Discovery of Potent and Fast-Acting Antimalarial Bis-1,2,4-triazines

Article abstract of DOI:10.1021/acs.jmedchem.1c00044

Novel 3,3′-disubstituted-5,5′-bi(1,2,4-triazine) compounds with potent in vitro activity against Plasmodium falciparum parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine 23, which exhibited significantly improved in vitro metabolic stability (8 and 42 μL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50 value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.

Full text of DOI:10.1021/acs.jmedchem.1c00044

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Article
Discovery of Potent and Fast-Acting Antimalarial Bis-1,2,4-triazines
Daniel L. Priebbenow, Mitch Mathiew, Da-Hua Shi, Jitendra R. Harjani, Julia G. Beveridge,
Marina Chavchich, Michael D. Edstein, Sandra Duffy, Vicky M. Avery, Robert T. Jacobs, Stephen Brand,
David M. Shackleford, Wen Wang, Longjin Zhong, Given Lee, Erin Tay, Helena Barker, Elly Crighton,
Karen L. White, Susan A. Charman, Amanda De Paoli, Darren J. Creek, and Jonathan B. Baell*
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ABSTRACT: Novel 3,3′-disubstituted-5,5′-bi(1,2,4-triazine) com-
pounds with potent in vitro activity against Plasmodium falciparum
parasites were recently discovered. To improve the pharmacoki-
netic properties of the triazine derivatives, a new structure−activity
relationship (SAR) investigation was initiated with a focus on
enhancing the metabolic stability of lead compounds. These efforts
led to the identification of second-generation highly potent
antimalarial bis-triazines, exemplified by triazine 23, which
exhibited significantly improved in vitro metabolic stability (8
and 42 μL/min/mg protein in human and mouse liver micro-
somes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50
value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of
considerable interest.
INTRODUCTION
■
With over 200 million cases resulting in 409 000 deaths
worldwide in 2019, malaria continues to have a significant
negative impact on communities across Africa and South East
Asia.1 The human form of malaria is predominantly caused by
Figure 1. 1,2,4-bistriazine identified as highly active against Pf in
previous studies.
the Plasmodium falciparum (Pf) parasite that infects red blood
cells (RBCs) and when left untreated can lead to death.2
Unfortunately, the emergence of Pf strains that are resistant to
existing antimalarial therapeutics has exacerbated the impact of
this devastating disease.3
The complexity of the malaria parasite lifecycle ensures that
the discovery and development of new preventions and
treatments remains a challenge.2 This was exemplified in a
recent review that highlighted the challenges associated with
the discovery and development of four new small molecule
antimalarial therapeutic agents (DSM265,4−8 KAE609,9−11
KAF156,12,13 and MMV04814−17).18
Despite exciting progress in the development of new
therapeutic strategies, there remains an ongoing need to
identify new antimalarial agents that act via a novel mode of
action.18 To this end, we recently reported the discovery of
3,3′-disubstituted-5,5′-bi(1,2,4-triazine) (1), active against Pf
with single-digit nanomolar activity and several thousand-fold
lower toxicity to mammalian cells, demonstrating excellent
selectivity (Figure 1).19
netic (PK) studies revealed rapid clearance and low exposure
of the parent compound in vivo, but excellent in vivo activity
was observed in the 4-day Peter’s test in the Plasmodium
berghei murine malaria model.20 The mechanism of action of
triazine 1 remains unknown; however, the unique structure
when compared with known antimalarial agents, as well as
equipotency against several resistant strains, suggests that
compounds of this type act at a novel biological target.
Previous investigations into this class of compounds revealed
that triazine 1 led to a rapid onset of action against all asexual
blood stages, resulting in the sustained suppression of
parasitaemia in vivo and demonstrated the inability to easily
select for resistance, which implied a fast-killing profile, all
Received: January 11, 2021
Published: March 24, 2021
Triazine 1 exhibited equipotent activity against chloroquine
and artemisinin-resistant laboratory strains of Pf and field
isolates of Pf and Plasmodium vivax. Subsequent pharmacoki-
https://doi.org/10.1021/acs.jmedchem.1c00044
© 2021 American Chemical Society
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highly attractive properties of antimalarial small molecule
therapeutics. To enable further development of these
compounds for the treatment of uncomplicated malaria, this
project involved a new structure−activity relationship (SAR)
study to interrogate the pharmacokinetic liabilities associated
with triazine 1, in particular the basis for rapid clearance.
improved pharmacokinetic properties. For the active com-
pounds previously identified (e.g., 1), multiple sites were
identified at which metabolic oxidation could occur, including
(i) the sulfur atom within the thioether motif; (ii) one of the
multiple heteroaryl nitrogen atoms; or (iii) the nitrogen within
the tethered amine group. To explore this further, initial
investigations focused on retaining the right hand side (RHS)
thioethylamine tether, and either (i) increasing the bulk
around the left hand side (LHS)-SMe motif to retard oxidation
at this site or (ii) replacing the thioether with a functional
group less susceptible to metabolic oxidation (Table 1).
RESULTS AND DISCUSSION
■
Chemistry. The monomers were synthesized as outlined in
Scheme 1 using well-established methods.19,21,22 Predom-
a
Scheme 1. Synthesis of Triazine Monomers
Table 1. SAR Exploring LHS Substitution within Thioether-
Derived 3,3′-Disubstituted-5,5′-Bi(1,2,4-triazine)s against
In Vitro Pf 3D7 Proliferation
a
compound
R1
3D7 IC50 (μM)
1
9
−SMe
−SiPr
0.0080
0.21
10
11
12
13
14
15
16
−S(CH2)2OMe
−OMe
−OnPr
0.26
0.85
0.011
0.081
0.013
0.12 0.085
0.0090 0.0004
−OiPr
a
Reagents and conditions: (a) RBr/NaI or RI, EtOH, 81 °C; (b) 40%
−OcyPr
−Et
aq glyoxal, NaHCO3, 0 °C then room temperature (rt); (c) RSH/
Na2CO3, MeCN, rt; (d) NaH/ROH or MeMgI/ROH, N,N-
dimethylformamide (DMF), rt.
−nBu
a
Values represent outcomes from one or two experiments (for two
experiments, the values represent the mean, with “ ” sign
representing the standard deviation (S.D.)) against Pf 3D7 strain,
erythrocyte stage (chloroquine control, half-maximal inhibitory
concentration (IC50) = 0.004 μM).23
inantly, 3-(alkylthio)-1,2,4-triazines 4 were accessed from S-
alkyl thiosemicarbizides 3 by employing a condensation
reaction with aqueous (aq) glyoxal. A condensation process
was also employed to access 3-alkyl-1,2,4-triazines 6 from the
corresponding hydrazonamides 5. Alternatively, 3-(alkylthio)-
1,2,4-triazines 4 could be prepared via the nucleophilic
substitution of 3-(methylsulfonyl)-1,2,4-triazines 7 with
mercaptans and 3-(alkoxy)-1,2,4-triazines 8 were prepared
using a similar strategy employing alkoxy nucleophiles derived
from alcohols.
To access the 3,3′-disubstituted-5,5′-bi(1,2,4-triazine) de-
rivatives, a cyanide-mediated dimerization process was
employed using equimolar mixtures of the requisite monomers
(Scheme 2). The use of structurally different monomers
typically afforded a product mixture comprising both the
heterodimer and the two possible homodimers (Scheme 2),
each of which was readily separated and subsequently
subjected to biochemical evaluation.
Using triazine 1 as a template, initial efforts to increase the
bulk of the thioether group to mitigate metabolism (if
occurring at this site) involved the synthesis of the isopropyl
(9, IC50 = 0.21 μM) and ethyl methyl ether (10, IC50 = 0.26
μM) derivatives; however, both substitution patterns led to a
significant decrease in antimalarial activity. O-Alkyl substitu-
tion at this position was then explored to mitigate the
oxidation of the thioether functionality, with the methoxy
analogue (11, IC50 = 0.85 μM) exhibiting relatively poorer
antimalarial activity when compared with the n-propoxy 12,
isopropoxy 13, and cyclopropoxy 14 derivatives, which all
showed very potent IC50 values of 0.011, 0.081, and 0.013 μM,
respectively, against the Pf 3D7 strain. Two additional
examples containing no heteroatoms within the LHS tether
were then prepared with the butyl analogue 16, exhibiting 13-
fold greater antimalarial activity (IC50 = 9.0 nM) than the
corresponding ethyl derivative (15, IC50 = 0.12 μM).
The overarching aim of this investigation was to gain
additional insight into the SAR of the triazine to discover new
compounds that were highly potent against Pf, yet possessed
Scheme 2. Cyanide-Mediated Dimerization Protocol was Employed to Prepare Both Hetero- and Homodimeric Triazines
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As the RHS thioethylamino chain (−S(CH2)2NMe2)
present in 1 was also previously identified as a possible site
of oxidation leading to poor metabolic stability, subsequent
efforts focused on retaining the SMe on LHS and varying the
RHS chain to remove the sulfur, amine, or both (Table 2).
Table 3. SAR Exploring LHS Substitution within Ether-
Derived 3,3′-Disubstituted-5,5′-Bi(1,2,4-triazine)s against
In Vitro Pf 3D7 Proliferation
Table 2. SAR Exploring RHS Substitution within Thioether-
Derived 3,3′-Disubstituted-5,5′-Bi(1,2,4-triazine)s against
In Vitro Pf 3D7 Proliferation
a
compound
R1
3D7 IC50 (μM)
19
22
23
24
25
26
−SMe
−SiPr
0.080
0.012
0.031 0.008
0.44 0.029
0.054 0.001
0.030 0.008
−OnPr
−OiPr
−O(CH2)2CF3
−nBu
a
compound
R2
3D7 IC50 (μM)
a
Values represent outcomes from one or two experiments (for two
17
18
19
20
21
−S−CH2CCH
0.032 0.017
0.0044 0.0002
0.080
0.032 0.083
0.13 0.030
experiments, the values represent the mean, with “ ” sign
representing the standard deviation) against Pf 3D7 strain,
erythrocyte stage (chloroquine control, IC50 = 0.004 μM).23
−S(CH2)3N3
−O(CH2)2NMe2
−OCH(Me)CH2NMe2
−O−CH2CCH
homodimeric analogues. To gain additional insight into SAR,
as a part of this study, these homodimers were screened for
activity with the results outlined in Table 4.
a
Values represent outcomes from one or two experiments (for two
experiments, the values represent the mean, with “ ” sign
representing the standard deviation) against Pf 3D7 strain,
erythrocyte stage (chloroquine control, IC50 = 0.004 μM).23
Table 4. SAR Exploring Homodimeric 3,3′-Disubstituted-
5,5′-Bi(1,2,4-triazine)s against In Vitro Pf 3D7 Proliferation
Substitution of the amino group with a propargyl group (17,
IC50 = 0.032 μM) led to a relatively active compound. The
activity was also retained when the sulfur atom was replaced
with an oxygen atom while keeping the tertiary amine tether
(19, IC50 = 0.080 μM and 20, IC50 = 0.032 μM). However,
when both the sulfur and amine components were modified
(21, IC50 = 0.13 μM), a 20-fold loss of potency was observed
in comparison to 1. Intriguingly, the replacement of the
ethylamino functionality with the propylazide afforded the
most potent triazine dimer 18, which exhibited exceptional
antimalarial activity against Pf 3D7, exhibiting an IC50 value of
4.4 nM.
Having shown that compound 19 (the oxygen analogue of
1) was relatively active against the 3D7 line, this compound
was used as the template for the next part of the investigation
(Table 3). In a manner similar to that outlined above, the RHS
oxyethylamine tether was kept constant and variations on the
LHS tether were explored.
Whereas increased bulk on the LHS thioether in the
previous series (Table 1) led to a loss of activity, for the oxy-
ethylamino derivatives changing from the methyl thioether
(19, IC50 = 0.080 μM) to the isopropyl thioether 22 on the
LHS increased activity with 22 exhibiting an excellent IC50
value of 0.012 μM against the Pf 3D7 cell line. The analogous
isopropoxy derivative (24, IC50 = 0.44 μM) was relatively
inactive; however, the n-propoxy compound 23 exhibited good
activity with an IC50 value of 0.031 μM. Removing the
heteroatom from this chain was also tolerated in the form of
the n-butyl derivative 26, which exhibited excellent activity at
0.030 μM. The trifluoromethyl derivative 25 possessing the
same chain length as n-propoxy analogue 23 also exhibited
good antimalarial activity (IC50 = 0.054 μM).
a
compound ID
R1 = R2
3D7 IC50 (μM)
27
28
29
30
31
32
33
34
35
36
37
38
39
−S(CH2)3N3
−nBu
0.031 0.002
0.34 0.043
0.11 0.014
2.0
−OnPr
−OiPr
−OcyPr
0.89
−O(CH2)2CF3
−O(CH2)2SiMe3
0.093 0.006
0.72 0.033
0.42 0.115
100%@40 μM
0.50 0.123
8.6 0.339
12 1.13
−OCH2CF3
−O(CH2)2OMe
−O−CH2CH2(4-methylthiazol-5-yl)
−O−(CH2)2NMe2
−O−CH(Me)CH2NMe2
−O−CH2CCH
1.0 0.111
a
Values represent outcomes from one or two experiments (for two
experiments, the values represent the mean, with “ ” sign
representing the standard deviation) against Pf 3D7 strain,
erythrocyte stage (chloroquine control, IC50 = 0.004 μM).23
We had previously observed that the symmetrical homo-
dimers containing thioether tethers were relatively active (up
to IC50 = 0.017 μM),19 whereas the bis(methoxy) triazine
dimer was inactive. For the series evaluated during this study, it
was observed that the thioethylazide dimer (27) was as potent
as some of the heterodimers, exhibiting an IC50 value of 0.031
μM. The n-propoxy (29) and corresponding trifluoromethyl
analogue (32) were also active against the Pf 3D7 cell line
(IC50 values of 0.11 and 0.093 μM, respectively); however, the
remainder of oxo-tethered homodimeric analogues screened
were all relatively inactive exhibiting values between 0.34 and
12 μM (Table 4, entries 4, 7−13).
A consequence of the synthetic strategy employed to access
the heterodimers discussed above (and those we have
previously reported19) was the formation of homodimers of
each monomer in the reaction system (refer to Scheme 2). As
such, across our research program, we accumulated a series of
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Table 5. Physicochemical Parameters and In Vitro Metabolism in Liver Microsomes of Selected Triazine Derivatives
partition
coefficient
b
solubility (μg/mL)
c
PSA
in vitro CLint (μL/min/mg protein)
in vitro degradation half-life (min)
a
cmpd
MW
(Å2)
(clog P)
pH 2.0
pH 6.5
[h/m]
[h/m]
1
309.41
291.38
319.43
305.34
359.31
303.36
82
82
82
100
100
91
1.0
0.4
1.3
0.5
0.1
0.7
6.3−12.5
50−100
50−100
>100
50−100
25−50
6.3−12.5
25−50
25−50
>100
50−100
25−50
15/138
9/118
56/330
8/42
7/33
25/109
113/13
199/15
31/5
229/42
232/53
69/16
15
16
23
25
26
a
b
c
Calculated using Chem Axon JChem software. Kinetic solubility determined by nephelometry. In vitro intrinsic clearance (CLint) determined in
human (h) or mouse (m) liver microsomes (HLM or MLM).
Figure 2. Major metabolites (based on peak area) detected in incubations with hepatic microsomes and cryopreserved hepatocytes. Metabolite M
+16 (III) appeared to be the main metabolite in both test systems based on the peak area. The dashed lines represent the region of the molecule
where oxidation is thought to occur; however, the exact site could not be confirmed.
Physicochemical Parameters and Predictive Absorp-
tion, Distribution, Metabolism, and Excretion (ADME).
The overarching aim of this investigation was to address the
relatively poor solubility and metabolic stability of the
previously identified lead compound. Five new compounds
with potent antimalarial activity against Pf were subjected to
the preliminary evaluation of drug-likeness with the results
compared to the previous lead compound 1 (Table 5).
As shown in Table 5, the molecular weight (MW) of each
compound was well below 500 g/mol, the topological polar
surface area (PSA) ranged from 82 to 100 Å2, which is within a
range that is likely to be conducive to good passive cellular
permeability24 and kinetic solubility in some cases was greater
than 100 μg/mL. Overall, the compounds evaluated
throughout this investigation demonstrated a much improved
solubility when compared with 1, predisposing these triazine
dimers favorably toward oral bioavailability.
To enable the further development of early leads,
compounds are ideally not rapidly metabolized, which would
improve the likelihood of good oral bioavailability and
reasonable in vivo half-life (T1/2). During this study, the
metabolic stability of selected triazines was evaluated initially
in human and mouse liver microsomes (referred to as HLM
and MLM, respectively) at 37°C (Table 5, see the Supporting
Information (SI) for methods). These data provide key
insights into the structural features of the 3,3′-disubstituted
triazine dimers that render such molecules less susceptible to
rapid hepatic metabolism. Selected compounds were also
evaluated using cryopreserved hepatocytes to determine the
possibility of metabolism by routes that are not present in the
microsomal fraction (see the SI for methods).
(IC50 = 0.12 μM). Extension of this alkyl chain to the n-butyl
group restored potency (triazine 16, IC50 = 0.009 μM);
however, this analogue was rapidly metabolized in both HLM
and MLM (Table 5). The ether version of this n-butyl
analogue 26, which exhibited an IC50 value of 0.030 μM
against Pf, was also more rapidly metabolized in both HLM
and MLM than triazine 1 (Table 5).
The best performing compounds identified from this
evaluation were the bis-ether triazine derivatives 23 and 25
(Table 5), which demonstrated potent antimalarial activity
with IC50 values of 0.031 and 0.054 μM, respectively, against
the Pf 3D7 strain. Inclusion of the terminal −CF3 group (25)
in place of the −CH3 group (23) offered no apparent
advantage, with triazine 23 exhibiting the best solubility and
metabolic stability in both HLM (CLint = 8 μL/min/mg
protein) and MLM (CLint = 42 μL/min/mg protein). The
CLint value was marginally higher in rat liver microsomes (66
μL/min/mg protein). These results represent a significant
improvement in microsomal stability for these second-
generation triazine dimers (in particular 23) with only a
minor loss in potency when compared to the previous lead
compound, triazine 1. The stability of 23 was also evaluated in
human, rat, and mouse cryopreserved hepatocytes, where the
in vitro CLint values were 3.6, 10, and 3.7 μL/min/106 cells,
respectively.
Metabolite predictions were conducted using the software
package Meteor Nexus (Lhasa Ltd., U.K.) and the predicted
plausible primary metabolites included three +16 oxygenation
metabolites (an amine oxide of a tertiary amine, hydroxylation
of a terminal methyl, hydroxylation of penultimate alkyl
methylene), oxidative N-demethylation, and oxidative O-
dealkylation, amongst others. Metabolite identification studies
with 23 were conducted using samples from incubations with
hepatic microsomes (see the SI for methods) with structure
confirmation supported by tandem mass spectrometry (MS/
Replacing the LHS thioether (−SMe) with an alkyl group
(−Et, triazine 15) resulted in an increase in microsomal
stability (HLM CLint = 9 μL/min/mg protein); however, this
modification also resulted in a considerable drop in potency
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MS) (collision-induced dissociation (CID)) spectral analysis
where possible. Three prominent oxygenated metabolites
(Figure 2, designated M+16 (I, II, and III)) were detected,
with M+16 (I and II) mainly forming in HLM and M+16 (III)
being the predominant metabolite across species based on the
peak area (authentic standards not available). Based on CID
spectra, M+16 (III) appears to be the N-oxide with M+16 (I
and II) being the terminal and penultimate hydroxylation sites.
Other metabolites included an N-demethylation product
(Figure 2, M−14) and O-dealkylation of the ethyl dimethyl-
amine tether (Figure 2, M−71); both primarily detected in
RLM and MLM but not in HLM. A further cleavage product
(M−69) was detected in all three species both in the absence
and presence of reduced nicotinamide adenine dinucleotide
phosphate (NADPH); however, the identity of this species has
not yet been determined. The metabolite profile was similar
when incubated with hepatocytes, with no conjugated
metabolites (e.g., glucuronides or sulfates) detected.
higher in microsomes than in hepatocytes, albeit that
transporter insufficiency in cryopreserved hepatocytes could
mask the true extent of intact hepatocyte metabolism.
In Vitro Parasite Reduction Ratio (PRR). Antimalarials
with a fast-killing profile are highly desirable as they reduce the
likelihood of the parasite developing resistance. Triazine 23
was evaluated against the P. falciparum 3D7A laboratory line in
the PRR model25 (two-point FACS) at a concentration of ten
times the IC50 value using artesunate, chloroquine, pyrimeth-
amine, and atovaquone for comparison, where it demonstrated
a fast-killing profile similar to that of both artesunate and
chloroquine (Figure 3).
Additional in vitro assays were conducted with 23 to
determine rat plasma protein binding (PPB), the blood-to-
plasma (B/P) partitioning ratio, blood and plasma stability,
Caco-2 permeability, and cytochrome (CYP) inhibition. The
results for these are summarized in Table 6 (see the SI for
Table 6. Predicted In Vivo Unbound Hepatic Intrinsic
Clearance (CLint,unb), Plasma Protein Binding (PPB),
Blood-to-Plasma (B/P) Partitioning Ratio, Caco-2
a
Permeability (Papp), and CYP Inhibition for 23
Figure 3. Percentage survival of P. falciparum 3D7A parasites after
treatment with 23 and standard antimalarials. Data points represent
the mean standard deviation (S.D.) (n = 3 replicates).
parameter
value
predicted in vivo CLint,unb (mL/min/kg)
microsomes [h/r/m]
hepatocytes [h/r/m]
PPB (% bound) [r]
B/P [r]
6.8/117/112
9.5/49/27
22
Activity against Drug-Resistant Strains. When the
antimalarial activity of 23 was assessed against a panel of
laboratory strains with mutations in recently identified targets
1.3
Papp (10−6 cm/s)
A − B
B − A
efflux ratio
40
46
1.1
26
3
using a standard H-incorporation growth inhibition assay,
23 was observed to be equipotent against all strains tested,
again indicating that the antimalarial activity is instigated via a
novel mechanism of action (Figure 4). The IC50 value for 23
obtained from this in vitro assay against NF54 was 0.021 μM.
Against the K1, 7G8, TM90C2B, RF12, MRA1240, and Dd2
lines, the IC50 value was determined to range from 0.023 to
CYP inhibition (IC50, μM)
CYP1A2, 2C9, 2C19, 2D6, 3A4 (midazolam 1′-
>20 for all
isoforms
hydroxylation), 3A4 (testosterone 6β-hydroxylation)
a
Where noted, values correspond to the human [h], rat [r], or mouse
[m] test systems.
methods). Plasma protein binding in rat plasma was low, B/P
indicated effective distribution into erythrocytes, and perme-
ability was high with no evidence of efflux. Ex vivo studies with
23 in rat and mouse blood indicated some instability, with 15
and 23% loss of parent, respectively, over a 4 h incubation.
There was no evidence of significant inhibition of the 5 major
CYP isoforms, suggesting a low likelihood of significant drug−
drug interactions of coadministered compounds that are
metabolized by these enzymes.
After scaling and correcting for compound binding, both
hepatic microsomes and hepatocytes predicted that 23 would
have a higher unbound hepatic intrinsic clearance (CLint,unb) in
rats and mice compared to humans (Table 6), although this
species difference was more marked in hepatocytes. There was
no evidence to suggest that 23 was subject to metabolism by
enzymes that were not present in the microsomal test system
(e.g., aldehyde oxidase, conjugative enzymes), as values for the
predicted unbound intrinsic clearance in rats and mice were
Figure 4. Antimalarial activity of triazine 23 against laboratory field
strains and laboratory-generated resistant strains.8,17,26−29 Data
represent the mean
range (n = 2 replicates). Compounds in
parentheses were used to generate the resistant strains.
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0.041 with a fold shift of 1.1−1.95 relative to its potency
against NF54, which is indicative of a lack of cross-resistance.
Dual Gamete Formation Assay (DGFA). Gametocytes
are quiescent in the human host and only undergo differ-
entiation into the active sexual stages upon insect blood
feeding and transfer into the mosquito midgut. The DGFA is a
convenient assay, which is highly predictive of the trans-
mission-blocking potential of compounds, and investigates the
“functional viability” of mature stage V gametocytes under-
going onward differentiation into male and female gametes.30
Intriguingly, early members of the triazine series previously
demonstrated activity against early-stage gametocytes but not
against mature gametocytes (stage V). In this study, triazine 23
was not particularly potent in the DGFA, compared to its
blood stage activity, exhibiting EC50 values of 1.2 and 2.9 μM
for male and female gametocytes, respectively. This would
suggest that at therapeutic (i.e., TCP-1, treatment) concen-
trations, the compound would have negligible transmission-
blocking potential. Compounds with good transmission
potential are generally considered as those that have
comparable potency in DGFA and blood stages, for example,
KAE609 and KAF156.31
Table 7. Pharmacokinetic Parameters for 23 in Male Swiss
Outbred Mice Following IV and Oral Administration
PO administration
parameter
IV administration
2.69
low dose
2.85
nd
high dose
calculated dose (mg/kg)
apparent t1/2 (h)
plasma Cmax (μM)
Tmax (h)
plasma AUC0‑inf (μM*h)
plasma CL (mL/min/kg)
plasma Vss (L/kg)
BA (%)
26.8
3.0
0.0820
1.0
a
b
0.20
0.0168
0.50
a
b
0.36
a
0.038
0.44
406
a
3.4
10
12
a
Values are approximate only as plasma concentrations were only
b
above the analytical limit of quantitation for 1 h postdose. The
terminal phase for the low dose per os (PO) group was not well-
defined; the terminal phase rate constant for the high dose PO group
was used to estimate AUC0‑inf
.
Building on these results, a pharmacokinetic study with 23
was then undertaken in Sprague Dawley rats with IV (3 mg/
kg) and oral (10 mg/kg) administration (see the SI for
methods). Plasma concentration versus time profiles are shown
in Figure 6, and pharmacokinetic parameters are presented in
Table 8.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Bromide (MTT) Assay to Assess Cytotoxicity. An MTT
assay was then conducted to assess the cytotoxicity of 23 (see
the SI for methods), which exhibited an EC50 value of 36 μM,
providing a “selectivity index” of >1000 (HEK CC50/3D7
IC50) and suggesting a low likelihood of interaction of 23 with
critical host cell metabolic processes.
Pharmacokinetic Properties. We then undertook mouse
and rat pharmacokinetic studies for the most promising
candidate, triazine 23 (see the SI for methods). As shown in
Figure 5 and Table 7, intravenous (IV) dosing of 23 to mice at
Figure 6. Plasma concentrations of 23 in male Sprague Dawley rats
following IV and oral administration.
Similar to the data in mice, measurable plasma concen-
trations after IV dosing to rats were only observed up to 2.5 h.
The estimated IV half-life was approximately 0.4 h and plasma
CL and Vss were both high. Given the blood-to-plasma ratio of
Figure 5. Plasma concentrations of 23 in male Swiss outbred mice
following IV and oral administration.
Table 8. Pharmacokinetic Parameters for 23 after IV (3 mg/
kg) and Oral (10 mg/kg) Administration to Male Sprague
Dawley Rats
2.7 mg/kg resulted in a rapid decline in plasma concentrations,
which were below the limit of quantitation by 2 h such that the
terminal phase was not well-defined (Figure 5). The estimated
plasma clearance was very high (∼400 mL/min/kg) and
considerably above hepatic blood flow in the mouse, suggestive
of extrahepatic clearance mechanisms. Given its very low
calculated Log D7.4 (−0.5), it was proposed that 23 might be
renally eliminated; however, urine was not collected following
dosing to mice.
Following oral administration at a low dose of 2.8 mg/kg, 23
was detected for 2 h after the dose was administered (Figure
5). After oral dosing at 27 mg/kg, 23 demonstrated plasma
concentrations below ∼30 mg/mL, an apparent half-life of ∼3
h, and a measurable but low oral bioavailability of 12% (Figure
5).
parameter
IV administration
3.0
PO administration
10
calculated dose (mg/kg)
apparent t1/2 (h)
plasma Cmax (μM)
Tmax (h)
plasma AUC0‑inf (μM*h)
plasma CL (mL/min/kg))
plasma Vss (L/kg)
BA (%)
a
b
0.39
nd
0.0894
1.3
a
0.658
a
nd
249
a
2.80
7.49
a
Values are approximate only as plasma concentrations were only
b
above the analytical limit of quantitation for 2.5 h postdose. Could
not calculate due to insufficient definition of the terminal elimination
phase.
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1.3, the apparent blood clearance (205 mL/min/kg) was
substantially higher than the nominal hepatic blood flow in the
rat (∼67.6 mL/min/kg), which again suggested that 23 is
subject to extrahepatic elimination. Renal elimination
represented a relatively minor in vivo clearance pathway with
less than 3% of the IV dose recovered unchanged in urine over
24 h postdose. Following oral administration to rats, maximum
plasma concentrations were observed at 0.25−2.5 h. The
apparent oral bioavailability (based on AUC0‑last) ranged from
5 to 10%.
Metabolite identification studies with selected plasma, urine,
and feces samples from the rat PK study were further analyzed
using high-resolution mass spectrometry (HRMS). Metabolite
screening was limited to the previously detected metabolites
(Figure 2) and selected phase II conjugation reactions (even
though these were not detected in vitro). Three oxygenation
products (M+16, Figure 2), a bis-oxygenation (M+32), and
demethylation (M−14) products were detected in urine and
fecal samples from both dosing routes. The major metabolite
based on the peak area, M+16 (III) (Figure 2), was also
detected in plasma samples. The previously unidentified
metabolite seen in vitro (M−69) was also detected at very
minor levels in the oral plasma samples.
In Vivo Evaluation of Efficacy: 4-day Peters’ Test in
the P. berghei-Rodent Model. The second-generation 23
was evaluated in the Peters 4-day blood schizonticidal test as
previously described.19 Following oral administration, the
mean ED50 and ED90 values of 23 were 1.85
0.37 and
4.10 0.13 mg/kg/day, respectively. In contrast to the
second-generation 23, the first-generation triazine 1 had ED50
and ED90 values of 1.47 0.01 and 3.44 0.40 mg/kg/day,
respectively. For comparison purposes, the mean ED50 values
of artesunate and chloroquine are 1.37 0.63 and 1.15 0.06
mg/kg/day, respectively. No adverse events, such as loss of
mobility or poor posture, were observed for 23 at the highest
dose tested of 32 mg/kg/day for 4 days. From this study, it can
be seen that despite its poor systemic exposure after oral
dosing, 23 displayed excellent oral efficacy in this rodent
malaria infection model.
In Vivo Evaluation of Efficacy: 4-day Peters’ Test in
the 3D7 Severe Combined Immunodeficient Mice
(SCID) Mouse Model. Triazine 23 was next evaluated for
its in vivo efficacy against P. falciparum in a 4-day model using
humanized NOD-SCID IL2Rγnull mice.32,33 These mice can be
engrafted daily with human erythrocytes as they are
immunodeficient. The mice are then inoculated with Pf
3D70087/N9, with the compound being administered for 4
consecutive days from the 3rd day at a once-daily oral dosing
of 50 mg/kg (Figure 7). It was observed that triazine 23
induced clearance of parasites from peripheral blood within 3
days of dosing.
Triazine 23 has high aqueous solubility (>100 μg/mL, Table
5), high permeability across Caco-2 cell monolayers (40 ×
10−6 cm/s, Table 6), and physicochemical parameters that are
within the ranges of compounds exhibiting good oral
absorption; therefore, it seems unlikely that bioavailability is
absorption limited. This is also consistent with the low fraction
(2%) of the oral dose recovered in feces as an intact parent
compound.
Additional mechanistic studies were conducted with 23 to
understand the basis for the very high in vivo clearance. First,
rats were administered 3 mg/kg 23 by intravenous infusion,
following a predose with the pan CYP inhibitor, 1-amino-
benzotriazine (ABT, see the SI for methods). Blood clearance
in the ABT-treated rats was comparable to that observed in
control animals (232 and 205 mL/min/kg, respectively),
suggesting that CYP-mediated metabolism is not a predom-
inant in vivo clearance process for 23 in rats. Second, the
potential for the elimination of 23 by direct biliary excretion
was evaluated in bile-cannulated rats. Only 0.1−0.2% of the IV
dose of 23 was recovered in bile, indicating that 23 was not
subject to biliary clearance to any meaningful extent. Further
studies are ongoing to understand the processes contributing
to the in vivo clearance of 23.
Figure 7. Efficacy of 23 against P. falciparum Pf3D70087/N9: Pf
acute in vivo model, 4-day test by Peters. The arrows indicate the days
of treatment (50 mg/kg PO) in the 4-day test by Peters. Values
correspond to the average parasitemia in the peripheral blood of n = 2
mice/group.
Overall, there remains a disconnect between the efficacy data
and the low exposure of parent 23, although the possibility of a
sex-based difference in exposure cannot be ruled out, as
efficacy was assessed in female mice, whereas pharmacokinetic
properties were defined in male mice. In terms of
pharmacokinetics, the low recovery of compounds in rat
urine and in bile suggests a minimal contribution of renal and
biliary elimination. It seems likely that the high-clearance
results from both hepatic metabolism and extrahepatic
elimination routes that are yet to be defined. In terms of
pharmacodynamics (PD), it cannot be ruled out that triazine
23 is fast-acting with a long duration of action even with rapid
systemic clearance and low exposure. Alternatively, bioactive
metabolites may contribute to this in vivo activity. We are
currently assembling a range of diverse variants of triazine 23
to determine the origins of the PK−PD disconnect.
The concentrations of triazine 23 in blood after oral
administration were measured in serial blood samples obtained
1, 2, 4, 6, and 24 h following the first dosing in all compound-
treated mice. Blood levels were measured at Swiss BioQuant
(Basel, Switzerland) by liquid chromatography (LC)-MS/MS.
Exposure of triazine 23 in this model was low with
concentrations in the range of 150−250 ng/mL for ∼6 h. By
24 h, concentrations were below the quantitation limit (Figure
8). The basis for the differences in exposure between the SCID
mouse study (50 mg/kg PO) and the PK study (27 mg/kg
PO) remains unclear.
CONCLUSIONS
■
The major limitations of our previously identified triazine 1
were poor PK properties (i.e., low exposure) and toxicity alerts
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(HPLC), 1200 series G1311A quaternary pump, 1200 series G1329A
thermostated autosampler, and 1200 series G1314B variable wave-
length detector. The conditions for liquid chromatography were:
reverse-phase HPLC analysis fitted with a Phenomenex Luna C8(2) 5
μm (50 mm × 4.6 mm) 100 Å column; column temperature, 30 °C;
injection volume, 5 μL; solvent, 99.9% acetonitrile, 0.1% formic acid;
gradient, 5−100% of solvent over 10 min; and detection, 254 nm. The
conditions for mass spectrometry were: a quadrupole ion source; ion
mode, multimode-ES; drying gas temp, 300 °C; vaporizer temper-
ature, 200 °C; capillary voltage, 2000 V (positive), 4000 V (negative);
scan range, 100−1000 m/z; step size, 0.1 s; acquisition time, 10 min;
or (ii) an Agilent UHPLC-MS 1260/6120 system with the following
technical information. Pump, 1260 Infinity G1312B binary pump;
autosampler, 1260 Infinity G1367E 1260 HiP ALS; and detector,
1290 Infinity G4212A 1290 DAD. LC conditions: reverse-phase
HPLC analysis; column, Poroshell 120 EC-C18 3.0 mm Å to 50 mm
2.7 μm; column temperature, 35 °C; injection volume, 1 μL; and flow
rate, 1 mL/min. Solvent A: 99.9% water and 0.1% formic acid. Solvent
B: 99.9% acetonitrile and 0.1% formic acid, gradient 5−100% of
solvent B in solvent A over 3.8 min. Gradient takes 4 min to get to
100% solvent B in solvent A, maintain for 3 min, and a further 3 min
to get back to the original 5% solvent B in solvent A. MS conditions:
an ion source, quadrupole; ion mode, atmospheric pressure
ionization-electrospray (API-ES); drying gas temp, 350 °C; capillary
voltage (V), 3000 (positive); capillary voltage (V), 3000 (negative);
scan 52 range, 100−1000; step size, 0.1 s; and acquisition time, 5 min.
HRMS. High-resolution MS was performed with an Agilent 6224
TOF LC/MS coupled to an Agilent 1290 Infinity LC. All data were
acquired, and reference mass corrected via a dual-spray electrospray
ionization (ESI) source. Each scan or data point on the total ion
chromatogram (TIC) is an average of 13 700 transients, producing a
spectrum every second. Mass spectra were obtained by averaging the
scans across each peak and subtracting the background from the first
10 s of the TIC. Acquisition was performed using the Agilent Mass
Hunter data acquisition software ver. B.05.00 build 5.0.5042.2, and
analysis was performed using Mass Hunter Qualitative Analysis ver.
B.05.00 build 5.0.519.13. Acquisition parameters: mode, ESI; drying
gas flow, 11 L/min; nebulizer pressure, 45 psi; and drying gas
temperature, 325 °C. Voltages: capillary, 4000 V; fragmentor, 160 V;
skimmer, 65 V; octapole RF, 750 V; scan range, 100−1500 m/z; and
positive ion mode internal reference ions, m/z 121.050873 and
922.009798. LC conditions: Agilent Zorbax SB-C18 Rapid Resolution
HT (2.1 mm Å to 50 mm, 1.8 mm column), 30 °C; sample (5 μL)
was eluted using a binary gradient (solvent A, 0.1% aq formic acid;
solvent B, 0.1% formic acid in acetonitrile; 5−100% B [3.5 min], 0.5
mL/min).
Interference Compounds. All final compounds have been
examined for the presence of substructures classified as Pan Assay
Interference Compounds (PAINS) using a KNIME workflow.34,35
General Synthetic Procedures. Triazines of three classes were
used in the cyanide-mediated dimerization process, namely, 3-
(alkylthio)-1,2,4-triazines, 3-(alkoxy)-1,2,4-triazines, and 3-(alkyl)-
1,2,4-triazines. The synthesis of each class has been previously
described by us or others in the literature. The synthesis of 3,3′-
disubstituted-5,5′-bi(1,2,4-triazine)s 9−39 was accomplished using
appropriate mixtures of 1,2,4-triazine monomers and potassium
cyanide for coupling reactions as described previously by Courcot
et al. and our group.19,21 While completing the synthesis of
heterodimers, homodimers of each monomer were also obtained.
Characterization data of the monomeric 1,2,4-triazines and dimeric
3,3′-disubstituted-5,5′-bi(1,2,4-triazine)s not previously described in
the literature are outlined below.
3-((3-Azidopropyl)thio)-1,2,4-triazine (4a). Isolated as a brown oil
in 77% yield. 1H NMR (400 MHz, CDCl3) δ 8.94 (d, J = 2.3 Hz, 1H),
8.38 (d, J = 2.3 Hz, 1H), 3.48 (t, J = 6.6 Hz, 2H), 3.34−3.30 (m, 2H),
2.07 (dd, J = 13.5, 6.6 Hz, 2H) ppm. 13C NMR (101 MHz, CDCl3) δ
174.0, 148.4, 145.6, 50.2, 28.5, 27.8 ppm. MS (m/z) 197.0 [M + H]+.
3-(Prop-2-yn-1-ylthio)-1,2,4-triazine (4b). Isolated as a brown oil
in 37% yield. 1H NMR (400 MHz, CDCl3) δ 8.98 (d, J = 2.4 Hz, 1H),
8.43 (d, J = 2.4 Hz, 1H), 4.01 (d, J = 2.6 Hz, 2H), 2.22 (t, J = 2.6 Hz,
Figure 8. Mean (n = 2) blood concentrations of 23 after oral
administration of 50 mg/kg to SCID mice.
associated with activated thioethers, which would limit the
progression of this compound. To address this limitation, new
SAR investigations led to the discovery of novel 3,3′-
disubstituted-5,5′-bi(1,2,4-triazine) derivatives with potent in
vitro activity against P. falciparum parasites. These second-
generation bis-triazines identified simple structural modifica-
tions that led to enhanced microsomal stability, good
solubility, and permeability while maintaining potency, which
is exemplified by triazine 23 with an IC50 value of 31 nM in Pf
3D7 parasites. However, these favorable in vitro properties did
not translate to an improved exposure profile in vivo and very
high clearance remains a key issue for this series.
Triazine 23 was also evaluated in the PRR model (two-point
FACS), where it demonstrated a fast-killing profile similar to
that of both artesunate and chloroquine, and when assessed
against a panel of laboratory strains with mutations in recently
identified targets, 23 was observed to be equipotent against all
strains tested, indicating that this substrate is acting via a novel
mechanism of action. Furthermore, in the 4-day Peters test in
the 3D7 SCID mouse model, it was observed that triazine 23
induced clearance of parasites from peripheral blood within 3
days of dosing.
The improved microsomal stability of the new compounds
with comparable oral efficacy to 1 despite lower in vitro
potency and poor systemic exposure is suggestive that this class
of orally active antimalarial bis-triazines can be further
optimized to improve the bioavailability, to afford a promising
preclinical candidate, and we will report on these efforts in due
course.
EXPERIMENTAL SECTION
■
Chemistry. General Experimental Methods. For all synthetic
reactions, commercially available reagents were used without further
purification. Column chromatography was performed using silica gel
(40−60 μm). The solvents utilized for chromatography were used as
supplied without purification. Predominantly, the reactions were
monitored by thin-layer chromatography (TLC) on Silica Gel 60F‑254
plates with detection by UV light and/or KMnO4 stain.
Purity. The purity of all compounds subjected for biological
testing was >95%, as determined using the methods described below.
1
NMR. H and 13C NMR spectra were recorded at 400 and 101
MHz, respectively, on a Bruker Avance III Nanobay spectrometer.
NMR solvents were obtained from Cambridge Isotope Laboratories.
Chemical shifts (δ, ppm) are reported relative to the solvent peak
(CDCl3), 7.26 ppm [1H] or 77.16 ppm [13C]. Proton resonances are
assigned as chemical shift (δ), multiplicity (s, singlet; d, doublet; m,
multiplet), coupling constant (J, Hz), and the number of protons.
LC/MS. Low-resolution mass spectrometry analyses were per-
formed on either (i) Agilent 6100 series single quad LC/MS coupled
with an Agilent 1200 series high-performance liquid chromatography
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1H) ppm. 13C NMR (101 MHz, CDCl3) δ 172.8, 148.4, 145.9, 78.6,
71.4, 19.3 ppm. MS (m/z) 152.0 [M + H]+.
3-(Methylthio)-3′-(prop-2-yn-1-ylthio)-5,5′-bi(1,2,4-triazine)
(17). Prepared according to General Method A. The title compound
was isolated as a yellow solid in 22% yield. H NMR (400 MHz,
1
3-Butyl-1,2,4-triazine (6a). Isolated as a yellow oil in 70% yield. 1H
NMR (400 MHz, CDCl3) δ 9.09−9.08 (m, 1H), 8.54−8.53 (m, 1H),
3.14−3.09 (m, 2H), 1.88−1.80 (m, 2H), 1.44−1.37 (m, 2H), 0.96−
0.91 (m, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 171.0, 148.8,
147.6, 37.2, 30.6, 22.5, 13.9 ppm. MS (m/z) 138.3 [M + H]+.
General Method A. Solid KCN (3.7 equiv) was added to a solution
of monomer A (1.0 equiv) and monomer B (1.0 equiv) in water (10
mL) and the solution was stirred at 40 °C for 1.5 h. The aqueous
solution was then extracted with diethyl ether (3 × 50 mL). The
combined ether phases were dried over anhydrous MgSO4 and the
solvent was evaporated. The residue was purified by column
chromatography on silica gel to afford the desired compounds.
2-((3′-(Isopropylthio)-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-N,N-dime-
thylethanamine (9). Prepared according to General Method A. The
CDCl3) δ 9.95 (s, 1H), 9.93 (s, 1H), 4.08 (d, J = 2.6 Hz, 2H), 2.77 (s,
3H), 2.25 (t, J = 2.6 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3) δ
174.6, 172.7, 150.4, 149.8, 142.6, 142.2, 78.2, 71.7, 19.7, 14.2 ppm.
MS (m/z) 277.1 [M + H]+, HRMS (m/z) calcd C10H8N6S2 [M + H]+
277.0325; found 277.0329. MP: 125−127 °C.
3-((3-Azidopropyl)thio)-3′-(methylthio)-5,5′-bi(1,2,4-triazine)
(18). Prepared according to General Method A. The title compound
1
was isolated as a yellow solid in 34% yield. H NMR (400 MHz,
CDCl3) δ 9.91 (s, 1H), 9.90 (s, 1H), 3.54 (t, J = 6.3 Hz, 2H), 3.44 (t,
J = 7.0 Hz, 2H), 2.77 (s, 3H), 2.27−1.99 (m, 2H) ppm. 13C NMR
(101 MHz, CDCl3) δ 174.6, 173.8, 150.3, 149.9, 142.4, 142.1, 50.2,
28.4, 28.1, 14.2 ppm. MS (m/z) 322.1 [M + H]+, HRMS (m/z) calcd
C10H11N9S2 [M + H]+ 322.0652; found 322.0655. MP: 67−69 °C.
N,N-Dimethyl-2-((3′-(methylthio)-[5,5′-bi(1,2,4-triazin)]-3-yl)-
oxy)propan-1-amine (20). Prepared according to General Method A.
1
title compound was isolated as a brown solid in 28% yield. H NMR
(400 MHz, CDCl3) δ 9.84 (s, 1H), 9.83 (s, 1H), 4.28−4.04 (m, 1H),
3.48 (t, J = 7.0 Hz, 2H), 2.75 (t, J = 7.0 Hz, 2H), 2.34 (s, 6H), 1.52
(d, J = 6.8 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ 174.5,
174.2, 150.1, 145.0, 142.1, 141.8, 57.7, 45.3, 36.4, 29.0, 22.7 ppm. MS
(m/z) 338.2 [M + H]+. MP: 98.9−101.8 °C.
1
The title compound was isolated as a yellow solid in 34% yield. H
NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 9.88 (s, 1H), 5.70 (m, 1H),
2.85 (dd, J = 13.1, 7.9 Hz, 1H), 2.75 (s, 3H), 2.48 (dd, J = 13.1, 4.3
Hz, 1H), 2.30 (s, 6H), 1.49 (d, J = 6.2 Hz, 3H) ppm. 13C NMR (101
MHz, CDCl3) δ 174.5, 165.2, 152.9, 150.1, 142.2, 141.5, 74.0, 64.6,
46.3, 18.2, 14.1 ppm. MS (m/z) 308.1 [M + H]+, HRMS (m/z) calcd
C12H17N7OS [M + H]+ 308.1288; found 308.1277.
2-((3′-((2-Methoxyethyl)thio)-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-
N,N-dimethylethanamine (10). Prepared according to General
Method A. The title compound was isolated as a brown oil in 4%
yield. 1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 9.87 (s, 1H), 3.78
(t, J = 6.2 Hz, 2H), 3.58 (t, J = 6.2 Hz, 2H), 3.52 (t, J = 7.0 Hz, 2H),
3.43 (s, 3H), 2.80 (t, J = 7.0 Hz, 2H), 2.38 (s, 6H) ppm. 13C NMR
(101 MHz, CDCl3) δ 173.2, 172.9, 149.0, 148.9, 141.2, 141.1, 69.3,
57.9, 56.6, 44.2, 29.6, 27.9 ppm. MS (m/z) 354.1 [M + H]+.
2-((3′-Methoxy-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-N,N-dimethyle-
thanamine (11). Prepared according to General Method A. The title
3-(Methylthio)-3′-(prop-2-yn-1-yloxy)-5,5′-bi(1,2,4-triazine) (21).
Prepared according to General Method A. The title compound was
isolated as a yellow solid in 41% yield. 1H NMR (400 MHz, CDCl3) δ
10.00 (s, 1H), 9.91 (s, 1H), 5.29 (d, J = 2.1 Hz, 2H), 2.76 (s, 1H),
2.59 (s, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 174.6, 164.4,
153.1, 149.8, 142.4, 142.2, 76.9, 76.4, 56.8, 14.2 ppm. MS (m/z)
261.1 [M + H]+, HRMS (m/z) calcd C10H8N6OS [M + H]+
260.0475; found 260.0465. MP: 116−118 °C.
1
compound was isolated as a brown oil in 7% yield. H NMR (400
2-((3′-(Isopropylthio)-[5,5′-bi(1,2,4-triazin)]-3-yl)oxy)-N,N-dime-
MHz, CDCl3) δ 9.91 (s, 1H), 9.89 (s, 1H), 4.30 (s, 3H), 3.49 (t, J =
7.0 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 2.35 (s, 6H) ppm. 13C NMR
(101 MHz, CDCl3) δ 174.2, 165.5, 152.8, 145.0, 142.1, 141.7, 56.5,
56.5, 45.3, 28.9 ppm. MS (m/z) 294.2 [M + H]+.
thylethanamine (22). Prepared according to General Method A. The
1
title compound was isolated as a brown solid in 7% yield. H NMR
(400 MHz, CDCl3) δ 9.90 (s, 1H), 9.85 (s, 1H), 4.77 (t, J = 5.6 Hz,
2H), 4.14 (m, 1H), 2.90 (t, J = 5.6 Hz, 2H), 2.39 (s, 6H), 1.51 (d, J =
6.8 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ 174.5, 165.1,
153.1, 149.9, 141.9, 141.8, 66.8, 57.5, 45.7, 36.5, 22.7 ppm. MS (m/z)
322.2 [M + H]+. MP: 63.4−67.9 °C.
N,N-Dimethyl-2-((3′-propoxy-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-
ethanamine (12). Prepared according to General Method A. The title
compound was isolated as a brown solid in 17% yield. 1H NMR (400
MHz, CDCl3) δ 9.91 (s, 1H), 9.90 (s, 1H) 4.62 (t, J = 6.7 Hz, 2H),
3.49 (t, J = 7.0 Hz, 2H), 2.76 (t, J = 7.0 Hz, 2H), 2.35 (s, 6H), 2.06−
1.89 (m, 2H), 1.11 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (101 MHz,
CDCl3) δ 174.2, 165.3, 152.7, 150.1, 142.2, 141.5, 71.2, 57.7, 45.3,
29.0, 22.1, 10.4 ppm. MS (m/z) 322.2 [M + H]+. MP: 80.9−85.9 °C.
2-((3′-Isopropoxy-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-N,N-dimethy-
lethanamine (13). Prepared according to General Method A. The
N,N-Dimethyl-2-((3′-propoxy-[5,5′-bi(1,2,4-triazin)]-3-yl)oxy)-
ethanamine (23). Prepared according to General Method A. The title
compound was isolated as a yellow solid in 19% yield. 1H NMR (400
MHz, CDCl3) δ 9.94 (s, 1H), 9.92 (s, 1H), 4.76 (t, J = 5.6 Hz, 2H),
4.61 (t, J = 6.7 Hz, 2H), 2.86 (t, J = 5.6 Hz, 2H), 2.36 (s, 6H), 2.08−
1.82 (m, 2H), 1.10 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (101 MHz,
CDCl3) δ 165.3, 165.2, 153.1, 152.8, 141.9, 141.7, 71.3, 67.2, 57.8,
45.9, 22.2, 10.5 ppm. MS (m/z) 306.2 [M + H]+. MP: 47−49 °C.
2-((3′-Isopropoxy-[5,5′-bi(1,2,4-triazin)]-3-yl)oxy)-N,N-dimethy-
lethanamine (24). Prepared according to General Method A. The
1
title compound was isolated as a brown solid in 3% yield. H NMR
(400 MHz, CDCl3) δ 9.89 (s, 1H), 9.89 (s, 1H), 5.71−5.54 (m, 1H),
3.51 (t, J = 7.1 Hz, 2H), 2.80 (t, J = 7.1 Hz, 2H), 2.38 (s, 6H), 1.53
(d, J = 6.2 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ 174.1,
164.8, 152.7, 150.2, 142.2, 141.2, 73.2, 57.6, 45.2, 28.8, 21.7 ppm. MS
(m/z) 322.2 [M + H]+. MP: 60.1−62.1 °C.
2-((3′-Ethyl-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-N,N-dimethylethan-
amine (15). Prepared according to General Method A. The title
compound was isolated as a yellow solid in 15% yield. 1H NMR (400
MHz, CDCl3) δ 10.08 (s, 1H), 9.96 (s, 1H), 3.50 (t, J = 6.8 Hz, 2H),
3.31 (dd, J = 14.8, 7.4 Hz, 2H), 2.76 (t, J = 6.7 Hz, 2H), 2.35 (s, 6H),
1.51 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 174.3,
171.3, 150.6, 150.5, 144.5, 142.3, 57.9, 45.4, 30.7, 29.2, 12.4 ppm. MS
(m/z) 292.2 [M + H]+. MP: 87−89 °C.
2-((3′-Butyl-[5,5′-bi(1,2,4-triazin)]-3-yl)thio)-N,N-dimethylethan-
amine (16). Prepared according to General Method A. The title
compound was isolated as a yellow solid in 28% yield. 1H NMR (400
MHz, CDCl3) δ 10.07 (s, 1H), 9.95 (s, 1H), 3.50 (t, J = 7.0 Hz, 2H),
3.38−3.17 (m, 2H), 2.77 (t, J = 7.0 Hz, 2H), 2.35 (s, 6H), 2.05−1.84
(m, 2H), 1.58−1.36 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H) ppm. 13C NMR
(101 MHz, CDCl3) δ 174.3, 170.7, 150.6, 150.5, 144.4, 142.3, 57.9,
45.4, 37.0, 30.5, 29.1, 22.5, 13.9 ppm. MS (m/z) 320.3 [M + H]+.
MP: 51−53 °C.
1
title compound was isolated as a yellow solid in 27% yield. H NMR
(400 MHz, CDCl3) δ 9.93 (s, 1H), 9.89 (s, 1H), 5.59 (dq, J = 11.7,
5.8 Hz, 1H), 4.75 (t, J = 5.5 Hz, 2H), 2.85 (t, J = 5.5 Hz, 2H), 2.35 (s,
6H), 1.52 (d, J = 6.1 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ
165.2, 164.8, 153.1, 152.8, 141.9, 141.4, 73.2, 67.2, 57.8, 45.9, 21.8
ppm. MS (m/z) 306.2 [M + H]+. MP: 81−83 °C.
N,N-Dimethyl-2-((3′-(3,3,3-trifluoropropoxy)-[5,5′-bi(1,2,4-tria-
zin)]-3-yl)oxy)ethanamine (25). Prepared according to General
Method A. The title compound was isolated as a yellow oil in 21%
1
yield. H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 9.95 (s, 1H),
4.91 (t, J = 6.4 Hz, 2H), 4.78 (t, J = 5.6 Hz, 2H), 2.88 (t, J = 5.6 Hz,
2H), 2.85−2.75 (m, 2H), 2.38 (s, 6H) ppm. 13C NMR (101 MHz,
CDCl3) δ 165.2, 164.7, 153.4, 152.7, 142.5, 141.9, 125.8 (q, J = 276.6
Hz), 67.3, 62.3 (d, J = 3.8 Hz), 57.8, 45.9, 33.7 (q, J = 29.5 Hz) ppm.
19F NMR (376 MHz, CDCl3) δ −64.77 ppm. MS (m/z) 360.2 [M +
H]+. MP: 50−52 °C.
2-((3′-Butyl-[5,5′-bi(1,2,4-triazin)]-3-yl)oxy)-N,N-dimethylethan-
amine (26). Prepared according to General Method A. The title
1
compound was isolated as a yellow oil in 30% yield. H NMR (400
MHz, CDCl3) δ 10.08 (s, 1H), 10.00 (s, 1H), 4.77 (t, J = 5.7 Hz,
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2H), 3.38−3.11 (m, 2H), 2.86 (t, J = 5.7 Hz, 2H), 2.37 (s, 6H),
2.02−1.79 (m, 2H), 1.57−1.36 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H)
ppm. 13C NMR (101 MHz, CDCl3) δ 170.6, 165.3, 153.6, 150.5,
144.5, 141.9, 67.2, 57.8, 45.9, 36.9, 30.5, 22.5, 13.9 ppm. MS (m/z)
304.3 [M + H]+.
J = 6.2 Hz, 4H), 2.47 (s, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ
165.0, 152.9, 150.5, 150.3, 142.1, 126.1, 69.0, 26.1, 15.1 ppm. HRMS
(m/z) calcd for C18H18N8O2S2 [M + H]+ 443.1067; found 443.1075.
MP: 168−170 °C.
2,2′-([5,5′-Bi(1,2,4-triazine)]-3,3′-diylbis(oxy))bis(N,N-dimethyle-
thanamine) (37). Prepared according to General Method A. The title
compound was isolated as a yellow solid in 27% yield. 1H NMR (400
MHz, CDCl3) δ 9.95 (s, 2H), 4.78 (t, J = 5.6 Hz, 4H), 2.87 (t, J = 5.6
Hz, 4H), 2.38 (s, 12H) ppm. 13C NMR (101 MHz, CDCl3) δ 165.2,
153.0, 142.0, 67.3, 57.9, 46.0 ppm. MS (m/z) 335.3 [M + H]+. MP:
80−82 °C.
3,3′-Bis((3-azidopropyl)thio)-5,5′-bi(1,2,4-triazine) (27). Prepared
according to General Method A. The title compound was isolated as a
1
yellow solid in 27% yield. H NMR (400 MHz, CDCl3) δ 9.92 (s,
2H), 3.53 (t, J = 6.3 Hz, 4H), 3.43 (t, J = 7.0 Hz, 4H), 2.39−1.84 (m,
2H) ppm. 13C NMR (101 MHz, CDCl3) δ 173.8, 150.1, 142.4, 50.2,
28.4, 28.1 ppm. MS (m/z) 391.1 (M + H)+. MP: 76−78 °C.
3,3′-Dibutyl-5,5′-bi(1,2,4-triazine) (28). Prepared according to
General Method A. The title compound was isolated as a yellow solid
in 50% yield. 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 2H), 3.28 (t, J
= 7.7 Hz, 4H), 2.03−1.84 (m, 4H), 1.49 (dq, J = 14.7, 7.3 Hz, 4H),
1.00 (t, J = 7.3 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ 170.7,
151.1, 144.6, 37.0, 30.6, 22.5, 14.0 ppm. MS (m/z) 273.2 [M + H]+.
MP: 45−47 °C.
2,2′-([5,5′-Bi(1,2,4-triazine)]-3,3′-diylbis(oxy))bis(N,N-dimethyl-
propan-1-amine) (38). Prepared according to General Method A.
1
The title compound was isolated as a yellow oil in 30% yield. H
NMR (400 MHz, CDCl3) δ 9.90 (s, 2H), 5.79−5.56 (m, 2H), 2.84
(dd, J = 13.1, 7.9 Hz, 2H), 2.48 (dd, J = 13.1, 4.4 Hz, 2H), 2.31 (s,
12H), 1.48 (t, J = 5.6 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ
165.2, 153.0, 141.6, 139.2, 73.9, 64.6, 46.3, 18.2 ppm. MS (m/z)
363.3 [M + H]+.
3,3′-Dipropoxy-5,5′-bi(1,2,4-triazine) (29). Prepared according to
General Method A. The title compound was isolated as a yellow solid
in 68% yield. 1H NMR (400 MHz, CDCl3) δ 9.96 (s, 2H), 4.64 (t, J =
6.7 Hz, 4H), 2.11−1.89 (m, 4H), 1.12 (t, J = 7.4 Hz, 6H) ppm. 13C
NMR (101 MHz, CDCl3) δ 165.4, 152.9, 141.7, 71.4, 22.2, 10.5 ppm.
MS (m/z) 277.2 [M + H]+, HRMS (m/z) calcd C12H16N6O2 [M +
H]+ 277.1408; found 277.1406. MP: 96−98 °C.
3,3′-Bis(prop-2-yn-1-yloxy)-5,5′-bi(1,2,4-triazine) (39). Prepared
according to General Method A. The title compound was isolated as a
1
yellow solid in 46% yield. H NMR (400 MHz, CDCl3) δ 10.05 (s,
2H), 5.31 (d, J = 2.3 Hz, 4H), 2.60 (t, J = 2.3 Hz, 2H) ppm. 13C
NMR (101 MHz, CDCl3) δ 164.5, 152.9, 142.5, 76.9, 76.5, 56.9 ppm.
MS (m/z) 269.1 [M + H]+. MP: 102−104 °C.
3,3′-Diisopropoxy-5,5′-bi(1,2,4-triazine) (30). Prepared according
to General Method A. The title compound was isolated as a yellow
ASSOCIATED CONTENT
■
1
solid in 41% yield. H NMR (400 MHz, CDCl3) δ 9.92 (s, 2H),
sı
* Supporting Information
5.72−5.52 (m, 2H), 1.55 (s, 6H), 1.54 (s, 6H) ppm. 13C NMR (101
MHz, CDCl3) δ 164.9, 153.1, 141.4, 73.3, 21.9 ppm. MS (m/z) 277.2
[M + H]+. MP: 114−116 °C.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00044.
3,3′-Dicyclopropoxy-5,5′-bi(1,2,4-triazine) (31). Prepared accord-
Structural biology methods are provided (PDF)
ing to General Method A. The title compound was isolated as a
1
yellow solid in 29% yield. H NMR (400 MHz, CDCl3) δ 10.00 (s,
SMILES strings are listed for all new compounds tested
(CSV)
2H), 4.75−4.53 (m, 2H), 0.99 (s, 8H) ppm. 13C NMR (101 MHz,
CDCl3) δ 166.4, 152.8, 142.2, 53.6, 6.2 ppm. MS (m/z) 273.2 [M +
H]+, HRMS (m/z) calcd C12H12N6O2 [M + H]+ 273.1095; found
273.1097. MP: 137−139 °C.
AUTHOR INFORMATION
■
3,3′-Bis(3,3,3-trifluoropropoxy)-5,5′-bi(1,2,4-triazine) (32). Pre-
pared according to General Method A. The title compound was
isolated as a yellow solid in 56% yield. 1H NMR (400 MHz, CDCl3) δ
10.02 (s, 2H), 4.93 (t, J = 6.3 Hz, 4H), 3.02−2.62 (m, 4H) ppm. 13C
NMR (101 MHz, CDCl3) δ 164.7, 153.0, 142.5, 125.8 (q, J = 276.7
Hz), 62.4 (d, J = 3.7 Hz), 33.7 (q, J = 29.6 Hz) ppm. 19F NMR (376
MHz, CDCl3) δ −64.77 ppm. MS (m/z) 385.1 [M + H]+. MP: 96−
98 °C.
Corresponding Author
Jonathan B. Baell − Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, Monash University (Parkville
Campus), Parkville, VIC 3052, Australia; ARC Centre for
Fragment-Based Design, Monash University (Parkville
Campus), Parkville, VIC 3052, Australia; orcid.org/
0000-0003-2114-8242; Email: jonathan.baell@
monash.edu
3,3′-Bis(2-(trimethylsilyl)ethoxy)-5,5′-bi(1,2,4-triazine) (33). Pre-
pared according to General Method A. The title compound was
isolated as a yellow solid in 24% yield. 1H NMR (400 MHz, CDCl3) δ
9.93 (s, 2H), 4.82−4.73 (m, 4H), 1.37−1.28 (m, 4H), 0.13 (s, 18H)
ppm. 13C NMR (101 MHz, CDCl3) δ 165.2, 153.0, 141.6, 68.3, 17.7,
−1.3 ppm. MS (m/z) 393.2 [M + H]+, HRMS (m/z) calcd for
C16H28N6O2Si2 [M + H]+ 393.1885; found 393.1883. MP: 91−93 °C.
3,3′-Bis(2,2,2-trifluoroethoxy)-5,5′-bi(1,2,4-triazine) (34). Pre-
pared according to General Method A. The title compound was
isolated as a yellow solid in 26% yield. 1H NMR (400 MHz, CDCl3) δ
10.11 (s, 2H), 5.12 (q, J = 8.0 Hz, 4H) ppm. 13C NMR (101 MHz,
CDCl3) δ 164.0, 153.1, 143.3, 122.8 (q, J = 277.5 Hz), 65.0 (q, J =
37.3 Hz) ppm. MS (m/z) 357.1 [M + H]+. MP: 157−159 °C.
3,3′-Bis(2-methoxyethoxy)-5,5′-bi(1,2,4-triazine) (35). Prepared
according to General Method A. The title compound was isolated as a
Authors
Daniel L. Priebbenow − School of Chemistry, The University
of Melbourne, Parkville, VIC 3010, Australia; orcid.org/
0000-0002-7840-0405
Mitch Mathiew − Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, Monash University (Parkville
Campus), Parkville, VIC 3052, Australia
Da-Hua Shi − Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, Monash University (Parkville
Campus), Parkville, VIC 3052, Australia
Jitendra R. Harjani − Medicinal Chemistry, Monash Institute
of Pharmaceutical Sciences, Monash University (Parkville
Campus), Parkville, VIC 3052, Australia
Julia G. Beveridge − Medicinal Chemistry, Monash Institute
of Pharmaceutical Sciences, Monash University (Parkville
Campus), Parkville, VIC 3052, Australia
Marina Chavchich − The Department of Drug Evaluation,
Australian Defence Force Malaria and Infectious Disease
Institute, Brisbane, QLD 4051, Australia
1
yellow solid in 42% yield. H NMR (400 MHz, CDCl3) δ 9.97 (s,
2H), 5.06−4.70 (m, 4H), 4.08−3.68 (m, 4H), 3.47 (s, 6H) ppm. 13C
NMR (101 MHz, CDCl3) δ 165.2, 153.0, 142.1, 70.3, 68.6, 59.3 ppm.
MS (m/z) 309.2 [M + H]+, HRMS (m/z) calcd for C12H16N6O4 [M
+ H]+ 309.1306; found 309.1297. MP: 104−106 °C.
3,3′-Bis(2-(4-methylthiazol-5-yl)ethoxy)-5,5′-bi(1,2,4-triazine)
(36). Prepared according to General Method A. The title compound
1
was isolated as a yellow solid in 43% yield. H NMR (400 MHz,
CDCl3) δ 9.96 (s, 2H), 8.61 (s, 2H), 4.84 (t, J = 6.3 Hz, 4H), 3.42 (t,
4159
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Michael D. Edstein − The Department of Drug Evaluation,
Australian Defence Force Malaria and Infectious Disease
Institute, Brisbane, QLD 4051, Australia
Sandra Duffy − Discovery Biology and School of Environment
& Science, Griffith University, Nathan, QLD 4111, Australia
Vicky M. Avery − Discovery Biology and School of
Environment & Science, Griffith University, Nathan, QLD
4111, Australia
S.B. all made key contributions to the array of biological testing
and analysis required. D.M.S., W.W., L.Z., G.L., E.T., H.B.,
E.C., K.L.W., and S.A.C. generated and interpreted the ADME
and PK data, and S.A.C. and D.M.S. contributed to manuscript
preparation. A.D.P. and D.J.C. generated and interpreted the
MTT assay data. J.B.B. was involved in leading and directing
the overall program and writing the manuscript.
Notes
Robert T. Jacobs − Medicines for Malaria Venture (MMV),
CH-1215 Geneva, Switzerland
The authors declare no competing financial interest.
Stephen Brand − Medicines for Malaria Venture (MMV),
CH-1215 Geneva, Switzerland
David M. Shackleford − Centre for Drug Candidate
Optimisation, Monash Institute of Pharmaceutical Sciences,
Monash University (Parkville Campus), Parkville, VIC 3052,
Australia
Wen Wang − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
Longjin Zhong − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
Given Lee − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
Erin Tay − Centre for Drug Candidate Optimisation, Monash
Institute of Pharmaceutical Sciences, Monash University
(Parkville Campus), Parkville, VIC 3052, Australia
Helena Barker − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
Elly Crighton − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
Karen L. White − Centre for Drug Candidate Optimisation,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
ACKNOWLEDGMENTS
■
D.L.P. acknowledges funding support from the Australian
Research Council (DE200100949). The National Health and
Medical Research Council of Australia (NHMRC) is thanked
for Research Support (#1030704, 1080146) and Fellowship
support for J.B.B. (2012−2016 Senior Research Fellowship
#1020411, 2017Principal Research Fellowship #1117602).
This work was also supported by an NHMRC Synergy grant
(#1185354). Also acknowledged are the Australian Federal
Government Education Investment Fund Super Science
Initiative and the Victorian State Government, Victoria Science
Agenda Investment Fund for infrastructure support and the
facilities and the scientific and technical assistance of the
Australian Translational Medicinal Chemistry Facility
(ATMCF), Monash Institute of Pharmaceutical Sciences
(MIPS). ATMCF and the Center for Drug Candidate
Optimisation (CDCO) are supported by Therapeutic
Innovation Australia (TIA). TIA is supported by the Australian
Government through the National Collaborative Research
Infrastructure Strategy (NCRIS) program. Delphine Baud,
Maelle Duffey, and Virginia Crivelli are acknowledged for their
contributions to the coordination of the various studies at
MMV partner facilities. Technical assistance from Jaya
Jayaseelan, Golnar Golbaghi, and Meiyu Hu is also gratefully
acknowledged. The ongoing support of the MMV to VMA is
also acknowledged. We are grateful to Kerryn Rowcliffe for
technical support in in vitro drug susceptibility assays and
thank the Australian Red Cross Blood Service for the provision
of human blood, plasma, and sera for in vitro cultivation of P.
falciparum lines at Monash University, ADFMIDI, and Griffith
University. We also thank Karin Van Breda, Ivor Harris,
Anthony Kent, and Stephen McLeod-Robertson for the in vivo
efficacy testing of 23 in the P. berghei-rodent model. The views
expressed in this article are those of the authors and do not
necessarily reflect those of the Australian Defence Force Joint
Health Command or any extant Australian Defence Force
policy.
Susan A. Charman − Centre for Drug Candidate
Optimisation, Monash Institute of Pharmaceutical Sciences,
Monash University (Parkville Campus), Parkville, VIC 3052,
Australia
Amanda De Paoli − Drug Delivery Disposition and Dynamics,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia
Darren J. Creek − Drug Delivery Disposition and Dynamics,
Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), Parkville, VIC 3052,
Australia; orcid.org/0000-0001-7497-7082
ABBREVIATIONS
■
ED50, half effective dose; SAR, structure−activity relationship;
DMF, N,N-dimethylformamide; MeCN, acetonitrile; rt, room
temperature; EtOH, ethanol; LHS, left hand side; RHS, right
hand side; Pf , Plasmodium falciparum; P., Plasmodium; Me,
n
i
n
methyl; Et, ethyl; Bu, n-butyl; Pr, isopropyl; Pr, n-propyl;
cyPr, cyclopropyl; ADMET, absorption, distribution, metabo-
lism, excretion and toxicity; CLint, intrinsic clearance; EH,
hepatic extraction ratio; IC50, half-maximal inhibitory concen-
tration; RBC, red blood cells; PRR, parasite reduction rate;
HLM, human liver microsomes; MLM, mouse liver micro-
somes; PK, Pharmacokinetics; MW, molecular weight; PSA,
polar surface area; TLC, thin-layer chromatography; UV,
ultraviolet
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00044
Author Contributions
D.L.P. and M.M. were involved in chemical synthesis and
manuscript preparation and D.-H.S., J.R.H., and J.G.B. assisted
with chemical synthesis. M.C., M.D.E, S.D., V.M.A., R.T.J., and
4160
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