Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity

Bioorganic Chemistry 115 (2021) 105244

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine

antimalarials with moderate to slow acting erythrocytic stage activity

,

Brodie L. Bailey^{a b}, William Nguyen^{a b}, Anna Ngo^a, Christopher D. Goodman^c,

Maria R. Gancheva^d, Paola Favuzza^{a b}, Laura M. Sanz^f, Francisco-Javier Gamo^f,

Kym N. Lowes^{a b}, Geoffrey I. McFadden^c, Danny W. Wilson^{d e}, Benoît Laleu^h, Stephen Brand^h,

,

Paul F. Jackson^g, Alan F. Cowman^{a b}, Brad E. Sleebs^a

,

,b,*

^aThe Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia

^bDepartment of Medical Biology, The University of Melbourne, Parkville 3010, Australia

^cSchool of Biosciences, University of Melbourne, Parkville, Victoria 3010, Australia

^dResearch Centre for Infectious Diseases, School of Biological Sciences, University of Adelaide, Adelaide, Australia

^eMacfarlane Burnet Institute for Medical Research and Public Health, Melbourne 3004, Australia

^fGlobal Health Pharma Research Unit, GlaxoSmithKline, Tres Cantos 28760, Spain

^gGlobal Public Health, Janssen Pharmaceuticals, San Diego, CA, United States

^hMedicines for Malaria Venture, Geneva 1215, Switzerland

A R T I C L E I N F O

A B S T R A C T

Keywords:

Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance

has been reported against all clinically available antimalarials, threatening our ability to control the disease and

therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identiﬁed

the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter

library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity rela-

tionship of the identiﬁed class and the optimisation of asexual stage activity while maintaining selectivity against

the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity

against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping

studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these

parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of

action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed

in in vitro ADME proﬁling. A signiﬁcant challenge remains to correct these liabilities for further advancement of

the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a

curative or prophylactic antimalarial treatment.

Malaria

Plasmodium

Antimalarial

Triazolopyrimidine

P. vivax has a signiﬁcant impact on human health in South East Asia and

the Americas [1]. P. knowlesi is a parasite of simian origin and infections

are becoming increasingly prevalent in Malaysia and more broadly in

South East Asia [2].

1. Introduction

Malaria is one of the most signiﬁcant parasitic diseases in human

history with approximately half of the world’s population at risk of

infection. Infected individuals are estimated to total 229 million annu-

ally with 409,000 succumbing to the disease in 2019 [1]. Malaria is

caused by the infection of the Apicomplexa parasite from the genus

Plasmodium and is transmitted to humans by the female Anopheles

mosquito. There are ﬁve Plasmodium species known to infect humans.

P. falciparum causes the greatest mortality in sub-Saharan Africa, while

The incidence of Malaria between 2000 and 2015 was reduced by

37% primarily through the use of interventions such as insecticide

treated bed nets and chemotherapeutics [1]. Currently, the front line

chemotherapeutic for P. falciparum and P. vivax, are Artemisinin Com-

bination Therapies (ACTs). Worryingly, reports of ACT resistance are

well established and spreading in Asia [3] and more recently in Africa

[4], posing an imminent threat to the control and treatment of Malaria.

* Corresponding author at: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3052, Victoria, Australia.

E-mail address: sleebs@wehi.edu.au (B.E. Sleebs).

https://doi.org/10.1016/j.bioorg.2021.105244

Received 2 July 2021; Received in revised form 29 July 2021; Accepted 2 August 2021

Available online 8 August 2021

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

throughput (HT) screening has been employed extensively for this

purpose, primarily on the asexual erythrocytic stage of P. falciparum as

culture of this form of the parasite is the most tractable in the laboratory

[7–9]. More recently, technological advancements have allowed both

the sexual stage [10] and the liver stage [11] of the malaria parasite to

be employed in HT screening campaigns, allowing the identiﬁcation of

compounds with target candidate proﬁles (TCPs) that have potential as

transmission blocking and prophylaxis agents [12]. This effort has

resulted in the identiﬁcation of many high-quality hits that have been

developed into candidates currently progressing towards the clinic or in

human clinical trials [13].

Abbreviations

ATP

ACT

adenosine 5^′-triphosphate

artemisinin combination therapy

CFDA-SE carboxylﬂuorescein diacetate succinimidyl ester

CL_int

cLogP

CQ

intrinsic clearance

calculated logP

chloroquine

HT

high-throughput

IPP

isopentenyl phosphate

lipophilic efﬁciency

medicines for malaria venture

Pharmaceutical compound libraries present an untapped source of

novel chemical matter for academic research in neglected tropical dis-

eases. The Janssen Jumpstarter library is a diverse 80,000 drug-like

compound library that has not been previously screened against any

form of the malaria parasite. We screened the Jumpstarter library

against the asexual form of the P. falciparum 3D7 parasite using the

previously reported Plasmodium lactate dehydrogenase (LDH) assay

format [14]. Two individual counterscreen approaches were conducted

to ensure compounds were genuinely reducing parasite viability and not

inhibiting parasite LDH or interfering with the LDH assay technology.

The high-throughput screen identiﬁed three structurally related hits 1–3

which have a [1,2,4]-triazolopyrimidine core with a 2,5-disubstituted

aryl group attached via a carboxamide (Fig. 1). The hit compounds

1–3 have EC₅₀values against asexual P. falciparum ranging from 0.21 to

LipE

MMV

NADPH nicotinamide adenine dinucleotide phosphate

Pf

Plasmodium falciparum

Pk

Plasmodium knowlesi

pLDH

SAR

SNAr

Plasmodium lactate dehydrogenase

structure activity relationship

nucleophilic aromatic substitution

TCAMS Tres Cantos antimalarial set

Therefore, there is an ongoing need for the development of novel classes

of antimalarial therapies. The World Health Organization has called for

the urgent development of new antimalarials with novel mechanisms of

action, targeting multiple stages of the parasite’s lifecycle to curb the

emergence of resistance [5]. Additionally, there is a need to develop

improved medicines which are proven to be safe for the treatment of

malaria in pregnant women, especially during the ﬁrst trimester since

quinine in combination with clindamycin remains the recommended

drug [1].

0.47 μM and did not inhibit human HepG2 cell growth (EC₅₀> 40 μM).

A literature search on the triazolopyrimidine scaffold found four

structurally related compounds 4–7 previously identiﬁed from the Tres

Cantos Antimalarial Set (TCAMS) originating from the high-throughput

screen of the GlaxoSmithKline two million drug-like compound diversity

library (Fig. 1) [8,15]. Subsequently one compound from the TCAMS

was selected for inclusion in the Medicines for Malaria Venture (MMV)

Pathogen Box (MMV019087, 7), an open-source collection of 400

compounds with activities against a variety of human pathogens [16].

Additional screening showed MMV019087 displayed modest activity

In the last 20 years, there has been a collaborative effort by many

organisations to identify novel chemical scaffolds for the development of

antimalarials against unique parasite targets [6]. Phenotypic high

Fig. 1. Three structurally related hits (1–3) with the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold were identiﬁed from the HT screen of the Janssen

Jumpstarter library against P. falciparum 3D7 asexual stage parasites. Structurally related compounds can also be found within the Tres Cantos Antimalarial Set

(TCAMS) and the MMV Pathogen Box (4–7).

2

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

against P. berghei sporozoites (EC₅₀1.2

μ

M), but no activity against

synthesis of the aniline moiety started with the S_NAr reaction between a

4-substituted 2-nitro aryl halide (8a-j) and a substituted amine. Subse-

quent reduction of the nitro functionality employing zinc and ammo-

nium chloride in ethanol, produced the corresponding aniline 9. In this

reaction, when a tertiary amine group was ortho to the nitro function-

ality a signiﬁcant by-product (12) was observed which impacted the

yields of the aniline product 9. The by-product 12 is known to form

through reductive cyclisation [20,21], and thus exclusion of oxygen

from the reaction mixture signiﬁcantly reduced the formation of

byproduct 12 and increased the yields of the aniline product 9. Finally,

an amide coupling between the aniline 9 and 2-carboxyl triazolopyr-

imidine or pyrazolopyrimidine (10a-d) using HATU, COMU or T3P was

performed to afford the desired carboxamide product 11.

P. falciparum stage V gametocytes (EC₅₀> 10

μ

M) (Table S1) [17,18]. To

the best of our knowledge, there has been no reports investigating the

structure activity relationship or parasite phenotype of the triazolopyr-

imidine antimalarial class.

Herein, we deﬁne the structure activity relationship of the 2-(N-

phenyl carboxamide) triazolopyrimidine scaffold towards improving

their antimalarial activity against asexual P. falciparum parasites and

maintaining a selectivity window against the human HepG2 cell line.

Physicochemical and in vitro metabolic parameters were also considered

in the design of analogues. A subset of analogues was subsequently

evaluated for their asexual stage of arrest, rate of kill and phenotyping

studies to infer potential antimalarial mechanistic information on the

compound class.

2.2. Structure activity relationship

2. Results and discussion

The investigation into the structure activity relationship of the 2-(N-

phenyl carboxamide) triazolopyrimidine scaffold started with deter-

mining the importance of functionality in the 2- and the 5- positions on

the exocyclic aniline ring for modulating P. falciparum asexual stage

activity (Table 1). The results show that the removal of both substituents

at the 2- and 5- positions (13) ablated activity, implying that sub-

stituents at both positions are making signiﬁcant contributions.

We next sought to determine the importance of the 5-triﬂuoromethyl

and 5-chloro substitution on parasite activity while maintaining N,N-

disubstitution in the 2-position that are similar to the groups in the hit

compounds 1–3 (Fig. 1). The removal of the substituent in the 5-position

(14–17) signiﬁcantly decreased the activity compared to the analogues

with 5-triﬂuoromethyl or 5-chloro substitution (19–22 and 24–27)

(Table 1). The analogues with 5-triﬂuoromethyl substitution (19–22)

consistently displayed greater potency in comparison to the 5-chloro

analogues (24–27). Preliminary activity trends with substitution in the

2-position were also noted from this study set of analogues, and there

was a correlation between increasing steric size of the N,N-substitution

with an increase in potency with the exception of morpholine. The N-

piperidine substitution in the 2-position (20 and 25) was consistently

the most potent from this study set (Table 1).

The primary goal of the research described herein was to establish

the structure activity relationship (SAR) by iterative variation of moi-

eties on the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold fol-

lowed by evaluation of P. falciparum asexual activity while monitoring

selectivity against the HepG2 human cell line. The hit compound 3 has

limited aqueous solubility (<1.6 µM at pH 6.5) and was rapidly

degraded in the presence of mouse liver microsomes (CL_int275 μL/min/

mg) (Table S3). This data was consistent with Pathogen Box data

collected on MMV019087 7 (Table S2). Therefore, improvement of these

two properties were factored into the optimisation of this compound

class. The P. falciparum 3D7 asexual activity of analogues was deter-

mined using a previously described Plasmodium LDH assay that derives

parasite viability from metabolic activity [14]. In this assay, human

erythrocytes infected with synchronized parasites are incubated in the

presence of the compound for 72 h followed by freeze–thaw lysis and

addition of LDH detection reagents. Human HepG2 cellular cytotoxicity

was iteratively monitored using ATP-dependent Cell TiterGlo as an

indication of cell growth [19].

2.1. Chemistry

The 5- and 7- position methyl substitution on the triazolopyrimidine

moiety present in the hit compounds (1–3) was next explored (Table 2).

It was found that analogues 19–22 with 5,7-dimethyl substitution were

signiﬁcantly more potent compared to compounds 28–31 with methyl

The general synthesis of triazolopyrimidine analogues involved

sequential building of the disubstituted aniline followed by an amide

coupling step with the 2-carboxyl triazolopyrimidine (Scheme 1). The

Scheme 1. General Synthetic Pathway to Access Triazolopyrimidine Analogues. ^aReagents and conditions: (a) (i) substituted amine or substituted alcohol, K₂CO₃or

NaH, DMF 80 ^◦C. (ii) Zn, NH₄Cl, EtOH, N₂, reﬂux. (b) HATU or COMU or T3P, DIPEA, DMF 50 ^◦C. The structure of the benzimidazole by-product 12 that was

observed when oxygen was not excluded from the zinc reduction of the nitro group in step (ii).

3

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

remainder of this study.

Table 1

Activity of anilino substituted analogues.

Further assessment of functional group tolerance at the 5-position of

the aniline ring was then investigated while maintaining either N-

piperidine or N-pyrrolidine substitution at the 2-position (Table 3).

These results show a range of groups were accommodated in the 5-posi-

tion, but analogues with the triﬂuoromethyl (20, 21), chloro (25, 26)

and triﬂuoromethoxy (44, 45) substituents were the most potent. In

contrast to chloro substitution in the 5-position, analogues with 5-ﬂuo-

rine substitution (36, 37) were signiﬁcantly less potent (EC₅₀0.80 and

Cmpd

R¹

R²

H

Pf parasite

HepG2 CC₅₀

PSA

cLogP ^c

LipE

1.17

moderately weaker (EC₅₀0.22 and 2.6

methyl substitution (40, 41) were approximately 2 to 3-fold weaker

(EC₅₀0.17 and 0.97 M) than 20 and 21 indicating electron neutral

hydrophobic groups were tolerated. Analogues with 5-methoxy substi-

μM) while the analogues with 5-bromine substitution (38, 39) were

EC₅₀(SD)

μ

M ^a

(SD) μ

M ^b

(Å²) ^c

μM). The analogues with 5-

13

14

H

> 10

–

72

2.9

3.8

–

μ

>10

–

75

tution (42, 43) were signiﬁcantly less potent (EC₅₀0.60 and 1.7 μM)

than 20 and 21, while analogues with 5-nitrile (46, 47) or 5-methyl

sulfone (48, 49) substitution resulted in a signiﬁcant reduction in anti-

15

16

H

0.90 (0.12)

> 40

75

3.9

3.5

2.17

parasitic activity (EC₅₀s > 0.45 μM), signifying polar groups are not

>10

–

tolerated at the 5-position of the aniline ring. Overall, the data from this

study set indicated small electron withdrawing and hydrophobic groups

such as the triﬂuoromethyl group at the 5-position were optimal. This is

reﬂected by the LipE values (~2) which are comparable across the

different analogues in Table 3. Although the potencies vary considerably

across analogues, the high cLogP necessary to obtain meaningful po-

tency is reﬂected in consistently low LipE values.

17

H

> 10

–

85

2.8

–

18

19

CF₃

H

> 10

–

72

75

3.8

4.7

–

1.15 (0.08)

>40

1.22

We next explored a greater breadth of functionality at the 2-position

while maintaining the triﬂuoromethyl group in the 5-position of the

aniline ring (Table 4). The addition of heterocycles with multiple

endocyclic nitrogens in the 2-position such as N-piperazine (54), N-4-

methylpiperazine (55), N-pyrazole (56) and N-[1,2,4]-triazole (57)

20

21

CF₃

0.07 (0.03)

0.36 (0.08)

> 40

75

4.8

4.4

2.39

2.07

signiﬁcantly decreased activity (EC₅₀s > 0.32 μM) in comparison to N-

piperidine substitution (20). Similarly, N-morpholine (22) at the 2-posi-

tion resulted in a 10-fold penalty to activity compared with N-piperidine

(20). Smaller acyclic amino groups in the 2-position such as N-dime-

thylamine (52) and N,N-diethylamine (19) also showed a decrease in

22

CF₃

0.68 (0.07)

> 40

85

3.7

2.47

23

24

Cl

H

> 10

–

72

75

3.4

4.4

–

potency (EC₅₀0.70 and 1.2

μ

M), while N,N-ethyl methylamine (53)

M). Unfortunately, access

substitution maintained potency (EC₅₀0.14

μ

3.37(1.6)

> 40

1.11

to a series of secondary amine substituents at the 2-position was

impeded by spontaneous ring closure at the carboxamide linkage

forming a benzimidazole (Scheme S7). It was noted that the benzimid-

azole by-product was not active against asexual P. falciparum (EC₅₀> 10

25

26

Cl

0.09 (0.01)

0.64 (0.20)

> 40

75

4.4

4.0

2.65

2.19

μ

M). Subsequent attempts to use protective group strategies in the

synthesis of secondary amine analogues were also unsuccessful, and thus

these analogues were not further pursued. Phenolic substituents were

also explored in the 2-position. n-Propyloxy (58), isobutoxy (59) and

benzyloxy (60) substituents resulted in a considerable reduction in ac-

27

Cl

1.01 (0.32)

> 40

85

3.3

2.67

tivity (EC₅₀1.4 to 3.3 μM) compared with N-piperidine substitution

a

EC₅₀data represents averages and SDs for three or more experiments

(20). Overall, there was a strong correlation between the size and hy-

drophobicity of substitution in the 2-position and parasite activity.

Consistent with this observation, the activity of the 2-N-homopiperidine

measuring LDH activity of P. falciparum 3D7 asexual parasites following treat-

ment with compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

b

EC₅₀data represents averages and SDs for 3 or more experiments following

analogue 50 (EC₅₀0.06 μM) and 2-N-nortropane 51 (EC₅₀0.08 μM)

exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell

growth inhibition.

exhibited comparable activity to the 2-N-piperidine analogue (20).

c

We next sought to introduce an endocyclic nitrogen into the aniline

aryl moiety (Table 5) to determine the effect on asexual parasite activity

and to also assist in improving aqueous solubility. While maintaining the

2,5-substitution pattern of the aniline ring, the endocyclic nitrogen at

the 3-position (61, 62) was the only endocyclic variant that was syn-

thetically tractable. Unfortunately, the addition of the endocyclic ni-

trogen in this position was detrimental to parasite activity (EC₅₀> 10

Determined using ChemAxon software [43].

substitution in the 7-position as well as compounds 32–35 with no

substitution in the 5- and 7- positions. As an example, removal of both

methyl groups at the 5- and 7-positions from compound 33 resulted in a

15-fold decrease in activity (EC₅₀1.02 μM) compared with the 5,7-

dimethyl substituted analogue 20 (EC₅₀0.07 μM), while the analogue

μ

M). To further explore the inclusion of an endocycle nitrogen at other

with no substitution at the 5-position and a methyl group in the 7-posi-

positions of the aniline ring, analogues with 4-triﬂuoromethyl substi-

tution (63, 64) were assessed. While these analogues (63, 64) were

signiﬁcantly less active, they did exhibit antiparasitic activity in the

tion (29) also resulted in a signiﬁcant reduction in potency (EC₅₀0.11

μ

M). Analogues with a methyl in the 5-position and no substitution at

the 7-position were not chemically tractable thus could not be assessed

for their activity. Collectively, this data suggested the 5,7-dimethyl

substitution was optimal and therefore was preserved for the

micromolar range (EC₅₀4.4 and 6.3 μM). The subsequent inclusion of an

endocyclic nitrogen in the 3- and 5- positions (65–68) while maintaining

4-triﬂuoromethyl substitution ablated parasite activity except for 65

4

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

Table 2

Activity of 5- and 7- methyl substituted analogues.

^aEC₅₀data represents averages and SDs for three or more experiments measuring LDH activity of P. falciparum 3D7 asexual parasites following treatment with

compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

5

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

^bEC₅₀data represents averages and SDs for 3 or more experiments following exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell growth

inhibition.

^cDetermined using ChemAxon software [34].

which had modest micromolar activity (EC₅₀3.3

μ

M). Collectively, this

was in the low micromolar range in both simulated gastrointestinal and

intestinal conditions and was consistent with the low aqueous solubility

observed with the MMV019087 7 (Table S2) from the MMV Pathogen

Box [16]. During exploration of the SAR and optimisation of the tri-

azolopyrimidine class, we attempted to address aqueous solubility by

inclusion of an endocyclic nitrogen in the aniline aryl ring and ionisable

groups in the 2-position of the aniline aryl ring, but these modiﬁcations

ablated parasite activity. The planar nature and high crystallinity of the

bicyclic triazolopyrimidine scaffold is a major contributor to the low

solubility of this antimalarial class, and in combination with the SAR

observed, overcoming low aqueous solubility will be a signiﬁcant

challenge.

data suggests that the incorporation of an endocyclic nitrogen in the

aniline ring is not tolerated.

We next investigated the necessity of the 3-position nitrogen in the

triazolopyrimidine ring while maintaining the 5,7-dimethyl substitution

on the triazolopyrimidine and either 5-triﬂuormethyl or 5-chloro 2-

piperdine substitution on the exocyclic aniline. The removal of endo-

cyclic nitrogen in the 3-position, to give the pyrazolopyrimidine ana-

logues, 69 and 70, resulted in a loss of antiparasitic activity (EC₅₀> 10

μ

M) (Table 6), implying this endocyclic nitrogen was involved with a

critical interaction with the target parasite protein. Analogues whereby

the endocyclic nitrogen was replaced with carbon at other positions on

the triazolopyrimidine core were not synthetically tractable.

The in vitro metabolism conducted in the presence of mouse liver

microsomes indicated a short half-life and high intrinsic clearance for

both 3 and 20 (Table S2). It was found that the degradation of 3 and 20

in liver microsomes was not dependent on the enzyme co-factor NADPH,

suggesting metabolism occurred via a non-oxidative mechanism. Sup-

porting this hypothesis, carboxamide hydrolysis metabolites were

identiﬁed by mass spectrometry (Table S2) that are consistent with

metabolic degradation by a hydrolase. Low stability in human liver

microsomes for the structurally related compound, MMV019087 7, was

also reported in the MMV Pathogen Box data (Table S2) [16]. Low sta-

bility in mouse plasma was also reported for MMV019087 7, consistent

with a non-NADPH mediated degradation event by a hydrolase. During

our optimisation study of the triazolopyrimidine class, we attempted to

address the metabolic hydrolysis of the carboxamide bond, by reversing

the orientation of the amide bond and N-methylation, but these changes

were not tolerated. Other chemical modiﬁcations were proposed, such

as amide bond isosteres, but despite our efforts, these iterations were not

synthetically tractable, and therefore addressing the metabolic stability

of the carboxamide moiety will be a major challenge in future optimi-

sation of the triazolopyrimidine antimalarial class.

The carboxamide bond was identiﬁed as a metabolic liability

(Table S3) (discussed in a later section) and therefore alternative itera-

tions of the carboxamide were explored in attempt to mitigate this

(Table 7). N-Methylation of the carboxamide amide nitrogen (71) was

trialled because N-methylation is known to suppress amide bond hy-

drolysis [22]. This modiﬁcation, however, led to a 100-fold decrease in

potency (EC₅₀7.4 μM) compared to the parent compound 20. Alteration

of the carboxamide bond in the opposite conﬁguration (72) was also

attempted, but this variation also resulted in a signiﬁcant loss of parasite

activity (EC₅₀2.3 μM) in comparison to analogue 20.

In summary of the structure activity relationship (Fig. 2), it was

found that the 3-endocyclic nitrogen present in the triazolopyrimidine

core is essential for maintaining potent P. falciparum asexual activity,

while presence of 5,7-dimethyl substitution signiﬁcantly improves ac-

tivity compared to the monomethyl substitution. 6- and 7-membered

aliphatic heterocycles such as a homo-piperidine or piperidine are

preferred at the 2-position of the aniline ring, while at the 5-position,

small hydrophobic substituents such as triﬂuoromethyl, chloro or tri-

ﬂuoromethoxy conferred superior potency, although there was toler-

ance for a range of functional groups. It was observed that addition of an

endocyclic nitrogen at various positions of the exocyclic aniline ring

signiﬁcantly impacted activity. Altering the carboxamide linkage be-

tween the aniline and triazolopyrimidine system was also detrimental to

potency. Overall, the structure activity relationship study deﬁned the

functionality required for asexual P. falciparum activity and engendered

2.5. Evaluation against P. knowlesi and multidrug resistant strains of

P. falciparum

To determine if the triazolopyrimidine class was active against

Plasmodium species other than P. falciparum, a representative selection

of potent analogues (20, 25, 44, and 50) were evaluated against

P. knowlesi, a human infective species endemic to Southeast Asia [23].

The activity of compounds 20, 25, 44, and 50 against P. knowlesi (EC₅₀

several potent analogues 20, 25, 44, and 50 (EC₅₀0.06–0.09 μM) for

further asexual phenotype proﬁling.

2.3. Evaluation of human cell cytotoxicity

0.37–1.2

μM) compared to P. falciparum (EC₅₀0.02–0.05 μM) was

considerably reduced with a 14- to 23- fold reduction in potency

(Table 8, Fig. S3). It was postulated, the difference in activity could be a

differentiation in the structure of the compound target protein between

the two species. Mechanism of action studies to identify the protein

target of the triazolopyrimidine antimalarial class is required to conﬁrm

this hypothesis.

Analogues were evaluated for human HepG2 cellular cytotoxicity

using Cell Titre-Glo as an ATP dependent marker of cell health

(Tables 1–6). All analogues evaluated had no observable cytotoxicity

within the titration range tested (EC₅₀> 40

μM). Overall, the tri-

azolopyrimidine antimalarial class was found to have a favourable

selectivity proﬁle against the human HepG2 cell line and implies the

parasite protein target of the triazolopyrimidine compounds is either not

present in or essential to the survival of human cells or has low ho-

mology to the homologous protein in human cells. Target identiﬁcation

studies with the triazolopyrimidine antimalarial class are required to

conﬁrm these hypotheses.

The representative compounds 20, 25, 44, and 50 were also evalu-

ated against a range of P. falciparum multidrug resistant strains. W2_mef

and its derivative DD2 is resistant to pyrimethamine, chloroquine,

meﬂoquine and quinine, with DD2 having additional copies of pfmdr1

that are reported to confer slightly higher meﬂoquine resistance [24].

7G8 is resistant to chloroquine and pyrimethamine [25], while Cam3.I is

an artemisinin resistant strain with mutations in the Kelch13 propellor

region [26]. The data using these resistant strains shows, the compounds

20, 25, 44, and 50 exhibited approximately equipotent activity against

P. falciparum multidrug resistant strains compared to 3D7 (Table 8,

Fig. S2). Overall, this data suggests the triazolopyrimidine antimalarial

class is not predisposed to the same resistance mechanisms as estab-

lished antimalarials. It is noted however, that Artemisinin resistance

cannot be monitored using the standard IC₅₀determination employed

2.4. Evaluation of aqueous solubility and in vitro metabolic stability

The aqueous solubility and in vitro liver microsome stability was

evaluated for selected focal analogues from this series to determine their

appropriateness for future in vivo studies. Kinetic aqueous solubility was

determined at simulated gastrointestinal (pH 2.0) and intestinal condi-

tions (pH 6.5) by nephelometry. The solubility of analogues 3 and 20

6

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

Table 3

Activity of 5^′-anilino substituted analogues.

7

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Bioorganic Chemistry 115 (2021) 105244

^aEC₅₀data represents averages and SDs for three or more experiments measuring LDH activity of P. falciparum 3D7 asexual parasites following treatment with

compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

^bEC₅₀data represents averages and SDs for 3 or more experiments following exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell growth

inhibition.

^cDetermined using ChemAxon software [34].

here, and therefore a ring survival assay would be required to further

investigate genuine artemisinin resistance.

third generation (120 h) of parasite development (Table 9, Fig. S4). The

results show no signiﬁcant change was observed between the two time

points for compounds 20, 25, 44, and 50. This indicates the tri-

azolopyrimidine antimalarial class are not killing asexual stage parasites

via a delayed death mechanism implying that the apicoplast is not the

target for this compound series.

2.6. Appraisal of compounds in the asexual stage parasite reduction ratio

assay

The asexual parasite reduction ratio assay [27] determines the rate of

parasite killing of antimalarials and is an important factor in deter-

mining their alignment with TCPs [12]. This method ﬁrstly involves

calculating the 3D7 (BEI Resource, Manassas) parasite growth inhibition

by [³H]-hypoxanthine incorporation after 48 and 72 h of treatment with

compound (Fig. 3A) [28]. In this assay, representative compounds 1 and

2 exhibited no inhibitory activity at 48 h, but potently inhibited parasite

2.8. Determination of stage of arrest in P. falciparum asexual parasites

Having established that the triazolopyrimidine class does not act

with a delayed death phenotype, we next sought to establish the precise

stage of parasite arrest in the asexual cycle. To determine the stage of

asexual arrest, synchronised ring stage parasites were treated with either

DMSO vehicle control or compound 20 at 10 times the EC₅₀. Giemsa-

stained blood smears were then prepared every 12 h to allow for para-

site stage determination, while ﬂow cytometry measurements deter-

mined parasitaemia over this time. These data showed that asexual cycle

arrest after early ring stage treatment with 20 occurred at trophozoite

stages within that same cycle of growth, while DMSO successfully

completed the remainder of the lifecycle leading to parasite multipli-

cation into new RBCs (Fig. 4A). Parasites treated with 20 failed to

replicate merozoites and thus could not multiply into new RBCs

(Fig. 4B). Combined, these data indicate that parasites treated at ring

stage were unable to develop past the equivalent of a trophozoite stage

parasite (e.g. parasites stop growing in size and merozoites fail to form).

However, the drug treated parasites were still capable of taking up hy-

poxanthine during the ﬁrst 48 h of development, leading to no change in

the 48 h hypoxanthine assay IC₅₀(Fig. 3A). Moreover, antimalarial

compounds acting very slowly could allow enough incorporation of

radiolabelled hypoxanthine before parasite growth is arrested preclud-

ing an accurate determination of the EC₅₀. Similar to pyrimethamine

and atovaquone that have a moderate/slow rate of kill respectively, the

growth inhibitory activity could be largely washed off with 24 h, but not

48 h of treatment (Fig. 3B). Collectively, the phenotype data shows the

triazolopyrimidine series has a moderate/slow onset of action and is

most effective at inhibiting parasite growth between 24 and 48 h post

ring stage parasite drug treatment. Mechanism of action studies will be a

focus of future studies on the triazolopyrimidine class to determine the

target protein responsible for the slow onset phenotype observed.

growth at 72 h (EC₅₀s 0.04 and 0.06

onset of parasite kill between 48 and 72 h. Compounds 1 and 2 are

shown to be less potent (EC₅₀s 0.47 and 0.21 M) in the 72 h LDH assay

μM). These results indicate a slow

μ

compared to the hypoxanthine assay. The discrepancies in half-maximal

inhibition between the [³H]-hypoxanthine and LDH assays likely result

from the signiﬁcant increase in parasite LDH levels prior to the onset of

growth inhibition in slower acting antimalarials, leading to elevated

EC₅₀values compared to the hypoxanthine assay [29,30].

The parasite reduction rate assay involves culturing asynchronous

parasites in the presence of compound (10 × hypoxanthine EC₅₀) for a

duration of 24 and 48 h [27]. Following compound washout, the par-

asites were cultured with uninfected erythrocytes stained with the

intracellular dye, carboxylﬂuorescein diacetate succinimidyl ester

(CFDA-SE). After a further 48 h of incubation parasite DNA was stained

with Hoechst 33342 and parasite viability (speciﬁcally new invasions)

was determined by two-colour ﬂow cytometry. The rate of kill in this

assay is deﬁned as fast (no viable parasites detected after 24 h of

treatment e.g., chloroquine and artemisinin), moderate (no viable par-

asites detected after 48 h of treatment e.g., pyrimethamine) and slow

(>48 h of treatment required to kill parasites e.g., atovaquone) [27]. In

this assay, compounds 1 and 2 exhibit a moderate to slow rate of parasite

kill similar to the rate of action observed for pyrimethamine and ato-

vaquone (Fig. 3B).

2.7. Investigation of delayed death phenotype

The moderate to slow onset of antimalarial activity observed by

compounds 1 and 2 in the parasite reduction rate assay suggested that

this triazolopyrimidine class could possibly be acting via mechanism

associated with a classic delayed death phenotype. The delayed death

phenotype is characterised by the absence of antimalarial activity in the

ﬁrst 48 h asexual cycle, but a time-dependent increase in growth inhi-

bition between the ﬁrst and the second daughter generation [31–33].

For example, azithromycin, a prokaryotic translational inhibitor, shows

3. Conclusions

The present study describes the investigation of the SAR and opti-

misation of the potency of the 2-(N-phenylcarboxamide) triazolopyr-

imidine scaffold identiﬁed from the P. falciparum phenotypic HT screen

of the Janssen Jumpstarter library. In this research, we deﬁned the SAR

and demonstrated each structural motif explored was relatively sensitive

to change. Despite this restrictive characteristic of this scaffold, com-

pounds were generated with potent activity against asexual stage

P. falciparum while showing no overt signs of cytotoxicity to the human

HepG2 cell line.

a 160-fold increase in potency from 48 to 96 h (8.6 μM to 0.05 μM) [34].

This increase in potency is still observed for delayed death antimalarials

even when drug pressure is removed after the ﬁrst cycle [35]. Delayed

death compounds speciﬁcally target the prokaryotic housekeeping

processes of an essential endosymbiotic organelle called the apicoplast

[34]. Chemical rescue studies using isopentenyl pyrophosphate (IPP)

supplementation has determined IPP biosynthesis is the only essential

function of the apicoplast during the blood stage [36]. The physiological

result of IPP depletion is compromised digestive vacuole function as a

result of impaired prenylation-dependent intracellular trafﬁcking [37].

To determine whether the slow onset of asexual parasite killing

observed with the triazolopyrimidine class was a result of delayed death,

we assessed the impact on parasite viability within the second (72 h) and

Several challenges remain for the further optimisation of this com-

pound series. The planarity and crystallinity of the bicyclic scaffold is an

inherent attribute that limits aqueous solubility. The issue with aqueous

solubility is further compounded by the requirement of lipophilic sub-

stituents to drive parasite potency. The ﬁne balance between function-

ality required for activity and the incorporation of groups that will break

crystallinity of the scaffold and improve LipE and solubility remains a

challenge. The non-NADPH dependent metabolic instability of the car-

boxamide, central to the activity this antimalarial class, has also been

8

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

Table 4

Activity of 2^′-alipahtic anilino analogues.

9

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

^aEC₅₀data represents averages and SDs for three or more experiments measuring LDH activity of P. falciparum 3D7 asexual parasites following treatment with

compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

^bEC₅₀data represents averages and SDs for 3 or more experiments following exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell growth

inhibition.

^cDetermined using ChemAxon software [34].

identiﬁed as a liability limiting further advancement of this scaffold.

From our cursory exploration of amide bond alternatives, this moiety

appears crucial to parasite activity and is not amenable to change. It is

foreseen that signiﬁcant development of complex syntheses will be

required to generate suitable non-hydrolysable carboxamide isosteres to

curtail metabolic stability and further advance the development of this

antimalarial class.

Aqueous Solubility. Aqueous solubility was determined kinetically

by nephelometry following the previously described protocol [19].

P. falciparum Multidrug Resistant and P. knowlesi Asexual

Parasite Viability Assays. Parasite viability assays were performed

according to the procedure previously described [39]. Brieﬂy, all para-

site lines were cultured according to standard conditions [26,40], and

parasites were synchronised using 5% ^D-sorbitol [41]. Synchronous ring

stage parasites were exposed to compound and parasite growth was

measured by ﬂow cytometry with ethidium bromide (EtBr) relative to

control following incubation to the second asexual cycle (72 h for drug

resistant and 48 h for P. knowlesi parasites). Dose response values and

error were determined using a nonlinear regression in GraphPad Prism

(version 8.0.1).

We characterised representative compounds from the triazolopyr-

imidine class and demonstrated they did not have cross-resistance to

multi-drug resistant P. falciparum lines which implies this scaffold is not

predisposed to resistance mechanisms of common antimalarials. Addi-

tionally, selected analogues were proﬁled against P. knowlesi and

showed reduced activity compared to P. falciparum parasites. We

postulate that amino acid differences in the target protein of this com-

pound class are responsible for the difference in activity observed be-

tween the two species of parasite. It is therefore possible structural

differences also exist in the compound target protein in other Plasmo-

dium species that infect humans, such as P. vivax.

Asexual Stage Parasite Reduction Ratio (PRR) Assay. This assay

was performed following the previously described method [27]. Four

antimalarial drugs (chloroquine, pyrimethamine, atovaquone and arte-

sunate) are included in each assay to validate the results produced and

allow comparative classiﬁcation of the killing behaviour of the drugs

tested.

We also investigated the asexual stage of arrest phenotype observed

in P. falciparum parasites with treatment of the triazolopyrimidine class.

The parasite reduction rate assay indicated that the compound series

had a moderate to slow onset of action within 24- and 48-hour window

of drug treatment, similar to the proﬁle observed with pyrimethamine

and atovaquone. This observation was supported by microscopy data

showing the triazolopyrimidine compounds arrest parasite development

within the ﬁrst 48-hour cycle of treatment at the trophozoite stage. In

contrast, parasites survived beyond the ﬁrst 48-hour cycle of treatment

in the [³H]-hypoxanthine assay, suggesting the trophozoite arrested

parasites are still metabolically active (detectable by the hypoxanthine

assay) until the end of the ﬁrst 48-hour asexual cycle, but are not

metabolically active in the second asexual cycle at 72 h. We subse-

quently showed that this killing proﬁle was not a classic delayed death

phenotype.

[³H]-Hypoxanthine P. falciparum Drug Susceptibility Assay.

P. falciparum 3D7A (BEI, Manassas) parasites were cultured in RPMI-

1640, Albumax II and 5 μM hypoxanthine at 0.5% parasitaemia and

2% haematocrit [40]. For 48 h determination, the culture was tightly

synchronised to early ring-stage with ^D-sorbitol synchronisation [41]

and incubated with compounds in a 9-point dilution series for 24 h. After

24 h of incubation [³H]-hypoxanthine was added and incubated for a

further 24 h. For 72 h determination, the culture was tightly

synchronised to early ring-stage with sorbitol synchronisation and

incubated with compounds in a 9-point dilution series for 48 h. After 48

h of incubation [³H]-hypoxanthine was added and incubated for a

further 24 h. Parasites were then harvested on a glass ﬁbre ﬁlter using a

TOMTEC Cell harvester 96. Filters were dried and melt-on scintillator

sheets were used to determine the incorporation of [³H]-hypoxanthine.

Radioactivity was measured using a microbeta counter. Data were

normalized using the incorporation of an untreated control. IC₅₀is

determined using Excel and Graﬁt software. The human biological

samples were sourced ethically, and their research use was in accord

with the terms of the informed consents under an IRB/EC approved

protocol.

In conclusion of this research, we deﬁned the structure activity

relationship and highlighted the challenges with further optimisation of

the triazolopyrimidine antimalarial class. The MMV Pathogen Box data

shows the triazolopyrimidine class has no gametocyte activity [18],

suggesting it will not be suitable for inclusion in a transmission popu-

lation control therapy. The modest liver stage activity exhibited by this

class [16], requires conﬁrmation, but the moderate to slow onset of

action and potent asexual stage activity suggests it may be suitable as a

partner agent in a prophylactic or curative treatment with a fast acting

antimalarial. To assist with further classiﬁcation and development of the

triazolopyrimidine antimalarial class, discovering the mechanism of

action is a key focus of future work.

Delayed-Death Asexual Assay. Parasite growth inhibition assays to

detect delayed-death effects were carried out using a modiﬁed version

the assay described in Rodríguez et al. [42] Brieﬂy, tightly synchronised

early ring stage parasites were cultured under standard conditions [41]

and seeded in 96-well plates at 0.5% (72 h) or 0.2% (120 h) parasitemia,

2% haematocrit [40]. Parasites were exposed to an 8-point dilution se-

ries of compounds dissolved in DMSO for 72 h, when the higher para-

sitemia plate was washed with PBS and the parasitemia was assessed

using a modiﬁed SYBR green method [35]. The growth media in the

lower parasitemia plate was replaced with fresh media lacking com-

pound and allowed to grow for a further 48 h before parasitemia was

assayed. IC₅₀values were calculated with the four-component model in

GraphPad Prism and the data presented is the mean and ± SEM of at

least three biological replicates.

4. Experimental

P. falciparum 3D7 Asexual Stage Parasite LDH Viability Assay.

The parasite viability assay using LDH was performed following the

previously described protocol [38]. Dose response values were deter-

mined using a 4-parameter log dose, non-linear regression analysis, with

sigmoidal dose response (variable slope) curve ﬁt in GraphPad Prism

(version 9.0.1).

Asexual Stage Arrest Assay. Plasmodium falciparum 3D7 parasite

cultures were synchronised twice in an 8 h interval then once again at

48 h following the initial synchronisation with 5% ^D-sorbitol [41]. 10 mL

cultures containing 700 nM of compound 20 or DMSO were established

below 1% parasitaemia and 5% haematocrit. Media and compound were

refreshed at 24 h intervals. Asexual stage was qualiﬁed by microscopic

examination of Giemsa-stained smears. Parasitaemia was quantiﬁed at

HepG2 Viability Assay. The previously described assay protocol

was followed [19]. A four-parameter logistic nonlinear regression model

was used to determine dose response values in GraphPad Prism (version

9.0.1).

In vitro Liver Microsome Stability. Metabolic stability was assessed

using the previously described method [19].

10

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

12 h intervals by ﬂow cytometry, withdrawing 50 µL samples in tripli-

cate. Parasites were ﬁxed with 50 µL of 0.25% glutaraldehyde diluted

with PBS at room temperature for 30 min. Samples were centrifuged at

1200 rpm for 2 min and the pellet was washed with 50 µL of PBS. After

removal of the PBS, parasites were stained with 50 µL of 2.5 × SYBR

Green (Invitrogen) for 30 min and diluted in 1 mL PBS. 100,000 cells

were counted by ﬂow cytometry using a Cytoﬂex Flow Cytometer

(Beckman Coulter). Parasite morphology was assessed by microscopy

using Giemsa-stained parasite infected RBCs at the time points

indicated.

Table 5

Activity of heterocyclic amide analogues.

4.1. Chemistry experimental

General Chemistry Methods. Chemistry methods are as described

in Nguyen et al. [39] Solvents were obtained commercially and used

without further puriﬁcation. Chromatography was performed with silica

gel 60 (particle size 0.040–0.063 μm) using an automated puriﬁcation

system. NMR spectra were collected on a Bruker Avance DRX300 or

Agilent MR400 or Varian INOVA 600. Chemical shifts are reported in

ppm on the δ scale and referenced to the appropriate solvent peak.

MeOD, DMSO‑d₆and CDCl₃contain H₂O. LCMS were recorded Agilent

LC-MS system composed of an Agilent G6120B Mass Detector, 1260

Inﬁnity G1312B Binary pump, 1260 Inﬁnity G1367E HiPALS autosam-

pler and 1260 Inﬁnity G4212B Diode Array Detector (Method B). Con-

ditions for LCMS Method A were as follows, column: Kinetex TM XB-C18

5 μm 4.6 × 50 mm, injection volume 10 μL, 5–100% B over 3 min

(solvent A: water 0.1% formic acid; solvent B: AcCN 0.1% formic acid),

ﬂow rate: 1.5 mL/min, detection: 100–600 nm, acquisition time: 6 min.

Unless otherwise noted, all compounds were found to be > 95% pure by

this method. HRMS were acquired through The Bio21 Mass Spectrom-

etry and Proteomics Facility using a Thermo Scientiﬁc™ nano-LC Q

Exactive™ Plus Mass spectrometer.

Compounds 8a-j, 10a-d, 17, 19, 21, 22 and 28 were purchased from

commercial vendors and used without further puriﬁcation.

4.2. Synthesis methods

General Protocol A: 5,7-Dimethyl-N-phenyl-[1,2,4]triazolo[1,5-a]

pyrimidine-2-carboxamide (13). 5,7-Dimethyl-[1,2,4]triazolo[1,5-a]py-

rimidine-2-carboxylic acid 10a (40 mg, 0.21 mmol), aniline (0.021 mL,

0.23 mmol), HATU (99 mg, 0.26 mmol) and DIPEA (0.15 mL, 0.83

mmol) in DMF (1 mL) were stirred at 50 ^◦C for 24 h. The reaction

mixture was diluted with EtOAc (15 mL) and washed with water (2 × 15

mL) and saturated brine (1 × 15 mL). The organic layer was separated

and dried (Na₂SO₄) before concentration in vacuo. To the crude residue

MeOH (5 mL) was added and the precipitate was ﬁltered and washed

with cold MeOH to obtain 13 as a white solid (11 mg, 20%). ¹H NMR

(DMSO‑d_6,400 MHz) δ 10.73 (s, 1H), 7.95–7.85 (m, 2H), 7.38 (t, J 7.8

Hz, 2H), 7.33 (d, J 1.2 Hz, 1H), 7.15 (t, J 7.4 Hz, 1H), 2.81 (s, 3H), 2.64

(s, 3H). ¹³C NMR (DMSO‑d₆, 101 MHz) δ 166.6 158.2, 158.2, 148.2,

138.7, 129.1, 124.7, 121.1, 112.7, 25.2, 17.0. HRMS Found: [M + H]⁺

268.1190; C₁₄H₁₃N₅O requires [M + H]⁺268.1193.

N-(2-(Diethylamino)phenyl)-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrim-

idine-2-carboxamide (14). General Protocol A was adapted using 73

(37.4 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 14 as a

beige solid (32 mg, 45%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.80 (s, 1H),

8.48 (dd, J 8.0, 1.6 Hz, 1H), 7.39 (dd, J 7.7, 1.6 Hz, 1H), 7.33 (s, 1H),

7.24 (td, J 7.7, 1.6 Hz, 1H), 7.18 (td, J 7.6, 1.6 Hz, 1H), 3.00 (q, J 7.1 Hz,

4H), 2.79 (s, 3H), 2.64 (s, 3H), 0.97 (t, J 7.1 Hz, 6H). ¹³C NMR

(DMSO‑d₆, 101 MHz) δ 166.8, 158.6, 156.7, 154.9, 148.3, 140.1, 136.0,

126.0, 124.7, 124.0, 118.6, 112.8, 49.3, 25.1, 16.9, 13.3. HRMS Found:

[M + H]⁺339.1928; C₁₈H₂₂N₆O requires [M + H]⁺339.1928.

^aEC₅₀data represents averages and SDs for three or more experiments measuring

LDH activity of P. falciparum 3D7 asexual parasites following treatment with

compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

^bEC₅₀data represents averages and SDs for 3 or more experiments following

exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell

growth inhibition.

5,7-Dimethyl-N-(2-(piperidin-1-yl)phenyl)-[1,2,4]triazolo[1,5-a]py-

rimidine-2-carboxamide (15). General Protocol A was modiﬁed using 74

(40 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 15 as a white

solid (29 mg, 40%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.43 (s, 1H), 8.40

^cDetermined using ChemAxon software [34].

11

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

Table 6

Activity of analogues with a pyrazolopyrimidine scaffold.

^aEC₅₀data represents averages and SDs for three or more experiments measuring LDH activity of P. falciparum 3D7 asexual parasites following treatment with

compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

^bEC₅₀data represents averages and SDs for 3 or more experiments following exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell growth

inhibition.

^cDetermined using ChemAxon software [34].

(dd, J 7.8, 1.8 Hz, 1H), 7.32 (s, 1H), 7.27 (dd, J 7.5, 1.8 Hz, 1H),

7.19–7.15 (m, 1H), 7.15–7.10 (m, 1H), 2.82 (t, J 5.2 Hz, 4H), 2.78 (s,

obtain 20 as a coral solid (19 mg, 22%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.36 (s, 1H), 8.75 (d, J 1.5 Hz, 1H), 7.52 (dd, J 8.4, 1.5 Hz, 1H), 7.46 (d,

J 8.4 Hz, 1H), 7.36 (s, 1H), 2.91 (t, J 5.2 Hz, 4H), 2.81 (s, 3H), 2.65 (s,

3H), 1.84 (quint, J 6.0 Hz, 4H), 1.64 (q, J 5.8 Hz, 2H). ¹³C NMR

(DMSO‑d₆, 151 MHz) δ 167.1, 158.2, 157.2, 155.1, 148.3, 147.0, 133.0,

125.6, 125.0, 121.8, 115.5, 113.1, 53.3, 26.6, 25.2, 24.0, 17.0 (¹J_C-Fnot

found). ¹⁹F (DMSO‑d₆, 282 MHz) ꢀ 60.66. HRMS Found: [M + H]⁺

419.1793, C₂₀H₂₁F₃N₆O requires [M + H]⁺419.1802.

3H), 2.63 (s, 3H), 1.80 (quint, J 5.5 Hz, 4H), 1.60 (d, J 7.4 Hz, 2H). ¹³

C

NMR (DMSO‑d₆, 101 MHz) δ 166.4, 158.2, 156.3, 154.6, 147.8, 143.0,

132.4, 124.6, 124.4, 120.8, 118.6, 112.5, 53.3, 26.3, 24.7, 23.7, 16.5.

HRMS Found: [M + H]⁺351.1927; C₁₉H₂₂N₆O requires [M + H]⁺

351.1928.

5,7-Dimethyl-N-(2-(pyrrolidin-1-yl)phenyl)-[1,2,4]triazolo[1,5-a]py-

rimidine-2-carboxamide (16). General Protocol A was altered using 75

(37.4 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 16 as a

N-(3-Chlorophenyl)-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-

carboxamide (23). General Protocol A was adapted using 3-chloroaniline

(0.02 mL, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 23 as a

white solid (20 mg, 32%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.97 (s, 1H),

8.07 (t, J 2 Hz, 1H), 7.86 (dd, J 8.2, 2.0 Hz, 1H), 7.41 (t, J 8.1 Hz, 1H),

1

brown solid (34 mg, 48%). H NMR (DMSO‑d_6,400 MHz) δ 10.14 (s,

1H), 7.95 (d, J 7.9 Hz, 1H), 7.32 (s, 1H), 7.14 (s, 1H), 7.13 (s, 1H), 7.00

(dt, J 8.4, 4.3 Hz, 1H), 3.15 (t, J 4 Hz, 4H), 2.79 (s, 3H), 2.64 (s, 3H),

1.96–1.90 (m, 4H). ¹³C NMR (DMSO‑d₆, 101 MHz) δ 166.3, 158.4,

156.8, 154.6, 147.8, 142.1, 129.0, 125.5, 122.7, 121.4, 118.0, 112.4,

51.0, 24.7, 24.4, 16.5. HRMS Found: [M + H]⁺337.1771; C₁₈H₂₀N₆O

requires [M + H]⁺337.1771.

7.34 (d, J 1.2 Hz, 1H), 7.21 (d, J 8 Hz, 1H), 2.81 (s, 3H), 2.64 (s, 3H). ¹³

C

NMR (DMSO‑d₆, 101 MHz) δ 166.7, 158.6, 158.4, 155.0, 148.2, 140.2,

133.41, 130.8, 124.4, 120.5, 119.5, 112.9, 25.3, 17.0. HRMS Found: [M

+ H]⁺302.0805; C₁₄H₁₂ClN₅O requires [M + H]⁺302.0803.

N-(5-Chloro-2-(diethylamino)phenyl)-5,7-dimethyl-[1,2,4]triazolo[1,5-

a]pyrimidine-2-carboxamide (24). General Protocol A was modiﬁed using

77 (45 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 24 as a

beige solid (31 mg, 40%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.78 (s, 1H),

8.52 (d, J 2.5 Hz, 1H), 7.44 (d, J 8.5 Hz, 1H), 7.35 (s, 1H), 7.24 (dd, J

8.5, 2.6 Hz, 1H), 3.00 (q, J 7.1 Hz, 4H), 2.79 (s, 3H), 2.64 (s, 3H), 0.97 (t,

J 7.1 Hz, 6H). ¹³C NMR (CDCl_3,75 MHz) δ 166.4, 159.6, 157.2, 155.2,

148.0, 138.4, 137.2, 131.2, 124.4, 124.3, 119.7, 112.0, 49.9, 25.3, 17.4,

12.9. HRMS Found: [M + H]⁺373.1538; C₁₈H₂₁ClN₅O requires [M +

H]⁺373.1538.

5,7-Dimethyl-N-(3-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]py-

rimidine-2-carboxamide (18). General Protocol A was adapted using 3-

(triﬂuoromethyl)aniline (0.03 mL, 0.26 mmol) and 10a (50 mg, 0.26

mmol) to obtain 18 as a white solid (12 mg, 13%). ¹H NMR (DMSO‑d₆,

400 MHz) δ 11.16 (s, 1H), 8.39 (s, 1H), 8.22 (d, J 8.4 Hz, 1H), 7.65 (t, J

8.0 Hz, 1H), 7.52 (d, J 8.0 Hz, 1H), 7.37 (s, 1H), 2.83 (s, 3H), 2.66 (s,

3H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 166.27, 158.16, 158.10, 154.57,

147.79, 139.07, 129.95, 129.41 (q, ²J_C-F32 Hz), 124.25, 124.14 (q, ¹J_C-F

272 Hz, two peaks observed), 120.60 (q, ³J_C-F4.3 Hz), 116.84 (q, ³J_C-F

4.0 Hz), 112.44, 24.82, 16.55. ¹⁹F (DMSO‑d₆, 282 MHz) ꢀ 61.24. HRMS

Found: [M + H]⁺336.1064, C₁₅H₁₂F₃N₅O requires [M + H]⁺336.1067.

5,7-Dimethyl-N-(2-(piperidin-1-yl)-5-(triﬂuoromethyl)phenyl)-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (20). General Protocol A was

modiﬁed using 76 (51 mg, 0.21 mmol) and 10a (40 mg, 0.21 mmol) to

N-(5-Chloro-2-(piperidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (25). General Protocol A was adapted

using 78 (48 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 25

as a beige solid (39 mg, 47%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.45 (s,

12

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Bioorganic Chemistry 115 (2021) 105244

Table 7

Activity of analogues with a carboxamide modiﬁcation.

^aEC₅₀data represents averages and SDs for three or more experiments measuring LDH activity of P. falciparum 3D7 asexual parasites following treatment with

compounds for 72 h. Chloroquine EC₅₀24 (2) nM.

^bEC₅₀data represents averages and SDs for 3 or more experiments following exposure to compounds for 48 h using Cell Titer-Glo to quantify HepG2 cell growth

inhibition.

^cDetermined using ChemAxon software [34].

Fig. 2. Summary of the structure activity relationships.

1H), 8.46 (d, J 2.5 Hz, 1H), 7.35 (s, 1H), 7.32 (d, J 8.5 Hz, 1H), 7.20 (dd,

J 8.5, 2.5 Hz, 1H), 2.89–2.74 (m, 7H), 2.65 (s, 3H), 1.81 (quint, J 5.5 Hz,

4H), 1.60 (t, J 4.7 Hz, 2H). ¹³C NMR (DMSO‑d₆, 100 MHz) δ 166.6,

157.8, 156.6, 154.6, 141.8, 136.1, 133.6, 128.5, 124.0, 122.6, 118.0,

112.6, 53.1, 26.3, 24.7, 23.6, 16.5. HRMS Found: [M + H]⁺385.1539;

requires [M + H]⁺371.1382.

N-(5-Chloro-2-morpholinophenyl)-5,7-dimethyl-[1,2,4]triazolo[1,5-a]

pyrimidine-2-carboxamide (27). General Protocol A was adapted using 80

(24 mg, 0.11 mmol) and 10a (20 mg, 0.10 mmol) to obtain 27 as a white

solid (6 mg, 8%). ¹H NMR (DMSO‑d_6,400 MHz) δ 10.51 (s, 1H), 8.46 (d,

J 2.5 Hz, 1H), 7.39 (s, 1H), 7.36 (s, 1H), 7.24 (dd, J 8.5, 2.5 Hz, 1H), 3.88

C

₁₉H₂₁ClN₆O requires [M + H]⁺385.1538.

N-(5-Chloro-2-(pyrrolidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

(t, J 4.4 Hz, 4H), 2.90 (t, J 4.4 Hz, 4H), 2.81 (s, 3H) 2.66 (s, 3H). ¹³

C

[1,5-a]pyrimidine-2-carboxamide (26). General Protocol A was modiﬁed

using 79 (45 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 26

as a beige solid (17 mg, 10%). ¹H NMR (DMSO‑d₆, 600 MHz) δ 10.21 (s,

1H), 7.94 (d, J 2.6 Hz, 1H), 7.34 (s, 1H), 7.18 (dd, J 8.8, 2.7 Hz, 1H),

7.12 (d, J 8.7 Hz, 1H), 2.79 (s, 3H), 2.64 (s, 3H), 1.94–1.89 (m, 4H), 3.16

(q, J 5.4, 4.5 Hz, 4H). ¹³C NMR (DMSO‑d₆, 151 MHz) 166.6, 159.0,

158.0, 147.8, 132.3, 129.6, 125.2, 124.4, 122.5, 119.3, 112.4, 51.0,

40.0, 24.7, 24.4, 16.5. HRMS Found: [M + H]⁺371.1379; C₁₈H₁₉ClN₆O

NMR (DMSO‑d₆, 100 MHz) δ 166.7, 157.8, 156.6, 154.6, 147.9, 140.5,

133.7, 129.1, 124.2, 122.9, 118.2, 112.7, 66.8, 52.0, 24.7, 16.6. HRMS

Found: [M + H]⁺387.1327; C₁₈H₁₉ClN₆O₂requires [M + H]⁺

387.1331.

7-Methyl-N-(2-(piperidin-1-yl)-5-(triﬂuoromethyl)phenyl)-[1,2,4]tri-

azolo[1,5-a]pyrimidine-2-carboxamide (29). General Protocol A was fol-

lowed using 76 (60 mg, 0.23 mmol) and 10b (40 mg, 0.22 mmol) to

1

obtain 29 as a bright yellow solid (47 mg, 52%). H NMR (DMSO‑d_6,

13

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

Table 8

Table 9

P. falciparum delayed death activity of representative compounds.

Activity of representative compounds against P. falciparum 3D7 multidrug

resistant strains and P. knowlesi asexual stage parasites.

3D7 inhibition at 72 h.

3D7 inhibition at 120 h.

Cmpd

EC₅₀(±SEM)

μ

M^a

EC₅₀(±SEM)

μ

M^a

ABS

Pk YH1

EC₅₀

ABS

ABS Pf resistant strains EC₅₀(SD) μM

Pf 3D7

W2mef^a

Dd2^a

7G8^a

Cam3.1^a,b

20

25

44

50

AZ

0.02 (<0.01)

0.03 (<0.01)

0.02 (<0.01)

0.05 (<0.01)

9.9 (0.10)

0.02 (<0.01)

0.04 (<0.01)

0.03 (<0.01)

0.06 (<0.01)

Cmpd

EC₅₀(SD)

(SD)

μ

M^a

μ

M^a

0.37

(0.14)

0.47

0.02

(0.01)

0.03

(0.02)

0.05

0.03

(0.002)

0.05

0.02

(0.008)

0.06

0.02

(0.001)

0.04

20

25

44

50

a

EC₅₀data are averages and ± SEMs for three or more independent experi-

ments measuring P. falciparum 3D7 parasite growth relative to control using

(0.08)

0.84

(0.02)

0.04

(0.03)

0.06

(0.006)

0.06

(0.02)

0.07

(0.02)

0.05

SYBR Green following exposure to compounds for 72 or 120 h. AZ

azithromycin.

=

(0.27)

1.18

(0.03)

0.05

(0.02)

0.08

(0.002)

0.07

(0.02)

0.07

(0.01)

0.06

(0.30)

0.03

(0.03)

0.01

(0.03)

0.16

(0.01)

0.13

(0.03)

0.45

(0.03)

0.16

CQ

a

(0.02)

(0.006)

(0.05)

(0.04)

(0.18)

(0.06)

EC₅₀data represents the means (SDs) of three experiments for P. falciparum

3D7 multidrug resistant strains and P. knowlesi (Pk YH1) asexual stage parasites

following treatment of compound for 72 and 48 h respectively measured by ﬂow

cytometry.

b

Artemisinin resistant Cam3.I DHA resistant (R539T) parasites.

Fig. 4. Early ring stage P. falciparum 3D7 parasites treated with compound 20

at a concentration of 0.70 M (10 times the EC₅₀) arrests asexual parasites at

μ

Fig. 3. Asexual rate of kill for compounds 1 and 2. (A) Differing EC₅₀values at

48 and 72 h incubation time points indicate a delay in parasite killing response.

EC₅₀data represents means and SDs for three independent experiments

measuring [³H] hypoxanthine incorporation by P. falciparum 3D7 parasites

following exposure to a 9-point dilution series for either 48 or 72 h. The

standard antimalarial chloroquine was used as a control. (B) Compounds 1 and

2 display a relatively slow parasite killing rate, comparable to atovaquone and

pyrimethamine. Parasite viability determined through exposure of P. falciparum

the trophozoite stage. (A) Morphology assessed by Giemsa-stained blood smears

indicate no progression beyond the trophozoite stage in compound 20 treated

parasites. (B) Parasites treated with compound 20 did not increase in para-

sitaemia over 48 h of incubation, indicating the stage of arrest occurred within

the ﬁrst asexual cycle. Parasitaemia was measured by FACS analysis of SYBR

Green DNA incorporation. (For interpretation of the references to colour in this

ﬁgure legend, the reader is referred to the web version of this article.)

3D7 parasites at previously determined 10 times the [³H] hypoxanthine EC₅₀

.

Parasite survival is measured by the ability to reinfect pre-labelled CFDA-SE

stained erythrocytes. After 48 h further incubation parasites are stained with

Hoechst 33342 and two-colour ﬂow cytometry measured survival compared to

an untreated control. Data represents means and SDs of 3 independent exper-

iments. Atovaquone (slow), pyrimethamine (moderate), chloroquine (fast) and

artesunate (fast) were used as controls.

modiﬁed using 81 (57 mg, 0.23 mmol) and 10b (40 mg, 0.22 mmol) to

1

obtain 30 as a bright yellow solid (16 mg, 18%). H NMR (DMSO‑d₆,

600 MHz) δ 10.45 (s, 1H), 8.87 (d, J 4.5 Hz, 1H), 7.56 (s, 1H), 7.43–7.41

(m, 2H), 6.94 (d, J 8.7 Hz, 1H), 3.34 (t, J 6.0 Hz, 4H), 2.83 (s, 3H), 1.84

(quint, J 3.2 Hz, 4H). ¹³C NMR (CDCl₃, 75 MHz) δ 159.4, 157.1, 155.5,

155.4, 149.5, 145.1, 128.4, 124.5 (q, ¹J_C-F272 Hz), 123.4 (q, ²J_C-F32

Hz), 122.8 (q, ³J_C-F4 Hz), 120.6 (q, ³J_C-F4 Hz), 117.4, 111.4, 51.6, 25.1,

17.6. HRMS Found: [M + H]⁺391.1486; C₁₈H₁₇F₃N₆O requires [M +

H]⁺391.1489.

400 MHz) δ 10.43 (s, 1H), 8.92 (d, J 4.5 Hz, 1H), 8.75 (d, J 2.1 Hz, 1H),

7.53 (dd, J 8.3, 2.1 Hz, 1H), 7.49–7.45 (m, 2H), 2.92 (t, J 5.1 Hz, 4H),

2.88 (s, 3H), 1.84 (q, J 5.6 Hz, 4H), 1.64 (d, J 7.1 Hz, 2H). ¹³C NMR

(CDCl₃, 75 MHz) δ 159.5, 157.0, 155.5, 155.4, 149.4, 146.5, 132.8,

126.6 (q, ²J_C-F32.6 Hz), 124.3 (q, ¹J_C-F272 Hz), 121.6 (q, ³J_C-F4.0 Hz),

7-Methyl-N-(2-morpholino-5-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (31). General Protocol A was adapted

using 82 (61 mg, 0.23 mmol) and 10b (40 mg, 0.22 mmol) to obtain 31

as a bright yellow solid (40 mg, 27%). ¹H NMR (DMSO‑d₆, 600 MHz) δ

10.47 (s, 1H), 8.93 (dd, J 4.5, 1.6 Hz, 1H), 8.72 (s, 1H), 7.56 (d, J 8.0 Hz,

1H), 7.53 (d, J 8.4 Hz, 1H), 7.48 (d, J 4.4 Hz, 1H), 3.91 (t, J 4.5 Hz, 4H),

2.99 (t, J 4.5 Hz, 4H), 2.89 (s, 3H). ¹³C NMR (DMSO‑d₆, 75 MHz)

158.35, 157.09, 156.87, 155.30, 149.86, 145.67, 133.07, 125.43 (q, ²J_C-

3

120.3, 117.0 (q, J_C-F4.0 Hz), 111.3, 53.6, 26.6, 24.2, 17.6. ¹⁹F

(DMSO‑d₆, 282 MHz) ꢀ 60.65. HRMS Found: [M + H]⁺405.1644;

C

₁₉H₁₉F₃N₆O requires [M + H]⁺405.1645.

7-Methyl-N-(2-(pyrrolidin-1-yl)-5-(triﬂuoromethyl)phenyl)-[1,2,4]tri-

azolo[1,5-a]pyrimidine-2-carboxamide (30). General Protocol A was

14

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

1

32 Hz), 124.7 (q, J_C-F272 Hz, two peaks observed) 122.15–121.81

147.8, 147.0 (d, ¹J_C-F238 Hz) 137.0 (d, ⁴J_C-F2.8 Hz), 132.4 (d, ³J_C-F12

Hz), 120.8 (d, ³J_C-F9 Hz), 112.5, 110.8 (d, ²J_C-F22 Hz), 107.1 (d, ²J_C-F

28 Hz) 51.90, 24.66, 24.10, 16.43. HRMS Found: [M + H]⁺355.1683;

F

(m), 115.80 (³J_C-F, J 4 Hz), 112.56, 67.12, 52.07, 17.22. ¹⁹F (DMSO‑d₆,

282 MHz) δ ꢀ 60.75. HRMS Found: [M + H]⁺407.1441; C₁₈H₁₇F₃N₆O₂

requires [M + H]⁺407.1438.

C

₁₈H₁₉FN₆O requires [M + H]⁺355.1677.

N-(2-(Diethylamino)-5-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]

pyrimidine-2-carboxamide (32). General Protocol A was followed using

83 (62 mg, 0.27 mmol) and 10c (40 mg, 0.24 mmol) to obtain 32 as a

white solid (5 mg, 5%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.67 (s, 1H),

9.56 (dt, J 6.9, 1.5 Hz, 1H), 9.04 (dt, J 2.9, 1.5 Hz, 1H), 8.80 (s, 1H), 7.60

(d, J 8.4 Hz, 1H), 7.56 (d, J 2.4 Hz, 1H), 7.54 (t, J 3.4 Hz, 1H), 3.08 (q, J

7.1 Hz, 4H), 1.01 (t, J 7.1 Hz, 6H). ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 60.80.

HRMS Found: [M + H]⁺379.1486; C₁₇H₁₇F₃N₆O requires [M + H]⁺

379.1489.

N-(5-Bromo-2-(piperidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (38). General Protocol A was adapted

using 86 (58 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 38

as a yellow solid (33 mg, 37%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.42 (s,

1H), 8.60 (d, J 2.3 Hz, 1H), 7.35–7.32 (m, 2H), 7.25 (d, J 8.5 Hz, 1H),

2.83 (t, 5.2 Hz, 4H), 2.80 (s, 3H), 2.65 (s, 3H), 1.81 (quint, J 5.2 Hz, 4H),

1.61 (d, J 5.2 Hz, 2H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 166.6, 157.8,

156.6, 154.6, 147.8, 142.3, 133.8, 127.0, 123.0, 120.9, 116.6, 112.6,

53.1, 26.2, 24.7, 23.5, 16.5. HRMS Found: [M + H]⁺429.1034;

N-(2-(Piperidin-1-yl)-5-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]

pyrimidine-2-carboxamide (33). General Protocol A was modiﬁed using

76 (58 mg, 0.24 mmol) and 10c (40 mg, 0.24 mmol) to obtain 33 as a

white solid (75 mg, 79%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.39 (s, 1H),

9.56 (dd, J 6.9, 1.9 Hz, 1H), 9.04 (dd, J 4.3, 1.9 Hz, 1H), 8.74 (d, J 2.1

Hz, 1H), 7.59–7.49 (m, 2H), 7.46 (d, J 8.4 Hz, 1H), 2.91 (t, J 5.4 Hz, 4H),

1.84 (quint, J 6.1 Hz, 4H), 1.64 (q, J 5.0 Hz, 2H). ¹³C NMR (DMSO‑d₆,

100 MHz) δ 158.9, 157.8, 156.9, 155.0, 147.0, 138.8, 132.9, 124.9 (q,

²J_C-F32 Hz), 123.4, 121.9 (q, ³J_C-F4 Hz), 121.8, 115.6 (q, ³J_C-F4 Hz),

113.1, 53.2, 26.5, 24.0 (¹J_C-Fnot found). ¹⁹F (DMSO‑d₆, 282 MHz) δ

ꢀ 60.68. HRMS Found: [M + H]⁺391.1485; C₁₈H₁₇F₃N₆O requires [M +

H]⁺391.1489.

C

₁₉H₂₁BrN₆O requires [M + H]⁺429.1033.

N-(5-Bromo-2-(pyrrolidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (39). General Protocol A was followed

using 87 (55 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 39

as a yellow solid (23 mg, 38%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.20 (s,

1H), 7.99 (d, J 2.4 Hz, 1H), 7.33 (d, J 0.8 Hz, 1H), 7.29 (dd, J 8.7, 3.7 Hz,

1H), 7.03 (d, J 8.8 Hz, 1H), 3.17 (t, J 6.3 Hz, 4H), 2.79 (s, 3H), 2.64 (s,

3H), 1.91 (quint, J 3.2 Hz, 4H). ¹³C NMR (DMSO‑d_6,75 MHz) δ 166.40,

158.07, 157.17, 154.56, 147.86, 142.01, 129.42, 128.29, 125.84,

119.51, 112.46, 111.67, 50.81, 24.73, 24.52, 16.53. HRMS Found: [M +

H]⁺415.0876; C₁₈H₁₉BrN₆O requires [M + H]⁺415.0876.

5,7-Dimethyl-N-(5-methyl-2-(piperidin-1-yl)phenyl)-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (40). General Protocol A was modiﬁed

using 88 (44 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 40

as a brown solid (13 mg, 17%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.48 (s,

1H), 8.27 (s, 1H), 7.34 (s, 1H), 7.18 (d, J 8.0 Hz, 1H), 6.96 (d, J 8.1 Hz,

1H), 2.85–2.77 (m, 7H), 2.65 (s, 3H), 2.32 (s, 3H), 1.81 (t, J 5.5 Hz, 4H),

1.61 (s, 2H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 166.4, 158.2, 156.2, 154.6,

147.8, 140.5, 133.7, 132.2, 124.8, 121.0, 119.0, 112.4, 53.4, 26.3, 24.7,

23.6, 21.0, 16.5. HRMS Found: [M + H]⁺365.2090; C₂₀H₂₄N₆O requires

[M + H]⁺365.2085.

N-(2-(Pyrrolidin-1-yl)-5-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-

a]pyrimidine-2-carboxamide (34). General Protocol A was adapted using

81 (52 mg, 0.022 mmol) and 10c (32 mg, 0.02 mmol) to obtain 34 as a

beige solid (3.3 mg, 4%). ¹H NMR (CDCl₃, 300 MHz) δ 9.68 (s, 1H), 9.03

(dd, J 6.7, 2.0 Hz, 1H), 8.96 (dd, J 4.3, 1.9 Hz, 1H), 8.48 (d, J 2.1 Hz,

1H), 7.37 (dd, J 8.6, 2.2 Hz, 1H), 7.29 (dd, J 6.9, 4.2 Hz, 1H), 7.10 (d, J

8.5 Hz, 1H), 3.28 (t, J 6.3 Hz, 4H), 2.04–2.00 (m, 4H).¹³C NMR (CDCl₃,

75 MHz) δ 160.1, 156.7, 156.3, 155.2, 145.2, 137.1, 128.4, 124.5 (q, ¹J_C-

_F271 Hz), 123.5 (q, ²J_C-F33 Hz), 122.9 (q, ⁴J_C-F3.8 Hz), 120.5 (q, ⁴J_C-F

3.8 Hz), 117.5, 111.9, 51.6, 25.1. HRMS Found: [M + H]⁺377.1330;

5,7-Dimethyl-N-(5-methyl-2-(pyrrolidin-1-yl)phenyl)-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (41). General Protocol A was adapted

using 89 (40 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 41

as a beige solid (8 mg, 11%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.15 (s,

1H), 7.95 (s, 1H), 7.32 (s, 1H), 7.09 (d, J 8.1 Hz, 1H), 6.93 (d, J 8.2 Hz,

1H), 3.08 (t, J 6.0 Hz, 4H), 2.79 (s, 3H), 2.63 (s, 3H), 2.29 (s, 3H), 1.94

(t, J 5.9 Hz, 4H). ¹³C NMR (DMSO‑d_6,75 MHz) δ 166.2, 158.3, 156.4,

154.5, 147.8, 138.8, 131.5, 130.3, 125.5, 121.5, 118.7, 112.3, 51.5,

24.7, 24.2, 20.7, 16.5. HRMS Found: [M + H]⁺351.1933; C₁₉H₂₂N₆O

requires [M + H]⁺351.1928.

C

₁₇H₁₅F₃N₆O requires [M + H]⁺377.1332.

N-(2-Morpholino-5-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]py-

rimidine-2-carboxamide (35). General Protocol A was modiﬁed using 82

(66 mg, 0.27 mmol) and 10c (32 mg, 0.24 mmol) to obtain 35 as a beige

solid (27 mg, 28%). ¹H NMR (DMSO‑d₆, 400 MHz) δ 10.44 (s, 1H), 9.59

(d, J 6.2 Hz, 1H), 9.05 (d, J 4.8 Hz, 1H), 8.71 (s, 1H), 7.54 (dd, J 6.8, 4.3

Hz, 1H), 7.57 (s, 1H), 7.52 (d, J 8.3 Hz, 1H), 3.91 (t, J 4.5 Hz, 4H), 2.99

(t, J 4.0 Hz, 4H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 158.5, 157.5, 156.6,

154.6, 145.4, 138.4, 132.6, 124.9 (q, ²J_C-F32 Hz), 124.3 (q, ¹J_C-F272

3

Hz), 121.8–121.6 (m, 2C, q J_C-Fand s), 115.5 (q, J_C-F4 Hz), 112.7,

66.7, 51.7. ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 60.75. HRMS Found: [M + H]⁺

393.1281; C₁₇H₁₅F₃N₆O₂requires [M + H]⁺393.1281.

N-(5-Methoxy-2-(piperidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (42). General Protocol A was modiﬁed

using 90 (20 mg, 0.10 mmol) and 10a (17 mg, 0.09 mmol) to obtain 42

as a yellow solid (2 mg, 6%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.60 (s,

1H), 8.08 (d, J 2.9 Hz, 1H), 7.34 (s, 1H), 7.24 (d, J 8.6 Hz, 1H), 6.71 (dd,

J 8.9, 2.6 Hz, 1H), 3.76 (s, 3H), 2.83–2.75 (m, 7H), 2.65 (s, 3H),

1.87–1.72 (m, 4H), 1.59 (s, 2H). ¹³C NMR (DMSO‑d_6,75 MHz) δ 166.4,

158.1, 156.3, 156.2, 154.6, 147.7, 136.1, 133.4, 121.8, 112.5, 108.7,

104.8, 55.3, 53.6, 26.4, 24.6, 23.6, 16.5. HRMS Found: [M + H]⁺

381.2039; C₂₀H₂₄N₆O₂requires [M + H]⁺381.2034.

N-(5-Fluoro-2-(piperidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (36). General Protocol A was adapted

using 84 (45 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 36

as a pale orange solid (44 mg, 58%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.58 (s, 1H), 8.23 (dd, J 10.8, 3.0 Hz, 1H), 7.40–7.30 (m, 2H), 6.98 (td,

J 8.8, 3.0 Hz, 1H), 2.88–2.76 (m, 7H), 2.65 (s, 3H), 1.81 (t, J 5.5 Hz, 4H),

1.61 (s, 2H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 166.5, 158.7 (d, ¹J_C-F239

Hz), 157.8, 156.6, 154.6, 147.8, 139.2 (d, ⁴J_C-F3 Hz), 133.7 (d, ³J_C-F12

Hz), 122.5 (d, ³J_C-F10 Hz), 112.6, 110.3 (d, ²J_C-F22 Hz), 105.5 (d, ²J_C-F

30 Hz), 53.4, 26.3, 24.7, 23.5, 16.5. ¹⁹F (DMSO‑d₆, 282 MHz) δ

ꢀ 115.84. HRMS Found: [M + H]⁺369.1839; C₁₉H₂₁FN₆O requires [M

+ H]⁺369.1834.

N-(5-Methoxy-2-(pyrrolidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (43). General Protocol A was adapted

using 91 (44 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 43

as a brown solid (23 mg, 30%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.37 (s,

1H), 7.98 (d, J 1.8 Hz, 1H), 7.34 (s, 1H), 7.26 (dd, J 8.8, 2.0 Hz, 1H),

6.73 (d, J 8.6 Hz, 1H), 3.76 (d, J 1.7 Hz, 3H), 3.01 (t, J 5.1 Hz, 4H), 2.80

(s, 3H), 2.64 (t, J 1.6 Hz, 3H), 1.98 (t, J 6.1 Hz, 4H). ¹³C NMR (DMSO‑d₆,

75 MHz,) δ 166.8, 158.6, 156.9, 156.1, 155.0, 148.2, 133.8, 133.6,

121.4, 112.9, 109.6, 106.2, 55.8, 52.8, 25.1, 24.5, 16.9. HRMS Found:

[M + H]⁺367.1881; C₁₉H₂₂N₆O₂requires [M + H]⁺367.1877.

5,7-Dimethyl-N-(2-(piperidin-1-yl)-5-(triﬂuoromethoxy)phenyl)-

N-(5-Fluoro-2-(pyrrolidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (37). General Protocol A was modiﬁed

using 85 (41 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 37

as a pale orange solid (37 mg, 50%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.32 (s, 1H), 8.05 (dd, J 10.4, 3.0 Hz, 1H), 7.38–7.26 (m, 2H), 6.98 (t, J

8.6 Hz, 1H), 3.07 (t, J 5.8 Hz, 4H), 2.80 (s, 3H), 2.64 (s, 3H), 1.98 (t, J

5.8 Hz, 4H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 166.4, 157.9, 156.7, 154.5,

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (44). General Protocol A

15

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

was followed using 92 (60 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol)

to obtain 44 as a pale-yellow solid (24 mg, 27%). ¹H NMR (DMSO‑d₆,

300 MHz) δ 10.49 (s, 1H), 8.42 (d, J 2.6 Hz, 1H), 7.40 (d, J 8.7 Hz, 1H),

7.36 (s, 1H), 7.15 (dd, J 8.7, 2.7 Hz, 1H), 2.92–2.74 (m, 7H), 2.65 (s,

3H), 1.82 (quint, J 5.7 Hz, 4H), 1.62 (q, J 4.7 Hz, 2H). ¹³C NMR

(DMSO‑d₆, 75 MHz,) δ 167.05, 158.18, 157.16, 155.08, 148.28, 145.04,

142.37, 133.98, 122.77, 116.95, 113.09, 111.56, 53.61, 26.72, 25.14,

24.00, 16.95 (¹J_C-Fnot found). ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 56.84.

HRMS Found: [M + H]⁺435.1750; C₂₀H₂₁F₃N₆O₂requires [M + H]⁺

435.1751.

Hz, 4H), 2.80 (s, 3H), 2.65 (s, 3H), 1.88–1.76 (m, 8H). MS m/z (%) [M +

H]⁺, 259 (100). ¹³C NMR (DMSO‑d₆, 75 MHz,) δ 166.5, 157.8, 157.0,

154.7, 148.9, 147.8, 132.1, 126.1, 123.9 (q, ²J_C-F32.0 Hz), 122.8, 121.6

(q, ³J_C-F4 Hz), 116.2 (q, ³J_C-F4 Hz), 112.6, 55.6, 29.0, 26.3, 24.7, 16.5

(¹J_C-Fnot observed). ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 60.57. HRMS Found:

[M + H]⁺433.1955; C₂₁H₂₃F₃N₆O requires [M + H]⁺433.1958.

General Protocol B: N-[2-(8-Azabicyclo[3.2.1]octan-8-yl)-5-(tri-

ﬂuoromethyl)phenyl]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-car-

boxamide (51). 10a (20 mg, 0.1 mmol), 99 (34 mg, 0.12 mmol) T3P

(0.08 mL, 0.13 mmol) and DIPEA (0.07 mL, 0.4 mmol) in DMF (0.5 mL)

◦

5,7-Dimethyl-N-(2-(pyrrolidin-1-yl)-5-(triﬂuoromethoxy)phenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (45). General Protocol A

was modiﬁed using 93 (56 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol)

to obtain 45 as a pale orange solid (16 mg, 18%). ¹H NMR (DMSO‑d₆,

300 MHz) δ 10.26 (s, 1H), 7.94 (d, J 2.2 Hz, 1H), 7.35 (s, 1H), 7.21 (d, J

8.9, 1H), 7.15 (dd, J 8.9, 2.6 Hz, 1H), 3.19 (t, J 6.1 Hz, 4H), 2.80 (s, 3H),

2.65 (s, 3H), 1.94 (quint, J 3.5 Hz, 4H). ¹³C NMR (DMSO‑d₆, 75 MHz,) δ

166.18, 162.96, 161.51, 156.68, 147.68, 129.87, 116.77, 112.62,

112.54, 112.46, 110.20, 44.37, 25.65, 24.75, 23.66, 16.52 (¹J_C-Fnot

found). ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 57.96. HRMS Found: [M + H]⁺

421.1593; C₁₉H₁₉F₃N₆O₂requires [M + H]⁺421.1594.

were stirred at 50 C for 72 h. The reaction mixture was diluted with

EtOAc (15 mL) and washed with water (2 × 15 mL) and saturated brine

(1 × 15 mL). The organic layer was separated and dried (Na₂SO₄) before

concentration in vacuo. The crude residue was puriﬁed using silica

chromatography eluting with a gradient from 10% EtOAc/heptane to

obtain 51 as a white solid (4 mg, 9%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.12 (s, 1H), 8.64 (d, J 1.9 Hz, 1H), 7.41 (dd, J 8.5, 1.8 Hz, 1H), 7.34 (s,

1H), 7.26 (d, J 8.5 Hz, 1H), 3.80 (s, 2H), 2.79 (s, 3H), 2.64 (s, 3H), 2.15

(q, J 4.0 Hz, 2H), 1.97 (d, J 5.3 Hz, 2H), 1.75 (q, J 7.5 Hz, 3H), 1.70–1.54

(m, 4H). HRMS Found: [M + H]⁺433.1958; C₂₁H₂₃F₃N₆O requires [M

+ H]⁺433.1958.

N-(5-Cyano-2-(piperidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (46). General Protocol A was adapted

using 94 (46 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 46

as a pale orange solid (12 mg, 15%). ¹H NMR (DMSO‑d₆, 400 MHz) δ

10.21 (s, 1H), 8.68 (d, J 1.9 Hz, 1H), 7.63 (dd, J 2.3 Hz, 1H), 7.41 (d, J

8.3 Hz, 1H), 7.36 (s, 1H), 2.92 (t, J 10.4 Hz, 4H), 2.80 (s, 3H), 2.65 (s,

3H), 1.83 (quint, J 11.1 Hz, 4H), 1.61 (d, J 4.4 Hz, 2H). ¹³C NMR

(DMSO‑d₆, 75 MHz,) δ 166.7, 157.6, 156.7, 154.6, 147.9, 147.5, 132.4,

128.9, 121.7, 121.6, 119.0, 112.7, 106.0, 52.5, 26.0, 24.7, 23.5, 16.5.

HRMS Found: [M + H]⁺376.1878; C₂₀H₂₁N₇O requires [M + H]⁺

376.1877.

N-(2-(Dimethylamino)-5-(triﬂuoromethyl)phenyl)-5,7-dimethyl-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (52). General Protocol A was

adapted using 100 (47 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to

obtain 52 as a white solid (4.4 mg, 6%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.26 (s, 1H), 8.64 (s, 1H), 7.53 (d, J 8.9 Hz, 1H), 7.47 (d, J 8.4 Hz, 1H),

7.36 (s, 1H), 2.80 (s, 3H), 2.78 (s, 6H), 2.64 (s, 3H). HRMS Found: [M +

H]⁺379.1488; C₁₇H₁₇F₃N₆O requires [M + H]⁺379.1489.

N-(2-(Ethyl(methyl)amino)-5-(triﬂuoromethyl)phenyl)-5,7-dimethyl-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (53). General Protocol A

was modiﬁed using 101 (50 mg, 0.23 mmol) and 10a (40 mg, 0.21

mmol) to obtain 53 as a pale pink solid (12 mg, 14%). ¹H NMR

(DMSO‑d₆, 300 MHz) δ 10.39 (s, 1H), 8.71 (s, 1H), 7.52–7.51 (m, 2H),

7.35 (d, J 0.8 Hz, 1H), 2.97 (q, J 7.1 Hz, 2H), 2.80 (s, 3H), 2.79 (s, 3H),

2.64 (s, 3H), 1.16 (t, J 7.1 Hz, 3H). ¹³C NMR (DMSO‑d₆, 75 MHz,) δ

166.6, 157.7, 156.8, 154.4, 147.9, 146.3, 133.0, 124.4 (q, ²J_C-F32 Hz),

N-(5-Cyano-2-(pyrrolidin-1-yl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (47). General Protocol A was modiﬁed

using 95 (43 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to obtain 47

as a bright yellow solid (9 mg, 12%). ¹H NMR (DMSO‑d₆, 600 MHz) δ

10.43 (s, 1H), 7.51 (d, J 1.8 Hz, 1H), 7.49 (dd, J 8.6, 2.0 Hz, 1H), 7.30 (s,

1H), 6.82 (d, J 8.8 Hz, 1H), 3.39 (t, J 6.2 Hz, 4H), 2.76 (s, 3H), 2.61 (s,

3H), 1.82 (td, J 6.3 Hz, 4H). ¹³C NMR (DMSO‑d₆, 75 MHz) δ 166.6,

158.8, 158.7, 155.1, 149.0, 148.3, 133.9, 131.9, 122.9, 120.2, 115.9,

112.8, 97.3, 50.2, 25.6, 25.2, 17.0. HRMS Found: [M + H]⁺362.1721;

1

3

124.2 (q, J_C-F, 272 Hz, two peaks observed), 122.2, 121.3 (q, J_C-F

4

Hz), 115.5 (q, J_C-F4 Hz), 112.5, 50.5, 40.42, 24.7, 16.5, 12.7. ¹⁹F

3

(DMSO‑d₆, 282 MHz) δ ꢀ 60.66 HRMS Found: [M + H]⁺393.1643;

C

₁₈H₁₉F₃N₆O requires [M + H]⁺393.1645.

5,7-Dimethyl-N-(2-(piperazin-1-yl)-5-(triﬂuoromethyl)phenyl)-[1,2,4]

C

₁₉H₁₉N₇O requires [M + H]⁺362.1724.

triazolo[1,5-a]pyrimidine-2-carboxamide (54). 10a (80 mg, 0.21 mmol),

102 (158 mg, 0.46 mmol), HATU (198 mg, 0.52 mmol) and DIPEA (0.30

mL, 1.67 mmol) in DMF (2 mL) were stirred at 50 ^◦C for 24 h. The re-

action mixture was diluted with EtOAc (15 mL) and washed with water

(2 × 15 mL) and saturated brine (1 × 15 mL). The organic layer was

separated and dried (Na₂SO₄) before concentration in vacuo. To the

crude residue MeOH (5 mL) was added and the precipitate was ﬁltered

and washed with cold MeOH to obtain a white solid. This was then

dissolved in a mixture of TFA (0.06 mL, 0.82 mmol) and DCM (2 mL) and

stirred at 20 ^◦C for 2 h. The reaction was diluted with saturated NaHCO₃

(1 × 5 mL) and extracted with DCM (3 × 5 mL). The organic layer was

separated and dried with anhydrous Na₂SO₄before being concentrated

in vacuo. To the crude residue MeOH (5 mL) was added and the pre-

cipitate was ﬁltered and washed with cold MeOH to obtain 54 as a

yellow solid (13 mg, 19%). ¹H NMR (DMSO‑d₆, 300 MHz) 10.41 (s, 1H),

8.75 (s, 1H), 7.53 (d, J 8.5 Hz, 1H), 7.47 (d, J 8.4 Hz, 1H), 7.36 (s, 1H),

3.03–3.00 (m, 4H), 2.89–2.86 (m, 4H), 2.81 (s, 3H), 2.67 (s, 3H), 1.45 (s,

1H). ¹³C NMR (DMSO‑d₆, 75 MHz,) δ 167.1, 158.2, 157.2, 155.0, 148.3,

146.4, 133.0, 125.0 (q, ²J_C-F32 Hz), 124.7 (q, ¹J_C-F272 Hz, two peaks

5,7-Dimethyl-N-(5-(methylsulfonyl)-2-(piperidin-1-yl)phenyl)-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (48). General Protocol A was

adapted using 96 (58 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to

obtain 48 as a white solid (4 mg, 4%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.32 (s, 1H), 8.92 (d, J 2.2 Hz, 1H), 7.72 (dd, J 8.4, 2.2 Hz, 1H), 7.50 (d,

J 8.4 Hz, 1H), 7.37 (d, J 1.0 Hz, 1H), 3.22 (s, 3H), 2.94 (t, J 5.2 Hz, 4H),

2.81 (s, 3H), 2.66 (s, 3H), 1.84 (q, J 6.0 Hz, 4H), 1.64 (d, J 5.3 Hz, 2H).

¹³C NMR (DMSO‑d₆, 75 MHz,) δ 166.62, 157.69, 156.64, 154.58,

147.87, 147.47, 136.01, 132.21, 123.64, 121.24, 117.06, 112.63, 52.67,

43.86, 26.05, 24.68, 23.51, 16.52. HRMS Found: [M + H]⁺429.1704;

C

₂₀H₂₄N₆O₃S requires [M + H]⁺429.1703.

5,7-Dimethyl-N-(5-(methylsulfonyl)-2-(pyrrolidin-1-yl)phenyl)-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (49). General Protocol A was

adjusted using 97 (55 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to

obtain 49 as a beige solid (1 mg, 1%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.48 (s, 1H), 7.69 (s, 1H), 7.63 (d, J 8.7 Hz, 1H), 7.33 (s, 1H), 6.94 (d, J

8.6 Hz, 1H), 3.41 (t, J 5.6 Hz, 4H), 3.12 (s, 3H), 2.79 (s, 3H), 2.64 (s, 3H),

1.86 (t, J 6.3 Hz, 4H). HRMS Found: [M + H]⁺415.1549; C₁₉H₂₂N₆O₃S

requires [M + H]⁺415.1547.

3

observed), 122.0–121.7 (m, s and J_C-F), 115.5 (q, J_C-F4 Hz), 113.1,

53.0, 46.4, 25.1, 17.0. ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 60.69. HRMS Found:

[M + H]⁺420.1749; C₁₉H₂₀F₃N₇O requires [M + H]⁺420.1754.

5,7-Dimethyl-N-(2-(4-methylpiperazin-1-yl)-5-(triﬂuoromethyl)phenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (55). General Protocol A

was adapted using 103 (59 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol)

N-(2-(Azepan-1-yl)-5-(triﬂuoromethyl)phenyl)-5,7-dimethyl-[1,2,4]tri-

azolo[1,5-a]pyrimidine-2-carboxamide (50). General Protocol A was

modiﬁed using 98 (59 mg, 0.23 mmol) and 10a (40 mg, 0.21 mmol) to

obtain 50 as a green solid (4 mg, 4%). ¹H NMR (DMSO‑d₆, 300 MHz) δ

10.48 (s, 1H), 8.63 (s, 1H), 7.54–7.45 (m, 2H), 7.35 (s, 1H), 3.12 (t, J 5.1

16

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

to obtain 55 as a pale orange solid (6 mg, 6%). ¹H NMR (DMSO‑d₆, 300

MHz) δ 10.29 (s, 1H), 8.73 (s, 1H), 7.57–7.44 (m, 2H), 7.36 (s, 1H), 2.97

(t, J 4.8 Hz, 4H), 2.82 (s, 3H), 2.71–2.56 (m, 7H), 2.30 (s, 3H). ¹³C NMR

(DMSO‑d₆, 75 MHz) δ 166.6, 159.3, 157.3, 155.4, 147.9, 145.1, 132.9,

127.3 (q, ²J_C-F33 Hz), 121.7 (q, ³J_C-F4 Hz), 120.5, 117.5 (q, ³J_C-F4 Hz),

112.2, 55.4, 51.8, 46.1, 25.4, 17.4 (¹J_C-Fnot observed). ¹⁹F (DMSO‑d₆,

282 MHz) δ ꢀ 62.25. HRMS Found: [M + H]⁺434.1911; C₂₀H₂₂F₃N₇O

requires [M + H]⁺434.1911.

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (61). General Protocol A

was modiﬁed using 109 (56 mg, 0.23 mmol) and 10a (40 mg, 0.21

mmol) to obtain 61 as a beige solid (5 mg, 6%). ¹H NMR (DMSO‑d₆, 300

MHz) δ 10.01 (s, 1H), 8.68 (s, 1H), 8.50 (s, 1H), 7.37 (s, 1H), 3.22 (t, J

5.4 Hz 4H), 2.81 (s, 3H), 2.66 (s, 3H), 1.78 (quint, J 5.8 Hz, 4H), 1.65 (q,

J 4.9 Hz, 2H). HRMS Found: [M + H]⁺420.1754; C₁₉H₂₀F₃N₇O requires

[M + H]⁺420.1754.

5,7-Dimethyl-N-(2-(pyrrolidin-1-yl)-5-(triﬂuoromethyl)pyridin-3-yl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (62). General Protocol A

was adjusted using 110 (53 mg, 0.23 mmol) and 10a (40 mg, 0.21

mmol) to obtain 62 as a green solid (4 mg, 5%). ¹H NMR (DMSO‑d₆, 300

MHz) δ 10.61 (s, 1H), 8.37 (s, 1H), 7.71 (s, 1H), 7.33 (s, 1H), 3.57 (t, J

6.2 Hz 4H), 2.79 (s, 3H), 2.64 (s, 3H), 1.83 (q, J 6.6 Hz, 4H). HRMS

Found: [M + H]⁺406.1599; C₁₈H₁₈F₃N₇O requires [M + H]⁺406.1598.

5,7-Dimethyl-N-(2-(piperidin-1-yl)-4-(triﬂuoromethyl)phenyl)-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (63). General Protocol B was

modiﬁed using 111 (38 mg, 0.16 mmol) and 10a (25 mg, 0.13 mmol) to

obtain 63 as a white solid (1 mg, 2%). ¹H NMR (DMSO‑d₆, 300 MHz,) δ

10.52 (s, 1H), 8.60 (d, J 8.7 Hz, 1H), 7.63–7.51 (m, 2H), 7.35 (s, 1H),

2.90 (t, J 5.2 Hz, 4H), 2.80 (s, 3H), 2.65 (s, 3H), 1.83 (quint, J 5.6 Hz,

4H), 1.63 (q, J 4.3 Hz, 2H). ¹³C NMR (DMSO‑d₆, 75 MHz,) δ 166.6,

157.7, 156.7, 154.6, 147.8, 143.3, 135.8, 124.4 (q, ²J_C-F32.0 Hz), 124.2

(q, ¹J_C-F272 Hz, two peaks observed) 121.9 (q, ³J_C-F4 Hz), 118.6, 117.7

(q, ³J_C-F4 Hz), 112.6, 52.9, 26.2, 24.7, 23.5, 16.5. ¹⁹F (DMSO‑d₆, 282

MHz) δ ꢀ 60.31. HRMS Found: [M + H]⁺419.1796; C₂₀H₂₁F₃N₆O re-

quires [M + H]⁺419.1802.

General Protocol C. N-(2-(1H-Pyrazol-1-yl)-5-(triﬂuoromethyl)

phenyl)-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide

(56). 10a (40 mg, 0.21 mmol), 105 (52 mg, 0.23 mmol) COMU (111 mg,

0.26 mmol) and DIPEA (0.15 mL, 0.83 mmol) in DMF (1 mL) were

stirred at 50 ^◦C for 24 h. The reaction mixture was diluted with EtOAc

(15 mL) and washed with water (2 × 15 mL) and saturated brine (1 × 15

mL). The organic layer was separated and dried (Na₂SO₄) before con-

centration in vacuo. To the crude residue diethyl ether (5 mL) was added

and the precipitate was ﬁltered and washed with cold MeOH to obtain

56 as a brown solid (6 mg, 7%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 12.49

(s, 1H), 9.01 (d, J 1.7 Hz, 1H), 8.55 (d, J 2.6 Hz, 1H), 8.09 (d, J 1.7 Hz,

1H), 7.97 (d, J 8.3 Hz, 1H), 7.71 (dd, J 8.5, 1.5 Hz, 1H), 7.34 (d, J 0.9 Hz,

1H), 6.72 (dd, J 2.5, 2.0 Hz, 1H), 2.80 (s, 3H), 2.64 (s, 3H). ¹³C NMR

(DMSO‑d₆, 75 MHz) δ 166.4, 157.6, 157.6, 154.7, 148.0, 142.7, 141.0

132.1, 131.1, 128.1 (q, ²J_C-F32 Hz, two peaks seen), 124.3 (q, ¹J_C-F272

Hz, two peaks observed) 123.7, 121.6 (q, ³J_C-F4 Hz), 119.8 (q, ³J_C-F

4

Hz), 112.6, 108.3, 25.2, 16.9. ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 61.14. HRMS

Found: [M + H]⁺402.1286; C₁₈H₁₄F₃N₇O requires [M + H]⁺402.1285.

N-(2-(1H-1,2,4-Triazol-1-yl)-5-(triﬂuoromethyl)phenyl)-5,7-dimethyl-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (57). General Protocol C

was followed using 10a (40 mg, 0.21 mmol), 106 (52 mg, 0.23 mmol)

COMU (111 mg, 0.26 mmol) and DIPEA (0.15 mL, 0.83 mmol) to obtain

57 as a brown solid (8 mg, 9%). ¹H NMR (DMSO‑d_6,300 MHz) δ 11.52

(s, 1H), 9.24 (s, 1H), 8.81 (d, J 1.5 Hz, 1H), 8.58 (d, J 3.8 Hz, 1H), 8.30

(s, 1H), 8.04 (d, J 8.7 Hz, 1H), 7.80 (dd, J 8.7, 2.1 Hz, 1H), 2.79 (s, 3H),

2.64 (s, 3H). HRMS Found: [M + H]⁺403.1240; C₁₇H₁₃F₃N₈O requires

[M + H]⁺403.1237.

5,7-Dimethyl-N-(2-(pyrrolidin-1-yl)-4-(triﬂuoromethyl)phenyl)-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (64). General Protocol B was

adapted using 112 (56 mg, 0.25 mmol) and 10a (40 mg, 0.21 mmol) to

obtain 64 as a brown crystalline solid (9 mg, 11%). ¹H NMR (DMSO‑d₆,

300 MHz) δ 10.33 (s, 1H), 7.97 (d, J 8.7 Hz, 1H), 7.34 (d, J 1.1 Hz, 1H),

7.30–7.24 (m, 2H), 3.29–3.24 (m, 4H), 2.80 (s, 3H), 2.64 (s, 3H),

1.96–1.91 (m, 4H).¹³C NMR (CDCl₃, 75 MHz) δ 166.6, 159.3, 157.3,

1

2

155.3, 148.0, 141.7, 133.9, 127.9 (q, J_C-F272 Hz) 127.0 (q, J_C-F32

Hz), 121.7, 120.2 (q, ³J_C-F3 Hz), 115.5 (q, ³J_C-F3 Hz), 112.2, 52.1, 25.4,

24.9, 17.4. HRMS Found: [M + H]⁺405.1642; C₁₉H₁₉F₃N₆O requires

[M + H]⁺405.1645.

5,7-Dimethyl-N-(2-propoxy-5-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (58). General Protocol C was adjusted

using 10a (40 mg, 0.21 mmol), 2-propoxy-5-(triﬂuoromethyl)aniline

(50 mg, 0.23 mmol), COMU (111 mg, 0.26 mmol) and DIPEA (0.15

mL, 0.83 mmol) to obtain 58 as a brown solid (2.3 mg, 3%). ¹H NMR

(DMSO‑d₆, 300 MHz) δ 10.05 (s, 1H), 8.70 (s, 1H), 7.53 (d, J 8.3 Hz, 1H),

7.35–7.32 (m, 2H), 4.21 (t, J 6.1 Hz, 2H), 2.79 (s, 3H), 2.64 (s, 3H), 1.88

(quint, J 7.2 Hz, 2H), 1.12 (t, J 7.4 Hz, 3H). HRMS Found: [M + H]⁺

394.1485; C₁₈H₁₈F₃N₅O₂requires [M + H]⁺394.1485.

5,7-Dimethyl-N-(2-(piperidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (65). General Protocol B

was modiﬁed using 113 (31 mg, 0.12 mmol) and 10a (20 mg, 0.10

mmol). The crude was puriﬁed using silica chromatography eluting with

a gradient of 50–100% EtOAc/heptane to obtain 65 as a white solid (10

mg, 23%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.10 (s, 1H), 8.74 (d, J 7.9

Hz, 1H), 7.66 (d, J 8.3 Hz, 1H), 7.38 (s, 1H), 3.11 (t, J 6.4 Hz, 4H), 2.82

N-(2-Isobutoxy-5-(triﬂuoromethyl)phenyl)-5,7-dimethyl-[1,2,4]triazolo

[1,5-a]pyrimidine-2-carboxamide (59). General Protocol B was modiﬁed

using 10a (20 mg, 0.1 mmol), 107 (36 mg, 0.15 mmol), T3P (0.1 mL,

0.11 mmol) and DIPEA (0.07 mL, 0.4 mmol) to obtain 59 as a white solid

(20.7 mg, 49%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.10 (s, 1H), 8.72 (d, J

2.1 Hz, 1H), 7.54 (dd, J 8.6, 1.6 Hz, 1H), 7.37–7.34 (m, 2H), 4.05 (d, J

6.3 Hz, 2H), 2.80 (s, 3H), 2.65 (s, 3H), 2.19 (quint, J 3.6 Hz, 1H), 1.12 (d,

J 6.7 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz) δ 166.44, 158.93, 157.11,

155.14, 150.14, 147.72, 127.56, 124.3 (q, ¹J_C-F272 Hz) 122.87 (q, ²J_C-F

32 Hz), 121.61 (q, ³J_C-F4 Hz), 117.04 (q, ³J_C-F4 Hz), 112.10, 110.65,

75.37, 28.16, 25.22, 19.25, 17.17. ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 61.61.

HRMS Found: [M + H]⁺408.1645; C₁₉H₂₀F₃N₅O₂requires [M + H]⁺

408.1642.

(s, 3H), 2.67 (s, 3H)1.80 (quint, J 3.8 Hz, 4H), 1.65 (q, J 4.9 Hz, 2H). ¹³

C

NMR (CDCl₃, 75 MHz) δ 166.81, 158.84, 157.93, 155.35, 154.36,

147.93, 141.34 (q, ²J_C-F34 Hz), 129.28, 127.96, 121.69 (q, ¹J_C-F274 Hz,

3

two peaks observed), 116.45 (q, J_C-F3 Hz), 112.40, 51.55, 26.03,

25.43, 24.32, 17.38. ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 62.11. HRMS Found:

[M + H]⁺420.1753; C₁₉H₂₀F₃N₇O requires [M + H]⁺420.1754.

5,7-Dimethyl-N-(2-(pyrrolidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (66). General Protocol B

was modiﬁed using 114 (31 mg, 0.14 mmol) and 10a (20 mg, 0.10

mmol). The crude was puriﬁed using silica chromatography eluting with

a gradient of 50–100% EtOAc/heptane to obtain 66 as a beige solid (7.5

mg, 18%). ¹H NMR (DMSO‑d₆, 300 MHz) 10.62 (s, 1H), 7.74 (d, J 7.5

Hz, 1H), 7.34 (s, 1H), 7.11 (d, J 7.8 Hz, 1H), 3.53 (t, J 6.4 Hz, 4H), 2.80

(s, 3H), 2.65 (s, 3H), 2.19 (t, J 6.6 Hz, 4H). ¹³C NMR (CDCl₃, 75 MHz) δ

166.9, 158.8, 157.6, 155.2, 152.9, 148.2, 142.2 (q, ²J_C-F34 Hz), 131.2,

124.1, 121.7 (q, ¹J_C-F274 Hz, middle two peaks seen), 112.5, 112.1 (q,

³J_C-F3 Hz), 50.3, 25.7, 25.5, 17.5. ¹⁹F (CDCl₃, 282 MHz) δ ꢀ 68.10.

HRMS Found: [M + H]⁺406.1612; C₁₈H₁₈F₃N₇O requires [M + H]⁺

406.1598.

N-(2-(Benzyloxy)-5-(triﬂuoromethyl)phenyl)-5,7-dimethyl-[1,2,4]tri-

azolo[1,5-a]pyrimidine-2-carboxamide (60). General Protocol C was

adjusted using 10a (40 mg, 0.21 mmol), 108 (61 mg, 0.23 mmol),

COMU (111 mg, 0.26 mmol) and DIPEA (0.15 mL, 0.83 mmol) to obtain

1

60 as a pale pink solid (8 mg, 9%). H NMR (DMSO‑d₆, 300 MHz) δ

10.14 (s, 1H), 8.67 (s, 1H), 7.60–7.54 (m, 3H), 7.45–7.35 (m, 5H), 5.42

(s, 2H), 2.75 (s, 3H), 2.64 (s, 3H). HRMS Found: [M + H]⁺442.1484;

5,7-Dimethyl-N-(4-(piperidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (67). General Protocol A

was adapted using 115 (15 mg, 0.06 mmol) and 10a (10 mg, 0.05 mmol)

C

₂₂H₁₈F₃N₅O₂requires [M + H]⁺442.1485.

5,7-Dimethyl-N-(2-(piperidin-1-yl)-5-(triﬂuoromethyl)pyridin-3-yl)-

17

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

to obtain 67 as a pale pink solid (1.7 mg, 8%). ¹H NMR (DMSO‑d₆, 300

MHz) δ 9.27 (s, 1H), 7.54 (s, 1H), 7.38 (s, 1H), 5.76 (s, 1H), 3.11 (t, J 5.4

Hz, 4H), 2.82 (s, 3H), 2.66 (s, 3H), 1.82–1.71 (m, 4H), 1.68–1.57 (m,

2H). HRMS Found: [M + H]⁺420.1751; C₁₉H₂₀F₃N₇O requires [M +

H]⁺420.1754.

mmol). The reaction was heated to reﬂux for 16 h before being ﬁltered

through Celite and washed with EtOH (30 mL). The reaction mixture

was concentrated to dryness and puriﬁed using silica chromatography

eluting with 25% EtOAc/heptane to obtain 73 as a brown oil (107.5 mg,

49%). ¹H NMR (CDCl_3,300 MHz) δ 7.02 (d, J 7.7 Hz, 1H), 6.93 (t, J 7.6

Hz, 1H), 6.79–6.66 (m, 2H), 2.95 (q, J 6.9 Hz, 4H), 0.98 (t, J 7.0 Hz, 6H).

MS m/z (%) [M + H]⁺165 (100).

5,7-Dimethyl-N-(4-(pyrrolidin-1-yl)-6-(triﬂuoromethyl)pyridin-3-yl)-

[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (68). General Protocol A

was adapted using 116 (12 mg, 0.05 mmol) and 10a (10 mg, 0.05 mmol)

to obtain 68 as a dark yellow solid (3.1 mg, 15%). ¹H NMR (DMSO‑d₆,

300 MHz) δ 10.70 (s, 1H), 8.10 (s, 1H), 7.34 (s, 1H), 6.98 (s, 1H), 3.50 (t,

J 6.3 Hz 4H), 2.80 (s 3H), 2.65 (s, 3H), 1.91–1.83 (m, 4H). HRMS Found:

[M + H]⁺406.1598; C₁₈H₁₈F₃N₇O requires [M + H]⁺406.1598.

5,7-Dimethyl-N-(2-(piperidin-1-yl)-5-(triﬂuoromethyl)phenyl)pyrazolo

[1,5-a]pyrimidine-2-carboxamide (69). General Protocol A was adapted

using 76 (56 mg, 0.23 mmol) and 10d (40 mg, 0.21 mmol) to obtain 69

2-(Piperidin-1-yl)aniline (74). General Protocol D was adapted using

1-ﬂuoro-2-nitrobenzene 8a (0.22 mL, 2.13 mmol), K₂CO₃(1.47 g, 12.0

mmol), piperidine (0.95 mL, 9.6 mmol), zinc (1.4 g, 21 mmol) and

ammonium chloride (1.14 g, 21 mmol) to obtain 74 as a brown crys-

talline solid (313 mg, 85%). ¹H NMR (MeOD, 300 MHz) δ 6.99 (dd, J 7.8,

1.5 Hz, 1H), 6.87 (ddd, J 7.8, 7.2, 1.7 Hz, 1H), 6.77 (dd, J 7.9, 1.7 Hz,

1H), 6.69 (ddd, J 7.8, 7.2, 1.6 Hz, 1H), 2.82 (t, J 5.0 Hz, 4H), 1.74 (quint,

J 5.5 Hz, 4H), 1.62 (q, J 4.5 Hz, 2H). MS m/z (%) [M + H]⁺177 (100)

2-(Pyrrolidin-1-yl)aniline (75). General Protocol D was adapted using

1-ﬂuoro-2-nitrobenzene 8a (0.22 mL, 2.13 mmol), K₂CO₃(1.47 g, 12.0

mmol), pyrrolidine (0.8 mL, 9.6 mmol), zinc (1.4 g, 21 mmol) and

ammonium chloride (1.14 g, 21 mmol) to obtain 75 as a black oil (253

mg, 75%). ¹H NMR (CDCl₃, 300 MHz) δ 7.01 (d, J 7.9 Hz, 1H), 6.90 (t, J

7.5 Hz, 1H), 6.77–6.72 (m, 2H), 3.84 (bs, 2H), 3.06 (t, J 5.9 Hz, 4H),

1.93 (quint, J 3.1 Hz, 4H). MS m/z (%) [M + H]⁺163 (100).

1

as a white solid (23 mg, 27%). H NMR (400 MHz, CDCl₃) δ H NMR

(CDCl_3,300 MHz) δ 10.36 (s, 1H), 8.62 (d, J 2.3 Hz, 1H), 7.17 (s, 1H),

7.07 (d, J 8.4 Hz, 1H), 7.01 (dd, J 8.5, 2.3 Hz, 1H), 6.70 (d, J 1.2 Hz, 1H),

2.84 (t, J 5.2 Hz, 4H), 2.79 (s, 3H), 2.58 (s, 3H), 1.85 (quint, J 5.7 Hz,

4H), 1.63 (s, 2H). ¹³C NMR (CDCl₃, 75 MHz,) δ 160.1, 159.6, 150.1,

149.5, 145.8, 145.3, 133.6, 127.0 (q, ²J_C-F32 Hz), 124.3 (q, ¹J_C-F272

3

Hz), 120.8 (q, J_C-F4 Hz), 120.5, 116.6 (q, J_C-F4.4 Hz), 110.6, 97.1,

53.6, 26.9, 24.9, 24.2, 17.1. ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 60.74. HRMS

Found: [M + H]⁺418.1844; C₂₁H₂₂F₃N₅O requires [M + H]⁺418.1849.

N-(5-Chloro-2-(piperidin-1-yl)phenyl)-5,7-dimethylpyrazolo[1,5-a]py-

rimidine-2-carboxamide (70). General Protocol A was adapted using 78

(48 mg, 0.23 mmol) and 10d (40 mg, 0.21 mmol) to obtain 70 as a white

solid (17 mg, 21%). ¹H NMR (DMSO‑d₆, 300 MHz) δ 10.38 (s, 1H), 8.48

(s, 1H), 7.30 (d, J 8.4 Hz, 1H), 7.15 (d, J 8.7 Hz, 1H), 7.11 (s, 1H), 7.05

(s, 1H), 2.82 (t, J 5.7 Hz, 4H), 2.77 (s, 3H), 2.54 (s, 3H), 1.81 (quint, J

5.1 Hz, 4H), 1.60 (d, J 5.1 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 159.8,

159.5, 150.2, 149.5, 145.3, 141.4, 134.5, 130.6, 123.6, 121.6, 119.5,

110.5, 97.0, 53.9, 27.0, 24.9, 24.2, 17.1. HRMS Found: [M + H]⁺

384.1587; C₂₀H₂₂ClN₅O requires [M + H]⁺384.1586.

2-(Piperidin-1-yl)-5-(triﬂuoromethyl)aniline (76). General Protocol D

was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene 8b

(0.34 mL, 2.4 mmol), K₂CO₃(1.65 g, 12.0 mmol), piperidine (1.1 mL,

10.8 mmol), zinc (1.6 g, 24.0 mmol) and ammonium chloride (1.3 g,

24.0 mmol) to obtain 76 as a pink crystalline solid (223 mg, 40%). ¹H

NMR (MeOD, 400 MHz) δ 7.03 (d, J 8.2 Hz, 1H), 6.97 (d, J 2.1 Hz, 1H),

6.89 (d, J 8.2, 2.2 Hz, 1H), 2.85 (t, J 5.6 Hz, 4H), 1.73 (quint, J 5.6 Hz,

4H), 1.63–1.57 (m, 2H). MS m/z (%) [M + H]⁺245 (100).

4-Chloro-N¹,N¹-diethylbenzene-1,2-diamine (77). General Protocol D

was adapted using 4-chloro-1-ﬂuoro-2-nitrobenzene 8c (0.28 mL, 2.85

mmol), K₂CO₃(1.65 g, 12.0 mmol), diethylamine (1.33 mL, 12.82

mmol), zinc (1.86 g, 28.5 mmol) and ammonium chloride (1.5 g, 28.5

mmol) to obtain 77 as a black oil (450.3 mg, 80%). ¹H NMR (CDCl₃, 300

MHz) δ 6.97 (d, J 8.3 Hz, 1H), 6.76 (d, J 1.9 Hz, 1H), 6.72 (dd, J 8.2, 1.7

Hz, 1H), 2.96 (q, J 7.1 Hz, 4H), 1.02 (t, J 7.1 Hz, 6H). MS m/z (%) [M +

H]⁺199 (100) 201 (30).

N,5,7-Trimethyl-N-[2-(1-piperidyl)-5-(triﬂuoromethyl)phenyl]-[1,2,4]

triazolo[1,5-a]pyrimidine-2-carboxamide (71). General Protocol B was

adapted using 118 (50 mg, 0.20 mmol) and 10a (25 mg, 0.13 mmol).

The crude was puriﬁed using silica chromatography eluting with 100%

DCM to 5% MeOH/DCM to obtain 71 as a beige solid (1.7 mg, 3%). ¹H

NMR (MeOD, 300 MHz) δ 7.56 (s, 1H), 7.49 (d, J 7.9 Hz, 1H), 7.10–7.05

(m, 2H), 3.58 (s, 3H), 2.92 (s, 2H), 2.69 (s, 2H), 2.62 (s, 3H), 2.53 (s,

3H), 1.71–1.55 (m, 6H). ¹⁹F (DMSO‑d₆, 282 MHz) δ ꢀ 64.20. HRMS

Found: [M + H]⁺433.1953; C₂₁H₂₃F₃N₆O requires [M + H]⁺433.1958.

N-(5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-2-(piperidin-1-

yl)-5-(triﬂuoromethyl)benzamide (72). 5,7-Dimethyl-[1,2,4]triazolo[1,5-

a]pyrimidin-2-amine (60 mg, 0.37 mmol), 119 (50 mg, 0.18 mmol), T3P

(0.22 mL, 0.37 mmol) and DIPEA (0.13 mL, 0.73 mmol) in DMF (1 mL)

was heated under microwave irradiation at 80 ^◦C for 2 h. The reaction

mixture was diluted with EtOAc (5 mL) and washed with water (2 × 5

mL) and saturated brine (1 × 5 mL). The organic layer was separated and

dried (Na₂SO₄) before concentration in vacuo. The crude residue was

puriﬁed by HPLC to obtain 72 as a white solid (1.3 mg, 2%). ¹H NMR

(DMSO‑d₆, 300 MHz) δ 12.44 (bs, 1H), 8.05 (s, 1H), 7.87 (d, J 7.7 Hz,

1H), 7.49 (d, J 5.5 Hz, 1H), 7.13 (s, 1H), 3.03 (s, 4H), 2.69 (s, 3H), 2.56

(s, 3H), 1.73 (s, 4H), 1.56 (s, 2H). HRMS Found: [M + H]⁺419.1797;

C₂₀H₂₁F₃N₆O requires [M + H]⁺419.1802.

5-Chloro-2-(piperidin-1-yl)aniline (78). General Protocol

D was

adapted using 4-chloro-1-ﬂuoro-2-nitrobenzene 8c (0.28 mL, 2.85

mmol), K₂CO₃(1.65 g, 12.0 mmol), piperidine (1.27 mL, 12.82 mmol),

zinc (1.86 g, 28.5 mmol) and ammonium chloride (1.5 g, 28.5 mmol) to

obtain 78 as a black crystalline solid (496 mg, 83%). ¹H NMR (CDCl₃,

400 MHz) δ 6.93 (d, J 8.4 Hz, 1H), 6.75 (d, J 2.3 Hz, 1H), 6.70 (dd, J 8.4,

2.3 Hz, 1H), 3.16–3.15 (m, 4H), 2.03–1.97 (m, 6H). MS m/z (%) [M +

H]⁺211 (100) 213 (30).

5-Chloro-2-(pyrrolidin-1-yl)aniline (79). General Protocol D was

adapted using 4-chloro-1-ﬂuoro-2-nitrobenzene 8c (0.06 mL, 0.57

mmol), K₂CO₃(0.4 g, 2.85 mmol), pyrrolidine (0.2 mL, 2.56 mmol), zinc

(0.37 g, 5.7 mmol) and ammonium chloride (0.3 g, 5.7 mmol) to obtain

79 as a brown crystalline solid (96 mg, 85%). ¹H NMR (CDCl₃, 400 MHz)

δ 6.93 (d, J 8.4 Hz, 1H), 6.75 (d, J 2.3 Hz, 1H), 6.70 (dd, J 8.4, 2.3 Hz,

1H), 3.16 (t, J 6.2 Hz, 4H), 2.03–1.98 (m, 4H). MS m/z (%) [M + H]⁺

197 (100) 199 (30).

5-Chloro-2-morpholinoaniline (80). General Protocol D was adapted

using 4-chloro-1-ﬂuoro-2-nitrobenzene 8c (0.75 mL, 7.6 mmol), K₂CO₃

(5.3 g, 38 mmol), morpholine (3 mL, 35 mmol), zinc (5 g, 77 mmol) and

ammonium chloride (4.1 g, 77 mmol) to obtain 80 as a black crystalline

solid (282 mg, 80%). ¹H NMR (CDCl₃, 400 MHz) δ 6.89 (d, J 8.4 Hz, 1H),

6.70 (d, J 2.4 Hz, 1H), 6.68 (dd, J 8.4, 2.4 Hz, 1H), 2.84 (t, J 4.5 Hz, 4H),

1.74 (t, J 4.5 Hz, 4H). MS m/z (%) [M + H]⁺213 (100) 215 (30).

2-(Pyrrolidin-1-yl)-5-(triﬂuoromethyl)aniline (81). General Protocol D

was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene 8b

(0.33 mL, 2.4 mmol), K₂CO₃(1.65 g, 12.0 mmol), pyrrolidine (0.9 mL,

10.8 mmol), zinc (1.6 g, 24 mmol) and ammonium chloride (1.3 g, 24

mmol) to obtain 81 as a brown oil (261 mg, 80%). ¹H NMR (CDCl₃, 600

General Protocol D. N,N-Diethylbenzene-1,2-diamine (73). K₂CO₃

(1.47 g, 12.0 mmol) was added to 1-ﬂuoro-2-nitrobenzene 8a (0.22 mL,

2.13 mmol) and N,N-diethylamine (0.99 mL, 9.6 mmol) in DMF (7.5 mL)

at 0 ^◦C. The reaction was stirred at 80 ^◦C for 4 h. The reaction was

diluted with EtOAc (20 mL), washed with water (2 × 10 mL) and then

saturated brine (20 mL). The organic layers were separated and dried

with MgSO₄and concentrated in vacuo. The orange crude residue was

added to a stirred solution of EtOH (20 mL). The reaction atmosphere

was evacuated and backﬁlled three times with nitrogen gas before the

addition of zinc (1.4 g, 21 mmol) and ammonium chloride (1.14 g, 21

18

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

MHz) δ 6.98–6.96 (m, 2H), 6.91 (s, 1H), 3.95 (bs, 2H), 3.11 (t, J 5.9 Hz,

4H), 1.98–1.91 (m, 4H). MS m/z (%) [M + H]⁺231 (100).

2-Morpholino-5-(triﬂuoromethyl)aniline (82). General Protocol D was

adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene 8b (0.34 mL,

2.4 mmol), K₂CO₃(1.65 g, 12.0 mmol), morpholine (0.94 mL, 10.8

mmol), zinc (1.6 g, 24 mmol) and ammonium chloride (1.3 g, 24 mmol)

to obtain 82 as brown solid (360 mg, 61%). ¹H NMR (MeOD, 400 MHz) δ

7.07 (d, J 8.2 Hz, 1H), 6.99 (d, J 2.1 Hz, 1H), 6.91 (dd, J 8.2, 2.1 Hz, 1H),

3.85 (t, J 4 Hz, 4H), 2.92 (t, J 4 Hz, 4H). MS m/z (%) [M + H]⁺247

(100).

EtOAc/heptane to obtain 89 as a pink oil (195 mg, 95%). ¹H NMR

(CDCl₃, 300 MHz) δ 6.90 (d, J 7.8 Hz, 1H), 6.57–6.53 (m, 2H), 3.02 (t, J

5.9 Hz, 4H), 2.24 (s, 3H), 1.94–1.90 (m, 4H). MS m/z (%) [M + H]⁺177

(100).

5-Methoxy-2-(piperidin-1-yl)aniline (90). General Protocol D was

adapted using 1-chloro-4-methoxy-2-nitrobenzene 8g (200 mg, 1.1

mmol), K₂CO₃(737 mg, 5.3 mmol), piperidine (0.5 mL, 4.8 mmol), zinc

(0.7 g, 11 mmol) and ammonium chloride (0.57 g, 11 mmol). The crude

was puriﬁed using silica chromatography eluting with 1% MeOH/DCM

to obtain 90 as a brown oil (20 mg, 9%). ¹H NMR (CDCl₃, 300 MHz) δ

6.92 (dd, J 8.6, 1.5 Hz, 1H), 6.35–6.23 (m, 2H), 3.74 (s, 3H), 2.78 (bs,

4H), 1.70 (quint, J 5.5 Hz, 4H), 1.56 (bs, 2H). MS m/z (%) [M + H]⁺207

(100).

N¹,N¹-Diethyl-4-(triﬂuoromethyl)benzene-1,2-diamine (83). General

Protocol D was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)ben-

zene 8b (0.34 mL, 2.4 mmol), K₂CO₃(1.65 g, 12.0 mmol), diethyl-

amine (1.1 mL, 10.8 mmol), zinc (1.6 g, 19 mmol) and ammonium

chloride (1.3 g, 19 mmol) to obtain 83 as an orange-red solid (229 mg,

47%). ¹H NMR (MeOD, 400 MHz) δ 7.10 (d, J 8.2 Hz, 1H), 7.00 (d, J 2.1

Hz, 1H), 6.89 (dd, J 8.2, 2.2, 1H), 2.99 (q, J 7.1 Hz, 4H), 0.99 (t, J 7.1 Hz,

6H). MS m/z (%) [M + H]⁺233 (100).

5-Methoxy-2-(pyrrolidin-1-yl)aniline (91). General Protocol D was

adapted using 1-chloro-4-methoxy-2-nitrobenzene 8g (200 mg, 1.1

mmol), K₂CO₃(737 mg, 5.3 mmol), pyrrolidine (0.4 mL, 4.8 mmol), zinc

(0.7 g, 11 mmol) and ammonium chloride (0.57 g, 11 mmol). The crude

was puriﬁed using silica chromatography eluting with 1% MeOH/DCM

to obtain 91 as a brown oil (85 mg, 42%). ¹H NMR (CDCl₃, 300 MHz) δ

6.95 (d, J 8.5 Hz, 1H), 6.35–6.23 (m, 2H), 3.74 (s, 3H), 2.98 (t, J 6.0 Hz,

4H), 1.91 (td, J 6.6, 3.5 Hz, 4H). MS m/z (%) [M + H]⁺193 (100).

2-(Piperidin-1-yl)-5-(triﬂuoromethoxy)aniline (92). General Protocol D

was adapted using 1-chloro-2-nitro-4-(triﬂuoromethoxy)benzene 8h

(0.15 mL, 0.83 mmol), K₂CO₃(472 mg, 4.1 mmol), piperidine (0.3 mL,

3.7 mmol), zinc (0.54 g, 8.3 mmol) and ammonium chloride (0.44 g, 8.3

mmol). The crude was puriﬁed using silica chromatography eluting with

25% EtOAc/heptane to obtain 92 as a brown oil (61 mg, 28%). ¹H NMR

(CDCl₃, 300 MHz) δ 6.94 (d, J 9.2 Hz, 1H), 6.57–6.55 (m, 2H), 4.10 (bs,

2H), 2.81 (t, J 5.5 Hz, 4H), 1.71 (quint, J 5.6 Hz, 4H), 1.59 (q, J 6.7 Hz,

2H). MS m/z (%) [M + H]⁺261 (100).

5-Fluoro-2-(piperidin-1-yl)aniline (84). General Protocol

D was

adapted using 1,4-diﬂuoro-2-nitrobenzene 8d (0.14 mL, 1.3 mmol),

K₂CO₃(871 mg, 6.3 mmol), piperidine (0.6 mL, 5.7 mmol), zinc (0.82 g,

12.6 mmol) and ammonium chloride (0.67 g, 12.6 mmol). The crude

was puriﬁed using silica chromatography eluting with 50% EtOAc/

heptane to obtain 84 as a brown solid (212.2 mg, 87%). ¹H NMR (CDCl₃,

300 MHz) δ 6.93–6.88 (m, 1H), 6.46–6.34 (m, 2H), 4.12 (bs, 2H), 2.77

(bs, 4H), 1.69 (quint, J 5.5 Hz, 4H), 1.60–1.55 (m, 2H). MS m/z (%) [M

+ H]⁺195 (100).

5-Fluoro-2-(pyrrolidin-1-yl)aniline (85). General Protocol D was

adapted using 1,4-diﬂuoro-2-nitrobenzene 8d (0.14 mL, 1.3 mmol),

K₂CO₃(871 mg, 6.3 mmol), pyrrolidine (0.5 mL, 5.7 mmol), zinc (0.82 g,

12.6 mmol) and ammonium chloride (0.67 g, 12.6 mmol). The crude

was puriﬁed using silica chromatography eluting with 50% EtOAc/

heptane to obtain 85 as a brown solid (132.8 mg, 59%). ¹H NMR (CDCl₃,

300 MHz) δ 6.97–6.89 (m, 1H), 6.46–6.36 (m, 2H), 4.05 (bs, 2H), 2.97

(t, J 5.4 Hz, 4H), 1.95–1.91 (m, 4H). MS m/z (%) [M + H]⁺181 (100).

2-(Pyrrolidin-1-yl)-5-(triﬂuoromethoxy)aniline (93). General Protocol

D was adapted using with 1-chloro-2-nitro-4-(triﬂuoromethoxy)benzene

8h (0.15 mL, 0.83 mmol), K₂CO₃(572 mg, 4.1 mmol), pyrrolidine (0.3

mL, 3.7 mmol), zinc (0.54 g, 8.3 mmol) and ammonium chloride (0.44 g,

8.3 mmol). The crude was puriﬁed using silica chromatography eluting

with 50% EtOAc/heptane to obtain 93 as a brown oil (193 mg, 95%). ¹H

NMR (CDCl₃, 300 MHz) δ 6.95 (d, J 9.4 Hz 1H), 6.59–6.56 (m, 2H), 3.95

(bs, 2H), 3.03 (t, J 5.5 Hz, 4H), 1.95–1.91 (m, 4H). MS m/z (%) [M + H]⁺

247 (100).

5-Bromo-2-(piperidin-1-yl)aniline (86). General Protocol

D was

adapted using 4-bromo-1-chloro-2-nitrobenzene 8e (200 mg, 0.85

mmol), K₂CO₃(585 mg, 4.2 mmol), piperidine (0.4 mL, 3.8 mmol), zinc

(0.55 g, 8.5 mmol) and ammonium chloride (0.45 g, 8.5 mmol). The

crude was puriﬁed using silica chromatography eluting with 20%

EtOAc/heptane to obtain 86 as a red solid (138 mg, 64%). ¹H NMR

(CDCl₃, 300 MHz) δ 6.84–6.82 (m, 3H), 4.03 (bs, 2H), 2.79 (t, J 5.2 Hz,

4H), 1.69 (quint, J 5.5 Hz, 4H), 1.57 (q, J 5.7 Hz, 2H). MS m/z (%) [M +

H]⁺255 (100) 257 (90).

3-Amino-4-(piperidin-1-yl)benzonitrile (94). General Protocol D was

adapted using with 4-chloro-3-nitrobenzonitrile 8i (200 mg, 1.1 mmol),

K₂CO₃(757 mg, 5.5 mmol), piperidine (0.5 mL, 4.9 mmol), zinc (0.72 g,

11 mmol) and ammonium chloride (0.6 g, 11 mmol) to obtain 94 as an

orange oil (105 mg, 48%). ¹H NMR (CDCl_3,300 MHz) δ 7.02–6.92 (m,

3H), 4.06 (bs, 2H), 2.85 (t, J 5.6 Hz, 4H), 1.70 (quint, J 5.4 Hz, 4H), 1.60

(q, J 5.7 Hz, 2H). MS m/z (%) [M + H]⁺202 (100).

5-Bromo-2-(pyrrolidin-1-yl)aniline (87). General Protocol D was

adapted using 4-bromo-1-chloro-2-nitrobenzene 8e (200 mg, 0.85

mmol), K₂CO₃(585 mg, 4.23 mmol), pyrrolidine (0.32 mL, 3.81 mmol),

zinc (0.55 g, 8.5 mmol) and ammonium chloride (0.45 g, 8.5 mmol). The

crude was puriﬁed using silica chromatography eluting with 50%

EtOAc/heptane to obtain 87 as a brown solid (136 mg, 67%). ¹H NMR

(CDCl₃, 300 MHz) δ 6.83–6.82 (m, 3H), 3.89 (bs, 2H), 3.00 (t, J 5.6 Hz,

4H), 1.91 (q, J 3.6 Hz, 4H). MS m/z (%) [M + H]⁺241 (90), 243 (100).

3-Amino-4-(pyrrolidin-1-yl)benzonitrile (95). General Protocol D was

adapted using with 4-chloro-3-nitrobenzonitrile 8i (200 mg, 1.1 mmol),

K₂CO₃(757 mg, 5.5 mmol), pyrrolidine (0.4 mL, 4.9 mmol), zinc (0.72 g,

11 mmol) and ammonium chloride (0.6 g, 11 mmol) to obtain 95 as a

red oil (157 mg, 71%). ¹H NMR (CDCl₃, 300 MHz) δ 7.02 (dd, J 8.1, 2.0

Hz, 1H), 6.93–6.84 (m, 2H), 3.82 (bs, 2H), 3.15 (t, J 6.2 Hz, 4H), 1.94

(quint, J 6.0 Hz, 4H). MS m/z (%) [M + H]⁺188 (100).

5-Methyl-2-(piperidin-1-yl)aniline (88). General Protocol

D was

adapted using 1-chloro-4-methyl-2-nitrobenzene 8f (0.15 mL, 1.2

mmol), K₂CO₃(806 mg, 5.8 mmol), piperidine (0.5 mL, 5.25 mmol), zinc

(0.76 g, 11.7 mmol) and ammonium chloride (0.62 g, 11.7 mmol). The

crude was puriﬁed using silica chromatography eluting with 50%

EtOAc/heptane to obtain 88 as an orange oil (201 mg, 91%). ¹H NMR

(CDCl_3,300 MHz) δ 6.89 (d, J 7.8 Hz, 1H), 6.59–6.52 (m, 2H), 4.01 (bs,

2H), 2.81 (t, J 5.1 Hz, 4H), 2.24 (s, 3H), 1.70 (quint, J 5.6 Hz, 4H), (q, J

5.2 Hz, 2H). MS m/z (%) [M + H]⁺191 (100).

5-(Methylsulfonyl)-2-(piperidin-1-yl)aniline (96). General Protocol D

was adapted using with 1-chloro-4-(methylsulfonyl)-2-nitrobenzene 8k

(200 mg, 0.85 mmol), K₂CO₃(587 mg, 4.2 mmol), piperidine (0.4 mL,

3.8 mmol), zinc (0.56 g, 8.5 mmol) and ammonium chloride (0.45 g, 8.5

mmol) to obtain 96 as a brown solid (155 mg, 72%). ¹H NMR (CDCl₃,

300 MHz) δ 7.23–7.21 (m, 2H), 6.98 (d, J 8.7 Hz, 1H), 4.20 (bs, 2H),

2.98 (s, 3H), 2.83 (t, J 5.1 Hz, 4H), 1.67 (quint, J 5.5 Hz, 4H), 1.56 (q, J

5.7 Hz, 2H). MS m/z (%) [M + H]⁺255 (100).

5-Methyl-2-(pyrrolidin-1-yl)aniline (89). General Protocol D was

adapted using 1-chloro-4-methyl-2-nitrobenzene 8f (0.15 mL, 1.2

mmol), K₂CO₃(806 mg, 5.8 mmol), pyrrolidine (0.4 mL, 5.25 mmol),

zinc (0.76 g, 11.7 mmol) and ammonium chloride (0.45 g, 8.5 mmol).

The crude was puriﬁed using silica chromatography eluting with 50%

5-(Methylsulfonyl)-2-(pyrrolidin-1-yl)aniline (97). General Protocol D

was adapted using with 1-chloro-4-(methylsulfonyl)-2-nitrobenzene 8k

(200 mg, 0.85 mmol), K₂CO₃(587 mg, 4.2 mmol), pyrrolidine (0.3 mL,

3.8 mmol), zinc (0.56 g, 8.5 mmol) and ammonium chloride (0.45 g, 8.5

mmol) to obtain 97 as a beige solid (136 mg, 67%). ¹H NMR (CDCl₃, 300

19

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

MHz,) δ 7.23 (dd, J 8.3, 2.2 Hz, 1H), 7.19 (d, J 2.2 Hz, 1H), 6.91 (d, J 8.3

Hz, 1H), 3.14 (t, J 6.5 Hz, 4H), 2.97 (s, 3H), 1.96–1.93 (m, 4H). MS m/z

(%) [M + H]⁺241 (100).

atmosphere was evacuated and backﬁlled three times with nitrogen gas

before the addition of zinc (390 g, 6 mmol) and ammonium chloride

(319 mg, 6 mmol). The reaction was heated at reﬂux for 24 h before

being ﬁltered through Celite and washed with EtOH (20 mL). The re-

action mixture was concentrated to dryness and puriﬁed using silica

chromatography eluting with 50% EtOAc/heptane to obtain 105 as an

orange solid (90 mg, 67%). ¹H NMR (CDCl_3,300 MHz) δ 7.77–7.76 (m,

2H), 7.29 (d, J 8.3 Hz, 1H), 7.07 (d, J 1.9 Hz, 1H), 7.01 (dd, J 8.3, 1.9 Hz,

1H), 6.48 (t, J 2.2 Hz, 1H), 4.92 (bs, 2H). MS m/z (%) [M + H]⁺228

(100).

2-(Azepan-1-yl)-5-(triﬂuoromethyl)aniline (98). General Protocol D

was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene 8b

(0.14 mL, 1 mmol), K₂CO₃(622 mg, 5 mmol), azepane (0.51 mL, 4.5

mmol zinc (0.66 g, 10 mmol) and ammonium chloride (0.54 g, 1 mmol).

The crude was puriﬁed using silica chromatography eluting with 25%

EtOAc/heptane to obtain 98 as a dark red solid (90 mg, 35%). ¹H NMR

(CDCl_3,300 MHz) δ 7.05 (d, J 8.0 Hz, 1H), 6.96–6.88 (m, 2H), 4.13 (bs,

2H), 3.08 (t, J 5.4 Hz, 4H), 1.80–1.68 (m, 8H). MS m/z (%) [M + H]⁺

259 (100).

2-(1H-1,2,4-Triazol-1-yl)-5-(triﬂuoromethyl)aniline (106). General

Protocol D was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)ben-

zene 8b (0.13 mL, 0.93 mmol), K₂CO₃(257 mg, 1.9 mmol), 1H-1,2,4-tri-

azole (71 mg, 1.02 mmol), zinc (0.61 g, 9.3 mmol) and ammonium

chloride (0.50 g, 9.3 mmol) to obtain 106 as a beige solid (108 mg,

51%). ¹H NMR (DMSO‑d_6,300 MHz) δ 8.96 (s, 1H), 8.30 (s, 1H), 7.50 (d,

J 8.2 Hz, 1H), 7.24 (d, J 1.4 Hz, 1H), 6.97 (dd, J 8.3, 1.4 Hz, 1H), 5.93

(bs, 2H). MS m/z (%) [M + H]⁺220 (100).

2-(8-Azabicyclo[3.2.1]octan-8-yl)-5-(triﬂuoromethyl)aniline

(99).

General Protocol D was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoro-

methyl)benzene 8b (0.03 mL, 0.21 mmol), K₂CO₃(132 mg, 0.96

mmol), 8-azabicyclooctane (50 mg, 0.45 mmol), zinc (140 mg, 2.1

mmol) and ammonium chloride (115 mg, 2.1 mmol). The crude material

was puriﬁed using silica chromatography eluting with 50% EtOAc/

heptane to obtain 99 as an orange solid (53 mg, 91%). ¹H NMR (CDCl₃,

300 MHz) δ 6.93–6.92 (m, 2H), 6.85 (d, J 8.8 Hz, 1H), 3.82 (bs, 2H),

2.05–1.58 (m, 12H). MS m/z (%) [M + H]⁺, 259 (100).

2-isoButoxy-5-(triﬂuoromethyl)aniline (107). 60% NaH in oil (191.3

mg, 4.8 mmol) was added to a solution of isobutanol (5 mL) and stirred

at room temperature for 30 min. 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)

benzene 8b (0.13 mL, 0.96 mmol) was then added and the mixture

was stirred at 80 ^◦C for 48 h. The reaction was quenched with 10% citric

acid (10 mL) and extracted with EtOAc (3 × 10 mL). The organics were

combined and washed with saturated brine (30 mL). The organic layer

was separated and dried with MgSO₄and concentrated in vacuo. The

orange crude residue was added to a stirred solution of EtOH (8 mL). The

reaction atmosphere was evacuated and backﬁlled three times with ni-

trogen gas before the addition of zinc (0.63 g, 9.6 mmol) and ammonium

chloride (0.51 g, 9.6 mmol). The reaction was heated at reﬂux for 24 h

before being ﬁltered through Celite and washed with EtOH (30 mL). The

reaction mixture was concentrated to dryness and puriﬁed using silica

chromatography eluting with 25% EtOAc/heptane to obtain 107 as a

yellow oil (171 mg, 78%). ¹H NMR (MeOD, 300 MHz) δ 6.99–6.96 (m,

1H), 6.94 (d, J 2.2 Hz, 1H), 6.79 (d, J 8.3 Hz, 1H), 4.00 (bs, 2H), 3.84 (d,

J 6.5 Hz, 2H), 2.15 (h, J 6.6 Hz, 1H), 1.09 (d, J 6.7 Hz, 6H). MS m/z (%)

[M + H]⁺234 (100).

N¹,N¹-Dimethyl-4-(triﬂuoromethyl)benzene-1,2-diamine (100). Gen-

eral Protocol D was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)

benzene 8b (0.14 mL, 1 mmol), K₂CO₃(613 mg, 4.8 mmol), dimethyl-

amine (33% in EtOH) (0.78 mL, 4.3 mmol), zinc (0.66 g, 10 mmol) and

ammonium chloride (0.54 g, 1 mmol) to obtain 100 as a brown solid

(127 mg, 65%). ¹H NMR (CDCl₃, 300 MHz) δ 7.09 (d, J 8.3 Hz, 1H), 6.99

(d, J 8.5 Hz, 1H), 6.96 (s, 1H), 2.82 (s, 6H). MS m/z (%) [M + H]⁺205

(100).

N¹-Ethyl-N¹-methyl-4-(triﬂuoromethyl)benzene-1,2-diamine

(101).

General Protocol D was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoro-

methyl)benzene 8b (0.14 mL, 1 mmol), K₂CO₃(691 mg, 4.8 mmol),

N-methylethanamine (0.4 mL, 4.5 mmol), zinc (0.66 g, 10 mmol) and

ammonium chloride (0.54 g, 1 mmol) to obtain 101 as a brown oil (33

mg, 15%). ¹H NMR (CDCl₃, 300 MHz) δ 7.03 (d, J 8.1 Hz, 1H), 6.99–6.90

(m, 2H), 4.09 (s, 2H), 2.93 (q, J 5.2 Hz, 2H), 2.66 (s, 3H), 1.09 (t, J 7.1

Hz, 3H). MS m/z (%) [M + H]⁺219 (100).

tert-Butyl 4-(2-amino-4-(triﬂuoromethyl)phenyl)piperazine-1-carbox-

ylate (102). General Protocol D was adapted using 1-ﬂuoro-2-nitro-4-

(triﬂuoromethyl)benzene 8b (0.14 mL, 1 mmol), K₂CO₃(661 mg, 4.8

mmol), 1-methylpiperazine (0.5 mL, 4.3 mmol) zinc (0.66 g, 10 mmol)

and ammonium chloride (0.54 g, 1 mmol) to obtain 101 as a beige solid

(330 mg, 99%). ¹H NMR (CDCl₃, 300 MHz) δ 7.01–6.92 (m, 3H), 4.10

(bs, 2H), 3.57 (t, J 4.7 Hz, 4H), 2.87 (t, J 5.1 Hz, 4H), 1.48 (s, 9H). MS m/

z (%) [M + H]⁺346 (100).

2-(Benzyloxy)-5-(triﬂuoromethyl)aniline (108). K₂CO₃(309 mg, 2.4

mmol) was added to 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene 8b

(0.06 mL, 0.5 mmol) and benzyl alcohol (0.22 mL, 2.2 mmol) in DMF (5

mL) at 0 ^◦C. The reaction was stirred at 80 ^◦C for 4 h. The reaction was

diluted with EtOAc (20 mL), washed with water (2 × 10 mL) and then

saturated brine (20 mL). The organic layers were separated and dried

with MgSO₄, concentrated in vacuo. The orange residue was added to a

stirred solution of EtOH (8 mL). The reaction atmosphere was evacuated

and backﬁlled three times with nitrogen before the addition of zinc (280

mg, 4.3 mmol) and ammonium chloride (229 mg, 4.3 mmol). The re-

action was stirred at reﬂux for 24 h before being ﬁltered through Celite

and washed with EtOH (30 mL). The reaction mixture was concentrated

to dryness to obtain 108 as a beige solid (111 mg, 83%). ¹H NMR

(DMSO‑d₆, 300 MHz) δ 7.50–7.32 (m, 5H), 7.00 (d, J 8.6 Hz, 1H), 6.93

(s, 1H), 6.80 (d, J 8.1 Hz, 1H), 5.18–5.17 (m, 4H). MS m/z (%) [M + H]⁺

268 (100).

2-(4-Methylpiperazin-1-yl)-5-(triﬂuoromethyl)aniline (103). General

Protocol D was adapted using 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)ben-

zene 8b (0.14 mL, 1 mmol), K₂CO₃(694 mg, 5 mmol), tert-butyl

piperazine-1-carboxylate (802 mg, 4.5 mmol), zinc (0.66 g, 10 mmol)

and ammonium chloride (0.54 g, 1 mmol) to obtain 102 as a beige solid

(247 mg, 95%). ¹H NMR (CDCl_3,300 MHz) δ 7.05 (d, J 8.2 Hz, 1H), 6.98

(d, J 8.5 Hz, 1H), 6.94 (s, 1H), 4.05 (s, 2H), 3.01 (t, J 4.8 Hz, 4H), 2.67

(bs, 4H), 2.42 (s, 3H). MS m/z (%) [M + H]⁺260 (100).

1-[2-Nitro-4-(triﬂuoromethyl)phenyl]pyrazole (104). K₂CO₃(1.47 g,

12.0 mmol) was added to 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene

8b (0.14 mL, 1 mmol) and pyrazole (306 mg, 1 mmol) in DMF (7.5

mL) at 0 ^◦C. The reaction was stirred at 80 ^◦C for 4 h. The reaction was

diluted with EtOAc (20 mL), washed with water (2 × 10 mL) and then

saturated brine (20 mL). The organic layers were separated and dried

with MgSO₄and concentrated in vacuo. The crude residue was puriﬁed

using silica chromatography eluting with DCM to obtain 104 as an or-

ange crystalline solid (153 mg, 62%). ¹H NMR (CDCl₃, 300 MHz) δ 7.93

(d, J 8.5 Hz, 1H), 7.83–7.72 (m, 3H), 6.54 (q, J 2.1 Hz, 1H), 5.29 (d, J

1.7 Hz, 1H). MS m/z (%) [M + H]⁺228 (100).

2-(Piperidin-1-yl)-5-(triﬂuoromethyl)pyridin-3-amine (109). General

Protocol D was adapted using 2-chloro-3-nitro-5-(triﬂuoromethyl)pyri-

dine (200 mg, 0.9 mmol), K₂CO₃(610 mg, 4.4 mmol), piperidine (0.4

mL, 4 mmol), zinc (0.58 g, 8.8 mmol) and ammonium chloride (0.47 g,

8.8 mmol) to obtain 109 as a green oil (126 mg, 58%). ¹H NMR (CDCl_3,

300 MHz) δ 8.02 (s, 1H), 7.05 (s, 1H), 3.90 (bs, 2H), 3.10 (t, J 5.3 Hz,

4H), 1.75–1.60 (m, 6H). MS m/z (%) [M + H]⁺246 (100).

2-(Pyrrolidin-1-yl)-5-(triﬂuoromethyl)pyridin-3-amine (110). General

Protocol D was adapted using 2-chloro-3-nitro-5-(triﬂuoromethyl)pyri-

dine (200 mg, 0.9 mmol), K₂CO₃(610 mg, 4.4 mmol), pyrrolidine

(0.33 mL, 4 mmol), zinc (0.58 g, 8.9 mmol) and ammonium chloride

(0.47 g, 8.9 mmol) to obtain 110 as a yellow oil (92 mg, 45%). ¹H NMR

(CDCl_3,300 MHz) δ 7.95 (s, 1H), 6.96 (s, 1H), 3.67 (bs, 2H), 3.51 (t, J

2-(1H-Pyrazol-1-yl)-5-(triﬂuoromethyl)aniline (105). 104 (153 mg,

0.6 mmol) was added to a stirred solution of EtOH (8 mL). The reaction

20

B.L. Bailey et al.

Bioorganic Chemistry 115 (2021) 105244

6.5 Hz, 4H), 1.99–1.94 (m, 4H). MS m/z (%) [M + H]⁺232 (100).

2-(1-Piperidyl)-4-(triﬂuoromethyl)aniline (111). General Protocol D

was adapted using 2-chloro-1-nitro-4-(triﬂuoromethyl)benzene (0.13

mg, 0.9 mmol), K₂CO₃(622 mg, 4.4 mmol), piperidine (0.33 mL, 4

mmol), zinc (0.58 g, 8.9 mmol) and ammonium chloride (0.47 g, 8.9

mmol) to obtain 111 as an orange oil (77 mg, 36%). ¹H NMR (CDCl_3,

300 MHz) δ 7.20–7.15 (m, 2H), 6.73 (d, J 8.2 Hz, 1H), 6.73 (d, J 8.2 Hz,

1H), 3.04–2.70 (m, 4H), 1.73 (t, J 6.0 Hz, 4H), 1.60 (d, J 6.7 Hz, 2H). MS

m/z (%) [M + H]⁺245 (100).

mL) and extracted with EtOAc (3 × 10 mL). The organic layers were

combined and dried (Na₂SO₄) before concentration in vacuo. The residue

was taken up in 4 M HCl in dioxane (3 mL) and stirred at reﬂux for 24 h.

The solution was concentrated in vacuo and puriﬁed using silica chro-

matography eluting with a gradient of 0 to 20% DCM/heptane to obtain

118 as a brown oil (87.2 mg, 43%). ¹H NMR (MeOD_,300 MHz) δ 7.01 (d,

J 8.0 Hz, 1H), 6.92 (dd, J 8.1, 2.1 Hz, 1H), 6.76 (d, J 2.0 Hz, 1H),

2.84–2.78 (m, 7H), 1.72 (quint, J 5.5 Hz, 4H), 1.60–1.59 (m, 2H). MS m/

z (%) [M + H]⁺259 (100).

2-Pyrrolidin-1-yl-4-(triﬂuoromethyl)aniline (112). General Protocol D

was adapted using 2-chloro-1-nitro-4-(triﬂuoromethyl)benzene (0.13

mL, 0.9 mmol), K₂CO₃(622 mg, 4.4 mmol), pyrrolidine (0.33 mL, 4

mmol), zinc (0.58 g, 8.9 mmol) and ammonium chloride (0.47 g, 8.9

mmol) to obtain 112 as a beige solid (136 mg, 66%). ¹H NMR (CDCl_3,

300 MHz) δ 7.20 (d, J 2.0 Hz, 1H), 7.14 (dd, J 8.2, 2.0 Hz, 1H), 6.73 (d, J

8.2 Hz, 1H), 4.14 (bs, 2H), 3.07 (t, J 5.8 Hz, 4H), 2.00–1.96 (m, 3.6 Hz,

4H). MS m/z (%) [M + H]⁺231 (100).

2-(1-Piperidyl)-5-(triﬂuoromethyl)benzoic acid (119). K₂CO₃(1.4 g,

9.6 mmol) was added to 1-ﬂuoro-2-nitro-4-(triﬂuoromethyl)benzene 8b

(400 mg, 1.9 mmol) and piperidine (0.85 mL, 8.65 mmol) in DMF (5 mL)

at 0 ^◦C. The reaction was stirred at 80 ^◦C for 4 h. The reaction was

diluted with EtOAc (20 mL), washed with water (2 × 10 mL) and then

saturated brine (20 mL). The organic layers were separated, dried with

MgSO₄and concentrated to dryness. The crude residue was puriﬁed

using silica chromatography eluting with a gradient from 10% MeOH/

DCM to obtain 119 as an orange solid (391 mg, 75%). ¹H NMR (CDCl_3,

300 MHz) δ 8.58 (d, J 2.2 Hz, 1H), 7.83 (dd, J 8.4, 2.3 Hz, 1H), 7.56 (d, J

8.4 Hz, 1H), 3.03 (t, J 5.4 Hz, 4H), 1.89 (quint, J 5.8 Hz, 4H), 1.71 (q, J 5.

6 Hz, 2H). MS m/z (%) [M + H] + 274 (100).

2-(1-Piperidyl)-6-(triﬂuoromethyl)pyridin-3-amine (113). General

Protocol D was adapted using 2-chloro-3-nitro-6-(triﬂuoromethyl)pyri-

dine (50 mg, 0.2 mmol), K₂CO₃(152 mg, 1.1 mmol), piperidine (0.1

mL, 1 mmol), zinc (144 mg, 2.2 mmol) and ammonium chloride (118

mg, 2.2 mmol) to obtain 113 as a red solid (57 mg, 31%). ¹H NMR

(CDCl₃, 300 MHz) δ 7.17 (d, J 7.9 Hz, 1H), 6.92 (d, J 8.0 Hz, 1H), 4.04

(bs, 2H), 3.07 (t, J 6.7 Hz, 4H), 1.71–1.58 (m, 6H). MS m/z (%) [M + H]⁺

246 (100).

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

2-Pyrrolidin-1-yl-6-(triﬂuoromethyl)pyridin-3-amine (114). General

Protocol D was adapted using 2-chloro-3-nitro-6-(triﬂuoromethyl)pyri-

dine (50 mg, 0.3 mmol), K₂CO₃(152 mg, 1.1 mmol), pyrrolidine (0.8

mL, 1 mmol zinc (144 mg, 2.2 mmol) and ammonium chloride (118 mg,

2.2 mmol) to obtain 114 as a red solid (31 mg, 60%). ¹H NMR (CDCl₃,

300 MHz) δ 7.09 (d, J 7.9 Hz, 1H), 6.90 (d, J 7.5 Hz, 1H), 3.87 (bs, 2H),

3.48 (t, J 5.9 Hz, 4H), 1.97–1.92 (m, 4H). MS m/z (%) [M + H]⁺232

(100).

Acknowledgments

This work was funded by the National Health and Medical Research

Council of Australia (Development Grant 1135421 to B.E.S. and A.F.C.;

Project Grant 1143974 to B.E.S., G.I.M, C.D.G. and D.W.W.; Program

Grant 1092789 to A.F.C.), the Australian Cancer Research Foundation,

the Victorian State Government Operational Infrastructure Support and

Australian Government NHMRC IRIISS. We thank and acknowledge the

Australian Red Cross Blood Bank for the provision of fresh red blood

cells, without which this research could not have been performed. We

would like to acknowledge BIO Ventures for Global Health (BVGH) for

facilitating a collaboration with Janssen: Pharmaceutical Companies of

Johnson & Johnson through WIPO and for sharing the Jumpstarter li-

brary for malaria research. A.F.C. is a Howard Hughes International

Scholar and an Australia Fellow of the NHMRC. B.E.S. is a Corin Cen-

tenary Fellow. D.W.W. is a Hospital Research Foundation Fellow. G.I.M.

is an Australian Research Council Laureate Fellow. We thank Dr Andrew

Powell for useful discussions.

4-(1-Piperidyl)-6-(triﬂuoromethyl)pyridin-3-amine (115). General

Protocol D was adapted using 4-chloro-5-nitro-2-(triﬂuoromethyl)pyri-

dine (75 mg, 0.3 mmol), K₂CO₃(229 mg, 1.7 mmol), piperidine (0.15

mL, 1.5 mmol), zinc (216 mg, 3.3 mmol) and ammonium chloride (177

mg, 3.3 mmol) to obtain 115 as a pale orange crystalline solid (81 mg,

64%). ¹H NMR (CDCl_3,300 MHz) δ 8.09 (s, 1H), 7.15 (s, 1H), 2.97 (t, J

5.2 Hz, 4H), 1.74 (quint, J 5.5 Hz, 4H), 1.64 (q, J 6.1 Hz, 2H). MS m/z

(%) [M + H]⁺246 (100).

4-Pyrrolidin-1-yl-6-(triﬂuoromethyl)pyridin-3-amine (116). General

Protocol D was adapted using 4-chloro-5-nitro-2-(triﬂuoromethyl)pyri-

dine (75 mg, 0.3 mmol), K₂CO₃(229 mg, 1.7 mmol), pyrrolidine

(0.12 mL, 1.5 mmol), zinc (216 mg, 3.3 mmol) and ammonium chloride

(177 mg, 3.3 mmol) to obtain 116 as a pale pink crystalline solid (40 mg,

52%). ¹H NMR (CDCl_3,300 MHz) δ 8.02 (s, 1H), 6.89 (s, 1H) 3.42 (t, J

6.7 Hz, 4H), 2.01–1.97 (m, 4H). MS m/z (%) [M + H]⁺232 (100).

tert-Butyl

N-[2-(1-piperidyl)-5-(triﬂuoromethyl)phenyl]carbamate

Appendix A. Supplementary material

(117). 60% NaH in oil (504 mg, 12.6 mmol) was added to a cooled

solution of 76 (514 mg, 2.1 mmol) in THF (20 mL) and stirred at room

temperature for 30 min. Di-tert-butyl decarbonate (1 mL, 4.6 mmol) was

added and stirred for 10 min before increasing the temperature to reﬂux

for 48 h. The reaction mixture was cooled before quenching with 10%

citric acid (20 mL) and extracted with EtOAc (3 × 10 mL). The organic

layers were combined and dried (Na₂SO₄) before concentration in vacuo.

The crude residue was puriﬁed using silica chromatography eluting with

0–70% EtOAc/DCM to obtain 117 as an orange solid (56 mg, 40%). ¹H

NMR (CDCl_3,300 MHz) δ 8.22 (s, 1H), 7.28 (m, 2H), 2.83 (t, J 4.1 Hz,

4H), 1.77 (q, J 4.0 Hz, 4H), 1.60 (d, J 5.0 Hz, 2H), 1.54 (s, 9H). MS m/z

(%) [M + H]⁺345 (100).

Supporting Information ﬁle contains dose response curves of focal

compounds against P. knowlesi and P. falciparum asexual parasites (3D7

and drug resistant lines), parasite stage arrest data, in vitro ADME data,

synthesis schemes for speciﬁc compounds and spectra for ﬁnal com-

pounds. Supplementary data to this article can be found online at https

://doi.org/10.1016/j.bioorg.2021.105244.

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22

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Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity

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