Exploring Heteroaromatic Rings as a Replacement for the Labile Amide of Antiplasmodial Pantothenamides

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Exploring Heteroaromatic Rings as a Replacement for the Labile

Amide of Antiplasmodial Pantothenamides

Jinming Guan,^††Christina Spry,^††Erick T. Tjhin,^††Penghui Yang, Tanakorn Kittikool,

Vanessa M. Howieson, Harriet Ling, Lora Starrs, Dustin Duncan, Gaetan Burgio, Kevin J. Saliba,*

and Karine Auclair *

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ABSTRACT: Malaria-causing Plasmodium parasites are developing resist-

ance to antimalarial drugs, providing the impetus for new antiplasmodials.

Although pantothenamides show potent antiplasmodial activity, hydrolysis by

pantetheinases/vanins present in blood rapidly inactivates them. We herein

report the facile synthesis and biological activity of a small library of

pantothenamide analogues in which the labile amide group is replaced with a

heteroaromatic ring. Several of these analogues display nanomolar

antiplasmodial activity against Plasmodium falciparum and/or Plasmodium knowlesi, and are stable in the presence of pantetheinase.

Both a known triazole and a novel isoxazole derivative were further characterized and found to possess high selectivity indices,

medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although they fail to suppress Plasmodium

berghei proliferation in vivo, the pharmacokinetic and contact time data presented provide a benchmark for the compound proﬁle

likely required to achieve antiplasmodial activity in mice and should facilitate lead optimization.

presence of pantetheinases. One approach consists of designing

pantetheinase inhibitors to be used in combination with

pantothenamides.^11,14Another strategy is to modify pantothe-

namides to protect them from pantetheinase-mediated

hydrolysis.^{8,9,12,13,15−21}Such alterations have been reported

at the secondary hydroxyl group,^17,20the geminal-dimethyl

group,^15,17,19the β-alanine moiety,^9,16,18and the labile amide

itself,^8,12,13,21sometimes at the cost of activity. Our team has

reported a series of analogues in which the labile amide group

is replaced with a 1,2,3-triazole ring that translates into

enhanced stability and potency.^12,21Our previous exploration

of structure−activity relationships (SARs) focused on the

triazole N-substituent, the length of the carbon linker between

the pantoyl group (Figure 1) and the triazole ring, and the

relative position of the two substituents on the ring. It was

found that the triazole 1,4-substitution pattern with a simple

methylene linker between the triazole and pantoyl moieties is

preferred for optimal antiplasmodial activity, and yields

compounds with low nanomolar antiplasmodial activity, e.g.,

1 and 2, which are proposed to mimic compounds with an

inverted amide group (Figure 1).^12,21We report herein the

synthesis and antiplasmodial activity of novel pantothenamide

1. INTRODUCTION

There were 229 million cases of malaria worldwide in 2019,

leading to 409 000 deaths.¹Malaria is caused by unicellular

protozoan Plasmodium parasites, of which six species infect

humans: Plasmodium falciparum, Plasmodium vivax, Plasmo-

dium malariae, Plasmodium ovale curtisi, Plasmodium ovale

wallikeri, and Plasmodium knowlesi.²Resistance to the currently

marketed antimalarial drugs, including standard artemisinin-

combination therapies, is spreading, making the discovery of

new antimalarials ever more crucial.³New structural classes of

antiplasmodials with a novel mechanism of action are

particularly desirable to minimize cross-resistance. The

antibacterial and antifungal activities of pantothenamides

(amides of pantothenate; Figure 1) were recognized in the

1970s,⁴whereas their antiplasmodial activity was demonstrated

more recently.⁵The unique mechanism of action of

pantothenamides involves metabolic activation by the

coenzyme A (CoA) biosynthetic pathway, which otherwise

uses pantothenate as a substrate. The resultant antimetabolites

are proposed to interfere with a subsequent CoA biosynthetic

step or downstream CoA-utilizing enzymes, thereby inhibiting

parasite proliferation.⁶⁻⁹Pantothenamides are easily accessible

synthetic targets, potent, and typically noncytotoxic.¹⁰Their

clinical use is, however, hindered by poor stability in human

serum; pantothenamides are rapidly hydrolyzed to pantothen-

ate and the corresponding amine by pantetheinases (also

known as vanins), which are present in serum (Figure 1).^5,11

We and others have previously explored several strategies to

improve the biological activity of pantothenamides in the

Received: October 16, 2020

Published: April 1, 2021

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J. Med. Chem. 2021, 64, 4478−4497

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Figure 1. (A) Hydrolysis of pantothenamides by serum pantetheinases to yield pantothenate and an amine. The pantoyl moiety is indicated in the

bracket. R₁is typically H and R₂is commonly an aliphatic or aromatic moiety. (B) Structure of compounds 1, 2, and CXP18.6-013 with their

respective 50% inhibitory concentrations (IC₅₀values) measured against intraerythrocytic P. falciparum in the presence of pantetheinase.^12,13

a

Scheme 1. Retrosynthetic Approach to the Target Heteroaromatic Pantothenamide Analogues 3a−v

a

X₁, X₂, X₃, and X₄are C, N, O, or S atoms; R is a butyl, pentyl, hexyl, or phenethyl group; and n = 1 or 2.

analogues containing diverse heteroaromatic rings instead of

the labile amide moiety, several of which show similar potency

to triazole compound 1. In addition, we report selectivity

indices, time of kill, Caco-2 cell permeability, metabolic

stability, and the ﬁrst in vivo evaluation, of heteroaromatic ring-

modiﬁed pantothenamides.

2B). The pentyl group was introduced at the ring N−H with 1-

bromopentane. The resulting compounds 6b−d were then

converted to the amides 7b−d with ammonium hydroxide and

ammonium chloride, before reduction with lithium aluminum

hydride to yield amines 8b−d. The reaction of each of these

amines with ^D-pantolactone generated 3b−d.

Synthesis of compounds 3e−i (Scheme 3A) ﬁrst entailed

producing oximes 10e−i by condensation of commercial

aldehydes 9e−i with hydroxylamine. The oximes 10e−i were

next chlorinated using N-chlorosuccinimide to form 11e−i,

followed by cycloaddition with the requisite alkyne at room

temperature. Boc-deprotection of isoxazoles 12e−i before

addition to ^D-pantolactone aﬀorded the desired 3e−i.

Compounds 3j−m were produced in a similar manner, but

from N-Boc-2-aminoacetaldehyde and using various alkynes

(Scheme 3B).

To access compound 3n (Scheme 3C), N-Boc-propargyl-

amine and trimethylsilyl azide were cycloadded to generate the

1,2,3-triazole 16. Compound 16 was next extended with 1-

bromopentane, before Boc-deprotection and condensation

with ^D-pantolactone to produce 3n.

To synthesize compound 3o (Scheme 4A), N-Boc-

glycinamide was ﬁrst converted to imidate 18 in the presence

of triethyloxonium hexaﬂuorophosphate. Compound 18 was

next condensed with hexanoic acid hydrazide to generate the

1,2,4-triazole 19. Finally, Boc-deprotection followed by ^D-

pantolactone ring opening aﬀorded compound 3o.

2. RESULTS

2.1. Synthesis. Heteroaromatic rings are attractive groups

due to their hydrolytic stability, their hydrogen-bond donors

and acceptors (as found in amides), and their ubiquity in

approved drugs.²²The speciﬁc target compounds 3a−v

(Schemes 1−5) were selected based on synthetic accessibility

and diversity. A feature common to 1, 2, and our synthetic

targets is the pantoyl moiety. We therefore envisaged a

synthetic scheme with the corresponding heteroaromatic ring-

containing amine undergoing a condensation with ^D-

pantolactone (Scheme 1). The desired primary amines were

prepared either directly or via the tert-butyloxycarbonyl (Boc)-

protected amine.

The pyrazole derivative 3a was assembled from 1-heptyne

(Scheme 2A). Deprotonation of the terminal alkyne using

ethylmagnesium bromide, followed by attack on the required

Weinreb amide yielded compound 4, which was condensed

with hydrazine to aﬀord the pyrazole 5. Finally, Boc-

deprotection produced the amine which was next used to

open the ^D-pantolactone ring, yielding the desired 3a.

Compounds 3b−d were prepared from commercial esters

already containing the requisite heteroaromatic ring (Scheme

1,3,4-Thiadiazole 3p was synthesized in three steps (Scheme

4B). First, N-Boc-glycine was coupled to hexanoic acid

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a

Scheme 2. Synthesis of Compound 3a (A) and Compounds 3b−d (B)

a

DCM: dichloromethane; DMF: N,N-dimethylformamide; TEA: triethylamine; TFA: triﬂuoroacetic acid; THF: tetrahydrofuran.

hydrazide in the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-

dihydroquinoline (EEDQ) to yield compound 20, before

thiation with Lawesson’s reagent and cyclization to generate

the 1,3,4-thiadiazole 21.²³Boc-deprotection and D-pantolac-

tone ring opening yielded 3p. The 1,3,4-oxadiazole 3q was

similarly prepared (Scheme 4C). After coupling N-Boc-glycine

and hexanoic acid hydrazide to yield 20, direct cyclization to

the 1,3,4-oxadiazole 22 was facilitated by CBr₄and PPh₃.

Finally, Boc-deprotection of 22, followed by condensation with

D-pantolactone, aﬀorded 3q.

condensation of the resulting amino oximes 23r−s with the

requisite carboxylic acid in the presence of a carbodiimide,

generated the 1,2,4-oxadiazoles 24r−s. Boc-deprotection,

followed by ^D-pantolactone ring opening yielded 3r−s.

Oxazoles 3t−u were prepared in three steps (Scheme 5B).

The ﬁrst step was the [2+2+1] annulation of a terminal alkyne,

a nitrile, and an oxygen atom from the oxidant 8-methylquino-

line N-oxide, catalyzed by BrettPhosAuNTf₂.^24,25This was

followed by Boc-deprotection and ^D-pantolactone ring opening

to yield 3t−u.

Compounds 3r and 3s, which diﬀer only by swapped

heteroaromatic N and O atoms (Scheme 5A), were accessed

by ﬁrst reacting the requisite nitrile with hydroxylamine. Next,

Thiophene 3v was synthesized in four successive steps

starting from 2-pentyl thiophene (Scheme 5C). Formylation

via the Vilsmeier−Haack reaction, amidation, and reduction of

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a

Scheme 3. Synthesis of Compounds 3e−i (A), Compounds 3j−m (B), and Compound 3n (C)

a

DCM: dichloromethane; DMF: N,N-dimethylformamide; TEA: triethylamine; TFA: triﬂuoroacetic acid.

the amide to the amine, followed by ring opening of D-

pantolactone aﬀorded the target compound 3v.

Interestingly, individual replacement of each of the ring N

atoms of compound 1 (positions X₁, X₂, or X₃, Scheme 1) with

a C atom leads to a substantial decrease in activity (see 3a−c),

with the largest eﬀect associated with replacement at the X₃

position. Consistent with this observation, 1,3,4-thiadiazole 3p

also shows higher potency than thiophene 3v. Furthermore, as

evidenced by the higher activity of 3e−f and 3j−k relative to

3a, replacing the ring N atom with an O atom is beneﬁcial at

both the X₂and X₃positions. Overall the preferred atoms are

O > N > C for both positions, correlating with electro-

negativity and size. By contrast, 3e, 3j, 3a, and 3b are more

2.2. Antiplasmodial Activity In Vitro. Compounds 3a−v

were evaluated for their antiplasmodial activity against

intraerythrocytic P. falciparum, the most virulent malaria

parasite.¹Isoxazoles 3e−f, 3j−l, and thiadiazole 3p were

found to inhibit proliferation of P. falciparum with IC₅₀values

in the nanomolar range, with compound 3k showing

comparable activity to lead compound 1 (Table 1 and Figure

S1).

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a

Scheme 4. Synthesis of Compound 3o (A), Compound 3p (B), and Compound 3q (C)

a

CDI: carbonyldiimidazole; DCM: dichloromethane; EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; TEA: triethylamine; TFA:

triﬂuoroacetic acid; THF: tetrahydrofuran.

potent than 3r, 3s, 3o, and 3d, respectively, implying that C is

preferred over N at the X₄position. Additionally, the activity

trend of 3p > 3a > 3o > 3q suggests a preference for S > C > N

> O at position X₄. The low tolerance for an O atom at this

position is also evidenced by the high IC₅₀values measured for

compounds 3t−u.

The substituent at X₁was found to also inﬂuence activity, as

revealed by the SARs observed for the 3e−h series, and the

3j−m series. It is apparent that a four-carbon chain is preferred

over a pentyl or a hexyl group. This contrasts with the trend

previously observed for the corresponding triazole pantothe-

namide analogues for which there is a very slight preference for

ﬁve- and six-carbon chains over four.¹²The diverging trend

may arise because replacement of the triazole with an isoxazole

aﬀects the mode of binding to a bioactivating or target protein.

Similar to what was previously reported for triazole

pantothenamide analogues,²¹alkyl chains are preferred over

the phenethyl group associated with high potency in

unmodiﬁed pantothenamides,⁵with phenethyl analogues 3h

and 3m showing lower activity than 3e−g and 3j−l,

respectively. A one-carbon linker between the pantoyl and

isoxazole moieties is also favored over a two-carbon linker, at

least within derivatives containing a 5-carbon chain (3e shows

improved activity relative to 3i), analogous to the optimal

spacing observed in the triazole series.²¹This is consistent with

our previous hypothesis that compounds with a one-carbon

linker better mimic pantothenamides with the labile amide

bond inverted, while those containing a two-carbon linker

simulate standard pantothenamides.²¹

As mentioned above, triazole 1 has been shown to be

resistant to degradation by pantetheinases.¹²To conﬁrm that

this is also true when the triazole moiety of compound 1 is

replaced with other heteroaromatic moieties, we compared the

activity of selected compounds (3f, j, k, p, and r) in standard

culture medium, which contains active pantetheinases, and

culture medium treated to inactivate pantetheinases. N5-Pan, a

representative unmodiﬁed pantothenamide susceptible to

pantetheinase-mediated degradation,⁵was 90-fold more active

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a

Scheme 5. Synthesis of Compounds 3r−s (A), Compounds 3t−u (B), and Compound 3v (C)

a

ACN: acetonitrile; DCM: dichloromethane; DMF: N,N-dimethylformamide; EDAC: N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

hydrochloride; TEA: triethylamine; TFA: triﬂuoroacetic acid.

when pantetheinases were inactivated (Table S1). By contrast,

the activities of pantothenamide analogues 3f, j, k, p, and r

(representative of pantothenamide analogues with four diﬀer-

ent heteroaromatic groups), like compound 1, were 1.1- to 1.4-

fold (Table S1) of those observed in the presence of

pantetheinases.

Previously, we have shown that the mechanism of action of

compound 1 against P. falciparum involves bioactivation by

pantothenate kinase (PanK), phosphopantetheine adenylyl-

transferase (PPAT), and dephospho-CoA kinase (DPCK) to

form a CoA derivative that is hypothesized to inhibit CoA-

utilizing enzymes.⁷To investigate whether the newly

discovered antiplasmodial isoxazole, thiadiazole, and oxadia-

zole derivatives exert their activity via a similar mechanism, the

antiplasmodial activity of a selection of compounds (3e, 3j, 3p,

and 3s) was also evaluated in the presence of excess

pantothenate (100 μM). Similar to compound 1, an increase

(>6-fold) in the IC₅₀values of all four compounds was

observed upon the addition of excess pantothenate (Table 1

and Figure S1), consistent with the isoxazole, thiadiazole and

oxadiazole derivatives also competing with pantothenate as

substrates of PanK.

Two compounds, 1 and 3j, were additionally evaluated for

their ability to inhibit proliferation of P. knowlesi, a recognized

cause of human malaria in South-East Asia.^26,27P. knowlesi is

the closest relative of P. vivax (a parasite responsible for ∼50%

of malaria cases in South-East Asia and the predominant

malaria parasite in the Americas) that is amenable to in vitro

continuous culturing.^1,28Both compounds were found to

inhibit proliferation of P. knowlesi at nanomolar concentrations.

Interestingly, in contrast to the ﬁndings for P. falciparum, 3j

showed superior activity compared to 1 against this species

(Table 1 and Figure S2).

The selectivity of compounds 3a−v was investigated by

measuring proliferation of human foreskin ﬁbroblast (HFF)

cells (a low-passage cell line) in the presence of each

compound. None of the compounds inhibited proliferation

of the cells by more than 50% at 100 μM and only compounds

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Table 1. Eﬀect of Compounds 3a−v on Proliferation of Intraerythrocytic P. falciparum, P. knowlesi, and Human Foreskin

Fibroblast (HFF) Cells in Growth Medium Containing Pantetheinases and 1 or 100 μM Pantothenate

a

IC₅₀values and percent proliferation are averages from three or more independent experiments, each performed in triplicate, and errors represent

b

SEM, unless otherwise indicated. Values that are in the nanomolar range are highlighted in red. The 100 μM pantothenate data are from two

experiments and where errors are shown, they are range/2. The selectivity index is a ratio of the IC₅₀value measured against HFF cells to that

c

measured against P. falciparum in the presence of 1 μM pantothenate. Except where otherwise indicated, proliferation of HFF cells remained above

50% even in the presence of 200 μM test compound, and so the lower limit of the selectivity index was calculated by dividing 200 μM by the IC₅₀

d

e

determined against P. falciparum. Data from two independent experiments, and the error value shown represents range/2. HFF cell proliferation

was inhibited by >50% at a concentration of 200 μM test compound, but <50% at a concentration of 100 μM. The selectivity index is therefore

reported as a range, with the upper limit calculated by dividing 200 μM by the IC₅₀determined against P. falciparum in the presence of 1 μM

pantothenate, and the lower limit calculated by dividing 100 μM by the IC₅₀determined against P. falciparum.

3t and 3v showed greater than 50% inhibition at a

concentration of 200 μM (Table 1). The selectivity indices

of the best parasite inhibitors are therefore well above 200

(Table 1).

2.3. Absorption and Metabolism In Vitro. Having

demonstrated that compounds 1 and 3j show selective

nanomolar antiplasmodial activity against P. falciparum and

P. knowlesi, we next investigated whether they are likely to be

absorbed from the intestine by measuring their apparent

permeability (P_app) across conﬂuent, diﬀerentiated Caco-2 cell

monolayers.²⁹Compound 3j permeated Caco-2 cell mono-

layers in the apical-to-basolateral (A → B) direction, with a

P_apphigher than that determined for propranolola highly

permeable reference compound (Table 2).²⁹Compound 1

shows moderate permeability, with a P_appapproximately 4-fold

lower than that determined for compound 3j (Table 2).

Neither compound 1 nor 3j was found to compromise the

integrity of the Caco-2 monolayer, as evidenced by the

exclusion of lucifer yellow,³⁰a nonpermeable reference

compound.

To explore the susceptibility of compounds 1 and 3j to

hepatic metabolism, stability of the compounds in the presence

of liver microsomes was measured. Minimal degradation of

compound 1 was observed during incubation with mouse or

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Table 2. Apparent Permeability (P_app) of Compounds 1 and

3j, as well as Reference Compounds, across Caco-2 Cell

Monolayers Assessed in the Apical to Basolateral (A → B)

Direction

a

compound

A → B P_app(10⁻⁶cm/s)

lucifer yellow

<1

propranolol

1

3j

49 2.3

13 1.8

55 0.6

a

Values reported are the mean standard deviation of measurements

from three transwells.

human microsomes (<15% over 60 min), corresponding to an

in vitro half-life (t_1/2) of >255 min (Table 3) and a low in vitro

intrinsic clearance (CL_{int,in vitro}; <7 μL/min/mg protein). In the

presence of mouse microsomes, some nicotinamide adenine

dinucleotide phosphate (NADPH)-independent degradation

(20−30% degradation over 60 min) and NADH-dependent

degradation of compound 3j was observed, with an estimated

t_1/2of 33 min. The compound was, however, more stable in

human than mouse microsomes. A t_1/2of 152 min was

Figure 2. Eﬀect of compounds 1 and 3j on the proliferation of P.

berghei in female BALB/c mice (A), and whole-blood concentrations

of each compound (B), following intraperitoneal administration with

100 mg/kg/day for 2 days. Groups of ﬁve P. berghei-infected mice

were administered with solvent (black circles), compound 1 (red

circles), or compound 3j (blue circles) at day 0 and day 1 (as

indicated by the arrows in (A)). Bars represent the average fold

increase in parasitemia in each treatment group, and error bars denote

standard deviations. Blood samples were collected 3 h after the ﬁrst

dose, and on day 1, blood samples were collected immediately before

the second dose. The whole-blood concentrations measured in each

of the ﬁve mice in each treatment group are averaged in (B). Errors

represent standard deviations. <LLQ: concentrations were below the

limit of quantitation of 16 or 17 nM (for compounds 1 and 3j,

respectively).

measured in the presence of human microsomes (CL_{int,in vitro}

=

11 μL/min/mg protein).

Table 3. Stability of Compounds 1 and 3j in Mouse and

Human Liver Microsomes In Vitro in the Presence of

NADPH

microsome

species

t_1/2

(min)

CL_{int,in vitro}(μL/min/mg

compound

protein)

1

mouse

human

mouse

human

>255

33

<7

53

11

3j

152

below the limit of quantitation. Fifteen times less compound 3j

(72 34 nM; Figure 2B) was detected in blood 3 h after the

ﬁrst dose relative to that detected of compound 1, a ﬁnding

consistent with the higher in vitro intrinsic clearance observed

in the microsome stability assays. In contrast to compound 1,

the concentration of compound 3j detected at 3 h did not

exceed the IC₅₀value determined against P. falciparum in vitro

(Table 1). As for compound 1, the concentration of 3j in

whole blood 24 h after drug administration was below the limit

of quantitation (Figure 2B).

To investigate whether the observed exposures were likely to

be suﬃcient for an antiplasmodial eﬀect or could instead

provide an explanation for why the antiplasmodial activity in

mice was not greater, we determined the contact time

necessary for compounds 1 and 3j to inhibit proliferation of

P. falciparum in vitro. Ring-stage P. falciparum-infected

erythrocytesthe same stage of the parasite that was initially

exposed to the compounds in the in vivo P. berghei

experimentswere exposed for pre-determined periods to

compounds 1 and 3j before the compounds were removed and

proliferation was monitored over a total of 96 h. As shown in

Figure 3, a 10 h exposure to the compounds was insuﬃcient

for nanomolar antiplasmodial activity. At least 24 h of contact

was needed to observe an antiplasmodial eﬀect at nanomolar

concentrations, and a full eﬀect required a 48 h exposure to the

compounds. These results, together with the whole-blood

concentrations detected 3 and 24 h post drug administration,

are consistent with compounds 1 and 3j not being available at

2.4. Antiplasmodial Activity In Vivo. Encouraged by the

permeability and stability of compounds 1 and 3j, the in vivo

antiplasmodial activity of these compounds was investigated

using a mouse model of malaria. Although the Caco-2 data

were consistent with the compounds being absorbed from the

gastrointestinal tractan important property of an antimalarial

drugwe chose to assess the activity of the compounds,

initially, using intraperitoneal rather than oral administration,

thereby bypassing the need for gastrointestinal absorption.

Female BALB/c mice were infected with Plasmodium berghei,

and when the average parasitemia reached ∼1%, they were

administered with 100 mg/kg/day of compound 1 or 3j, or the

corresponding solvent control, for 2 days. Unfortunately,

neither of the compounds were found to inhibit proliferation of

P. berghei in mice, with the average parasitemia increasing at a

similar rate in all treatment groups (Figure 2A).

Three and 24 h after each dose of compound was

administered to mice, blood samples were collected and, in

an attempt to shed light on the reasons for the lack of

antiplasmodial activity in vivo, the whole-blood concentration

of the test compound was determined. Three hours after the

ﬁrst dose, compound 1 was detected in whole blood at a

concentration of 1.1 0.2 μM (20 times the IC₅₀value against

P. falciparum reported in Table 1), but within 24 h of drug

administration, this decreased to below the limit of

quantitation (16 nM) (Figure 2B). Twenty-four hours after

the second dose, the whole-blood concentration remained

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Figure 3. Eﬀect of exposure time on the antiplasmodial activity of 1 and 3j in vitro. P. falciparum-infected erythrocytes were incubated in the

presence of compound 1 or 3j for 5 h (closed circles), 10 h (open circles), 24 h (closed triangles), 48 h (open triangles), or 96 h (closed squares)

before the compounds were removed and parasite proliferation was monitored over a total of 96 h. Values are averages from three independent

experiments that were initiated with “triple-synchronized” ring-stage parasites, each performed in triplicate. Error bars represent SEM and are not

visible when smaller than the symbols. For clarity, only negative or positive error bars are shown for the 48 and 96 h data, respectively.

a suﬃciently high concentrations for long enough to

irreversibly inhibit parasite proliferation under the conditions

of the in vivo experiment.

Schalkwijk et al. (2019) previously reported a series of

pantothenamide analogues with an inverted amide bond (e.g.,

CXP18.6-013, Figure 1), which show stability toward serum

pantetheinases. One of these derivatives was also reported to

clear parasites (following oral administration) in a humanized

rodent infection model, despite parasite killing not being

instantaneous and the plasma concentration decreasing to

below the IC₉₉within 24 h, as observed for 1 and 3j.⁸It was

demonstrated that the inverted pantothenamide analogues are

bioactivated to the corresponding CoA analogues by

uninfected erythrocytes, rendering erythrocytes inhospitable

to P. falciparum. It is proposed that this phenomenon may lead

Figure 4. Eﬀect of preexposure of erythrocytes to compounds 1, 3j, or

to accumulation of the compounds within erythrocytes (with

the CoA analogues being less permeable to membranes than

pantothenamides) and could explain why parasite elimination

in mice continues after the concentration of the inverted

pantothenamide is undetectable in plasma. To explore further

the reasons for the lack of antiplasmodial activity of

compounds 1 and 3j in mice, we examined whether or not

preexposure to compounds 1 and 3j similarly aﬀects the ability

of erythrocytes to support proliferation of P. falciparum.

Uninfected erythrocytes were incubated with compounds 1, 3j,

chloroquine (an antimalarial shown previously to accumulate

in uninfected erythrocytes),^8,31dihydroartemisinin (an anti-

malarial that does not accumulate in uninfected erythrocytes),⁸

or the corresponding solvent control. The pretreatment was

performed as described before,⁸prior to mixing with enriched

P. falciparum-infected erythrocytes (parasitemia >95%).

Proliferation of P. falciparum in erythrocytes pretreated with

100 μM chloroquine was inhibited in some experiments.

However, variability between erythrocyte batches was observed

and the averaged inhibition was not statistically signiﬁcant

(Figure 4, 95% conﬁdence intervals, CI, include 100%

proliferation). Proliferation of parasites in erythrocytes treated

with solvent (0.1%, v/v, dimethyl sulfoxide, DMSO),

dihydroartemisinin (25 μM), compound 1 (100 μM), or

compound 3j (100 μM) was unaﬀected (Figure 4). These data

are consistent with exposure of uninfected erythrocytes to

compounds 1 and 3j not contributing to their antiplasmodial

activity, unlike what was proposed for the inverted

pantothenamides. Hence, antiplasmodial activity of these

reference compounds on the proliferation of P. falciparum. Bars show

the average proliferation measured in three to ﬁve independent

experiments (using blood from three diﬀerent donors: black circles,

donor 1; gray symbols, donor 2; white symbols, donor 3) each

performed in duplicate. Error bars represent the standard error of the

mean. Proliferation in erythrocytes preexposed to test or reference

compounds is shown as a percentage of proliferation in erythrocytes

exposed to 0.1% (v/v) DMSO, which was present in all treatments.

Although proliferation was reduced following chloroquine preexpo-

sure of erythrocytes from certain donors, the averaged decrease was

not statistically signiﬁcant (95% CI: 8.5−100.9% proliferation).

Preexposure to dihydroartemisinin or compounds 1 or 3j did not

result in a reduction in parasite proliferation (95% CI: dihydroarte-

misinin = 73.9−107.4, compound 1: 87.8−107.0, compound 3j =

81.5−117.8% proliferation).

compounds is unlikely to continue after they have been

cleared.

3. DISCUSSION

A key goal of this study was to explore the range of

heteroaromatic rings that could be used to replace the labile

amide bond of pantothenamides and thereby stabilize these

compounds in serum. Not all heteroaromatic rings present in

compounds 3a−v are good substitutes for the labile amide

group. For example, some, such as the oxadiazoles harbored by

compounds 3q−s, are missing a hydrogen atom that can mimic

the amide N−H. It is interesting that among the new

compounds reported here, the most potent antiplasmodials,

isoxazoles 3e−f, j−l and thiadiazole 3p, may simulate

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Figure 5. Isoxazole rings of 3e and 3j and the thiadiazole ring of 3p may mimic the inverted amide bond of pantothenamide CXP18.6-013. Key

electron donors are shown in red, while key electron acceptors are highlighted in blue.

pantothenamide analogues with an inverted amide bond.¹³As

shown in Figure 5, we propose that N-2 of 3e and O-1 of 3j

may mimic the carbonyl oxygen of CXP18.6-013, while H-4 of

both 3e and 3j may replace the N-H of CXP18.6-013.

Compound 3p is somewhat diﬀerent; while its ring N-3 may

imitate the carbonyl oxygen of CXP18.6-013, it does not have

a hydrogen atom on the thiadiazole ring. The low-lying σ*

orbital of the C−S bond is however known to interact with

electron donors like amide N−H bonds do.³²It is therefore

possible that this σ* orbital in 3p may emulate the N−H of

CXP18.6-013. Our postulate also correlates well with the

ﬁndings that (1) a N atom is preferred at position X₁

(numbering of Scheme 1), (2) N or O atoms are preferred

at positions X₂and X₃, and (3) C and S atom are preferred at

position X₄.

The antiplasmodial activity of pantothenamides with an

inverted amide has been optimized by replacing the pentyl

substituent with various groups, of which a phenethyl moiety

was especially promising.¹³In contrast, our current results with

isoxazoles, and those previously reported for triazole-modiﬁed

pantothenamides,^12,21reveal that replacement of the pentyl

chain with a phenethyl moiety (as in compounds 3h and 3m)

is detrimental to antiplasmodial activity. Here, the only

substituent that proved advantageous over the pentyl chain

was a linear butyl chain (as in compounds 3f and 3k), which

interestingly was associated with marginally lower activity than

the pentyl moiety in pantothenamides with an inverted

amide¹³and 1,2,3-triazole-modiﬁed pantothenamides.¹²One

possible explanation for the divergent SAR is that the

antimetabolites of the diﬀerent pantothenamide analogues

may bind to their target(s) in slightly diﬀerent poses, or even

act on distinct targets. Nonetheless, a more expansive

exploration of additional substituents that have enhanced

antiplasmodial activity in other pantothenamide analogue

series is warranted in the context of both triazole- and

isoxazole-modiﬁed pantothenamides.

drug susceptibility of P. knowlesi and P. falciparum have

previously been reported for other drug classes.³³The

diﬀerence observed in this study could reﬂect an inherent

diﬀerence in the biology of the two parasites.

The potency and selectivity of the known triazole-modiﬁed

pantothenamide 1¹²and of the potent isoxazoles reported here

warranted an investigation into the absorption, distribution,

metabolism, and excretion (ADME) properties of these

compounds. The Caco-2 permeability (A → B) of triazole 1

and isoxazole 3j compare favorably to other modiﬁed

pantothenamides, including the pantothenamide analogue

with an inverted amide shown to possess antiplasmodial

activity in mice (P_appof 3.5 × 10⁻⁶cm/s for the inverted amide

pantothenamide vs 13 × 10⁻⁶and 55 × 10⁻⁶cm/s for

compounds 1 and 3j, respectively).^8,9The same is true of

stability to in vitro metabolic clearance, where compound 1

showed similarly high stability to the above-mentioned

inverted amide (CL_{int,in vitro}= 3 μL/min/10⁶cells⁸vs <7 μL/

min/mg protein for the inverted amide and 1, respectively,

which, using physiological-based scaling factors³⁴to enable a

comparison, are predicted to correspond approximately to 384

and <329 μL/min/g liver, respectively). Compound 3j was less

stable, but still more stable than other pantothenamide

analogues.⁹Despite compound 1’s stability, it failed to

suppress P. berghei proliferation in mice following intra-

peritoneal administration, as did 3j. At 3 h post-administration,

the whole-blood concentration of compound 3j, was below the

IC₅₀value measured against P. falciparum over 96 h, and this

limited exposure of P. berghei is likely to explain the lack of

activity of this compound in vivo. By contrast, compound 1

exceeded the IC₉₉concentration at 3 h post-administration.

This diﬀerence in the whole-blood concentration of the

compounds, which was consistent with the slower microsomal

clearance observed for compound 1 in vitro, highlights the

impact of simply changing the triazole ring to an isoxazole ring

on the ADME properties. The identiﬁcation of metabolites of

3j generated in the presence of microsomes will be important

for determining the mechanism of clearance for this (and

possibly other) compound(s). Contact time experiments

revealed that exposure to compound 1 for 24 h was required

for submicromolar inhibition of P. falciparum proliferation

(and 48 ha complete intraerythrocytic P. falciparum cycle

was required for full eﬀect). By comparison, whole-blood

serum measurements showed that the concentration of

This study is the ﬁrst to demonstrate nanomolar

antiplasmodial activity of pantothenamide analogues against

Plasmodium species infecting humans other than P. falciparum.

The sensitivity of P. knowlesi to compounds 1 and 3j suggests

that proliferation of the closely related parasite P. vivax is likely

to also be inhibited by these compounds. Interestingly, while

compound 1 is more potent than 3j against P. falciparum, the

opposite is true for P. knowlesi. Such variations between the

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proliferation assays never exceeded 0.1% (v/v). Reagents were from

Sigma-Aldrich, unless otherwise speciﬁed. Gentamicin and Albumax II

were purchased from Thermo Fisher Scientiﬁc.

compound 1 had decreased to below the limit of quantitation

by 24 h post-administration (one intraerythrocytic P. berghei

cycle). Hence, insuﬃcient exposure may also have contributed

to the failure of compound 1.

5.1.2. Cell culture. P. falciparum (3D7 strain) parasites were

maintained in vitro essentially as described previously,¹²in human O⁺

erythrocytes suspended in HEPES- and Glutamax-supplemented

RPMI 1640 (Thermo Fisher Scientiﬁc, order number 72400120) to

which 11 mM ^D-glucose, 200 μM hypoxanthine, 24 μg/mL

gentamicin and 0.6% (w/v) Albumax II had been added. Parasites

were kept synchronous by regular sorbitol synchronization of

predominantly ring-stage cultures.^35,36

P. knowlesi parasites (A1.H1 clone) were maintained under the

same conditions as P. falciparum, except that the culture medium was

additionally supplemented with 10% (v/v) pooled human serum and

the cultures were maintained at a lower hematocrit (2%), as described

elsewhere.²⁸

Moving forward, it will also be important to establish

whether other in vivo malaria models may be more suitable for

evaluating the antimalarial activity of pantothenamide

analogues. Mice are known to have a high serum concentration

of pantothenate, which competes for metabolic transformation.

This was recently determined to be approximately 2 μM,⁸6-

fold higher than the concentration present in human serum,

and 2-fold higher than that present in the in vitro proliferation

assays performed to measure antiplasmodial activity. The in

vitro microsomal stability assay data presented here, is also

consistent with 5 times faster clearance, at least of compound

3j, in mice relative to humans. In addition, an inherently lower

sensitivity of P. berghei to the test compounds (relative to P.

falciparum) cannot be ruled out as a factor contributing to the

observed lack of antiplasmodial activity in vivo.

HFF cells were maintained in vitro in DMEM supplemented with

10% (v/v) bovine calf serum, 50 units/mL penicillin, 50 μg/mL

streptomycin, 10 μg/mL gentamicin, 0.2 mM ^L-glutamine, and 0.25

μg/mL amphotericin B, as described elsewhere.³⁷

5.2. In Vitro Antiplasmodial Activity Assays. In vitro

antiplasmodial activity against P. falciparum and P. knowlesi was

assessed over 96 and 72 h, respectively, using a modiﬁcation⁵of the

malaria SYBR Green I-based ﬂuorescence assay reported elsewhere.³⁸

P. falciparum assays were performed starting with ring-stage P.

falciparum-infected erythrocytes at a parasitemia and hematocrit of 0.5

and 1%, respectively, as previously described.⁵P. knowlesi assays were

initiated with asynchronous P. knowlesi-infected erythrocytes, also at a

parasitemia and hematocrit of 0.5 and 1%, respectively. Assays were

performed using the same medium used to culture each of the

parasites. Where speciﬁed, 100 μM sodium pantothenate (Yick-Vic

Chemicals and Pharmaceuticals, Hong Kong) was added to the

medium. For testing of pantothenamide analogues in pantetheinase-

inactivated culture medium (where speciﬁed), the Albumax II-

supplemented parasite culture medium was incubated at 37 °C in the

presence of light for 40 h prior to experiments as described

previously.⁵Infected erythrocytes incubated in the presence of

chloroquine (0.25−25 μM for P. falciparum assays and 2.5 μM for P.

knowlesi assays) served as zero proliferation controls. At the end of the

72 or 96 h incubation, assay plates were frozen and stored at −20 °C.

The plates were subsequently thawed, and the lysed cells in each well

were mixed with SYBR Safe DNA gel stain in 20 mM Tris, pH 7.5,

containing 5 mM ethylenediaminetetraacetic acid (EDTA), 0.008%

(w/v) saponin, and 0.08% (v/v) Triton X-100. The ﬂuorescence in

each well was measured as previously described.⁵

Here, we did not observe a reduced capacity of erythrocytes

pretreated with compounds 1 and 3j to support P. falciparum

proliferation, as reported for a pantothenamide analogue with

an inverted amide, where antiplasmodial activity was observed

in vivo despite the plasma concentration decreasing to below

the IC₉₉within 24 h.⁸It will be important not only to

understand why, but also to gauge the contribution of

erythrocyte accumulation of pantothenamide analogue anti-

metabolites to in vivo eﬃcacy. Evaluating the Caco-2

permeability and microsome stability of some of the other

potent pantothenamide analogues presented here (e.g.,

compounds 3f and 3p) will also be important for selecting/

designing the best preclinical candidates.

4. CONCLUSIONS

Triazole, isoxazole, and thiadiazole rings are among the most

commonly used scaﬀolds in drugs.²²We report herein that, as

established for the triazole ring,^12,21replacement of the labile

amide bond of pantothenamides with either an isoxazole or a

thiadiazole ring considerably enhances potency toward P.

falciparum in the presence of pantetheinase. This is also the

ﬁrst report of heteroaromatic ring-containing pantothenamide

analogues with activity against P. knowlesi. Furthermore, as for

the triazole-modiﬁed pantothenamides, the isoxazoles and

thiadiazoles possess high selectivity indices. Favorable ADME

properties were determined for the triazole and isoxazole

investigated, with isoxazole 3j showing high Caco-2 perme-

ability, consistent with good bioavailability, and triazole 1

showing low microsomal clearance. Although we observed here

that compounds 1 and 3j fail to suppress proliferation of P.

berghei in vivo following intraperitoneal administration, the

pharmacokinetic analyses and contact time experiments

provide a benchmark for the compound proﬁle likely required

to achieve antiplasmodial activity in mice, which should

facilitate lead optimization. In addition to isoxazole 3j, several

novel compounds reported here show potent activity against P.

falciparum and provide additional leads for the development of

novel antimalarials. Their straightforward synthesis further

increases the appeal.

5.3. In Vitro HFF Cell Proliferation Assays. Activity against

HFF cells was assessed over a period of 3−4 days using a SYBR safe-

based assay, as previously reported.^9,17To facilitate the comparison of

antiplasmodial activity and antiproliferative eﬀects on HFF cells, HFF

cell assays were performed in RPMI 1640 (rather than DMEM)

supplemented with 10% (v/v) bovine calf serum, 50 units/mL

penicillin, 50 μg/mL streptomycin, 10 μg/mL gentamicin, 0.2 mM ^L-

glutamine, and 0.25 μg/mL amphotericin B.

5.4. Caco-2 Permeability. Caco-2 permeability was assessed as

described previously.^9,39The concentration of compounds 1 and 3j in

donor solutions were conﬁrmed to be within 5% of that intended (20

μM). At the end of the permeability assay the mass balance was

calculated according to the following equation: Mass balance (%) =

(Mass_{final‑donor}+ Mass_{final‑acceptor})/Mass_initial× 100. Mass balances of

104

3% (mean

standard deviation) and 105

1%, were

determined for compound 1 and 3j, respectively.

5.5. In Vitro Microsomal Stability. Stability to degradation by

mouse and human liver microsomes was assessed in vitro in the

presence and absence of an NADPH-regenerating system as described

elsewhere.^9,40

5.6. In Vivo Antiplasmodial Activity Assays. The antiplasmo-

dial activity of compounds 1 and 3j was assessed in P. berghei-infected

female BALB/c mice. To initiate experiments, stock suspensions of P.

berghei ANKA-infected erythrocytes (stored at −80 °C) were rapidly

warmed to 37 °C, before injection (250 μL) into the intraperitoneal

5. EXPERIMENTAL SECTION

5.1. Biology Methods. 5.1.1. Materials. All pantothenamide

analogues tested were initially dissolved to a concentration of 200

mM in DMSO. The ﬁnal concentration of DMSO in in vitro

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cavity of “donor” SJL/J mice. Once the parasitemia reached between

1−10%, the donor mice were ethically culled and the infected

erythrocytes harvested from one mouse via cardiac puncture. The

parasitized erythrocytes collected were immediately diluted to 5 ×

10⁵/mL in Krebs−Ringer buﬀered saline solution, containing 0.2%

(w/v) glucose, as previously described.⁴¹Fifteen female BALB/c

mice, between 8 and 12 weeks of age, were then infected

intraperitoneally with 1 × 10⁵P. berghei-infected erythrocytes (in

200 μL of Krebs−Ringer buﬀered saline solution). Tail bleeds were

performed daily and the parasitemia of the collected blood analyzed

by giemsa-stained thin blood smear or ﬂow cytometry (as described

below). When the parasitemia of all 15 mice reached between 0.4 and

1.5%, ﬁve mice were administered (via intraperitoneal injection) 100

mg/kg of compound 1 (dissolved in 150 mM NaCl containing 20.9%,

v/v, DMSO), another ﬁve mice were administered 100 mg/kg 3j

(dissolved as for compound 1), and the ﬁnal ﬁve mice were

administered a corresponding volume of the vehicle (160 μL per 20 g

mouse).

For determination of the whole-blood concentration of the test

compounds, 3 h after treatment, mice were tail bled again, this time

with 10 μL of blood collected using a Mitra microsampling device

(Neoteryx). The microsampling devices were allowed to dry

overnight and processed as described below. Approximately 24 h

after the ﬁrst dose, blood was collected for determination of

parasitemia and whole-blood inhibitor concentration (as already

described) and immediately thereafter, all mice were administered a

second dose. Blood collection was repeated again 48 h after the ﬁrst

dose, and the mice were ethically culled at 72 h post-administration of

the ﬁrst dose.

μm) using a 4 min gradient of acetonitrile:water with 0.05% (v/v)

formic acid, at a ﬂow rate of 0.4 mL/min. To enable quantitation of

the amounts of compound 1 and 3j detected in the blood of mice

administered with the compounds, a standard curve was generated by

spiking 20 μL samples of mouse blood (in quadruplicate) with each

compound at 10 diﬀerent concentrations, ranging from 5 ng/mL to

20 μg/mL. A 10 μL aliquot of each sample was then collected using a

Mitra microsampling device. Devices were dried overnight, and the

absorbed analytes were extracted and analyzed as for the experimental

samples.

5.7. Onset of Action Experiments. The length of exposure to

compounds 1 and 3j required for irreversible inhibition of P.

falciparum proliferation was investigated using a modiﬁcation of a

previously reported method.⁴³Test compound (at one of ten

concentrations tested) or 0.5 μM chloroquine was added to

erythrocytes infected with ring-stage parasites suspended in parasite

culture medium (at 0.5% parasitemia and 1% hematocrit) in T25

culture ﬂasks. A T25 ﬂask containing a suspension of infected

erythrocytes at the same parasitemia and hematocrit was prepared in

the absence of test compound or chloroquine to serve as a control for

uninhibited parasite proliferation. Aliquots (200 μL) of each ﬂask

were immediately transferred in triplicate to the wells of a 96-well

microtiter plate to serve as the 96 h incubation time point. All T25

ﬂasks and plates were incubated stationarily at 37 °C under an

atmosphere of 96% N₂, 3% CO₂, and 1% O₂. After 5 h, the cells from

each ﬂask were resuspended and a 1 mL aliquot transferred to a

microcentrifuge tube and centrifuged (10 000 ×g, 30 s) before the

supernatant was removed. The infected erythrocytes were thereafter

washed three times in 1 mL of inhibitor-free culture medium before

being resuspended in 1 mL of inhibitor-free culture medium or, in the

case of the chloroquine-treated control, medium containing 0.5 μM

chloroquine. Aliquots (200 μL) of the washed and resuspended cells

were then transferred in triplicate to the wells of a 96-well microtiter

plate, and the plate was incubated as before. This procedure was then

repeated 10, 24, and 48 h after the assay was initiated, and all infected

erythrocyte suspensions were incubated for a total of 96 h. The

contents of the plates were then frozen at −20 °C prior to thawing

and processing as described for the SYBR Safe-based in vitro

antiplasmodial activity assays.

Onset of action experiments were performed with parasites

synchronized by the triple synchronization method described

previously.⁴⁴Brieﬂy, 48 h prior to experiments, P. falciparum cultures

were sorbitol-synchronized as described above. Sorbitol synchroniza-

tion was then repeated 12 h later and immediately prior to the assay.

5.8. Erythrocyte Preloading Experiments. The eﬀect of

preincubating uninfected erythrocytes with compounds 1 and 3j on

proliferation of P. falciparum was investigated using a modiﬁcation of

a method previously described.⁸Uninfected erythrocytes were

incubated (at a hematocrit of 5%) in P. falciparum culture medium

to which a test compound (at 100 μM) or reference compound

(chloroquine at 100 μM or dihydroartemisinin at 25 μM) had been

added. The incubation was performed in T25 culture ﬂasks, at 37 °C

(with shaking) under an atmosphere of 96% N₂, 3% CO₂, 1% O₂. A

ﬂask of erythrocytes to which no test/reference compound was added,

but that was otherwise treated under identical conditions, served as a

“no inhibitor” control. The concentration of DMSO in all ﬂasks was

0.1% (v/v). After 3 h, the cells were transferred to centrifuge tubes

and collected by centrifugation (500 ×g, 5 min). The cell pellets were

subsequently washed with 10 mL of culture medium (500 ×g, 5 min)

and then with 1 mL of culture medium three times (each time

centrifuging at 10 000 ×g for 2 min) to remove unaccumulated test/

reference compound. Finally, the pelleted erythrocytes were

suspended in culture medium (at a hematocrit of 5%) without test/

reference compound and incubated in T25 culture ﬂasks, as before,

for 24 h.

Flow cytometry was used to assess mouse parasitemia according to

a modiﬁcation of a previously reported method.⁴²Immediately after

collection, the 5 μL samples of tail blood were mixed with 20 μL of

ice-cold staining solution (MT-Ringer Complete; 154 mM NaCl, 5.6

mM KCl, 1 mM MgCl₂, 2.2 mM CaCl₂, 20 mM HEPES, 10 mM

glucose, 0.5%, w/v, bovine serum albumin, 4 mM EDTA, pH 7.4, 0.22

μm ﬁlter sterilized) containing 12 μM JC-1 (Life Technologies,

Carlsbad, CA), 5 μM Hoechst 33342 (Sigma-Aldrich, St Louis, MO),

1 μg/mL anti-CD45 APC eFluor 780 (clone 30-F11) (eBioscience,

San Diego, CA), 1 μg/mL anti-CD71 APC (clone R17217)

(eBioscience, San Diego, CA) and 2 μg/mL anti-TER119 PE-

Cyanine7 (BioLegend, San Diego, CA). The cells were stained on ice

for 30 min, before being washed (twice) with 500 μL of MT-Ringer

Complete (800 ×g, 5 min) and ﬁnally resuspended in 1 mL of MT-

Ringer Complete. All samples were analyzed using a BD LSRII

Fortessa Flow Cytometer operated using the BD FACS Diva software

(BD Bioscience, Franklin Lakes, NJ), with 1 000 000 total events

collected per sample. The ﬁnal gating strategy was based on FSC/SSC

properties; single-cell gating was applied using FSC(area):FSC-

(height), before the mature erythrocyte population (CD45⁻/

CD71^low/TER119⁺) was selected and gated for the presence of

Hoechst and JC-1 (indicative of nuclear and mitochondrial staining,

respectively), with CD45⁻/CD71^low/TER119⁺/Hoechst⁺/JC1⁺cells

deﬁned as mature, infected erythrocytes. The CD45⁻/CD71^high

/

TER119⁺immature erythrocytic population was also examined for

parasitemia via Hoechst⁺/JC1⁺staining. Final population analyses

were performed using FlowJo software (BD Bioscience, Franklin

Lakes, NJ).

Analytes were extracted from the mouse blood samples (now

dried) collected with microsampling devices, using 80% (v/v)

acetonitrile in water. The blood-ﬁlled absorbent tip from each device

was transferred to a microcentrifuge tube to which 80% (v/v)

acetonitrile (100 μL) and the internal standard diazepam (10 μL of 5

μg/mL in 50% (v/v) acetonitrile in water) was added. Each tube was

then vortexed, sonicated for 5 min, and mixed on an orbital platform

(operating at 500 rpm) for 1 h, prior to centrifugation (10 000 ×g, 3

min). An aliquot of the supernatant (3 μL) was subsequently analyzed

by LC-MS performed on a Waters Xevo TQS micro coupled to a

Waters Acquity UPLC operating in positive electrospray ionization

multiple-reaction monitoring mode. Analytes were separated on a

Supelco Ascentis Express RP Amide column (50 mm × 2.1 mm, 2.7

After 24 h, the erythrocytes were again transferred to centrifuge

tubes, collected by centrifugation (500 ×g, 5 min), and then washed

with 1 mL of culture medium (10 000 ×g, 2 min). During the

incubation of uninfected erythrocytes, P. falciparum-infected eryth-

rocytes were enriched using a Miltenyi Biotec VarioMACS magnet,

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using a modiﬁcation of a previously described method.⁴⁵Brieﬂy, ∼30

mL of a predominantly trophozoite/schizont-stage P. falciparum

culture (4% hematocrit, 5−10% parasitemia) was passed through an

LS column (Miltenyi Biotec; previously rinsed with 2 mL of culture

medium) mounted in the magnet. The P. falciparum-infected

erythrocytes retained on the column were subsequently washed

with culture medium (∼30 mL), before the column was removed

from the magnet and the infected erythrocytes eluted with 5 mL of

medium. The process was repeated (typically 3 times) until a

suﬃcient quantity of P. falciparum-infected erythrocytes was obtained.

The P. falciparum-infected erythrocytes, which were conﬁrmed by

Giemsa-stained smear to be at a parasitemia >95%, were thereafter

harvested by centrifugation at 500 ×g (5 min), washed with 1 mL of

culture medium (1000 ×g, 3 min), and the supernatant was removed.

To generate suspensions of P. falciparum-infected erythrocytes at a

parasitemia and hematocrit of 1 and 2%, respectively, the washed and

pelleted uninfected erythrocytes were diluted in culture medium and

mixed with the pelleted >95% parasitemia trophozoite/schizont-stage

P. falciparum-infected parasites.

Aliquots (100 μL) of each of the suspensions were mixed in

triplicate with 100 μL of culture medium or 100 μL of culture

medium containing 0.5 μM chloroquine, in the wells of a 96-well

microtitre plate. This resulted in P. falciparum-infected erythrocytes at

a ﬁnal parasitemia and hematocrit of 1%, in the presence or absence of

0.25 μM chloroquine, in a ﬁnal volume of 200 μL. Subsequently,

plates were incubated for 72 h at 37 °C in an atmosphere of 96% N₂,

3% CO₂, and 1% O₂, before being processed (without freezing) as

described for the SYBR Safe-based in vitro antiplasmodial activity

assays. Initially, the average ﬂuorescence measured across the

triplicate wells for each cell suspension in the presence of chloroquine

was subtracted from the ﬂuorescence measurements obtained for each

cell suspension in the absence of chloroquine. These “background-

adjusted” measurements were subsequently expressed as a percentage

of the background-adjusted measurements obtained for the wells

containing parasites grown in the “no inhibitor control” erythrocytes.

The conﬁdence intervals associated with the mean % proliferation

determined for P. falciparum parasites in each pretreated erythrocyte

suspension were calculated using GraphPad Prism software.

5.9. Chemistry. 5.9.1. Materials. All reagents and solvents were

purchased either from Chem Impex International, Inc. or Sigma-

Aldrich, Canada, and used without further puriﬁcation. Dry solvents

were obtained from an Innovative Tech Pure Solve MD-7 Solvent

puriﬁcation system. Milli-Q-quality water was used in all experiments.

Unless noted otherwise, all reactions were performed under a nitrogen

atmosphere. Flash chromatography was performed on RediSep Rf

Gold Silica Flash Chromatography Columns from Teledyne ISCO.

Analysis by thin-layer chromatography (TLC) was performed with 60

Å silica gel F-254 TLC plates from Silicycle. High-resolution mass

spectra (HRMS) were acquired at the McGill University Mass

Spectral Facility on an EXACTIVE TM Plus Orbitrap Mass

Spectrometer from Thermo Fisher Scientiﬁc or a MaXis Impact

HD Mass Spectrometer from Bruker. The NMR chemical shifts (δ)

are reported in parts per million (ppm) and are referenced to the

internal standard tetramethylsilane. Compound purity was deter-

mined by reversed-phase HPLC analyses using an Agilent 6120

Quadrupole LCMS system (see the Supporting Information for the

methods used).

5.9.2. General Protocol 1 for Synthesis of Compounds 3a and

3e−u. The synthesis of compounds 3a and 3e−u followed the

previously reported procedure with modiﬁcations.¹²The Boc-

protected heteroaromatic compound (0.19−0.82 mmol, 1.0 equiv)

was dissolved in a mixture of DCM and triﬂuoroacetic acid (1:1, v/v,

2−4 mL). The reaction was stirred at room temperature for 30 min to

2 h, or until completion of the deprotection as judged by TLC. An

aqueous NaOH solution (1 M, 10−20 mL) was used to quench the

reaction, making sure that the ﬁnal pH was higher than 9. The

product was extracted in EtOAc (4 × 10 mL). The combined organic

layers were dried over anhydrous sodium sulfate. The solid was

removed by ﬁltration, and the solvent was evaporated in vacuo to

aﬀord the crude free amine, which was used directly in the next step.

In a pressure vessel (5 mL), the free amine dissolved in anhydrous

methanol or ethanol (2 mL) was mixed with ^D-pantolactone (0.75−

3.27 mmol, 4.0 equiv) and triethylamine (0.75−3.27 mmol, 4.0

equiv). The reaction was heated to 115 °C for 12−16 h in an oil bath.

The reaction mixture was directly loaded on a silica gel column and

puriﬁcation was achieved with a gradient of 0−100% EtOAc in

hexanes, followed by 0−50% methanol in EtOAc.

5.9.3. General Protocol 2 for Synthesis of Compounds 3b−d.

The synthesis of compounds 3b−d followed the previously reported

procedure with modiﬁcations.^12,46,47Compound 6b, 6c, or 6d (0.95

mmol, 1.0 equiv) was added to an aqueous solution (14 mL) of

ammonium hydroxide (100.66 mmol, 105.8 equiv), before addition of

ammonium chloride (0.26 mmol, 0.3 equiv). The mixture was heated

to 105 °C for 16 h. The reaction mixture was cooled to room

temperature and extracted with EtOAc (4 × 10 mL). The organic

layers were dried over anhydrous sodium sulfate and concentrated in

vacuo to get the crude amide.

The crude amide was dissolved in THF (10 mL) and lithium

aluminum hydride (4.75 mmol, 5.0 equiv) in THF (2 mL) was slowly

added. The mixture was stirred at room temperature for 16 h, before

quenching with water (0.18 mL), a 15% aqueous NaOH solution

(0.18 mL), and water (10 mL) sequentially. The product was

extracted with EtOAc (4 × 10 mL). The combined organic layers

were dried over anhydrous sodium sulfate and concentrated in vacuo

to get the crude amine.

To the crude amine were added ^D-pantolactone (3.80 mmol, 4.0

equiv), triethylamine (3.80 mmol, 4.0 equiv), and ethanol (2 mL).

The reaction was stirred at 115 °C for 12 h. The reaction mixture was

puriﬁed by ﬂash chromatography on silica gel using a gradient of 0−

100% EtOAc in hexanes, followed by 0−50% methanol in EtOAc.

A solution of compound 27 (0.78 mmol, 1.0 equiv) in dried THF

was added dropwise to a suspension of lithium aluminum hydride

(2.30 mmol, 3.0 equiv) in dried THF over 3 min at 0 °C. The mixture

was then reﬂuxed for 4 h. Water (3 mL) was added, and the mixture

was stirred for 3 min at room temperature. The product was extracted

with EtOAc (4 × 10 mL), dried with sodium sulfate, and evaporated

to aﬀord amine 28. To the amine in EtOH (2 mL) was added ^D-

pantolactone (1.17 mmol, 1.5 equiv) followed by triethylamine (1.19

mmol, 1.5 equiv). The mixture was then heated in a microwave

reactor at 100 °C for 2.5 h. Subsequently, the reaction mixture was

concentrated in vacuo and the residue was puriﬁed by ﬂash

chromatography on silica gel using a gradient of 0−100% EtOAc in

hexanes, followed by 0−50% methanol in EtOAc.

5.9.4. Synthesis of Compound 4. The synthesis of compound 4

followed the previously reported procedure with modiﬁcations.^48,491-

Heptyne (2.75 mmol, 1.2 equiv) was added dropwise to a round-

bottom ﬂask, which contained ethylmagnesium bromide (2.75 mmol,

1.2 equiv) in THF (4 mL) at 50 °C. The reaction mixture was stirred

at 50 °C for 30 min, before N-(tert-butoxycarbonyl)glycine N’-

methoxy-N’-methylamide (2.29 mmol, 1.0 equiv) in THF (4 mL) was

added. The resulting mixture was stirred at 50 °C for 16 h, before

being cooled and poured into water (20 mL). The product was

extracted with EtOAc (4 × 20 mL). The combined organic layers

were dried over anhydrous sodium sulfate and concentrated in vacuo.

Puriﬁcation was achieved on silica gel using a gradient of 0−100%

EtOAc in hexanes.

5.9.5. Synthesis of Compound 5. The synthesis of compound 5

followed the previously reported procedure with modiﬁcations.⁵⁰To

compound 4 (0.87 mmol, 1.0 equiv) dissolved in methanol (3 mL)

was added hydrazine monohydrate (2.95 mmol, 3.4 equiv). The

reaction was stirred at room temperature for 1 h. The product was

puriﬁed by ﬂash chromatography on silica gel using a gradient of 0−

100% EtOAc in hexanes.

5.9.6. General Protocol 3 for Synthesis of Compounds 6b−d.

The synthesis of compounds 6b−d followed the previously reported

procedure with modiﬁcations.⁵¹To the commercial heteroaromatic

compound (2.10 mmol, 1.0 equiv) in dry DMF (4.5 mL) were added

1-bromopentane (2.31 mmol, 1.1 equiv) and sodium carbonate (4.20

mmol, 2.0 equiv) sequentially. The mixture was stirred at 80 °C for 16

h, before being diluted in EtOAc (40 mL), and washed with water (15

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mL). The organic layer was dried over anhydrous sodium sulfate and

concentrated in vacuo. Puriﬁcation was achieved on silica gel using a

gradient of 0−100% EtOAc in hexanes.

5.9.11. Synthesis of Compound 22. The synthesis of compound

22 followed the previously reported procedure with modiﬁcations.⁵⁷

At 0 °C, carbonyldiimidazole (0.92 mmol, 1.1 equiv) in DCM (1 mL)

was mixed with N-Boc glycine (0.88 mmol, 1.0 equiv) in DCM (4

mL). The mixture was stirred at 0 °C for 30 min. Hexanoic acid

hydrazide (0.88 mmol, 1.0 equiv) in DCM (1 mL) was next added.

After stirring at 0 °C for 45 min, CBr₄(1.76 mmol, 2.0 equiv) and

PPh₃(1.76 mmol, 2.0 equiv) in DCM (2 mL) were added at 0 °C

sequentially. The reaction mixture was stirred for 16 h while allowing

the mixture to warm up to room temperature. The reaction mixture

was loaded on silica gel directly and puriﬁed with a gradient of 0−

100% EtOAc in hexanes.

5.9.12. General Protocol 5 for Synthesis of Compounds 24r−s.

The synthesis of compounds 24r−s followed the previously reported

procedure with modiﬁcations.^58,59To a stirring solution of the

commercial nitrile (1.92−2.06 mmol, 1.0 equiv) in EtOH (2 mL)

were added hydroxylamine (9.60−0.29 mmol, 5.0 equiv) and

triethylamine (2.11−2.26 mmol, 1.1 equiv). The reaction was heated

to 80 °C for 16 h. The reaction mixture was diluted in EtOAc (20

mL) and washed with water (10 mL). The organic layer was dried

over anhydrous sodium sulfate and concentrated in vacuo and used

directly for the next step.

To a stirring solution of 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide (1.92−2.06 mmol, 1.0 equiv) in dry acetonitrile (2−5

mL) was added the requisite carboxylic acid (1.92−2.06 mmol, 1.0

equiv). The mixture was stirred at room temperature for 20 min,

before adding the crude product from the ﬁrst step in DMF (2 mL).

The reaction mixture was stirred at room temperature for 4 h and

then heated at 110 °C for 1.5−12 h. The reaction residue was loaded

on silica gel directly and puriﬁed with a gradient of 0−100% EtOAc in

hexanes.

5.9.13. General Protocol 6 for Synthesis of Compounds 25t−u.

The synthesis of compounds 25t−u followed the previously reported

procedure with modiﬁcations.²⁴The commercial alkyne (0.38−0.73

mmol, 1.0 equiv), nitrile (0.73−2.28 mmol, 1.0−6.0 equiv), 8-

methylquinoline N-oxide (0.49−0.94 mmol, 1.3 equiv), and the

catalyst BrettPhosAuNTf₂(0.02−0.04 mmol, 0.05 equiv) were added

to a pressure vessel (5 mL). The reaction was heated at 60 °C for 1−3

h in a microwave reactor. The cloudy mixture was loaded on silica gel

directly and puriﬁed with a gradient of 0−100% EtOAc in hexanes.

5.9.14. Synthesis of Compound 26. DMF (3.30 mmol, 1.3 equiv)

in DCM (3 mL) was cooled to 0 °C, and POCl₃(3.20 mmol, 1.3

equiv) was added dropwise. The mixture was stirred for 30 min at 0

°C. Next, 2-pentyl thiophene (2.50 mmol, 1.0 equiv) dissolved in

DCM (5 mL) was added to the mixture dropwise over 45 min at 0

°C. After warming to room temperature, the mixture was heated to 70

°C and stirred for 1.5 h. Upon cooling to room temperature, the

mixture was poured into iced water and neutralized with sodium

bicarbonate. The mixture was extracted with diethyl ether (4 × 10

mL) and dried with sodium sulfate. After evaporation, the reaction

residue was loaded on silica gel directly and puriﬁed with a gradient of

0−100% EtOAc in hexanes, followed by 0−50% methanol in EtOAc.

5.9.15. Synthesis of Compound 27. To a mixture of compound 26

(1.50 mmol, 1.0 equiv), NH₂OH·HCl (1.50 mmol, 1.0 equiv), and

NaOAc (1.65 mmol, 1.10 equiv) in a dried round-bottom ﬂask was

added CuSO₄·5H₂O (0.08 mmol, 0.05 equiv). The mixture was

heated at 110 °C on an oil bath with thorough stirring for 5 h (TLC

monitoring revealed that the reaction was complete). After cooling

the reaction mixture to room temperature, water (5 mL) was added to

quench the reaction followed by extraction with EtOAc (3 × 10 mL).

The combined extracts were washed with brine (2 × 5 mL), dried

(anhydrous sodium sulfate), and concentrated under reduced

pressure. The crude product was loaded on silica gel directly and

puriﬁed with a gradient of 0−100% EtOAc in hexanes, followed by 0−

50% methanol in EtOAc.

5.9.7. General Protocol 4 for Synthesis of Compounds 12e−i

and 15j−m. The synthesis of compounds 12e−i and 15j−m followed

the previously reported procedure with modiﬁcations.⁵²The

commercial aldehyde (1.20−2.00 mmol, 1.0 equiv) was added to

hydroxylamine (1.25−2.10 mmol, 1.1 equiv) in t-BuOH/H₂O (1:1,

v/v, 4 mL). After stirring for 30 min to 12 h at room temperature or

80 °C, TLC analysis indicated that oxime formation was complete.

The product was extracted with EtOAc (4 × 10 mL). The combined

organic layers were dried over anhydrous sodium sulfate and

concentrated in vacuo to get the crude oxime.

To a stirred solution of oxime in DMF (2 mL), was added N-

chlorosuccinimide (1.55−2.60 mmol, 1.3 equiv). The reaction

mixture was stirred at room temperature for 3 h. The pale green

solution was then diluted in brine (10 mL), before extraction of the

product was in diethyl ether (3 × 10 mL). The combined organic

layers were dried with anhydrous sodium sulfate and concentrated in

vacuo to aﬀord the crude 11e−i or 14j−m.

To a stirred solution of the requisite alkyne (0.90−1.29 mmol, 0.7

equiv) in DCM (2 mL), triethylamine (1.10−1.84 mmol, 0.9 equiv)

was added. A solution of 11e−i or 14j−m in DCM (2 mL) was added

dropwise. The reaction was stirred at room temperature for 16 h. The

reaction mixture was washed with brine (10 mL), dried over

anhydrous sodium sulfate, and concentrated in vacuo. Puriﬁcation was

achieved on silica gel using a gradient of 0−100% EtOAc in hexanes.

5.9.8. Synthesis of Compound 17. The synthesis of compound 17

followed the previously reported procedure with modiﬁcations.^51,53

To a solution of Boc-protected propargylamine (1.93 mmol, 1.0

equiv) in DMF (2 mL) and methanol (0.45 mL) were added CuI

(0.19 mmol, 0.1 equiv) and trimethylsilyl azide (2.90 mmol, 1.5

equiv). After heating at 100 °C for 16 h, the reaction was cooled to

room temperature and ﬁltered through Celite. This crude reaction

mixture was used directly in the following step.

To the crude residue were added sodium carbonate (3.87 mmol,

2.0 equiv) and 1-bromopentane (2.13 mmol, 1.1 equiv) sequentially.

The reaction was heated to 80 °C for 16 h. The reaction mixture was

cooled to room temperature and ﬁltered through Celite. The mixture

was then loaded on silica gel directly and puriﬁed with a gradient of

0−100% EtOAc in hexanes.

5.9.9. Synthesis of Compound 19. The synthesis of compound 19

followed the previously reported procedure with modiﬁcations.⁵⁴⁻⁵⁶

To N-Boc-glycinamide (2.18 mmol, 1.0 equiv) in DCM (5 mL) was

added triethyloxonium hexaﬂuorophosphate (2.40 mmol, 1.1 equiv)

in DCM (2 mL). After stirring at room temperature for 24 h, the

reaction mixture was poured into an ice-cold aqueous sodium

carbonate solution (1 M, 10 mL). The product was extracted in DCM

(4 × 10 mL). The combined organic layers were dried over

anhydrous sodium sulfate and concentrated in vacuo. The crude

residue was used directly for the following step.

To hexanoic acid hydrazide (2.18 mmol, 1.0 equiv) were added

triethylamine (2.18 mmol, 1.0 equiv) and an ethanolic solution (2

mL) of the residue from the previous step. The reaction mixture was

stirred at 115 °C for 16 h. The mixture was loaded on silica gel

directly and puriﬁed with a gradient of 0−100% EtOAc in hexanes.

5.9.10. Synthesis of Compound 21. The synthesis of compound

21 followed the previously reported procedure with modiﬁcations.²³

To N-Boc glycine (1.14 mmol, 1.0 equiv) dissolved in DCM (3 mL)

was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.14

mmol, 1.0 equiv), followed by hexanoic acid hydrazide (1.37 mmol,

1.2 equiv). The mixture was stirred at room temperature for 16 h. The

mixture was ﬁltered and concentrated in vacuo to provide the requisite

coupling product 20, which was used directly in the next step.

To the crude residue of 20 in dry THF (5 mL) was added

Lawesson’s reagent (1.26 mmol, 1.1 equiv). The reaction mixture was

stirred at 70 °C for 16 h, before concentration in vacuo. The

concentrated crude product was loaded on silica gel directly.

Puriﬁcation was achieved using a gradient of 0−100% EtOAc in

hexanes.

5.9.16. Compound Characterization. 5.9.16.1. (R)-2,4-Dihydroxy-

3,3-dimethyl-N-((5-pentyl-1H-pyrazol-3-yl)methyl)butanamide

(3a). Compound 3a was prepared from 5 (0.48 mmol) using general

protocol 1. Yield: 31%, R_f= 0.50 (10% MeOH in EtOAc). Purity is

>95% with methods A and B, R_t= 4.87 min with method A (see the

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Supporting Information); R_t= 5.27 min with method B. H NMR

(500 MHz, CDCl₃) δ 7.55 (m, 1H), 5.93 (s, 1H), 4.52 (dd, J = 15.4,

6.2 Hz, 1H), 4.32 (dd, J = 15.5, 5.0 Hz, 1H), 4.03 (s, 1H), 3.47 (m,

2H), 2.55 (t, J = 7.7 Hz, 2H), 1.58 (m, 2H), 1.37−1.26 (m, 4H), 0.97

(s, 3H), 0.91 (s, 3H), 0.87 (t, J = 6.7 Hz, 3H). ¹³C NMR (126 MHz,

CDCl₃) δ 174.18, 147.71, 147.20, 102.27, 77.28 (based on HSQC

and HMBC), 70.51, 39.38, 36.06, 31.40, 28.82, 26.23, 22.36, 21.49,

20.67, 13.96. HRMS for C₁₅H₂₇N₃O₃Na [M + Na]⁺calcd 320.1945,

found 320.1937.

5.9.16.7. (R)-N-((3-Hexylisoxazol-5-yl)methyl)-2,4-dihydroxy-3,3-

dimethylbutanamide (3g). Compound 3g was prepared from 12g

(1.09 mmol) using general protocol 1. Yield: 63%, R_f= 0.39 (100%

EtOAc). Purity is >95% with methods B and C, R_t= 11.41 min with

1

method B; R_t= 14.24 min with method C. H NMR (500 MHz,

CDCl₃) δ 7.45 (t, J = 5.9 Hz, 1H), 6.06 (s, 1H), 4.56 (d, J = 6.0 Hz,

2H), 4.10 (s, 1H), 3.54 (d, J = 11.2, 1H), 3.50 (d, J = 11.2, 1H), 2.63

(t, J = 7.7 Hz, 2H), 1.60−1.66 (m, 2H), 1.29−1.39 (m, 6H), 1.02 (s,

3H), 0.94 (s, 3H), 0.89 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz,

CDCl₃) δ 173.4, 168.1, 164.4, 101.9, 77.7, 71.4, 39.3, 34.8, 31.4, 28.8,

28.1, 26.0, 22.5, 21.1, 20.4, 14.0. HRMS for C₁₆H₂₈N₂O₄Na [M +

Na]⁺calcd 335.1941, found 335.1943.

5.9.16.2. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((1-pentyl-1H-imida-

zol-4-yl)methyl)butanamide (3b). Compound 3b was prepared from

6b (0.95 mmol) using general protocol 2. Yield: 19%, R_f= 0.24 (10%

MeOH in EtOAc). Purity is >95% with methods A and B, R_t= 3.27

5.9.16.8. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((3-phenethylisoxa-

zol-5-yl)methyl)butanamide (3h). Compound 3h was prepared

from 12h (0.40 mmol) using general protocol 1. Yield: 54%, R_f=

0.43 (100% EtOAc). Purity is >95% with methods B and C, R_t=

1

min with method A; R_t= 6.35 min with method B. H NMR (500

MHz, CDCl₃) δ 7.62 (m, 1H), 7.36 (d, J = 1.1 Hz, 1H), 6.83 (s, 1H),

4.40 (dd, J = 15.0, 6.1 Hz, 1H), 4.32 (dd, J = 14.9, 5.5 Hz, 1H), 4.05

(s, 1H), 3.86 (t, J = 7.2 Hz, 2H), 3.49 (d, J = 11.4 Hz, 1H), 3.46 (d, J

= 11.4 Hz, 1H), 1.75 (m, 2H), 1.33 (m, 2H), 1.25 (m, 2H), 1.05 (s,

3H), 0.93 (s, 3H), 0.89 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz,

CDCl₃) δ 173.55, 138.48, 136.54, 116.36, 77.50, 70.80, 47.31, 39.70,

36.18, 30.57, 28.63, 22.46, 22.12, 20.28, 13.86. HRMS for

C₁₅H₂₈N₃O₃[M + H]⁺calcd 298.21252, found 298.21185.

5.9.16.3. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((1-pentyl-1H-pyra-

zol-4-yl)methyl)butanamide (3c). Compound 3c was prepared

from 6c (0.95 mmol) using general protocol 2. Yield: 23%, R_f=

0.59 (10% MeOH in EtOAc). Purity is >95% with methods A and B,

R_t= 3.30 min with method A; R_t= 5.96 min with method B. ¹H NMR

(400 MHz, CDCl₃) δ 7.40 (s, 1H), 7.36 (s, 1H), 6.95 (m, 1H), 4.33

(d, J = 5.7 Hz, 2H), 4.07−4.04 (m, 3H), 3.52 (d, J = 11.2 Hz, 1H),

3.49 (d, J = 11.2 Hz, 1H), 1.83 (m, 2H), 1.39−1.20 (m, 4H), 1.02 (s,

3H), 0.93−0.86 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 172.56,

138.37, 128.30, 117.85, 77.72, 71.42, 52.31, 39.47, 33.55, 30.08, 28.74,

22.21, 21.51, 20.10, 13.92. HRMS for C₁₅H₂₇N₃O₃Na [M + Na]⁺

calcd 320.19446, found 320.19396.

1

10.46 min with method B; R_t= 13.22 min with method C. H NMR

(500 MHz, CDCl₃) δ 7.21−7.33 (m, 6H), 6.00 (s, 1H), 4.57 (d, J =

2.2 Hz, 1H), 4.56 (d, J = 2.3 Hz, 1H), 4.12 (s, 1H), 3.56 (d, J = 11.1

Hz, 1H), 3.52 (d, J = 11.1 Hz, 1H), 2.99 (m, 4H), 1.04 (s, 3H), 0.95

(s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.1, 168.2, 163.5, 140.4,

128.5, 128.3, 126.4, 102.0, 77.8, 71.5, 39.3, 34.8, 34.2, 27.8, 21.1, 20.4.

HRMS for C₁₈H₂₄N₂O₄Na [M + Na]⁺calcd 355.1628, found

355.1630.

5.9.16.9. (R)-2,4-Dihydroxy-3,3-dimethyl-N-(2-(3-pentylisoxazol-

5-yl)ethyl)butanamide (3i). Compound 3i was prepared from 12i

(1.15 mmol) using general protocol 1. Yield: 47%, R_f= 0.39 (100%

EtOAc). Purity is >95% with methods B and C, R_t= 10.06 min with

1

method B; R_t= 12.91 min with method C. H NMR (500 MHz,

CDCl₃) δ 7.06 (bs, 1H), 5.96 (s, 1H), 4.05 (s, 1H), 3.67−3.72 (m,

1H), 3.60−3.66 (m, 1H), 3.53 (d, J = 11.4, 1H), 3.50 (d, J = 11.2,

1H), 3.01 (t, J = 6.6 Hz, 2H), 2.63 (t, J = 7.1 Hz, 2H), 1.62−1.68 (m,

4H), 1.34−1.37 (m, 4H), 1.02 (s, 3H), 0.90−0.93 (m, 6H). ¹³C NMR

(126 MHz, CDCl₃) δ 173.4, 170.0, 164.4, 101.7, 77.6, 71.3, 39.2, 37.0,

31.3, 27.9, 27.1, 26.0, 22.3, 21.3, 20.2, 13.9. HRMS for

C₁₆H₂₈N₂O₄Na [M + Na]⁺calcd 335.1941, found 335.1943.

5.9.16.4. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((1-pentyl-1H-1,2,4-

triazol-3-yl)methyl)butanamide (3d). Compound 3d was prepared

from 6d (1.14 mmol) using general protocol 2. Yield: 4%, R_f= 0.62

(20% MeOH in EtOAc). Purity is >90% with methods A and B, R_t=

5.9.16.10. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentylisoxazol-

3-yl)methyl)butanamide (3j). Compound 3j was prepared from 15j

(0.43 mmol) using general protocol 1. Yield: 74%, R_f= 0.50 (100%

EtOAc). Purity is >95% with methods C and D, R_t= 12.48 min with

1

3.66 min with method A; R_t= 2.14 min with method B. H NMR

(500 MHz, CDCl₃) δ 7.99 (s, 1H), 7.54 (bs, 1H), 4.56 (m, 2H),

4.09−4.05 (m, 3H), 3.9 (d, J = 11.3 Hz, 1H), 3.47 (d, J = 11.3 Hz,

1H), 1.84 (m, 2H), 1.37−1.21 (m, 4H), 1.03 (s, 3H), 0.95 (s, 3H),

0.88 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.72,

160.98, 143.42, 77.64, 70.54, 49.92, 39.51, 36.62, 29.31, 28.55, 22.07,

21.90, 20.78, 13.83. HRMS for C₁₄H₂₆N₄O₃Na [M + Na]⁺calcd

321.1897, found 321.1895.

5.9.16.5. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((3-pentylisoxazol-5-

yl)methyl)butanamide (3e). Compound 3e was prepared from 12e

(0.35 mmol) using general protocol 1. Yield: 44%, R_f= 0.39 (100%

EtOAc). Purity is >95% with methods A and B, R_t= 4.56 min with

method A; R_t= 6.66 min with method B. ¹H NMR (500 MHz,

CDCl₃) δ 7.36 (m, 1H), 6.04 (s, 1H), 4.55 (d, J = 6.1 Hz, 2H), 4.09

(s, 1H), 3.53 (d, J = 11.1 Hz, 1H), 3.50 (d, J = 11.1 Hz, 1H), 2.62 (m,

2H), 1.62 (m, 2H), 1.38−1.28 (m, 4H), 1.02 (s, 3H), 0.92 (s, 3H),

0.89 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.19,

168.10, 164.44, 101.92, 77.82, 71.47, 39.32, 34.77, 31.30, 27.86, 25.95,

22.30, 21.13, 20.35, 13.92. HRMS for C₁₅H₂₅N₂O₄[M + H]⁺calcd

297.18198, found 297.18244.

1

method C; R_t= 13.57 min with method D. H NMR (500 MHz,

CDCl₃) δ 7.41 (m, 1H), 5.95 (s, 1H), 4.53 (dd, J = 15.9, 6.1 Hz, 1H),

4.47 (dd, J = 15.8, 5.9 Hz, 1H), 4.07 (s, 1H), 3.50 (m, 2H), 2.69 (t, J

= 7.6 Hz, 2H), 1.66 (m, 2H), 1.37−1.28 (m, 4H), 1.01 (s, 3H), 0.94

(s, 3H), 0.89 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

174.67, 173.48, 160.98, 100.01, 77.81, 71.11, 39.38, 35.02, 31.17,

27.10, 26.66, 22.24, 21.39, 20.55, 13.88. HRMS for C₁₅H₂₆N₂O₄Na

[M + Na]⁺calcd 321.17848, found 321.17918.

5.9.16.11. (R)-N-((5-Butylisoxazol-3-yl)methyl)-2,4-dihydroxy-

3,3-dimethylbutanamide (3k). Compound 3k was prepared from

15k (0.16 mmol) using general protocol 1. Yield: 77%, R_f= 0.45

(100% EtOAc). Purity is >95% with methods B and C, R_t= 10.12 min

with method B; R_t= 12.97 min with method C. ¹H NMR (500 MHz,

CDCl₃) δ 7.41 (bs, 1H), 5.98 (s, 1H), 4.56 (dd, J = 6.1, 15.9 Hz, 1H),

4.50 (dd, J = 5.85, 15.9 Hz, 1H), 4.11 (s, 1H), 3.54 (s, 2H), 3.20 (bs,

2H), 2.73 (t, J = 7.6 Hz, 2H), 1.64−1.70 (m, 2H), 1.35−1.43 (m,

2H), 1.05 (s, 3H), 0.97 (s, 3H), 0.95 (t, J = 7.35 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 174.7, 173.4, 161.0, 100.0, 77.9, 71.1, 39.4, 35.0,

29.5, 26.4, 22.1, 21.4, 20.6, 13.6. HRMS for C₁₄H₂₄N₂O₄Na [M +

Na]⁺calcd 307.1628, found 307.1622.

5.9.16.6. (R)-N-((3-Butylisoxazol-5-yl)methyl)-2,4-dihydroxy-3,3-

dimethylbutanamide (3f). Compound 3f was prepared from 12f

(0.32 mmol) using general protocol 1. Yield: 38%, R_f= 0.35 (100%

EtOAc). Purity is >95% with methods B and C, R_t= 9.78 min with

5.9.16.12. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentylisoxazol-

3-yl)methyl)butanamide (3l). Compound 3l was prepared from 15l

(0.30 mmol) using general protocol 1. Yield: 77%, R_f= 0.48 (100%

EtOAc). Purity is >95% with methods B and C, R_t= 11.75 min with

1

method B; R_t= 12.25 min with method C. H NMR (400 MHz,

CDCl₃) δ 7.44(bs, 1H), 6.04 (s, 1H), 4.54 (d, J = 6.0 Hz, 2H), 4.08

(s, 1H), 3.52 (d, J = 11.5, 1H), 3.48 (d, J = 11.5, 1H), 3.22 (bs, 2H),

2.61 (t, J = 7.7 Hz, 2H), 1.56−1.61 (m, 2H), 1.30−1.40 (m, 2H),

0.99 (s, 3H), 0.89−0.93 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ

173.3, 168.1, 164.4, 101.9, 77.7, 71.4, 39.3, 34.8, 30.2, 25.6, 22.2, 21.1,

20.4, 13.7. HRMS for C₁₄H₂₃N₂O₄[M − H]⁻calcd 283.1663, found

283.1662.

1

method B; R_t= 14.56 min with method C. H NMR (500 MHz,

CDCl₃) δ 7.35 (bs, 1H), 5.98 (s, 1H), 4.59 (dd, J = 6.1, 15.9 Hz, 1H),

4.51 (dd, J = 5.9, 15.9 Hz, 1H), 4.11 (s, 1H), 3.56 (s, 2H), 2.73(t, J =

7.6 Hz, 2H), 1.66−1.72 (m, 2H), 1.30−1.40 (m, 6H), 1.07 (s, 3H),

0.99 (s, 3H), 0.91 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)

δ 174.7, 173.2, 161.0, 100.0, 80.0, 71.2, 39.4, 35.1, 31.4, 28.7, 27.4,

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26.7, 22.5, 21.5, 20.6, 14.0. HRMS for C₁₆H₂₈N₂O₄Na [M + Na]⁺

calcd 335.1941, found 335.1952.

2H), 1.71 (m, 2H), 1.36−1.29 (m, 4H), 1.03 (s, 3H), 0.97 (s, 3H),

0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 175.89,

173.90, 170.77, 77.89, 71.07, 39.53, 35.34, 31.30, 26.61, 25.92, 22.34,

21.28, 20.96, 13.99. HRMS for C₁₄H₂₄N₃O₄[M − H]⁻calcd

298.17723, found 298.17721.

5.9.16.13. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-phenethylisoxa-

zol-3-yl)methyl)butanamide (3m). Compound 3m was prepared

from 15m (0.55 mmol) using general protocol 1. Yield: 21%, R_f=

0.50 (100% EtOAc). Purity is >95% with methods B and C, R_t=

5.9.16.19. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentyl-1,2,4-ox-

adiazol-3-yl)methyl)butanamide (3s). Compound 3s was prepared

from 24s (0.82 mmol) using general protocol 1. Yield: 75%, R_f= 0.62

(10% MeOH in EtOAc). Purity is >95% with methods A and B, R_t=

1

10.69 min with method B; R_t= 13.46 min with method C. H NMR

(500 MHz, CDCl₃) δ 7.29−7.33 (m, 3H), 7.23−7.26 (m, 1H), 7.18−

7.20 (m, 2H), 5.94 (s, 1H), 4.58 (dd, J = 6.1, 15.9 Hz, 1H), 4.49 (dd,

J = 5.9, 15.9 Hz, 1H), 4.11 (d, J = 5.2 Hz, 1H), 3.72 (d, J = 5.2 Hz,

1H), 3.55 (d, J = 4.9 Hz, 1H), 3.00−3.09 (m, 5H), 1.07 (s, 3H), 0.98

(s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.34, 173.09, 161.01,

139.85, 128.60, 128.24, 126.52, 100.47, 78.06, 71.24, 39.43, 35.06,

33.46, 28.46, 21.57, 20.60. HRMS for C₁₈H₂₄N₂O₄Na [M + Na]⁺

calcd 335.1628, found 335.1627.

1

3.78 min with method A; R_t= 6.45 min with method B. H NMR

(500 MHz, CDCl₃) δ 7.39 (m, 1H), 4.67 (dd, J = 16.7, 6.2 Hz, 1H),

4.56 (dd, J = 16.7, 5.7 Hz, 1H), 4.10 (s, 1H), 3.55 (m, 2H), 2.86 (t, J

= 7.6 Hz, 2H), 1.80 (m, 2H), 1.39−1.31 (m, 4H), 1.07 (s, 3H), 0.98

(s, 3H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

180.97, 173.41, 167.73, 78.12, 71.24, 39.63, 35.18, 31.23, 26.64, 26.30,

22.28, 21.71, 20.81, 13.96. HRMS for C₁₄H₂₅N₃O₄Na [M + Na]⁺

calcd 322.17373, found 322.17430.

5.9.16.14. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((2-pentyl-2H-1,2,3-

triazol-4-yl)methyl)butanamide (3n). Compound 3n was prepared

from 17 (0.78 mmol) using general protocol 1. Yield: 45%, R_f= 0.65

(10% MeOH in EtOAc). Purity is >95% with methods A and B, R_t=

5.9.16.20. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((2-pentyloxazol-5-

yl)methyl)butanamide (3t). Compound 3t was prepared from 25t

(0.34 mmol) using general protocol 1. Yield: 40%, R_f= 0.50 (10%

MeOH in EtOAc). Purity is >95% with methods A and B, R_t= 3.76

1

3.49 min with method A; R_t= 2.31 min with method B. H NMR

(500 MHz, CDCl₃) δ 7.50 (s, 1H), 7.18 (bs, 1H), 4.59 (dd, J = 15.4,

6.0 Hz, 1H), 4.51 (dd, J = 15.4, 5.7 Hz, 1H), 4.36 (t, J = 7.2 Hz, 2H),

4.08 (d, J = 4.7 Hz, 1H), 3.53 (m, 2H), 1.93 (m, 2H), 1.39−1.23 (m,

4H), 1.04 (s, 3H), 0.94 (s, 3H), 0.89 (t, J = 7.2 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 172.75, 144.59, 132.41, 77.91, 71.33, 54.99,

39.47, 34.44, 29.41, 28.62, 22.12, 21.44, 20.38, 13.88. HRMS for

C₁₄H₂₆N₄O₃Na [M + Na]⁺calcd 321.1897, found 321.1899.

1

min with method A; R_t= 4.76 min with method B. H NMR (500

MHz, CDCl₃) δ 7.19 (m, 1H), 6.79 (s, 1H), 4.50 (dd, J = 15.8, 6.0

Hz, 1H), 4.44 (dd, J = 15.8, 5.8 Hz, 1H), 4.07 (s, 1H), 3.52 (d, J =

11.1 Hz, 1H), 3.49 (d, J = 11.1 Hz, 1H), 2.70 (t, J = 7.6 Hz, 2H), 1.72

(m, 2H), 1.35−1.29 (m, 4H), 1.01 (s, 3H), 0.91 (s, 3H), 0.88 (t, J =

7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 173.14, 165.48, 148.04,

124.00, 77.94, 71.59, 39.42, 33.81, 31.36, 28.23, 26.73, 22.38, 21.26,

20.46, 14.03. HRMS for C₁₅H₂₅N₂O₄[M − H]⁻calcd 297.18198,

found 297.18170.

5.9.16.15. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentyl-4H-1,2,4-

triazol-3-yl)methyl)butanamide (3o). Compound 3o was prepared

from 19 (0.74 mmol) using general protocol 1. Yield: 24%, R_f= 0.54

(20% MeOH in EtOAc). Purity is >95% with methods A and B, R_t=

5.9.16.21. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentyloxazol-2-

yl)methyl)butanamide (3u). Compound 3u was prepared from 25u

(0.25 mmol) using general protocol 1. Yield: 25%, R_f= 0.50 (8%

MeOH in EtOAc). Purity is >95% with methods A and B, R_t= 3.93

1

3.73 min with method A; R_t= 3.892 min with method B. H NMR

(500 MHz, CD₃OD) δ 4.47 (m, 2H), 3.96 (s, 1H), 3.46 (d, J = 11.0

Hz, 1H), 3.43 (d, J = 11.0 Hz, 1H), 2.73 (m, 2H), 1.72 (m, 2H),

1.41−1.27 (m, 4H), 0.95 (s, 3H), 0.94 (s, 3H), 0.91 (t, J = 7.1 Hz,

3H). ¹³C NMR (126 MHz, CD₃OD) δ 176.20, 161.52, 159.42, 77.43,

70.07, 40.50, 37.10, 32.32, 28.73, 27.06, 23.31, 21.53, 21.00, 14.25.

HRMS for C₁₄H₂₅N₄O₃[M − H]⁻calcd 297.19321, found

297.19359.

1

min with method A; R_t= 6.75 min with method B. H NMR (400

MHz, CDCl₃) δ 7.56 (m, 1H), 6.61 (s, 1H), 4.63 (dd, J = 16.8, 6.4

Hz, 1H), 4.48 (dd, J = 16.8, 5.5 Hz, 1H), 4.07 (s, 1H), 3.51 (d, J =

11.2 Hz, 1H), 3.48 (d, J = 11.2 Hz, 1H), 2.59 (t, J = 7.6, 2H), 1.60

(m, 2H), 1.36−1.27 (m, 4H), 1.04 (s, 3H), 0.99 (s, 3H), 0.89 (t, J =

6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 173.87, 159.97, 154.15,

121.75, 78.10, 70.72, 39.63, 36.30, 31.32, 27.24, 25.53, 22.41, 21.99,

21.21, 14.05. HRMS for C₁₅H₂₆N₂O₄Na [M + Na]⁺calcd 321.1785,

found 321.1783.

5.9.16.16. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentyl-1,3,4-

thiadiazol-2-yl)methyl)butanamide (3p). Compound 3p was

prepared from 21 (0.69 mmol) using general protocol 1. Yield:

41%, R_f= 0.16 (100% EtOAc). Purity is >90% with methods A and B,

1

R_t= 12.25 min with method A; R_t= 2.14 min with method B. H

5.9.16.22. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentylthiophen-

2-yl)methyl)butanamide (3v). Compound 3v was prepared from 27

(0.78 mmol) as described. Yield: 51%, R_f= 0.76 (10% MeOH in

EtOAc). Purity is >90% with methods B and C, R_t= 12.76 min with

NMR (500 MHz, CDCl₃) δ 7.66 (m, 1H), 4.88 (dd, J = 15.9, 6.3 Hz,

1H), 4.81 (dd, J = 15.9, 6.1 Hz, 1H), 4.12 (s, 1H), 3.54 (m, 2H), 3.06

(t, J = 7.6, 2H), 1.78 (m, 2H), 1.42−1.30 (m, 4H), 1.04 (s, 3H), 0.98

(s, 3H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

173.51, 172.51, 166.77, 78.10, 71.45, 39.59, 38.07, 31.23, 30.21, 29.80,

22.38, 21.40, 20.91, 14.02. HRMS for C₁₄H₂₅N₃O₃SNa [M + Na]⁺

calcd 338.1509, found 338.1509.

1

method B; R_t= 15.64 min with method C. H NMR (500 MHz,

CDCl₃) δ 7.08 (bs, 1H, -NH), 6.78 (d, J = 3.35 Hz, 1H), 6.61 (d, J =

3.35 Hz, 1H), 4.53−4.64 (m, 2H), 4.09 (s, 1H), 3.66 (m, 1H), 3.58

(d, J = 11.15 Hz, 1H), 3.51 (d, J = 11.15 Hz, 1H), 2.77 (t, J = 7.70

Hz, 2H), 1.65−1.68 (m, 2H), 1.33−1.36 (m, 4H), 1.06 (s, 3H), 0.95

(s, 3H), 0.91 (t, J = 6.95 Hz, 3H). ¹³C NMR (126 MHz, CDCl3) δ

172.47, 146.22, 137.71, 125.79, 123.58, 77.73, 71.45, 39.52, 38.18,

31.33, 31.25, 30.14, 22.40, 21.45, 20.14, 14.00. HRMS for

C₁₆H₂₇NNaO₃S [M + Na]⁺calcd 336.1604, found 336.1611.

5.9.16.17. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((5-pentyl-1,3,4-ox-

adiazol-2-yl)methyl)butanamide (3q). Compound 3q was prepared

from 22 (0.26 mmol) using general protocol 1. Yield: 39%, R_f= 0.47

(10% MeOH in EtOAc). Purity is >95% with methods A and B, R_t=

1

4.82 min with method A; R_t= 9.52 min with method B. H NMR

(500 MHz, CDCl₃) δ 7.73 (t, J = 5.8 Hz, 1H), 4.66 (m, 2H), 4.11 (s,

1H), 3.54 (d, J = 11.1 Hz, 1H), 3.46 (d, J = 11.1 Hz, 1H), 2.79 (t, J =

7.6 Hz, 2H), 1.75 (m, 2H), 1.38−1.29 (m, 4H), 0.99 (s, 3H), 0.97 (s,

3H), 0.89 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 174.12,

168.05, 163.98, 77.68, 70.91, 39.47, 34.09, 31.18, 26.11, 25.36, 22.25,

21.21, 21.04, 13.93. HRMS for C₁₄H₂₅N₃O₄Na [M + Na]⁺calcd

322.17373, found 322.17248.

5.9.16.23. tert-Butyl (2-Oxonon-3-yn-1-yl)carbamate (4). Com-

pound 4 was prepared from N-(tert-butoxycarbonyl)glycine N’-

methoxy-N’-methylamide (2.29 mmol) as described. Yield: 22%. R_f

= 0.62 (30% EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.15

(bs, 1H), 4.11 (d, J = 5.0 Hz, 2H), 2.38 (t, J = 7.1 Hz, 2H), 1.59 (m,

2H), 1.45 (s, 9H), 1.42−1.29 (m, 4H), 0.91 (t, J = 7.2 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 183.10, 155.41, 97.87, 80.01, 78.79,

52.28, 30.96, 28.30, 27.24, 22.07, 19.03, 13.86. HRMS for

C₁₄H₂₃NO₃Na [M + Na]⁺calcd 276.1570, found 276.1573.

5.9.16.18. (R)-2,4-Dihydroxy-3,3-dimethyl-N-((3-pentyl-1,2,4-ox-

adiazol-5-yl)methyl)butanamide (3r). Compound 3r was prepared

from 24r (0.41 mmol) using general protocol 1. Yield: 50%, R_f= 0.67

(10% MeOH in EtOAc). Purity is >90% with methods A and B, R_t=

5.9.16.24. tert-Butyl ((5-Pentyl-1H-pyrazol-3-yl)methyl)-

carbamate (5). Compound 5 was prepared from 4 (0.87 mmol) as

1

3.19 min with method A; R_t= 3.92 min with method B. H NMR

described. Yield: 56%. R_f= 0.23 (50% EtOAc in hexanes). H NMR

(500 MHz, CDCl₃) δ 7.62 (m, 1H), 4.75 (dd, J = 17.3, 6.3 Hz, 1H),

4.63 (dd, J = 17.3, 5.8 Hz, 1H), 4.10 (s, 1H), 3.50 (m, 2H), 2.68 (m,

(500 MHz, CDCl₃) δ 5.96 (s, 1H), 5.15 (bs, 1H), 4.28 (d, J = 5.7 Hz,

2H), 2.60 (t, J = 7.9 Hz, 2H), 1.63 (m, 2H), 1.45 (s, 9H), 1.35−1.29

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(m, 4H), 0.89 (t, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

156.19, 147.79, 147.79 (based on HSQC and HMBC), 102.23, 79.62,

37.68, 31.43, 28.89, 28.41, 26.45, 22.40, 13.97. HRMS for

C₁₄H₂₅N₃O₂Na [M + Na]⁺calcd 290.1839, found 290.1843.

5.9.16.25. Ethyl 1-Pentyl-1H-imidazole-4-carboxylate (6b). Com-

pound 6b was prepared from ethyl imidazole-4-carboxylate (2.10

mmol) using general protocol 3. Yield: 37%. R_f= 0.32 (100% EtOAc).

¹H NMR (500 MHz, CDCl₃) δ 7.59 (s, 1H), 7.46 (s, 1H), 4.35 (q, J

= 7.1 Hz, 2H), 3.94 (t, J = 7.1 Hz, 2H), 1.79 (m, 2H), 1.37 (t, J = 7.1

Hz, 3H), 1.35−1.22 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 162.51, 137.81, 134.13, 124.94, 60.50, 47.56,

30.55, 28.52, 22.08, 14.42, 13.82. HRMS for C₁₁H₁₈N₂O₂Na [M +

Na]⁺calcd 233.1260, found 233.1262.

5.9.16.26. Ethyl 1-Pentyl-1H-pyrazole-4-carboxylate (6c). Com-

pound 6c was prepared from ethyl 4-pyrazolecarboxylate (2.10 mmol)

using general protocol 3. Yield: 48%, R_f= 0.55 (30% EtOAc in

hexanes). ¹H NMR (500 MHz, CDCl₃) δ 7.89 (s, 1H), 7.86 (s, 1H),

4.28 (q, J = 7.1 Hz, 2H), 4.11 (t, J = 7.2 Hz, 2H), 1.87 (m, 2H),

1.37−1.31 (m, 5H), 1.26 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). ¹³C NMR

(126 MHz, CDCl₃) δ 163.12, 140.89, 132.30, 114.85, 60.11, 52.67,

29.76, 28.61, 22.15, 14.40, 13.87. HRMS for C₁₁H₁₈N₂O₂Na [M +

Na]⁺calcd 233.1260, found 233.1268.

(bs, 1H), 3.50−3.46 (m, 2H), 2.95 (t, J = 6.6 Hz, 2H), 2.64 (t, J = 7.6

Hz, 2H), 1.63−1.69 (m, 2H), 1.46 (s, 9H), 1.34−1.37 (m, 4H), 0.92

(t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.2, 164.3,

155.8, 101.5, 79.6, 38.5, 31.4, 28.4, 28.0, 27.6, 26.0, 22.3, 13.9. HRMS

for C₁₅H₂₆N₂O₃Na [M + Na]⁺calcd 305.1836, found 305.1832.

5.9.16.33. tert-Butyl ((5-Pentylisoxazol-3-yl)methyl)carbamate

(15j). Compound 15j was prepared from commercial 1-heptyne

(0.90 mmol) using general protocol 4. Yield: 23%, R_f= 0.77 (50%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.96 (s, 1H), 4.99

(bs, 1H), 4.34 (d, J = 5.8 Hz, 2H), 2.70 (t, J = 7.6 Hz, 2H), 1.68 (m,

2H), 1.46 (s, 9H), 1.35−1.32 (m, 4H), 0.90 (t, J = 7.0 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 174.30, 161.57, 155.78, 99.86, 79.93,

36.69, 31.20, 28.35, 27.14, 26.68, 22.27, 13.90. HRMS for

C₁₄H₂₄N₂O₃Na [M + Na]⁺calcd 291.16791, found 291.16834.

5.9.16.34. tert-Butyl ((5-Butylisoxazol-3-yl)methyl)carbamate

(15k). Compound 15k was prepared from commercial 1-hexyne

(2.50 mmol) using general protocol 4. Yield: 8%, R_f= 0.63 (50%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.98 (s, 1H), 5.01

(bs, 1H), 4.37 (d, J = 5.6 Hz, 2H), 2.74 (t, J = 7.1 Hz, 2H), 1.65−1.71

(m, 2H), 1.48 (s, 9H), 1.37−1.44 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 174.3, 161.6, 155.8, 99.9, 79.9, 36.7,

29.5, 29.4, 26.4, 22.2, 13.7. HRMS for C₁₃H₂₂N₂O₃Na [M + Na]⁺

calcd 277.1523, found 277.1526.

5.9.16.27. Methyl 1-Pentyl-1H-1,2,4-triazole-3-carboxylate (6d).

Compound 6d was prepared from methyl 1,2,4-triazole-3-carboxylate

(3.93 mmol) using general protocol 3. Yield: 55%, R_f= 0.15 (50%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 8.12 (s, 1H), 4.22

(t, J = 7.3 Hz, 2H), 3.98 (s, 3H), 1.92 (m, 2H), 1.39−1.26 (m, 4H),

0.88 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 160.20,

154.80, 144.29, 52.74, 50.65, 29.42, 28.49, 22.04, 13.81. HRMS for

C₉H₁₅N₃O₂Na [M + Na]⁺calcd 220.1056, found 220.1059.

5.9.16.35. tert-Butyl ((5-Hexylisoxazol-3-yl)methyl)carbamate

(15l). Compound 15l was prepared from commercial 1-octyne

(4.09 mmol) using general protocol 4. Yield: 11%, R_f= 0.65 (50%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.98 (s, 1H), 5.02

(bs, 1H), 4.37 (d, J = 5.3 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H), 1.66−1.72

(m, 2H), 1.48 (s, 9H), 1.35−1.40 (m, 2H), 1.30−1.33 (m, 4H), 0.91

(t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 174.3, 161.6,

155.8, 99.9, 79.9, 36.7, 31.4, 28.7, 28.4, 27.4, 26.7, 22.5, 14.0. HRMS

for C₁₅H₂₆N₂O₃Na [M + Na]⁺calcd 305.1836; found 305.1836.

5.9.16.36. tert-Butyl ((5-Phenethylisoxazol-3-yl)methyl)-

carbamate (15m). Compound 15m was prepared from commercial

4-phenyl-1-butyne (6.00 mmol) using general protocol 4. Yield: 10%,

5.9.16.28. tert-Butyl ((3-Pentylisoxazol-5-yl)methyl)carbamate

(12e). Compound 12e was prepared from N-Boc-propargylamine

(1.29 mmol) using general protocol 4. Yield: 99%, R_f= 0.32 (20%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 6.01 (s, 1H), 4.97

(bs, 1H), 4.38 (d, J = 5.6 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 1.64 (m,

2H), 1.45 (s, 9H), 1.36−1.31 (m, 4H), 0.90 (t, J = 7.0 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 169.08, 164.29, 155.47, 101.45, 80.23,

36.62, 31.33, 28.32, 27.92, 25.97, 22.32, 13.93. HRMS for

C₁₄H₂₄N₂O₃Na [M + Na]⁺calcd 291.1679, found 291.1679.

5.9.16.29. tert-Butyl ((3-Butylisoxazol-5-yl)methyl)carbamate

(12f). Compound 12f was prepared from N-Boc-propargylamine

(1.29 mmol) using general protocol 4. Yield: 26%, R_f= 0.30 (20%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 6.03 (s, 1H), 5.01

(bs, 1H), 4.41 (d, J = 5.2 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H), 1.61−1.67

(m, 2H), 1.47 (s, 9H), 1.37−1.42 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 169.1, 164.3, 155.5, 101.4, 90.2, 35.6,

1

R_f= 0.66 (50% EtOAc in hexanes). H NMR (500 MHz, CDCl₃) δ

7.28−7.32 (m, 2H), 7.19−7.25 (m, 3H), 5.96 (s, 1H), 5.11 (bs, 1H),

4.35 (d, J = 5.8 Hz, 2H), 3.00−3.07 (m, 4H), 1.48 (s, 9H). ¹³C NMR

(126 MHz, CDCl₃) δ 172.93, 161.70, 155.81, 139.99, 128.57, 128.26,

126.47, 100.42, 79.90, 36.63, 33.52, 28.51, 28.36. HRMS for

C₁₇H₂₂N₂O₃Na [M + Na]⁺calcd 325.1522, found 325.1522.

5.9.16.37. tert-Butyl ((2-Pentyl-2H-1,2,3-triazol-4-yl)methyl)-

carbamate (17). Compound 17 was prepared from N-Boc-propargyl-

amine (1.93 mmol) as described. Yield: 34%, R_f= 0.45 (30% EtOAc

1

in hexanes). H NMR (500 MHz, CDCl₃) δ 7.49 (s, 1H), 4.94 (bs,

1H), 4.38−4.34 (m, 4H), 1.93 (m, 2H), 1.45 (s, 9H), 1.38−1.23 (m,

4H), 0.89 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 155.71,

145.38, 132.33, 79.74, 54.92, 36.16, 29.43, 28.64, 28.37, 22.13, 13.88.

HRMS for C₁₃H₂₄N₄O₂Na [M + Na]⁺calcd 291.1791, found

291.1799.

30.3, 28.3, 25.7, 22.3, 13.7. HRMS for C₁₃H₂₂N₂O₃Na [M + Na]⁺

calcd 277.1523, found 277.1516.

5.9.16.30. tert-Butyl ((3-Hexylisoxazol-5-yl)methyl)carbamate

(12g). Compound 12g was prepared from N-Boc-propargylamine

(1.50 mmol) using general protocol 4. Yield: 72%, R_f= 0.30 (20%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 6.00 (s, 1H), 5.05

(bs, 1H), 4.36 (d, J = 5.8 Hz, 2H), 2.60 (t, J = 7.7 Hz, 2H), 1.57−1.65

(m, 2H), 1.43 (s, 9H), 1.27−1.34 (m, 6H), 0.86 (t, J = 7.1 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 169.1, 164.3, 155.5, 101.4, 80.2, 36.6,

5.9.16.38. tert-Butyl ((5-Pentyl-4H-1,2,4-triazol-3-yl)methyl)-

carbamate (19). Compound 19 was prepared from N-Boc-

glycinamide (2.18 mmol) as described. Yield: 52%, R_f= 0.15 (60%

1

EtOAc in hexanes). H NMR (500 MHz, CDCl₃) δ 5.42 (bs, 1H),

4.40 (d, J = 5.7 Hz, 2H), 2.74 (t, J = 7.7 Hz, 2H), 1.75 (m, 2H), 1.44

(s, 9H), 1.37−1.32 (m, 4H), 0.89 (t, J = 7.1 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 160.79, 158.20, 156.50, 80.20, 37.67, 31.56, 28.47,

27.88, 27.36, 22.44, 14.05. HRMS for C₁₃H₂₅N₄O₂[M + H]⁺calcd

269.19720, found 269.19651.

5.9.16.39. tert-Butyl ((5-Pentyl-1,3,4-thiadiazol-2-yl)methyl)-

carbamate (21). Compound 21 was prepared from N-Boc glycine

(1.14 mmol) as described. Yield: 60%, R_f= 0.38 (50% EtOAc in

hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.27 (bs, 1H), 4.67 (d, J =

6.1 Hz, 2H), 3.06 (t, J = 7.6, 2H), 1.78 (m, 2H), 1.46 (s, 9H), 1.41−

1.32 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)

δ 172.11, 167.87, 155.71, 80.65, 39.91, 31.26, 30.24, 29.86, 28.46,

22.39, 14.02. HRMS for C₁₃H₂₃N₃O₂SNa [M + Na]⁺calcd 308.1403,

found 308.1401.

31.5, 28.8, 28.3, 28.2, 26.0, 22.5, 14.0. HRMS for C₁₅H₂₆N₂O₃Na [M

+ Na]⁺calcd 305.1835, found 305.1829.

5.9.16.31. tert-Butyl ((3-Phenethylisoxazol-5-yl)methyl)-

carbamate (12h). Compound 12h was prepared from N-Boc-

propargylamine (1.50 mmol) using general protocol 4. Yield: 27%, R_f

1

= 0.35 (20% EtOAc in hexanes). H NMR (500 MHz, CDCl₃) δ

7.33−7.30 (m, 2H), 7.26−7.21 (m, 3H), 5.99 (s, 1H), 4.99 (bs, 1H),

4.40 (d, J = 5.6 Hz, 2H), 3.01−2.97 (m, 4H), 1.48 (s, 9H). ¹³C NMR

(126 MHz, CDCl₃) δ 169.3, 163.4, 155.5, 140.6, 128.5, 128.3, 126.3,

101.6, 80.3, 36.6, 34.3, 28.3, 27.9. HRMS for C₁₇H₂₂N₂O₃Na [M +

Na]⁺calcd 325.1523, found 325.1520.

5.9.16.32. tert-Butyl (2-(3-Pentylisoxazol-5-yl)ethyl)carbamate

(12i). Compound 12i was prepared from N-Boc-3-butynylamine

(2.20 mmol) using general protocol 4. Yield: 72%, R_f= 0.33 (20%

EtOAc in hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.92 (s, 1H), 4.75

5.9.16.40. tert-Butyl ((5-Pentyl-1,3,4-oxadiazol-2-yl)methyl)-

carbamate (22). Compound 22 was prepared from N-Boc glycine

(0.88 mmol) as described. Yield: 31%, R_f= 0.41 (50% EtOAc in

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J. Med. Chem. 2021, 64, 4478−4497

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Article

hexanes). ¹H NMR (500 MHz, CDCl₃) δ 5.10 (bs, 1H), 4.52 (d, J =

5.1 Hz, 2H), 2.82 (t, J = 7.6, 2H), 1.78 (m, 2H), 1.46 (s, 9H), 1.39−

1.33 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)

δ 167.89, 163.98, 155.53, 80.70, 36.01, 31.25, 28.43, 26.24, 25.44,

22.31, 13.98. HRMS for C₁₃H₂₃N₃O₃Na [M + Na]⁺calcd 292.16316,

found 292.16219.

AUTHOR INFORMATION

Corresponding Authors

■

Kevin J. Saliba − Research School of Biology and Medical

School, The Australian National University, Acton, ACT

2601, Australia; orcid.org/0000-0003-3345-8440;

Email: kevin.saliba@anu.edu.au

Karine Auclair − Department of Chemistry, McGill University,

Montreal, QC H3A 0B8, Canada; orcid.org/0000-0003-

0933-7956; Email: k.auclair@mcgill.ca

5.9.16.41. tert-Butyl ((3-Pentyl-1,2,4-oxadiazol-5-yl)methyl)-

carbamate (24r). Compound 24r was prepared from hexanenitrile

(2.06 mmol) using general protocol 5. Yield: 36%, R_f= 0.57 (40%

1

EtOAc in hexanes). H NMR (500 MHz, CDCl₃) δ 5.13 (bs, 1H),

4.55 (d, J = 5.3 Hz, 2H), 2.71 (t, J = 7.7, 2H), 1.75 (m, 2H), 1.46 (s,

9H), 1.37−1.32 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 176.18, 170.89, 155.50, 80.81, 37.29, 31.39, 28.42,

26.75, 26.04, 22.41, 14.05. HRMS for C₁₃H₂₃N₃O₃Na [M + Na]⁺

calcd 292.1632, found 292.1639.

5.9.16.42. tert-Butyl ((5-Pentyl-1,2,4-oxadiazol-3-yl)methyl)-

carbamate (24s). Compound 24s was prepared from N-(tert-

butoxycarbonyl)-2-aminoacetonitrile (1.92 mmol) using general

protocol 5. Yield: 45%, R_f= 0.70 (50% EtOAc in hexanes). ¹H

NMR (500 MHz, CDCl₃) δ 5.12 (bs, 1H), 4.44 (d, J = 5.1 Hz, 2H),

2.86 (t, J = 7.6 Hz, 2H), 1.80 (m, 2H), 1.45 (s, 9H), 1.39−1.31 (m,

4H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 180.72,

168.00, 155.67, 80.27, 37.00, 31.25, 28.46, 26.65, 26.34, 22.28, 13.96.

HRMS for C₁₃H₂₃N₃O₃Na [M + Na]⁺calcd 292.1632, found

292.1641.

5.9.16.43. tert-Butyl ((2-Pentyloxazol-5-yl)methyl)carbamate

(25t). Compound 25t was prepared from N-Boc-propargylamine

(0.38 mmol) using general protocol 6. Yield: 36%, R_f= 0.39 (40%

EtOAc in hexanes). ¹H NMR (400 MHz, CDCl₃) δ 6.80 (s, 1H), 4.81

(bs, 1H), 4.30 (d, J = 5.2 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H), 1.74 (m,

2H), 1.45 (s, 9H), 1.37−1.31 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H). ¹³C

NMR (126 MHz, CDCl₃) δ 165.15, 155.56, 148.55, 123.94, 80.15,

35.70, 31.43, 28.49, 28.30, 26.77, 22.42, 14.06. HRMS for

C₁₄H₂₄N₂O₃Na [M + Na]⁺calcd 291.1679, found 291.1666.

5.9.16.44. tert-Butyl ((5-Pentyloxazol-2-yl)methyl)carbamate

(25u). Compound 25u was prepared from 1-heptyne (0.72 mmol)

using general protocol 6. Yield: 21%, R_f= 0.56 (50% EtOAc in

hexanes). ¹H NMR (500 MHz, CDCl₃) δ 6.64 (s, 1H), 5.11 (bs, 1H),

4.39 (d, J = 5.0 Hz, 2H), 2.60 (t, J = 7.4 Hz, 2H), 1.61 (m, 2H), 1.46

(s, 9H), 1.36−1.30 (m, 4H), 0.90 (t, J = 7.0 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 159.94, 155.70, 153.78, 122.09, 80.15, 38.27, 31.36,

28.47, 27.29, 25.58, 22.44, 14.08. HRMS for C₁₄H₂₄N₂O₃Na [M +

Na]⁺calcd 291.1679, found 291.1669.

Authors

Jinming Guan − Department of Chemistry, McGill University,

Montreal, QC H3A 0B8, Canada

Christina Spry − Research School of Biology, The Australian

National University, Acton, ACT 2601, Australia;

orcid.org/0000-0002-8156-7070

Erick T. Tjhin − Research School of Biology, The Australian

National University, Acton, ACT 2601, Australia;

orcid.org/0000-0003-1711-0815

Penghui Yang − Department of Chemistry, McGill University,

Montreal, QC H3A 0B8, Canada; College of Chemistry and

Chemical Engineering, Xi’an Shiyou University, Xi’an

710065, China

Tanakorn Kittikool − Department of Chemistry, McGill

University, Montreal, QC H3A 0B8, Canada

Vanessa M. Howieson − Research School of Biology, The

Australian National University, Acton, ACT 2601,

Australia; orcid.org/0000-0001-9052-2037

Harriet Ling − Research School of Biology, The Australian

National University, Acton, ACT 2601, Australia

Lora Starrs − John Curtin School of Medical Research, The

Australian National University, Acton, ACT 2601, Australia

Dustin Duncan − Department of Chemistry, McGill

University, Montreal, QC H3A 0B8, Canada

Gaetan Burgio − John Curtin School of Medical Research, The

Australian National University, Acton, ACT 2601,

Australia; orcid.org/0000-0002-7434-926X

Complete contact information is available at:

5.9.16.45. 5-Pentylthiophene-2-carbaldehyde (26). Compound

26 was prepared from 2-pentyl thiophene (2.50 mmol) as described.

Yield: 82%. The characterization data is as previously reported.⁶⁰¹H

NMR (500 MHz, CDCl₃) δ 9.84 (s, 1H), 7.63 (d, J = 3.75 Hz, 1H),

6.93 (d, J = 3.75 Hz, 1H), 2.89 (t, J = 7.70 Hz, 2H), 1.71−1.77 (m,

2H), 1.36−1.39 (m, 4H), 0.93 (t, J = 7.05 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 182.66, 157.80, 141.60, 137.02, 125.83, 31.17, 30.96,

30.82, 22.33, 13.94.

https://pubs.acs.org/10.1021/acs.jmedchem.0c01755

Author Contributions

^††J.G., C.S., and E.T.T. contributed equally to the work. The

manuscript was drafted by J.G. and C.S., and all authors

contributed to editing and ﬁnalizing the document. We

conﬁrm that all authors have approved submission of the

ﬁnal version of this manuscript.

5.9.16.46. 5-Pentylthiophene-2-carboxamide (27). Compound

1

27 was prepared from 26 (1.50 mmol) as described. Yield: 52%. H

Funding

NMR (500 MHz, CDCl₃) δ 7.39 (d, J = 3.7 Hz, 1H), 6.80 (d, J = 3.7

Hz, 1H), 5.66 (bs, 2H), 2.84 (t, J = 7.6 Hz, 2H), 1.68−1.74 (m, 2H),

1.35−1.39 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz,

CDCl₃) δ 163.66, 152.59, 134.59, 129.52, 125.05, 31.16, 31.13, 30.36,

22.36, 13.96. HRMS for C₁₀H₁₆NOS [M + H]⁺calcd 198.0947, found

198.0952.

This work was ﬁnancially supported by grants from the

Australian National Health and Medical Research Council (to

K.J.S. and K.A.; APP1129843) and the Canadian Institute of

Health Research (to K.A.; grant no. 89784).

Notes

The authors declare no competing ﬁnancial interest.

ASSOCIATED CONTENT

■

ACKNOWLEDGMENTS

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01755.

The China Scholarship Council is thanked for providing a

scholarship to J.G. The Australian Government provided

scholarships for E.T.T. and V.M.H., whereas C.S. was funded

by an NHMRC Overseas Biomedical Fellowship (1016357).

The authors thank the Centre for Drug Candidate

Optimisation, Monash Institute of Pharmaceutical Sciences,

Monash University, for ADME analyses. They are also grateful

Biological results, purity studies, and NMR spectra

(PDF)

SMILES (CSV)

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Sivonen, K.; Li, X. X.; Auclair, K.; Saliba, K. J. Triazole substitution of

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antiplasmodial potency. Antimicrob. Agents Chemother. 2016,

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bacterial activity. Bioorg. Med. Chem. 2011, 19, 2696−2706.

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Developing pantetheinase-resistant pantothenamide antibacterials:

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to the Canberra branch of the Australian Red Cross Blood

Service for providing red blood cells and human serum.

ABBREVIATIONS

■

ACN, acetonitrile; ADME, absorption, distribution metabo-

lism, and excretion; CDI, carbonyldiimidazole; CoA, coenzyme

A; DCM, dichloromethane; DMF, N,N-dimethylformamide;

DHA, dihydroartemisin; EDAC, N-(3-dimethylaminopropyl)-

N’-ethylcarbodiimide hydrochloride; EEDQ, 2-ethoxy-1-ethox-

ycarbonyl-1,2-dihydroquinoline; HFF, human foreskin ﬁbro-

blast; SAR, structure−activity relationship; TEA, triethylamine;

TFA, triﬂuoroacetic acid; THF, tetrahydrofuran

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Exploring Heteroaromatic Rings as a Replacement for the Labile Amide of Antiplasmodial Pantothenamides

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