Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and in Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model

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Identiﬁcation of 2,4-Disubstituted Imidazopyridines as Hemozoin

Formation Inhibitors with Fast-Killing Kinetics and In Vivo Eﬃcacy in

the Plasmodium falciparum NSG Mouse Model

́

Andre Horatscheck, Ana Andrijevic, Aloysius T. Nchinda, Claire Le Manach, Tanya Paquet,

Lutete Peguy Khonde, Jean Dam, Kailash Pawar, Dale Taylor, Nina Lawrence, Christel Brunschwig,

̈

Liezl Gibhard, Mathew Njoroge, Janette Reader, Mariette van der Watt, Kathryn Wicht,

Ana Carolina C. de Sousa, John Okombo, Keletso Maepa, Timothy J. Egan, Lyn-Marie Birkholtz,

Gregory S. Basarab, Sergio Wittlin, Paul V. Fish, Leslie J. Street, James Du ﬀ y, and Kelly Chibale *

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Supporting Information

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ABSTRACT: A series of 2,4-disubstituted imidazopyridines,

originating from a SoftFocus Kinase library, was identiﬁed from

a high throughput phenotypic screen against the human malaria

parasite Plasmodium falciparum. Hit compounds showed moderate

asexual blood stage activity. During lead optimization, several

issues were ﬂagged such as cross-resistance against the multidrug-

resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were

addressed through structure−activity and structure−property

relationship studies. Pharmacokinetic properties were assessed in

mice for compounds showing desirable in vitro activity, a selectivity

window over cytotoxicity, and microsomal metabolic stability.

Frontrunner compound 37 showed good exposure in mice

combined with good in vitro activity against the malaria parasite,

which translated into in vivo eﬃcacy in the P. falciparum NOD-scid IL-2Rγ^null(NSG) mouse model. Preliminary mechanistic studies

suggest inhibition of hemozoin formation as a contributing mode of action.

hemozoin. However, certain mutations in the Plasmodium

falciparum chloroquine resistance transporter (Pf CRT) lead to

chloroquine eﬄux and resistance to the drug.⁵Numerous eﬀorts

are being made to develop drugs targeting the chloroquine mode

of action without being susceptible to the Pf CRT mode of

resistance.

We previously reported the development of a fast-killing and

highly eﬃcacious 2,6-disubstituted imidazopyridine series from

hits identiﬁed during the screening of a SoftFocus Kinase (SFK)

library provided by BioFocus (now Charles River).^6,7We herein

report the development of a fast-killing 2,4-disubstituted

imidazopyridine series originating from the same screen. The

new series was characterized by good in vitro activity against

Plasmodium falciparum strains, high aqueous solubility, and

INTRODUCTION

■

Despite a reduction in mortality from 585,000 people in 2010 to

405,000 people in 2018 according to the World Health

Organization (WHO) estimates, malaria remains one of the

world’s deadliest diseases. Children under the age of ﬁve are the

most vulnerable population, especially in sub-Saharan Africa.¹

The continuous emergence of resistant Plasmodium parasites

and mosquitoes against frontline antimalarials and insecticides,

respectively, further hampers current attempts to control

malaria.¹⁻³This warrants an urgent search for new treatment

options with either novel modes of action or known modes of

action without cross-resistance against clinical isolates. Some of

the most eﬃcient modes of action to kill Plasmodium parasites as

seen, for example, with chloroquine, target its digestive vacuole

(DV), where the parasite breaks down hemoglobin as a source of

amino acids.⁴One of the end products of this catabolic process is

free heme that can oxidize to Fe (III) hematin, both of which are

toxic to the parasite. In order to eliminate free heme, the parasite

has developed a detoxiﬁcation process to convert it to an

insoluble crystal called hemozoin. This detoxiﬁcation process is

prevented by chloroquine that accumulates in the DV, binds to

free heme, and thereby prevents heme conversion to crystalline

Received: August 12, 2020

https://dx.doi.org/10.1021/acs.jmedchem.0c01411

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A

Journal of Medicinal Chemistry

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a

Scheme 1. General Synthetic Route for Synthesis of the Imidazopyridines

a

Conditions: (a) R²COOH (R²= Ar), Eaton’s reagent, 16 h, 100 °C; (b) POBr₃, 3 h, 120 °C; (c) amine (R¹), EtOH, 6 h, mw 140 °C; (d) primary

or secondary amine (R¹), n-BuOH, 16 h, 140 °C.

desirable physicochemical space. Therefore, a hit-to-lead drug

discovery program was initiated that successfully reduced cross-

resistance seen with earlier hits of this series against the

multidrug-resistant P. falciparum isolate K1 and signiﬁcantly

improved cytotoxicity and hERG margins over antiplasmodium

activity. This led to the identiﬁcation of lead compound 37,

which demonstrated an in vivo proof-of-concept in a NOD-scid

IL-2Rγ^null(NSG) mouse model of P. falciparum infection.

RESULTS AND DISCUSSION

■

Antiplasmodium Activity, Cytotoxicity, Absorption,

Distribution, Metabolism, and Excretion (ADME) Proﬁl-

ing. All compounds were evaluated for antiplasmodium activity

against the asexual blood stages using the drug-sensitive NF54

strain of P. falciparum. Compounds with IC₅₀values lower than

0.50 μM were further tested against the multidrug-(chloroquine,

pyrimethamine, and sulfadoxine)-resistant K1 strain to check for

cross-resistance. Chloroquine and artesunate were used as

reference compounds in these experiments. A selection of

compounds was also tested against the liver stages of the rodent

parasite P. berghei and early- and late-stage gametocytes of P.

falciparum to evaluate their potential against other parasite life

cycle stages.

Aqueous solubility and cytotoxicity were monitored through-

out the project as they represent key parameters that can

indicate limitations for either absorption from the intestines and

drug safety, respectively. A benchmark for criteria to progress

compounds in the diﬀerent discovery stages of malaria drug

development can be found on the Medicines for Malaria

Venture (MMV) website.⁸Equilibrium aqueous solubility was

determined at an upper intestinal pH of 6.5 for all compounds

using a miniaturized shake ﬂask method. Compounds with

aqueous solubility greater than 10 μM and in vitro NF54 activity

lower than 0.50 μM were assessed in a 30 min microsomal

metabolic stability assay by incubating with human, rat, and

mouse liver microsomes. Cytotoxicity was determined in

Chinese hamster ovary (CHO) cells for compounds with

NF54 IC₅₀values lower than 0.50 μM with the goal of

identifying compounds with selective antiparasitic activity. In

order to get an early assessment of potential cardiotoxicity risks,

the inhibitory activity of selected compounds against the human

CHEMISTRY

■

The general synthetic route to the imidazopyridine scaﬀold II

was achieved by condensing the desired benzoic acid derivatives

and commercially available 2-chloro-3,4-diaminopyridine I,

using Eaton’s reagent (P₂O₅in MeSO₃H) at 100 °C for 16 h,

in good yields up to 99% (Scheme 1). The chloro-

imidazopyridine II was converted to the bromide III using

phosphoryl bromide before being coupled to the corresponding

amines via nucleophilic aryl substitution (S_NAr) at 140 °C under

microwave irradiation. In a diﬀerent approach, II was directly

converted to IV after being heated at 140 °C with the

corresponding amines in n-butanol in a sealed tube.

Commercially available amines were used unless otherwise

indicated. In the case of primary or secondary bis-amines, tert-

butyl carbamate (Boc)- or benzyl carbamate (Cbz)-protected

precursors were used for the S_NAr reactions. The Boc protecting

group was subsequently removed under acidic conditions. The

Cbz group was removed by reﬂuxing in TFA or by hydro-

genolysis.

Commercially unavailable aminomethyl-4-piperidine build-

ing blocks VII that were introduced onto the imidazopyridine

scaﬀold were synthesized from the Boc-protected piperidine-4-

yl methanol V (Scheme 2). Conversion of alcohol V to the

̀

ether-a-go-go related gene (hERG) channel was determined in a

patch-clamp assay. All methods for the assays mentioned above

are described further in the Supporting Information.

Scheme 2. Synthesis of Aminomethyl-4-piperazine Building

Blocks

a

In Vitro Antiplasmodium Activity and Cytotoxicity.

Following a single-point high throughput screen of a SFK library

against NF54, hit compounds harboring a 2,4-disubstituted

imidazopyridine scaﬀold, with IC₅₀values less than 0.50 μM,

were identiﬁed as exempliﬁed by 1 (IC₅₀= 0.24 μM) and 2 (IC₅₀

= 0.49 μM, Figure 1). Among these, R¹groups containing a basic

aliphatic functionality were a common feature, and derivatives

with 3,4-diﬂuorophenyl and 3-triﬂuoromethoxyphenyl groups at

the R²position had the highest activity.

a

Conditions: (a) CBr₄, PPh₃, DCM, 25 °C, 8 h; (b) K₂CO₃, HNR₂,

MeCN, 60 °C, 24 h; (c) HCl/dioxane, DCM, 25 °C, 6 h.

Studies to determine the minimum pharmacophore (Table 1)

showed that a basic functionality on the R¹substituent predicted

to be protonated at physiological pH was beneﬁcial for NF54

activity as seen for morpholine 4 and piperazinone 3 with

considerably weaker activity (NF54 IC₅₀= 3.1 μM and > 10 μM,

respectively) relative to the hit piperazine 1 (NF54 IC₅₀= 0.24

μM). The basicity of the pyridine nitrogen in the scaﬀold also

appeared to be beneﬁcial for activity. Hence, removing the

nitrogen that links the substituent to the scaﬀold as with 5

bromide VI using carbon tetrabromide and triphenylphosphine

followed by a nucleophilic substitution with the desired amine

(e.g., substituted piperazines or morpholines) and the removal

of the Boc-protecting group using HCl in dioxane yielded the

desired building blocks. The detailed syntheses of other R¹

building blocks are described in the Supporting Information.

B

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however, that the predicted pK_aof the imidazopyridines is more

than one log unit higher than the measured values,

demonstrating the limitations of the calculations. Nonetheless,

the calculations can be useful for estimating relative pK_avalues

within an analogue series. Substituted R¹piperazines having

slightly larger substituents such as the pyrrolidopiperazine 7

(NF54 IC₅₀= 0.22 μM) and the hydroxyethyl derivative 8

(NF54 IC₅₀= 0.57 μM) maintained their activity, unless their

basicity was lowered by the substituent as with the triﬂuoroethyl

analogue 9 (NF54 IC₅₀= 2.3 μM) and the N-phenyl analogue 10

(NF54 IC₅₀> 1 μM). N-demethylation of 1 did not have a

signiﬁcant eﬀect (6: NF54 IC₅₀= 0.17 μM) on activity.

Figure 1. Hit compounds 1 and 2 from a phenotypic screen against the

asexual blood stages of P. falciparum.

After establishing that a basic center was required for activity,

SAR studies on the R¹substituent were further expanded (Table

2). The addition of methyl groups to obtain 3,5-dimethyl

piperazine derivative 11 (NF54 IC₅₀= 0.076 μM) led to the ﬁrst

compound with sub-100 nanomolar activity, while its N-methyl

analogue 12 (NF54 IC₅₀= 0.26 μM) was signiﬁcantly less active.

Interestingly, this trend was reversed when expanding the six-

membered piperazine ring to a seven-membered diazepane ring.

The desmethyl diazepane 13 (NF54 IC₅₀= 0.21 μM) was

(NF54 IC₅₀> 10 μM) abolished activity at the highest

concentration tested (compared to 6, NF54 IC₅₀= 0.17 μM).

The change from R¹being an amino to alkyl group was predicted

to decrease the pK_aof the scaﬀold pyridine nitrogen from 8.3 to

5.5 (ACD Labs Percepta v 2019.1.3), which could explain the

loss of activity. Further structure-activity relationship (SAR)

supports the importance of the scaﬀold pK_a: the pyrrolopyr-

idines (SAR) to support the importance of the scaﬀold pK_a, the

pyrrolopyridines with a measured pK_aone log unit higher than

the imidazopyridines (pK_aof ∼8 vs ∼7) were slightly more

active (Supporting Information, Table S1). It should be noted,

signiﬁcantly less active than the methylated (14, NF54 IC₅₀

=

0.061 μM), cyclopropylmethyl (15, NF54 IC₅₀= 0.020 μM),

Table 1. Studies to Determine the Minimum Pharmacophore at the R¹Substituent

a

Mean from 3 values of ≥2 independent experiments with multidrug-resistant (K1) and multidrug-sensitive (NF54) strains of P. falciparum using

the parasite lactate dehydrogenase (pLDH) assay. The majority of the individual values varied less than 2× (maximum 3×). A set of compounds

b

was conﬁrmed in the [³H]-hypoxanthine incorporation assay. Mean of 3 values performed on one occasion. Individual replicates varied less than

2×.

C

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Table 2. SAR and SPR Studies with Variations on the R¹

Substituent

Table 2. continued

compounds was conﬁrmed in the [³H]-hypoxanthine incorporation

b

assay. Mean of 3 values performed on one occasion. Individual

replicates varied less than 2×.

and bridged tricyclic (16, NF54 IC₅₀= 0.079 μM) analogues.

Relative to the initial hit compound 1, the cyclopropylmethyl

diazepane 15 gained more than 10-fold activity while

maintaining high aqueous solubility at pH 6.5 (200 μM) and

low in vitro cytotoxicity (CC₅₀= 17 μM), resulting in a selectivity

index (SI) of 850 over NF54 activity.

One drawback of this series became evident as cross-

resistance was observed in the multidrug-resistant K1 strain

where IC₅₀values were 4-fold or higher compared to NF54. For

example, the 4-aminopiperidine 25 showed a cross-resistance

ratio of 16 (NF54 IC₅₀= 0.073 μM, K1 IC₅₀= 1.2 μM for a K1/

NF54 = 16) comparable to that of chloroquine (K1/NF54 =

35). Minimizing the activity shift between drug-sensitive and

drug-resistant strains is important for potential clinical utility

since clinically used antimalarial drugs must be active against

drug-resistant strains. A higher IC₅₀for a given ﬁeld isolate

would require a higher drug dose and a higher blood exposure,

which in turn would increase the probability of adverse eﬀects.

The mechanistic reason for the variable cross-resistance in this

series is unknown, but the working hypothesis was that drug

resistance occurred due to eﬄux of certain chemotypes, e.g., by

Pf CRT, and could be overcome by structural modiﬁcations.

Comparison of cross-resistance ratios in tertiary, secondary, and

primary amines highlighted that the cross-resistance ratio in

tertiary amines, such as methylpiperazine 1 (K1/NF54 = 2.1),

diazepanes 14 (K1/NF54 = 1.6) and 15 (K1/NF54 = 3.9), and

piperazinyl-4-piperidine 30 (K1/NF54 = 1.4) was lower

compared to their des-alkyl secondary amine analogues 6

(K1/NF54 = 5.7), 13 (K1/NF54 = 5), and 31 (K1/NF54 = 13).

N-Dimethylation of the 4-aminopiperidine 25 to give 26 led to a

reduction in the cross-resistance ratio from 16 to 1.1, with only a

minimal loss in activity (NF54 IC₅₀= 0.14 μM). Furthermore,

the insertion of a methylene group in 25 (K1/NF54 = 16) to

obtain 28 (NF54 IC₅₀= 0.068 μM, K1/NF54 = 2.8), thereby

pushing out the distal basic center, also signiﬁcantly reduced the

cross-resistance ratio while maintaining activity. Encouraged by

these observations, we focused on the aminopiperidine

derivatives that displayed overall favorable properties for further

optimization. The diazepane analogues (e.g., 14) were

discontinued due to microsomal metabolic instability (mouse

liver microsomes (MLM) CL_int,app= 145 μL/min/mg) and

hERG inhibitory potency (hERG IC₅₀= 0.4 μM) (see

Supporting Information, Tables S1 and S5).

A successful strategy to reduce cross-resistance and improve

NF54 activity was the extension of the basic center by

introducing a N-methylpiperazine substituent on the amino-

piperidine, creating the piperazinylpiperidine of 27 and 18

(NF54 IC₅₀s = 0.064 and 0.046 μM, respectively). However,

cytotoxicity margins for the latter compounds were low (SI =

47−110). Based on the promising activity and cross-resistance

trends of the aminomethyl-piperidine 28 and the gains of

activity observed by addition of a piperazine leading to 27, the

corresponding methyl piperazine analogues 22 and 29 were

synthesized and shown to possess signiﬁcantly higher activity

with NF54 IC₅₀values of less than 0.020 μM and comparable

values against K1. Further SAR studies indicated that

modulation of basicity could improve cytotoxicity margins, as

a

Mean from 3 values of ≥2 independent experiments with multidrug-

resistant (K1) and multidrug-sensitive (NF54) strains of P. falciparum

using the parasite lactate dehydrogenase (pLDH) assay. The majority

of the individual values varied less than 2× (maximum 3×). A set of

D

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Table 3. SAR Exploration of the R²Substituent

exempliﬁed by the morpholine 24 that exhibited only a slight

loss in activity (NF54 IC₅₀= 0.051 μM) while cytotoxicity

margins improved from 370-fold for piperazine 22 to 690-fold.

The inversed piperidinylpiperazine 19 was nearly equipotent

and showed higher cytotoxicity margins (CC₅₀= 33 μM, SI =

460) compared to 27 (CC₅₀= 3.0 μM) and 18 (CC₅₀= 5.0 μM).

In addition, 19 displayed the lowest intrinsic clearance in a

mouse PK study (see section further below) compared to other

compounds. As mentioned, alkylation of diazepanes increased

activity as opposed to methylation of piperazines, which

decreased activity, and the addition of a distant basic center

was beneﬁcial for NF54 activity and hERG (see section below).

Hence, a diazepane was used as a linker leading to 20 with the

goal of combining these positive trends. While the activity and

the cytotoxicity margin for 20 were high (NF54 IC₅₀= 0.035

μM, SI = 490), microsomal stability (MLM CL_int,app> 200 μL/

min/mg) was still low. The attempt to improve microsomal

stability by lowering the electron density in the diazepane

through the introduction of ﬂuorines (21: NF54 IC₅₀= 0.059

μM, CC₅₀= 33 μM) led to signiﬁcant improvements (MLM

CL_int,app= 33 μL/min/mg), albeit not suﬃciently so for

progression toward an optimized lead.

In order to further assess the inﬂuence of R²substitution on

antiplasmodium activity and improve the cytotoxicity SI, the

most promising R¹substituents in terms of activity,

piperazinylpiperidine 27 and piperazinylmethylpiperidine 29,

were used to explore diﬀerent R²substituents (Table 3). In

general, electron-withdrawing substituents on R²such as

ﬂuorine (e.g., 18, 32, 30, 38), nitrile (33), and ester (35)

showed high NF54 activity with IC₅₀values of less than 0.1 μM.

The saturated cyclopentanes of 36 and 44 (NF54 IC₅₀> 10 and

9.3 μM, respectively) and more polar substituents, such as the

amide of 41 (NF54 IC₅₀= 1.0 μM), the amine of 42 (NF54 IC₅₀

= 2.2 μM), and the carboxylic acid of 43 (NF54 IC₅₀> 10 μM),

were detrimental to activity.

Interestingly, the R²substitution pattern had a signiﬁcant

impact on cytotoxicity. While 3-triﬂuoromethoxy- and 3,4-

diﬂuorophenyl derivatives 27, 29, 18, and 22 displayed low

cytotoxicity margins (SI ranging from 47−370), the 3,5-

diﬂuorophenyl analogues exempliﬁed by 37 showed an

improvement (SI = 1040) while retaining good NF54 activity

(IC₅₀= 0.023 μM).

a

Mean from 3 values of ≥2 independent experiments with multidrug-

resistant (K1) and multidrug-sensitive (NF54) strains of P. falciparum

using the parasite lactate dehydrogenase (pLDH) assay. The majority

of the individual values varied less than 2× (maximum 3×). A set of

compounds was conﬁrmed in the [³H]-hypoxanthine incorporation

b

assay. Mean of 3 values performed on one occasion. Individual

replicates varied less than 2×.

Keeping the R²3,5-diﬂuorophenyl substituent constant,

further variations of the R¹substituent were done investigating

diﬀerent linkers (Table 4), such as the 1,4-diaminohexyl linkers

in compounds 46 and 47 as well as 3-position piperazinome-

thylpiperidines of 48 and 49. Several derivatives with modulated

basicity in the R¹substituent were also synthesized. However,

derivatives with replacements of the piperazine without a distal

Encouraged by the favorable proﬁle of 37, including high

NF54 and K1 activity and an excellent cytotoxicity margin,

changes to the scaﬀold imidazopyridine were investigated while

maintaining the R¹and R²substituents of 37 with the aim of

improving PK properties (shown later for select compounds), as

shown in Table 5. Omission of the pyridine nitrogen of the

scaﬀold as for the benzimidazole 57 was detrimental to activity

(NF54 IC₅₀= 0.83 μM), while replacement of one of the

imidazole nitrogens with a carbon atom, exempliﬁed by

pyrrolopyridine 58 (NF54 IC₅₀= 0.014 μM), maintained high

activity. Methylation at the N¹-position as in 59 was detrimental

(NF54 IC₅₀> 3.0 μM) to activity. None of the scaﬀold

modiﬁcations, such as the isomeric 60 and 61 or the pyridazine

64 and the pyrimidine 65 (NF54 IC₅₀values between 0.13 and

1.8 μM), led to any improvements in activity. There was a 2-fold

improvement in NF54 activity for the 6-Me analogue 62 (IC₅₀=

basic center, such as thiomorpholinedioxide 50 (NF54 IC₅₀

=

1.4 μM), diﬂuoropiperidine 51 (NF54 IC₅₀= 0.20 μM),

morpholine 52 (NF54 IC₅₀= 0.29 μM), and spiromorpholine

54 (NF54 IC₅₀= 0.18 μM), were detrimental to activity, further

highlighting the importance of the distal basic center. The N-

triﬂuoroethylated derivative 53 maintained NF54 activity below

0.1 μM despite the expectation that the distal piperazine

nitrogen would not be protonated at physiological pH. Bulkier

modiﬁcations on the distal piperazine nitrogen, such as tert-butyl

and methylimidazole (56 and 55), maintained activity and high

aqueous solubility. The cytotoxicity margin for 55 (SI = 1140)

was on par with the methyl substituted derivative 37, making it

interesting for further in vivo studies.

E

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that mitigated its activity (Supporting Information, Table S1).⁹

Herein, it was found that hERG activity was mainly inﬂuenced

by substitution changes on the scaﬀold and on the R¹

substituent.

Table 4. Activity and Cytotoxicity of Compounds with 3,5-

Diﬂuorophenyl R²Substituent

While the scaﬀold change from imidazopyridine to

pyrrolopyridine had little impact on NF54 activity, hERG

activity was up to 7-fold higher for the pyrrolopyridines

(compare hERG IC₅₀s of imidazopyridines/pyrrolopyridines,

e.g., 23/S2: 12 μM/3 μM; 32/S5: >30 μM/4 μM; and 37/58:

>30 μM/5 μM) (see Supporting Information, Table S1). It is

noteworthy that pyrrolopyridines in general showed a higher

measured pK_afor the core, as exempliﬁed by pyrrolopyridine S7

(pK_a= 8.2) versus imidazopyridine S6 (pK_a= 7.4), suggesting

that the higher basicity of the core heterocycle increased hERG

binding. Relative to the R¹substituent wherein there is a second

basic moiety, diazepanes (e.g., 14 and S4) as well as the

structurally similar piperazine 6 showed low hERG IC₅₀values

(0.4, 0.4, and 3.4 μM, respectively), which led to their

deprioritization. In general, R¹substituents with a second

protonation site closer to the core (e.g., 14, 24, 53, S8; all with

hERG IC₅₀s ≤ 3 μM) had a higher hERG liability compared to

compounds with a protonation site further away from the core

(e.g., 32 and 37, all >30 μM).

Overall, hERG activity was the lowest for imidazopyridine

compounds with a protonation site on the R¹substituent further

away from the core. As 37 (hERG IC₅₀> 30 μM) showed the

highest hERG margin (>1300-fold over the NF54 strain) along

with high NF54 activity and a high SI relative to cytotoxicity, it

was selected for advanced proﬁling as a frontrunner compound

(see below).

Parasitological and Life Cycle Proﬁling of Selected

Compounds. The frontrunner compound 37 was shown to be

fast acting without lag time in a parasite reduction ratio (PRR)

assay¹⁰at 10 × IC₅₀exhibiting a 4.9 log reduction of parasitemia

within the 48 h life cycle of P. falciparum (Figure 2). This boded

well for the compound and the series, as fast-killing kinetics of

the asexual blood stage parasites in the clinical setting are key to

a fast relief of symptoms.

Proﬁling of 37 against a panel of lab-generated resistant

mutants less susceptible to antimalarials in clinical development

showed no signs of cross-resistance (see Supporting Informa-

tion, Table S3). Hence, its mode of action or mode of resistance

most likely diﬀers from those ascribed to the other compounds

of Table S3, including inhibition of or binding to Pf eukaryotic

elongation factor 2 (PfeEF2),¹¹Pf phosphatidylinositol-4-kinase

(Pf PI4K),¹²Pf dihydroorotate dehydrogenase (Pf DHODH),¹³

Pf cyclic amine resistance locus (Pfcarl),¹⁴and Pf cytochrome B

(PfcytB).¹⁵While there is no information on the clinical

relevance of these lab mutants, it was encouraging to see that

they were equally susceptible to the tested imidazopyridine. In

addition, no cross-resistance was found when tested against a

panel of clinical ﬁeld isolates (see Supporting Information, Table

S4) although the higher IC₅₀against the K1 strain sets a

potential bar for which coverage would be needed for 37 in the

clinical setting. When 37 was assessed against a synchronized

culture of asexual parasites, it showed comparable activity on the

ring and schizont stages (IC₅₀= 0.027 and 0.060 μM,

respectively), similar to what is observed for chloroquine.¹⁶

Four compounds, including frontrunner 37, were selected for

proﬁling against the liver and transmissible sexual gametocyte

stages of the Plasmodium parasite life cycle (Table 6) in order to

assess their potential for prophylaxis and transmission blocking,

respectively. Data from the early-stage (stage I-III) and late-

a

Mean from 3 values of ≥2 independent experiments with multidrug-

resistant (K1) and multidrug-sensitive (NF54) strains of P. falciparum

using the parasite lactate dehydrogenase (pLDH) assay. The majority

of the individual values varied less than 2× (maximum 3×). A set of

compounds was conﬁrmed in the [³H]-hypoxanthine incorporation

assay. Mean of 3 values performed on one occasion. Individual

replicates varied less than 2×.

b

0.012 μM) over 37; however, the cytotoxicity margin (SI = 160)

was lower.

hERG Activity. As hERG activity has been determined for a

variety of compounds and served as an indicator of potential

cardiotoxicity risks, we set out to identify the chemical features

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Table 5. SAR Studies on Scaﬀold Changes to the Imidazopyridine Core

a

Mean from 3 values of ≥2 independent experiments with multidrug-resistant (K1) and multidrug-sensitive (NF54) strains of P. falciparum using

the parasite lactate dehydrogenase (pLDH) assay. The majority of the individual values varied less than 2× (maximum 3×). A set of compounds

b

was conﬁrmed in the [³H]-hypoxanthine incorporation assay. Mean of 3 values performed on one occasion. Individual replicates varied less than

2×.

stage (stage IV/V) sexual gametocytes were obtained from P.

falciparum. Liver stage data was obtained from the rodent

parasite P. berghei in vitro assay.¹⁷While all of the compounds

showed liver stage activity, only 24 showed a lower IC₅₀of 0.29

μM that was within 6-fold of its NF54 IC₅₀. With respect to the

transmission stages,¹⁸all of the compounds only showed weak to

moderate activity against early- and late-stage gametocytes with

25 being most notable (stages I−III IC₅₀= 0.78 μM and stages

IV/V IC₅₀= 2.8 μM). Interestingly, the relative activities against

the diﬀerent life cycle stages can diﬀer quite signiﬁcantly for each

compound. 37 only showed high activity against the asexual

stages of the parasite life cycle, therefore showing a similar

proﬁle to chloroquine in PRR,¹⁰stage speciﬁcity,¹⁹and life cycle

activity,²⁰albeit cross-resistance to chloroquine-resistant strains

was minimal.

In view of the aforementioned proﬁle similarity to

chloroquine, investigation of chloroquine-related inhibition of

hemozoin formation for 37 was carried out using a β-hematin

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hematin assay (IC₅₀= 25 μM), where compound accumulation

is not a requirement for potency, while being 25-fold less active

against whole cell NF54 parasites. Despite having an electron-

withdrawing group adjacent to the core nitrogen, the nitrile

derivative 63 maintained reasonable NF54 activity (IC₅₀= 0.077

μM), which may be a result of its greater ability to inhibit

hemozoin formation (βH IC₅₀= 8.5 μM). In contrast to that,

methyl derivative 62 was slightly less potent (β-hematin IC₅₀

=

15 μM), but a more basic core nitrogen could explain the higher

NF54 activity (IC₅₀= 0.012 μM) compared to the nitrile-

substituted derivative 63. The aminopiperidine 25, which

showed a high K1 cross-resistance ratio, was less active in the

β-hematin assay (IC₅₀= 148 μM) relative to the frontrunner 37,

suggesting a potential mode of action in the DV. To further

probe the eﬀect of these derivatives on hemozoin formation in

whole parasites, 25 was tested alongside 37 in a cell fractionation

assay, which determines dose response levels of both free heme

and hemozoin intracellularly. An increase in free heme with a

corresponding decrease in hemozoin, correlated with parasite

survival, would be expected for a true hemozoin formation

inhibitor. 25 showed statistically signiﬁcant dose response

increasing free heme and decreasing hemozoin (Figure 3),

indicative of a mode of action similar to that of chloroquine.

Surprisingly, no signiﬁcant change in absolute free heme levels

was observed with increasing concentrations of 37. However, a

decrease in hemozoin indicated that 37 could be targeting an

alternative digestive vacuole process, even though it may not be a

direct hemozoin inhibitor.

Figure 2. Results of the in vitro parasite reduction ratio (PRR) assay for

37 in comparison to known antimalarials.

(βH) inhibition assay.²¹This assay uses pyridine to detect

unreacted hematin during the formation of synthetic hemozoin

(β-hematin) mediated by the detergent Nonidet P-40 and oﬀers

a robust method to ﬁnd β-hematin formation inhibitors as a

surrogate to what occurs in the digestive vacuole (DV) of the

parasite. Herein, 37 showed an IC₅₀of 24 μM comparable to

chloroquine (IC₅₀= 21 μM). Basic compounds targeting the

processes in the DV presumably accumulate to high

concentrations in this acidic vacuole via pH trapping. Therefore,

both pH trapping and hemozoin inhibition may contribute to

NF54 activity. As a consequence, the core pyridazine analogue

64 without a basic core nitrogen was equipotent in the β-

In Vivo Pharmacokinetics. Compounds that showed high

NF54 activity, cytotoxicity margins, and microsomal stability

(Supporting Information, Table S5) were proﬁled for in vivo

Table 6. Activity of Selected Compound against the Plasmodium Life Cycle: Asexual Blood Stage (ABS), Liver Stage (Pb

c

Sporozoite), Stages I−III (Early), and Stages IV/V (Late) Gametocytes

a

Mean from 3 values of ≥2 independent experiments with multidrug-resistant (K1) and multidrug-sensitive (NF54) strains of P. falciparum using

the parasite lactate dehydrogenase (pLDH) assay. The majority of the individual values varied less than 2× (maximum 3×). A set of compounds

b

was conﬁrmed in the [³H]-hypoxanthine incorporation assay. Mean of 3 technical replicates using the luciferase assay. The individual values varied

c

less than 2×. n.a. = not active (<50% inhibition at 5 μM).

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Figure 3. (a) Correlation of parasite survival with intracellular free heme and (b) hemozoin levels, with increasing inhibitor concentration of 25 and 37.

a

Table 7. Mouse Pharmacokinetic Parameters Calculated from Non-compartmental Analysis

nominal dose

(mg/kg)

C_max

(μM)

T_max

(h)

t

_1/2terminal

(h)

mouse CL_b

(mL/min/kg)

V_ss

(L/kg)

AUC_0−t

(min·μmol/L)

C

_av,u,24h/NF54

entry route

F (%)

IC₅₀

1

i.v.

p.o.

i.v.

p.o.

i.v.

p.o.

i.v.

p.o.

i.v.

p.o.

i.v.

p.o.

i.v.

p.o.

2

5

2

20

2

20

2

20

2

20

2

20

2

10

1

5.9

5.4

8.2

27.3

11.4

19.6

34.8

20.2

9

4.3

20

30

31

6

215

195

406

398

250

2074

117

711

145

1198

473

1141

58

1.1

1.7

1.1

0.3

3.7

0.3

1.5

0.6

1.5

1.6

1.7

0.1

0.4

0.7

2.3

0.7

1.5

1.7

2.3

3.5

36

11

83

61

83

24

73

0.1

0.9

4.6

2.5

4.5

1.5

0.3

19

24

32

37

55

60

10.8

25.8

13.5

12.6

9.8

23.1

32.7

12.2

9.2

6.9

4.9

71.8

51

214

a

i.v., intravenous; p.o., oral; CL_b, blood clearance; V_ss, volume at the steady state; AUC, area under the concentration−time curve from 0 h to t_last

;

F, oral bioavailability.

pharmacokinetics in mice (Table 7), to identify compounds for

an in vivo eﬃcacy study in a mouse model of P. falciparum

infection (the NSG model). The selection for an in vivo eﬃcacy

study was made based on the best combination of having a high

ratio of unbound average drug concentration to NF54 activity

(C_av,u,24h/NF54 IC₅₀) (Table 7), as well as a high cytotoxicity

and hERG margin relative to NF54 activity. C_av,u,24hrepresents

an approximation of the average unbound drug concentration

and is determined from the unbound area under the curve

(AUC_uor AUC multiplied by unbound fraction f_u) divided by

the time course of the PK experiment, in this case 24 h

(Supporting Information, Table S9). It provides a convenient

yardstick that summarizes the aim of maximizing both drug

exposure and antiplasmodium activity. Total blood clearance

was low (<30% of hepatic blood ﬂow) to moderate for most

compounds shown in Table 7, and a variable and high volume of

distribution (8−96 L/kg) resulted in long plasma half-lives

ranging from 6 to 26 h. Generally, compounds showed high oral

bioavailability ranging from 24 to 83%. As an exception, 19

showed low bioavailability (11%), which, despite the lowest in

vivo intrinsic clearance of all the compounds (CL_int= 54 mL/

min/kg, compare Supporting Information Table S9), limited its

blood exposure. 37 showed low clearance and high oral

bioavailability (83%); however, its exposure in the blood was

relatively modest due to a very high volume of distribution (V_ss=

37 L/kg). It was mainly excreted as an unchanged parent in

urine, and only minor metabolites were identiﬁed in mouse

blood including the product of N-demethylation and a

combination of oxidation and glucuronidation products

(Supporting Information, Table S11). The N-demethylated

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Figure 4. Results of the in vivo eﬃcacy study using P. falciparum-infected NSG mice in a single-dose regimen of 37. (a) Percent parasitemia observed

after treatment. (b) Compound concentration in plasma over time following administration.

a

Table 8. Pharmacokinetic Data for 37 of P. falciparum Infected NSG and Healthy Balb/c Mice on PO Dosing

mouse PK parameter

dose (mg/kg)

T_max(h)

C

_max(μM)

C_max/dose (μM ·kg/mg) AUC₀₋₂₄(μM·min) AUC₀₋₂₄/dose (μM·min·kg/mg)

P. falciparum-infected NSG mice

1.5

5

15

1.5

2.5

6.0

12.5

1.0

4.0

0.04

0.13

0.81

2.84

6.68

2.84

1.52

0.03*

0.05

0.06

0.07

0.06

0.08

46

170

969

2856

5142

3102

1198

31**

34**

65

57

51

50

100

4*50

20

62

healthy Balb/c mice

1.7

60

a

Values statistically diﬀerent within columns according to ANOVA (*p < 0.05; **p < 0.01).

metabolite 45 was equipotent against NF54 to the parent but

showed 6-fold lower activity against the K1 strain. As the

metabolite concentrations were minor, they were not expected

to impact the activity of the parent compound in a mouse disease

model. Sustained exposure of 37 was also seen in rats with an

oral bioavailability of 84% (Supporting Information, Table S8),

oﬀering conﬁdence that PK would be similarly favorable in

higher species including, most importantly, humans. It was

hypothesized that renal clearance of the compound was due to a

lack of reabsorption from the renal tubules since, based on pK_a

predictions, it would be doubly protonated at a mouse urine pH

of 6. log D was measured to be 2.6 at pH 7.4 and is estimated to

be around 0.4 at pH 6. Therefore, compounds aiming at a lower

pK_aat the R¹substituent had been synthesized but none of them

showed a suﬃciently good proﬁle to be progressed into PK

studies. Exemplary for the core changes with a modulated, lower

core pK_a, 60 was assessed in a PK study but no improvement in

clearance was observed, and, due to low activity, C_av,u,24h/NF54

IC₅₀was low (0.3). While 24 was on par with 37 in terms of

expected eﬃcacy, 37 had a much superior hERG margin and was

therefore assessed for in vivo eﬃcacy in a NSG mouse model of

P. falciparum infection.

In vivo Eﬃcacy Studies. 37 was initially tested for eﬃcacy

in the NSG infection model via daily dosing over 4 days at 50

mg/kg three days following inoculation with P. falciparum. By

day 6 post-infection, parasitemia was reduced to below the limit

of detection (LOD) (Supporting Information, Scheme S1).

Hence, 37 achieved an important proof-of-concept (PoC)

milestone. A second study was carried out with escalating single

doses of 37 at 1.5, 5, 15, 50, and 100 mg/kg (Figure 4).

Parasitemia was reduced to below the LOD only at the 100 mg/

kg dose, and the ED₉₀was calculated to be 62 mg/kg. The ED₉₀

after a single or quadruple oral dose of chloroquine had

previously been determined to be ∼10 mg/kg (SW personal

communication) or ∼5 mg/kg, respectively.²²This suggests that

37 is less potent than chloroquine. In addition, the PK analysis

from the NSG infection model showed dose proportionality

between 15 and 100 mg/kg and good correlation between

healthy and infected mice data (Table 8). The ED₉₀dose of 62

mg/kg amounts to an approximate AUC = 3500 μM·min,

oﬀering a benchmark of what would be needed to be improved

with further analogue optimization work. Although 37 showed

comparable activity against NF54 to compounds in clinical

development including, for example, MMV390048 (NF54 IC₅₀

= 0.028 μM), its exposure on PO dosing was considerably lower

wherein the latter showed an AUC = 27912 μM·min on PO

dosing in healthy Balb/c mice, a value over 20-fold higher.¹²

Hence, less favorable PK has limited 37 from considerations for

further development.

CONCLUSIONS

■

Starting from hit compound 1, antiplasmodium activity was

improved by repositioning the basic center using diﬀerent R¹

substituents such as diazepanes and 4-aminopiperidines.

However, cross-resistance against the K1 strain was observed

for some compounds. During these structure−activity relation-

ship studies, tertiary amines were found to show the lowest

cross-resistance ratio (NF54/K1 mostly between 1 and 4), while

those of secondary and primary amines were increasingly higher

(up to 16-fold). The extension of the R¹basic center further

away from the core mitigated hERG activity increasing margins

from 6-fold in diazepanes to 1300-fold in 37. Cytotoxicity could

be regulated through the modulation of basicity of the R¹

substituent and the ﬂuorine substitution pattern of the R²

substituent. However, decreasing R¹basicity was detrimental

to NF54 activity thereby also compromising hERG margins. In

summary, hit-to-lead progression based on structure−activity

relationship studies and modulation of physicochemical proper-

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ﬂash chromatography using a gradient of (0.5 M NH₃) MeOH in DCM

to aﬀord the desired product.

ties led to 37 with signiﬁcantly improved in vitro antiplasmo-

dium (NF54 and K1) activity, cytotoxicity SI, and hERG

margins (Figure 5).

General Procedure B for the Amination. To a suspension of the

4-chloro-2-aryl-1H-imidazo[4,5-c]pyridine (1 equiv) and the corre-

sponding amine (3 equiv) in a minimum volume of n-BuOH (1.5 mL

for 100 mg of chloride starting material) was added DIPEA (5 equiv) in

a seal pressure reaction tube. The resulting reaction mixture was stirred

and heated at a temperature of 140 °C. After 16 h, the mixture was

cooled to 25 °C and concentrated in vacuo. The crude reaction mixture

was puriﬁed by ﬂash chromatography using a gradient of (0.5 M NH₃)

MeOH in DCM (0−30% gradient) aﬀording the desired product.

2-(3,4-Diﬂuorophenyl)-4-(4-methylpiperazin-1-yl)-3H-imidazo-

[4,5-c]pyridine (1). To a stirred solution of 4-bromo-2-(3,4-

diﬂuorophenyl)-1H-imidazo[4,5-c]pyridine (0.600 g, 1.93 mmol) and

N-methyl piperazine (0.290 g, 2.90 mmol) in MeCN (10 mL) was

added DIPEA (0.612 g, 4.75 mmol). The reaction mixture was

irradiated at 150 °C for 3 h, and the solvent was evaporated under

vacuum. The crude product was puriﬁed by column chromatography

using neutral alumina and 2% MeOH in DCM to yield 1.

Figure 5. Summary of key SAR and SPR learnings.

Yield: 0.130 g (20%). ¹H NMR (600 MHz, DMSO-d₆) δ 13.11 (s,

1H), 8.14−8.07 (m, 1H), 8.00−7.94 (m, 1H), 7.81 (d, J = 5.5 Hz, 1H),

7.63 (dt, J = 10.6, 8.5 Hz, 1H), 6.87 (d, J = 5.6 Hz, 1H), 4.11 (s, 4H),

2.46 (t, J = 5.0 Hz, 4H), 2.22 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆)

δ 151.1, 150.3 (dd, J = 249.5, 12.4 Hz), 149.7 (dd, J = 246.0, 13.0 Hz),

146.0, 140.9, 140.2, 128.4, 127.3, 123.3, 118.4 (d, J = 17.7 Hz), 115.2

(d, J = 18.8 Hz), 98.4, 54.8, 45.9, 45.7. LC/MS (ESI⁺): found m/z =

330.2 [M + H]⁺(calc. for C₁₇H₁₇F₂N₅m/z = 330.2 [M + H]⁺).

4-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

piperazin-2-one (3). General procedure B. Yield: 50%. ¹H NMR (300

MHz, DMSO-d₆) δ 13.26 (s, 1H), 8.14 (ddd, J = 11.8, 7.8, 2.2 Hz, 1H),

7.98 (s, 1H), 7.86 (d, J = 5.6 Hz, 1H), 7.74−7.57 (m, 1H), 6.95 (d, J =

5.7 Hz, 1H), 4.59 (s, 2H), 4.39 (t, J = 5.5 Hz, 2H), 3.36 (m, 4H). LC/

MS (ESI⁺): found m/z = 330.1 (M + H)⁺(calc. for C₁₆H₁₃F₂N₅O m/z =

330.1 [M + H]⁺).

4-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

morpholine (4). General procedure A using MeCN as solvent. Yield:

22%. ¹H NMR (400 MHz, DMSO-d₆): δ 13.19 (s, 1H), 8.13 (t, J = 7.60

Hz, 1H), 7.97 (s, 1H), 7.84 (d, J = 5.20 Hz, 1H), 7.65 (q, J = 8.40 Hz,

1H), 6.93 (d, J = 5.60 Hz, 1H), 4.09 (t, J = 4.40 Hz, 4H), 3.77 (t, J = 4.40

Hz, 4H). LC/MS (APCI⁺): found m/z 317.0 (M + H)⁺(calc. for

C₁₆H₁₄F₂N₄O m/z = 317.1 [M + H]⁺).

Compound 37 showed fast-killing kinetics in the in vitro PRR

assay. It did not show any hint of cross-resistance against panels

of lab-generated resistant mutants and ﬁeld isolates, which oﬀers

a clear advantage over chloroquine. With a favorable in vitro

proﬁle, 37 was prioritized for in vivo pharmacokinetic and

eﬃcacy studies, from which it demonstrated PoC in the NSG

mouse model with an ED₉₀of 62 mg/kg. Hence, 37 has

considerable value as a lead antimalarial compound without

issues of pre-existing resistance in the ﬁeld and the potential of

quick alleviation of symptomology in the clinic. Work still needs

to be done to fully understand the mode of action, which would

be of great value to identify an exploitable Plasmodium target for

drug design. Initial attempts to generate drug-resistant mutants

using 37 were unsuccessful. These “irresistible” compounds are

seen as a high priority for malaria drug development since

generation of resistance in the ﬁeld is deemed less likely.

Furthermore, improvement of PK attributes, antiplasmodium

activity, and/or both is still needed to deliver an optimized lead

that would have a suﬃciently low predicted dose in humans to

warrant further development.

2-(3,4-Diﬂuorophenyl)-4-(piperazin-1-yl)-1H-imidazo[4,5-c]-

pyridine (6). Step 1: General procedure A. tert-Butyl-4-(2-(3,4-

diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)piperazine-1-carba-

mate. Yield: 41%. LC/MS (ESI⁺): found m/z = 416.2 (M + H)⁺(calc.

for C₂₁H₂₃F₂N₅O₂m/z = 416.2 [M + H]⁺).

EXPERIMENTAL SECTION

■

All commercially available chemicals were purchased from either

Sigma-Aldrich, Combi-Blocks, Enamine, or Fluorochem. ¹H-NMR

spectra were recorded on a Bruker spectrometer at 300 MHz. ¹³C-NMR

spectra were recorded on either a Bruker spectrometer at 400 MHz (¹H

400.2 MHz; ¹³C 100.6 MHz) or a Bruker-600 (¹H 600.3 MHz; ¹³C

150.9 MHz). Chemical shifts (δ) are given in parts per million (ppm)

referenced to the respective residual solvent peak. Coupling constants,

J, are recorded in Hertz (Hz). Standard acronyms representing

multiplicity are used as follows: br s = broad singlet, s = singlet, d =

doublet, t = triplet, m = multiplet. Analytical thin-layer chromatography

(TLC) was performed on aluminum-backed silica gel 60 F254 (70−

230 mesh) plates. Column chromatography was performed with Merck

silica gel 60 (70−230 mesh) using a Teledyne ISCO Combiﬂash Rf.

Purity was determined using an Agilent LC/MS system consisting of an

Agilent 1260 Inﬁnity binary pump, an Agilent 1260 Inﬁnity diode array

detector (DAD), an Agilent 1290 Inﬁnity column compartment, an

Agilent 1260 Inﬁnity standard autosampler, and an Agilent 6120

quadrupole (single) mass spectrometer, equipped with an APCI and

ESI multimode ionization source, and all compounds tested for

biological activity were conﬁrmed to have ≥95% purity. The syntheses

of building blocks and further ﬁnal compounds are described in the

Supporting Information.

Step 2: To a suspension of tert-butyl-4-(2-(3,4-diﬂuorophenyl)-1H-

imidazo[4,5-c]pyridin-4-yl)piperazine-1-carbamate (58 mg, 0.140

mmol) in DCM (1 mL) was added TFA (0.1 mL) at 25 °C. After 3

h, the product was precipitated with Et₂O and desalted using an SCX-2

column.

Yield: 96%. ¹H NMR (300 MHz, DMSO-d₆) δ 8.10 (ddd, J = 11.8,

7.8, 2.2 Hz, 1H), 7.97 (m, 1H), 7.81 (d, J = 5.5 Hz, 1H), 7.62 (dt, J =

10.6, 8.5 Hz, 1H), 6.86 (d, J = 5.6 Hz, 1H), 4.07−3.98 (m, 4H), 2.94−

2.78 (m, 4H). LC/MS (ESI⁺): found m/z = 316.1 (M + H)⁺(calc. for

C₁₆H₁₅F₂N₅m/z = 316.1 [M + H]⁺).

2-(3,4-Diﬂuorophenyl)-4-(hexahydropyrrolo[1,2-a]pyrazin-

2(1H)-yl)-1H-imidazo[4,5-c]pyridine (7). General procedure A. Yield:

27%. ¹H NMR (300 MHz, DMSO-d₆) δ 8.11 (ddd, J = 11.8, 7.7, 2.1 Hz,

1H), 7.98 (ddd, J = 8.8, 4.4, 1.9 Hz, 1H), 7.82 (d, J = 5.6 Hz, 1H), 7.64

(dt, J = 10.5, 8.5 Hz, 1H), 6.87 (d, J = 5.5 Hz, 1H), 5.35 (d, J = 12.4 Hz,

2H), 3.16−2.99 (m, 3H), 2.73 (t, J = 11.1 Hz, 1H), 2.26 (s, 1H), 2.10

(d, J = 11.6 Hz, 2H), 1.96−1.79 (m, 1H), 1.79−1.61 (m, 2H), 1.57−

1.33 (m, 1H). LC/MS (ESI⁺): found m/z = 356.1 (M + H)⁺(calc. for

C₁₉H₁₉F₂N₅m/z = 356.2 [M + H]⁺).

2-(4-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

1

piperazin-1-yl)ethan-1-ol (8). General procedure B. Yield: 65%. H

General Procedure A for the Amination. To 4-bromo-2-aryl-

1H-imidazo[4,5-c]pyridine (1 equiv) and the corresponding amine (2

equiv) in EtOH, DIPEA (2.5 equiv) was added. The mixture was stirred

at 140 °C for 4.5−6 h in a microwave, cooled to 25 °C, and puriﬁed by

NMR (300 MHz, DMSO-d₆) δ 8.11 (ddd, J = 11.8, 7.8, 2.1 Hz, 1H),

7.97 (ddt, J = 8.2, 3.7, 1.6 Hz, 1H), 7.82 (d, J = 5.6 Hz, 1H), 7.63 (dt, J =

10.5, 8.5 Hz, 1H), 6.88 (d, J = 5.6 Hz, 1H), 4.46 (s, 1H), 4.12 (t, J = 4.7

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Hz, 4H), 3.57 (t, J = 6.2 Hz, 2H), 2.60 (t, J = 4.9 Hz, 4H), 2.53−2.41

(m, 3H). LC/MS (ESI⁺): found m/z = 360.1 [M + H]⁺(calc. for

C₁₈H₁₉F₂N₅O m/z = 360.2 [M + H]⁺).

NMR (300 MHz, DMSO-d₆) δ 12.99 (s, 1H), 8.15−8.04 (m, 1H),

8.01−7.92 (m, 1H), 7.81 (d, J = 5.6 Hz, 1H), 7.63 (dt, J = 10.5, 8.5 Hz,

1H), 7.43−7.19 (m, 6H), 6.85 (d, J = 5.6 Hz, 1H), 5.17 (d, J = 12.8 Hz,

2H), 5.03 (s, 2H), 3.71−3.55 (m, 1H), 3.15 (t, J = 11.6 Hz, 2H), 1.86

(d, J = 9.8 Hz, 2H), 1.45 (dd, J = 20.3, 11.3 Hz, 2H). LC/MS (ESI⁺):

found m/z = 464.2 [M + H]⁺(calc. for C₂₅H₂₃F₂N₅O₂m/z = 464.2 [M

+ H]⁺).

Step 2: To a suspension of benzyl (1-(2-(3,4-diﬂuorophenyl)-1H-

imidazo[4,5-c]pyridin-4-yl)piperidin-4-yl)carbamate (205 mg, 0.442

mmol) in MeOH (5 mL), 10% Pd/C (23 mg, 0.022 mmol) was added

under nitrogen. Hydrogen was bubbled through the solution using a

balloon with a needle for 3.5 h. The reaction mixture was ﬁltered

through a pad of Celite and washed with MeOH (3 × 8 mL). The

ﬁltrate was puriﬁed by preparative HPLC and subsequently desalted

using an SCX-2 column.

2-(3,4-Diﬂuorophenyl)-4-(4-(2,2,2-triﬂuoroethyl)piperazin-1-yl)-

1

1H-imidazo[4,5-c]pyridine (9). General procedure A. Yield: 20%. H

NMR (300 MHz, DMSO-d₆) δ 8.13 (ddd, J = 11.7, 7.8, 2.2 Hz, 1H),

7.98 (ddt, J = 10.5, 3.9, 1.6 Hz, 1H), 7.82 (d, J = 5.7 Hz, 1H), 7.64 (dt, J

= 10.6, 8.5 Hz, 1H), 6.92 (d, J = 5.7 Hz, 1H), 4.15 (t, J = 4.8 Hz, 4H),

3.24 (q, J = 10.2 Hz, 2H), 2.79 (t, J = 4.9 Hz, 4H). LC/MS (ESI⁺):

found m/z = 398.1 (M + H)⁺(calc. for C₁₈H₁₆F₅N₅m/z = 398.1 [M +

H]⁺).

2-(3,4-Diﬂuorophenyl)-4-(4-phenylpiperazin-1-yl)-1H-imidazo-

[4,5-c]pyridine (10). General procedure A. Yield: 15%. ¹H NMR (300

MHz, DMSO-d₆) δ 8.13 (ddd, J = 11.8, 7.8, 2.2 Hz, 1H), 8.05−7.93 (m,

1H), 7.85 (d, J = 5.6 Hz, 1H), 7.64 (dt, J = 10.6, 8.5 Hz, 1H), 7.30−7.17

(m, 2H), 7.01 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 5.6 Hz, 1H), 6.79 (t, J =

7.2 Hz, 1H), 4.27 (t, J = 4.9 Hz, 4H), 4.02 (s, 2H), 3.28 (2H, covered by

water peak). LC/MS (ESI⁺): found m/z = 392.1 (M + H)⁺(calc. for

C₂₂H₁₉F₂N₅m/z = 392.2 [M + H]⁺).

2-(3,4-Diﬂuorophenyl)-4-((3S,5R)-3,5-dimethylpiperazin-1-yl)-

1H-imidazo[4,5-c]pyridine (11). General procedure A. Yield: 47%. ¹H

NMR (300 MHz, methanol-d₄) δ 7.99 (ddd, J = 11.6, 7.6, 2.2 Hz, 1H),

7.91 (ddt, J = 8.0, 3.8, 1.7 Hz, 1H), 7.80 (d, J = 5.8 Hz, 1H), 7.44 (dt, J =

10.3, 8.4 Hz, 1H), 6.92 (d, J = 5.8 Hz, 1H), 5.19−5.05 (m, 2H), 3.03

(dtd, J = 12.8, 6.4, 2.8 Hz, 2H), 2.65 (dd, J = 12.9, 10.7 Hz, 2H), 1.22 (s,

3H), 1.20 (s, 3H). LC/MS (ESI⁺): found m/z = 344.2 [M + H]⁺(calc.

for C₁₈H₁₉F₂N₅m/z = 344.2 [M + H]⁺).

2-(3,4-Diﬂuorophenyl)-4-((3S,5R)-3,4,5-trimethylpiperazin-1-yl)-

1H-imidazo[4,5-c]pyridine (12). General procedure B. Yield: 66%. ¹H

NMR (300 MHz, DMSO-d₆) δ 13.41 (s, 1H), 8.29−8.13 (m, 1H), 8.02

(s, 1H), 7.89 (d, J = 5.6 Hz, 1H), 7.66 (dt, J = 10.5, 8.5 Hz, 1H), 7.01 (d,

J = 5.6 Hz, 1H), 5.42 (d, J = 13.4 Hz, 2H), 3.68−3.55 (m, 1H), 3.40 (m,

4H) 3.17−3.05 (m, 1H), 2.82 (s, 3H), 1.43 (6, 5H). LC/MS (ESI⁺):

found m/z = 358.2 [M + H]⁺(calc. for C₁₉H₂₁F₂N₅m/z = 358.2 [M +

H]⁺).

Yield: 45%. ¹H NMR (300 MHz, DMSO-d₆) δ 8.29−8.04 (m, 1H),

7.99 (br, 1H), 7.78 (d, J = 4.4 Hz, 1H), 7.71−7.48 (m, 1H), 6.84 (d, J =

5.2 Hz, 1H), 5.15 (d, J = 11.7 Hz, 2H), 3.10 (t, J = 11.6 Hz, 2H), 2.90

(br, 1H), 1.96−1.72 (m, 2H), 1.53−1.07 (m, 2H). LC/MS (ESI⁺):

found m/z = 330.1 [M + H]⁺(calc. for C₁₇H₁₇F₂N₅m/z = 330.2 [M +

H]⁺).

2-(3,4-Diﬂuorophenyl)-4-(4-(4-methylpiperazin-1-yl)piperidin-1-

yl)-1H-imidazo[4,5-c]pyridine (18). General procedure A. Yield: 32%.

¹H NMR (300 MHz, DMSO-d₆) δ 13.12 (s, 1H), 8.11 (ddd, J = 11.8,

7.8, 2.2 Hz, 1H), 7.98 (ddd, J = 8.8, 4.3, 1.9 Hz, 1H), 7.81 (d, J = 5.5 Hz,

1H), 7.64 (dt, J = 10.6, 8.5 Hz, 1H), 6.85 (d, J = 5.6 Hz, 1H), 5.32 (d, J =

11.8 Hz, 2H), 2.96 (t, J = 12.3 Hz, 2H), 2.33 (s, 4H), 2.15 (s, 3H), 1.88

(dd, J = 12.1, 8.8 Hz, 2H), 1.55−1.37 (m, 2H), 1.24 (d, J = 7.4 Hz, 1H).

Four protons hidden underneath the DMSO peak (by HH-COSY).

LC/MS (ESI⁺): found m/z = 413.2 [M + H]⁺(calc. for C₂₂H₂₆F₂N₆m/

z = 413.2 [M + H]⁺).

2-(3,4-Diﬂuorophenyl)-4-(4-(1-methylpiperidin-4-yl)piperazin-1-

yl)-1H-imidazo[4,5-c]pyridine (19). General procedure A. Yield: 46%.

¹H NMR (400 MHz, DMSO-d₆) δ 13.14 (s, 1H), 8.16−8.06 (m, 1H),

8.00−7.92 (m, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.64 (q, J = 9.0 Hz, 1H),

6.87 (d, J = 5.6 Hz, 1H), 4.08 (s, 4H), 2.83 (d, J = 11.1 Hz, 2H), 2.62 (t,

J = 4.9 Hz, 4H), 2.17 (s, 4H), 1.99−1.86 (m, 2H), 1.77 (d, J = 12.3 Hz,

2H), 1.55−1.38 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 151.0,

150.3 (dd, J = 248.7, 12.1 Hz), 149.7 (dd, J = 246.3, 13.6 Hz), 146.0,

140.9, 140.2, 128.3, 127.4, 123.3, 118.4 (d, J = 17.5 Hz), 115.3 (d, J =

18.8 Hz), 98.4, 60.7, 54.7, 49.0, 46.2, 45.6, 27.7. LC/MS (ESI⁺): found

m/z = 413.2 [M + H]⁺(calc. for C₂₂H₂₆F₂N₆m/z = 413.2 [M + H]⁺).

2-(3,4-Diﬂuorophenyl)-4-[4-(1-methyl-4-piperidyl)-1,4-diazepan-

1-yl]-1H-imidazo[4,5-c]pyridine (20). In a sealed tube, 4-(1,4-

diazepan-1-yl)-2-(3,4-diﬂuorophenyl)-1H-imidazo[4,5-c]pyridine 13

(113 mg, 0.340 mmol) and 1-methyl-4-piperidone (0.04 mL, 0.340

mmol) were dissolved in Ti(ⁱOPr)₄(0.12 mL, 0.410 mmol) and stirred

at 25 °C for 1 h. Solid NaCNBH₃(21.6 mg, 0.340 mmol) was then

added to the reaction mixture, and the reaction mixture was stirred at 25

°C 18 h. The reaction mixture was then concentrated in vacuo and

puriﬁed by ﬂash column chromatography using a gradient of 5−20%

(0.5 M NH₃) MeOH in DCM.

4-(1,4-Diazepan-1-yl)-2-(3,4-diﬂuorophenyl)-1H-imidazo[4,5-c]-

pyridine (13). General procedure B using Boc-diazepane. Thermal in

situ Boc deprotection. Yield: 18%. ¹H NMR (300 MHz, DMSO-d₆) δ

8.14 (ddd, J = 11.8, 7.8, 2.1 Hz, 1H), 8.03−7.93 (m, 1H), 7.83 (d, J =

5.6 Hz, 1H), 7.66 (dt, J = 10.6, 8.5 Hz, 1H), 6.87 (d, J = 5.6 Hz, 1H),

4.37 (t, J = 5.1 Hz, 2H), 4.24 (t, J = 6.1 Hz, 2H), 3.41 (t, J = 5.3 Hz, 2H),

3.22−3.16 (m, 2H), 2.18−2.07 (m, 2H). Note: The NH proton could

not be seen clearly due to water in DMSO-d₆. LC/MS (ESI⁺): found m/

z = 330.1 [M + H]⁺(calc. for C₁₇H₁₇F₂N₅m/z = 330.2 [M + H]⁺).

2-(3,4-Diﬂuorophenyl)-4-(4-methyl-1,4-diazepan-1-yl)-1H-

1

imidazo[4,5-c]pyridine (14). General procedure B. Yield: 75%. H

NMR (300 MHz, DMSO-d₆) δ 13.03 (s, 1H), 8.09 (ddd, J = 11.8, 7.8,

2.1 Hz, 1H), 7.95 (ddd, J = 8.2, 4.2, 2.0 Hz, 1H), 7.78 (d, J = 5.6 Hz,

1H), 7.63 (dt, J = 10.6, 8.5 Hz, 1H), 6.76 (d, J = 5.6 Hz, 1H), 4.27 (t, J =

4.9 Hz, 2H), 4.13 (t, J = 6.4 Hz, 2H), 2.78 (t, J = 4.8 Hz, 2H), 2.55 (d, J

= 6.9 Hz, 2H), 2.31 (s, 3H), 2.05−1.95 (m, 2H). LC/MS (ESI⁺): found

m/z = 344.1 [M + H]⁺(calc. for C₁₈H₁₉F₂N₅m/z = 344.2 [M + H]⁺).

4-(4-(Cyclopropylmethyl)-1,4-diazepan-1-yl)-2-(3,4-diﬂuoro-

phenyl)-1H-imidazo[4,5-c]pyridine (15). General procedure B. Yield:

31%. ¹H NMR (300 MHz, methanol-d₄) δ 8.02 (ddd, J = 11.5, 7.6, 2.1

Hz, 1H), 7.98−7.86 (m, 1H), 7.81 (d, J = 5.8 Hz, 1H), 7.44 (dt, J = 10.3,

8.4 Hz, 1H), 6.91 (d, J = 5.8 Hz, 1H), 4.60−4.41 (m, 2H), 4.22 (t, J =

6.3 Hz, 2H), 3.67−3.51 (m, 2H), 2.96 (d, J = 7.0 Hz, 2H), 2.48−2.27

(m, 2H), 1.30 (s, 1H), 1.19−0.84 (m, 2H), 0.82−0.63 (m, 2H), 0.47−

0.30 (m, 2H). LC/MS (ESI⁺): found m/z = 384.2 [M + H]⁺(calc. for

C₂₁H₂₃F₂N₅m/z = 384.2 [M + H]⁺).

1

Yield: 118 mg (81%). H NMR (300 MHz, methanol-d₄) δ 8.01

(ddd, J = 11.6, 7.7, 2.1 Hz, 1H), 7.91 (ddt, J = 7.9, 4.0, 1.7 Hz, 1H), 7.75

(d, J = 5.9 Hz, 1H), 7.44 (dt, J = 10.3, 8.4 Hz, 1H), 6.86 (d, J = 5.9 Hz,

1H), 4.37 (t, J = 5.2 Hz, 2H), 4.24 (t, J = 6.2 Hz, 2H), 3.22−3.07 (m,

4H), 2.96−2.75 (m, 3H), 2.47 (s, 3H), 2.40 (d, J = 11.8 Hz, 2H), 2.11

(q, J = 6.0 Hz, 2H), 1.96 (d, J = 13.1 Hz, 2H), 1.84−1.63 (m, 2H). LC/

MS (ESI⁺): found m/z = 427.1 [M + H]⁺(calc. for C₂₃H₂₉F₂N₆m/z =

427.2 [M + H]⁺).

4-[6,6-Diﬂuoro-4-(1-methyl-4-piperidyl)-1,4-diazepan-1-yl]-2-

(3,4-diﬂuorophenyl)-1H-imidazo[4,5-c]pyridine (21). General proce-

dure B (reaction mixture heated to 150 °C for 3 d). Yield: 4%. ¹H NMR

(300 MHz, methanol-d₄) δ 7.99 (t, J = 9.8 Hz, 1H), 7.89 (br s, 1H), 7.79

(d, J = 5.7 Hz, 1H), 7.43 (q, J = 8.8 Hz, 1H), 6.88 (d, J = 5.7 Hz, 1H),

4.86 (d, J = 2.5 Hz, 2H), 4.28 (d, J = 5.5 Hz, 2H), 3.17 (t, J = 5.5 Hz,

2H), 3.01 (t, J = 13.4 Hz, 2H), 2.90−2.80 (m, 1H), 2.77−2.66 (m, 2H),

2.63 (s, 3H), 1.95 (d, J = 13.4 Hz, 2H), 1.82−1.66 (m, 2H), 1.45−1.32

(m, 2H). LC/MS (ESI⁺): found m/z = 463.1 [M + H]⁺(calc. for

C₂₃H₂₇F₄N₆m/z = 463.1 [M + H]⁺).

4-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-1,4-

diazabicyclo[3.2.2]nonane (16). General procedure B. Yield: 34%. ¹H

NMR (300 MHz, DMSO-d₆) δ 8.23−8.06 (m, 1H), 8.06−7.91 (m,

1H), 7.84 (d, J = 5.6 Hz, 1H), 7.65 (dd, J = 19.1, 8.6 Hz, 1H), 6.87 (d, J

= 5.6 Hz, 1H), 5.63−5.44 (m, 1H), 4.70−4.57 (m, 2H), 3.46−3.40 (m,

5H), 3.21−3.15 (m, 2H), 2.34−2.15 (m, 2H), 2.15−1.94 (m, 2H). LC/

MS (ESI⁺): m/z = 356.0 [M + H]⁺(calc. for C₁₉H₁₉F₂N₅m/z = 356.2

[M + H]⁺).

1-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

piperidin-4-amine (17). Step 1: General procedure B. Yield: 75%. ¹H-

L

https://dx.doi.org/10.1021/acs.jmedchem.0c01411

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2-(3,4-Diﬂuorophenyl)-4-(4-((4-methylpiperazin-1-yl)methyl)-

piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (22). General procedure A.

Yield: 5%. ¹H NMR (300 MHz, methanol-d₄) δ 8.01 (ddd, J = 11.6, 7.7,

2.2 Hz, 1H), 7.92 (ddt, J = 8.0, 3.7, 1.8 Hz, 1H), 7.77 (d, J = 5.8 Hz,

1H), 7.44 (dt, J = 10.4, 8.4 Hz, 1H), 6.91 (d, J = 5.8 Hz, 1H), 5.15 (d, J =

13.1 Hz, 2H), 3.10 (t, J = 12.2 Hz, 2H), 2.64 (s, 7H), 2.34 (s, 3H), 2.30

(d, J = 6.5 Hz, 2H), 2.01−1.83 (m, 3H), 1.46−1.28 (m, 3H). LC/MS

(ESI⁺): found m/z = 427.2 [M + H]⁺(calc. for C₂₃H₂₈F₂N₆m/z = 427.2

[M + H]⁺).

(1-(2-(3-(Triﬂuoromethoxy)phenyl)-1H-imidazo[4,5-c]pyridin-4-

yl)piperidin-4-yl)methanamine (28). Step 1: Using tert-butyl-(piper-

idin-4-ylmethyl)carbamate. General procedure A. Yield: 99%. LC/MS

(ESI⁺): found m/z = 492.2 [M + H]⁺(calc. for C₂₄H₂₈F₃N₅O₃m/z =

492.2 [M + H]⁺).

Step 2: To a suspension of tert-butyl-((1-(2-(3-(triﬂuoromethoxy)-

phenyl)-1H-imidazo[4,5-c]pyridin-4-yl)piperidin-4-yl)methyl)-

carbamate (180 mg, 0.366 mmol) in DCM (2.2 mL) was added TFA

(0.22 mL) and stirred for 2 h at 25 °C. Et₂O (30 mL) was added, and

the precipitate was ﬁltered oﬀ and desalted using an SCX-2 column.

Yield: 68%. ¹H NMR (300 MHz, DMSO-d₆) δ 8.16 (dt, J = 8.0, 1.2

Hz, 1H), 8.09−8.02 (m, 1H), 7.79 (d, J = 5.6 Hz, 1H), 7.69 (t, J = 8.0

Hz, 1H), 7.47 (ddt, J = 8.2, 2.3, 1.1 Hz, 1H), 6.84 (d, J = 5.5 Hz, 1H),

5.32 (d, J = 13.0 Hz, 2H), 2.96 (td, J = 12.8, 2.5 Hz, 2H), 2.47 (d, J = 6.4

Hz, 2H), 1.85−1.72 (m, 2H), 1.63−1.45 (m, 1H), 1.29−1.08 (m, 2H).

LC/MS (ESI⁺): found m/z = 392.2 [M + H]⁺(calc. for C₁₉H₂₀F₃N₅O

m/z = 392.2 [M + H]⁺).

(1-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

piperidin-4-yl)(4-methylpiperazin-1-yl)methanone (23). General

1

procedure B. Yield: 12%. H NMR (300 MHz, DMSO-d₆) δ 10.24

(s, 1H), 8.17 (t, J = 9.8 Hz, 1H), 8.00 (s, 1H), 7.73 (dd, J = 23.6, 7.9 Hz,

2H), 7.07 (s, 1H), 5.21 (d, J = 13.0 Hz, 2H), 3.75−3.25 (m, 8H), 3.12

(m, 2H), 2.80 (s, 3H), 2.33 (s, 3H), 1.94−1.58 (m, 4H). LC/MS

(ESI⁺): found m/z = 441.2 [M + H]⁺(calc. for C₂₃H₂₆F₂N₆O m/z =

441.2 [M + H]⁺).

4-(4-((4-Methylpiperazin-1-yl)methyl)piperidin-1-yl)-2-(3-

(triﬂuoromethoxy)phenyl)-1H-imidazo[4,5-c]pyridine (29). General

procedure A. Yield: 8%. ¹H NMR (300 MHz, methanol-d₄) δ 8.11 (d, J

= 8.5 Hz, 1H), 8.03 (s, 1H), 7.78 (d, J = 5.8 Hz, 1H), 7.63 (dd, J = 8.0

Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 5.9 Hz, 1H), 5.17 (d, J =

13.1 Hz, 2H), 3.11 (t, J = 12.4 Hz, 2H), 2.55 (s, 7H), 2.31 (d, J = 11.1

Hz, 5H), 2.01−1.81 (m, 3H), 1.50−1.26 (m, 3H). LC/MS (ESI⁺)

found m/z = 475.2 [M + H]⁺(calc. for C₂₄H₂₉F₃N₆O m/z = 475.5 [M +

H]⁺).

4-((1-(2-(3,4-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

piperidin-4-yl)methyl)morpholine (24). General procedure B. Yield:

46%. ¹H NMR (400 MHz, DMSO-d₆) δ 13.14 (s, 1H), 8.16−8.06 (m,

1H), 8.00−7.92 (m, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.64 (q, J = 9.0 Hz,

1H), 6.87 (d, J = 5.6 Hz, 1H), 4.08 (s, 4H), 2.83 (d, J = 11.1 Hz, 2H),

2.62 (t, J = 4.9 Hz, 4H), 2.17 (s, 3H), 2.17−2.11 (m, 1H), 1.99−1.86

(m, 2H), 1.77 (d, J = 12.3 Hz, 2H), 1.46 (qd, J = 11.9, 11.3, 3.4 Hz, 2H).

¹³C NMR (151 MHz, DMSO-d₆) δ 151.2, 150.2 (dd, J = 248.2, 12.1

Hz), 149.7 (dd, J = 245.6, 12.7 Hz), 145.7, 140.9, 140.3, 128.3, 127.4,

123.2, 118.4 (d, J = 17.7 Hz), 115.1 (d, J = 19.0 Hz), 97.8, 66.2, 64.6,

53.8, 45.9, 32.9, 30.5. LC/MS (ESI⁺): found m/z = 414.2 [M + H]⁺

(calc. for C₂₂H₂₅F₂N₅O m/z = 414.4 [M + H]⁺).

1-(2-(3-(Triﬂuoromethoxy)phenyl)-1H-imidazo[4,5-c]pyridin-4-

yl)piperidin-4-amine (25). Step 1: Benzyl (1-(2-(3-

(triﬂuoromethoxy)phenyl)-1H-imidazo[4,5-c]pyridin-4-yl)piperidin-

4-yl)carbamate. General procedure A. Deviating from the routine

workup, a few drops of MeCN were added to the crude reaction mixture

followed by 80 mL of H₂O and a little MeOH to aﬀord the product as a

white precipitate. Yield: 78%. LC/MS (ESI⁺): found m/z = 512.2 [M +

H]⁺(calc. for C₂₆H₂₄F₃N₅O₃m/z = 512.2 [M + H]⁺).

2-(4-Fluoro-3-(triﬂuoromethyl)phenyl)-4-(4-(4-methylpiperazin-

1-yl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (30). General proce-

dure B. Yield: 49%. ¹H NMR (300 MHz, methanol-d₄) δ 8.48−8.38 (m,

2H), 7.80 (d, J = 5.8 Hz, 1H), 7.52 (dd, J = 10.1, 8.7 Hz, 1H), 6.93 (d, J

= 5.8 Hz, 1H), 5.28 (d, J = 13.2 Hz, 2H), 3.07 (dd, J = 13.2, 11.2 Hz,

2H), 2.85−2.65 (m, 4H), 2.65−2.40 (m, 5H), 2.30 (s, 3H), 2.07 (d, J =

11.6 Hz, 2H), 1.66 (qd, J = 12.2, 4.0 Hz, 2H). LC/MS (ESI⁺): found m/

z = 463.2 [M + H]⁺(calc. for C₂₃H₂₆F₄N₆m/z = 463.2 [M + H]⁺).

2-(4-Fluoro-3-(triﬂuoromethyl)phenyl)-4-(4-(piperazin-1-yl)-

piperidin-1-yl)-1H-imidazo[4,5-c]pyridine dihydrochloride (31).

Step 1: General procedure B using tert-butyl-4-(piperidin-4-yl)-

1

piperazine-1-carbamate. Yield: 90%. H NMR (300 MHz, methanol-

Step 2: To benzyl (1-(2-(3-(triﬂuoromethoxy)phenyl)-1H-imidazo-

[4,5-c]pyridin-4-yl)piperidin-4-yl)carbamate (374 mg, 0.731 mmol)

was added TFA (4.2 mL), and the solution was heated to 100 °C for 16

h. The TFA was removed in vacuo. The residue was sonicated with

Et₂O, and the solvent was removed in vacuo. The yellow solid was

dissolved in MeOH, puriﬁed by HPLC (NH₄OAc), and subsequently

desalted using an SCX-2 column.

d₄) δ 8.44 (d, J = 5.8 Hz, 1H), 8.43−8.38 (m, 1H), 7.80 (d, J = 5.7 Hz,

1H), 7.57−7.48 (m, 1H), 6.93 (d, J = 5.8 Hz, 1H), 5.29 (d, J = 13.0 Hz,

2H), 3.46 (t, J = 5.1 Hz, 4H), 3.14−3.01 (m, 2H), 2.68−2.57 (m, 5H),

2.04 (d, J = 12.4 Hz, 2H), 1.67 (qd, J = 12.2, 4.0 Hz, 2H), 1.48 (s, 9H).

LC/MS (ESI⁺): found m/z = 549.3 [M + H]⁺(calc. for C₂₇H₃₂F₄N₆O₂

m/z = 549.3 [M + H]⁺).

Step 2: To a solution of tert-butyl-4-(1-(2-(4-ﬂuoro-3-

(triﬂuoromethyl)phenyl)-1H-imidazo[4,5-c]pyridin-4-yl)piperidin-4-

yl)piperazine-1-carbamate (138 mg, 0.252 mmol) in DCM (7 mL) was

added HCl in dioxane (4 M, 0.377 mL, 1.51 mmol). The mixture was

stirred for 3 h at 25 °C, Et₂O (20 mL) was added, and the precipitate

was ﬁltered oﬀ and washed with Et₂O (3×).

1

Yield: 67%. H NMR (400 MHz, methanol-d₄) δ 8.12−8.04 (m,

1H), 8.01 (dd, J = 2.4, 1.2 Hz, 1H), 7.77 (d, J = 5.8 Hz, 1H), 7.60 (t, J =

8.0 Hz, 1H), 7.37 (ddt, J = 8.2, 2.3, 1.1 Hz, 1H), 6.90 (d, J = 5.8 Hz,

1H), 5.21−5.09 (m, 2H), 3.13 (ddd, J = 13.3, 12.0, 2.5 Hz, 2H), 2.97

(tt, J = 10.9, 4.1 Hz, 1H), 2.03−1.93 (m, 2H), 1.61−1.43 (m, 2H). LC/

MS (ESI⁺): found m/z = 378.1 [M + H]⁺(calc. for C₁₈H₁₈F₃N₅O m/z =

378.2 [M + H]⁺).

1

Yield: 90% as 2HCl. H NMR (300 MHz, DMSO-d₆) δ 13.47 (s,

1H), 9.60 (s, 2H), 8.71−8.53 (m, 2H), 7.84−7.75 (m, 2H), 7.27 (d, J =

6.8 Hz, 1H), 5.44 (d, J = 13.6 Hz, 2H), 3.79−3.43 (m, 11H), 2.37 (d, J =

12.2 Hz, 2H), 2.06−1.87 (m, 2H). LC/MS (ESI⁺): found m/z = 449.2

[M + H]⁺(calc. for C₂₂H₂₄F₄N₆m/z = 449.2 [M + H]⁺).

N,N-Dimethyl-1-(2-(3-(triﬂuoromethoxy)phenyl)-1H-imidazo-

[4,5-c]pyridin-4-yl)piperidin-4-amine (26). General procedure A.

1

Yield: 75%. H NMR (300 MHz, DMSO-d₆) δ 13.20 (s, 1H), 8.16

(dt, J = 7.8, 1.2 Hz, 1H), 8.06 (s, 1H), 7.82 (d, J = 5.6 Hz, 1H), 7.70 (t, J

= 8.0 Hz, 1H), 7.49 (ddt, J = 8.3, 2.4, 1.2 Hz, 1H), 6.88 (d, J = 5.6 Hz,

1H), 5.38 (d, J = 13.2 Hz, 2H), 3.06−2.91 (m, 2H), 2.74 (s, 1H), 2.39

(s, 6H), 2.00−1.88 (m, 2H), 1.58−1.40 (m, 2H). LC/MS (ESI⁺):

found m/z = 406.2 [M + H]⁺(calc. for C₂₀H₂₂F₃N₅O m/z = 406.2 [M +

H]⁺).

4-(4-(4-Methylpiperazin-1-yl)piperidin-1-yl)-2-(3-

(triﬂuoromethoxy)phenyl)-1H-imidazo[4,5-c]pyridine (27). General

procedure A. Yield: 17%. The product was converted to the HCl salt for

better handling. ¹H NMR (300 MHz, DMSO-d₆) δ 14.80 (s, 1H), 13.52

(s, 1H), 11.91 (s, 2H), 8.29 (dt, J = 7.9, 1.2 Hz, 1H), 8.20 (s, 1H), 7.79

(d, J = 1.8 Hz, 1H), 7.76 (s, 1H), 7.62−7.56 (m, 1H), 7.26 (d, J = 6.9

Hz, 1H), 5.47 (d, J = 13.5 Hz, 2H), 3.87−3.48 (m, 11H), 2.84 (s, 3H),

2.46−2.30 (m, 2H), 2.07−1.88 (m, 2H). LC/MS (ESI⁺): found m/z =

461.2 [M + H]⁺(calc. for C₂₃H₂₇F₃N₆O m/z = 461.2 [M + H]⁺).

2-(3,5-Diﬂuorophenyl)-4-(4-(4-methylpiperazin-1-yl)piperidin-1-

yl)-1H-imidazo[4,5-c]pyridine (32). General procedure B. Yield: 46%.

¹H NMR (600 MHz, DMSO-d₆) δ 13.18 (s, 1H), 7.81 (d, J = 5.7 Hz,

1H), 7.80−7.77 (m, 2H), 7.38 (tt, J = 9.2, 2.4 Hz, 1H), 6.85 (d, J = 5.6

Hz, 1H), 5.32 (d, J = 13.0 Hz, 2H), 3.01−2.93 (m, 2H), 2.64−2.50 (m,

4H), 2.49−2.45 (m, 1H), 2.33 (s, 4H), 2.15 (s, 3H), 1.92−1.82 (m,

2H), 1.44 (qd, J = 12.2, 4.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 162.7 (dd, J = 246.1, 13.6 Hz), 151.0, 145.5, 140.9, 140.7, 133.1,

128.2, 109.7−108.7 (m), 104.9 (t, J = 26.1 Hz), 98.0, 61.4, 55.1, 48.4,

45.6, 45.3, 28.0. LC/MS (ESI⁺): found m/z = 413.2 [M + H]⁺(calc. for

C₂₂H₂₆F₂N₆m/z = 413.2 [M + H]⁺).

3-(4-(4-(4-Methylpiperazin-1-yl)piperidin-1-yl)-1H-imidazo[4,5-

c]pyridin-2-yl)benzonitrile (33). General procedure B. Yield: 24%. ¹H

NMR (300 MHz, methanol-d₄) δ 8.45 (td, J = 1.7, 0.6 Hz, 1H), 8.40

(ddd, J = 7.9, 1.8, 1.2 Hz, 1H), 7.84 (dt, J = 7.7, 1.4 Hz, 1H), 7.81 (d, J =

M

https://dx.doi.org/10.1021/acs.jmedchem.0c01411

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

5.8 Hz, 1H), 7.72 (td, J = 7.8, 0.6 Hz, 1H), 6.94 (d, J = 5.8 Hz, 1H), 5.31

(d, J = 13.2 Hz, 2H), 3.09 (td, J = 12.6, 1.8 Hz, 2H), 2.94−2.72 (m, 4H),

2.72−2.51 (m, 5H), 2.38 (s, 3H), 2.08 (d, J = 12.2 Hz, 2H), 1.68 (qd, J

= 12.3, 4.0 Hz, 2H), 1.43−1.28 (m, 1H). LC/MS (ESI⁺): found m/z =

402.2 [M + H]⁺(calc. for C₂₃H₂₇N₇m/z = 402.2 [M + H]⁺).

1H), 8.16 (dt, J = 9.2, 2.0 Hz, 1H), 7.74−7.64 (m, 2H), 7.21 (d, J = 6.9

Hz, 1H), 5.26 (d, J = 13.7 Hz, 2H), 3.64−3.47 (m, 7H), 3.28 (br s, 4H),

2.97 (s, 3H), 2.85 (d, J = 7.1 Hz, 2H), 2.39−2.22 (m, 1H), 2.15 (d, J =

13.3 Hz, 2H), 1.67−1.46 (m, 2H). LC/MS (ESI⁺): found m/z = 477.1

[M + H]⁺(calc. for C₂₄H₂₈F₄N₆m/z = 477.2 [M + H]⁺).

N,N-Dimethyl-3-(4-(4-((4-methylpiperazin-1-yl)methyl)piperidin-

1-yl)-1H-imidazo[4,5-c]pyridin-2-yl)benzamide (41). General proce-

dure B. Yield: 25%. ¹H NMR (300 MHz, DMSO-d₆) δ 13.10 (s, 1H),

8.23−8.09 (m, 2H), 7.96 (s, 1H), 7.79 (d, J = 5.5 Hz, 1H), 7.61 (t, J =

7.7 Hz, 1H), 7.54−7.44 (m, 1H), 6.83 (d, J = 5.6 Hz, 1H), 5.30 (d, J =

12.9 Hz, 2H), 4.12 (t, J = 12.6 Hz, 2H), 3.64 (d, J = 13.7 Hz, 2H), 3.08−

2.92 (m, 6H), 2.80−2.63 (m, 4H), 2.42 (s, 3H), 2.23 (m, 1H), 1.78 (m,

3H), 1.31−1.09 (m, 4H). LC/MS (ESI⁺): found m/z = 462.2 [M + H]⁺

(calc. for C₂₆H₃₅N₇O m/z = 462.3 [M + H]⁺).

4-(4-(4-Methylpiperazin-1-yl)piperidin-1-yl)-2-(3-

(methylsulfonyl)phenyl)-1H-imidazo[4,5-c]pyridine (34). General

1

procedure B. Yield: 45%. H NMR (300 MHz, methanol-d₄) δ 8.69

(dt, J = 1.9, 1.0 Hz, 1H), 8.45 (ddd, J = 7.9, 1.8, 1.1 Hz, 1H), 8.07 (ddd, J

= 7.9, 1.9, 1.1 Hz, 1H), 7.81 (t, J = 7.7 Hz, 2H), 7.81 (d, J = 5.8 Hz, 2H),

6.95 (d, J = 5.8 Hz, 1H), 5.31 (d, J = 13.2 Hz, 2H), 3.22 (s, 3H), 3.15−

3.00 (m, 2H), 2.72 (s, 4H), 2.65−2.42 (m, 5H), 2.30 (s, 3H), 2.07 (d, J

= 11.3 Hz, 2H), 1.66 (qd, J = 12.2, 4.0 Hz, 2H). LC/MS (ESI⁺): found

m/z = 455.2 [M + H]⁺(calc. for C₂₃H₃₀N₆O₂S m/z = 455.2 [M + H]⁺).

Methyl 3-(4-(4-(4-Methylpiperazin-1-yl)piperidin-1-yl)-1H-

imidazo[4,5-c]pyridin-2-yl)benzoate (35). General procedure B.

2-(3-(4-(4-((4-Methylpiperazin-1-yl)methyl)piperidin-1-yl)-1H-

imidazo[4,5-c]pyridin-2-yl)phenoxy)ethan-1-amine (42). General

1

Yield: 17%. H NMR (300 MHz, methanol-d₄) δ 8.76 (t, J = 1.8 Hz,

procedure B. Yield: 8%. H NMR (300 MHz, methanol-d₄) δ 7.84−

1H), 8.36 (dt, J = 7.8, 1.5 Hz, 1H), 8.13 (dt, J = 7.8, 1.4 Hz, 1H), 7.81

(d, J = 5.8 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 6.94 (d, J = 5.8 Hz, 1H),

5.29 (d, J = 13.2 Hz, 2H), 3.07 (t, J = 12.5 Hz, 2H), 2.73 (s, 4H), 2.66−

2.46 (m, 5H), 2.31 (s, 3H), 2.07 (d, J = 12.4 Hz, 2H), 1.68 (qd, J = 12.4,

3.6 Hz, 2H). LC/MS (ESI⁺): found m/z = 435.2 [M + H]⁺(calc. for

C₂₄H₃₀N₆O₂m/z = 435.3 [M + H]⁺).

2-Cyclopentyl-1-(4-methoxybenzyl)-4-(4-(4-methylpiperazin-1-

yl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (36). Step 1: General

procedure B using PMB-protected intermediate. Yield: 35%. LCMS

(ESI⁺): found m/z = 489.3 [M + H]⁺(calc. for C₂₉H₄₀N₆O m/z = 489.3

[M + H]⁺).

7.75 (m, 2H), 7.66 (d, J = 6.9 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.30−

7.15 (m, 2H), 5.27 (d, J = 13.6 Hz, 2H), 4.46−4.31 (m, 2H), 3.63−3.41

(m, 8H), 3.22 (s, 4H), 2.96 (s, 3H), 2.80 (d, J = 7.0 Hz, 2H), 2.25 (s,

0H), 2.13 (d, J = 13.6 Hz, 2H), 1.66−1.46 (m, 2H). LC/MS (ESI⁺):

found m/z = 450.2 [M + H]⁺(calc. for C₂₅H₃₅N₇O m/z = 450.3 [M +

H]⁺).

3-(4-(4-((4-Methylpiperazin-1-yl)methyl)piperidin-1-yl)-1H-

imidazo[4,5-c]pyridin-2-yl)benzoic acid (43). General procedure B.

Yield: 25%. ¹H NMR (300 MHz, DMSO-d₆) δ 8.70 (d, J = 1.8 Hz, 1H),

8.24 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 5.5 Hz,

1H), 7.57 (t, J = 7.7 Hz, 1H), 6.86 (d, J = 5.5 Hz, 1H), 5.30 (d, J = 13.0

Hz, 2H), 2.98 (t, J = 12.2 Hz, 5H), 2.35 (m, 6H), 2.15 (s, 3H), 1.93 (d, J

= 11.7 Hz, 2H), 1.78 (m, 2H), 1.30−1.11 (m, 2H). LC/MS (ESI⁺):

found m/z = 435.1 [M + H]⁺(calc. for C₂₄H₃₀N₆O₂m/z = 435.3 [M +

H]⁺).

2-Cyclopentyl-4-(4-((4-methylpiperazin-1-yl)methyl)piperidin-1-

yl)-1H-imidazo[4,5-c]pyridine (44). Step 1: General procedure B using

PMB-protected intermediate. Yield: 29%. LC/MS (ESI⁺): found m/z =

503.4 [M + H]⁺(calc. for C₃₀H₄₂N₆O m/z = 503.4 [M + H]⁺).

Step 2: A suspension of 2-cyclopentyl-1-(4-methoxybenzyl)-4-(4-

((4-methylpiperazin-1-yl)methyl)piperidin-1-yl)-1H-imidazo[4,5-c]-

pyridine (43 mg, 0.086 mmol) in TFA (2.0 mL) was heated at 100 °C

overnight, cooled to 25 °C, and concentrated in vacuo. The residue was

puriﬁed by ﬂash chromatography using a gradient of 0−25% (0.5 M

NH₃) MeOH in DCM.

Step 2: A suspension of 2-cyclopentyl-1-(4-methoxybenzyl)-4-(4-(4-

methylpiperazin-1-yl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (50

mg, 0.102 mmol) in TFA (2.0 mL) was heated to 100 °C overnight,

cooled to RT, and concentrated in vacuo. The residue was puriﬁed by

ﬂash chromatography using a gradient of 0−20% (0.5 M NH₃) MeOH

in DCM.

Yield: 77%. ¹H NMR (300 MHz, DMSO-d₆) δ 7.66 (d, J = 6.6 Hz,

1H), 7.09 (d, J = 6.5 Hz, 1H), 5.09 (d, J = 13.0 Hz, 2H), 3.31 (m, 3H),

3.01 (m, 9H), 2.75 (m, 4H), 2.22−1.42 (m, 12H). LC/MS (ESI⁺):

found m/z = 368.9 [M + H]⁺(calc. for C₂₁H₃₂N₆m/z = 369.3 [M +

H]⁺).

2-(3,5-Diﬂuorophenyl)-4-(4-((4-methylpiperazin-1-yl)methyl)-

piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (37). General procedure B.

1

Yield: 22%. H NMR (400 MHz, DMSO-d₆) δ 13.19 (s, 1H), 7.85−

7.73 (m, 3H), 7.38 (t, J = 9.3 Hz, 1H), 6.83 (d, J = 5.6 Hz, 1H), 5.27 (d,

J = 12.9 Hz, 2H), 2.96 (t, J = 12.5 Hz, 2H), 2.47−2.17 (m, 8H), 2.14 (s,

3H), 2.12 (d, J = 6.8 Hz, 2H), 1.79 (d, J = 12.3 Hz, 3H), 1.25−1.06 (m,

2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.7 (dd, J = 246.0, 13.4 Hz),

151.2, 145.4, 141.0, 140.6, 133.2, 128.2, 109.9−108.2 (m), 104.8 (t, J =

26.0 Hz), 97.9, 64.2, 54.8, 53.1, 45.9, 45.8, 33.3, 30.6. LC/MS (ESI⁺):

found m/z = 427.1 [M + H]⁺(calc. for C₂₃H₂₈F₂N₆m/z = 427.2 [M +

H]⁺).

Yield: 89%. ¹H NMR (300 MHz, DMSO-d₆) δ 7.65 (d, J = 6.7 Hz,

1H), 7.08 (d, J = 6.9 Hz, 1H), 5.13−4.97 (m, 2H), 3.32 (m, 9H), 2.99

(m, 4H), 2.77 (m, 3H), 2.28 (m, 2H), 2.20−2.00 (m, 2H), 1.99−1.65

(m, 7H), 1.27 (d, J = 13.3 Hz, 3H). LC/MS (ESI⁺): found m/z = 382.9

[M + H]⁺(calc. for C₂₂H₃₄N₆m/z = 383.3 [M + H]⁺).

2-(3,5-Diﬂuorophenyl)-4-(4-(piperazin-1-ylmethyl)piperidin-1-

yl)-1H-imidazo[4,5-c]pyridine (45). Step 1: General procedure B using

1-benzyl-4-(piperidin-4-ylmethyl)piperazine hydrochloride. Yield:

42%. ¹H NMR (300 MHz, DMSO-d₆) δ 13.16 (br s, 1H), 7.88−7.72

(m, 3H), 7.48−7.15 (m, 6H), 6.84 (d, J = 5.7 Hz, 1H), 5.28 (d, J = 12.7

Hz, 2H), 3.57−3.42 (m, 3H), 2.99 (t, J = 12.7 Hz, 2H), 2.48−2.31 (m,

6H), 2.27−2.05 (m, 2H), 1.89−1.69 (m, 3H), 1.27−0.99 (m, 3H). LC/

MS (ESI⁺): found m/z = 503.1 [M + H]⁺(calc. for C₂₉H₃₃F₂N₆m/z =

503.3 [M + H]⁺).

Step 2: N₂was bubbled through MeOH (10 mL) for 20 min in a two-

neck ﬂask equipped with a stirrer bar. 10% Pd/C (27.5 mg, 0.026

mmol) was then carefully introduced under an inert atmosphere. Once

all the powder was safely washed into the MeOH, the N₂was removed

and 4-(4-((4-benzylpiperazin-1-yl)methyl)piperidin-1-yl)-2-(3,5-di-

ﬂuorophenyl)-1H-imidazo[4,5-c]pyridine (130 mg, 0.259 mmol) was

added to the suspension, which was then stirred under a H₂atmosphere

at 25 °C for 16 h. The reaction mixture was ﬁltered through Celite and

washed with MeOH. After concentration in vacuo, the residue was

puriﬁed by ﬂash column chromatography with an eluent of 5−15% (0.5

M NH₃) in MeOH/DCM to yield 2-(3,5-diﬂuorophenyl)-4-(4-

(piperazin-1-ylmethyl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine as

an oﬀ-white solid.

2-(3-Chloro-4-ﬂuorophenyl)-4-(4-((4-methylpiperazin-1-yl)-

methyl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (38). General pro-

cedure B. Yield: 42%. ¹H NMR (400 MHz, methanol-d₄) δ 8.20 (d, J =

7.0 Hz, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.75 (d, J = 5.7 Hz, 1H), 7.38 (t, J

= 8.7 Hz, 1H), 6.89 (d, J = 5.8 Hz, 1H), 5.13 (d, J = 13.0 Hz, 2H), 3.07

(t, J = 12.6 Hz, 2H), 2.55 (s, 8H), 2.33 (s, 3H), 2.27 (d, J = 6.7 Hz, 3H),

1.91 (d, J = 12.2 Hz, 3H), 1.35 (q, J = 12.7 Hz, 2H). LC/MS (ESI⁺):

found m/z = 443.1 [M + H]⁺(calc. for C₂₃H₂₈ClFN₆m/z = 443.2 [M +

H]⁺).

2-(4-Fluoro-3-(triﬂuoromethyl)phenyl)-4-(4-((4-methylpiperazin-

1-yl)methyl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (39). General

procedure B. Yield: 45%. ¹H NMR (300 MHz, methanol-d₄) δ 8.49−

8.36 (m, 2H), 7.76 (d, J = 5.9 Hz, 1H), 7.51 (dd, J = 10.3, 8.6 Hz, 1H),

6.92 (d, J = 5.9 Hz, 1H), 5.16 (d, J = 12.9 Hz, 2H), 3.18−3.03 (m, 2H),

2.55 (s, 7H), 2.32 (s, 3H), 2.29 (d, J = 6.5 Hz, 2H), 1.93 (d, J = 12.0 Hz,

3H), 1.42−1.27 (m, 2H). LC/MS (ESI⁺): found m/z = 477.0 [M + H]⁺

(calc. for C₂₄H₂₈F₄N₆m/z = 477.2 [M + H]⁺).

2-(3-Fluoro-5-(triﬂuoromethyl)phenyl)-4-(4-((4-methylpiperazin-

1-yl)methyl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (40). General

procedure B. Yield: 32%. ¹H NMR: (300 MHz, methanol-d₄) δ 8.28 (s,

N

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Yield: 45%. ¹H NMR (300 MHz, DMSO-d₆) δ 14.62 (s, 1H), 9.45 (s,

1H), 7.84 (h, J = 5.4 Hz, 2H), 7.74 (d, J = 6.9 Hz, 1H), 7.47 (td, J = 9.2,

4.6 Hz, 1H), 7.23 (d, J = 6.9 Hz, 1H), 5.25−5.3 (d, 2H), 3.62−3.40 (m,

9H), 3.22−3.11 (m, 2H), 2.39−2.26 (m, 1H), 2.07 (d, J = 13.1 Hz,

2H), 1.47 (q, J = 11.8, 11.2 Hz, 2H), 1.22 (s, 1H). LC/MS (ESI⁺):

found m/z = 412.9 [M + H]⁺(calc. for C₂₂H₂₆F₂N₆m/z = 413.2 [M +

H]⁺).

(m, 3H), 7.37 (tt, J = 9.2, 2.4 Hz, 1H), 6.86 (d, J = 5.7 Hz, 1H), 5.28 (d,

J = 13.0 Hz, 2H), 4.08−4.01 (m, 1H), 3.65 (t, J = 4.6 Hz, 4H), 3.03 (t, J

= 12.4 Hz, 2H), 2.39−2.26 (m, 4H), 1.95−1.78 (m, 3H), 1.21 (d, J =

10.9 Hz, 3H). LC/MS (ESI⁺): found m/z = 414.2 [M + H]⁺(calc. for

C₂₂H₂₅F₂N₅O m/z = 414.2 [M + H]⁺).

2-(3,5-Diﬂuorophenyl)-4-(4-((4-(2,2,2-triﬂuoroethyl)piperazin-1-

yl)methyl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (53). General

1

procedure B. Yield: 33%. H NMR (400 MHz, DMSO-d₆): δ 13.16

2-(3,5-Diﬂuorophenyl)-N-((1S,4S)-4-(4-methylpiperazin-1-yl)-

cyclohexyl)-1H-imidazo[4,5-c]pyridin-4-amine (46) and 2-(3,5-Di-

ﬂuorophenyl)-N-((1R,4R)-4-(4-methylpiperazin-1-yl)cyclohexyl)-1H-

imidazo[4,5-c]pyridin-4-amine (47). To a solution of 4-(4-methyl-

piperazin-1-yl)cyclohexanamine (128 mg, 0.650 mmol) in n-BuOH (2

mL) were added 4-chloro-2-(3,5-diﬂuorophenyl)-1H-imidazo[4,5-

c]pyridine (145 mg, 0.550 mmol) and DIPEA (1.14 mL, 6.55 mmol)

at 25 °C. The resulting reaction mixture was heated at 140 °C for 4 days.

The reaction mixture was concentrated in vacuo, and the residue was

puriﬁed by ﬂash chromatography eluting a gradient of (0.5 M NH₃)

MeOH in DCM 5:95 to 30:70 to yield two diastereoisomers, eluting

ﬁrst the cis-product 46 and then the trans-product 47.

(s, 1H), 7.78−7.81 (m, 3H), 7.39 (t, J = 9.16 Hz, 1H), 6.82 (d, J = 5.52

Hz, 1H), 5.28 (d, J = 12.6 Hz, 2H), 3.15 (q, J = 10.2 Hz, 2H), 2.97 (t, J =

11.8 Hz, 2H), 2.59−2.61 (m, 4H), 2.36 (br s, 4H), 2.13 (d, J = 6.48 Hz,

2H), 1.79 (d, J = 11.2 Hz, 3H), 1.13−1.19 (m, 2H). LC/MS (ESI⁺):

found m/z = 495.4 [M + H]⁺(calc. for C₂₄H₂₇F₅N₆m/z = 495.2 [M +

H]⁺).

9-(2-(3,5-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-4-

methyl-1-oxa-4,9-diazaspiro[5.5]undecane (54). Step 1: tert-Butyl-1-

oxa-6-azaspiro[2.5]octane-6-carboxylate (2.00 g, 9.38 mmol) was

dissolved in methanamine (8.83 g, 94.0 mmol), 33% solution in

EtOH. The mixture was stirred at 50 °C overnight followed by TLC.

The solvent was removed under reduced pressure to yield 54 as a

yellowish oil, which solidiﬁed upon standing.

46. Yield: 7%. ¹H NMR (300 MHz, methanol-d₄) δ 7.79−7.59 (m,

3H), 7.12 (tdd, J = 1.6, 3.6, 9.1 Hz, 1H), 6.88 (d, J = 6.2 Hz, 1H), 4.26

(br s, 1H), 3.59−3.50 (m, 1H), 3.11−3.05 (m, 1H), 2.83 (br s, 2H),

2.72 (br s, 2H), 2.70 (s, 3H), 2.43 (s, 2H), 2.08 (d, J = 10.6 Hz, 2H),

1.89−1.76 (m, 4H), 1.35−1.26 (m, 2H), 1.18 (t, J = 7.0 Hz, 1H), 0.99−

0.83 (m, 2H). LC/MS (ESI⁺): found m/z = 427.1 [M + H]⁺(calc. for

C₂₃H₂₈F₂N₆m/z = 427.2 [M + H]⁺).

Yield: 1.77 g (77%). ¹H NMR (300 MHz, chloroform-d) δ 3.86 (d, J

= 13.2 Hz, 2H), 3.30−3.09 (m, 2H), 2.70−2.59 (m, 2H), 2.57 (s, 2H),

2.53 (s, 3H), 1.59 (q, J = 2.9 Hz, 1H), 1.47 (s, 9H), 1.26 (d, J = 5.3 Hz,

1H).

1

Step 2: General procedure B. Yield: 51%. H NMR (300 MHz,

47. Yield: 16%. ¹H NMR (300 MHz, methanol-d₄) δ 7.84−7.59 (m,

3H), 7.14 (td, J = 4.5, 9.0 Hz, 1H), 6.94 (d, J = 6.4 Hz, 1H), 3.97 (br s,

1H), 3.01 (br s, 8H), 2.70 (s, 3H), 2.63 (br s, 2H), 2.28 (d, J = 11.2 Hz,

2H), 2.10 (s, 2H), 1.87 (t, J = 11.4 Hz, 2H), 1.57 (d, J = 12.8 Hz, 2H),

1.04−0.87 (m, 1H). LC/MS (ESI⁺): found m/z = 427.1 [M + H]⁺

(calc. for C₂₃H₂₈F₂N₆m/z = 427.2 [M + H]⁺).

DMSO-d₆) δ 13.27 (s, 1H), 7.86−7.76 (m, 3H), 7.37 (tt, J = 9.2, 2.4 Hz,

1H), 6.85 (d, J = 5.6 Hz, 1H), 4.70 (dd, J = 13.2, 4.4 Hz, 2H), 3.76−3.55

(m, 4H), 2.36 (s, 2H), 2.27 (s, 2H), 2.19 (s, 3H), 1.92 (d, J = 13.7 Hz,

2H), 1.61 (ddd, J = 14.0, 10.6, 4.0 Hz, 2H). LC/MS (ESI⁺): found m/z

= 400.1 [M + H]⁺(calc. for C₂₁H₂₃F₂N₅O m/z = 400.3 [M + H]⁺).

2-(3,5-Diﬂuorophenyl)-4-[4-[[4-(1-methylimidazol-2-yl)-

piperazin-1-yl]methyl]-1-piperidyl]-1H-imidazo[4,5-c]pyridine (55).

General procedure B. Yield: 24%. ¹H NMR (600 MHz, DMSO-d₆) δ

13.12 (s, 1H), 7.80−7.72 (m, 3H), 7.34 (t, J = 9.2 Hz, 1H), 6.82 (d, J =

1.5 Hz, 1H), 6.80 (d, J = 5.5 Hz, 1H), 6.55 (d, J = 1.5 Hz, 1H), 5.26 (d, J

= 13.0 Hz, 2H), 3.39 (s, 3H), 2.97 (t, J = 12.4 Hz, 2H), 2.94−2.89 (m,

4H), 2.52−2.47 (m, 4H), 2.24−2.15 (m, 2H), 1.88−1.76 (m, 3H),

1.18−1.11 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.8 (dd, J =

246.3, 13.4 Hz), 151.6, 151.3, 145.4, 140.9, 140.7, 133.1, 128.3, 124.0,

118.6, 109.2 (dd, J = 21.6, 5.2 Hz), 105.2−104.6 (m), 97.8, 64.2, 53.0,

50.4, 45.9, 33.2, 31.6, 30.6. LC/MS (ESI⁺): found m/z = 493.2 [M +

H]⁺(calc. for C₂₆H₃₀F₂N₈m/z = 493.3 [M + H]⁺).

1-{[(3S)-1-[2-(3,5-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-

yl]piperidin-3-yl]methyl}-4-methylpiperazine (48). General proce-

dure B. Yield: 32%. ¹H NMR (400 MHz, DMSO-d₆): δ 13.15 (s, 1H),

7.80−7.82 (m, 3H), 7.40 (t, J = 9.08 Hz, 1H), 6.82 (d, J = 5.48 Hz, 1H),

5.50 (d, J = 11.5 Hz, 2H), 4.99 (d, J = 12.6 Hz, 1H), 2.93 (t, J = 11.4 Hz,

1H), 2.74 (t, J = 10.9 Hz, 1H), 2.23−2.29 (m, 8H), 2.07−2.11 (m, 4H),

1.70−1.79 (m, 3H), 1.52−1.58 (m, 1H), 1.13−1.22 (m, 1H). LC/MS

(ESI⁺): found m/z = 427.3 [M + H]⁺(calc. for C₂₃H₂₈F₂N₆m/z = 427.2

[M + H]⁺). Speciﬁc rotation: [+42.20°] at 25 °C (c = 0.47% solution in

CHCl₃; λ = 589 nm; d = 100 mm).

11-{[(3R)-1-[2-(3,5-diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-

yl]piperidin-3-yl]methyl}-4-methylpiperazine (49). General proce-

dure B. Yield: 2%. ¹H NMR (400 MHz, DMSO-d₆): δ 13.18 (s, 1H),

7.80−7.82 (m, 3H), 7.39 (t, J = 9.12 Hz, 1H), 6.82 (d, J = 5.56 Hz, 1H),

5.43−5.47 (m, 1H), 4.99−5.02 (m, 1H), 2.95−2.97 (m, 1H), 2.77 (t, J

= 10.3 Hz, 1H), 2.28−2.40 (m, 8H), 2.16−2.21 (m, 4H), 1.70−1.80

(m, 3H), 1.52−1.55 (m, 1H), 1.18−1.23 (m, 2H). LC/MS (ESI⁺):

found m/z = 427.4 [M + H]⁺(calc. for C₂₃H₂₈F₂N₆m/z = 427.2 [M +

H]⁺). Speciﬁc rotation: [−29.20°] at 25 °C (c = 0.34% solution in

CHCl₃; λ = 589 nm; d = 100 mm).

4-(4-((4-(tert-Butyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(3,5-

diﬂuorophenyl)-1H-imidazo[4,5-c]pyridine (56). Step 1: K₂CO₃(350

mg, 2.53 mmol), tert-butyl 4-(bromomethyl)piperidine-1-carboxylate

(400 mg, 1.44 mmol), and 1-(tert-butyl)piperazine (180 mg, 1.27

mmol) were placed in MeCN (4 mL), sealed, and heated at 75 °C

overnight. The contents were diluted with EtOAc (100 mL), washed

with H₂O (50 mL) and NH₄Cl (50 mL), dried over anhydrous MgSO₄,

ﬁltered, and concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography, eluting with 0−10% MeOH/DCM.

4-((1-(2-(3,5-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

Yield: 180 mg (42%). ¹H NMR (300 MHz, methanol-d₄) δ 4.07 (d, J

= 13.2 Hz, 2H), 3.09−2.91 (m, 3H), 2.77 (t, J = 12.9 Hz, 3H), 2.70−

2.53 (m, 3H), 2.28 (d, J = 6.7 Hz, 2H), 1.77 (d, J = 12.1 Hz, 3H), 1.47

(s, 9H), 1.27 (s, 9H), 1.17−0.98 (m, 3H).

piperidin-4-yl)methyl)thiomorpholine 1,1-dioxide (50). General

1

procedure B. Yield: 56%. H NMR (300 MHz, DMSO-d₆) δ 13.17

(s, 1H), 7.85−7.73 (m, 3H), 7.45−7.31 (m, 1H), 6.85 (d, J = 5.7 Hz,

1H), 5.29 (d, J = 13.0 Hz, 2H), 3.12−3.04 (m, 5H), 3.03−3.00 (m,

1H), 2.93−2.87 (m, 4H), 2.34 (d, J = 6.5 Hz, 2H), 1.83 (d, J = 11.6 Hz,

3H), 1.27−1.12 (m, 2H). LC/MS (ESI⁺): found m/z = 462.2 [M + H]⁺

(calc. for C₂₂H₂₅F₂N₅O₂S m/z = 462.2 [M + H]⁺).

2-(3,5-Diﬂuorophenyl)-4-(4-((4,4-diﬂuoropiperidin-1-yl)methyl)-

piperidin-1-yl)-1H-imidazo[4,5-c]pyridine (51). General procedure B.

Yield: 8.3% ¹H NMR (300 MHz, methanol-d₄) δ 7.83−7.61 (m, 3H),

7.07 (tt, J = 9.0, 2.2 Hz, 1H), 6.91 (d, J = 5.9 Hz, 1H), 5.17 (d, J = 13.6

Hz, 2H), 3.11 (t, J = 11.9 Hz, 2H), 2.66−2.52 (m, 4H), 2.33 (d, J = 6.6

Hz, 2H), 2.10−1.92 (m, 6H), 1.46−1.31 (m, 3H). LC/MS (ESI⁺):

found m/z = 448.2 [M + H]⁺(calc. for C₂₃H₂₅F₄N₅m/z = 448.2 [M +

H]⁺).

1

Step 2: General procedure B. Yield: 10%. H NMR (300 MHz,

methanol-d₄) δ 7.83−7.64 (m, 3H), 7.06 (tt, J = 9.0, 2.4 Hz, 1H), 6.90

(d, J = 5.9 Hz, 1H), 5.18 (d, J = 13.2 Hz, 2H), 3.19−3.03 (m, 2H),

2.97−2.78 (m, 4H), 2.70−2.51 (m, 3H), 2.30 (d, J = 6.5 Hz, 2H), 1.93

(d, J = 13.1 Hz, 3H), 1.50−1.27 (m, 3H), 1.20 (s, 9H). LC/MS (ESI⁺):

found m/z = 469.2 [M + H]⁺(calc. for C₂₆H₃₄F₂N₆m/z = 469.3 [M +

H]⁺).

In Vitro P. falciparum Assay. Compounds were screened against

multidrug-resistant (K1) and multidrug-sensitive (NF54) strains of P.

falciparum in vitro using the modiﬁed [³H]-hypoxanthine incorporation

assay and the parasite lactate dehydrogenase assay. Both assays showed

excellent alignment and are fully described in the Supporting

Information providing a side-by-side comparison for a selection of

compounds (Table S2).

4-((1-(2-(3,5-Diﬂuorophenyl)-1H-imidazo[4,5-c]pyridin-4-yl)-

piperidin-4-yl)methyl)morpholine (52). General procedure B. Yield:

27%. ¹H NMR (300 MHz, DMSO-d₆) δ 13.20 (br s, 1H), 7.88−7.74

O

https://dx.doi.org/10.1021/acs.jmedchem.0c01411

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Ethics. For the in vivo pharmacokinetics and eﬃcacy studies, all

animal experiments performed in the manuscript were conducted in

compliance with institutional guidelines.

Mathew Njoroge − Drug Discovery and Development Centre

(H3D), Division of Clinical Pharmacology, University of Cape

Town, Rondebosch 7701, South Africa; orcid.org/0000-

0003-1276-2224

Janette Reader − Department of Biochemistry, Genetics and

Microbiology, Institute for Sustainable Malaria Control,

University of Pretoria, Pretoria 0028, South Africa

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01411.

̈

Mariette van der Watt − Department of Biochemistry, Genetics

and Microbiology, Institute for Sustainable Malaria Control,

University of Pretoria, Pretoria 0028, South Africa

Kathryn Wicht − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Ana Carolina C. de Sousa − Department of Chemistry,

University of Cape Town, Rondebosch 7701, South Africa

John Okombo − Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa

Synthetic procedures of building blocks and scaﬀold

changes and procedures used for the in vitro and in vivo

antimalarial studies as well as PK and metabolism studies

(PDF)

SMILES ﬁle with NF54, K1 and cytotoxicity IC50 values

(CSV)

AUTHOR INFORMATION

Corresponding Author

■

Keletso Maepa − Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa

Kelly Chibale − South African Medical Research Council, Drug

Discovery and Development Research Unit, Department of

Chemistry and Institute of Infectious Disease and Molecular

Medicine, University of Cape Town, Rondebosch 7701, South

Africa; orcid.org/0000-0002-1327-4727; Phone: +27-21-

6502553; Email: Kelly.Chibale@uct.ac.za; Fax: +27-21-

65045215

Timothy J. Egan − Department of Chemistry and Institute of

Infectious Disease and Molecular Medicine, University of Cape

Town, Rondebosch 7701, South Africa; orcid.org/0000-

0001-7720-8473

Lyn-Marie Birkholtz − Department of Biochemistry, Genetics and

Microbiology, Institute for Sustainable Malaria Control,

University of Pretoria, Pretoria 0028, South Africa

Gregory S. Basarab − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Authors

́

Andre Horatscheck − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Sergio Wittlin − Swiss Tropical and Public Health Institute, 4002

Basel, Switzerland; University of Basel, 4002 Basel, Switzerland

Paul V. Fish − Alzheimer’s Research UK, UCL Drug Discovery

Institute, The Cruciform Building, University College London,

London WC1E 6BT, U.K.; orcid.org/0000-0002-2117-

2173

Leslie J. Street − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Ana Andrijevic − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Aloysius T. Nchinda − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Claire Le Manach − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa; orcid.org/0000-0002-

9310-0048

Tanya Paquet − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

James Duﬀy − Medicines for Malaria Venture, ICC, 1215 Geneva,

Switzerland

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01411

Lutete Peguy Khonde − Drug Discovery and Development

Centre (H3D), Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa

Jean Dam − Drug Discovery and Development Centre (H3D),

Department of Chemistry, University of Cape Town, Rondebosch

7701, South Africa

Kailash Pawar − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Dale Taylor − Drug Discovery and Development Centre (H3D),

Division of Clinical Pharmacology, University of Cape Town,

Rondebosch 7701, South Africa

Nina Lawrence − Drug Discovery and Development Centre

(H3D), Division of Clinical Pharmacology, University of Cape

Town, Rondebosch 7701, South Africa

Christel Brunschwig − Drug Discovery and Development Centre

(H3D), Division of Clinical Pharmacology, University of Cape

Town, Rondebosch 7701, South Africa

Liezl Gibhard − Drug Discovery and Development Centre (H3D),

Division of Clinical Pharmacology, University of Cape Town,

Rondebosch 7701, South Africa

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Medicines for Malaria Ventures (MMV), South

African Technology Innovation Agency (TIA), and the Strategic

Health Innovation Partnerships (SHIP) unit of the South

African Medical Research Council (SAMRC) for ﬁnancial

support of this research (Project MMV09/0002). The

University of Cape Town (UCT), SAMRC, and South African

Research Chairs Initiative of the Department of Science and

Innovation, administered through the South African National

Research Foundation, are gratefully acknowledged for support

(KC and the chair initiative to LMB, UID84627). At Swiss TPH,

we thank Sibylle Sax and Christian Scheurer for technical

assistance with the [³H]-hypoxanthine incorporation assay and

Ursula Lehmann, Sibylle Sax, and Christoph Fischli with the

SCID mouse model. At UCT, we thank Carmen de Kock, Virgil

Verhoog, and Sumaya Salie for running the antimalarial assays,

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01411

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Circumvent Atypical Dose-Response Curves against Multidrug

Resistant Parasite Strains. J. Med. Chem. 2018, 61, 9371−9385.

(8) Medicines for Malaria Venture (MMV). https://www.mmv.org/

research-development/information-scientists (accessed Sept 16,

2020).

Nesia Barnes and Warren Olifant for running the ADME assays,

and Trevor Finch for assistance with the animal work. We thank

Stephan Meister at UCSD for the test against Pb liver forms and

Dennis Smith for advice on pharmacokinetics. We would also

like to thank Syngene and TCGLS for synthetic support. TJE

and ACCdS acknowledge the National Institute of Allergy and

Infectious Diseases of the National Institutes of Health under

Award Number R01AI143521. The content is solely the

responsibility of the authors and does not necessarily represent

the oﬃcial views of the National Institutes of Health.

(9) Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. Medicinal

Chemistry of HERG Optimizations: Highlights and Hang-Ups. J. Med.

Chem. 2006, 49, 5029−5046.

́

(10) Sanz, L. M.; Crespo, B.; De-Cozar, C.; Ding, X. C.; Llergo, J. L.;

Burrows, J. N.; García-Bustos, J. F.; Gamo, F.-J. P. Falciparum in Vitro

Killing Rates Allow to Discriminate between Different Antimalarial

Mode-of-Action. PLoS One 2012, 7, e30949.

(11) Baragana, B.; Hallyburton, I.; Lee, M. C. S.; Norcross, N. R.;

̃

ABBREVIATIONS

■

Grimaldi, R.; Otto, T. D.; Proto, W. R.; Blagborough, A. M.; Meister, S.;

Wirjanata, G.; Ruecker, A.; Upton, L. M.; Abraham, T. S.; Almeida, M.

J.; Pradhan, A.; Porzelle, A.; Martínez, M. S.; Bolscher, J. M.; Woodland,

A.; Norval, S.; Zuccotto, F.; Thomas, J.; Simeons, F.; Stojanovski, L.;

Osuna-Cabello, M.; Brock, P. M.; Churcher, T. S.; Sala, K. A.;

ABS, asexual blood stage; ADME, absorption, distribution,

metabolism, excretion; APCI, atmospheric pressure chemical

ionization; βH, β-hematin; Boc, tert-butylcarbamate; carl, cyclic

amine resistance locus; Cbz, benzylcarbamate; CHO cells,

Chinese hamster ovarian cells; CL, clearance; cytB, cytochrome

B; DCM, dichloromethane; DHODH, dihydroorotate dehy-

drogenase; DIPEA, diisopropylethylamine; DMSO, dimethyl-

sulfoxide; DV, digestive vacuole; eEF2, eukaryotic elongation

factor 2; ESI, electrospray ionization; hERG, human ether-a-go-

go related gene; HPLC, high pressure liquid chromatography;

i.v., intravenous administration; LC/MS, liquid chromatogra-

phy/mass spectrometry; LOD, limit of detection; MetID,

metabolite identiﬁcation; MLM, mouse liver microsomes;

NSG, NODscidIL2Rγnull; Pb, Plasmodium berghei; PCT99.9,

parasite clearance time (99.9%); PI4K, phosphatidylinositol-4-

kinase; Pf, Plasmodium falciparum; pLDH, parasite lactate

dehydrogenase; p.o., oral administration; PoC, proof of concept;

PRR, parasite reduction ratio; SAR, structure−activity relation-

ship; SFK, SoftFocus Kinase; SI, selectivity index; S_NAr,

nucleophilic aromatic substitution; TFA, triﬂuoroacetic acid;

TLC, thin-layer chromatography; WHO, World Health

Organization

́

Zakutansky, S. E.; Jimenez-Díaz, M. B.; Sanz, L. M.; Riley, J.; Basak, R.;

Campbell, M.; Avery, V. M.; Sauerwein, R. W.; Dechering, K. J.;

Noviyanti, R.; Campo, B.; Frearson, J. A.; Angulo-Barturen, I.; Ferrer-

Bazaga, S.; Gamo, F. J.; Wyatt, P. G.; Leroy, D.; Siegl, P.; Delves, M. J.;

Kyle, D. E.; Wittlin, S.; Marfurt, J.; Price, R. N.; Sinden, R. E.; Winzeler,

E. A.; Charman, S. A.; Bebrevska, L.; Gray, D. W.; Campbell, S.;

Fairlamb, A. H.; Willis, P. A.; Rayner, J. C.; Fidock, D. A.; Read, K. D.;

Gilbert, I. H. A Novel Multiple-Stage Antimalarial Agent That Inhibits

Protein Synthesis. Nature 2015, 522, 315−320.

(12) Paquet, T.; Le Manach, C.; Cabrera, D. G.; Younis, Y.; Henrich,

P. P.; Abraham, T. S.; Lee, M. C. S.; Basak, R.; Ghidelli-Disse, S.;

Lafuente-Monasterio, M. J.; Bantscheff, M.; Ruecker, A.; Blagborough,

A. M.; Zakutansky, S. E.; Zeeman, A.-M.; White, K. L.; Shackleford, D.

M.; Mannila, J.; Morizzi, J.; Scheurer, C.; Angulo-Barturen, I.; Martínez,

M. S.; Ferrer, S.; Sanz, L. M.; Gamo, F. J.; Reader, J.; Botha, M.;

Dechering, K. J.; Sauerwein, R. W.; Tungtaeng, A.; Vanachayangkul, P.;

Lim, C. S.; Burrows, J.; Witty, M. J.; Marsh, K. C.; Bodenreider, C.;

́

Rochford, R.; Solapure, S. M.; Jimenez-Díaz, M. B.; Wittlin, S.;

Charman, S. A.; Donini, C.; Campo, B.; Birkholtz, L.-M.; Hanson, K. K.;

Drewes, G.; Kocken, C. H. M.; Delves, M. J.; Leroy, D.; Fidock, D. A.;

Waterson, D.; Street, L. J.; Chibale, K. Antimalarial Efficacy of

MMV390048, an Inhibitor of Plasmodium Phosphatidylinositol 4-

Kinase. Sci. Transl. Med. 2017, 9, eaad9735.

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Tomchick, D. R.; Deng, X.; Laird, T.; Bhatia, S. N.; March, S.; Ng, C. L.;

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and in Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model

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