Development of new highly potent imidazo[1,2-b[pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1

Article abstract of DOI:10.1016/j.ejmech.2015.10.004

Using a structure-based design approach, we have developed a new series of imidazo[1,2-b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity relationships and docking studies confirmed the binding mode of these inhibitors within the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar concentrations, with a good selectivity profile against a panel of mammalian kinases. The potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC₅₀ values of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to mammalian cells and were selected for further in vivo investigations on murine model of acute toxoplasmosis.

Full text of DOI:10.1016/j.ejmech.2015.10.004

Accepted Manuscript
Development of new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma
gondii Calcium-Dependent Protein Kinase 1
Espérance Moine, Isabelle Dimier-Poisson, Cécile Enguehard-Gueiffier, Cédric Logé,
Mélanie Pénichon, Nathalie Moiré, Claire Delehouzé, Béatrice Foll-Josselin, Sandrine
Ruchaud, Stéphane Bach, Alain Gueiffier, Françoise Debierre-Grockiego, Caroline
Denevault-Sabourin
PII:
S0223-5234(15)30286-5
10.1016/j.ejmech.2015.10.004
EJMECH 8139
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 July 2015
Revised Date: 2 October 2015
Accepted Date: 3 October 2015
Please cite this article as: E. Moine, I. Dimier-Poisson, C. Enguehard-Gueiffier, C. Logé, M. Pénichon,
N. Moiré, C. Delehouzé, B. Foll-Josselin, S. Ruchaud, S. Bach, A. Gueiffier, F. Debierre-Grockiego, C.
Denevault-Sabourin, Development of new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma
gondii Calcium-Dependent Protein Kinase 1, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.10.004.
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IC (nM)
16a
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16f
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Compound 16a
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TgCDPK1
T. gondii
HFF
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70
42500
21300

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Development of new highly potent imidazo[1,2-b]pyridazines targeting
Toxoplasma gondii Calcium-Dependent Protein Kinase 1
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Espérance Moine , Isabelle Dimier-Poisson
, Cécile Enguehard-Gueiffier
,
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a, b
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Cédric Logé , Mélanie Pénichon , Nathalie Moiré , Claire Delehouzé , Béatrice
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a, b
Foll-Josselin , Sandrine Ruchaud , Stéphane Bach , Alain Gueiffier , Françoise
a, b, 2
a, b, , 2, 3
Debierre-Grockiego
, Caroline Denevault-Sabourin
*
.
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Université François-Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F-
37000 Tours, France
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INRA, UMR1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France
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Université de Nantes, Nantes Atlantique Universités, Laboratoire de Chimie
Thérapeutique, Cibles et Médicaments des Infections et du Cancer, IICIMED-EA 1155,
UFR Sciences Pharmaceutiques, F-44035 Nantes, France
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USR3151 CNRS/UPMC, Plate-forme de criblage KISSf (Kinase Inhibitor Specialized
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Screening facility), Station Biologique de Roscoff, F-29688 Roscoff, France
* Corresponding author, Department of Recherche et Innovation en Chimie Médicinale,
University François Rabelais of Tours, 31 avenue Monge, F-37200 Tours, France. Tel.:
+33 2 47367035.
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E-mail address: caroline.sabourin@univ-tours.fr (C. Denevault-Sabourin).
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These authors contributed equally to this work.
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Co-senior authors.
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Present address: UMR7292 GICC CNRS, Team 4, Innovation Moléculaire et
Thérapeutique, University François Rabelais of Tours, F-37200 Tours, France

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Abstract
Using a structure-based design approach, we have developed a new series of imidazo[1,2-
b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from
Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity
relationships and docking studies confirmed the binding mode of these inhibitors within
the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then
identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar
concentrations, with a good selectivity profile against a panel of mammalian kinases. The
potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC values
5
0
of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to
mammalian cells and were selected for further in vivo investigations on murine model of
acute toxoplasmosis.
Keywords
imidazo[1,2-b]pyridazine; anti-apicomplexan; Toxoplasma gondii; Toxoplasma gondii
calcium-dependent protein kinase 1.
1
Abbreviations
1
IC , 50% inhibitory concentration; EC , 50% effective concentration; CC , 50% cytotoxic
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concentration; SAR, structure-activity relationships; HFF, human foreskin fibroblasts; T. g., Toxoplasma
gondii; TgCDPK1, Toxoplasma gondii calcium-dependent protein kinase 1; CK1, casein kinase 1; GSK3,
glycogen synthase kinase 3; CDK5, cyclin-dependent kinase 5; RIPK3, receptor-interacting protein kinase
3
.

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1. Introduction
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Toxoplasmosis is a wide-spread ubiquitous zoonosis (nearly one-third of humanity has
been exposed to this parasite) caused by Toxoplasma gondii, an obligate intracellular
protozoan of the phylum Apicomplexa [1]. The biological success of T. gondii could be
explained by its wide intermediate host range as it can infect almost all homeothermic
animals including humans. Human infection occurs by ingestion of oocysts shed in the
feces of infected felids (definitive hosts), via contaminated food or water, or by ingestion
of tissue cysts found in undercooked or raw meat from infected animals. The infesting
stages release sporozoites or bradyzoites, respectively, which differentiate into the
tachyzoite stage. This form of the parasite disseminates rapidly, infecting a wide range of
nucleated cells (acute infection), before being controlled by innate and adaptive immune
responses. Approximately 3 weeks after infection, tachyzoites differentiate into a slow
growing stage (bradyzoites), establishing a reservoir of tissue cysts, mostly found in
brain, eyes and muscles of hosts. This cystic stage allows the parasite to escape the
immune system and to remain in a latent way in the body (chronic infection) [2].
In immunocompetent individuals, this infection is, most of the time, asymptomatic. But it
represents a major health concern during pregnancy, since the transplacental passage of
the tachyzoite form can be responsible for fetus contamination, leading to abortion, fetal
growth retardation or birth defects like neurologic and ocular disorders (congenital
toxoplasmosis) [3]. Severe or fatal toxoplasmosis can also be observed in
immunocompromised individuals, by reactivation of their latent infection, which can be
responsible for cerebral toxoplasmosis, sometimes resulting in encephalitis [4].
Moreover, there is an increasing number of symptomatic forms in immunocompetent
host, generated by atypical strains, leading to a variety of symptoms including severe
systemic disorders [5].
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Unfortunately, the therapeutic arsenal is constituted of a restricted number of molecules.
The current therapy is a combination of pyrimethamine with a sulfonamide drug, such as
sulfadiazine [6]. These drugs, targeting parasite folate metabolism enzymes, are not
parasite-specific and demonstrate important toxicity (e.g. frequent allergic reactions and
bone marrow toxicity). Consequently, because of pyrimethamine hematologic side

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effects, a co-administration of folinic acid during treatment is necessary. This molecule is
also not recommended during the first trimester of pregnancy [7–9]; spiramycin can then
be used but have only a parasitostatic activity. Moreover these drugs are not effective
once the parasite has differentiated into the cyst form (chronic stage of the disease).
Therefore, new therapeutic alternatives need to be identified to develop drugs (i)
targeting more specifically parasite proteins and (ii) with a better toxicity profile.
T. gondii, as an obligate intracellular parasite, cannot replicate without host cells. Thus,
tachyzoite egress from the host cell and subsequent invasion into another cell are key
steps that could be targeted for drug therapy. Different studies have demonstrated that
these parasite functions were regulated by calcium-regulated signaling enzymes,
including a family of plant-like calcium-dependent protein kinases (CDPKs), which are
present in plants, ciliates and apicomplexans but not in animals, making them interesting
targets for drug discovery [10]. In T. gondii, CDPK1 has been found to control
microneme secretion, gliding motility, host cell invasion and egress and is therefore
essential for parasite survival [11].
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The vast majority of protein kinase inhibitors (PKIs) target the ATP-binding pocket, by
occupying the adenine-binding region and extending into surrounding regions not
occupied by ATP, such as the hydrophobic back pocket to achieve target selectivity. The
size of this pocket is mainly determined by a residue called gatekeeper which is not
conserved [12]. About three-quarters of kinases have a bulky side chain at this position
(methionine, leucine or phenylalanine) and, thus, a relatively small back pocket. Crystal
structures of TgCDPK1 revealed that this enzyme possesses a gatekeeper of particularly
small side chain, a glycine residue, which is extremely rare [13] and permits access to an
enlarged back pocket, which can be exploited to develop selective bulky inhibitors with
large hydrophobic substituents [14].
In a preliminary work, we performed an in vitro screening of our chemical library on T.
gondii, allowing the identification of imidazo[1,2-b]pyridazine 1 as a new lead compound
(Fig. 1). This compound was able to inhibit the TgCDPK1 enzymatic activity at low
nanomolar concentrations.
Docking studies, using program GOLD (GOLD version 4.0; CCDC, Cambridge, UK),
were performed with the lead compound 1 into the ATP pocket of TgCDPK1 (3T3V.pdb)

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(Fig. 2). Data analysis suggested that this molecule can make a key hydrogen-bond
interaction between the pyridin-4-yl moiety and the ATP hinge residue Tyr131, while the
3-methoxy group is oriented towards the large hydrophobic pocket, adjacent to the rare
glycine gatekeeper position. Additionally, the protonated N-methylpiperazine solubilizing
group, which is partially exposed to the solvent, can form a salt bridge with the
carboxylate side chain of Glu135. These studies demonstrated interesting interactions
between compound 1 and several amino acids of the ATP binding site of TgCDPK1,
orienting the R substituent inside the back pocket.
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This molecule was however part of a drug discovery program concerning new anti-
CK1δ/ε compounds [15], suggesting that its selectivity profile has to be improved. In this
purpose, twenty new compounds were synthesized, exploiting the unique sequence of
TgCDPK1 ATP-binding cleft to probe the back pocket. We report herein the design,
synthesis, structure-activity relationships (SAR) and in vitro evaluations of this new class
of imidazo[1,2-b]pyridazine derivatives.
2. Chemistry
The preparation of imidazo[1,2-b]pyridazines 16a-p was performed as shown in scheme
6 by condensation between 3-amino-6-iodopyridazine 3 and the appropriate α-
halogenocarbonyl compounds 12a-m obtained by bromination of the corresponding
ketones 5a-d, 7, 10a-b and 11a-d.
The synthesis of 3-amino-6-iodopyridazine 3 was achieved in two steps from the
commercial 3,6-dichloropyridazine according to literature procedures [16,17] (Scheme
1). First, the 3,6-dichloropyridazine was aminated in ammonium hydroxide under
pressure to give compound 2, which was then converted into the corresponding iodide
derivative 3, using hydriodic acid.
Access to ketones was performed in parallel using different synthetic pathways (Schemes
2, 3, 4 and 5).
Ketones 5a-d were prepared according to the Grignard reaction by addition of
methylmagnesium bromide to a commercially available aldehyde in anhydrous THF, to

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give corresponding alcohols 4a-d, which were then oxidised using Dess-Martin
periodinane reagent in dichloromethane [18,19] (Scheme 2).
For the synthesis of ketone 7, the Weinreb amide 6 was prepared from the commercial
benzothiazole-6-carboxylic acid, and was reacted with methylmagnesium bromide,
affording the desired ketone [20] (Scheme 3).
The synthesis of naphthylketones 10a-b was achieved in three steps using the recently
described protocol of Kišić et al. [21] (Scheme 4). Thus 6-hydroxy-1-naphthoic acid was
treated with the appropriate alkyl iodide in acetone at 50 °C to give alkylated products 8a
and 8b which were then saponified using a 85% in water solution of potassium hydroxide
in THF at 100 °C, leading to the corresponding carboxylic acids 9a-b. Action of
methyllithium on these intermediates in anhydrous diethyl ether at 0 °C afforded the
desired ketones 10a-b.
Finally ketones 11a-d were prepared from commercial 1-(5-bromothien-2-yl)ethanone or
1-(3-bromophenyl)ethanone via a Suzuki-Miyaura cross-coupling reaction under
microwave irradiation in classical conditions (1.5 eq of boronic acid, 2 mol% of
3 4 2 3 2
Pd(PPh ) , 2 eq of Na CO , in DME/H O) [22] (Scheme 5).
Bromination of ketones 5a-d, 7, 10a-b, 11a-d and of commercial 1-acetonaphthone and
4-phenylacetophenone was achieved using phenyltrimethylammonium tribromide in THF
to give the corresponding acyl bromides 12a-m [23] (Scheme 6). These intermediates
were then condensed with 3-amino-6-iodopyridazine 3 in refluxing n-butanol to yield
imidazo[1,2-b]pyridazines 13a-m [24].
Amination of the imidazo[1,2-b]pyridazine scaffold in position 6 was performed
according to two different methods. Prolonged heating of the compounds 13a-m at 170
°C with N-methylpiperazine in sealed tubes afforded derivatives 14a-m. The introduction
of 3,4,5-trimethylpiperazin-1-yl group was achieved according to a modified literature
procedure [25]. Thus, 1,2,6-trimethylpiperazine [26] was reacted with imidazo[1,2-
b]pyridazine 13a using diisopropylethylamine in 1,4-dioxane under microwave
irradiation to give compound 14n.
Iodination in position 3 of compounds 14a-n using iodine monochloride in chloroform
afforded derivatives 15a-n [27]. Finally access to the imidazo[1,2-b]pyridazines 16a-p

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was performed via a Suzuki-Miyaura cross-coupling reaction under microwave
irradiation [22].
The previous synthetic pathway permitted access to the majority of desired
imidazoazines. However, it requires the need of acyl bromide derivatives. Thus, for the
preparation of derivatives 24a-b, an alternative synthetic route, implying a triflate
intermediate, was developed according to a modified literature procedure previously
described in imidazo[1,2-a]pyridine series [28] (Scheme 7). In the first step, the amino
group of pyridazine 3 was protected with a tert-butyloxycarbonyl (Boc) group using di-
tert-butyldicarbonate in presence of triethylamine in dichloromethane to give derivative
17 which was aminated in position 6 with N-methylpiperazine and deprotected using
trifluoroacetic acid in dichloromethane to yield compound 19. The triflate intermediate
21 was then synthesized in two steps: compound 19 was reacted with ethyl bromoacetate
to give a crude mixture containing intermediate 20, which was subsequently treated with
N-phenyl-bis(trifluoromethanesulfonimide), leading to compound 21. This intermediate
was used for the next step without further purification due to its instability under
chromatography. Functionalization of the triflate by a Suzuki-Miyaura cross-coupling
reaction under microwave irradiation gave compounds 22a-b. As previously, an
iodination and a Suzuki-Miyaura cross-coupling reaction in position 3 afforded
compounds 24a-b.
Finally, compounds 29 and 33 were synthesized in order to complete the SAR studies
(Schemes 8 and 9). To explore the importance of the nitrogen of the pyridazine ring for
activity, an analogue of compound 16a in imidazo[1,2-a]pyridine series was synthesized
in four steps leading to compound 29 (Scheme 8): condensation between 2-amino-5-
iodopyridine 25 [29] and the acyl bromide derivative 12a in refluxing absolute ethanol
gave the imidazo[1,2-a]pyridine 26, which was afterwards aminated in position 6 using
3 4
Buchwald conditions (1.1 eq of N-methylpiperazine, 15 mol% of CuI, 2 eq of K PO in
ethylene glycol/isopropanol) leading to intermediate 27 [30]. Successive iodination and
Suzuki-Miyaura cross-coupling reaction in position 3 gave the desired imidazo[1,2-
a]pyridine 29.
Finally, to study influence of the substitution in position 6, an analogue of compound
16a, non-substituted in this position, was prepared. Compound 33 was thus obtained in

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four steps (Scheme 9): dehalogenation of compound 2, using palladium on carbon
catalyst under hydrogen at atmospheric pressure [31], gave derivative 30, which was then
condensed with intermediate 12a to afford compound 31. As previously, an iodination
followed by a Suzuki-Miyaura cross-coupling reaction in position 3, gave the desired
compound 33.
3. Results and discussion
3.1. Enzymatic Assays
3.1.1. TgCDPK1 enzymatic activity inhibition
Compounds were first evaluated for their efficacy to inhibit the in vitro enzymatic
activity of wild type TgCDPK1, using a luminescence-based kinase assay, according to a
slightly modified literature procedure [13].
We first decided to structurally vary the substitution patterns of the imidazo[1,2-
b]pyridazine scaffold of 1 in position 2, introducing bulky hydrophobic substituents
oriented towards the back pocket adjacent to the ATP binding site, trying to probe this
pocket (Table 1). As shown in Table 1, a large variety of aryl substituents can be
accommodated in this area. Indeed, 10 compounds from this series demonstrated potent
inhibition of TgCDPK1, with IC50 < 1 µM. Furthermore, two of them (16a and 16f)
displayed low nanomolar inhibition (IC50 of 14 nM and 8 nM, respectively). Replacement
of the methoxyphenyl group of lead compound 1, by a non-planar bicycle led to a loss of
activity in almost every case (Table 1, entries 1, 3, 4 and 5). Thus, compounds 16b, 16c
and 16d were less potent (16.5-fold, 137-fold and 12-fold, respectively) than compound 1
(IC50 of 9 nM). However, compound 16a bearing a benzo[d][1,3]dioxol-4-yl moiety in
position 2 maintained low nanomolar inhibition of TgCDPK1 enzymatic activity with an
IC of 14 nM. Interestingly, introduction of a supplementary methylene into the dioxolyl
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ring of 16a led to a drastic loss of potency (compound 16c, IC of 1.23 µM), suggesting
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that an increased steric bulk at this position was not favorable for activity and should
probably clash with residues of the back pocket. It also appears that orientation of the
dioxol ring has important repercussions on compounds activity, since passage from

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ortho/meta (16a) to meta/para (16b) position reduced significantly the inhibitory potency
(16b, IC of 0.15 µM). Replacement of the dioxol ring of compound 16b by a thiazol led
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group in position 2, respecting the same orientation as the benzodioxolyl substituent of
16a (Table 1, entries 7, 8 and 9). While compound 16f maintained a very high potency
level, with an IC of 8 nM, we observed a decreased of activity when a methoxy (16g) or
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respectively. This activity reduction was proportional to the substituent size, suggesting
again that too bulky groups at this position should induce steric clashes within the back
pocket. Finally, replacement of the methoxy moiety in meta position of compound 1
phenyl ring by phenyl (16j), thien-2-yl (16k), thien-3-yl (16l), or morpholine-4-carbonyl
(24b) groups led to a significant loss of potency (Table 1, entries 11, 12, 13 and 16). Para
substitution of this phenyl ring was also not favorable for the activity, as shown by
derivatives 16i and 24a, which were respectively 8-fold and > 2-fold less active than their
“meta” analogues 16j and 24b, respectively. As previously, these data suggested that
orientation of the substituents of the phenyl ring in position 2 of imidazo[1,2-
b]pyridazine scaffold had important repercussions on inhibitors potency.
To get closer insight into the potential binding mode of our target structures within the
ATP binding pocket of TgCDPK1, we decided, in a second time, to pharmacomodulate
one of our best inhibitors, compound 16a (Table 2). Docking studies carried out on lead
compound 1 suggested that the pyridin-4-yl moiety in position 3 of the imidazo[1,2-
b]pyridazine scaffold realized a key hydrogen-bond interaction into the hinge region of
the TgCDPK1 ATP binding pocket. To confirm this hypothesis, we prepared two
analogues of 16a bearing a pyridin-3-yl (16o) or a phenyl (16p) group in position 3 of the
imidazopyridazine scaffold. As expected, both analogues displayed a dramatic loss of
TgCDPK1 enzymatic activity inhibition with IC about 400-fold lower.
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6 of the imidazopyridazine ring of 1 could be able to form a salt bridge with a
carboxylate side chain of the TgCDPK1 ATP binding pocket. Not surprisingly, deletion
of the entire N-methylpiperazinyl moiety of compound 16a (compound 33) markedly

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reduced TgCDPK1 activity, with a decrease in potency of 16-fold. Moreover, addition of
two methyl on N-methylpiperazine group of 16a also led to a decrease of inhibitory
activity (16n, IC50 45-fold lower), suggesting that the N-methylpiperazine ring would
enhance potency of the designed inhibitors against TgCDPK1 by increasing molecular
interactions with residues of the TgCDPK1 ATP binding pocket. These interactions could
be hindered in the case of the trimethylpiperazine analogue 16n, especially as this
compound is a mixture of several isomers.
Finally, we decided to explore the role of the nitrogen in position 5 of the pyridazine ring.
While docking studies did not highlight any role of this nitrogen, we observed a drastic
loss of activity with compound 29 (IC50 61-fold lower), analogue of 16a in series
imidazo[1,2-a]pyridine.
3.1.2. Selectivity over a panel of protein kinases
To get a selectivity profile of the synthesized inhibitors, compounds were further
evaluated in an expanded panel of mammalian serine/threonine protein kinases
containing bulky gatekeeper residues like phenylalanine (HsCDK5/p25, HsHaspin),
leucine (SscGSK3) or methionine (SscCK1δ/ε) or a small gatekeeper residue like
threonine (HsRIPK3). Inhibition values were determined using a radioactive kinase
assay. As expected, all TgCDPK1 inhibitors did not potently inhibit kinases with bulky
phenylalanine or leucine gatekeepers. Indeed, similar inhibition trends were observed for
HsCDK5/p25 and HsHaspin, with IC > 10 µM in nearly every case and for SscGSK3
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0
(IC50 > 2 µM) (Tables 1 and 2). Importantly, we observed > 380-fold and > 1000-fold
differences between IC50 values for TgCDPK1 over these mammalian kinases for our
lead inhibitors 16a and 16f, respectively.
Concerning SscCK1δ/ε, if most of the imidazoazines tested have limited activity against
this kinase, four of them still displayed submicromolar inhibition (Table 1, entries 2, 3, 6
and 7). Our leads 16a and 16f exhibited nevertheless IC values at least 25-fold higher
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for SscCK1δ/ε than for TgCDPK1.
Not surprisingly, compounds inhibition of the small gatekeeper enzyme HsRIPK3 was
sometimes slightly increased (Table 1, entries 7-9 and 11-13). However, 16a and 16f
were again > 100-fold selective over this kinase.

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3.2. Docking studies
The binding pose found by the docking program GOLD (GOLD version 4.0; CCDC,
Cambridge, UK) for lead compound 16a into the ATP pocket of TgCDPK1, CK1ε and
RIPK3 is shown in Figure 3. Overall selectivity for TgCDPK1 over CK1ε and RIPK3 can
be visually compared according to the sizes of each hydrophobic back pockets. As
expected for TgCDPK1, this molecule displayed a similar binding mode than compound
1 and shared similar interactions. In particularly, the sterically encumbered benzodioxolyl
group was oriented towards the large hydrophobic back pocket lined by the small sized
gatekeeper residue Gly128 (Figure 3A). Interestingly, this molecule was also able to
adopt a similar conformation in both CK1ε and RIPK3 (Figures 3B and 3C), despite the
presence of a methionine and a threonine gatekeeper residue, respectively. Even if
threonine is another small sized gatekeeper, methionine has a known flexible side chain
that could allow compounds to still access and occupy the back pocket according to an
induced-fit type of mechanism [32]. The presence of the charged residue Glu135 in the
solvent exposed area of TgCDPK1 compared to the corresponding Ser88 and Ser102 in
both CK1ε and RIPK3 enzymes, may also play an important role in binding and
positioning inhibitors in the cavity. Thus, the favorable salt-bridge formed between the
protonated N-methylpiperazine of 16a and this glutamate residue could partially
contribute to the observed differences in the activity profiles.
3.3. In vitro T. gondii invasion/proliferation inhibition
We then tested inhibitors for their ability to inhibit invasion of T. gondii tachyzoites into
human foreskin fibroblasts (HFF) host cells. This evaluation was made indirectly by the
measure of parasite growth inhibition, according to a colorimetric microtiter assay,
previously described [33], using a recombinant T. gondii strain expressing bacterial β-
galactosidase. Compounds that displayed T. gondii growth inhibition with EC50 > 5 µM
were considered inactive. Over the 20 new inhibitors tested, 15 compounds demonstrated
a significant anti-parasite activity against T. gondii (EC < 5 µM), with a submicromolar
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potency for 7 of them (Tables 1 and 2). In a general manner, a strong correlation was
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potency. Indeed, compounds with a good level of activity on TgCDPK1 (IC50 of 0.11 to
1.23 µM) were also effective on T. gondii growth in vitro with EC values ranged from
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nM and 8 nM, respectively), were also the most potent anti-T. gondii compounds (EC50
of 0.10 ± 0.05 µM and 0.07 ± 0.03 µM, respectively), with a higher anti-T. gondii activity
than the reference drug pyrimethamine (EC50 of 0.34 ± 0.02 µM). In the same way,
compounds with a very low level of potency (16i, 16m, 16o, 24a and 24b) in the
TgCDPK1 enzymatic test demonstrated a poor profile in the cellular assay with EC > 3
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µM in every case (Table 1, entries 10, 14, 15 and 16 and Table 2, entry 2). Compound
16p was an exception: in spite of a very weak activity on TgCDPK1, it still exhibited a
moderate anti-T. gondii activity (Table 2, entry 3), suggesting that this inhibitor could act
via another target. Another remarkable exception was observed with the imidazo[1,2-
a]pyridine derivative 29, which displayed an IC50 of 0.85 µM on TgCDPK1 but was
inactive against T. gondii (Table 2, entry 6).
3.4. In vitro non-specific cytotoxicity
To prove that compounds inhibition of T. gondii growth in vitro was not the result of a
non-specific toxicity effect on host cells, we finally determine the toxicity of our
inhibitors on HFF, using a previously described Alamar Blue reduction assay designed to
measure quantitatively the cell proliferation [33] (Tables 1 and 2). Interestingly, the two
most potent compounds 16a and 16f demonstrated low toxicity to these mammalian cells
(CC50 of 42.5 ± 7.9 and 21.3 ± 2.6, respectively), suggesting a specific effect of these
compounds and making them good candidates for further in vivo investigations. Not
surprisingly, a cytotoxic effect (CC50 < 10 µM) was observed for compounds with LogP
values close to 5.0 (data not shown) (Table 1, entries 9 and 12-14 and Table 2, entry 33).
Two exceptions were however observed with compounds 16d and 24b, which were
cytotoxic against HFF despite a LogP value < 3.0 (data not shown).

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4. Conclusion
In summary, using structure-based design, we have developed a very promising
imidazo[1,2-b]pyridazine series, with highly potent anti-T. gondii activity, targeting
TgCDPK1. Among the 20 compounds synthesized, 14 significantly blocked TgCDPK1
(IC < 1 µM) and 7 of them were able to inhibit T. gondii tachyzoites invasion in
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human fibroblasts with EC50 values below 1 µM. The strong correlation between
enzyme inhibition and growth inhibition suggests that TgCDPK1 is probably the main
target of these inhibitors. Two lead compounds, 16a and 16f, were then identified. They
demonstrated low nanomolar potency on TgCDPK1 and a good selectivity profile
against mammalian kinases with gatekeeper residues of different sizes. In vitro studies
confirmed their interest with an anti-T. gondii activity at submicromolar to nanomolar
concentrations and a low toxicity to mammalian cells. Their high therapeutic index
make them good candidates for further in vivo investigations on a murine model of
acute toxoplasmosis.
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5. Experimental section
5.1. General remarks
All solvents were anhydrous reagents from commercial sources. Unless otherwise
noted, all chemicals and reagents were obtained commercially and used without
purification. Microwave heating was carried out with a single-mode Discover (CEM)
unit. Melting points (Mp) were determined on a Stuart capillary apparatus and are
uncorrected. All MS spectra were acquired using a Thermo Scientific Q Exactive
quadrupole-orbitrap mass spectrometer. Parameters of ion source were set as follows:
ion source electrospray, scanning mode positive and/or negative, ion source temperature
200 °C, capillary voltage 3.50 kV, capillary temperature 320 °C, probe temperature 100
1
13
°C. NMR spectra were recorded at 300 MHz ( H) or 75 MHz ( C) on a Bruker Avance
(300 MHz) spectrometer. The chemical shifts are reported in parts per million (ppm, δ)
relative to residual deuterated solvent peaks. The abbreviations s = singlet, d = doublet, t
= triplet, q = quadruplet, m = multiplet and bs = broad signal were used throughout. For

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the peak assignation, the following abbreviations were used: Pyr = pyridinyl; the
position of the considered proton on the corresponding heterocycle was indicated by its
number after a dash; proton numbering was assigned according to IUPAC
nomenclature. The possible inversion of two values in the NMR spectra is expressed by
an asterisk. Known compounds were prepared according to literature procedures: 2-(3-
methoxyphenyl)-6-(4-methylpiperazin-1-yl)-3-(pyridin-4-yl)imidazo[1,2-b]pyridazine 1
[15], 3,4,5-trimethylpiperazine [26] and 2-amino-5-iodopyridine 25 [29]. Due to limited
capacity of microwave vials or sealed tubes, some reactions were equally divided into n
vials (n will be defined for each concerned reaction).
5.2. Chemistry
5.2.1. 3-amino-6-chloropyridazine (2).
In a Parr vessel was introduced 2,6-dichloropyridazine (20 g, 130 mmol) in an ammonia
solution 30% (200 mL). The mixture was stirred at 150 °C for 4 h and then cooled at
room temperature overnight. The resulting precipitate was filtered and washed with
water to give compound 2 (13.5 g, 77%) as a beige solid.
1
Mp 221.1 °C. H NMR (300 MHz, DMSO-d ) δ 7.36 (d, 1H, J = 9.3 Hz, H5), 6.83 (d,
6
13
1H, J = 9.3 Hz, H4), 6.63 (bs, 2H, NH ). C NMR (75 MHz, DMSO-d6) δ 160.3,
2
145.0, 129.0, 117.6.
5.2.2. 3-amino-6-iodopyridazine (3).
A mixture of compound 2 (11.30 g, 87 mmol) in a 57% hydriodic acid solution (115
mL, 0.87 mol) was stirred magnetically at 110 °C for 18 h. After cooling, a 10 M
sodium hydroxide solution was added to obtain a slightly basic solution and water was
then added. The resulting mixture was cooled to room temperature and stirred for an
additional hour. The resulting precipitate was filtered and washed with water to obtain
derivative 3 (14.58 g, 76%) as a beige solid.
1
Mp 160.5 °C. H NMR (300 MHz, DMSO-d ) δ 7.57 (d, 1H, J = 9.3 Hz, H5), 6.68 (bs,
6
1
3
2H, NH ), 6.60 (d, 1H, J = 9.3 Hz, H4). C NMR (75 MHz, DMSO-d ) δ 159.0, 138.1,
2
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5.2.3. 1-(benzo[d][1,3]dioxol-4-yl)ethanol (4a).
Method A: to a solution of 3 M methylmagnesium bromide (8.33 mL, 24.98 mmol) in
anhydrous THF (25 mL) stirred at -10 °C under argon was added dropwise a solution of
benzo[d][1,3]dioxol-4-carbaldehyde (2.5 g, 16.65 mmol) in anhydrous THF (25 mL).
The mixture was stirred at -10 °C for 3 h, quenched with a cold saturated solution of
ammonium chloride (100 mL), and extracted with Et O. The combined organic layers
2
were dried over MgSO , filtered, and evaporated under reduced pressure. Purification
4
by silica column chromatography using CH Cl as eluent gave the desired alcohol 4a
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(1.76 g, 64%) as a pale yellow oil.
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H NMR (300 MHz, CDCl ) δ 6.89-6.75 (m, 3H, H5, H6, H7), 5.97 (dd, 2H, J = 3.6,
3
1.2 Hz, CH ), 5.00 (qd, 1H, J = 6.3, 4.8, CHOH), 2.13 (d, 1H, J = 4.8 Hz, OH), 1.53 (d,
2
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3
3H, J = 6.3 Hz, CH ). C NMR (75 MHz, CDCl ) δ 147.3, 143.9, 127.2, 121.7, 118.7,
3
3
107.7, 100.9, 66.2, 23.3.
5.2.4. 1-(benzo[d][1,3]dioxol-5-yl)ethanol (4b).
The title compound was synthesized according to the general method A from
methylmagnesium bromide (16.65 mL, 49.96 mmol) in anhydrous THF (50 mL) and
benzo[d][1,3]dioxol-5-carbaldehyde (5.0 g, 33.30 mmol) in anhydrous THF (50 mL). A
saturated solution of ammonium chloride (200 mL) was used to quench the reaction.
Compound 4b was obtained (4.54 g, 82%) as a yellow oil.
1
H NMR (300 MHz, CDCl ) δ 6.90 (s, 1H, H4), 6.84-6.76 (m, 2H, H6, H7), 5.95 (s, 2H,
3
CH ), 4.82 (q, 1H, J = 6.3 Hz, CHOH), 1.82 (bs, 1H, OH), 1.46 (d, 3H, J = 6.3 Hz,
2
1
3
CH ). C NMR (75 MHz, CDCl ) δ 147.9, 146.8, 138.1, 119.8, 108.0, 106.4, 100.9,
3
3
74.1, 24.7.
5.2.5. 1-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)ethanol (4c).
The title compound was synthesized according to the general method A from
methylmagnesium bromide (3.08 mL, 9.23 mmol) in anhydrous THF (10 mL) and 2,3-
dihydrobenzo[b][1,4]dioxin-5-carbaldehyde (1.01 g, 6.15 mmol) in anhydrous THF (10
mL). A saturated solution of ammonium chloride (100 mL) was used to quench the
reaction. Compound 4c was obtained (708 mg, 64%) as a yellow oil.

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H NMR (300 MHz, CDCl ) δ 6.93 (dd, 1H, J = 6.9, 2.1 Hz, H6*), 6.81 (dd, 1H, J =
3
8.1, 6.9 Hz, H7), 6.77 (dd, 1H, J = 8.1, 2.1 Hz, H8*), 5.06 (qd, 1H, J = 6.3, 4.5 Hz,
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6.3 Hz, CH ). C NMR (75 MHz, CDCl ) δ 143.4, 140.6, 134.0, 121.1, 118.0, 116.3,
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65.9, 64.3, 64.1, 23.0.
5.2.6. 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethanol (4d).
The title compound was synthesized according to the general method A from
methylmagnesium bromide (6.66 mL, 19.98 mmol) in anhydrous THF (20 mL) and 2,3-
dihydrobenzo[b][1,4]dioxine-6-carbaldehyde (2.0 g, 12.18 mmol) in anhydrous THF
(20 mL). A saturated solution of ammonium chloride (200 mL) was used to quench the
reaction. Compound 4d was obtained (1.82 g, 83%) as a yellow oil.
1
H NMR (300 MHz, CDCl ) δ 6.87-6.78 (m, 3H, H5, H7, H8), 4.72 (qd, 1H, J = 6.3,
3
2.4 Hz, CHOH), 4.19 (s, 4H, 2×CH O), 2.74 (bs, 1H, OH), 1.41 (dd, 3H, J = 6.3, 1.5
2
1
3
Hz, CH ). C NMR (75 MHz, CDCl ) δ 143.4, 142.8, 139.3, 118.4, 117.2, 114.4, 69.9,
3
3
64.4, 64.3, 24.9.
5.2.7. 1-(benzo[d][1,3]dioxol-4-yl)ethanone (5a).
Method B: to a solution of compound 4a (1.65 g, 9.94 mmol) in anhydrous CH Cl (100
2
2
mL) stirred at 0 °C under argon was added Dess-Martin reagent (5.06 g, 11.93 mmol).
The mixture was allowed to warm to room temperature after 10 min and stirred for 16 h.
The reaction was quenched with a saturated sodium bicarbonate solution and salts were
filtered off. The filtrate was extracted with CH Cl The combined organic layers were
2
2.
dried over MgSO , filtered and evaporated under reduced pressure. Purification by silica
4
column chromatography using CH Cl as eluent gave the desired ketone 5a (1.42 g,
2
2
87%) as a pale yellow solid.
1
Mp 93.2 °C. H NMR (300 MHz, CDCl ) δ 7.37 (d, 1H, J = 8.1 Hz, H5), 6.98 (d, 1H, J
3
= 7.5 Hz, H7), 6.88 (dd, 1H, J = 8.1, 7.5 Hz, H6), 6.10 (s, 2H, CH ), 2.61 (s, 3H, CH ).
2
3
1
3
C NMR (75 MHz, CDCl ) δ 195.5, 148.6, 148.0, 121.4, 121.2, 120.3, 112.5, 101.5,
3
30.2.
5.2.8. 1-(benzo[d][1,3]dioxol-5-yl)ethanone (5b).

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The title compound was synthesized according to the general method B from compound
4b (4.54 g, 27.35 mmol) and Dess-Martin reagent (13.92 g, 32.82 mmol) in anhydrous
CH Cl (200 mL). Compound 5b was obtained (3.77 g, 84%) as a yellow solid.
2
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Mp 84.4 °C. H NMR (300 MHz, CDCl ) δ 7.54 (dd, 1H, J = 8.1, 1.8 Hz, H6), 7.42 (d,
3
1H, J = 1.8 Hz, H4), 6.83 (d, 1H, J = 8.1 Hz, H7), 6.03 (s, 2H, CH ), 2.53 (s, 3H, CH ).
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C NMR (75 MHz, CDCl ) δ 196.1, 151.7, 148.1, 132.0, 124.7, 107.9, 107.7, 101.8,
3
26.4.
5.2.9. 1-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)ethanone (5c).
The title compound was synthesized according to the general method B from compound
4c (708 mg, 3.93 mmol) and Dess-Martin reagent (2.0 g, 4.72 mmol) in anhydrous
CH Cl (40 mL). Compound 5c was obtained (651 mg, 93%) as a pale yellow solid.
2
2
1
Mp 135.5 °C. H NMR (300 MHz, CDCl ) δ 7.18 (dd, 1H, J = 7.8, 1.5 Hz, H6), 6.89
3
(dd, 1H, J = 8.1, 1.5 Hz, H8), 6.74 (dd, 1H, J = 8.1, 7.8 Hz, H7), 4.24 (m, 2H, CH O*),
2
13
4.17 (m, 2H, CH O*), 2.49 (s, 3H, CH ). C NMR (75 MHz, CDCl ) δ 198.2, 143.6,
2
3
3
143.3, 127.9, 121.7, 120.9, 120.2, 64.0, 63.4, 31.3.
5.2.10. 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethanone (5d).
The title compound was synthesized according to the general method B from compound
4d (1.82 g, 10.13 mmol) and Dess-Martin reagent (5.16 g, 12.16 mmol) in anhydrous
CH Cl (100 mL). Compound 5d was obtained (1.47 g, 82%) as a pale yellow solid.
2
2
1
Mp 83.7 °C. H NMR (300 MHz, CDCl ) δ 7.52-7.48 (m, 2H, H5, H8), 6.91 (dd, 1H, J
3
1
3
= 9.0, 3.0 Hz, H7), 4.35-4.28 (m, 4H, 2×CH O), 2.54 (s, 3H, CH ). C NMR (75 MHz,
2
3
CDCl ) δ 196.7, 148.0, 143.2, 131.1, 122.5, 117.8, 117.2, 64.7, 64.1, 26.4.
3
5.2.11. N-methoxy-N-methylbenzo[d]thiazole-6-carboxamide (6).
To a solution of benzo[d]thiazole-6-carboxylic acid (2.5 g, 13.95 mmol) in anhydrous
CH Cl (30 mL) was added triethylamine (1.95 mL, 13.95 mmol). The mixture was
2
2
stirred at 10 °C and a solution of ethyl chloroformate (1.33 mL, 13.95 mmol) in CH Cl2
2
(3 mL) was added dropwise. The resulting solution was stirred at 10 °C for an hour
before addition of N-methoxymethylamine (1.36 g, 22.32 mmol) and triethylamine
(1.95 mL, 13.95 mmol). The mixture was stirred 1 h 30 at 10 °C, quenched with water,

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and extracted with CH Cl . The combined organic layers were dried over MgSO ,
2
2
4
filtered, and evaporated under reduced pressure. Purification of the residue was
performed by alumina column chromatography using CH Cl as eluent to give
2
2
compound 6 (1.22 g, 39%) as a pale yellow oil.
1
H NMR (300 MHz, CDCl ) δ 9.10 (s, 1H, CHS), 8.35 (d, 1H, J = 1.5 Hz, H7), 8.14 (d,
3
1H, J = 8.7 Hz, H4), 7.86 (dd, 1H, J = 8.7, 1.5 Hz, H5), 3.55 (s, 3H, CH O), 3.41 (s,
3
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3H, CH N). C NMR (75 MHz, CDCl ) δ 169.0, 156.1, 154.4, 133.4, 131.2, 126.4,
3
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123.0, 122.6, 61.1, 33.7.
5.2.12. 1-(benzo[d]thiazol-6-yl)ethanone (7).
The title compound was synthesized according to the general method A from
methylmagnesium bromide (2.41 mL, 7.23 mmol) in anhydrous THF (10 mL) and a
solution of compound 6 (1.07 g, 4.82 mmol) in anhydrous THF (10 mL). The mixture
was stirred at -10 °C for 30 min, warmed to room temperature, and stirred for 16 h. A
purification by alumina column chromatography using petroleum ether/CH Cl (50:50
2
2
v/v) as eluent gave compound 7 (590 mg, 69%) as a yellow solid.
1
Mp 98.6 °C. H NMR (300 MHz, CDCl ) δ 9.17 (s, 1H, CHS), 8.61 (d, 1H, J = 0.9 Hz,
3
H7), 8.19 (d, 1H, J = 8.7 Hz, H4), 8.11 (dd, 1H, J = 8.7, 0.9 Hz, H5), 2.71 (s, 3H, CH3).
1
3
C NMR (75 MHz, CDCl ) δ 197.0, 157.6, 155.9, 134.4, 134.1, 126.2, 123.6, 122.9,
3
26.9.
5.2.13. Methyl 6-methoxy-1-naphthoate (8a).
Method C: to a solution of 6-hydroxy-1-naphthoic acid (1.54 g, 8.19 mmol) and
potassium carbonate (3.39 g, 24.57 mmol) in acetone (20 mL) was added slowly
iodomethane (1.52 ml, 24.57 mmol). The mixture was stirred at 50 °C for 16 h and
cooled to room temperature. Salts were then filtered and washed with acetone. The
filtrate was evaporated and the resulting residue was suspended in water and extracted
with CH Cl . The combined organic layers were then dried over MgSO , filtered, and
2
2
4
evaporated under reduced pressure to obtain the desired ester 8a (1.70 g, 96%) as a light
brown oil.
1
H NMR (300 MHz, CDCl ) δ 8.85 (d, 1H, J = 9.6 Hz, H8), 8.04 (dd, 1H, J = 8.7, 1.2
3
Hz, H2), 7.91 (dd, 1H, J = 7.2, 1.2 Hz, H4), 7.46 (dd, 1H, J = 8.7, 7.2 Hz, H3), 7.28

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(dd, 1H, J = 9.6, 2.7 Hz, H7), 7.16 (d, 1H, J = 2.7 Hz, H5), 4.00 (s, 3H, CH OCO), 3.93
3
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3
(s, 3H, CH O). C NMR (75 MHz, CDCl ) δ 168.1, 157.5, 135.3, 132.1, 127.8, 127.4,
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126.9, 126.8, 125.0, 120.3, 106.3, 55.2, 52.1.
5.2.14. Methyl 6-ethoxy-1-naphthoate (8b).
The title compound was synthesized according to the general method C from 6-
hydroxy-1-naphthoic acid (2.00 g, 10.63 mmol), potassium carbonate (4.40 g, 31.88
mmol) and iodoethane (2.55 ml, 31.88 mmol) in acetone (25 mL). Compound 8b was
obtained (2.07 g, 80%) as a yellow solid.
1
Mp 47.5 °C. H NMR (300 MHz, CDCl ) δ 8.84 (d, 1H, J = 9.3 Hz, H8), 8.03 (dd, 1H,
3
J = 7.2, 1.2 Hz, H2), 7.88 (dd, 1H, J = 8.4, 1.2 Hz, H4), 7.45 (dd, 1H, J = 8.4, 7.2 Hz,
H3), 7.28 (dd, 1H, J = 9.3, 2.7 Hz, H7), 7.15 (d, 1H, J = 2.7 Hz, H5), 4.47 (q, 2H, J =
7.2 Hz, CH OCO), 4.16 (q, 2H, J = 7.2 Hz, CH O), 1.50 (t, 3H, J = 7.2 Hz,
2
2
1
3
CH CH O*), 1.47 (t, 3H, J = 7.2 Hz, CH CH OCO*). C NMR (75 MHz, CDCl ) δ
3
2
3
2
3
167.7, 156.8, 135.3, 131.9, 127.6, 127.4, 127.3, 126.7, 125.0, 120.5, 107.1, 63.4, 61.0,
14.7, 14.3.
5.2.15. 6-methoxy-1-naphthoic acid (9a).
Method D: to a solution of compound 8a (1.70 g, 7.87 mmol) in THF (6 mL) was added
a 85% aqueous potassium hydroxide solution (3 mL). The mixture was stirred at 100 °C
for 4 h, cooled to room temperature, and acidified with a 1 M HCl solution. The
resulting precipitate was filtered and washed with water to afford the desired acid 9a
(1.51 g, 95%) as a-pink solid.
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Mp 95.3 °C. H NMR (300 MHz, DMSO-d ) δ 8.77 (d, 1H, J = 9.3 Hz, H8), 8.04 (dd,
6
1H, J = 8.1, 1.2 Hz, H2), 7.97 (dd, 1H, J = 8.4, 1.2 Hz, H4), 7.53 (dd, 1H, J = 8.4, 8.1
Hz, H3), 7.42 (d, 1H, J = 2.7 Hz, H5), 7.28 (dd, 1H, J = 9.3, 2.7 Hz, H7), 3.89 (s, 3H,
13
CH ). C NMR (75 MHz, DMSO-d ) δ 168.8, 157.1, 135.2, 131.9, 127.7, 127.5, 127.2,
3
6
126.1, 125.5, 120.0, 106.7, 55.3.
5.2.16. 6-ethoxy-1-naphthoic acid (9b).

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The title compound was synthesized according to the general method D from compound
8b (2.05 g, 8.40 mmol) in THF (7 mL) using a 85% aqueous potassium hydroxide
solution (3.2 mL). Compound 9b was obtained (1.71 g, 94%) as a pale yellow solid.
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Mp 176.9 °C. H NMR (300 MHz, DMSO-d ) δ 8.63 (d, 1H, J = 9.6 Hz, H8), 7.90 (dd,
6
1H, J = 8.1, 1.2 Hz, H2), 7.83 (dd, 1H, J = 7.2, 1.2 Hz, H4), 7.39 (dd, 1H, J = 8.1, 7.2
Hz, H3), 7.27 (d, 1H, J = 2.7 Hz, H5), 7.14 (dd, 1H, J = 9.6, 2.7 Hz, H7), 4.03 (q, 2H, J
13
= 7.2 Hz, CH ), 1.27 (t, 3H, J = 7.2 Hz, CH ). C NMR (75 MHz, DMSO-d ) δ 168.9,
2
3
6
156.4, 135.3, 131.9, 127.7, 127.5, 127.2, 126.1, 125.5, 120.3, 107.4, 63.3, 14.7.
5.2.17. 1-(6-methoxynaphth-1-yl)ethanone (10a).
Method E: to a solution of compound 9a (667 mg, 3.30 mmol) in anhydrous Et O (40
2
mL) stirred at 0 °C under argon was slowly added a 1.6 M methyllithium solution (8.26
mL, 13.20 mmol). The resulting solution was stirred at 0 °C for 16 h. After that, two
supplementary equivalents of the methyllithium solution were added and the mixture
was stirred at 0 °C for additional 24 h. The reaction was quenched with a saturated
ammonium chloride solution and extracted with Et O. The combined organic layers
2
were dried over MgSO , filtered, and evaporated under reduced pressure. A purification
4
by silica column chromatography using CH Cl as eluent gave the desired ketone 10a
2
2
(324 mg, 49%) as an orange oil.
1
H NMR (300 MHz, CDCl ) δ 8.69 (d, 1H, J = 9.3 Hz, H8), 7.89 (dd, 1H, J = 8.4, 1.2
3
Hz, H2), 7.81 (dd, 1H, J = 7.5, 1.2 Hz, H4), 7.46 (dd, 1H, J = 8.4, 7.5 Hz, H3), 7.27
(dd, 1H, J = 9.3, 2.7 Hz, H7), 7.16 (d, 1H, J = 2.7 Hz, H5), 3.93 (s, 3H, CH O), 2.74 (s,
3
13
3H, CH CO). C NMR (75 MHz, CDCl ) δ 201.9, 157.6, 135.5, 135.2, 131.8, 127.6,
3
3
126.5, 125.5, 124.9, 120.4, 106.3, 55.2, 29.8.
5.2.18. 1-(6-ethoxynaphth-1-yl)ethanone (10b).
The title compound was synthesized according to the general method E from compound
9b (1.71 g, 7.92 mmol) in anhydrous Et O (95 mL) and a 1.6 M methyllithium solution
2
(19.80 mL, 31.66 mmol). Compound 10b was obtained (1.58 g, 93%) as a pale yellow
oil.
1
H NMR (300 MHz, CDCl ) δ 8.69 (d, 1H, J = 9.6 Hz, H8), 7.87 (dd, 1H, J = 8.4, 1.2
3
Hz, H2), 7.80 (dd, 1H, J = 7.2, 1.2 Hz, H4), 7.45 (dd, 1H, J = 8.4, 7.2 Hz, H3), 7.27

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(dd, 1H, J = 9.6, 2.7 Hz, H7), 7.15 (d, 1H, J = 2.7 Hz, H5), 4.16 (q, 2H, J = 6.9 Hz,
1
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CH CH ), 2.74 (s, 3H, CH CO), 1.49 (t, 3H, J = 6.9 Hz, CH CH ). C NMR (75 MHz,
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CDCl ) δ 201.9, 157.0, 135.5, 135.2, 131.8, 127.6, 126.5, 125.4, 124.8, 120.7, 107.1,
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63.4, 29.7, 14.7.
5.2.19. 1-([1,1’-biphenyl]-3-yl)ethanone (11a).
Method F: into four microwave vials were equally distributed 1-(3-
bromophenyl)ethanone (5 g, 25.12 mmol), phenylboronic acid (4.59 g, 37.68 mmol),
sodium carbonate (5.33 g, 50.24 mmol) and tetrakis(triphenylphosphine)palladium(0)
(0.58 g, 0.50 mmol) in 1,2-dimethoxyethane (20 mL) and water (10 mL). The reaction
mixture was stirred under microwave irradiation at 100 °C for 30 min. The resulting
mixture was taken up in water and extracted with CH Cl . The combined organic layers
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2
were dried over MgSO , filtered, and evaporated under reduced pressure. Purification
4
by alumina column chromatography using petroleum ether as eluent gave compound
11a (1.23 g, 25%) as a colourless oil.
1
H NMR (300 MHz, CDCl ) δ 8.20 (s, 1H, H2), 7.95 (d, 1H, J = 7.8 Hz, H4), 7.81 (d,
3
1
3
1H, J = 7.8 Hz, H6), 7.66-7.38 (m, 6H), 2.68 (s, 3H, CH ). C NMR (75 MHz, CDCl )
3
3
δ 198.1, 141.7, 140.1, 137.6, 131.7, 129.0, 128.9 (2×C), 127.8, 127.2 (3×C), 126.9,
26.7.
5.2.20. 1-(3-(thien-2-yl)phenyl)ethanone (11b).
The title compound was synthesized according to the general method F from 1-(3-
bromophenyl)ethanone (2 g, 10.05 mmol), thien-2-ylboronic acid (1.93 g, 15.07 mmol),
sodium carbonate (2.13 g, 20.10 mmol), Pd(PPh ) (0.23 g, 0.20 mmol) in 1,2-
3
4
dimethoxyethane (8 mL) and water (4 mL) equally distributed into 2 vials. The reaction
mixture was stirred under microwave irradiation at 100 °C for 1 h. A purification by
silica column chromatography using petroleum ether/CH Cl (50:50 v/v) as eluent gave
2
2
compound 11b (1.18 g, 58%) as a yellow oil.
1
H NMR (300 MHz, CDCl ) δ 8.19 (t, 1H, J = 1.8 Hz, H2), 7.86 (td, 1H, J = 7.8, 1.8
3
Hz, H6), 7.80 (td, 1H, J = 7.8, 1.8 Hz, H4), 7.48 (t, 1H, J = 7.8 Hz, H5), 7.40 (dd, 1H, J
= 4.8, 1.2 Hz, H5’), 7.33 (dd, 1H, J = 5.1, 1.2 Hz, H3’), 7.11 (dd, 1H, J = 5.1, 4.8 Hz,

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H4’), 2.65 (s, 3H, CH ). C NMR (75 MHz, CDCl ) δ 197.8, 143.1, 137.6, 134.9,
3
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130.3, 129.1, 128.1, 127.2, 125.5, 125.4, 123.8, 26.7.
5.2.21. 1-(3-(thien-3-yl)phenyl)ethanone (11c).
The title compound was synthesized according to the general method F from 1-(3-
bromophenyl)ethanone (2 g, 10.05 mmol), thien-3-ylboronic acid (1.93 g, 15.07 mmol),
sodium carbonate (2.13 g, 20.10 mmol), Pd(PPh ) (0.23 g, 0.20 mmol) in 1,2-
3
4
dimethoxyethane (8 mL) and water (4 mL) equally distributed into 2 vials. The reaction
mixture was stirred under microwave irradiation at 100 °C for 1 h. A purification by
silica column chromatography using petroleum ether/CH Cl (50:50 v/v) as eluent gave
2
2
compound 11c (1.38 g, 68%) as a yellow oil.
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H NMR (300 MHz, CDCl ) δ 8.19 (t, 1H, J = 1.8 Hz, H2), 7.87 (td, 1H, J = 7.8, 1.8
3
Hz, H6), 7.79 (td, 1H, J = 7.8, 1.8 Hz, H4), 7.53 (dd, 1H, J = 5.7, 1.8 Hz, H5’), 7.50 (t,
1
3
1H, J = 7.8 Hz, H5), 7.45-7.40 (m, 2H, H2’, H4’), 2.65 (s, 3H, CH ). C NMR (75
3
MHz, CDCl ) δ 198.0, 141.2, 137.6, 136.2, 130.9, 129.0, 127.0, 126.8, 126.1, 126.0,
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121.1, 26.7.
5.2.22. 1-(5-phenylthien-2-yl)ethanone (11d).
The title compound was synthesized according to method F from 1-(5-bromothien-2-
yl)ethanone (5 g, 24.39 mmol), phenylboronic acid (4.46 g, 36.58 mmol), sodium
carbonate (5.17 g, 48.78 mmol), Pd(PPh ) (0.56 g, 0.49 mmol) in 1,2-dimethoxyethane
3
4
(20 mL) and water (10 mL) equally distributed into 5 vials. The reaction mixture was
stirred under microwave irradiation for 1 h at 130 °C. A purification by silica column
chromatography using CH Cl as eluent gave compound 11d (4.93 g, 100%) as a yellow
2
2
solid.
1
Mp 115.8 °C. H NMR (300 MHz, CDCl ) δ 7.72-7.61 (m, 3H), 7.50-7.30 (m, 4H), 2.58
3
1
3
(s, 3H, CH ). C NMR (75 MHz, CDCl ) δ 190.6, 152.7, 143.0, 133.4, 133.3, 129.1
3
3
(2×C), 129.0, 126.2 (2×C), 123.9, 26.5.
5.2.23. 1-(benzo[d][1,3]dioxol-4-yl)-2-bromoethanone (12a).
Method G: a solution of compound 5a (210 mg, 1.28 mmol) in anhydrous THF (5 mL)
was stirred at 0 °C under argon before addition of phenyltrimethylammonium

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tribromide (481 mg, 1.28 mmol). The mixture was then stirred for 15 min at 0 °C and
warmed to room temperature. The reaction mixture was stirred for 16 h and evaporated
to dryness. The residue was suspended in water and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried over MgSO , filtered,
4
and evaporated under reduced pressure. A purification by silica column chromatography
using petroleum ether/CH Cl (9:1 v/v) as eluent gave the desired bromoethanone 12a
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(239 mg, 77%) as a white solid.
1
Mp 68.2 °C. H NMR (300 MHz, CDCl ) δ 7.43 (dd, 1H, J = 8.4, 1.2 Hz, H5), 7.02 (dd,
3
1H, J = 7.5, 1.2 Hz, H7), 6.91 (dd, 1H, J = 8.4, 7.5 Hz, H6), 6.12 (s, 2H, CH O), 4.50
2
1
3
(s, 2H, CH Br). C NMR (75 MHz, CDCl ) δ 188.2, 148.5, 147.8, 121.9, 121.6, 117.0,
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3
113.3, 101.8, 35.0.
5.2.24. 1-(benzo[d][1,3]dioxol-5-yl)-2-bromoethanone (12b).
The title compound was synthesized according to the general method G from compound
5b (3.50 g, 21.34 mmol) and phenyltrimethylammonium tribromide (8.02 g, 21.34
mmol) in anhydrous THF (60 mL). The reaction mixture was stirred for 5 h. Compound
12b was obtained (4.12 g, 79%) as a light pink solid.
1
Mp 93.6 °C. H NMR (300 MHz, CDCl ) δ 7.57 (dd, 1H, J = 8.4, 1.8 Hz, H6), 7.42 (d,
3
1H, J = 1.8 Hz, H4), 6.86 (d, 1H, J = 8.4 Hz, H7), 6.06 (s, 2H, CH O), 4.37 (s, 2H,
2
13
CH Br). C NMR (75 MHz, CDCl ) δ 189.4, 152.4, 148.3, 128.5, 125.5, 108.4, 108.0,
2
3
102.0, 30.7.
5.2.25. 2-bromo-1-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)ethanone (12c).
The title compound was synthesized according to the general method G from compound
5c (617 mg, 3.47 mmol) and phenyltrimethylammonium tribromide (1.30 g, 3.47 mmol)
in anhydrous THF (15 mL). The reaction mixture was stirred for 2 h 30. Compound 12c
was obtained (729 mg, 84%) as a yellow oil.
1
H NMR (300 MHz, CDCl ) δ 7.25 (dd, 1H, J = 7.8, 1.5 Hz, H6), 6.94 (dd, 1H, J = 7.8,
3
1.5 Hz, H8), 6.77 (t, 1H, J = 7.8 Hz, H7), 4.49 (s, 2H, CH Br), 4.29 (m, 2H, CH O*),
2
2
1
3
4.21 (m, 2H, CH O*). C NMR (75 MHz, CDCl ) δ 190.7, 143.6, 143.3, 124.3, 122.5,
2
3
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5.2.26. 2-bromo-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethanone (12d).
The title compound was synthesized according to the general method G from compound
5d (1.47 g, 8.65 mmol) and phenyltrimethylammonium tribromide (3.25 g, 8.65 mmol)
in anhydrous THF (30 mL). The reaction mixture was stirred for 16 h. Compound 12d
was obtained (604 mg, 28%) as a white solid.
1
Mp 119.5 °C. H NMR (300 MHz, CDCl ) δ 7.55-7.51 (m, 2H, H5, H8), 6.94 (dd, 1H, J
3
1
3
= 9.0, 2.1 Hz, H7), 4.39 (s, 2H, CH Br), 4.36-4.28 (m, 4H, 2×CH O). C NMR (75
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MHz, CDCl ) δ 189.8, 148.8, 143.5, 127.7, 123.2, 118.4, 117.5, 64.7, 64.1, 30.7.
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5.2.27. 1-(benzo[d]thiazol-6-yl)-2-bromoethanone (12e).
The title compound was synthesized according to the general method G from compound
7 (563 mg, 3.18 mmol) and phenyltrimethylammonium tribromide (1.20 g, 3.18 mmol)
in anhydrous THF (15 mL). The reaction mixture was stirred for 16 h. For purification
CH Cl was used as eluent. Compound 12e was obtained (300 mg, 37%) as a yellow
2
2
solid.
1
Mp > 320 °C (dec). H NMR (300 MHz, CDCl ) δ 9.22 (s, 1H, CHS), 8.68 (d, 1H, J =
3
1.8 Hz, H7), 8.23 (d, 1H, J = 8.7 Hz, H4), 8.14 (dd, 1H, J = 8.7, 1.8 Hz, H5), 4.54 (s,
1
3
2H, CH Br). C NMR (75 MHz, CDCl ) δ 190.5, 158.3, 156.4, 131.2 (2×C), 126.7,
2
3
123.9, 123.8, 30.6.
5.2.28. 2-bromo-1-(naphth-1-yl)ethanone (12f).
The title compound was synthesized according to the general method G from 1-
acetonaphthone (2.68 mL, 17.63 mmol) and phenyltrimethylammonium tribromide
(5.30 g, 14.10 mmol) in anhydrous THF (60 mL). The reaction mixture was stirred for
16 h. The crude compound 12f was obtained (3.83 g) as a yellow oil and used without
purification.
1
H NMR (300 MHz, CDCl ) δ 8.66 (dd, 1H, J = 8.7, 1.2 Hz, H8), 8.03 (d, 1H, J = 8.4
3
1
3
Hz, H2), 7.93-7.87 (m, 2H), 7.67-7.47 (m, 3H), 4.58 (s, 2H, CH Br). C NMR (75
2
MHz, CDCl ) δ 194.3, 135.4, 133.9, 132.2, 128.5 (4×C), 126.8, 125.6, 124.2, 33.8.
3
5.2.29. 2-bromo-1-(6-methoxynaphth-1-yl)ethanone (12g).

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The title compound was synthesized according to the general method G from compound
10a (525 mg, 2.63 mmol) and phenyltrimethylammonium tribromide (789 mg, 2.10
mmol) in anhydrous THF (12 mL). The reaction mixture was stirred for 5 h. Compound
12g was obtained (438 mg, 75%) as an orange oil.
1
H NMR (300 MHz, CDCl ) δ 8.57 (d, 1H, J = 9.6 Hz, H8), 7.85 (dd, 1H, J = 8.4, 1.2
3
Hz, H2), 7.69 (dd, 1H, J = 8.4, 1.2 Hz, H4), 7.38 (t, 1H, J = 8.4 Hz, H3), 7.24 (dd, 1H,
J = 9.6, 2.7 Hz, H7), 7.10 (d, 1H, J = 2.7 Hz, H5), 4.53 (s, 2H, CH ), 3.86 (s, 3H, CH ).
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3
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C NMR (75 MHz, CDCl ) δ 194.0, 157.6, 135.3, 132.5, 131.6, 127.0, 126.3, 125.6,
3
124.6, 120.7, 106.3, 55.0, 34.0.
5.2.30. 2-bromo-1-(6-ethoxynaphth-1-yl)ethanone (12h).
The title compound was synthesized according to the general method G from compound
10b (1.56 g, 7.29 mmol) and phenyltrimethylammonium tribromide (2.19 g, 5.83 mmol)
in anhydrous THF (35 mL). The reaction mixture was stirred for 5 h. For purification
petroleum ether was used as eluent. Compound 12h was obtained (1.73 g, 100%) as a
bright yellow oil.
1
H NMR (300 MHz, CDCl ) δ 8.56 (d, 1H, J = 9.3 Hz, H8), 7.92 (dd, 1H, J = 8.1, 1.2
3
Hz, H2), 7.78 (dd, 1H, J = 7.2, 1.2 Hz, H4), 7.47 (dd, 1H, J = 8.1, 7.2 Hz, H3), 7.28
(dd, 1H, J = 9.3, 2.7 Hz, H7), 7.16 (d, 1H, J = 2.7 Hz, H5), 4.57 (s, 2H, CH Br), 4.16
2
13
(q, 2H, J = 6.9 Hz, CH O), 1.50 (t, 3H, J = 6.9 Hz, CH ). C NMR (75 MHz, CDCl ) δ
2
3
3
194.3, 157.3, 135.6, 132.7, 132.1, 127.3, 126.3, 125.9, 124.7, 121.2, 107.2, 63.5, 33.9,
14.7.
5.2.31. 1-([1,1’-biphenyl]-4-yl)-2-bromoethanone (12i).
The title compound was synthesized according to the general method G from 4-
phenylacetophenone (5 g, 25.48 mmol) and phenyltrimethylammonium tribromide (9.58
g, 25.48 mmol) in anhydrous THF (80 mL). The reaction mixture was stirred for 16 h.
For purification petroleum ether was used as eluent. Compound 12i was obtained (7.00
g, 100%) as a white solid.
1
Mp 128.2 °C. H NMR (300 MHz, CDCl ) δ 8.07 (dd, 2H, J = 8.7, 2.1 Hz, H3, H5),
3
7.72 (dd, 2H, J = 8.7, 2.1 Hz, H2*, H6*), 7.65 (dd, 2H, J = 8.7, 2.1 Hz, H2’*, H6’*),
1
3
7.52-7.40 (m, 3H, H3’, H4’, H5’), 4.49 (s, 2H, CH ). C NMR (75 MHz, CDCl ) δ
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190.9, 146.6, 139.5, 132.6, 129.5 (2×C), 129.0 (2×C), 128.4, 127.4 (2×C), 127.2 (2×C),
30.9.
5.2.32. 1-([1,1’-biphenyl]-3-yl)-2-bromoethanone (12j).
The title compound was synthesized according to the general method G from compound
11a (1.2 g, 6.12 mmol) and phenyltrimethylammonium tribromide (2.30 g, 6.12 mmol)
in anhydrous THF (20 mL). The reaction mixture was stirred for 3 h. For purification
petroleum ether was used as eluent. Compound 12j was obtained (1.03 g, 61%) as a
yellow oil.
1
H NMR (300 MHz, CDCl ) δ 8.22 (t, 1H, J = 1.8 Hz, H2), 7.97 (td, 1H, J = 7.8, 1.8
3
13
Hz, H4), 7.84 (td, 1H, J = 7.8, 1.8 Hz, H6), 7.65-7.39 (m, 6H), 4.52 (s, 2H, CH ). C
2
NMR (75 MHz, CDCl ) δ 191.3, 142.0, 139.8, 134.4, 132.6, 129.3, 129.0 (2×C), 128.0,
3
127.7, 127.6, 127.2 (2×C), 30.9.
5.2.33. 2-bromo-1-(3-(thien-2-yl)phenyl)ethanone (12k).
The title compound was synthesized according to the general method G from compound
11b (0.89 g, 4.41 mmol) and phenyltrimethylammonium tribromide (1.66 g, 4.41 mmol)
in anhydrous THF (20 mL). The reaction mixture was stirred for 16 h. For purification
petroleum ether was used as eluent. Compound 12k was obtained (782 mg, 63%) as a
pale yellow oil.
1
H NMR (300 MHz, CDCl ) δ 8.22 (t, 1H, J = 1.8 Hz, H2), 7.87 (td, 1H, J = 7.8, 1.8
3
Hz, H6), 7.84 (td, 1H, J = 7.8, 1.8 Hz, H4), 7.51 (t, 1H, J = 7.8 Hz, H5), 7.40 (dd, 1H, J
= 4.8, 1.2 Hz, H5’), 7.35 (dd, 1H, J = 5.1, 1.2 Hz, H3’), 7.12 (dd, 1H, J = 5.1, 4.8 Hz,
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3
H4’), 4.50 (s, 2H, CH ). C NMR (75 MHz, CDCl ) δ 191.0, 142.7, 135.3, 134.5,
2
3
131.1, 129.4, 128.2, 127.7, 126.1, 125.8 124.1, 30.9.
5.2.34. 2-bromo-1-(3-(thien-3-yl)phenyl)ethanone (12l).
The title compound was synthesized according to the general method G from compound
11c (1.3 g, 6.44 mmol) and phenyltrimethylammonium tribromide (2.42 g, 6.44 mmol)
in anhydrous THF (25 mL). The reaction mixture was stirred for 16 h. For purification
petroleum ether was used as eluent. Compound 12l was obtained (929 mg, 51%) as a
colourless oil.

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H NMR (300 MHz, CDCl ) δ 8.21 (t, 1H, J = 1.8 Hz, H2), 7.89 (td, 1H, J = 7.8, 1.8
3
Hz, H6), 7.84 (td, 1H, J = 7.8, 1.8 Hz, H4), 7.57-7.49 (m, 2H, H5, H5’), 7.46-7.41 (m,
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2H, H2’, H4’), 4.50 (s, 2H, CH ). C NMR (75 MHz, CDCl ) δ 191.2, 140.9, 136.6,
2
3
134.4, 131.7, 129.3, 127.5, 126.8, 126.7, 126.1, 121.4, 30.9.
5.2.35. 2-bromo-1-(5-phenylthien-2-yl)ethanone (12m).
The title compound was synthesized according to the general method G from compound
11d (1 g, 4.95 mmol) and phenyltrimethylammonium tribromide (1.86 g, 4.95 mmol) in
anhydrous THF (20 mL). The reaction mixture was stirred for 16 h. For purification
petroleum ether/CH Cl (50:50 v/v) was used as eluent. Compound 12m was obtained
2
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(1.14 g, 82%) as a yellow solid.
1
Mp 117.3 °C. H NMR (300 MHz, CDCl ) δ 7.78 (d, 1H, J = 4.2 Hz, H3), 7.67 (dd, 2H,
3
J = 8.1, 1.5 Hz, H2’, H6’), 7.48-7.40 (m, 3H, H3’, H4’, H5’), 7.36 (d, 1H, J = 4.2 Hz,
13
H4), 4.37 (s, 2H, CH ). C NMR (75 MHz, CDCl ) δ 184.2, 154.4, 139.2, 134.6, 132.9,
2
3
129.4, 129.2 (2×C), 126.3 (2×C), 124.1, 30.2.
5.2.36. 2-(benzo[d][1,3]dioxol-4-yl)-6-iodoimidazo[1,2-b]pyridazine (13a).
Method H: to a solution of compound 12a (788 mg, 3.24 mmol) in n-butanol (12 mL)
was added compound 3 (717 mg, 3.24 mmol). The mixture was refluxed for 16 h,
evaporated to dryness, and the residue was suspended in CHCl . The solution was made
3
alkaline with a 30% ammonium hydroxide solution and extracted with CHCl . The
3
combined organic layers were dried over MgSO , filtered, and evaporated under
4
reduced pressure to give the desired imidazo[1,2-b]pyridazine 13a (1.20 g, 100%) as a
brown solid.
1
Mp 218.4 °C. H NMR (300 MHz, CDCl ) δ 8.44 (d, 1H, J = 0.6 Hz, H3), 7.68 (dd, 1H,
3
J = 8.1, 1.2 Hz, H5’), 7.65 (dd, 1H, J = 9.6, 0.6 Hz, H8), 7.32 (d, 1H, J = 9.6 Hz, H7),
6.97 (dd, 1H, J = 8.1, 7.8 Hz, H6’), 6.85 (dd, 1H, J = 7.8, 1.2 Hz, H7’), 6.13 (s, 2H,
1
3
CH ). C NMR (75 MHz, CDCl ) δ 147.8, 144.7, 140.8, 137.3, 127.2, 125.2, 122.0,
2
3
119.3, 115.7, 115.3, 110.4, 108.5, 101.3.
5.2.37. 2-(benzo[d][1,3]dioxol-5-yl)-6-iodoimidazo[1,2-b]pyridazine (13b).

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The title compound was synthesized according to the general method H from compound
12b (1 g, 4.12 mmol) and compound 3 (909 mg, 4.12 mmol) in n-BuOH (15 mL). The
reaction mixture was stirred for 3 h. Compound 13b was obtained (981 mg, 65%) as a
yellow solid.
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Mp 232.9 °C. H NMR (300 MHz, DMSO-d ) δ 8.73 (s, 1H, H3), 7.77 (d, 1H, J = 9.3
6
Hz, H8), 7.49 (dd, 1H, J = 8.1, 1.5 Hz, H6’), 7.48 (d, 1H, J = 1.5 Hz, H4’), 7.43 (d, 1H,
1
3
J = 9.3 Hz, H7), 6.93 (d, 1H, J = 8.1 Hz, H7’), 6.00 (s, 2H, CH ). C NMR (75 MHz,
2
DMSO-d ) δ 147.9, 147.6, 144.6, 137.4, 127.2, 127.1, 125.3, 119.7, 113.1, 112.7, 108.8,
6
106.0, 101.3.
5.2.38. 2-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)-6-iodoimidazo[1,2-b]pyridazine (13c).
The title compound was synthesized according to the general method H from compound
12c (670 mg, 2.69 mmol) and compound 3 (595 mg, 2.69 mmol) in n-BuOH (10 mL).
The reaction mixture was stirred for 5 h. Compound 13c was obtained (424 mg, 42%) as
a yellow solid.
1
Mp 216.5 °C. H NMR (300 MHz, CDCl ) δ 8.56 (d 1H, J = 0.9 Hz, H3), 7.88 (dd, 1H,
3
J = 7.8, 1.8 Hz, H6’), 7.63 (dd, 1H, J = 9.3, 0.9 Hz, H8), 7.30 (d, 1H, J = 9.3 Hz, H7),
6.98 (dd, 1H, J = 8.1, 7.8 Hz, H7’), 6.90 (dd, 1H, J = 8.1, 1.8 Hz, H8’), 4.44 (m, 2H,
13
CH *), 4.36 (m, 2H, CH *). C NMR (75 MHz, CDCl ) δ 143.8, 141.2, 141.0, 136.6,
2
2
3
126.9, 125.1, 121.7, 121.2, 120.5, 117.5, 117.0, 110.1, 64.5, 64.0.
5.2.39. 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-iodoimidazo[1,2-b]pyridazine (13d).
The title compound was synthesized according to the general method H from compound
12d (600 mg, 2.41 mmol) and compound 3 (533 mg, 2.41 mmol) in n-BuOH (10 mL).
The reaction mixture was stirred for 3 h. Compound 13d was obtained (551 mg, 60%)
as a brown solid.
1
Mp 193.3 °C. H NMR (300 MHz, CDCl ) δ 8.14 (s, 1H, H3), 7.61 (d, 1H, J = 9.3 Hz,
3
H8), 7.47 (d, 1H, J = 2.1 Hz, H5’), 7.45 (dd, 1H, J = 8.1, 2.1 Hz, H7’), 7.29 (d, 1H, J =
1
3
9.3 Hz, H7), 6.95 (d, 1H, J = 8.1 Hz, H8’), 4.31 (s, 4H, 2×CH ). C NMR (75 MHz,
2
CDCl ) δ 145.8, 144.3, 143.9, 137.8, 126.9, 126.3, 125.0, 119.5, 117.8, 115.1, 112.2,
3
110.1, 64.5, 64.4.