In vitro and in vivo evaluation of the antimalarial MMV665831 and structural analogs

Article abstract of DOI:10.1016/j.bmcl.2020.127348

Antimalarial candidates possessing novel mechanisms of action are needed to control drug resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure–activity relationship. Modulation of the diethyl amino group was fruitful, producing compound 25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide. Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC₅₀ ≤ 200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation. Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P. berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen relative to control. These results are discussed in the context of measured plasma and hepatocyte stabilities, with reference to structurally-related, orally-efficacious antimalarials.

Full text of DOI:10.1016/j.bmcl.2020.127348

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In vitro and in vivo evaluation of the antimalarial MMV665831 and structural
analogs
Sha Ding, Katherine R. Fike, Michael Klemba, Paul R. Carlier
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Reference:
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BMCL 127348
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Bioorganic & Medicinal Chemistry Letters
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9 June 2020
Please cite this article as: Ding, S., Fike, K.R., Klemba, M., Carlier, P.R., In vitro and in vivo evaluation of the
antimalarial MMV665831 and structural analogs, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://
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malarial MMV665831 and structural analogs
Sha Ding, Katherine R. Fike, Michael Klemba, and Paul R. Carlier

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
In vitro and in vivo evaluation of the antimalarial MMV665831 and structural
analogs
Sha Dinga, Katherine R. Fikeb Michael Klembab, and Paul R. Carliera,
a Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, United States
b Department of Biochemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Antimalarial candidates possessing novel mechanisms of action are needed to control drug
resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by
virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure-
activity relationship. Modulation of the diethyl amino group was fruitful, producing compound
25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the
phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide.
Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC50
≤
200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation.
Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both
the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P.
berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen
relative to control. These results are discussed in the context of measured plasma and hepatocyte
stabilities, with reference to structurally-related, orally-efficacious antimalarials.
Keywords:
malaria
parasite
PAINs
Phenolic Mannich Base
Malaria Box
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +1 540 231 9219; e-mail: pcarlier@vt.edu (P. R. Carlier).

Malaria remains a deadly disease, leading to approximately 228
million malaria cases worldwide and killing over 400,000 in
2018.1 Artemisinin combination therapies have contributed in
large part to a decline in malaria mortality over the last 15 years;
however, resistance to these treatments is emerging, and the
estimated malaria caseload has been on the rise in the last two
years. Thus, the need for novel antimalarial compounds remains
urgent.1, 2 Malaria symptoms in humans are caused by the blood-
stage growth of Plasmodium spp., of which P. falciparum is
responsible for ~96% of malaria cases worldwide. To accelerate
the development of novel antimalarial drugs, a collection known
as the “Malaria Box” was assembled from over 20,000 compounds
with confirmed in vitro activity against blood-stage P. falciparum,
and low general cytotoxicity.3 From the 20,000 hits, 400
compounds were selected using several criteria, including
commercial availability, oral bioavailability, and chemical
diversity. This final set contained 200 “drug-like” and 200 “probe-
like” compounds, classified according to their perceived
druglikeness.
Scheme 1. Synthesis of benzothiophene 3. Reagents and conditions: (i)
piperidine, r.t., 6 h. (ii) PhCOCl, TEA, DCM, r.t., 1 day. (iii) K2S2O8, CuSO4,
H2O/MeCN, 80 ºC, overnight. (iv) Bu4NBr3, DCM, MeOH, r.t., 18 h. (v)
Li2CO3, LiBr, DMF, 120 ºC, 2 h.
Since the 2-ethyl ester in 1 was viewed as a potential hydrolytic
liability in vivo, we desired to prepare and evaluate amide analogs.
Compound 3 dissolved poorly in ethanolic methylamine (33 wt%)
at 25 °C, and no conversion was observed after 24 h; the mixture
was heated to 60 ºC in a sealed tube overnight, but instead of the
ethyl ester 8, amine 9 was isolated in 45% yield due to aminolysis
of the benzamide (Scheme 2). The silyl ether 10 was prepared to
improve substrate solubility, and subsequent aminolysis at 35 ºC
gave full conversion. Under these conditions the desired methyl
amide 11 was isolated in 40% yield, together with 42% of
debenzoylated byproduct 12. The factors contributing to the
stability of the ester group towards aminolysis remain unclear, but
byproduct 12 proved useful as shown below. Silyl ether 11 was
easily deprotected with TBAF to give phenol Mannich precursor
8 in 88% yield.
One of us (Klemba) identified compound 1 (Figure 1,
MMV665831) as having excellent in vitro growth inhibition of
asexual blood-stage P. falciparum, and similar high potency in
inhibiting amino acid efflux4 from parasitized erythrocytes.5 We
have previously observed such behavior in a study of mefloquine
(MQ) and its non-piperidine analogs, and suggested that the
antimalarial action of MQ may stem from inhibition of
hemoglobin endocytosis.2 Repeat assay with purchased authentic
samples of 1 and its N,N-dimethyl analog 2 (ChemDiv) confirmed
these activities. Thus, we sought to explore the structural
determinants of antimalarial action of 1 and 2. In particular, we
wanted to establish whether potency could be sustained when the
phenolic Mannich base functionality (a likely trigger of the probe-
like designation6) and C3-carboxylic ester were converted to less-
reactive moieties (Figure 1).
Figure 1. Structure of MMV665831 (1) and its N,N-dimethyl analog (2), with
their phenolic Mannich base functionality highlighted in red. The carboxylic
ester at C3 is also a potential metabolic liability.
Scheme 2. Synthesis of amide derivative 8. Reagents and conditions: (i)
MeNH2, EtOH, sealed tube, 60 ºC 16 h. (ii) TIPSCl, TEA, DCM, 0 ºC to r.t.,
1 h. Material used directly for Mannich reaction (Table 1) (iii) MeNH2,
EtOH, sealed tube, 35 ºC, 16 h. (iv) TBAF, THF, 0 ºC to r.t., 30 min.
To enable the synthesis of such analogs of 1/2, we developed a
gram-scale synthesis of the core 7-hydroxybenzothiophene 3,
which serves as a Mannich reaction precursor for 1 and its analogs.
(Scheme 1) A facile Gewald reaction from cyclohexanone, sulfur,
and ethyl cyanoacetate7 constructed the known thiophene 4 in 73%
yield, which was benzoylated to give 5 in 95% yield.
Regioselective oxidation at C7 was achieved with
CuSO4/potassium persulfate8 to give 6 with 58% yield.
To provide isosteric comparison with the methyl amide 8, the
methyl ester 13 was prepared from methyl cyanoacetate, using the
method for 3 (Scheme 3). The byproduct 2-aminobenzothiophene
12 obtained from the aminolysis of 10 was acetylated with 4-
chlorobenzoyl chloride to give 14, which was desilylated to give
phenol Mannich precursor 15. Curiously, the reaction of 12 with
4-methoxybenzoyl chloride, even with 4-DMAP catalysis, did not
To achieve the conversion of ketone 6 to the desired phenol 3,
we employed an α-halogenation/dehydrohalogenation sequence.
Whereas bromination of 6 with Br2 or NBS resulted in a mixture
of mono- and di-brominated product, regioselective mono-
give any conversion. Fortunately, thiophene
4 could be
benzoylated with the 4-methoxybenzoyl chloride to give 16, which
was converted to precursor 17 (Scheme 3).
9
bromination was achieved with Bu4NBr3 affording 7 in 82%
yield. Treatment of 7 with lithium carbonate in DMF at 120 ºC for
2 h cleanly eliminated HBr;10 addition of water precipitated phenol
3 in 94% yield, which was sufficiently pure for further steps.

of the parasite was used to select compounds for the Malaria Box,3
we employed it as well; MQ was used as positive control. Within
error compound 1 is equipotent with MQ, with a P. falciparum
growth inhibition EC50 values of 27 ± 8 and 16 ± 6 nM,
respectively. (Entries 1 and 2, Table 2). Our limited series
modifying the para-position of the benzamide (Y, Table 2, entries
2, 4, 5) shows Y = H (1, 27 ± 8 nM) is favorable to Y = Cl (19, 70
± 18 nM), although Y = OCH3 (20, 66 ± 35 nM) is
indistinguishable from Y = H (1). Similarly, our limited
modification of the 3-carboxy substituent (X) indicates that an
amide is not tolerated (22, Table 2, Entry 7, > 1,000 nM). Methyl
ester 21 is equipotent with 1, within error (Entry 6, Table 2, 53 ±
25 nM). Thus, the poor potency of the methyl amide 22 is not steric
in origin.
To our delight, varying the NR1R2 moiety is well-tolerated, and
can result in improved potency. Whereas dimethylamine analog 2
is equipotent to 1 (Entry 3, Table 2, 24 ± 8 nM); dipropylamine
analog 23 and pyrrolidine analog 24 appear slightly more potent
than 1 (Entries 8 and 9, Table 2, 16 ± 2, 16 ± 5 nM, respectively);
piperidine analog 25 is the most potent in this series (Entry 10,
Table 2, 13 ± 2 nM). Interestingly, the isosteric morpholine analog
26 is 7- to 8-fold less potent than piperidine analog 25 (Entry 11,
Table 2, 100 ± 50 nM). It is tempting to speculate that the reduced
basicity of morpholine related to piperidine may drive this reduced
potency.11 Compound 27 with its 1-adamantyl (1-Ad) group was
assessed to probe the effect of increased lipophilicity in the high
potency of 25. The significant (14-fold) loss of potency compared
to 1 (Entry 12, Table 2, 388 ± 116 nM), may well be a consequence
of the significant steric bulk of the 1-Ad group.
Scheme 3. Synthesis of C2-methyl ester analog 13, and C3-Ar analog 15, 17.
Reagents and conditions: (i) 4-Cl-benzoyl chloride or 4-OMe-benzoyl
chloride, TEA, DCM, r.t., overnight. (ii) TBAF, THF, 0 ºC to r.t., 30 min.
Phenols 3, 8, 13, 15, and 17 readily underwent Mannich
reaction with a series of secondary amines in the presence of
formalin, giving the desired products 1, 19-26 in 44-92% yield.
(Table 2) Reaction of 3 with the 1° amine 1-adamantylamine and
formalin initially gave hemiaminal ether 18; treatment with
aqueous acid gave the desired product 27.
Table 1. Mannich reactions of phenols 3, 8, 13, 15, and 17.
Compound 1 is classified as a “probe-like” member of the
Malaria Box, but no specific explanation for this designation was
given in the original publication.3 As mentioned above, we
suspected the phenolic Mannich base functionality of 1 was to
blame, and application of the PAINs (Pan Assay Interference
Compounds) filter6 in SwissADME12 did generate an alert for this
functionality.
Entry Product
(phenol)
X
Y
NR1R2
Yield
1
2
3
4
5
6
7
8
9
10
1 (3)
OEt
OEt
OEt
OMe
NHMe
OEt
OEt
OEt
OEt
OEt
H
NEt2
72%
48%
90%
82%
44%
92%
81%
80%
60%
45%
19 (15)
20 (17)
21 (13)
22 (8)
23 (3)
24 (3)
25 (3)
26 (3)
27 (3)
Cl
OMe
H
NEt2
NEt2
NEt2
H
NEt2
Figure 2. Phenolic Mannich base moiety in 1 is a quinone methide precursor.
H
NPr2
H
N(CH2)4
N(CH2)5
N(CH2CH2)2O
NHAda
Elimination of the phenolic Mannich base moiety to a highly
electrophilic quinone methide13,14 and subsequent reaction with
biological nucleophiles15-17 may be responsible for promiscuous
binding sometimes exhibited by this class of compounds (Figure
2). More importantly, formation of this electrophile in vivo could
lead to idiosyncratic toxicity.
H
H
H
aAd designates 1-adamantyl; isolated as HCl salt.
The P. falciparum growth inhibition potencies of compound 1,
19-27 were tested in a 48-hour growth assay. Since the 3D7 strain
Table 2. P. falciparum growth inhibitory activity

Entry
Compound
X
Y
NR1R2
R3
P. falciparum
3D7 straina
Growth
inhibition EC50
(nM)
1
Mefloquine
(MQ)
NA
NA
NA
NA
16 ± 6
2
1
OEt
OEt
OEt
OEt
OMe
NHMe
OEt
OEt
OEt
OEt
OEt
na
H
NEt2
H
H
H
H
H
H
H
H
H
H
H
na
Me
27 ± 8
3
2b
H
NMe2
NEt2
24 ± 8
4
19
Cl
OMe
H
70 ± 18
66 ± 35
53 ± 25
>1000
5
20
NEt2
6
21b
22b
23b
24b
25b
26b
27b
28
NEt2
7
H
NEt2
8
H
NnPr2
16 ± 2
9
H
N(CH2)4
N(CH2)5
N(CH2CH2)2O
NH-1-Adc
na
16 ± 5
10
11
12
13
14
H
13 ± 2
H
100 ± 50
388 ± 116
182 ± 72
> 1,000
H
na
H
29b
OEt
NEt2
aThis strain is sensitive to chloroquine, pyrimethamine, and mefloquine. bTested as HCl salt. c1-Ad designates 1-adamantyl.
To block this potential reactivity, we proposed two different
analogs: a) one resulting from insertion of an additional methylene
i.e., compound 28, and b) the other resulting from methylation of
the phenol, i.e., compound 29.
To synthesize the desired homologated analog, phenol 3 was
formylated selectively at the ortho- position with
paraformaldehyde and MgCl2.18,19 (Scheme 4) However,
attempted Henry reaction of 30 was unsuccessful, presumably due
to its poor solubility in nitromethane. We thus prepared the silyl
ether 31 to improve solubility as well as reduce polarity of the
products. Henry reaction of silyl ether 31 catalyzed by AcOH and
pyridine20 was successful, affording the desired product 32 in 56%
yield.21 The nitrostyrene 32 was reduced with sodium borohydride
at 0 ºC to give 33,22 and the silyl ether was deprotected with TBAF
to give nitro alkane 34 in 83% yield. The nitro alkane 34 was
reduced under catalytic hydrogenation conditions, and
a
subsequent Eschweiler-Clarke reaction gave the homolog 28.
Unfortunately, insertion of the additional methylene in 28 caused
an approximate 7-fold loss of potency compared to 2 (Entry 13,
Table 2, 182 ± 72 nM). However, the fact that 28 retained
significant growth inhibition potency strongly suggests that
quinone methide formation is not the basis of the antimalarial
activity of this compound class.
Scheme 4. Synthesis of homolog 28. Reagents and conditions: (i)
paraformaldehyde, MgCl2, TEA, MeCN, 80 ºC, overnight. (ii) TIPSCl, TEA,
DCM, 0 ºC to r.t., 1 h. (iii) MeNO2, HOAc, pyridine, DCM, 0 ºC to r.t.,
overnight. (iv) NaBH4, MeOH/THF, 0 ºC, 30 min. (v) TBAF, THF, 0 ºC to
r.t., 30 min (vi) Pd/C, H2, MeOH, r.t. overnight. (vii) HCHO, NaBH3CN,
HOAc, MeCN, 1 h.
The o-formyl phenol 30 was readily methylated using methyl
iodide/K2CO3, giving 35 in 81% yield (Scheme 5). Interestingly,
the standard reductive amination protocol (sodium
triacetoxyborohydride in DCE23) was not effective for reaction of
35
with
diethylamine.
Fortunately
the
alternative
Ti(OiPr)4/NaBH3CN protocol24 recommended by Abdel-Magid et
al.23 did afford 29 in 69% yield, with 25% recovered starting
material 35. Unfortunately, methylation of the phenol completely
abrogated potency (Entry 14, Table 2, EC50 > 1,000 nM).

Scheme 5. Synthesis of 7-methoxy analog 29. Reagents and conditions: (i)
K2CO3, MeI, DMF/THF, r.t., overnight. (ii) Et2NH, Ti(OiPr)4, 2 h; NaBH3CN,
EtOH, 16 h.
Since structural modifications to prevent quinone methide
formation from 1 or 2 significantly reduced potency, we thought it
would be worthwhile to investigate the thermal stability of these
compounds. After all, two WHO-approved and several
investigational antimalarials25-29 contain the phenolic Mannich
base functionality (Figure 3). To our knowledge, formation of
quinone methide intermediates has not been implicated for any of
the phenolic Mannich base antimalarials in Figure 3, or others
reported to have in vitro activity.30,31 But studies of phenolic
Mannich bases directed at other diseases/targets have
Scheme 6. Stability test of 2•HCl
Thus, the ethyl ester moiety of 2 (and by extension 1) is
remarkably stable to acidic hydrolysis. Together these experiments
indicated that 1 might be stable enough to exert antimalarial
activity in vivo, and thus we tested 1 (40 mg/kg po) in a mouse
model of blood-stage malaria (P. berghei). Unfortunately,
compound 1 did not reduce parasitemia in vivo compared to
vehicle control (See Figures S1, S2 in Supplementary Material).
Plasma (mouse) stability studies of 1 showed no loss of parent over
120 minutes, indicating that the ethyl ester moiety was quite stable
towards plasma carboxylesterases. Mouse hepatocyte stability
experiments showed rapid loss of parent with a t1/2 of 11 min.
However, a number of other phenolic Mannich Base antimalarials
have similar rates of liver metabolism, and yet retain in vivo
efficacy (e.g. 36, 39-44, see Supplementary Material, Table S3).26-
29
Without performing oral pharmacokinetics and identifying
metabolites (and their antimalarial efficacy), it is impossible to
know whether the failed efficacy of 1 is due to poor plasma
exposure, or to a (rare) species difference (P. berghei vs P.
falciparum) in drug sensitivity.33,34
In conclusion, we prepared a series of analogs of 1 featuring
modifications at the C2-benzamide, C3-ester, C6-Mannich base,
and C7-phenol moieties. In vitro P. falciparum growth inhibition
potencies revealed that neither an electron-withdrawing group or
electron-donating group on the C2-benzamide improved potency
(cf. 19, 20 with 1), that an amide cannot substitute for an ester at
the C3 position (cf. 22 with 1, 21), and that the C7-phenol is
essential for potency (cf. 29, 1). Increasing lipophilicity of the NR2
unit of the C6 Mannich base can double potency (cf. 23-25 with
1,2), but steric constraints eventually limit this effect (cf. 27 with
1,2). Attempts to block quinone methide formation by
homologation of 1 to 28 reduced, but did not eliminate antimalarial
potency (cf. Table 2). Thus, antimalarial action of 1 does not
appear to require formation of the quinone methide. In fact, the
Mannich base and ester functionalities of 2 (and by extension 1)
were found to be quite stable under neutral and acidic conditions,
and no loss of 1 was seen in mouse plasma after 2 h. Unfortunately,
1 was rapidly consumed in mouse hepatocytes, and it failed to
show oral efficacy at 40 mg/kg in a mouse model of malaria.
Figure 3. WHO-approved (36, 37) and orally-active investigational25-29 (38-
44) antimalarial drugs containing the phenolic Mannich base moiety.
demonstrated quinone methide formation in compounds ranging
from quite reactive14-17 to fairly robust.13,32 Thus we subjected 2 to
a series of tests (Scheme 6). Heating 2•HCl in methanolic
diethylamine at 40 ºC for 2 days did not result in any observable
1
change by H NMR or HRMS. Not only did the dimethylamino
group remain intact, so did the ethyl ester, consistent with our
observation of the low reactivity of 3 towards amidation (Scheme
2). Compound 2•HCl also proved stable in D2O at 50 ºC after 7
days, but heating at 90 ºC overnight resulted in the formation of a
precipitate, which was insoluble in D2O or CDCl3. This result
suggests the quinone methide intermediate did form under neutral
conditions at 90 °C, and subsequently polymerized. However
under acidic conditions (0.1 M DCl in D2O), 2•HCl was quite
stable after 1 day at 90 ºC, only undergoing H-D exchange
selectively at C4, forming 45.
Acknowledgments
We acknowledge the National Institutes of Health (AI128362
to PRC) and the Virginia Tech Center for Drug Discovery for
financial support. In vivo studies of 1 in P. berghei-infected mice
were carried out at the New York University Anti-infectives Core
Facility. Plasma and microsomal stability studies of 1 were
performed by Pharmaron.

Jacobus, D. P.; Edstein, M. D. Antimicrob. Agents. Chemother. 2016,
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The yield of 32 was reduced in part by the addition of a
second
equivalent of
nitromethane,
due to further
nitro
addition
aldol
to
nitrostyrene
32.
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interests/personal relationships which may be
considered as potential competing interests:
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