Results in Chemistry 10 (2024) 101699

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Results in Chemistry
journal homepage: www.sciencedirect.com/journal/results-in-chemistry

Development of novel antibiotics derived from pyridazine: Synthesis,

spectroscopic characterization, in vitro antimicrobial activity and
molecular docking studies
Mouad Lahyaoui a, * , Mouad Filali a , Khadija Benamar b , Riham Sghyar a ,
Kawtar Fikri-Benbrahim b , Amal Haoudi a , Ahmed Mazzah c , Souad El khattabi d ,
El Mestafa El Hadrami a , Youssef Kandri Rodi a , Nada Kheira Sebbar e, f, *
a
Laboratory of Applied Organic Chemistry, Faculty of Science and Techniques, Sidi Mohamed Ben Abdellah University, B.P. 2202, Route d’Imouzzer, Fez 30050,
Morocco
b
Laboratory of Microbial Biotechnology and Bioactive Molecules, Faculty of Science and Techniques, Sidi Mohamed Ben Abdellah University, B.P. 2202, Route
d’Imouzzer, Fez 30050, Morocco
c
University of Lille, CNRS, USR 3290, MSAP, Miniaturization for Synthesis, Analysis and Proteomics, Lille, France
d
Laboratory of Engineering, Systems and Applications, National School of Applied Sciences, Sidi Mohamed Ben Abdellah-Fez University, BP Box 72, Fez, Morocco
e
Laboratory of Plant Chemistry, Organic and Bioorganic Synthesis, Faculty of Sciences, Mohammed V University in Rabat, 4 Avenue Ibn Battouta B.P. 1014 RP, Morocco
f
Laboratory of Organic and Physical Chemistry, Applied Bioorganic Chemistry Team, Faculty of Sciences, Ibnou Zohr University, Agadir, Morocco

A R T I C L E I N F O A B S T R A C T

Keywords: In light of the increasing antibiotic resistance, it is crucial to discover new antimicrobial medications. Pyridazines
Pyridazine are frequently investigated in research for the development of new chemical entities with enhanced therapeutic
Amino Esters properties. Their structure allows for significant flexibility in the attachment of various functional groups, which
Antimicrobial activity
is essential for drug discovery.
Docking study
Coupling
Our work focuses on synthesizing new pyridazine derivatives (7a, 7b, 7c and 7d) through coupling reactions
with various amino esters. The structures of these compounds have been analyzed using spectroscopic mea-
surements including 1H, 13C NMR, IR, and mass spectroscopy. The compounds showed positive effects against
two bacterial strains: Escherichia coli: ATCC25922 and Staphylococcus aureus: CIP543154. Molecular docking
results of the protein 7AZ5 indicated significant affinities with these molecules through hydrogen bonds.
Compounds 7a and 7d demonstrated the highest binding energies and the best antimicrobial activity, suggesting
their potential as a model for creating novel and potent antimicrobial compounds.

1. Introduction herbicidal and fungicidal activities [4–8]. A significant challenge in the

synthesis of new synthetic heterocyclic-like pyridazine is the search for
This century has seen a significant increase in the development and promising drug candidates with strong pharmacological and therapeutic
use of antibiotics based on heterocyclic pharmacophores due to their properties.
potential to treat bacterial infections. However, bacteria have developed The major amino acids have been employed as primary constituents
resistance to these antibiotics, posing a global public health threat of many drugs, including β-lactams and glutamate antagonists [9,10]. In
known as the growing antibiotic resistance phenomenon [1]. Pyridazine particular, phenylalanine and tryptophan are known to play important
is a commonly synthesized antibiotic that scientists have extensively roles in living organisms and transmit a vast array of therapeutic ac-
used to create new antibacterial agents [2,3]. These compounds have a tivities [11]. In recent years, amino acid modifications have been
range of effects, including antispasmodic, antihypertensive, antihista- explored by coupling them with other bioactive compounds such as
mine, analgesic, anti-inflammatory, and cardiotonic properties with cinnamic acid or coumarin, to study their biological and pharmacolog-
vasodilator activity. Additionally, they have been found to have ical activities [12,13]. To provide some intriguing biological activities,

* Corresponding authors.
E-mail addresses: [email protected] (M. Lahyaoui), [email protected] (N. Kheira Sebbar).

https://doi.org/10.1016/j.rechem.2024.101699
Received 6 June 2024; Accepted 31 July 2024
Available online 3 August 2024
2211-7156/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Lahyaoui et al. Results in Chemistry 10 (2024) 101699

we attempted to graft new pyridazine heterocyclic compounds to a se- bacteria with molecular docking studies for all compounds.
ries of amino esters groups in our case study (Scheme 1).
In this study and due to the growing interest in pyridazine de- 2. Materials and methods
rivatives, it was expected that new pyridazine derivatives would be
required for biological activity research. The compound 3,6-di(2-pyr- The reaction completion was supervised by TLC using 60-F254 flat
idyl)-1,2,4,5-tetrazine serves as a suitable starting material to pursue silica gel and visualized under UV and iodine light. Column chroma-
this objective through its interaction with various amino esters. tography was performed using silica gel 60 (Merck 230–400 mesh) and a
Developing substances that can prevent the action of DNA poly- hexanes-ethyl acetate eluent mixture. 1H and 13C NMR spectra were
merase enzymes in microbes is necessary to use DNA polymerase as a taken in CDCl3 on Bruker spectrometers (300 MHz for 1H and 75 MHz for
13
target for pyridazine derivatives with antibacterial activity. The mi- C) with chemical shift values (δ) given in part per million (ppm)
crobes may eventually die or experience growth inhibition as a result of relative to TMS (δ 0.00) as the internal standard. Coupling constants (J)
this inhibition, which may interfere with DNA replication. In both pro- are expressed in Hz and multiplicities are denoted by s (singlet),
karyotic and eukaryotic microbes, DNA polymerase is essential for DNA d (doublet), dd (doublet of doublet), t (triplet), dt (doublet of triplet), q
replication. By including nucleotides that are complementary to the (quartet) and m (multiplet). Mass spectra (MS) were performed in ESI
template strand, it creates new DNA strands. By attaching to the DNA mode and data were reported as m/z (100 % intensity). Infrared (IR) was
polymerase active site, pyridazine derivatives can function as competi- carried out by Interface Regional University Center in Fez, Morocco.
tive inhibitors by blocking the binding of natural nucleotides. Moreover,
these substances can attach to allosteric regions, changing the enzyme’s 1. General procedure for the synthesis of 4 and 5
structure and lowering its activity.
The process of molecular docking occupies computational technique A mixture of 3,6-Bis(pyridin-2-yl)-1,2,4,5-tetrazine 1 (4 mmol) was
scores to assess the binding affinity of docked molecules to receptors. It dissolved in toluene (20 mL), and then 1 equiv. of 1-ethynyl-4-methyl-
is well known that this method works very well for in silico drug benzene 2 or methyl 4-ethynyl benzoate 3 was added and their action
development [14–18]. mixture was stirred and refluxed at temperatures between 140 and
However, to observe the binding mode of pyridazine derivatives with 180 ◦ C. The solvent was then evaporated. The product obtained was
antimicrobial activity on the active site of 7AZ5 in vitro, a study using separated by chromatography on a column of silica gel. The isolated
molecular docking on the target protein was conducted. We present the solid was recrystallized from hexane–dichloromethane (1:1v/v)
synthesis of four new pyridazine compounds, their structural charac- [19,20].
terizations, and well as antibacterial activity against two types of

Scheme 1. Synthesis of some new pyridazine derivatives.

2
M. Lahyaoui et al. Results in Chemistry 10 (2024) 101699

2. General saponification procedure for the synthesis of 6 Autodock software, which was used to calculate and provide scores.
Using ChemDraw (18.2), the chemical structures were built. The target
The given procedure involves mixing 1 mmol of compound 4 with crystal structure for antibacterial activity named DNA polymerase
10 mL of a dioxane-water mixture (9:1). This mixture is stirred while 0.5 sliding clamp (PDB code = 7AZ5) was retrieved and constructed using
N sodium hydroxide solution is slowly added until reaching a neutral the Protein Data Bank. After being separated from the protein structure
pH. After a 6-hour reaction at room temperature, the dioxane is evap- and fixed with hydrogen atoms, all of the ligands and cofactors that bind
orated and 15 mL of water is added. The resulting mixture is then water were optimized. one by one eliminating the active sites to create
extracted three times with CH2Cl2. The aqueous phase is neutralized fictitious atoms. A switch was pressed, assigning all parameters and
using a 1 N hydrochloric acid solution and then extracted with ethyl charges to the MMFF94x force field. The module was utilized to dock the
acetate. The organic phase is dried using concentrated Na2SO4. structural model of the molecules to the surface of the protein within the
alpha site spheres that were assembled through the use of the site search
3. General oxidation procedure for the synthesis of 6 module. The upgrading was done using two separate refining proced-
ures, while the docking scoring was done using the London dG scoring
We start by dissolving 3 mol of potassium permanganate (KMnO4) in function. The best 30 dock poses that were chosen for inspection to get
one liter of water, and then adding about 5 mol of NaOH to make the the highest score afterward received access to self-turning docks. The
solution basic (pH>7). Then, we add this solution to 1 equivalent of binding pose’s RMSD was then obtained by matching the docking pos-
compound 5 to oxidize it. The reaction is stirred for 15 min at 95 ◦ C. tures with the ligand in the co-crystallized structure utilizing the data-
After that, we cool the solution slightly and add 10 mL of ethanol to react base browser. The compounds’ ability to attach to the protein molecules
with the excess potassium permanganate. The resulting manganese di- under study was then classified using the calculation of hydrogen bonds
oxide is removed by suction filtration. The remaining solution is then and binding free energy between the produced molecules and the amino
neutralized by adding 2 M hydrochloric acid until a neutral pH is acid residues of the receptors. The default-docking model was estab-
reached. The precipitate is left overnight and then filtered by suction lished by comparing the interaction types with the RMSD of the native
ligand of the receptor structure.
4. General coupling amino esters procedure for synthesizing 7a, 7b, 7c
and 7d 3. Results and discussion

We solubilized 1 mol of methyl 2-amino-2-phenylacetate or methyl 1. Synthesis and characterization

2-aminopropanoate or methyl 2-amino-3-hydroxypropanoate or methyl
2-aminoacetate in 20 ml of DMF and gradually added 2.2 mol of trie- Compound 5 was produced in 90 % yield following the thermal
thylamine over 15 min at 0 ◦ C. Then, we added 1 mol of compound 6 cycloaddition reaction [19,20] and after purification by silica gel col-
with 1.1 mol of hexafluorophosphate benzotriazole tetramethyl uro- umn chromatography. The identification of the synthesized derivative
nium (HBTU). The reaction was left to stir at room temperature for 1 h. was performed by 1H, 13C NMR, IR and mass spectroscopy. The analysis
After the reaction, we removed the DMF by evaporation and performed of the 1H NMR spectrum revealed a signal around 2.34 ppm related to
an extraction process with ethyl acetate. Finally, we purified the product CH3 bound to phenyl, a massive between 7.09 and 7.18 ppm which
by chromatography on silica gel. shows the presence of benzene and a singlet around 8.65 ppm reflects
the presence of the carbon of the aromatic ring in ortho position con-
5. Antimicrobial activity cerning benzene. On the 13C NMR spectrum, we note the presence of a
signal around 21.24 ppm corresponding to the carbon of CH3, 2 signals
Using the broth microdilution method [21] the antibacterial activity at 128.87 ppm, 129.19 ppm due to the 4 CH of benzene, a signal around
of the compounds was evaluated against one gram-positive (Staphylo- 121.87 ppm corresponding to the carbon of the aromatic ring in ortho
coccus aureus CIP543154) and one gram-negative (Escherichia coli position concerning benzene, and 3 signals related to the quaternary
ATCC25922). 100 μL of each chemical was added to the wells of the first carbons at 133.87 ppm (C- CH3), 137.40 ppm (C-Ph) and 140.44 ppm
colon of the 96-well microplate at a concentration of 20 μg/mL. Sub- (C).
sequently, the investigated compounds were diluted twice in Luria- Under the same conditions as above, the action of compound 3 on
Bertani (LB) liquid media. Suspensions of bacterial strains adjusted to compound 1 resulted in compound 4 with a yield of 90 % after purifi-
106 UFC/mL were introduced in 50 μL. Only the Luria-Bertani (LB) cation by silica gel column chromatography. The compound was char-
medium enriched with the strain was present in the positive and nega- acterized by the spectroscopic methods of 1H, 13C NMR, mass
tive controls, respectively. The microplate was then incubated for 24 h at spectrometry and X-ray crystallography [19,20].
37 ◦ C. After adding 15 μL of 0.15 % resazurin, the microplate was Compound 6 is obtained by different methods. On the one hand, the
incubated for 30 min at 37 ◦ C to examine color changes. Pink-colored oxidation reaction gives a low yield compared to the other saponifica-
wells indicated the vitality of the cells, while wells that maintained tion reaction which has a rather high yield. Both methods in the end give
the resazurin-blue hue indicated the suppression of cell growth [22]. the desired product which was characterized by 1H and 13C NMR
The minimum bactericidal concentration (MBC), which was found spectroscopy. Compounds 7a, 7b, 7c and 7d were produced in good
by inoculating Petri dishes with the contents of the wells where bacterial yields following the general procedure described above and after puri-
inhibition was found, presents the minimal concentration at which fication by silica gel column chromatography, the identification of the
bacteria were killed, while the minimal inhibitory concentration (MIC) synthesized derivatives was performed by 1H, 13C NMR spectroscopy.
presents the minimal concentration at which bacterial growth was The analysis of the 1H NMR spectrum revealed a signal around 3.77
inhibited. Following a 24-hour incubation period at 37 ◦ C, the results ppm related to CH3 of amino ester in compound 7a, a signal around
were observed. The MBC is shown in the lowest concentration at which 3.765 ppm for the O-CH3 group of compound 7b and another one
no bacterial growth was observed. around 4.775 ppm which shows the presence of CH function that is next
to CH3. A massive 4.117 ppm shows the presence of the OH function for
6. Molecular docking studies compound 7c and a doublet in the region of 4.15 indicates the CH2
function. A massive in the range of 7.30 ppm demonstrates the presence
Molecular docking has been the most popular way to support ligand of NH function for all compounds in the coupling. On the 13C NMR
behavior against a target of interest and conduct structure-based virtual spectrum, we note the presence of a signal around 172 ppm corre-
screening campaigns. The molecular docking method was applied with sponding to the carbon of CO of amino ester for all compounds 7a, 7b,

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M. Lahyaoui et al. Results in Chemistry 10 (2024) 101699

7c and 7d. At the end, a signal around 166 ppm corresponds to the demonstrated that compounds containing pyridazine ring exert different
carbon of CO linked to the NH function. biological activities including anti-cancer [28], anti-inflammatory [29]
and antimicrobial effects [30,31].
The mechanism of antimicrobial action of compounds including
2. Antimicrobial activity
pyridazine was attributed to the inhibition of bacterial DNA gyrase [31],
the key enzyme involved in DNA replication. Hence, the combination of
Table 1 displays the findings from the broth microdilution method
these two important antimicrobial units (Pyridazine and amino-esters)
used to evaluate the antibacterial activity of the compounds under
resulted in a high antimicrobial effect as demonstrated in Table 1.
study. As we can see, at the highest tested dose (10 μg/mL), none of the
Moreover, nitrogen atoms of amino-esters and pyridazine ring are also
substances showed any bactericidal action against S. aureus or E. coli.
probably contributing to the antimicrobial effect related to different
However, an important bacteriostatic activity was observed against the
intra and extra-cellular interactions including hydrogen bonding [32].
targeted bacteria with minimal inhibitory concentrations (MICs) lower
These interactions may cause an occupation of microbial cells active
than 10 μg/mL for most of the tested compounds. Moreover, it appears
sites and also a disruption of bacterial permeability and therefore induce
that the bacterial growth was inhibited at very low concentrations (<1
microbial cell death or growth or inhibition.
μg/mL) with compounds 7a and 7d: MIC Vs S. aureus = 0.62 ± 0.00 μg/
mL and MIC Vs S. aureus and E. coli = 0.07 ± 0.00 μg/mL, respectively.
Similar results with MIC<10 mg/ml were obtained by Mustafa and 3. Molecular docking studies
Mostafa (2020) testing the activity of pyridazine derivatives (obtained
by coupling various acetophones or benzaldehydes with hydrazide) Molecular docking provides a powerful method for predicting and
against different gram negative and gram positive bacteria, namely identifying the type of interaction and binding sites with interacting
Bacillus cereus, Solibacillus isronensis, Escherichia coli, Pseudomonas molecules. The binding sites and docking scores of the encoded com-
aeruginosa and Serratia marcens [23]. An interesting antibacterial ac- pounds for the encoded protein 7AZ5 were viewed and calculated using
tivity was also obtained by Chen et al. (2023), investigating pyridazine Autodock software. The binding energy with the lowest activity was
associated with chalcone derivates. Indeed, a high inhibitory activity of identified based on all computed energies, as shown by the ranking
bacterial growth was obtained at the concentrations of 6.29 ± 0.36, 6.39 positions generated by the scoring functions given in Table 2.
± 6.10 and 6.76 ± 0.72 µg/mL, against Pseudomonas syringae pv. 7a and 7d had the highest scores, corresponding to − 6.17 and − 6.23
Actinidiae, Xanthomonas oryzae pv. Oryzae and Xanthomonas axono- kcal/mol, respectively. The table above lists the hydrogen bonds that
podis pv. Citri, respectively [24]. exist between the chemicals and the selected protein coenzymes. Figs. 1
More recently, Alatawi et al. (2024) revealed the antiviral, antifungal and 2 show the best-fitting positions adopted by the enzyme-calmed
and antibacterial activity of pyridazine derivatives resulting from the come the same pound, 7AZ5. Autodock is the most commonly used
reaction of hydrazothiazole with different active methylene groups. molecular docking method for determining a precise docking study
Their results showed a MIC ranging from 9 to 92 ppm for E. coli, and between compounds and target proteins. The measured LD30 of the
from 12 to 100 ppm for S. aureus [25]. Moreover, another pyrimidine- compounds against the target protein matched the interaction types and
pyrazolo-pyridazine (4-amino-5-benzoyl-6-phenyl-phenanthro[9,10-e] docking position.
pyrimidino[1,2-b]pyrazo-lo [3,4-c] pyridazine) showed a good antimi- Using an H-bond with SER 346 and hydrogen π-stacking with the 6-
crobial activity against S. aureus, E. coli and Aspergillus niger membered ring of VAL247, compound 7a had a high docking score. The
(Respective MIC values of 50, 12.5 and 2,5 μg/mL) (Fayed et al., 2015) energy stabilization was between − 0.9 and − 2.9 kcal/mol and the
[26]. interaction distance ranged between 3.18 and 3.87A◦ .
Overall, all and these studies showed the important antimicrobial Through an H-bond with both ARG246 and SER346, compound 7d
activity of pyridazine based compounds, however, it’s the first time that demonstrated a strong docking score. The energy stabilization was close
the antibacterial activity of compounds obtained by coupling pyridazine to − 1 kcal/mol and the interaction distance was between 2.92 and 3.3
derivatives with amino esters is tested in the present study. A◦ .
As regards compounds obtained by coupling pyridazine derivatives The structure of the target receptor was stabilized by these bonds
with amino esters (7a, 7b, 7c and 7d), it was claimed that amino-acid that formed with the essential amino acid residues of the binding
scaffold presents an extremely important interest in the development pockets identified by the previously mentioned data. The lowest binding
of several antimicrobial agents such those including amino-phosphonate energy docked poses are considered to be the best-docked conformation
or amino-boronated groups. These amino-acids-based compounds because they all have the highest affinity. In addition, the docking
exhibit a potential antimicrobial effect since they can inhibit strongly software made it possible to match binding modes that were observed
enzymes involved in the metabolism of proteins and the incorporation of experimentally, which provided insight into the precise conformation of
amino acids as components of peptidoglycan that constitutes the cell the ligand and the target of these crucial molecular interactions.
wall of bacteria especially of gram-negative ones. In the present work,
the antimicrobial activity of compounds obtained by coupling amino- Conclusion
esters to pyridazine may be attributed to disorders affecting microbial
biochemical pathways due to the obtained modified amino-esters that We synthesized the acid 6 from pyridazine 1 in two ways: oxidation
mimic the natural function of the original-amino-esters but lead to and saponification. The coupling process successfully obtained four new
different changes in cellular metabolism. However, it’s important to compounds, namely 7a, 7b, 7c and 7d. The desired products were ob-
mention that the pyridazine ring is also considered one of the best tained in good yields and their structures were validated by 1H and 13C
heterocycles that can be used for drug design [27]. Several studies have NMR spectral techniques. The assessment of the antimicrobial activity of

Table 1
Antimicrobial activity of compounds against E. coli and S. aureus.
Bacterial species Antimicrobial activity of compounds
MIC (μg/mL) / MBC (μg/mL)
Compound 6 Compound 7a Compound 7b Compound 7c Compound 7d

E. coli 1.25 ± 0.00/ND >10 ± 0.00/ND 5.00 ± 0.00/ND 10.00 ± 0.00/ND 0.07 ± 0.00/ND
S. aureus 5.00 ± 0.00/ND 0.62 ± 0.00/ND 5.00 ± 0.00/ND 5.00 ± 0.00/ND 0.07 ± 0.00/ND

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M. Lahyaoui et al. Results in Chemistry 10 (2024) 101699

Table 2
Docking score and interaction between the compounds and 7AZ5 protein.
Compounds Binding energy (kcal/mol) Ligand receptor interaction distance E (kcal/mol)

6 − 5.57 O 40 SER 345 H-donor 3.50 − 0.7

O 39 LYS 250 H-acceptor 3.09 − 2.9
6-ring VAL 247 pi-H 3.84 − 0.8
7a − 6.17 O6 SER 346 H-acceptor 3.18 − 2.9
O6 SER 346 H-acceptor 3.26 − 0.9
6-ring VAL 247 pi-H 3.87 − 1.2
7b − 6.03 O 47 LYS 250 H-acceptor 3.09 − 5.4
7c − 6.00 O7 THR 341 H-acceptor 3.01 − 2.2
O 46 PRO 249 H-acceptor 3.65 − 0.6
O 46 LYS 250 H-acceptor 3.08 − 5.1
6-ring VAL 247 pi-H 3.85 − 0.8
7d − 6.23 O9 ARG 246 H-donor 2.92 − 1.2
O 51 SER 346 H-acceptor 3.30 − 0.7

Fig. 1. Molecular docking analysis of 7AZ5. Protein-ligand interactions between 7AZ45 and compound 7a.

Fig. 2. 3D Molecular docking analysis of 7AZ5. Protein-ligand interactions between 7AZ45 and compound 7d.

pyridazine derivatives revealed their potential as agents against 4H, CHAr); 7.82–8.02 (m, 5H, CHAr); 8.36–8.38 ppm (d,1H, J=6 Hz,
Escherichia coli and Staphylococcus aureus. These findings imply that CHAr); 8.69–8.79 ppm (m, 3H, CHAr). 13C NMR (CDCl3) δ (ppm): 52.28
these substances could significantly advance the synthesis of potent (CH3-O); 121.87; 123.65; 124.81; 125.01; 125.61; 128.91; 129.62;
antimicrobial agents. It’s important to retain the fact that the docking 129.83; 136.82; 137.30; 148.97; 14.51 (CHAr); 128.91; 139.62; 141.88;
study’s objective was to determine how the most potent substances 153.04; 155.24; 157.76; 158.00; 166.62(CAr).
interacted with the target protein 7AZ5. The outcomes corroborate the SM (FABþ): m/z = 369.13[MH]+ (See Figures S1, S2 and S3 Sup-
experimental findings by demonstrating that hydrogen bonds have a porting Information).
significant influence on the activity values under investigation. 7a and
7d were found to possess the highest binding affinities to the target 3,6-di (pyridin-2-yl)-4-(p-tolyl)pyridazine: (5)
1
protein based on the computational calculations. In light of the previ- H NMR δ (CDCl3) (ppm): 2.34 ppm (s, 3H); 7.1–7.18 (m, 4H);
ously provided information, those chemicals could be important com- 7.26–7.3 ppm (m, 1H);7.40 ppm (dd, 1H, J3 = 7.6, J4 = 4.8 Hz);
ponents in the development of novel, potent antimicrobial medications. 7.76–7.94 ppm (m, 3H); 8.50 ppm (d, 1H, J=3.9 Hz); 8.65 ppm (s, 1H);
8.75 ppm (d,1H, J=3.3 Hz); 8.79 ppm (d, 1H, J=7.2 Hz). 13C NMR δ
Spectral data (CDCl3) (ppm): 21.24 (CH3-Ph); 121.87, 123.28, 124.74, 124.91,
125.46, 128.87, 129.19, 136.50, 137.17, 149.08, 149.43 (CHAr);
Methyl 4-(3,6-di(pyridin-2-yl)pyridazin-4-yl)benzoate: (4) 133.87, 138.48, 140.04, 153.49, 156.07, 157.69, 158.44 (CAr). SM
1
H NMR (CDCl3) δ (ppm): 3.89 (s, 3H, O-CH3); 7.25–7.47 ppm (m, (FABþ): m/z = 325.14[MH]+; IR: ν (cm-1): 1570 (C=CAr); 2930 (CH3-

5
M. Lahyaoui et al. Results in Chemistry 10 (2024) 101699

Ph) (See Figures S4, S5, S6 and S7 Supporting Information). Formal analysis. Riham Sghyar: Investigation, Methodology, Writing –
original draft. Kawtar Fikri-Benbrahim: Writing – original draft,
4-(3,6-di(pyridin-2-yl)pyridazin-4-yl)benzoic acid: (6) Methodology. Amal Haoudi: Writing – original draft, Validation,
1
H NMR δ (CDCl3) (ppm): 7.403–7.449 ppm (m, 3H, 2CH=CHN, Methodology. Ahmed Mazzah: Writing – original draft, Validation.
CH=CqN); 7.577–7.621 ppm (dq, 1H, CH=CHCq, J=0.9); 7.873–7.901 Souad El khattabi: Writing – original draft, Formal analysis. El Mestafa
ppm (d, 2H, CH=Cq, J=8.4); 7.987–8.010 ppm (m, 2H, CH=Cq–CO2H); El Hadrami: Writing – original draft, Methodology. Youssef Kandri
8.053–8.110 ppm (ddd, 1H, CH=CH-Cq, J=1.8); 8.375–8.390 ppm (m, Rodi: Writing – original draft, Validation, Supervision, Methodology.
1H, CH-Cq-N); 8.553 ppm (s, 1H, CH-Cq-N); 8.656–8.682 ppm (d, 1H, Nada Kheira Sebbar: Writing – original draft, Validation, Supervision,
CH-N, J=7.8); 8.780–8.793 ppm (d, 1H, CH-N, J=3.9); Methodology, Investigation.
13
C NMR δ (CDCl3) (ppm): 121.75, 124.42, 125.30, 125.56, 125.95,
129.46, 129.76, 137.64, 138.30, 149.15, 150.32 (CHAr); 130.91,
139.53, 141.62, 152.82, 155.41, 157.80, 158.55 (CAr); 167.40 (CO2H). Declaration of competing interest
(See Figures S8 and S9 Supporting Information).
The authors declare that they have no known competing financial
methyl 2-(4-(3,6-di(pyridin-2-yl)pyridazin-4-yl)benzamido)-2- interests or personal relationships that could have appeared to influence
phenylacetate: (7a) the work reported in this paper.
1
H NMR δ (CDCl3) (ppm): 3.77 ppm (s, 3H, CH3); 5.77 ppm (d, 1H,
J=6.9, CH); 7.26–7.46 ppm (m, 10H, CHAr); 7.76–7.96 ppm (m, 5H, Appendix A. Supplementary data
4CHAr, NH); 8.41–8.43 ppm (d, 1H, J=6, CHAr); 8.63 ppm (s, 1H, CHAr);
8.72–8.78 ppm (m, 2H, CHAr). 13C NMR δ (CDCl3) (ppm): 52.97 (CH3); Supplementary data to this article can be found online at https://doi.
56.93 (CH-NH); 121.95, 123.71, 124.84, 124.90, 125.02, 125.68, org/10.1016/j.rechem.2024.101699.
127.36, 127.41, 128.71, 129.06, 129.15, 136.90, 137.02, 137.33,
149.06, 149.53 (CHAr); 133.14, 136.34, 139.59, 140.69, 153.06, References
155.14, 157.81, 157.95 (CAr); 166.01 (CO-NH); 171.47 (CO2Bz). (See
Figures S10 and S11 Supporting Information). [1] E.A. Ashley, M. Dhorda, R.M. Fairhurst, A.R. Keramati, M.D. Mohsen Fathzadeh,
A. Mani, Spread of artemisinin resistance in plasmodium falciparum malaria,
N. Engl. J. Med. 371 (2014) 411–423.
methyl (4-(3,6-di(pyridin-2-yl)pyridazin-4-yl)benzoyl)-D-alaninate: (7b) [2] H. Frank, G. Heinisch, Pharmacologically active pyridazines part I, In Progress in
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H NMR δ (CDCl3) (ppm): 1.493 ppm (d, 3H, J=7.2, CH3); 3.765 Medicinal Chemistry. 27 (1990) 1–49.
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