Antiplasmodial imidazopyridazines: structure-activity relationship studies lead to the identification of analogues with improved solubility and hERG profiles

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Lawrence, D. Taylor, S. Wittlin and K. Chibale, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00382C.

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Antiplasmodial imidazopyridazines: structure-activity relationship

studies lead to the identification of analogues with improved

solubility and hERG profiles^†

Received 00th January 20xx,

Accepted 00th January 20xx

Peter Mubanga Cheuka,^aNina Lawrence,^bDale Taylor,^bSergio Wittlin^cdand Kelly Chibale^abef

*

DOI: 10.1039/x0xx00000x

www.rsc.org/

3,6-Diarylated imidazopyridazines have recently been shown to possess good in vitro antiplasmodial and in vivo

antimalarial activity. However, frontrunner compounds have been associated with poor solubility and a hERG (human

ether-a-go-go-related gene) inhibition liability raising concerns for potential cardiotoxicity risks. Herein, we report the

synthesis and structure-activity relationship studies of new imidazopyridazines aimed at improving aqueous solubility and

countering hERG inhibition while maintaining antiplasmodial potency. While we identified new analogues with potent

antiplasmodial activity (IC₅₀= 0.031 μM against the NF54 drug-sensitive strain, and IC₅₀= 0.0246 μM against the K1

multidrug resistant strain), hERG inhibition remained an issue. Excitingly, on the other hand, new analogues with a

substantially improved hERG inhibition profile (IC₅₀= 7.83 – 32.3 μM) with sub-micromolar antiplasmodial activity (NF54,

IC₅₀= 0.151 – 0.922 μM) were identified. Similarly, the introduced molecular features also resulted in analogues with

moderate to high solubility (60 – 200 μM) while also displaying sub-micromolar antiplasmodial potency (NF54, IC₅₀= 0.136

– 0.99 μM).

the bites of infected female Anopheles mosquitoes.¹Of the five

species known to infect humans, Plasmodium falciparum and

Plasmodium vivax are the most virulent. On the African continent,

P. falciparum is the most prevalent and causes the most malaria-

related deaths worldwide while P. vivax dominates most countries

outside of sub-Saharan Africa.¹

Once effective antimalarial drugs like chloroquine and

sulfadoxine-pyrimethamine have suffered widespread resistance,

which has undermined malaria control efforts.^2–5Therefore,

antimalarial chemotherapy has shifted towards the use of

artemisinin combination therapies (ACTs). Regrettably, emerging

resistance to artemisinins has been reported recently in 5 countries

of the Greater Mekong sub-region, which includes Cambodia, Lao

People’s Democratic Republic, Myanmar, Thailand and Viet Nam.¹

Therefore, with this threat of resistance to first line treatment

options, there is a critical need to intensify research efforts into

novel, affordable and structurally diverse antimalarials.

Introduction

According to the World Health Organization (WHO)

estimates, malaria was responsible for 445 000 deaths and 216

million cases in 2016.¹The WHO African region accounted for 90%

of all malaria cases and 91% of malaria deaths. Children under the

age of 5 years are particularly susceptible to infection, illness and

death in areas with intense transmission of malaria. Although the

under-5 malaria death rate has declined by 29% worldwide, malaria

remains a major killer of children in this age group claiming the life

of a child every 2 minutes.¹

Malaria is caused by five species of protozoan parasites of

the genus Plasmodium which are transmitted to humans through

Imidazopyridazines possess a range of biological activities

including kinase inhibition,^6,7anxiety treatment⁸and anticancer

potential.⁹These compounds have also been investigated as

potential novel antimalarial agents.^10–13More recently, 3,6-

diarylated imidazopyridazines with potent antiplasmodial activity

were identified through a high throughput screening (HTS) of a

SoftFocus kinase library.¹⁴Further medicinal chemistry optimization

led to the identification of, among others, the lead compound 1

(figure 1) with high in vitro potency against both the NF54 (IC₅₀= 7.3

nM) and the K1 (IC₅₀= 6.3 nM) strains of P. falciparum.¹⁴Compound

1 also showed good oral efficacy (98% at 4 × 50 mg/kg) in the P.

berghei-infected mouse model.¹⁴However, the compound

exhibited poor solubility (< 5 μΜ at pH 6.5) and a hERG liability (IC₅₀

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= 0.9 μM). Additionally, the compound only exhibited a mean

survival days (MSD) time of 7 days.

In a quest to address solubility issues associated with the

recently studied antimalarial imidazopyridazines, we employed

known strategies, which included introducing polar water-

solubilizing groups (analogues 6 – 12 and 14 – 27) and disruption of

molecular planarity to discourage the solubility-suppressing effect

of π – π stacking (analogues 19 – 21).^19–21

While changes were introduced at positions 3 and 6 of the

imidazopyridazine core-scaffold, the 4-methylsulfinylphenyl and 3-

methylsulfinylphenyl groups were fixed at these positions in most

analogues (analogues 3, 5 – 8, and 10 – 27). Le Manach and co-

workers have recently employed such a prodrug strategy by

synthesizing solubility-enhancing sulfoxides which were shown to

be metabolized in vivo to the corresponding active sulfones.¹⁵In

two cases, sulfone-containing derivatives, 4 and 9, were also

synthesized for comparison purposes. For derivatives bearing

sulfoxide-containing phenyl rings at the point of variation, sulfone

counterparts were synthesized for the same reason in some cases

(e.g., analogues 3 vs 5, 14 vs 15, 16 vs 17, 24 vs 27, and 25 vs 26).

P. f, NF54 IC₅₀= 7.3 nM

P. f, K1 IC₅₀= 6.3 nM

P. f, NF54 IC₅₀= 1.1 nM

In vivo P. berghei (po) at 4

× 50 mg/kg, 99.8%, > 30

MSD, 3 out of 3 mice cured

In vivo P. berghei (po) at 4 ×

50 mg/kg, 98%, 7 MSD

hERG IC₅₀= 0.9 μM

Results and Discussion

Fig. 1 Previously explored antimalarial imidazopyridazine analogues

Chemistry

Analogues 1, 4 – 12, 14 – 23 and 29 – 32 were synthesized

In an article published in the same year, Le Manach and

colleagues¹⁵further explored the structure-activity relationship

(SAR) of this class of molecules in the hope of addressing the

aforementioned shortcomings. Although compound 2 (figure 1),

identified from this study, completely cured P. berghei-infected

mice at 4 × 50 mg/kg/day, better efficacy at lower doses was not

achieved. The lead compound also retained sub-optimal solubility

(< 5 μM at pH 6.5) and some off-target activities such as hERG

inhibition. It is noteworthy that this sulfoxide-substituted analogue

has no intrinsic hERG potency. But it is rapidly metabolized in vivo

to the sulfone analogue which has potent hERG activity (IC₅₀= 0.4

μΜ).¹⁵In a more recent paper, further expansion of SAR based on

modifications to the imidazopyridazine core aimed at improving

pharmacokinetics (PK), in vivo efficacy and selectivity over hERG

were reported.¹⁶This led to the identification of the

pyrazolopyridine analogue in which the two substituents in

compound 2 were retained. Despite being able to completely cure

P. berghei-infected mice at 4 × 50 mg/kg, the scaffold-hoped

analogue still exhibited a hERG liability and poor solubility.¹⁶

Recently, there has been a significant proportion of drug

withdrawals arising from their unwanted prolongation of the time

between the start of the Q wave and the end of the T wave (QT

interval) in an electrocardiogram.¹⁷Such a phenomenon, which can

lead to malignant ventricular arrhythmia and sometimes sudden

cardiac death, is known to arise from, mainly, the blockade of the

potassium ion channel (I_Kr) encoded by the hERG gene.¹⁷There are

a number of strategies used to address the hERG liability in drug

molecules.¹⁸Herein, we report the application of some of these

strategies in designing imidazopyridazine analogues (figure 2) with

potentially reduced in vitro hERG inhibition and improved solubility,

which also served to expand the antiplasmodial SAR around this

class of molecules. These approaches include subtle modifications

(analogues 3 – 5, 13 and 28 – 32); replacing aromatic rings with

saturated systems to discourage π - π stacking with aromatic

residues in the large cavity of the hERG channel (analogues 19 –

21); introducing polar functionalities into the molecule to

potentially disrupt lipophilic drug-hERG interactions (analogues 14 –

27).

according to scheme 1. The intermediate 2a was obtained by

condensation/cyclization

of

commercially

available

6-

chloropyridazin-3-amine (1a) with bromoacetaldehyde diethylacetal

in the presence of hydrobromic acid (HBr).¹⁴A regioselective

electrophilic aromatic iodination using N-iodosuccinimide (NIS)

afforded the iodinated intermediate, 3a, in 81% yield.¹⁴Sequential

Suzuki-Miyaura cross coupling reactions on intermediate 3a

delivered the diaryl-imidazopyridazines (1, 4 - 12 and 29 - 32) in 13

– 80% yield.¹⁴The aminated target molecules, 14 – 23, were

realized, in poor to low yields (8 – 30%), via a palladium-mediated

Buchwald-Hartwig amination^22,23of the chloro-bearing intermediate

4a. The aniline starting materials 5d and 5h, which were used to

synthesize analogues 22 and 23 respectively, were not available

commercially. These were synthesized in-house according to the

synthetic schemes in the electronic supplementary information.

Schemes 2A and 2B were used to synthesize analogues 3,

13, 24 – 27 and 28. Using an excess (2.2 equivalents) of the

respective boronic acid, analogues 3 and 28 were obtained by a one

pot Suzuki-Miyaura cross-coupling on both reaction sites of the

previously synthesized intermediate 3a (scheme 2A).¹⁴Analogues

13 and 24 – 27 were obtained via a common bromo-substituted

intermediate 4i according to scheme 2B. Intermediate 4i was

synthesized from

a previously synthesized imidazopyridazine

intermediate 2a by a Suzuki-Miyaura cross coupling followed by

regioselective electrophilic aromatic substitution in presence of N-

bromosuccinimide (NBS).¹⁴A Suzuki-Miyaura cross-coupling on

intermediate 4i furnished analogue 13 in 40% yield. The aminated

derivatives 24 – 27 were obtained in poor yields (9 – 17%) via

Buchwald-Hartwig amination.^22,23

2 | J. Name., 2012, 00, 1-3

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N

R³

N

R¹

N

H

N

R²

HN

R⁴

SO_nMe

SOMe

3: R¹= 4-MeOS-Ph, n = 1

4: R¹= 4-MeOS-Ph, n = 2

14: R³= 3-MeO₂S-Ph

15: R³= 3-MeOS-Ph

16: R³= 4-MeOS-Ph

17: R³= 4-MeO₂S-Ph

18: R³= 2-pyrazinyl

19: R³= 4-tetrahydro-2H-thiopyran 1,1-dioxide

20: R³= 1-methylpiperid-4-yl

10: R²= 4-((diethylamino)methyl)-2-F-Ph

24: R⁴= 4-MeO₂S-Ph

11: R²= 4-((diethylamino)methyl)-3-F-Ph

12: R²= 3-((diethylamino)methyl)-4-F-Ph

13: R²= 3-MeOS-Ph

25: R⁴= 3-MeO₂S-Ph

26: R⁴= 3-MeOS-Ph

27: R⁴= 4-MeOS-Ph

5: R¹= 4-MeO₂S-Ph, n = 1

6: R¹= 4-((diethylamino)methyl)-2-F-Ph, n = 1

7: R¹= 4-((diethylamino)methyl)-3-F-Ph, n = 1

8: R¹= 3-((diethylamino)methyl)-4-F-Ph, n = 1

9: R¹= 4-((diethylamino)methyl)-3-F-Ph, n = 2

21: R³= 1-(MeSO₂)piperid-4-yl

22: R³= 3-((diethylamino)methyl)-4-F-Ph

23: R³= 2-((diethylamino)methyl)phenol-4-yl

Other subtle changes: Also important for addressing hERG liability

N

1

N

MeO₂S

SO₂Me

N

SO₂Me

29

SO₂Me

28

N

MeO₂

S

MeOS

SOMe

SO₂Me

SOMe

30

32

31

Fig. 2 Structural modifications made around the imidazopyridazine core

Scheme 1 General synthetic approach for analogues 1, 4 – 12, 14 – 23 and 29 – 32. Reagents and conditions: (i) BrCH₂CH(OEt)₂, aq HBr, EtOH/H₂O, reflux,

103 °C, 22 h; (ii) NIS, DMF, rt (21 °C), 5 d, 81%; (iii) appropriate boronic acid or boronic acid pinacol ester, Pd(PPh₃)₂Cl₂, 1 M aq K₂CO₃, DMF, 80 °C, 3.5 - 46 h,

26 – 77%; (iv) appropriate boronic acid or boronic acid pinacol ester, Pd(PPh₃)₂Cl₂, 1 M aq K₂CO₃, DMF, 100 °C, 4 – 21 h, 13 – 80%; (v) R₃NH₂, Pd₂(dba)₃, (R)-

BINAP for 17, BrettPhos for 14, 22 & 23, XPhos for 15, 16 & 18 – 21, K₂CO₃for 17, Cs₂CO₃for 14, 22 & 23, NaOt-Bu for 15, 16 & 18 - 21, toluene/1,4-dioxane

for 17, 1,4-dioxane for all other analogues, 100 °C for 20, 120 °C for all other analogues, sealed tube, inert atmosphere (N₂), 6 – 43 h, 8 – 30%.

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Scheme 2 General synthetic approach for analogues 3, 13, 28 and 24 – 27. Reagents and conditions: (i) appropriate boronic acid (excess, 2.2 eq), Pd(PPh₃)₂Cl₂,

1 M aq K₂CO₃, DMF, 100 °C, 15 h for 3 and 24 h for 28, 39% for 3 and 62% for 28; (ii) 3-methylsulfinylphenylboronic acid, Pd(PPh₃)₂Cl₂, 1 M aq K₂CO₃, DMF,

100 °C, 3.25 h, 48%; (iii) NBS, DMF, rt (23 °C), 22 h, 59%; (iv) 3-methylsulfinylphenylboronic acid, Pd(PPh₃)₂Cl₂, 1 M aq K₂CO₃, DMF, 100 °C, 20 h, 40%; (v) R₄-

NH₂, Pd₂(dba)₃, BrettPhos, Cs₂CO₃, 1,4-dioxane, 120 °C, sealed tube, 5 – 39 h, 9 – 17%.

Biology and solubility

A total of 31 imidazopyridazine analogues were synthesized

exhibited superior solubility (200 μM) compared to the sulfone-

substituted analogue 5 (5 μM). Amongst the analogues substituted

with a fluoro and a basic side chain (6, 7 and 8, table 1), 8 proved

the most potent (IC₅₀= 0.136 μM) signifying the importance of the

para-fluoro and meta-diethylaminomethyl substitution pattern to

potency. It is noteworthy that compound 8 was also the most

potent in the entire series of analogues in table 1. Gratifyingly, we

also managed to significantly improve solubility in such basic amine-

substituted analogues (180 – 200 μM). Although potency was,

generally, compromised for the aminated imidazopyridazines, most

of them still retained sub-micromolar potency. Notable amongst

and tested in biological and solubility assays. All the synthesized

analogues were evaluated for in vitro antiplasmodial activity against

the drug sensitive strain (NF54) of the P. falciparum malaria

parasites. Analogues exhibiting sub-micromolar (IC₅₀< 1 μM)

potency were further tested in vitro against the multi drug-resistant

strain (K1) of P. falciparum. In parallel, the solubility of all the

synthesized analogues was determined. Significantly potent (IC₅₀

<

1 μM) analogues were advanced to cytotoxicity testing using the

Chinese Hamster Ovary (CHO) cell line (ATCC). The selectivity index

(SI) for each compound was calculated as a ratio of cytotoxic IC₅₀

value to corresponding antiplasmodial (P. f NF54) IC₅₀value. The

SAR explorations are discussed with reference to IC₅₀values on the

NF54 strain. Representative compounds were selected and

screened for hERG activity on the QPatch gigaseal automated patch

clamp platform.

these is 3-methylsulfonylaniline-substituted analogue 14 (IC₅₀

0.274 μM) which was 3-fold more potent than the reduced form 15

(IC₅₀= 0.820 μM). However, the para-substituted versions 16 (IC₅₀

=

0.552 μM) and 17 (IC₅₀= 0.423 μM) were equipotent. In this series,

we also designed analogues with saturated cyclic groups (19 – 21)

which we hypothesized could help discourage π - π stacking thereby

improving aqueous solubility.²⁴Strikingly, the sulfone group in

analogue 21 (IC₅₀= 0.498 μM) was important in retaining sub-

micromolar potency – analogue 20 lacking this group exhibited 5-

fold less potency (IC₅₀= 2.67 μM). All the aminated analogues,

generally exhibited improved solubility with sulfone-substituted

analogues showing inferior solubility relative to their sulfoxide

counterparts.

In vitro antiplasmodial activity and solubility. The in vitro

antiplasmodial (IC₅₀values), and solubility values for all the

synthesized compounds are shown in tables 1 – 3.

We set out by fixing a 4-methylsulfinylphenyl group at

position 3 while introducing saturation, subtle modifications and

water-solubilizing groups at position 6 (table 1). The antiplasmodial

potency of all analogues in this series was generally compromised

(IC₅₀= 0.136 – 2.67 μM) compared to the previously reported¹⁴lead

compound 1 (IC₅₀= 0.0034 μM). All analogues evaluated for

antiplasmodial activity against both the NF54 and K1 parasites were

equipotent on these two strains, suggesting the absence of cross-

resistance. This is in conformity with previously reported

imidazopyridazine derivatives.¹⁵Para substitution with either a

sulfone or sulfoxide group at 6-phenyl ring proved detrimental to

potency as reflected in analogues 3 (IC₅₀= 0.673 μM) and 5 (IC₅₀

=

0.974 μM). As anticipated, the sulfoxide-substituted analogue 3

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Table 1 In vitro antiplasmodial activity against P. falciparum (NF54 and K1) and solubility of pyridazine-substituted analogues

P. f IC₅₀, μM^c,d

Solubility, μM,

Compound code

R⁵

pH 6.5

NF54

K1

3

5

6

7

8

0.673

0.499

200

0.974

1.88

0.82

ND^e

5

180

195

200

F

1.67

ND^e

Et₂N

0.136

0.114

22

23

14

15

1.62

0.922

0.274

0.820

ND^e

200

195

20

0.774

0.236

0.854

190

16

17

0.552

0.423

0.488

0.236

195

< 5

18

19

20

0.468

0.524

2.67

0.436

0.492

ND^e

25

10

200

21

0.498

0.439

70

^cMean from n values of ≥ 2 independent experiments with multidrug resistant (K1) and sensitive (NF54) strains of P. falciparum. The majority of the

individual values differed less than 2-fold (maximum 2-fold).

^dArtesunate [IC₅₀= 2.2 ng/mL (NF54), 0.93 ng/mL (K1)] and chloroquine [IC₅₀= 4.3 ng/mL (NF54), 83 ng/mL (K1)] were used as reference drugs.

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^eND, not determined.

from compound 13, which exhibited promising activity against both

the NF54 (IC₅₀= 0.151 μM) and K1 (IC₅₀= 0.0866 μM) strains,

generally, this series represented modifications which compromised

potency the most (IC₅₀= 0.99 – 7.55 μM). However, the introduced

water-solubilizing groups imparted moderate to high solubility (35 –

200 μM) in these analogues. An increase in dihedral angle which

compromises π – π stacking following ortho substitution of bi-aryl

systems could explain the notable difference in solubility between

the ortho-fluorinated analogue 10 (200 μM) and the meta-

fluorinated regioisomer 11 (35 μM).²⁴As previously observed,

sulfoxide-containing aminated analogues 26 (150 μM) and 27 (165

μM) were more soluble than the corresponding sulfone analogues

25 (40 μM) and 24 (60 μM).

Inspired by previous studies¹⁴which found meta-

substitution at the phenyl ring on position 6 optimal for potency,

we kept this group fixed as 3-methylsulfinylphenyl group while

exploring other changes at position 3 of the core-scaffold. The

antiplasmodial and solubility results are shown in table 2. Apart

Table 2 In vitro antiplasmodial activity against P. falciparum (NF54 and K1) and solubility of imidazo-substituted analogues

P. f IC₅₀, μM^c,d

Solubility, μM,

Compound code

R⁶

pH 6.5

NF54

K1

10

3.2

ND^e

200

11

12

1.05

0.99

ND^e

35

0.558

200

13

24

0.151

3.62

0.0866

ND^e

200

60

25

26

7.55

4.71

ND^e

40

150

27

3.81

ND^e

165

^cMean from n values of ≥ 2 independent experiments with multidrug resistant (K1) and sensitive (NF54) strains of P. falciparum. The majority of the

individual values differed less than 2-fold (maximum 2-fold).

^dArtesunate [IC₅₀= 2.2 ng/mL (NF54), 0.93 ng/mL (K1)] and chloroquine [IC₅₀= 4.3 ng/mL (NF54), 83 ng/mL (K1)] were used as reference drugs.

^eND, not determined.

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compounds. Interestingly, compound 28, a regioisomer of the

previously reported¹⁴lead compound 1 was highly potent (IC₅₀

=

0.031 μM) indicating that the para-to-meta positional change of the

right-hand-side sulfone was tolerated albeit solubility (< 5 μM) was

still an issue. However, this isomer is still 9-fold less potent than the

lead compound 1 (IC₅₀= 0.0034 μM) indicating a slight decrease in

potency accompanies this positional change. While maintaining the

sulfone at the meta position on the right-hand-side phenyl ring,

para substitution on the left-hand-side phenyl ring, irrespective of

the nature of substituent as shown in analogues 29 (IC₅₀= 0.396

μM) and 30 (IC₅₀= 0.264 μM) was detrimental to potency. Similarly,

keeping the sulfoxide fixed at the meta position of the right-hand-

side phenyl ring while introducing the sulfone and sulfoxide groups

at the para position of the left-hand-side phenyl ring proved

detrimental to potency (see analogues 31, IC₅₀= 1.68 μM and 32,

IC₅₀= 0.870 μΜ). It is also noteworthy that the sulfoxide-substituted

analogue 32 was twice as potent as the sulfone-substituted

counterpart 31. Furthermore, analogue 4 was 7-fold less potent

than its previously reported¹⁵regioisomer further confirming that

para-substitution on the left-hand-side phenyl ring is detrimental to

activity. Save for analogue 9, whose excellent solubility (200 μM)

could be attributed to the water-solubilizing basic side chain, all

analogues in this series having at least one sulfone group were

poorly soluble (< 5 – 10 μM). As expected, analogue 32 with

sulfoxide-substituted phenyl rings, exhibited excellent solubility

(200 μM).

In another strategy to address the hERG liability, we

designed analogues 4, 28 – 32 (table 3), 3, 5 and 13 (table 1) with

subtle modifications, a strategy that has also been used by other

drug discovery programmes to diminish hERG activity.¹⁸These

included peripheral positional changes of the sulfone and sulfoxide

groups on the two phenyl rings as well as switching sulfones with

sulfoxide groups. The sulfone version of compound 7 in table 1,

compound

9 (table 3), was also included in this series of

Table 3 In vitro antiplasmodial activity against P. falciparum (NF54 and K1) and solubility for discreetly-modified analogues

P. f IC₅₀, μM^c,d

Solubility, μM,

Compound code

Structure

pH 6.5

NF54

K1

4

0.278

0.207

10

9

1

0.822

ND^e

200

10

N

0.0034

0.00225

SO₂Me

28

29

0.031

0.396

0.0246

0.314

< 5

30

31

0.264

1.68

0.189

ND^e

< 5

N

MeO₂S

SOMe

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P. f IC₅₀, μM^c,d

Solubility, μM,

pH 6.5

Compound code

32

Structure

NF54

K1

0.870

0.872

200

^cMean from n values of ≥ 2 independent experiments with multidrug resistant (K1) and sensitive (NF54) strains of P. falciparum. The majority of the

individual values differed less than 2-fold (maximum 2-fold).

^dArtesunate [IC₅₀= 2.2 ng/mL (NF54), 0.93 ng/mL (K1)] and chloroquine [IC₅₀= 4.3 ng/mL (NF54), 83 ng/mL (K1)] were used as reference drugs.

^eND, not determined.

in compound 28 were switched to sulfoxides as shown in compound

13 (IC₅₀= 8.45 μM), hERG potency was found to diminish by almost

4-fold. Moreover, positional change of the left-hand-side sulfoxide

group in 13 to para in analogue 32 (IC₅₀= 21.1 μM) significantly

lowers hERG activity. When compared to the lead compound 1,

para sulfoxide substitution on the left-hand-side phenyl ring, as

shown in analogue 4 (IC₅₀= 17.9 μM), reduced hERG potency by 5-

fold. Analogues with basic side chains, 8 (IC₅₀= 0.59 μM), 9 (IC₅₀

=

5.2 μM) and 23 (IC₅₀= 7.83 μM) were potent on the hERG channel.

This is not unexpected since most ligands that block the hERG

channel contain a basic nitrogen,¹⁸which can get protonated at

In vitro mammalian cytotoxicity and hERG inhibition. physiological pH thereby facilitating hERG binding through cation -

Analogues with in vitro antiplasmodial potency IC₅₀< 1 μM were π interactions between the protonated nitrogen and the π system

evaluated for in vitro cytotoxicity against the mammalian CHO cell of the aromatic residues in the channel cavity.¹⁸For the aniline

line. Analogues screened for hERG inhibition were, generally, derivatives, 14 – 17, meta sulfonylation seems to enhance hERG

selected based on demonstrated sub-micromolar antiplasmodial activity (14, IC₅₀= 2.36 μM). Strikingly, switching the sulfone in 14

activity. However, for derivatives representing discreet peripheral with the sulfoxide diminishes hERG activity by about 8-fold as

changes (e.g., 3 - 5, 13, 28 – 30 and 32) with sub-micromolar demonstrated in analogue 15 (IC₅₀= 18.2 μM). The regioisomer of

antiplasmodial activity, only representative compounds were tested 15, compound 16 (IC₅₀= 32.3 μM), which is para substituted

for hERG inhibition. Generally, most analogues were non-cytotoxic showed further decrease in hERG inhibition. Interestingly,

a

with selectivity indices in the range 72 - > 874 (table 4). Strikingly, positional change from meta to para (14 to 17) also led to a 11-fold

compounds 8, 9, 12 and 23, containing the basic side chains, were reduction in hERG activity. We also hypothesized that the

particularly cytotoxic with lower selectivity (SI = 10 – 30) (table 4).

introduced saturated rings in 19 and 21 would reduce hERG binding

The hERG inhibition results for selected analogues are by disrupting π – π interactions between phenyl rings in our

summarized in table 4. As previously reported,¹⁴compound 1 was molecules and those embedded in the channel cavity.¹⁸

potent on hERG (IC₅₀= 3.61 μM). Its isomers, 28 (IC₅₀= 2.35 μM) Interestingly, both analogues 19 (IC₅₀= 28.8 μM) and 21 (IC₅₀= 25.9

and 29 (IC₅₀= 4.0 μM), still retained hERG potency. When sulfones μM) indeed displayed significantly weaker hERG inhibition activity.

Table 4 In vitro mammalian cytotoxicity and hERG inhibition profiling for selected analogues

Cytotoxicity^f

CHO cells

Compound code

Structure

hERG IC₅₀, μM (SD)^g

IC₅₀, μM (SD)

SI^h

1

> 234

> 69,000

3.61 (0.623)

28

13

11.7 (0.89)

31.1 (0.2)

377

206

2.35 (0.46)

8.45 (0.8)

8 | J. Name., 2012, 00, 1-3

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Cytotoxicity^f

CHO cells

Compound code

Structure

hERG IC₅₀, μM (SD)^g

IC₅₀, μM (SD)

SI^h

29

181 (5.2)

457

4 (0.83)

30

32

18.9 (1.5)

106 (21)

72

ND^e

122

21.1 (2.8)

3

5

4

8

> 253

> 243

> 376

> 249

> 874

30

ND^e

N

ND^e

MeO₂

S

SOMe

> 243

17.9 (5.56)

0.59 (0.02)

4.08 (0.96)

23

14

15

16

17

9.41 (3.0)

123 (0.4)

ND^e

10

7.83 (1.21)

2.36 (0.21)

18.2 (8.45)

32.3 (1.54)

26.1 (6.30)

449

ND^e

> 244

> 442

> 553

> 234

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Cytotoxicity^f

CHO cells

Compound code

Structure

hERG IC₅₀, μM (SD)^g

IC₅₀, μM (SD)

SI^h

18

277 (18)

592

7.84 (0.76)

25.9 (1.83)

28.8 (3.64)

21

19

> 231

> 247

> 464

> 471

9

9.44 (2.1)

12 (2.7)

11

12

5.2 (0.44)

12

ND^e

^eND, not determined.

^fMean from n = 3 independent experiments; SD, standard deviation; Emetine was used as a reference drug (IC₅₀= 0.033 ± 0.006 μM).

^hSI = selectivity index = IC₅₀(CHO)/IC₅₀(P. f NF54).

^gMean from n = 3 independent experiments; Verapamil was used as a positive control (IC₅₀= 0.560 ± 0.0961 μM).

Conclusion

In our efforts to improve solubility and counter hERG nm). An Agilent LC-MS instrument with the following components

inhibition, we identified a regioisomer of lead 1, compound 28 was used for compound purity checks and reaction monitoring:

(tables 3 and 4) with potent in vitro antiplasmodial activity (IC₅₀

=

Agilent 1260® Infinity Binary Pump, Agilent 1260® Infinity Diode

0.031 μM) and high selectivity (SI = 377) against the CHO cell line. Array Detector, Agilent 1290® Infinity Column Compartment,

However, hERG inhibition (IC₅₀= 2.35 μM) and poor solubility (< 5 Agilent 1260® Infinity Autosampler, Agilent 6120® Quadrupole LC-

μM) remain issues. Interestingly, the sulfoxide version, compound MS, and Peak Scientific® Genius 1050 Nitrogen Generator. An X-

13 (tables 2 and 4), exhibited excellent solubility (200 μM) with bridge® (C18, 2.5 µm, 3.0 mm (ID) x 50 mm length) column

reduced hERG inhibition (IC₅₀

= 8.45 μM). Additionally, we maintained at 35 °C or 40 °C was used. The chromatographic mobile

successfully managed to substantially reduce hERG activity in phase was composed of 10 mM aqueous ammonium acetate

molecules 4, 13, 15 - 19, 21, 23 and 32 (IC₅₀= 7.83 – 32.3 μM, table (NH₄Ac) spiked with 0.4% acetic acid while the organic phase was

4). Similarly, the introduced molecular features also resulted in composed of 10 mM NH₄Ac in methanol spiked with 0.4% acetic

analogues 3, 6 – 10, 12, 13, 15, 16, 20 – 24, 26, 27 and 32 (tables 1 - acid. The mass spectra were acquired using electrospray ionisation

3) with moderate to high solubility (60 – 200 μM).

(ESI) and/or atmospheric pressure chemical ionization (APCI) in the

positive ionisation mode unless otherwise stated.

Biotage grade silica gel was employed for column

chromatographic purifications on the Biotage Isolera One Flash

Chromatography system. Additionally, Analtech Uniplate

preparative TLC (prep-TLC) plates (silica gel GF, 20 × 20 cm, 2000

microns) were used for prep-TLC purifications. The mobile phase

solvents were AR grade and were used without further distillation.

All yields reported are isolated and correspond to individual

synthetic steps in a scheme.

Experimental procedures

Chemistry

All reagents and chemicals used in all reactions were

purchased from various commercial sources and used without

further purification. All the solvents used in the reactions were

anhydrous save for ethanol which was absolute (99.9%). Reactions

¹H-NMR and ¹³C-NMR spectra were acquired on either

Bruker AV 400 (¹H 400.0, ¹³C 101 MHz) or Varian Mercury 300 (¹H

300.1 MHz) spectrometers.

were monitored by

a combination of analytical thin layer

chromatography (TLC) and liquid chromatography mass

spectrometry (LC-MS). The TLC plates were sourced from Merck

(TLC Silica gel 60 F₂₅₄aluminium-backed) and were developed in a

100 mL beaker covered with either a watch glass or aluminium foil.

The plates were visualized under ultraviolet light (UV 254 and 366

All final compounds were subjected to purity check

experiments using LC-MS to ensure acceptable purity (≥ 95%).

Melting points were measured using the Automatic Stuart Melting

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Point Apparatus SMP40 and are uncorrected. Purification and washed with deionized water (8 ×), saturated aqueous solutions of

characterization for some selected intermediates and analogues are NaHCO₃(3 ×), NH₄Cl (3 ×) and NaCl (1 ×). For analogues 6 – 12, with

reported in electronic supplementary information.

basic side chains, washing with NH₄Cl (aq) was avoided. The organic

6-Chloroimidazo[1,2-b]pyridazine (2a). Bromoacetaldehyde layer was dried (MgSO₄for 1 and 6 - 12; Na₂SO₄for 4, 5 and 29 – 32)

diethylacetal (12.0 mL, 78 mmol) was added to a suspension of 3- after which the solvent was removed in vacuo and the resulting

amino-6-chloropyridazine (1a) (5.00 g, 39 mmol) in absolute crude mixture subjected to further purification procedures.

ethanol (99.9%) (78 mL) and deionized water (50 mL). A 48%

6-(4-(Methylsulfinyl)phenyl)-3-(4-

aqueous solution of HBr (4.4 mL, 39 mmol) was added after which (methylsulfonyl)phenyl)imidazo[1,2-b]pyridazine (4). Purified by

the suspension cleared. The reaction mixture was refluxed at 103 °C prep-TLC (developed in 4% CH₃OH/DCM) and trituration in ethyl

for 22 hours. The resulting brown solution was extracted with ethyl acetate. Yellow solid (0.0683 g, 57%), mp 236.6 – 239.0 °C (from

acetate (EtOAc) (100 mL × 3). The organic layers were combined ethyl acetate); R_f(CH₃OH : CH₂Cl₂4 : 96) 0.18; ¹H-NMR δ_H(400 MHz;

after which the solvent was removed in vacuo to obtain 2a as a CDCl₃) 8.41 (d, J = 8.8 Hz, 2H), 8.27 (s, 1H), 8.24 (d, J = 9.5 Hz, 1H),

brown solid which was used in the next step without further 8.19 (d, J = 8.7 Hz, 2H), 8.13 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.7 Hz,

purification (LC-MS, ESI/APCI⁺: m/z [M + H]⁺= 154.0, calculated 2H), 7.70 (d, J = 9.5 Hz, 1H), 3.15 (s, 3H), 2.84 (s, 3H); ¹³C-NMR δ_C

exact mass = 153.0094, t_r= 1.5 min).

(101 MHz; CDCl₃) 151.23, 148.38, 140.09, 139.43, 137.92, 134.52,

6-Chloro-3-iodoimidazo[1,2-b]pyridazine (3a).

A

reddish 133.74, 128.08 (3C), 127.97 (2C), 127.05 (2C), 126.65, 124.49 (2C),

solution of 6-chloroimidazo[1,2-b]pyridazine (2a) (4.60 g, 30 mmol) 116.66, 44.58, 44.00; LC-MS, APCI⁺: m/z [M + H]⁺= 412.0, calculated

in 30 mL of N,N-dimethylformamide (DMF) was purged with exact mass = 411.0711, purity: 98.8%, t_r= 3.4 min.

nitrogen for 30 minutes. NIS (7.41 g, 33 mmol) was added followed

N-Ethyl-N-(3-fluoro-4-(3-(4-

by the addition of another 45 mL of DMF. The reaction mixture was (methylsulfinyl)phenyl)imidazo[1,2-b]pyridazin-6-

then left to magnetically stir at room temperature (21 °C) for 5 days yl)benzyl)ethanamine (6). Purified by flash chromatography (0 – 4%

(initially a suspension, the reaction mixture cleared after 2 days of CH₃OH/DCM) and trituration in diethyl ether. Yellow solid (0.128 g,

stirring). DMF was then removed in vacuo. The resulting brown 59%), mp 110.7 – 113.0 °C (from diethyl ether); R_f(CH₃OH : CH₂Cl₂6

residue was taken up in 200 mL dichloromethane (DCM) and : 94) 0.32; ¹H-NMR δ_H(300 MHz; CDCl₃) 8.35 (d, J = 8.3 Hz, 2H), 8.20

washed with deionized water (100 mL × 3), a saturated aqueous (s, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.94 – 7.75 (m, 3H), 7.62 (d, J = 9.2

solution of a mixture of sodium metabisulfite (Na₂S₂O₅) and sodium Hz, 1H), 7.46 – 7.34 (m, 2H), 3.76 (s, 2H), 2.81 (s, 3H), 2.70 (br s,

bisulfite (NaHSO₃) (100 mL × 2). The solvent from the organic layer 4H), 1.17 (t, J = 6.8 Hz, 6H); ¹³C-NMR δ_C(100.6 MHz; CDCl₃) 162.1,

was removed in vacuo to afford 3a as a brown solid (6.82 g, 81%); 159.4, 148.8, 144.8, 134.1, 131.6, 130.5, 127.3 (4C), 125.8 (2C),

¹H-NMR δ_H(300 MHz; CDCl₃) 7.92 (d, J = 9.4 Hz, 1H), 7.88 (s, 1H), 124.0 (3C), 119.0, 117.4, 56.6, 46.9 (2C), 44.0, 11.2 (2C); LC-MS,

7.14 (d, J = 9.4 Hz, 1H); LC-MS, ESI/APCI⁺: m/z [M + H]⁺= 279.8, ESI/APCI⁺: m/z [M + H]⁺= 437.1, calculated exact mass = 436.1733,

calculated exact mass = 278.9060, t_r= 3.0 min.

purity: 99.6%, t_r= 2.4 min.

3-(3-(Methylsulfonyl)phenyl)-6-(4-

General procedure for synthesis of chloro-substituted

intermediates (4a – g). A suspension of 6-chloro-3-iodoimidazo[1,2- (methylsulfonyl)phenyl)imidazo[1,2-b]pyridazine (29). Purified by

b]pyridazine (3a) (1.0 eq), an appropriate boronic acid or boronic prep-TLC (developed in 4% CH₃OH/DCM). Yellow solid (0.0223 g,

acid pinacol ester (1.1 eq), and Pd(PPh₃)₂Cl₂(0.05 eq) in DMF (3 20%), mp 264.0 – 267.5 °C (from 7% MeOH in DCM); R_f(CH₃OH :

mL/mmol of 3a) was purged with nitrogen for 20 minutes. A 1 M CH₂Cl₂4 : 96) 0.38; ¹H-NMR δ_H(400 MHz; DMSO-d₆) 9.12 – 9.08 (m,

aqueous solution of K₂CO₃(1.05 eq) was then added after which the 1H), 8.56 (s, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.50 (d, J = 8.4 Hz, 2H), 8.45

reaction mixture was heated to 80 °C and left to magnetically stir at (d, J = 9.6 Hz, 1H), 8.13 (d, J = 8.5 Hz, 2H), 8.09 (d, J = 9.6 Hz, 1H),

that temperature for 3.5 – 46 hours. The reaction mixture was 8.00 – 7.96 (m, 1H), 7.86 (t, J = 7.9 Hz, 1H), 3.33 (m, 6H); ¹³C-NMR δ_C

diluted with DCM, washed with deionized water (8 ×), saturated (101 MHz; DMSO-d₆) 150.20, 142.63, 142.04, 140.26, 140.07,

aqueous solutions of NaHCO₃(3 ×), NH₄Cl (3 ×) and NaCl (1 ×). For 135.36, 131.32, 130.51, 129.90, 128.49 (2C), 128.17 (2C), 127.33,

intermediates 4e – g, containing the basic side chain, washing with 126.10, 126.38, 124.60, 116.99, 44.08, 43.90; LC-MS, APCI⁺: m/z [M

aq NH₄Cl was avoided. After drying the organic layer (MgSO₄), the + H]⁺= 428.0, calculated exact mass = 427.0660, purity: 95.8%, t_r=

solvent was removed in vacuo and the resulting residue subjected 3.5 min.

to automated column chromatography on silica to give the chloro-

substituted derivatives in 26 – 77% yield.

General procedure for synthesis of aminated analogues

(15, 16, 18 and 21). suspension of 6-chloro-3-(4-

(methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine (4a) (1.0 eq), an

A

6-Chloro-3-(4-(methylsulfinyl)phenyl)imidazo[1,2-

b]pyridazine (4a). Purified by flash chromatography (0 – 3% appropriate amine (1.1 eq), Pd₂(dba)₃(0.08 eq), XPhos (0.12 eq) and

CH₃OH/DCM). Yellow solid (0.587 g, 77%); ¹H-NMR δ_H(300 MHz; sodium tert-butoxide (2.0 eq) in anhydrous 1,4-dioxane (6.0

CDCl₃) 8.24 (d, J = 8.2 Hz, 2H), 8.18 (s, 1H), 8.11 (d, J = 9.4 Hz, 1H), mL/mmol of 4a) in a sealed tube was flashed with nitrogen for 20

7.82 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 9.4 Hz, 1H) 2.81 (s, 3H); LC-MS, minutes. The reaction mixture was then heated to 120 °C and

ESI/APCI⁺: m/z [M + H]⁺= 292.0, calculated exact mass = 291.0233, stirred for 6 - 20 hours. The solvent was removed in vacuo after

t_r= 3.0 min.

which the resulting residue was taken up in DCM, washed with

General procedure for synthesis of diarylated saturated aqueous solutions of NaHCO₃(2 ×) and NaCl (2 ×). The

imidazopyridazines (1, 4 – 12 and 29 - 32). A suspension of the organic layer was dried (MgSO₄) (Na₂SO₄for 16), where after the

relevant chloro-substituted intermediate (4a

– g) (1.0 eq), solvent was removed in vacuo and obtained residue further

appropriate boronic acid or boronic acid pinacol ester (1.1 eq) and purified.

Pd(PPh₃)₂Cl₂(0.05 eq) in DMF (3 mL/mmol of 4a – g) was purged

N-(3-(Methylsulfinyl)phenyl)-3-(4-

with nitrogen for 20 minutes. A 1 M aqueous solution of K₂CO₃(methylsulfinyl)phenyl)imidazo[1,2-b]pyridazin-6-amine

(15).

(1.05 eq) was then added after which the reaction mixture was Purified by prep-TLC (developed twice in 6% CH₃OH/DCM). Brown

heated to 100 °C and magnetically stirred at that temperature for 4 solid (0.0175 g, 18%); R_f(CH₃OH : CH₂Cl₂6 : 94) 0.19; ¹H-NMR δ_H

– 21 hours. The reaction mixture was then diluted with DCM, (400 MHz; DMSO-d₆) 9.81 (s, 1H), 8.28 (d, J = 8.5 Hz, 2H), 8.11 (s,

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1H), 8.05 (s, 1H), 8.03 (d, J = 9.7 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.77 Hz, 1H), 7.78 (dt, J = 7.8, 1.5 Hz, 1H), 7.74 (td, J = 7.6, 0.6 Hz, 1H),

(dd, J = 8.1, 1.3 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.31 (d, J = 7.7 Hz, 7.69 (d, J = 9.5 Hz, 1H), 2.84 (s, 3H); LC-MS, ESI⁺: m/z [M + H]⁺

1H), 7.02 (d, J = 9.7 Hz, 1H), 2.82 (s, 3H), 2.73 (s, 3H); ¹³C-NMR δ_C258.1, calculated exact mass = 257.0623, t_r= 3.1 min.

=

(101 MHz; DMSO-d₆) 151.09, 147.73, 145.44, 141.43, 138.21,

3-Bromo-6-(3-(methylsulfinyl)phenyl)imidazo[1,2-

132.23, 131.62, 130.32, 127.41 (2C), 127.34 (2C), 124.69 (2C), b]pyridazine

(4i).

A

brown

solution

of

6-(3-

121.14, 117.01, 113.89, 113.50, 43.83, 43.73; LC-MS, APCI⁺: m/z [M (methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine (4h) (1.20 g, 4.7

+ H]⁺= 411.1, calculated exact mass = 410.0871, purity: 98.9%, t_r= mmol) in anhydrous DMF (4 mL) was purged with nitrogen for 30

3.8 min.

3-(4-(Methylsulfinyl)phenyl)-N-(pyrazin-2-yl)imidazo[1,2-

minutes. NBS (0.912 g, 5.1 mmol) was added where after the

reaction mixture was stirred at room temperature (23 °C) for 22

b]pyridazin-6-amine (18). Purified by prep-TLC (developed in 5% hours. The reaction mixture, containing a yellow precipitate at this

CH₃OH/DCM). Light yellow solid (0.0242 g, 29%); R_f(CH₃OH : CH₂Cl₂point, was taken up in 100 mL DCM and washed with deionized

1

6 : 94) 0.3; H-NMR δ_H(400 MHz; DMSO-d₆) 10.43 (s, 1H), 9.23 (d, J water (75 mL × 8), saturated aqueous solutions of NaHCO₃(75 mL ×

= 1.3 Hz, 1H), 8.38 (dd, J = 2.6, 1.5 Hz, 1H), 8.35 (d, J = 8.5 Hz, 2H), 2), NH₄Cl (75 mL × 2) and NaCl (75 mL × 1). The organic layer was

8.26 (d, J = 2.6 Hz, 1H), 8.16 – 8.09 (m, 2H), 7.83 (d, J = 8.5 Hz, 2H), dried (MgSO₄) after which the solvent was removed in vacuo. The

7.43 (d, J = 9.7 Hz, 1H), 2.82 (s, 3H); ¹³C-NMR δ_C(101 MHz; DMSO- obtained oily residue was crystallized and triturated with ethyl

d₆) 150.20, 149.95, 145.70, 142.75, 138.50, 137.91, 135.82, 132.81, acetate for 30 minutes. The solid was filtered and washed with

1

131.46, 127.50, 127.35 (2C), 127.09, 124.45 (2C), 113.48, 43.68; LC- ethyl acetate to give 4i as a yellow solid (0.939 g, 59%); H-NMR δ_H

MS, APCI⁺: m/z [M + H]⁺= 351.1, calculated exact mass = 350.0950, (300 MHz; CDCl₃) 8.44 – 8.35 (m, 2H), 8.29 – 8.23 (m, 1H), 7.93 (s,

purity: 96.5%, t_r= 3.7 min.

General procedure for synthesis of diarylated 336.0 & 338.0, calculated exact mass = 334.9728, t_r= 3.9 min.

imidazopyridazines and 28. suspension of 6-chloro-3- 3,6-Bis(3-(methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine

iodoimidazo[1,2-b]pyridazine (3a) (1.0 eq), an appropriate boronic (13). suspension of 3-bromo-6-(3-

1H), 7.90 – 7.73 (m, 3H), 2.85 (s, 3H); LC-MS, ESI⁺: m/z [M + H]⁺=

3

A

acid (2.2 eq), and Pd(PPh₃)₂Cl₂(0.10 eq) in anhydrous DMF (2.6 mL) (methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine (4i) (0.100 g, 0.30

was purged with nitrogen for 30 minutes. A 1 M aqueous solution mmol), 3-methylsulfinylphenylboronic acid (0.0602 g, 0.33 mmol),

of K₂CO₃(2.1 eq) was then added. The reaction mixture was heated and Pd(PPh₃)₂Cl₂(0.0105 g, 0.015 mmol) in anhydrous DMF (2.0 mL)

to 100 °C and left to magnetically stir at this temperature for 24 was purged with nitrogen for 30 minutes. A 1 M aqueous solution

hours (28) and 15 hours (3). The reaction mixture was then diluted of K₂CO₃(0.0435 g, 0.32 mmol) was then added. The reaction

with DCM (60 mL), washed with deionized water (40 mL × 7), mixture was heated to 100 °C and magnetically stirred at this

saturated aqueous solutions of NaHCO₃(40 mL × 3), NH₄Cl (40 mL × temperature for 20 hours. The reaction mixture was then diluted

3), and NaCl (40 mL × 1). The organic layer was dried (Na₂SO₄), after with DCM (60 mL), washed with deionized water (40 mL × 6),

which the solvent was removed in vacuo. The resulting crude saturated aqueous solutions of NaHCO₃(40 mL × 3), NH₄Cl (40 mL ×

mixture was subjected to prep-TLC purification to get the title 3) and NaCl (40 mL × 1). The organic layer was dried (MgSO₄),

compounds.

3,6-Bis(4-(methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine

where after the solvent was removed in vacuo. The resulting crude

mixture was subjected to prep-TLC (developed twice in 4%

(3). Purified by prep-TLC (developed twice in 5% CH₃OH/DCM). CH₃OH/DCM) to get 13 as an oil. Crystallization with diethyl ether

Crystallized in diethyl ether. Yellow solid (0.0665 g, 39%); mp 180.4 gave 13 as a yellow solid (0.0481 g, 40%); R_f(CH₃OH : CH₂Cl₂4 : 96)

1

– 182.3 °C (from diethyl ether); R_f(CH₃OH : CH₂Cl₂4 : 96) 0.21; H- 0.17; H-NMR δ_H(400 MHz; DMSO-d₆) 8.80 (m, 1H), 8.48 – 8.42 (m,

NMR δ_H(400 MHz; DMSO-d₆) 8.48 (d, J = 8.8 Hz, 2H), 8.45 (s, 1H), 2H), 8.42 – 8.35 (m, 2H), 8.33 (dt, J = 7.6, 1.6 Hz, 1H), 8.03 (d, J = 9.5

8.39 (d, J = 9.6 Hz, 1H), 8.35 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 9.6 Hz, Hz, 1H), 7.94 – 7.88 (m, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.76 (t, J = 7.7

1H), 7.91 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.8 Hz, 2H), 2.84 (s, 3H), 2.83 Hz, 1H), 7.73 – 7.69 (m, 1H), 2.88 (s, 3H), 2.86 (s, 3H) ¹³C-NMR δ_C

(s, 3H); ¹³C-NMR δ_C(101 MHz; DMSO-d₆) 151.04, 148.96, 145.89, (101 MHz; DMSO-d₆) 150.75, 148.34, 147.67, 140.06, 136.44,

140.15, 137.75, 135.09, 131.10, 128.43 (2C), 127.23 (3C), 127.13, 134.81, 130.65, 130.14, 129.89, 129.73, 128.72, 127.15, 127.08,

124.91 (2C), 124.68 (2C), 116.98, 43.67 (2C); LC-MS, APCI⁺: m/z [M + 125.63, 123.28, 122.60, 121.19, 116.75, 43.97, 43.82; LC-MS, APCI⁺:

H]⁺= 396.1, calculated exact mass = 395.0762, purity: 98.5%, t_r= 3.7 m/z [M + H]⁺= 396.1, calculated exact mass = 395.0762, purity:

min.

99.8%, t_r= 3.8 min.

General procedure for synthesis of aminated analogues (24

27). suspension of 3-bromo-6-(3-

6-(3-(Methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine (4h). A

suspension of 6-chloroimidazo[1,2-b]pyridazine (2a) (1.50 g, 9.8

-

A

mmol), 3-methylsulfinylphenylboronic acid (1.78 g, 9.8 mmol), and (methylsulfinyl)phenyl)imidazo[1,2-b]pyridazine (4i) (1.0 eq), an

Pd(PPh₃)₂Cl₂(0.344 g, 0.49 mmol) in anhydrous DMF (30 mL) was appropriate amine (1.3 eq), Pd₂(dba)₃(0.2 eq), BrettPhos (0.12 eq)

purged with nitrogen for 30 minutes. A 1 M aqueous solution of and Cs₂CO₃(2.0 eq) in anhydrous 1,4-dioxane (2 mL) in a sealed

K₂CO₃(1.42 g, 10.29 mmol) was then added. The reaction mixture tube was flashed with nitrogen for 20 minutes. The reaction

was heated to 100 °C and magnetically stirred at this temperature mixture was then heated to 120 °C and stirred for 5 - 39 hours. The

for 3.25 hours. The black reaction mixture was then diluted with solvent was removed in vacuo after which the resulting black

DCM (150 mL), washed with deionized water (75 mL × 8), saturated residue was taken up in DCM (60 mL), washed with saturated

aqueous solutions of NaHCO₃(75 mL × 3), NH₄Cl (75 mL × 1) and aqueous solutions of NaHCO₃(40 mL × 3) and NaCl (40 mL × 1). The

NaCl (50 mL × 5). After drying (MgSO₄) the organic layer, the solvent organic layer was dried (Na₂SO₄), after which the solvent was

was removed in vacuo and the resulting crude mixture subjected to removed in vacuo and the crude mixture further purified.

flash column chromatography (0 – 5% CH₃OH/DCM) to afford the

6-(3-(Methylsulfinyl)phenyl)-N-(4-

phenylated intermediate, 4h which was further crystallized in (methylsulfonyl)phenyl)imidazo[1,2-b]pyridazin-3-amine

(24).

diethyl ether to obtain a yellow solid (1.20 g, 48%); ¹H-NMR δ_H(400 Purified by prep-TLC (developed thrice in 2.5% CH₃OH/DCM).

MHz; CDCl₃) 8.36 (td, J = 1.8, 0.6 Hz, 1H), 8.25 (d, J = 9.6 Hz, 1H), Crystallized in diethyl ether. Brown solid (0.0121 g, 9%); mp 105.0 –

8.15 (dt, J = 7.7, 1.7 Hz, 1H), 8.10 (d, J = 1.3 Hz, 1H), 7.89 (d, J = 1.4 107.2 °C (from diethyl ether); R_f(CH₃OH : CH₂Cl₂4 : 96) 0.23; ¹H-

12 | J. Name., 2012, 00, 1-3

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ARTICLE

NMR δ_H(400 MHz; DMSO-d₆) 9.13 (s, 1H), 8.32 – 8.25 (m, 2H), 8.18

(d, J = 7.7 Hz, 1H), 7.91 – 7.80 (m, 3H), 7.78 – 7.68 (m, 3H), 6.99 (d, J

= 8.4 Hz, 2H), 3.11 (s, 3H), 2.80 (s, 3H); ¹³C-NMR δ_C(101 MHz;

DMSO-d₆) 150.57, 149.91, 148.01, 136.65, 135.99, 130.46, 130.33,

129.64, 129.31 (2C), 127.66, 127.04, 126.95, 125.62, 122.61,

7686–7698.

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(11)

Osborne, S.; Chapman, T.; Large, J.; Bouloc, N.; Wallace, C.

Fused Heterocyclic Compounds for Use in the Treatment of

Malaria. WO 2011101640, 2011.

Lemercier, G.; Fernandez-Montalvan, A.; Shaw, J. P.;

Kugelstadt, D.; Bomke, J.; Domostoj, M.; Schwarz, M. K.;

Scheer, A.; Kappes, B.; Leroy, D. Identification and

Characterization of Novel Small Molecules as Potent

Inhibitors of the Plasmodial Calcium-Dependent Protein

Kinase 1. Biochemistry 2009, 48, 6379–6389.

115.86, 114.13 (2C), 44.75, 43.63; LC-MS, APCI⁺: m/z [M + H]⁺

=

427.1, calculated exact mass = 426.0820, purity: 97.7%, t_r= 3.4 min.

Conflicts of interest

There are no conflicts to declare.

(12)

(13)

Sahu, N. K.; Kohli, D. V. Structural Insight for

Imidazopyridazines as Malarial Kinase PfPK7 Inhibitors

Using QSAR Techniques. Med. Chem. (Los. Angeles). 2012,

8, 636–648.

Acknowledgements

We gratefully acknowledge the support of the University of Cape

Town, South African Medical Research Council and the South

African Research Chairs Initiative of the Department of Science and

Technology administered through the South African National

Research Foundation (K.C.). At Swiss TPH, we thank Christoph

Fischli, Christin Gumpp, Sibylle Sax and Christian Scheurer for

assistance in performing the antimalarial assays.

McNamara, C. W.; Lee, M. C. S.; Lim, C. S.; Lim, S. H.;

Roland, J.; Nagle, A.; Simon, O.; Yeung, B. K. S.; Chatterjee,

A. K.; McCormack, S. L.; Manary, M. J.; Zeeman, A.-M.;

Dechering, K. J.; Kumar, T. R. S.; Henrich, P. P.; Gagaring, K.;

Ibanez, M.; Kato, N.; Kuhen, K. L.; Fischli, C.; Rottmann, M.;

Plouffe, D. M.; Bursulaya, B.; Meister, S.; Rameh, L.;

Trappe, J.; Haasen, D.; Timmerman, M.; Sauerwein, R. W.;

Suwanarusk, R.; Russell, B.; Renia, L.; Nosten, F.; Tully, D.

C.; Kocken, C. H. M.; Glynne, R. J.; Bodenreider, C.; Fidock,

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Le Manach, C.; Gonzàlez Cabrera, D.; Douelle, F.; Nchinda,

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Imidazopyridazine Hits from High Throughput Screening of

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Antiplasmodial imidazopyridazines: structure-activity relationship studies lead to the identification of analogues with improved solubility and hERG profiles

DOI: 10.1039/C8MD00382C

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