55 (m, 2H, Ph-3', 5'-H), 7.87-7.90 (d, 1H, J= 9.84 Hz, pyridazine-4H),
8.48 (s, 1H, -NH), 8.52-8.54 (m, 2H, Ph-2', 6'-H); 13C NMR (100 MHz, DMSO-d6) δ: 114.96,
123.51, 126.86, 126.95, 128.30, 129.08, 143.11, 145.03, 156.35. Anal. Calcd. for C11H10N6: C,
58.40; H, 4.46; N, 37.15. Found: C, 58.12; H, 4.14, N, 37.39.

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2.2.2. 6-Hydrazino-3-(2'-chlorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (2b)
1
Yield 69%; m.p. >315 ºC; IR (KBr, cm-1): 3448 (br NH str.), 1610 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.10 (bs, 2H, -NH2), 6.90-6.92 (d, 1H, J=9.88 Hz, pyridazine-5H), 7.48-7.52
(dt, 1H, Jo=7.44 Hz, Jm=1.4 Hz, Ph-4'-H), 7.54-7.59 (dt, 1H, Jo=7.42 Hz, Jm=1.8 Hz, Ph-5'-H),
7.62-7.65 (dd, 1H, Jo=8.02 Hz, Jm=1.24 Hz, Ph-3'-H), 7.71-7.73 (dd, 1H, Jo=7.54 Hz, Jm=1.76
Hz, Ph-6'-H ), 7.93-7.95 (d, 1H, J=9.84 Hz, pyridazine-4H), 8.42 (s, 1H, -NH); 13C NMR (100
MHz, DMSO-d6) δ: 115.52, 123.41, 126.24, 126.89, 129.73, 131.36, 132.46, 133.63, 143.16,
145.58, 156.45. MS: m/z 261.05 [M+1]+, 263.04 [M+1+2]+ (3:1). Anal. Calcd. for C11H9ClN6: C,
50.68; H, 3.48; N, 32.24. Found: C, 50.54; H, 3.83, N, 32.06.
2.2.3. 6-Hydrazino-3-(4'-chlorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (2c)
1
Yield 70%; m.p. 231 ºC; IR (KBr, cm-1): 3336 (br NH str.), 1612 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.32 (bs, 2H, -NH2), 6.85-6.88 (d, 1H, J=9.88 Hz, pyridazine-5H), 7.53-7.56
(dd, 2H, Jo=7.0 Hz, Jm=1.72 Hz, Ph-3', 5'-H), 7.89-7.91 (d, 1H, J=9.84 Hz, pyridazine-4H), 8.53
(s, 1H, -NH), 8.56-8.58 (m, 2H, Ph-2', 6'-H); 13C NMR (100 MHz, DMSO-d6) δ: 115.16, 123.63,
125.71, 128.42, 128.59, 133.96, 143.98, 145.05, 156.46. Anal. Calcd. for C11H9ClN6: C, 50.68;
H, 3.48; N, 32.24 Found: C, 50.23; H, 3.10, N, 32.06.
2.2.4. 6-Hydrazino-3-(3'-bromophenyl)-1,2,4-triazolo[4,3-b]pyridazine (2d)
1
Yield 68%; m.p. 263 ºC; IR (KBr, cm-1): 3244 (br NH str.), 1605 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.35 (bs, 2H, -NH2), 6.88-6.90 (d, 1H, J=9.88 Hz, pyridazine-5H), 7.47-7.51
(t, 1H, J=7.96 Hz, Ph-5'-H), 7.63-7.65 (m, 1H, Jo=8.02 Hz, Jm=1.0 Hz, Ph-6'-H), 7.92-7.95 (d,
1H, J=9.88 Hz, pyridazine-4H), 8.57 (s, 1H, -NH), 8.59-8.61 (m, 1H, Jo=8.0 Hz, Jm=1.28 Hz,
Ph-4'-H), 8.66-8.67 (m, 1H, Jm=1.72 Hz, Ph-2'-H); 13C NMR (100 MHz, DMSO-d6) δ: 115.34,
121.83, 123.78, 125.64, 128.91, 129.03, 130.93, 132.06, 144.17, 144.50, 156.60. MS: m/z 305.03

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[M+1]+, 307.03 [M+1+2]+ (1:1). Anal. Calcd. for C11H9BrN6: C, 43.30; H, 2.97; N, 27.54.
Found: C, 43.75; H, 3.12, N, 27.28.
2.2.5. 6-Hydrazino-3-(4'-bromophenyl)-1,2,4-triazolo[4,3-b]pyridazine (2e)
1
Yield 71%; m.p. 224.5 ºC; IR (KBr, cm-1): 3248 (br NH str.), 1636 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.25 (bs, 2H, -NH2), 6.86-6.88 (d, 1H, J=9.84 Hz, pyridazine-5H), 7.66-7.68
(d, 2H, J=8.56 Hz, Ph-3', 5'-H), 7.85-7.88 (d, 1H, J=9.8 Hz, pyridazine-4H), 8.48-8.50 (d, 2H,
J=8.6 Hz, Ph-2', 6'-H), 8.51 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6) δ: 115.16, 122.65,
123.59, 126.06, 128.62, 131.48, 144.00, 145.13, 156.44. MS: m/z 305.01 [M+1]+, 307.02
[M+1+2]+ (1:1). Anal. Calcd. For C11H9BrN6: C, 43.30; H, 2.97; N, 27.54. Found: C, 43.15; H,
2.45, N, 27.24.
2.2.6. 6-Hydrazino-3-(2'-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (2f)
1
Yield 66%; m.p. 252.5 ºC; IR (KBr, cm-1): 3564 (br NH str.), 1601 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.34 (bs, 2H, -NH2), 6.83-6.86 (d, 1H, J=9.84 Hz, pyridazine-5H), 7.23-7.25
(m, 1H, Ph-5'-H), 7.65-7.67 (m, 1H, Ph-3'-H), 7.88-7.91 (d, 1H, J=9.84 Hz, pyridazine-4H),
8.29-8.30 (m, 1H, Ph-4'-H), 8.51 (s, 1H, -NH), 8.57 (m, 1H, Ph-6'-H); 13C NMR (100 MHz,
DMSO-d6) δ: 114.98, 115.47, 123.41, 125.21, 127.05, 127.60, 132.01, 143.15, 144.29, 156.54,
159.11. Anal. Calcd. for C11H9FN6: C, 54.10; H, 3.71; N, 34.41. Found: C, 54.24; H, 3.80, N,
34.56.
2.2.7. 6-Hydrazino-3-(4'-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (2g)
1
Yield 69%; m.p. 258.5 ºC; IR (KBr, cm-1): 3672 (br NH str.), 1607 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.32 (bs, 2H, -NH2), 6.84-6.86 (d, 1H, J=9.84 Hz, pyridazine-5H), 7.29-7.33
(t, 2H, Jo=8.84 Hz, Ph-3', 5'-H), 7.89-7.92 (d, 1H, J=9.88 Hz, pyridazine-4H), 8.51 (s, 1H, -NH),
13
8.58-8.61 (dd, 2H, Jo=8.8 Hz, J(m) HF=5.6 Hz, Ph-2', 6'-H); C NMR (100 MHz, DMSO-d6) δ:

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115.04, 115.38-115.59 (d, 2JC-F=21 Hz, Ph-3', 5'-C), 123.48, 123.64, 129.10-129.18 (d, 3JC-F=8.0
Hz, Ph-2', 6'-C), 143.81, 145.21, 156.44, 161.28-163.74 (d, 1JC-F=246 Hz, Ph-4'-C). Anal. Calcd.
for C11H9FN6: C, 54.10; H, 3.71; N, 34.41. Found: C, 54.42; H, 3.44, N, 34.10.
2.2.8. 6-Hydrazino-3-(4'-methoxyphenyl)-1,2,4-triazolo[4,3-b]pyridazine (2h)
1
Yield 68%; m.p. 258.5 ºC; IR (KBr, cm-1): 3317 (br NH str.), 1609 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 3.85 (s, 3H, -OCH3), 4.29 (bs, 2H, -NH2), 6.81-6.83 (d, 1H, J=9.88 Hz,
pyridazine-5H), 7.06-7.08 (d, 2H, J=8.96 Hz, Ph-3', 5'-H), 7.86-7.88 (d, 1H, J=9.84 Hz,
pyridazine-4H), 8.44 (s, 1H, -NH), 8.47-8.49 (d, 2H, J=8.92 Hz, Ph-2', 6'-H); 13C NMR (100
MHz, DMSO-d6) δ: 55.11, 113.86, 114.67, 119.47, 123.65, 128.44, 143.41, 148.89, 156.32,
159.41. Anal. Calcd. for C12H12N6O: C, 56.24; H, 4.72; N, 32.79. Found: C, 56.58; H, 4.40, N,
32.25.
2.2.9. 6-Hydrazino-3-(2',5'-dimethoxyphenyl)-1,2,4-triazolo[4,3-b]pyridazine (2i)
1
Yield 67%; m.p. 125 ºC; IR (KBr, cm-1): 3317 (br NH str.), 1612 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 3.71 (s, 3H, Ph-5'-OCH3), 3.77 (s, 3H, Ph-2'-OCH3), 4.16 (bs, 2H, -NH2),
6.86-6.89 (d, 1H, J= 9.84 Hz, pyridazine-5H), 7.05-7.12 (m, 3H, Ph-3', 4', 6'-H), 7.87-7.90 (d,
13
1H, J= 9.88 Hz, pyridazine-4H), 8.34 (s, 1H, -NH); C NMR (100 MHz, DMSO-d6) δ: 55.51,
56.22, 113.28, 115.13, 116.45, 116.59, 116.65, 123.40, 143.08, 145.64, 152.07, 152.77, 156.22.
Anal. Calcd. for C13H14N6O2: C, 54.54; H, 4.93; N, 29.35. Found: C, 54.16; H, 4.62, N, 29.79.
2.2.10. 6-Hydrazino-3-(3',4'-dimethoxyphenyl)-1,2,4-triazolo[4,3-b]pyridazine (2j)
Yield 69%; m.p. 192 ºC; IR (KBr, cm-1): 3217(br NH str.), 1624 (C=N str.); 1H NMR (400 MHz,
DMSO-d6) δ: 3.87 (s, 3H, Ph-3'-OCH3), 3.91 (s, 3H, Ph-4'-OCH3), 4.17 (bs, 2H, -NH2), 6.81-
6.84 (d, 1H, J=9.8 Hz, pyridazine-5H), 7.06-7.08 (d, 1H, Jo=8.36 Hz, Ph-5'-H), 7.86-7.88 (d, 1H,
J=9.92 Hz, pyridazine-4H), 8.13-8.16 (dd, 1H, Jo=8.48 Hz, Jm=1.88 Hz, Ph-6'-H), 8.18 (d, 1H,

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Jm=1.88 Hz, Ph-2'-H), 8.48 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6) δ: 55.36, 55.48,
109.93, 111.40, 114.64, 119.51, 119.77, 123.65, 143.62, 145.82, 148.36, 149.57, 156.30. Anal.
Calcd. for C13H14N6O2: C, 54.54; H, 4.93; N, 29.35. Found: C, 54.25; H, 5.04, N, 29.67.
2.2.11. 6-Hydrazino-3-(thiophen-2'-yl)-1,2,4-triazolo[4,3-b]pyridazine (2k)
1
Yield 67%; m.p. 248.5 ºC; IR (KBr, cm-1): 3402 (br NH str.), 1630 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 4.38 (bs, 2H, -NH2), 6.83-6.86 (d, 1H, J=9.88 Hz, pyridazine-5H), 7.24-7.26
(dd, 1H, J4', 5' =5.0 Hz, J3', 4' =3.72 Hz, thiophene-4'-H), 7.69-7.71 (dd, 1H, J4', 5' =5.0 Hz, J3', 5'
=1.0 Hz, thiophene-5'-H), 7.92-7.94 (d, 1H, J=9.88 Hz, pyridazine-4H), 8.30-8.32 (m, 1H, J3', 4'
=3.64 Hz, J3', 5' =1.0 Hz, thiophene-3'-H), 8.58 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6) δ:
115.00, 123.48, 127.12, 127.50, 127.62, 127.69, 143.13, 143.32, 156.58. MS: m/z 233.09 [M]+.
Anal. Calcd. for C9H8N6S: C, 46.54; H, 3.47; N, 36.18. Found: C, 46.25; H, 3.86, N, 36.52.
2.2.12. 6-Hydrazino-3-(furan-2'-yl)-1,2,4-triazolo[4,3-b]pyridazine (2l)
1
Yield 65%; m.p. 226 ºC; IR (KBr, cm-1): 3232 (br NH str.), 1615 (C=N str.); H NMR (400
MHz, DMSO-d6) δ: 3.33 (bs, 2H, -NH2), 6.70-6.71 (dd, 1H, J3', 4' =3.38 Hz, J4', 5' =1.8 Hz, furan-
4'-H), 6.87-6.89 (d, 1H, J=9.88 Hz, pyridazine-5H), 7.65-7.66 (d, 1H, J3', 4' =3.32 Hz, furan-3'-
H), 7.84-7.85 (d, 1H, J3', 5' =1.08 Hz, furan-5'-H), 7.90-7.93 (d, 1H, J=9.88 Hz, pyridazine-4H),
13
8.67 (s, 1H, -NH); C NMR (100 MHz, DMSO-d6) δ: 105.01, 107.15, 116.23, 125.70, 142.30,
143.21, 154.12, 163.14. Anal. Calcd. for C9H8N6O: C, 50.00; H, 3.73; N, 38.87. Found: C, 49.85;
H, 3.80, N, 38.60.
2.3. Synthesis of 6-arylidenehydrazino-3-aryl-1,2,4-triazolo[4,3-b]pyridazine (3a-l)
6-Hydrazino-3-aryl/heteroaryl-1,2,4-triazolo[4,3-b]pyridazine (2a-l) (3.0 mmol) was
dissolved in 15 mL of ethanol and the solution was heated for 10 min. After complete dissolution
of 2a-l in ethanol, corresponding aldehyde (0.31 g, 3.0 mmol) was added. The reaction mixture

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was then refluxed for 30 min. The reaction was monitored by TLC. On completion of the
reaction, the crude product was filtered, washed with cold ethanol, and recrystallized using
ethanol to afford 3a-l.
2.3.1. 6-Benzylidenehydrazino-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine (3a)
Yield 75%; m.p. 266.5 ºC; Lit m.p. [23]. 13C NMR (100 MHz, DMSO-d6) δ: 113.13, 124.84,
126.23, 126.64, 126.92, 128.19, 128.37, 128.96, 129.29, 134.43, 142.24, 146.20, 153.53. Anal.
Calcd. For C18H14N6: C, 68.78; H, 4.49; N, 26.93. Found: C, 68.23; H, 3.99; N, 26.75.
2.3.2.
6-(2'-Chlorobenzylidenehydrazino)-3-(2'-chlorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3b)
Yield 70%; m.p. 293.5 ºC; IR (KBr, cm-1): 3152 (NH str.), 1602 (C=N str.); 1H NMR (400 MHz,
DMSO-d6) δ: 7.37-7.39 (m, 2H, Ph-H), 7.46-7.48 (m, 1H, Ph-H), 7.54-7.58 (m, 1H, Ph-H), 7.62-
7.66 (m, 1H, Ph-H), 7.70-7.74 (m, 3H, Ph-H), 8.04-8.06 (d, 1H, J=9.36 Hz, pyridazine-5H),
8.30-8.32 (d, 1H, J=10 Hz, pyridazine-4H), 8.44 (s, 1H, -CH), 11.99 (s, 1H, -NH); 13C NMR
(100 MHz, DMSO-d6) δ: 113.44, 125.09, 126.17, 126.31, 126.90, 126.99, 129.55, 130.09,
131.61, 131.81, 132.44, 132.54, 134.06, 138.14, 143.36, 145.71, 153.87. MS: m/z 383.01
[M+1]+, 385.02 [M+1+2]+, 387.01 [M+1+4]+ (9:6:1). Anal. Calcd. for C18H12Cl2N6: C, 56.41; H,
3.16; N, 21.93. Found: C, 56.14; H, 3.20, N, 21.67.
2.3.3.
6-(4'-Chlorobenzylidenehydrazino)-3-(4'-chlorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3c)
1
Yield 72%; m.p. 187 ºC; IR (KBr, cm-1): 3355 (NH str.), 1606 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 7.53-7.55 (d, 2H, J=8.64 Hz, Ph-H), 7.63-7.65 (d, 2H, J=8.56 Hz, Ph-H), 7.74-7.76
(d, 1H, J=8.4 Hz, pyridazine-5H), 7.88-7.90 (d, 2H, J=8.64 Hz, Ph-H), 8.19-8.21 (d, 1H, J=9.8
Hz, pyridazine-4H), 8.52-8.54 (d, 2H, J=8.44 Hz, Ph-H), 8.69 (s, 1H, -CH), 11.03 (s, 1H, -NH);

ACCEPTED MANUSCRIPT
13C NMR (100 MHz, DMSO-d6) δ: 113.55, 124.76, 127.87, 128.23, 128.59, 128.89, 129.84,
132.47, 134.22, 136.08, 139.92, 143.24, 145.11, 160.46. Anal. Calcd. for C18H12Cl2N6: C, 56.41;
H, 3.16; N, 21.93. Found: C, 56.64; H, 3.45, N, 21.46.
2.3.4.
6-(3'-Bromobenzylidenehydrazino)-3-(3'-bromophenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3d)
1
Yield 73%; m.p. 305 ºC; IR (KBr, cm-1): 3155 (NH str.), 1608 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 7.35-7.38 (t, 1H, J=7.84 Hz, Ph-H), 7.51-7.55 (m, 4H, Ph-H), 7.67-7.69 (d, 1H,
J=7.96 Hz, pyridazine-5H), 7.73-7.75 (d, 1H, J=7.76 Hz, Ph-H), 7.95-7.97 (m, 1H, Ph-H), 8.13
(s, 1H, -CH), 8.14-8.17 (d, 1H, J=9.9 Hz, pyridazine-4H), 8.48-8.50 (m, 1H, Ph-H), 8.84 (s, 1H,
-NH). MS: m/z 470.96 [M+1]+, 472.94 [M+1+2]+, 474.95 [M+1+4]+ (1:2:1); Anal. Calcd. for
C18H12Br2N6: C, 45.79; H, 2.56; N, 17.80. Found: C, 45.33; H, 2.21, N, 17.31.
2.3.5.
6-(4'-Bromobenzylidenehydrazino)-3-(4'-bromophenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3e)
1
Yield 76%; m.p. 211 ºC; IR (KBr, cm-1): 3124 (NH str.), 1611 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 7.59-7.61 (d, 2H, J=8.32 Hz, Ph-H), 7.66-7.69 (m, 4H, Ph-H), 7.75-7.77 (d, 1H,
J=8.4 Hz, pyridazine-5H), 7.80-7.82 (d, 2H, J=8.32 Hz, Ph-H), 8.45-8.47 (d, 1H, J=8.36 Hz,
pyridazine-4H), 8.66 (s, 1H, -CH), 11.77 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6) δ:
113.11, 124.71, 125.39, 125.93, 128.23, 128.46, 130.06, 131.56, 131.63, 131.84, 133.80, 144.38,
145.44, 153.16. Anal. Calcd. for C18H12Br2N6: C, 45.79; H, 2.56; N, 17.80. Found: C, 45.91; H,
2.80, N, 17.60.
2.3.6.
6-(2'-Fluorobenzylidenehydrazino)-3-(2'-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3f)

ACCEPTED MANUSCRIPT
1
Yield 72%; m.p. 235 ºC; IR (KBr, cm-1): 3132 (NH str.), 1615 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 7.01-7.02 (m, 3H, Ph-H), 7.30-7.62 (m, 5H, Ph-H), 8.02-8.04 (d, 1H, J=9.6 Hz,
pyridazine-5H), 8.28-8.30 (d, 1H, J=9.2 Hz, pyridazine-4H), 8.37 (s, 1H, -CH) 11.61 (s, 1H, -
NH); 13C NMR (100 MHz, DMSO-d6) δ: 113.15, 115.22, 115.35, 115.43, 115.56, 124.97,
128.17, 128.26, 128.96, 129.04, 131.12, 141.16, 143.98, 144.04, 145.21, 161.39. Anal. Calcd. for
C18H12F2N6: C, 61.71; H, 3.45; N, 23.99. Found: C, 61.36; H, 3.89, N, 23.56.
2.3.7.
6-(4'-Fluorobenzylidenehydrazino)-3-(4'-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3g)
1
Yield 76%; m.p. 249 ºC; IR (KBr, cm-1): 3132 (NH str.), 1620 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 7.19-7.24 (t, 2H, J=8.76 Hz, Ph-H), 7.35-7.39 (t, 2H, J=8.84 Hz, Ph-H), 7.59-7.61
(d, 1H, J=9.68 Hz, pyridazine-5H), 7.75-7.79 (dd, 2H, Jo=8.6 Hz, J(m) HF=5.6 Hz, Ph-H), 8.14 (s,
1H, -CH), 8.16-8.19 (d, 1H, J=10 Hz, pyridazine-4H), 8.53-8.56 (dd, 2H, Jo=8.8 Hz, J(m) HF=5.6
Hz, Ph-H), 11.62 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6) δ: 113.29, 115.39-115.61 (d,
2
2JC-F=22 Hz, Ph-3', 5'-C), 115.49-115.71 (d, JC-F=22 Hz, Ph-3', 5'-C), 123.28, 125.17, 128.32-
3
3
128.41 (d, JC-F=8.0 Hz, Ph-2', 6'-C), 128.98-129.06 (d, JC-F=9.0 Hz, Ph-2', 6'-C), 131.13,
141.23, 144.04, 145.21, 153.40, 161.11, 162.92. Anal. Calcd. for C18H12F2N6: C, 61.71; H, 3.45;
N, 23.99. Found: C, 61.63; H, 3.71, N, 23.62.
2.3.8. 6-(4'-Methoxybenzylidenehydrazino)-3-(4'-methoxyphenyl)-1,2,4-triazolo[4,3-b]pyridazine
(3h)
1
Yield 73%; m.p. 278 ºC; IR (KBr, cm-1): 3155 (NH str.), 1614 (C=N str.); H NMR (400 MHz,
CDCl3) δ: 3.86 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 6.94-6.96 (d, 2H, Jo=8.8 Hz, Ph-H), 7.04-
7.06 (dd, 2H, Jo=8.88 Hz, Jm=1.92 Hz, Ph-H), 7.61-7.64 (d, 2H, Jo=8.8 Hz, Ph-H), 7.63-7.65 (d,
1H, J=9.96 Hz, pyridazine-5H), 7.82 (s, 1H, -CH), 8.04-8.06 (d, 1H, J=9.96 Hz, pyridazine-4H),

ACCEPTED MANUSCRIPT
8.38-8.39 (m, 2H, Ph-H), 8.40 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6) δ: 54.98, 55.01,
112.89, 113.62, 113.90, 119.23, 124.73, 127.14, 127.43, 128.41, 142.13, 160.12, 160.16. Anal.
Calcd. for C20H18N6O2: C, 64.16; H, 4.85; N, 22.45. Found: C, 64.32; H, 4.95, N, 22.80.
2.3.9.
6-(2',5'-Dimethoxybenzylidenehydrazino)-3-(2',5'-dimethoxyphenyl)-1,2,4-triazolo[4,3-
b]pyridazine (3i)
Yield 74%; m.p. 250.5 ºC; IR (KBr, cm-1): 3240 (NH str.), 1606 (C=N str.); 1H NMR (400 MHz,
DMSO-d6) δ: 3.71-3.79 (m, 12H, -OCH3), 6.90-7.04 (m, 4H, Ph-H), 7.33-7.34 (d, 1H, J=2.84
Hz, Ph-H), 7.48-7.49 (d, 1H, J=2.84 Hz, Ph-H), 7.56-7.58 (d, 1H, J=9.88 Hz, pyridazine-5H),
13
7.99-8.01 (d, 1H, J=9.96 Hz, pyridazine-4H), 8.29 (s, 1H, -CH), 11.42 (s, 1H, -NH); C NMR
(100 MHz, DMSO-d6) δ: 55.29, 55.46, 56.03, 56.20, 112.66, 113.01, 113.12, 116.08, 116.53,
116.69, 119.04, 122.22, 124.69, 137.40, 143.25, 145.60, 151.50, 152.15, 152.87, 153.06, 153.22,
156.40. MS: m/z 435.19 [M+1]+. Anal. Calcd. for C22H22N6O4: C, 60.82; H, 5.10; N, 19.34.
Found: C, 60.51; H, 4.98, N, 19.22.
2.3.10. 6-(3',4'-Dimethoxybenzylidenehydrazino)-3-(3',4'-dimethoxyphenyl)-1,2,4-triazolo[4,3-
b]pyridazine (3j)
Yield 72%; m.p. 247 ºC; IR (KBr, cm-1): 3233 (NH str.), 1621 (C=N str.); 1H NMR (400 MHz,
CDCl3) δ: 3.93 (s, 3H, -OCH3), 3.95 (s, 3H, -OCH3), 3.98 (s, 3H, -OCH3), 3.99 (s, 3H, -OCH3),
6.88-6.90 (d, 1H, J=8.32 Hz, Ph-H), 6.97-6.99 (d, 1H, J=8.48 Hz, Ph-H), 7.10-7.13 (dd, 1H,
Jo=8.3 Hz, Jm=1.8 Hz, Ph-H), 7.34-7.35 (d, 1H, Jm=1.76 Hz, Ph-H), 7.61-7.64 (d, 1H, J=9.96 Hz,
pyridazine-5H), 7.84 (s, 1H, -CH), 7.96-7.97 (d, 1H, Jm=1.8 Hz, Ph-H), 8.05-8.07 (d, 1H, J=9.96
13
Hz, pyridazine-4H), 8.09-8.12 (dd, 1H, Jo=8.42 Hz, Jm=1.96 Hz, Ph-H); 8.55 (s, 1H, -NH); C
NMR (100 MHz, DMSO-d6) δ: 55.39, 55.46, 55.48, 55.71, 107.63, 110.51, 110.67, 110.74,

ACCEPTED MANUSCRIPT
113.07, 119.34, 120.07, 120.76, 124.50, 127.34, 143.83, 146.22, 148.37, 148.83, 149.78, 149.95.
Anal. Calcd. for C22H22N6O4: C, 60.82; H, 5.10; N, 19.34. Found: C, 60.25; H, 5.36, N, 19.56.
2.3.11.
3-(Thiophen-2'-yl)-6-(2-(thiophen-2'-ylmethylidene)hydrazino)-1,2,4-triazolo[4,3-
b]pyridazine (3k)
1
Yield 72%; m.p. 90 ºC; IR (KBr, cm-1): 3122 (NH str.), 1603 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 7.08-7.10 (t, 1H, J4', 5' =4.8 Hz, J3', 4' =3.8 Hz, thiophene-4'-H), 7.26-7.28 (m, 1H,
J4', 5' =4.8 Hz, J3', 4' =3.8 Hz, thiophene-4'-H), 7.33-7.34 (d, 1H, J3', 4' =3.2 Hz, thiophene-3'-H),
7.43-7.46 (d, 1H, J=9.8 Hz, pyridazine-5H), 7.50-7.51 (d, 1H, J4', 5' =4.9 Hz, thiophene-5'-H),
7.68-7.69 (d, 1H, J4', 5' =4.64 Hz, thiophene-5'-H), 8.14-8.15 (d, 1H, J= 6.04 Hz, pyridazine-4H),
8.28-8.29 (d, 1H, J3', 4' =3.12 Hz, thiophene-3'-H), 8.36 (s, 1H, -CH), 11.55 (s, 1H, -NH); 13C
NMR (100 MHz, DMSO-d6) δ: 113.07, 125.12, 126.89, 127.52, 127.69, 128.95, 137.87, 139.46,
143.54. Anal. Calcd. for C14H10N6S2: C, 51.52; H, 3.09; N, 25.75. Found: C, 51.36; H, 3.36, N,
23.54.
2.3.12. 3-(Furan-2'-yl)-6-(2-(furan-2'-ylmethylidene)hydrazino)-1,2,4-triazolo[4,3-b]pyridazine
(3l)
1
Yield 70%; m.p. 94 ºC; IR (KBr, cm-1): 3122 (NH str.), 1618 (C=N str.); H NMR (400 MHz,
DMSO-d6) δ: 6.59 (m, 1H, furan-4'-H), 6.77 (m, 1H, furan-4'-H), 6.82-6.83 (d, 1H, J3', 4' =3.08
Hz, furan-3'-H), 7.49-7.51 (m, 2H, pyridazine-5H, furan-H), 7.75 (s, 1H, furan-H), 7.91 (s, 1H, -
13
CH), 8.05 (s, 1H, furan-H), 8.18-8.21 (d, 1H, J=10 Hz, pyridazine-4H), 11.60 (s, 1H, -NH); C
NMR (100 MHz, DMSO-d6) δ: 111.43, 111.72, 112.14, 113.37, 125.31, 132.98, 140.28, 140.94,
143.40, 144.47, 144.58, 149.53, 153.56. MS: m/z 295.10 [M+1]+. Anal. Calcd. for C14H10N6O2:
C, 57.14; H,3.43; N, 28.56. Found: C, 57.31 H, 3.11, N, 28.30.
2.4. Synthesis of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4a-l)

ACCEPTED MANUSCRIPT
To a solution of 6-benzylidenehydrazino-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine (3a)
(0.62 g, 2.0 mmol) in dichloromethane (20 mL), iodobenzene diacetate (0.70 g, 2.2 mmol) was
added in small portion and the reaction mixture was stirred at room temperature for 1-2 hour or
until the completion of reaction as monitored by TLC. Solid separated out was filtered, washed
and recrystallized with ethanol to afford 4a.
2.4.1. 3,6-Diphenyl-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4a)
Yield 82%; m.p. 274 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1638 (C=N str.);
1H NMR (400 MHz, CDCl3) δ: 7.06-7.17 (m, 3H, 2×Ph-3', 4', 5'-H), 7.29-7.32 (m, 2H, 2×Ph-2',
6'-H), 7.71 (s, 1H, 2×pyridazine-H); 13C NMR (100 MHz, CDCl3) δ: 118.10, 126.30, 128.55,
128.76, 130.73, 146.70, 148.25. Anal. Calcd. for C18H12N6: C, 69.22; H, 3.87; N, 26.91. Found:
C, 69.49; H, 3.42, N, 26.63.
2.4.2. 3,6-Di-(2'-chlorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4b)
Yield 78%; m.p. 201.5 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1602 (C=N str.);
1H NMR (400 MHz, CDCl3) δ: 6.93-6.95 (d, 1H, J=8.0 Hz, 2×Ph-H), 7.09-7.13 (dt, 1H, J=7.94
Hz, J=1.4 Hz, 2×Ph-H), 7.17-7.21 (dt, 1H, J=7.56 Hz, J=0.84 Hz, 2×Ph-H), 7.74 (s, 1H,
2×pyridazine-H), 7.79-7.81 (dd, 1H, J=7.62 Hz, J=1.48 Hz, 2×Ph-2'-H); 13C NMR (100 MHz,
CDCl3) δ: 118.16, 126.07, 127.17, 129.32, 132.17, 132.91, 134.42, 145.55, 146.27. MS: m/z
381.01 [M+1]+, 383.02 [M+1+2]+, 385.06 [M+1+4]+, (9:6:1). Anal. Calcd. for C18H10Cl2N6: C,
56.71; H, 2.64; N, 22.05. Found: C, 56.92; H, 2.51, N, 22.39.
2.4.3. 3,6-Di-(4'-chlorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4c)
Yield 74%; m.p. 244.5 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1623 (C=N str.);
1H NMR (400 MHz, DMSO-d6) δ: 7.12-7.14 (d, 2H, J=8.4 Hz, 2×Ph-H), 7.39-7.41 (d, 2H, J=8.4
13
Hz, 2×Ph-H), 8.06 (s, 1H, 2×pyridazine-H); C NMR (100 MHz, DMSO-d6) δ: 118.13, 125.57,

ACCEPTED MANUSCRIPT
128.26, 130.77, 135.35, 146.11, 146.98. Anal. Calcd. for C18H10Cl2N6: C, 56.71; H, 2.64; N,
22.05. Found: C, 56.39; H, 2.30, N, 22.20.
2.4.4. 3,6-Di-(3'-bromophenyl)-bis-1,2,4-triazolo-[4,3-b][3’,4’-f]pyridazine (4d)
Yield 72%; m.p. 190 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1613 (C=N str.);
1H NMR (400 MHz, CDCl3) δ: 7.08-7.12 (t, 1H, J=7.84 Hz, 2×Ph-H), 7.34-7.36 (dd, 1H, J=8.12
Hz, J=0.96 Hz, 2×Ph-H), 7.38 (m, 1H, 2×Ph-H), 7.41-7.43 (m, 1H, 2×Ph-H), 7.75 (s, 1H,
2×pyridazine-H); 13C NMR (100 MHz, CDCl3) δ: 118.34, 123.08, 127.12, 127.92, 130.37,
131.39, 132.92, 146.51, 146.74. Anal. Calcd. for C18H10Br2N6: C, 45.99; H, 2.14, N, 17.88.
Found: C, 45.72; H, 2.01, N, 17.46.
2.4.5. 3,6-Di-(4'-bromophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4e)
Yield 74%; m.p. 197 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1629 (C=N str.);
1H NMR (400 MHz, CDCl3) δ: 7.15-7.17 (d, 2H, J=8.52 Hz, 2×Ph-H), 7.29-7.31 (d, 2H, J=8.52
13
Hz, 2×Ph-H), 7.72 (s, 1H, 2×pyridazine-H); C NMR (100 MHz, DMSO-d6) δ: 117.98, 124.41,
125.73, 130.71, 131.18, 146.21, 146.84. MS: m/z 468.92 [M+1]+, 470.93 [M+1+2]+, 473.93
[M+1+4]+, (1:2:1). Anal. Calcd. for C18H10Br2N6: C, 45.99; H, 2.14, N, 17.88. Found: C, 45.69;
H, 2.33, N, 17.59.
2.4.6. 3,6-Di-(2'-fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4f)
Yield 70%; m.p. 257.5 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1631 (C=N str.);
1H NMR (400 MHz, DMSO-d6) δ: 6.82-7.62 (m, 4H, 2×Ph-H), 7.98 (s, 1H, 2×pyridazine-H);
2
13C NMR (100 MHz, DMSO-d6) δ: 115.62-115.82 (d, JC-F=20 Hz), 118.20-118.29 (d, J=9 Hz),
124.54-126.62 (d, J=8 Hz), 130.73, 131.79, 133.62-133.70 (d, J=8 Hz), 141.97, 142.35, 146.54,
1
157.48-159.96 (d, JC-F=248 Hz). Anal. Calcd. for C18H10F2N6: C, 62.07; H, 2.89, N, 24.13.
Found: C, 62.36; H, 2.99, N, 24.33.

ACCEPTED MANUSCRIPT
2.4.7. 3,6-Di-(4'-fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4g)
Yield 72%; m.p. 292.5 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1610 (C=N str.);
1H NMR (400 MHz, DMSO-d6) δ: 6.87-6.92 (t, 2H, Jo=8.8 Hz, 2×Ph-3', 5'-H), 7.45-7.48 (dd,
2H, Jo=8.76 Hz, J(m) HF=5.32 Hz, 2×Ph-2', 6'-H), 8.17 (s, 1H, 2×pyridazine-H); 13C NMR (100
MHz, DMSO-d6) δ: 115.15-115.37 (d, 2JC-F=22 Hz, Ph-3', 5'-C), 118.04, 123.31-123.34 (d, J=3),
3
131.47-131.56 (d, JC-F=9 Hz, Ph-2', 6'-C), 146.23, 146.89, 161.49-163.96 (d, 1JC-F=247 Hz, Ph-
4'-C). Anal. Calcd. for C18H10F2N6: C, 62.07; H, 2.89, N, 24.13. Found: C, 62.50; H, 2.61, N,
24.01.
2.4.8. 3,6-Di-(4'-methoxyphenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4h)
Yield 75%; m.p. 225 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1612 (C=N str.);
1H NMR (300 MHz, CDCl3) δ: 3.76 (s, 3H, 2×Ph-4'-OCH3), 6.57-6.59 (m, 2H, 2×Ph-3', 5'-H),
7.04-7.07 (m, 2H, 2×Ph-2', 6'-H), 7.64 (s, 1H, 2×pyridazine-H); 13C NMR (100 MHz, DMSO-d6)
δ: 54.89, 113.53, 117.60, 118.89, 130.42, 146.52, 147.23, 160.15. Anal. Calcd. for C20H16N6O2:
C, 64.51; H, 4.33, N, 22.57. Found: C, 64.24; H, 4.25; N, 22.22.
2.4.9. 3,6-Di-(2',5'-dimethoxyphenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4i)
Yield 73%; m.p. >315 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1617 (C=N str.);
1H NMR (300 MHz, CDCl3) δ: 3.44 (s, 3H, 2×Ph-5'-OCH3), 3.70 (s, 3H, 2×Ph-2'-OCH3), 6.33-
6.36 (d, 1H, Jo=9.3 Hz, 2×Ph-3̍-H), 6.66-6.70 (dd, 1H, Jo=9.3 Hz, Jm=3Hz, 2×Ph-4'-H), 7.13-
7.14 (d, 1H, Jm=3Hz, 2×Ph-6'-H), 7.62 (s, 1H, 2×pyridazine-H); 13C NMR (100 MHz, CDCl3)
δ: 55.25, 56.02, 111.20, 115.05, 116.41, 117.87, 118.69, 146.31, 146.62, 151.26, 153.43. MS:
m/z 433.16 [M+1]+. Anal. Calcd. for C22H20N6O4: C, 61.10; H, 4.66, N, 19.43. Found: C, 61.38;
H, 4.39, N, 19.68.
2.4.10. 3,6-Di-(3',4'-dimethoxyphenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4j)

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Yield 73%; m.p. 226 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1606 (C=N str.);
1H NMR (400 MHz, CDCl3) δ: 3.78 (s, 3H, 2×Ph-4'-OCH3), 3.83 (s, 3H, 2×Ph-3'-OCH3), 6.51-
6.53 (d, 1H, Jo=8.32 Hz, 2×Ph-5'-H), 6.74-6.75 (d, 1H, Jm=1.8 Hz, 2×Ph-2'-H), 6.98-7.01 (dd,
1H, Jo=8.28 Hz, Jm=1.88 Hz, 2×Ph-6'-H), 7.67 (s, 1H, 2×pyridazine-H); 13C NMR (100 MHz,
CDCl3) δ: 55.87, 55.92, 110.87, 111.16, 117.95, 118.67, 121.35, 146.59, 148.14, 148.33, 150.71.
Anal. Calcd. for C22H20N6O4: C, 61.10; H, 4.66, N, 19.43. Found: C, 61.15; H, 4.90, N, 19.23.
2.4.11. 3,6-Di-(thiophen-2'-yl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4k)
Yield 72%; m.p. 231 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1611 (C=N str.);
1H NMR (400 MHz, DMSO-d6) δ: 6.72-6.74 (t, 1H, J4', 5' =4.8 Hz, J3', 4' =4.0 Hz, 2×thiophene-4'-
H), 7.16-7.18 (d, 1H, J3', 4' =3.6 Hz, 2×thiophene-3'-H), 7.53-7.54 (d, 1H, J4', 5' =5.2 Hz,
2×thiophene-5'-H), 7.86 (s, 1H, 2×pyridazine-H); 13C NMR (100 MHz, DMSO-d6) δ: 118.11,
126.48, 127.76, 129.26, 131.10, 142.64, 146.83. Anal. Calcd. for C14H8N6S2: C, 51.84; H, 2.49;
N, 25.91. Found: C, 51.52; H, 2.88, N, 25.61.
2.4.12. 3,6-Di-(furan-2'-yl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine (4l)
Yield 70%; m.p. 225 ºC; IR (KBr, cm-1): transparent in the region of –NH str., 1610 (C=N str.);
1H NMR (400 MHz, DMSO-d6) δ: 6.38 (m, 1H, 2×furan-4'-H), 6.89-6.90 (d, 1H, J3', 4' =3.08 Hz,
2×furan-3'-H), 7.17 (m, 1H, 2×furan-5'-H), 8.22 (s, 1H, 2×pyridazine-H). Anal. Calcd. for
C14H8N6O2: C, 57.54; H, 2.76; N, 28.76. Found: C, 57.23; H, 2.40, N, 28.53.
2.5. X-ray crystallography
To study the effect of substituents on the planarity of the molecule three candidates viz.
3,6-di-(2'-fluorophenyl)-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine 4f, 3,6-di-(4'-fluorophenyl)-
bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine 4g and 3,6-di-(2',5'-dimethoxyphenyl)-bis-1,2,4-
triazolo-[4,3-b][3',4'-f]pyridazine 4i were selected for X-ray diffraction studies. Compounds 4f

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and 4g have fluorine as substituent at ortho and para position of aryl ring, respectively. In
compound 4i two methoxy groups are present at ortho and meta positions thereby further
increasing the steric crowding. Single crystals of 4f, 4g and 4i were obtained by slow
evaporation of a DCM-ethanol (7:3) solution containing 2-3 drops of petroleum ether of the
initial solid product at ambient temperature over a period of about 3 days. The molecular
structures of 4f and 4g were determined by single crystal X-ray diffraction. However,
unfortunately the crystals of the compound 4i did not diffract X-rays and its structure could not
be obtained through X-ray.
Crystallographic data for compounds 4f and 4g, can be obtained from CAI de Difracción
de Rayos-X, Universidad Complutense de Madrid (Madrid, Spain).
2.5.1. X-Ray data collection and structure refinement
Data collection for 4f and 4g was carried out at room temperature on a Bruker Smart
CCD diffractometer using in all cases graphite-monochromated Mo-Kα radiation (λ = 0.71073
Å) operating at 50 kV and 35 mA, with exposure times of 20 s in omega. The details of the
fundamental crystal structure and data refinement parameters of the compounds 4f and 4g are
given in Table 1. The structures were solved by direct methods and refined by full-matrix least-
squares procedures on F2 using the SHELXL-97 [31]. All non-hydrogen atoms were refined
anisotropically. The hydrogen atoms were included in their calculated positions and refined
riding on the respective carbon atoms.
Table 1
Crystal data and structure refinement details for compounds 4f and 4g.
Crystal Data
4f
4g
1850032
C18H10F2 N6
1850033
C18H10F2 N6
CCDC code
Empirical formula
Formula weight
348.32
348.32

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Crystal system
Space group
a/Å
Orthorhombic
P212121
10.293(1)
10.989(1)
13.939(2)
90.0
Orthorhombic
P212121
6.620(2)
14.886(3)
15.601(4)
90.0
b/Å
c/Å
α
β
γ
(°)
(°)
(°)
90.0
90.0
90.0
90.0
V/Å3
1578.6(3)
4
1537.3(6)
4
Z
Dc /g/cm3
µ(Mo-Kα) /mm-1
F (000)
1.467
1.505
0.110
0.113
712
712
2.36 to 25.0
-12,-13,-14 to
12, 12, 16
12050
1.89 to 26.00
-8,-18,-19 to
7, 18, 16
12688
θ
range (°)
Index ranges
Reflections collected
Unique reflections
[R(int)]
2769
3001
[R(int) = 0.0535]
99.8%
[R(int) = 0.0531]
99.3%
Completeness to theta %
Data/restraints/parameters
Goodness-of-fit on F2
R1 (reflns obsd) [I>2σ
(I)] a
wR2 (all data) b
2769 / 0 / 235
0.996
3001/ 0 / 235
0.998
0.0332 (1720)
0.0517
0.0310 (2131)
0.0657
a R1=Σ||Fo|-|Fc||/ Σ|Fo|
2
2 2
bwR2={Σ[w(Fo -Fc2)2]/ Σ[w(Fo ) ]}
CCDC 1850032 and 1850033 contain the supplementary crystallographic data for the
compound 4f and 4g, respectively. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
2.6. Cytotoxic activity
2.6.1. Cell Culture

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Human cervical carcinoma (HeLa) cell lines were cultured in RPMI-1640 medium
supplemented with 10% fetal serum albumin (FBS), 100 units/ml penicillin, and 100 mg/ml
streptomycin. The cell lines were maintained in an incubator containing 5% of CO2 at 37 ºC.
2.6.2. Cell Viability Assay
The cells were seeded at a density of 2 × 105 per mL in RPMI solution by transferring
200 µL into each well on a 96-well plate and incubated for 24 hr. Cells were treated with various
compounds at different concentration (50 µM and 100 µM in DMSO) and incubated over a
period of 48 h at 37 ºC. After this 20 ꢀL of MTT reagent (5 mg/mL in phosphate-buffered saline)
was added to each well and incubated for 4 hr at 37 °C. 200 ꢀL DMSO (MTT solvent) was
added and absorbance was read using a plate reader, with the optical density of the purple
formazan A550 being proportional to the number of viable cells. The mean values and the
standard error of the mean (SEM) of all the experiments for each compound were then used to
plot graphs using GraphPad Prism program and Excel. The concentration required for 50%
inhibition of cell viability (IC50) was calculated using the GraphPad Prism.
3. Results and Discussion
3.1. Chemistry
Synthesis of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazine derivatives (4a-
l) is depicted in Scheme 1. Precursor 6-hydrazino-3-aryl/heteroaryl-1,2,4-triazolo[4,3-
b]pyridazines (2a-l) were prepared from reaction of different substituted 6-chloro-1,2,4-
triazolo[4,3-b]pyridazines (1a-l) with hydrazine hydrate in refluxing ethanol. The key
intermediate 6-arylidenehydrazino-3-aryl-1,2,4-triazolo[4,3-b]pyridazines (3a-l) were obtained
by condensation of compounds 2a-l with aldehydes in ethanol. Hydrazones (3a-l) upon oxidative

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cyclization with 1.1 equivalents of IBD in dichloromethane at room temperature afforded the
corresponding bis-1,2,4-triazolo-[4,3-b][3',4'-f]pyridazines (4a-l).
Scheme 1. IBD-mediated synthesis of 3,6-disubstituted-bis-1,2,4-triazolo-[4,3-b][3',4'-
f]pyridazines (4).
The structures and purity of all the compounds were established on the basis of a careful
1
13
1
comparison of their spectral data (IR, H NMR, C NMR) as well as high resolution mass. H
NMR spectra of compounds 2a-l exhibited a broad singlet signal at δ 3.33-4.38 ppm for two
proton intensity corresponding to NH2 and a sharp singlet signal at δ 8.34-8.67 ppm for one
1
proton intensity corresponding to NH proton. In addition, H NMR spectra of compounds 2a-l

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displayed two doublets of one proton intensity at δ 6.81-6.92 ppm and 7.85-7.95 ppm attributed
to pyridazine protons H-5 and H-4, respectively having the coupling constant J= ~ 9.8 Hz.
IR spectra of compounds 3a-l showed characteristic absorption band in the region ~3122-
1
3355 cm-1 for N-H stretching vibration and 1602-1621 cm-1 for C=N stretching vibration. H
NMR spectra of compounds 3a-l exhibited a sharp singlet at δ 7.82-8.69 ppm for aldehydic
proton and a broad singlet at δ 8.40-11.99 ppm for NH. Moreover, in compounds 3 a pair of
doublets of one proton intensity was obtained at δ 7.43-8.06 ppm and 7.99-8.47 ppm
corresponding to pyridazine protons H-4 and H-5, respectively. IR spectra of 4a-l were found to
be transparent in the region of NH stretch and bend, thus confirm the oxidation of 3 to 4. An
1
important characteristic feature in the H NMR spectra of 4a-l was disappearance of the signals
at δ 7.82-8.69 ppm and 8.40-11.99 ppm for aldehydic proton and for NH, respectively, which
were present in the spectra of the intermediate hydrazones 3a-l, thus suggesting the cyclization
I
of hydrazones 3 into bis-triazoles 4. Interestingly, in H NMR spectra of compounds 4 a sharp
singlet signal was observed at δ 7.62-8.22 ppm for pyridazine protons H-4 and H-5 in contrast to
1
13
two doublets of one proton intensity as obtained in 2 and 3. In conjunction with H NMR, C
NMR spectra also exhibited symmetrical pattern of 4.
The most striking feature of NMR (1H and13C) spectra of bis-triazoles 4 indicated that the
molecule is totally symmetrical. It is evident particularly from the spectra of 4h, 4i and 4j, all
having methoxy substituents that number of expected peaks in aliphatic region is just half.
Similarly pyridazine proton and aryl protons also appear in half proportion. This observation
seems to be quite unusual as the two aryl groups are present in a very narrow space and it is
expected that they will be distorted in space to accommodate each other. Therefore, it was
planned to study the structure of bis-triazoles by X-ray diffraction studies.

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3.2. Crystallographic studies
To rationalize the planarity of tricyclic system 4, perspective MM2-minimum energy
conformational analysis of 4f, 4g and 4i was carried out using the software ChemBioDrawUltra,
Cambridge Soft, 2014 [32]. It seems that tricyclic ring is not planar and phenyl rings are parallel
(Figure 2). In case of 4i, triazolopyridazine rings are oriented differently due to more steric bulk
of methoxy substituents on phenyl ring.
Figure 2. (a), (b) and (c) Perspective MM2-mimimum energy conformational analysis of
compound 4f, 4g and 4i, respectively.
Suitable crystals of 4f and 4g were studied by single crystal X-ray diffraction.
Experimental details for structural analyses are summarized in Table 1. The selected bond
distances and bond angles for compound 4f and 4g are given in Table 2. Both compounds
crystallize in orthorhombic P212121 space group, containing one single molecule per asymmetric
unit. The ORTEP plot of 4f and 4g with the labelling of their asymmetric units is depicted in
Figures 3 and 4, respectively.
The structures for compounds 4f and 4g show similarities. The molecules in both
compounds are not planar, as expected [33], because of the twist of the aromatic rings on the
bistriazole units in order to minimize their steric hindrance (Figure 5). In that way, the phenyl
rings form a dihedral angle of about 50º with their corresponding triazole rings, concretely
56.0(2)º and 51.9(2)º in compound 4g, while they are a bit lower for 4f, 48.2(3)º and 47.5(3)º.

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The presence of the fluorine atoms in the ortho position in the later compound seem not to affect
significantly to this angle, as they are located opposite each other and towards the outside of the
theoretical molecular plane.
Table 2
Selected bond length and bond angles (Å, °) of compound 4f and 4g with esd’s in parenthesis.
Bond length 4f
4g
Bond angle
4f
4g
C(1)-N(2)
C(1)-N(3)
C(2)-N(1)
C(2)-N(3)
C(2)-C(3)
C(3)-C(4)
C(4)-C(5)
C(5)-N(4)
C(5)-N(6)
C(6)-N(5)
C(6)-N(6)
N(3)-N(6)
N(2)-N(1)
N(4)-N(5)
1.316(3) 1.321(2)
1.371(3) 1.380(2)
1.311(3) 1.314(2)
1.383(3) 1.380(2)
1.427(4) 1.421(3)
1.326(3) 1.327(3)
1.432(4) 1.435(3)
1.312(3) 1.312(2)
1.380(3) 1.378(2)
1.318(3) 1.318(2)
1.377(2) 1.372(2)
N(2)-C(1)-N(3)
N(2)-C(1)-C(7)
N(3)-C(1)-C(7)
N(1)-C(2)-N(3)
N(1)-C(2)-C(3)
N(3)-C(2)-C(3)
C(4)-C(3)-C(2)
C(3)-C(4)-C(5)
N(4)-C(5)-N(6)
N(4)-C(5)-C(4)
N(6)-C(5)-C(4)
108.8(2) 107.23(17)
120.3(2) 122.79(18)
130.8(2) 129.68(16)
109.4(2) 109.04(17)
129.9(3) 129.8(2)
120.5(3) 120.1(2)
119.8(3) 119.7(2)
119.7(3) 119.49(18)
109.4(2) 108.73(16)
129.8(3) 130.24(17)
120.6(3) 119.96(18)
108.2(2) 107.96(15)
120.5(2) 124.52(16)
131.1(2) 127.07(16)
117.4(2) 119.40(19)
122.9(2) 121.68(17)
119.4(2) 118.59(18)
1.388(2) 1.3818(19) N(5)-C(6)-N(6)
1.381(3) 1.379(2)
1.375(3) 1.390(2)
N(5)-C(6)-C(13)
N(6)-C(6)-C(13)
C(8)-C(7)-C(12)
C(8)-C(7)-C(1)
C(12)-C(7)-C(1)
C(14)-C(13)-C(18) 117.3(2) 119.09(16)
C(14)-C(13)-C(6) 123.1(2) 120.30(16)
C(18)-C(13)-C(6) 118.8(2) 120.37(17)
C(1)-N(3)-C(2)
C(1)-N(3)-N(6)
C(2)-N(3)-N(6)
C(1)-N(2)-N(1)
C(2)-N(1)-N(2)
C(6)-N(6)-C(5)
C(6)-N(6)-N(3)
C(5)-N(6)-N(3)
C(5)-N(4)-N(5)
C(6)-N(5)-N(4)
105.6(2) 106.65(15)
135.1(2) 133.66(15)
119.2(2) 119.37(15)
108.6(2) 109.62(16)
107.4(2) 107.32(16)
105.8(2) 106.84(15)
135.2(2) 134.21(14)
119.0(2) 118.86(15)
107.5(2) 107.59(15)
109.0(2) 108.68(14)

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Figure 3. ORTEP plot (20% probability for the ellipsoids) of 4f showing the labelling scheme of
its asymmetric unit.
Figure 4. ORTEP plot (20% probability for the ellipsoids) of 4g showing the labelling scheme of
its asymmetric unit.

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Additionally, the phenyl rings on both triazole units are very close, inducing a distortion
in the bistriazolopyridazine that deviates from the planarity; this feature agrees with the bond
distances and angles found for both compounds, due to a partial electronic delocalization
observed for this moiety. In this sense, in compound 4f, one of the triazole and the pyridazine
rings are almost coplanar. This moiety forms a dihedral angle about 12º with the other triazole
ring, being the electronic delocalization disrupted in the C6N6N3C1 fragment (Figure 5a). In
compound 4g the distortion is more accused with dihedral angles of 13.2(2)º and 14.3(2)º
between each triazole fragment and the pyridazine ring, and a greater deviation from the
planarity located on the N4N5C6 fragment (Figure 5b).
In both derivatives the phenyl rings are almost parallel, in order to minimize their steric
hindrance, since they are linked to C1 and C6, two atoms separated by a distance comparable to
the sum of their van der Waals radii. For this reason, the substituting ortho or para fluorine
atoms do not have influence in the molecular structure and only seem to do it on the
intermolecular interactions. In this context, compound 4f shows the fluorine atoms pointing
towards opposite directions in order to avoid very short intramolecular contacts, giving rise
instead to the formation of weak F···F contacts (distance of 2.882(3) Å) between adjacent
molecules leading to the formation of chains along a axis, as depicted in Figure 6. These
intermolecular fluorine interactions can be considered as weak if they are compared with the sum
of the van der Waals radii of 2.94 Å that it is mostly considered the borderline, but they are in the
usual range for this type of contacts. They are centrosymmetric in cis disposition (Type I, cisoid)
and CF···FC angles of 144.8(3)º and 146.6(3)º [34]. No significant interaction between the
chains has been found. Nevertheless, in compound 4g the fluorine atoms in para positions
precludes the intermolecular halogen bonding interactions. Moreover, no significant contacts

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between the molecules of 4g have been found apart from the usual van der Waals forces (Figure
7).
Figure 5. Different views of the molecules for (a) compound 4f and (b) for 4g.

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Figure 6. Perspective view of the chains formed along a axis in 4f showing weak F···F
interactions.
A significant distortion of the valence angles is also observed due to the steric hindrance
between the aryl substituents. For instance, in compound 4f the exocyclic angles N(3)C(1)C(7)
130.8(2)º and N(6)C(6)C(13) 131.1(2)º are appreciably greater, and the angles N(2)C(1)C(7)
120.3(2)º and N(5)-C(6)-C(13) 120.5(2)º are appreciably smaller than the ideal value of 126º for
the five-membered ring. The C(1)-N(3)-N(6) 135.1(2)º and C(6)-N(6)-N(3) 135.2(2)º angles are
also increased. The angles C(1)-N(3)-C(2) 105.6(2)º and C(6)-N(6)-C(5) 105.8(2)º in the triazole