Synthesis and antifungal activity of imidazo[1,2-b]pyridazine derivatives against phytopathogenic fungi

Article abstract of DOI:10.1016/j.bmcl.2020.127139

A series of 3,6-disubstituted imidazo[1,2-b]pyridazine derivatives have been synthesized and characterized with spectroscopic analyses. The antifungal activities of these compounds against nine phytopathogenic fungi were evaluated by the mycelium growth rate method. The in vitro antifungal bioassays indicated that most of compounds displayed excellent and broad-spectrum antifungal activities. Especially, compounds 4a, 4c, 4d, 4l and 4r exhibited 1.9–25.5 fold more potent than the commercially available fungicide hymexazol against Corn Curvalaria Leaf Spot (CL), Alternaria alternate (AA), Pyricularia oryzae (PO) and Alternaria brassicae (AB) strains. Structure-activity relationship analysis showed that the enhanced antifungal activity is significantly affected by the substituents on the benzene ring and pyridazine ring.

Full text of DOI:10.1016/j.bmcl.2020.127139

Journal Pre-proofs
Synthesis and antifungal activity of imidazo[1,2-b]pyridazine derivatives
against phytopathogenic fungi
Lingling Fan, Zhongfu Luo, Yi Li, Xinyun Liu, Judi Fan, Wei Xue, Lei Tang,
Yong Li
PII:
S0960-894X(20)30232-8
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127139
BMCL 127139
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 January 2020
20 March 2020
23 March 2020
Please cite this article as: Fan, L., Luo, Z., Li, Y., Liu, X., Fan, J., Xue, W., Tang, L., Li, Y., Synthesis and
antifungal activity of imidazo[1,2-b]pyridazine derivatives against phytopathogenic fungi, Bioorganic &
Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127139
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Synthesis and antifungal activity of imidazo[1,2-b]pyridazine
derivatives against phytopathogenic fungi
Lingling Fana, Zhongfu Luoa, Yi Lia, Xinyun Liua, Judi Fana, Wei Xueb, Lei Tanga*,
Yong Lia*
a State Key Laboratory of Functions and Applications of Medicinal Plants, College of
Pharmacy, Guizhou Provincial Engineering Technology Research Center for
Chemical Drug R&D, Guizhou Medical University, Guiyang 550004, PR China
b
Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang, 550025, PR China.
*Corresponding author. Tel.: +86-851-690-8318; fax: +86-851-690-8318; e-mail:
tlei1974@hotmail.com (L. Tang), liyong 19851016@126.com (Y. Li).
1

Abstract: A series of 3,6-disubstituted imidazo[1,2-b]pyridazine derivatives have
been synthesized and characterized with spectroscopic analyses. The antifungal
activities of these compounds against nine phytopathogenic fungi were evaluated by
the mycelium growth rate method. The in vitro antifungal bioassays indicated that
most of compounds displayed excellent and broad-spectrum antifungal activities.
Especially, compounds 4a, 4c, 4d, 4l and 4r exhibited 1.9-25.5 fold more potent than
the commercially available fungicide hymexazol against Corn Curvalaria Leaf Spot
(CL), Alternaria alternate (AA), Pyricularia oryzae (PO) and Alternaria brassicae
(AB) strains. Structure-activity relationship analysis showed that the enhanced
antifungal activity is significantly affected by the substituents on the benzene ring and
pyridazine ring.
Keywords: imidazo[1,2-b]pyridazine, pathogenic fungi, synthesis, antifungal activity
2

Phytopathogenic fungi present a serious problem of agricultural production
worldwide, since they often quickly infect many crops and cause significant yield
reduction[1]. Particularly, many phytopathogenic fungi produce mycotoxins that are
harmful to animal and human health if they enter the food chain[2]. Therefore, many
commercial fungicides with high activity and broad antifungal spectrum have been
extensively used to ensure agricultural production and food safety while controlling a
variety of plant diseases [3]. However, the continuing use of commercial fungicides
has led to the cross-resistance of phytopathogenic fungi to fungicides[4]. Thus, it is
necessary to develop new fungicides with novel molecular frameworks to efficiently
control these agricultural fungi.
Pyridazines are important heterocycle scaffolds that display diverse biologiacl
activites in the fields of medicine[5-11] and agriculture[12]. For example (Fig. 1),
Minaprine is a psychotropic drug which has proved to be effective in the treatment of
various depressive states[13]; pyridazomy is an antifungal and antibiotic compound,
which is the first pyridazine derivative isolated from a natural source[14]; pyridaben is
widely used as an acaricide with a long residual action; and chloridazone has been
used as a herbicide with a long history[15]. Furthermore, imidazole is an important
class of nitrogen containing heterocycle, and some heterocycle-linked imidazole
derivatives exhibited potential antimicrobial and antifungal activities, such as
imidazo[1,2-a]pyrazines[16], imidazo[1,2-a]pyridine[17], imidazo[1,2-a]pyrimidine[18]
and pyrazolo[1,5-a]pyrimidine[19] derivatives showed significant antifungal activity
against human pathogenic microorganisms, and [1,2,4]triazolo[1,5-a]pyrimidines[20]
3

derivatives exhibited certain inhibition activity against phytopathogenic fungi (Fig. 1).
Consequently, in the continuation of our research aimed at the discovery and
development of novel antifungal candidates[21-24]
,
here we selected
imidazo[1,2-b]pyridazine[25] skeleton composed of imidazole and pyridazine rings as
the lead compound, a series of 3,6-disubstituted imidazo[1,2-b]pyridazine derivatives
(Fig. 2) were designed, synthesized and evaluated for their antifungal activities
against nine phytopathogenic fungi.
O
Cl
O
N
S
O
N
O
H2N
NH2
Cl
Cl-
N
N
N
N
N
N
N
COOH
N
H
H2N
pyridazomycin
minaprine
pyridaben
chloridazone
N
N
N
N
N
N
N
N
N
imidazo[1,2-a]pyridine
[1,2,4]Triazolo[1,5-a]pyrimidines
imidazo[1,2-a]pyrazine
Antibacterial activity (Gram-positive and
Gram-negative), Antifungal activity
(Aspergillus niger, Chrysosporium
tropicum, Rhizopus oryzae,Fusarium
moniliforme and Curvularia lunata)
Herbicidal activity, antibacterial and
antifungal activity (against G. zeae, F.
oxysporum, C. mandshurica, P.sasakii
and R. solani)(plant pathogenic fungi)
Antibacterial activity( Gram-
positive and Gram-negative )
Antifungal activity (C. albicans,
C. parapsilosis, A. flavus and
M. gypsuem. Ampicillin)
N
N
N
N
N
N
R1
N
N
N
R2
imidazo[1,2-b]pyrazine
imidazo[1,2-a]pyrimidine
pyrazolo[1,5-a]pyrimidine
Antiproliferative, anticancer,
anti-malarial, antiviral and
anti-apicomplexan activity
Antibacterial activity( Gram-
positive and Gram-negative )
Antibacterial activity( Gram-
positive and Gram-negative )
Fig. 1. Chemical structures of containing pyridazine or imidazole
As shown in Scheme 1, 6-chloroimidazo[1,2-b]pyridazine (2) was obtained by the
cyclization of 3-amino-6-chloropyridazine (1) and chloroacetaldehyde at reflux in
butanol for 16 h, followed by N-bromosuccinimide (NBS) bromination to afford
3-bromo-6-chloro-imidazo[1,2-b]pyridazines (3). Afterwards, a series of C-3
substituted 6-chloro-imidazo[1,2-b]pyridazine derivatives (4a–n) were smoothly
4

obtained by the Suzuki cross-coupling reaction. In order to investigate the impact of
C-6 substituents on the antifungal activity, we selected
6-chloro-3-phenylimidazo[1,2-b]pyridazine (4a) with the higher activity as a
secondary parent compound and designed a series of its derivatives. As depicted in
Scheme 2, several C-6 substituted 3-phenylimidazo[1,2-b]pyridazine derivatives were
prepared by fluorination (4o), palladium carbon dechlorination (4p), Suzuki reaction
(4q) and etherification (4r-v). The structures of all synthesized compounds were
confirmed by 1H NMR, 13C NMR and HRMS.
O
Cl
N
NH2
N
NBS, CHCl3,
9 h, 20 oC
H
N
N
N
(1) n-BuOH, 16h, reflux;
(2) H2O, NaOH
Cl
N
Cl
N
Cl
N
Br
3
1
2
N
Bronic acid, K2CO3, Pd(PPh3)4
Dioxane/H2O, 80oC
N
Cl
N
R1
4a-n
R1=
Cl
F
Cl
Br
CHO
Cl
4a
4b
4c
4d
4e
4f
4g
S
S
N
CF3
4n
CH2OH
OH
NH2
4h
4i
4j
4k
4l
4m
Scheme 1. Synthesis of C-3 substituted 6-chloro-imidazo[1,2-b]pyridazines (4a–n)
5

N
N
N
N
N
KF, DMSO/toluene=1:1
180 oC, 10 h
MeONa, CH3OH
R2O
F
N
N
N
Cl
N
or NaH, THF, R2OH
acid
4o
c
4r-4v
4a
R2= : CH3
4r
2CO3
K
ylboroni
,
4s
: CH2CH=CHCH3
)
4
O
3
phen
Pd(PPh
Dioxa
Pd-C/H2, THF, 40oC, 10h
N
H2
4t
:
ne,
N
CH2
4u
N
:
N
N
H
N
N
N
O
4v
:
4p
4q
Scheme 2. Synthesis of C-6 substituted imidazo[1,2-b]pyridazines (4m-v)
According to the mycelium linear growth rate method, a series of compounds 4a–v
were screened in vitro for their antifungal activities at 50 μg/mL against nine
phytopathogenic fungi [e.g., Fusarium solani (FS), Botryosphaeria berengriana f. sp.
Piricola (BP), Corn Curvalaria Leaf Spot (CL), Fusarium bulbigenum (FB),
Fusarium graminearum (FG), Alternaria alternata (AA), Pyricularia oryzae (PO),
Fusarium oxysporum f. sp. Vasinfectum (FV) and Alternaria brassicae (AB)].
Hymexazol and carbendazim, two commercial agricultural fungicides, were used as
the positive control.
Table 1 Antifungal activities of 4a-v against nine phytopathogenic fungi at 50 μg/mL.
Antifungal activities (inhibition % ± SE) a
Compounds
FS
FV
FB
FG
CL
BP
AA
PO
AB
3
46.2 ± 0.6 41.9 ± 0.7 65.3 ± 2.0 42.5 ± 0.7 12.6 ± 1.5 44.3 ± 0.8 28.1 ± 0.7 11.4 ± 1.4
98.9 ± 1.1 74.2 ± 1.4 76.1 ± 2.2 81.3 ± 0.8 97.8 ± 1.8 96.7 ±1.2 91.1 ± 0.8 87.4 ± 1.6
11.9 ± 3.3 5.8 ± 3.4 12.3 ± 2.5 9.5 ± 2.2 53.3 ± 0.5 37.7 ± 1.4 49.6 ± 1.3 40.2 ± 3.1
87.4 ± 1.3 64.2 ± 1.7 93.6 ± 1.3 78.1 ± 2.2 95.6 ± 0.4 85.2 ± 1.6 89.4 ± 2.6 88.0 ± 4.2
84.8 ± 0.6 52.5 ± 4.3 54.7 ± 0.6 68.9 ± 0.7 93.3 ± 0.2 84.2 ± 0.7 91.7 ± 1.3 87.6 ± 2.7
68.0 ± 1.2
92.8 ± 1.3
33.3 ± 2.3
86.9 ± 1.3
85.5 ± 2.7
52.9 ± 1.3
20.3 ± 3.2
27.5 ± 1.3
13.0 ± 2.2
62.3 ± 3.3
4a
4b
4c
4d
4e
4f
8.5 ± 1.7
12.5 ± 0.6
13.0 ±2.2
31.9 ±1.6
72.6 ±1.4
5.6 ± 1.9
54.1 ±1.6
5.5 ± 2.5
56.3 ±1.3
41.0 ±2.7
20.0 ± 2.2 27.1 ± 0.7 35.9 ± 1.1 16.0 ± 1.5
11.9 ± 1.9 17.1 ± 1.6
4g
4h
4i
12.2 ± 1.1
18.9 ± 1.9
3.3 ± 3.8
7.9 ± 1.9
8.0 ± 1.3
29.3 ± 0.7 30.4 ± 1.3 20.2 ± 2.5 57.0 ± 1.3 43.6 ± 2.7
4.4 ± 2.2 23.5 ± 1.9 10.4 ± 4.3 13.7 ± 1.6
21.7 ± 2.2 18.6 ± 1.4
37.8 ± 3.8 58.3 ± 2.9 68.8 ± 2.5 32.4 ± 1.6 57.0 ± 1.3 61.2 ± 0.9 49.6 ± 1.3 45.3 ± 1.6
6

4j
30.4 ± 3.4 57.5 ± 1.1 61.2 ± 1.0 14.8 ± 2.2 61.4 ± 1.4 51.9 ± 1.9 44.7 ± 0.7 43.6 ± 0.9
41.5 ± 3.2 37.9 ±2.3 41.8 ±0.8 29.0 ±1.6 29.6 ±1.9 61.2 ±0.9 35.1 ±1.1 24.8 ±1.6
56.5 ± 2.2
35.5 ±3.3
84.1 ± 1.3
30.4 ± 2.0
81.4 ± 1.2
74.6 ± 2.2
72.5 ± 3.6
28.3 ± 3.2
94.2 ± 1.3
73.9 ± 2.2
17.4 ± 2.8
8.7 ± 0.6
4k
4l
98.1 ± 0.6 92.9 ± 1.4 94.3 ± 1.7 73.8 ± 2.2 96.7 ± 0.6 96.6 ± 0.4 98.9 ± 1.3 91.7 ± 2.7
30.0 ± 1.1 46.4 ± 1.1 49.7 ± 1.0 25.2 ± 2.2 58.6 ± 2.6 36.6 ± 0.6 31.9 ± 2.1 20.5 ± 1.0
62.1 ± 1.1 17.1 ± 1.2 52.6 ± 1.2 72.0 ± 0.7 82.0 ± 1.5 77.0 ± 0.8 84.5 ± 0.7 77.2 ± 1.4
68.1 ± 3.4 64.4 ± 0.6 66.7 ± 1.0 62.4 ± 0.8 90.1 ± 1.1 67.2 ± 4.6 76.2 ± 0.6 71.8 ± 2.7
56.3 ± 2.4 57.5 ± 1.2 57.6 ± 1.0 69.5 ± 0.8 88.3 ± 1.1 80.3 ± 1.6 63.8 ± 0.8 65.8 ± 1.6
4m
4n
4o
4p
4q
31.9 ± 3.3
38.6 ±1.1
39.7 ± 0.5
4.8 ± 1.6
36.4 ± 2.8 31.7 ± 0.8 22.0 ± 1.2 15.4 ± 5.4
4r
98.7 ± 1.3 79.8 ± 1.1 84.9 ± 0.7 81.7 ± 1.4 99.7 ± 0.5 81.4 ± 0.9 92.4 ± 1.6 86.3 ± 3.1
67.5 ± 1.5 42.2 ± 0.6 39.2 ± 0.2 42.9 ± 1.8 84.3 ± 1.3 62.3 ± 3.3 70.9 ± 2.6 70.7 ± 0.1
4s
4t
4u
2.6 ± 2.6
5.4 ± 1.2
2.0 ± 2.0
2.2 ± 2.7
7.7 ± 1.0
19.3 ± 1.3 30.1 ± 1.4 19.1 ± 0.8 24.4 ± 2.7
20.0 ± 3.8 26.8 ± 2.5 14.2 ± 1.3 24.4 ± 0.5
15.1 ± 1.3 15.6 ± 1.2 10.5 ± 2.3
4v
38.1 ± 2.2 23.1 ± 0.8 14.4 ± 1.1 25.6 ± 2.1 18.5 ± 2.4 25.7 ± 0.9 22.0 ± 3.4 33.3 ± 4.1
3.6 ± 1.3
Hymexazol
63.5 ± 1.4 42.8 ± 2.5 59.3 ± 2.2 42.8 ± 2.2 63.0 ± 3.4
100 ± 1.0 99.8 ± 0.2 99.6 ± 0.9 99.8 ± 1.2 9.9 ± 2.1
44.8 ±1.9
93.4 ± 1.6
78.4 ± 1.3 72.4 ± 1.6
1.4 ± 1.2 7.0 ± 0.5
74.6 ±2.8
Carbendazim
2.2 ± 1.2
a Values are the mean ± SE of three replicates;
As described in Table 1, the results revealed that all the compounds displayed
varying degrees of antifungal activity against each of the tested plant pathogens.
Among them, the key intermediate compound 3 exhibited certain antifungal activity,
especially for the FB and AB strains. Therefore, compounds 4a-4n were synthesized
by replacing the bromine atom of compound 3 with aromatic rings and heterocycles.
It is noteworthy that compounds 4a, 4c, 4d and 4l exhibited broad spectrum
antifungal activity against nine phytopathogenic fungi than the positive control
hymexazol, and also showed remarkable antifungal activity against CL, AA, PO and
AB compared with carbendazim. For example, at the concentration of 50 μg/mL,
compound 4a demonstrated over 91.1% inhibition of FS, CL, BP, AA and AB strains;
compound 4c displayed 93.6% and 95.6% inhibition of FB and CL strains,
respectively; compound 4d displayed 93.3% and 91.7% inhibition of CL and AA
strains, respectively; and the compound 4l demonstrated over 91.7% inhibition of FS,
7

FV, FB, CL, BP, AA and AB strains. Additionally, compounds 4i, 4j, 4k and 4n
showed similar activities as hymexazol. Unfortunately, compounds 4b, 4e, 4f, 4g and
4h exhibited weak antifungal activity to some tested fungi. Subsequently, in order to
further explore the influence of C-6 substituents on the antifungal activity, compounds
4o-4v were synthesized by derivatization from compound 4a. To our delight,
compound 4r possessed significant antifungal activity compared with other
compounds and the two positive control compounds. Meanwhile, compounds 4o, 4p
and 4s showed more activity than the positive control hymexazol and more potent
than carbendazim against CL, AA, PO and AB, but compounds 4q, 4t, 4u and 4v
displayed weak antifungal activity against nine strains of phytopathogenic fungi.
Based on the bioassay results in Table 1, some interesting structure-activity
relationships was observed. Firstly, compared with the intermediate compound 3,
introduction of phenyl ring (4a) and thiofuran ring (4l) on the C-3 position
significantly improved the antifungal activity. For example, compound 3 showed less
than 68.0% inhibition against nine phytopathogenic fungi, but compounds 4a and 4l
displayed more than 73.8% inhibition at the concentration of 50 μg/mL.
Secondly, compared with the unsubstituted phenyl ring compound 4a,
introduction of either electron-donating or electron-withdrawing groups on the phenyl
ring invariably reduced the antifungal activity. Generally, some of the title
compounds bearing halogen atoms (F, Cl, CF3) on the phenyl ring, such as
compounds 4c, 4d and 4n, displayed better antifungal activity than those bearing
hydrogen bond donor groups of CH2OH, OH and NH2 (4i, 4j and 4k). Whereas,
8

compounds 4b and 4e (containing the 2,3-dichloro and 2-bromo groups on the phenyl
ring) exhibited weak antifungal activity to four phytopathogenic fungi (FS, FV, FB
and FG). The ortho formyl and para pheny substitution on the C3 phenyl ring
abrogated the antifungal activity, such as compounds 4f and 4g.
Unlike thiofuran group (4l), introducing 2-benzthiophenyl group (4h) and
3-pyridyl group (4m) at R1 position overall decreased the antifungal effects. For
example, compound 4l showed more than 73.8% inhibition against nine
phytopathogenic fungi, but compounds 4h and 4m displayed less than 58.6%
inhibition at the concentration of 50 μg/mL.
Substitution of chlorine atom of compound 4a with hydrogen, a benzene ring or
alkoxy substituent resulted in loss of activity, except the small methoxy substituent
bearing compound 4r retained the antifungal activity. For instance, the removal of
chlorine atom from the C-6 position of compound 4a afforded the moderate potent
compound 4p (56.3% < inhibition rate < 88.3%), which suggested that the chlorine
atoms was essential for the antifungal activities. Meanwhile, introduction of fluorine
atom on the C-6 position afforded the slightly more potent compound 4o (62.4% <
inhibition rate < 90.1%) and introduction of OCH2CH=CHCH3 group afforded the
slightly less potent compound 4f (39.2% < inhibition rate < 84.3%) than compound
4p. However, introduction of the methoxy group on the 6-position of compound 4p
afforded compound 4r (6-OCH3), which showed highly similar inhibition rate
(>79.8%) and broad-spectrum of antifungal activities as compound 4a (6-Cl) against
all phytopathogenic fungi at the concentration of 50 μg/mL. Unfortunately,
9

introducing a benzene ring, cycloalkanes or heterocycloalkanes at the C-6 position
obviously decreased the antifungal effects, such as compounds 4q, 4t, 4u and 4v.
Subsequently, the median effective concentrations (EC50) values of compounds
4a, 4c, 4d, 4l and 4r against nine phytopathogenic fungi were calculated, with
carbendazim and hymexazol used as positive controls. As shown in Table 2, it was
clearly seen that all of the five tested compounds showed excellent activity, with EC50
values of 1.3-64.1 μg/mL. As for FS strain, all the compounds showed the superior
activity (EC50= 5.1-17.9 μg/mL) than hymexazol (EC50 = 27.3 mg/mL), especially
compound 4l and 4r were found to have EC50 values of 5.1 μg/mL and 8.1 μg/mL
against this pathogen, respectively. As for FV strain, compound 4l (EC50 =8.4 μg/mL)
exhibited 11.3 fold more potent activity than hymexazol (EC50 = 95.3 μg/mL).
Regarding FB strain, all the compounds exhibited better activity than hymexazol
(EC50 = 29.4 μg/mL) except for compound 4d (EC50 = 44.3 μg/mL). Regarding FG
and BP strains, compounds 4a, 4c, 4d, 4l and 4r displayed much superior antifungal
activity to hymexazol, but failed to exceed carbendazim. For CL, AA, PO and AB
strains, compounds 4a, 4c, 4d, 4l and 4r exhibited 1.9-25.5 fold more potent activities
than hymexazol (EC50 = 16.7-33.2 μg/mL). Particularly, it was worth to note that the
EC50 values of compounds 4c and 4d against CL, AA , PO and AB strains reached
2.3/1.3, 4.2/2.9, 5.7/4.9 and 4.8/6.0 μg/mL, respectively. The effects of compounds 4a
and 4r on the growth of CL and FB at different concentrations were shown in Fig.2. It
can be seen that all the tested compounds exhibited a concentration dependent
antifungal activity. The morphological changing of Fusarium solani (FS) was then
10

viewed under the light microscope. As shown in Fig.3, the mycelium of the control
group had an eel shape, smooth surface, uniform size and much-branched, while the
mycelium appeared invagination, shriveling and few-branched after treated with 25
μg/mL of compound 4l. These results indicated that compound 4l may exert its
antifungal effect by preventing fungal nutrient uptake and metabolism.
Table 2 EC50 values of some selected compounds against nine phytopathogenic fungi.
EC50±SD values (μg/mL)a
Compounds
FS
FV
FB
FG
CL
BP
AA
PO
AB
4a
13.5 ± 0.3 25.6 ± 1.9 18.6 ± 1.6 13.2 ± 0.9
10.7 ± 0.4 28.6 ± 0.8 16.0 ± 0.3 14.8 ± 0.5
17.9 ± 1.3 64.1 ± 0.9 44.3 ± 0.6 16.6 ± 0.4
5.6 ± 0.7
2.3 ± 0.8
1.3 ± 0.8
6.8 ± 0.5
6.4 ±0.2
13.8 ± 1.0
5.9 ± 0.1
7.1 ± 0.3
9.1 ± 2.2
11.8 ± 3.8
6.7 ± 1.5
4.2 ± 1.1
2.9 ± 2.5
8.9 ± 1.6
7.8 ± 1.5
13.5 ± 0.7 10.0 ± 1.1
4c
5.7 ± 1.2
4.9 ± 0.9
4.8 ± 1.5
6.0 ± 1.4
4d
4l
5.1 ± 0.3
8.1 ± 0.1
8.4 ± 1.8
7.5 ± 0.2
14.3 ± 0.7
11.8 ± 1.4 13.2 ± 3.1
12.0 ± 1.9 11.3 ± 0.9
4r
18.5 ± 2.2 13.0 ± 1.4 10.6 ± 1.5
Hymexazol
Carbendazim
27.3 ± 0.5 95.3 ± 1.6 29.4 ± 2.0 62.3 ± 4.7 33.2 ± 3.5 65.5 ± 0.5 16.7 ± 3.5 25.5 ± 2.7 33.9 ± 0.2
0.7 ± 0.2 1.2 ± 0.3 0.8 ± 0.6 0.5 ± 0.1 >100 0.2 ± 0.1 >100 >100 >100
[a] 50% Effective concentration: concentration of compound that inhibits the fungi growth
Figs.2 Effects of compounds 4a and 4r on the growth of CL and FB at different
concentrations (CK: blank control group)
11

CK
Compound 4l
Figs.3 The effects of compound 4l on the growth of Fusarium solani (FS) at 25 μg/mL
(CK: blank control group)
In summary, we have synthesized
a
series of 3,6-disubstitued
imidazo[1,2-b]pyridazine derivatives and evaluated their antifungal activities against
nine phytopathogenic fungi. Among them, compounds 4a, 4c, 4d, 4l and 4r exhibited
excellent and broad-spectrum of antifungal activities compared with the two positive
control hymexazol and carbendazim; especially against CL, AA, PO and AB strains,
some derivatives exhibited 1.9-25.5 fold more potent activities than hymexazol.
Structure-activity relationship analysis showed that substituents on the benzene ring
and pyridazine ring could have significant effects on the activity. Furthermore, to the
best of our knowledge, this is the first report on the antifungal activity against plant
pathogens of the imidazo[1,2-b]pyridazine skeleton, which will pave the way for
further preparation and application of imidazo[1,2-b]pyridazine derivatives as
antifungal agents.
Acknowledgements
This research was supported by the Guizhou Provincial Engineering Laboratory for
Chemical Drug R&D (No. [2016]5402), Guizhou Provincial Science and Technology
Support Project [No. [2016]2848 and [2017]2835], Guizhou Provincial Natural
12

Science Foundation (No. [2019]1269), Guiyang Science and Technology Fund (No.
[2017]30-28) and The Opening Foundation of The Key Laboratory of Green Pesticide
and Agricultural Bioengineering, Ministry of Education, Guizhou University (No.
2018GDGP0101).
References
[1] Brauer, V. S.; Rezende, C. P.; Pessoni, A. M.; De Paula, R. G.; Rangappa, K.S.;
Nayaka, S. C.; Gupta, V. K.; Almeida, F. Biomole. 2019, 10(9), 1-21.
[2] Brase, S.; Encinas, A.; Keck, J.; Nising, C. F. Chem Rev. 2009, 109, 3903-3990.
[3] Savary, S.; Teng, P. S.; Willocquet, L.; Nutter, F. W. Annu. Rev Phytopathol.
2009, 44, 89-112.
[4] Ma, Z. H.; Michailides, T. J.; Crop Prot. 2005, 24, 853-863.
[5] Saquib, M.; Ansari, M.; Johnson C. R.; Khatoon, S.; Hussain, M. K.; Coop, A.
Eur J Med Chem. 2019, 182, 111657.
[6] Islam, M.; Siddiqui, A. A.; Rajesh, R.; Goyal, S. Acta Pol Pharm. 2008, 65(4),
441-447.
[7] Gokce, M.; Utku, S.; Kupeli, E. Eur J Med Chem. 2009, 44(9), 3760-3764.
[8] Rubat, C.; Coudert, P.; Tronche, P.; Bastide, J.; Bastide, P.; Privat, A. M. Chem
Pharm Bull. 1989, 37(10), 2832-2835.
[9] Rathish, I. G.; Javed, K.; Bano, S.; Ahmad, S.; Alam, M. S.; Pillai, K. K. Eur J
Med Chem. 2009, 44(6), 2673-2678.
[10] Abouzid, K.; Hakeem, M. A.; Khalil, O.; Maklad, Y. Bioorg Med Chem. 2008,
16, 382-389.
13

[11] Asif, M.; Singh, A.; Siddiqui, A. A. Med Chem Res. 2012, 21, 3336-3346.
[12] Cao, S.; Qian, X.; Song, G.; Chai, B.; Jiang, Z. J Agric Food Chem. 2003, 51(1),
152-155.
[13] Contreras, J. M.; Rival, Y. M.; Chayer, S.; Bourguignon, J. J.; Wermuth, C. G. J
Med Chem. 1999, 42(4), 730-741.
[14] Grote R, Chen Y, Zeeck A, Chen Z, Zähner, H.; Mischnick-Lübbecke, P.; König,
W. A. J Antibiot. 1988, 41, 595.
[15] Horkel, E. Top. Heterocycl Chem. 2013, 31, 223-268.
[16] Jyothi, B.; Madhavi, N. Asian J Chem. 2020, 32, 84-90.
[17] Al-Tel, T. H.; Al-Qawasmeh R. A.; Zaarour R. Eur J Med Chem. 2011, 46,
1874-1881.
[18] Abdelhamid, A. O.; Abdelall, E. K. A.; Zaki, Y. H. J Heterocyclic Chem. 2010,
47, 477 -482.
[19] Hussein, A. M. J Suadi Chem Soc. 2010, 14, 61-68.
[20] Chen, Q.; Zhu X. L.; Jiang L. L.; Liu Z. M.; Yang, G. F. Eur J Med Chem.
2008, 43, 595-603.
[21] Xu, H.; Fan, L. L. Eur J Med Chem. 2011, 46, 364-369.
[22] Xu, H.; Fan, L .L. Eur J Med Chem. 2011, 46, 1919-1925.
[23] Fan, L. L.; Luo, B. L.; Luo, Z. F.; Zhang, L.; Fan, J. D.; Xue, W.; Tang, L.; Li,
Y. Z Naturforsch B. 2019, 74(11-12), 811-818.
[24] Xu, H. Y.; Jia, A.; Hou, E. H.; Liu, Z. Y.; Yang, R. ; Yang, R. G.; Guo, Y. Chem
Biodivers. 2020, 17, e1900570.
14

[25] Bendjeddou, L. Z.; Loaëa, N.; Villiers, B.; Prina, E.; Späth, G. F.; Galons, H.;
Meijer, L.; Oumata, N. Eur J Med Chem. 2017, 125, 696-709.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
none
15

Graphical Abstract
N
N
3,6-Disubstitution
Antifungal activities
N
R2
N
N
N
R1
1
4a
4c
4d
: R = Ph,
R2 = Cl
R2 = Cl
Imidazo[1,2-b]pyridazine
1
: R = 2-F-Ph,
1
: R = 2-Cl-Ph,
R2 = Cl
1
2
4l
: R = 3-thiophen, R = Cl
1
R2 = OCH3
4r
: R = Ph,
Highlights：

Some novel 3,6-disubstituted imidazo[1,2-b]pyridazine derivatives were
prepared.
This is the first report on the antifungal activity against plant pathogens of
imidazo[1,2-b]pyridazine skeleton.
Compounds 4a, 4c, 4d, 4l and 4r exhibited more potent antifungal activities


compared with the two positive controls hymexazol and carbendazim.
16