Robenidine Analogues Are Potent Antimalarials in Drug-Resistant Plasmodium falciparum

Article abstract of DOI:10.1021/acsinfecdis.1c00001

Robenidine is a veterinary drug used in the poultry industry to treat coccidiosis caused by parasites in the Eimeria genus. Though this compound and related aminoguanidines have recently been studied in other pathogens, the chemotype has not been systematically explored to optimize antimalarial activity despite the close genetic relationship between Eimeria and Plasmodium (both are members of the Apicomplexa phylum of unicellular, spore-forming parasites). In this study, a series of aminoguanidine robenidine analogues was prepared and tested in vitro against Plasmodium falciparum, including multidrug-resistant strains. Selected compounds were further evaluated in vivo against murine Plasmodium yoelii in mice. Iterative structure-activity relationship studies led to the discovery of 1, an aminoguanidine with excellent activity against drug-resistant malaria in vitro and impressive in vivo efficacy with an ED50 value of 0.25 mg/kg/day in a standard 4-day test.

Full text of DOI:10.1021/acsinfecdis.1c00001

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Robenidine Analogues Are Potent Antimalarials in Drug-Resistant
Plasmodium falciparum
Alina Krollenbrock,* Yuexin Li, Jane Xu Kelly, and Michael K. Riscoe*
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ABSTRACT: Robenidine is a veterinary drug used in the poultry
industry to treat coccidiosis caused by parasites in the Eimeria
genus. Though this compound and related aminoguanidines have
recently been studied in other pathogens, the chemotype has not
been systematically explored to optimize antimalarial activity
despite the close genetic relationship between Eimeria and
Plasmodium (both are members of the Apicomplexa phylum of
unicellular, spore-forming parasites). In this study, a series of
aminoguanidine robenidine analogues was prepared and tested in
vitro against Plasmodium falciparum, including multidrug-resistant
strains. Selected compounds were further evaluated in vivo against
murine Plasmodium yoelii in mice. Iterative structure−activity
relationship studies led to the discovery of 1, an aminoguanidine with excellent activity against drug-resistant malaria in vitro and
impressive in vivo efficacy with an ED50 value of 0.25 mg/kg/day in a standard 4-day test.
KEYWORDS: robenidine, Plasmodium, Eimeria, aminoguanidine, malaria, coccidiosis
alaria remains a devastating parasitic disease responsible
M
for 229 million infections and 409 000 deaths in 2019.1
Among the species of malaria parasites that can infect humans,
Plasmodium falciparum is the most virulent and deadly.
Pregnant women and children are the most vulnerable to
mortality from malaria, and most cases and deaths occur in
sub-Saharan Africa, where P. falciparum is the dominant
species. Widespread resistance to existing therapies for malaria
is an increasingly significant concern for global health. Drug
resistance continues to spread for frontline malaria therapies,
and resistance has emerged in the past decade in Cambodia
and neighboring countries for current “last line of defense”
drugs such as artemisinin.2 The spread of artemisinin
resistance outside of southeast Asia seems inevitable and
threatens to reverse the recent progress that has been made in
decreasing malaria deaths worldwide, i.e., from the year 2000
to 2019, deaths have fallen from 736 000 to 409 000.
Figure 1. (a) Robenidine, a commercial veterinary drug; (b) 1, an
aminoguanidine with potent antimalarial activity.
Development of new malaria drug classes that can evade
existing resistance mechanisms is an urgent global need if
elimination and eradication goals are to be achieved.3 One
frequently proven successful approach to the development of
novel therapeutics is to begin with a drug for a related parasite
or pathogen and refine its structure using medicinal chemistry
techniques.
Robenidine (Figure 1a), formerly called robenzidene, is a
veterinary drug developed in the early 1970s to treat
coccidiosis caused by parasites in the Eimeria genus.4−10
Robenidine is frequently added to animal feed to prevent and
treat coccidiosis in chickens,11 other fowl,12 and rabbits.13
Special Issue: In Celebration of Jonathan Venner-
strom: A Pioneer in Neglected Tropical Diseases
Received: January 1, 2021
© XXXX The Authors. Published by
American Chemical Society
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Eimeria and Plasmodium, the parasite responsible for malaria
infections, are both members of the Apicomplexan phylum,
distinguished by the presence of the apical complex and
frequent presence of the apicoplast organelle.14
of concentrations of robenidine, and 72 h parasite growth was
measured by SYBR green staining relative to untreated
controls.38 In this assessment, robenidine exhibited an average
in vitro IC50 of 324 nM (Figure 2). This value represents an
Robenidine and other aminoguanidines have been evaluated
for efficacy against several other protozoan parasites, including
Toxoplasma gondii (in vitro IC50 0.03 μg/mL for robeni-
dine),15−18 Leishmania donovani (in vitro IC50 18 μM for the
analogue CGP 40215A),19 Babesia microti (murine in vivo
nonrecrudescence dose 100 mg/kg/day for oral robeni-
dine),20,21 and the trypanosomes, T. brucei and T. cruzi (in
vitro IC50 20 μM for the analogue CGP 40215A).22−24 It has
also been tested against microorganisms such as Lactobacil-
lus,25 E. coli,26 Acanthamoeba polyphaga,27 Goussia carpelli,28
and several other Gram-positive and Gram-negative bacterial
pathogens.29,30
Recently, work by Trott and McClusky31−36 has further
explored the aminoguanidine chemotype of robenidine,
applying a medicinal chemistry approach to repurpose the
drug for various bacterial pathogens. Their ongoing research
has demonstrated that this chemical scaffold is amenable to
synthetic modification and can successfully be refined for
activity against pathogens other than Eimeria.
Despite significant genetic similarity between Eimeria and
Plasmodium, there has been relatively little research into the
efficacy of robenidine against malaria in its 50-year history.
Robenidine was evaluated against the murine species P. berghei
during its initial development in 1970 and found to have
moderate activity in vivo.4 An immine analogue of robenidine,
CGP 40215A, was tested against the human pathogen P.
falciparum in vitro with low micromolar IC50 value.37 To date,
no medicinal chemistry efforts have focused on refining and
optimizing the aminoguanidine scaffold for antimalarial
activity.
The current study represents the first attempt to evaluate
structure−activity relationships (SAR) of the aminoguanidine
chemotype against malaria parasites using in vitro assays and an
in vivo murine model. A library of 38 structurally diverse
aminoguanidines was created and compared for in vitro
antiplasmodial activity as well as mammalian cell cytotoxicity.
Compounds with promising selective activity were further
evaluated in vivo in a murine model of malaria. Multiple
aminoguanidines from this library were found to have high
potency in vitro, which translated to robust efficacy in vivo. One
compound, 1 (Figure 1b), exhibits single-digit nanomolar IC50
values against P. falciparum strains with an in vivo ED50 value
below 1 mg/kg/day vs murine malaria.
Figure 2. IC50 curve of robenidine in D6 compared with chloroquine.
Y values represent fluorescence relative to untreated controls.
excellent starting point for further evaluation of structure−
activity relationships of robenidine analogues. Notably, the
concentration−response curve was characterized by a steep
slope at the IC50 inflection point, similar to the control drug
chloroquine.
Synthesis of Aminoguanidine Robenidine Ana-
logues. Given the successful initial evaluation of robenidine
in vitro, a series of 38 robenidine analogues was designed and
synthesized to determine the potential for improved
antimalarial activity within the aminoguanidine chemotype.
Robenidine analogues were synthesized in a single step route
starting from commercially available substituted benzaldehydes
or acetophenones with the synthetic approach utilized in
robenidine’s initial discovery.4 Two equivalents of each starting
material were refluxed in ethanol with 1,3-diaminoguanidine
hydrochloride (Scheme 1). The resulting symmetrical aryl
Scheme 1. A General Synthesis of Aminoguanidine
a
Robenidine Analogues
RESULTS AND DISCUSSION
■
The Aminoguanidine Robenidine Is an Effective
Antimalarial in Vitro. Robenidine is a symmetrical amino-
guanidine drug originally developed for the treatment of
Eimeria-derived coccidiosis in poultry and livestock.4 Recent
investigations by Trott, McCluskey, and others have
demonstrated that the aminoguanidine chemotype can be
modified and reapplied to pathogens other than Eimeria.31
Prior to the present study, robenidine had not been tested for
antimalarial activity against P. falciparum despite the close
genetic relationship between Plasmodium and Eimeria.
a
Compounds were prepared by refluxing benzaldehydes or
acetophenones with 1,3-diaminoguanidine hydrochloride in ethanol.
aminoguanidine products (formed as HCl salts) were crashed
out of solution using diethyl ether, filtered, and washed with
diethyl ether. Compounds were purified by recrystallization
from ethanol.
As evidenced by the continued commercial success of
robenidine as a veterinary drug, this synthetic approach is
highly amenable to affordable, large scale synthesis, an
important attribute given that any new antimalarial drug
To gain an initial assessment of robenidine as a potential
antimalarial drug, it was evaluated in vitro for the ability to
inhibit the growth of P. falciparum strain D6, a drug-sensitive
strain. Blood stage parasite cultures were incubated in a range
B
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a
Table 1. Aminoguanidine Halogen Series and Antimalarial Drugs
compound
R1
R2
IC50 D6 (nM)
IC50 Dd2 (nM)
IC50 Tm90−C2B (nM)
IC50 A6 (nM)
IC50 cytotoxicity (μM)
IVTI
2
3
4
5
2-F
3-F
4-F
H
H
H
H
H
H
H
H
2866
767
1016
334
202
324
280
572
277
<1
4593
>5000
3792
961
567
814
715
1516
876
<1
70
>5000
>5000
4904
1219
664
1317
974
1838
899
1893
426
369
348
237
410
876
502
269
>5000
16
>200
49
24
>200
35
30
94
62
38
72
>100
64
24
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
N/A
N/A
>100
>100
93
>100
>100
>100
>100
>100
6
robenidine
7
8
9
atovaquone
chloroquine
H
N/A
N/A
>5000
297
10
181
a
See Scheme 1 for the aminoguanidine scaffold and sites of modification. P. falciparum IC50 values are the average of two to four determinations,
each carried out in quadruplicate (a more granular view of this data is provided in the Supporting Information). D6, P. falciparum pan-sensitive
strain; Dd2, multidrug-resistant P. falciparum strain; Tm90−C2B, multidrug-resistant P. falciparum clinical isolate that is also resistant to
atovaquone; A6, P. falciparum in-house derived mutant line resistant to respiratory antagonists.40 Cytotoxicity assays were carried out with human
hepatoma derived HepG2 cells and performed in quadruplicate. IVTI = in vitro therapeutic index, defined as cytotoxicity/D6 IC50. N/A = not
applicable.
a
Table 2. Aminoguanidine Methyl, Methoxy, Fluoromethyl, Fluoromethoxy, and Nitrile Series
compound
R1
R2
IC50 D6 (nM)
IC50 Dd2 (nM)
IC50 Tm90−C2B (nM)
IC50 A6 (nM)
IC50 cytotoxicity (μM)
IVTI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1
4-CH3
4-OCH3
4-CF3
H
H
H
CH3
H
H
H
CH3
H
CH3
H
905
263
614
211
1080
670
39
140
71
99
1024
7
58
47
4
2427
1602
1536
414
2328
1571
83
526
191
403
2097
20
2529
>5000
2742
451
>5000
2988
114
458
193
315
1990
31
>5000
289
567
283
391
689
52
182
87
171
969
24
43
>200
40
114
156
61
11
9
19
3
48
>100
65
>100
>100
91
>100
64
>100
30
4-CF3
2-OCF3
3-OCF3
4-OCF3
4-OCF3
4-OCHF2
4-OCHF2
2-CN
3-CN
4-CN
3-CN
3-CN, 4-F
31
7
>200
9
30
H
H
CH3
H
>100
>100
>100
>100
166
70
12
312
104
16
76
17
14
8
a
See Table 1 legend and Methods.
a
Table 3. Other Aminoguanidines
IC50A6
(nM)
compound
R1
R2
IC50 D6 (nM) IC50 Dd2 (nM) IC50 Tm90−C2B (nM)
IC50 cytotoxicity (μM) IVTI
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
4-OH
4-Ph
4-NO2
4-NMe2
4-COOH
4-CONH2
4-SO2NH2
4-propynoxy
4-morpholino
3-tetrazole
4-tetrazole
(2,2-difluoro) 2,3-dioxazole
2,4-OMe, 5-Cl
H
705
143
15
96
118
>5000
527
>5000
102
131
1645
838
4298
240
87
198
264
>5000
405
>5000
199
>5000
286
71
247
213
>5000
727
>5000
376
295
>5000
>5000
905
1180
>5000
581
278
33
85
122
>5000
516
>5000
153
>200
39
>200
>200
20
>200
42
>200
>200
>200
107
>100
>100
>100
>100
>100
N/A
80
N/A
>100
>100
65
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
329
116
>5000
>5000
800
1247
2202
4790
>5000
235
216
833
>200
>200
3
N/A
>100
8
250
360
1028
b
mono 2-F
>200
>100
a
b
See Table 1 legend and Methods. Compound 38 has a 2-fluoro substituent at only one R1 site. The other R1 site is unsubstituted (4-H).
must be inexpensive for use in resource poor regions where the
disease is endemic. It is noteworthy that the aminoguanidines
can be prepared in a single step from commercially available
starting materials, and no chromatographic separations are
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required for their purification. This is a significant advantage
over other antimalarial drug candidates requiring multiple steps
and/or complex separations.
value of 7 nM, much lower than any other aminoguanidine
evaluated up to that point. The R2-methyl analogue (23) of
this compound was prepared and found to be less active than
the R2-H original.
A few general trends were observed for in vitro antimalarial
activity. In general, substitution at the ortho position
dramatically reduced antimalarial activity, possibly by sterically
constraining rotation of the aryl rings. The carbonate 29 and
tetrazoles 34 and 35 were inactive against all tested P.
falciparum strains. Electron withdrawing substituents appear to
have a positive effect on antimalarial activity, though the
biphenyl analogue 26 is unusually potent and the dimethyla-
mino (28) and propynoxy (32) compounds also exhibit
respectable antiparasitic activity.
All aminoguanidines synthesized were also evaluated against
three multidrug-resistant P. falciparum strains (Dd2, Tm90−
C2B, and A6) and for mammalian cell cytotoxicity. Robenidine
and the lead compounds 16, 21, and 22 were further evaluated
in vivo to guide optimization, resulting in the design of 1, the
overall series lead. The results of these experiments are listed in
Tables 1−3 and discussed below.
Aminoguanidines Retain in Vitro Activity in Drug-
Resistant Strains of Malaria. In addition to the drug-
sensitive P. falciparum D6 strain, the aminoguanidines were
assessed against three drug-resistant strains (Tables 1−3). P.
falciparum Dd2 is a multidrug-resistant strain sensitive to
atovaquone but resistant to chloroquine as well as the
antifolate combination of pyrimethamine + sulfadoxine. P.
falciparum Tm90−C2B is a multidrug-resistant clinical isolate
including resistance to both atovaquone and chloroquine.39 P.
falciparum A6 is derived from D6 and is resistant to respiratory
antagonists such as atovaquone and antimycin A but sensitive
to chloroquine.40
The degree of cross resistance observed for the MDR strains
Dd2 and C2B with the tested aminoguanidine series ranged
from extensive (e.g., ∼19-fold for 11), to intermediate (e.g.,
6.5-fold for 3), to modest (2−4-fold for 1 and 23) relative to
the drug sensitive D6 strain of P. falciparum. The general lack
of significant cross-resistance is consistent with expectations
given that robenidine and other aminoguanidines are not
clinically prescribed for malaria or even administered directly
to humans for any indication (it is possible that trace amounts
of robenidine have passed into humans via the consumption of
poultry treated for coccidiosis, though these trace amounts are
unlikely to drive antimalarial resistance).
Aminoguanidines Have High in Vitro Therapeutic
Indices. The aminoguanidines were evaluated for cytotoxicity
against human hepatoma derived HepG2 cells (Tables 1-3). In
this assay, HepG2 cells were incubated with test compounds
for 24 h, followed by a 24-h recovery period and subsequent
staining to evaluate for cytotoxic effects with resazurin. The
ratio of the resulting HepG2 IC50 value to the P. falciparum D6
IC50 value can be considered an “in vitro therapeutic index” or
IVTI.
Structure−Activity Relationships of Aminoguani-
dines in Vitro. The in vitro activity of each compound in
the aminoguanidine library was assessed against asynchronous
cultures of Plasmodium falciparum parasites replicating within
human erythrocytes (Tables 1−3). As the chemical develop-
ment of robenidine was not discussed in its original publication
in 19704 and limited structural information is available
regarding its mechanism of action,10 profiling was guided
primarily by in vitro potency against the P. falciparum D6 strain
cultured in human erythrocytes as described above.
Robenidine is structured as a diarylaminoguanidine with
symmetrical 4-chloro (para relative to the aminoguanidine
moiety) substituents on its two phenyl rings. Our SAR studies
primarily focused on the potential to improve antimalarial
activity by exchanging these 4-chloro substituents for different
functional groups at the same position. All compounds are
symmetrical and take the form shown in Scheme 1 unless
otherwise noted. A halogen series (Table 1) was prepared to
examine the effects of both halide type and position on
antiplasmodial activity. Within this series, chloro and bromo
substituted aminoguanidines were more active than fluoro
substituted compounds, with position effects varying among
the halides. The ortho-fluoro compound 2 displayed much
lower activity than robenidine, and the para-bromo compound
9 showed moderately improved activity.
Other replacement functional groups for robenidine’s 4-
chloro substituents were varied widely for size, lipophilicity,
and electron withdrawing vs donating effects on the adjacent
phenyl ring. Compounds with promising activity were also
prepared as their ortho and meta isomers to investigate
positional effects for these functional groups. Similarly,
promising compounds were prepared with additional methyl
groups on the benzyl carbons of the aminoguanidine moieties
as shown in Scheme 1 (the R2 position) by starting from the
analogous acetophenones rather than benzaldehydes.
The 4-methoxy robenidine derivative 11 was among the
earliest compounds to show improved potency over
robenidine. To further pursue this activity, the methyl (10),
trifluoromethyl (12), trifluoromethoxy (16), and difluorome-
thoxy (18) derivatives were also prepared along with their R2-
methyl analogues (Table 2). Among these compounds 16 was
the most potent, quickly becoming the frontrunner with a
nearly 10-fold reduction in IC50 value (39 nM vs D6) relative
to robenidine (324 nM vs D6). Conversion of the R2 moiety
from H to methyl did not have a consistent effect, improving
activity for the trifluoromethyl derivative (12 vs 13) while
reducing activities of the trifluoromethoxy and difluorome-
thoxy derivatives (16 vs 17 and 18 vs 19, respectively). The
ortho (14) and meta (15) variants of 16 were also prepared
and demonstrated significantly reduced antimalarial activity in
comparison with the para isomer.
The early success of 16 led to an interest in exploring other
electron withdrawing groups at the para position. The nitro
(27) and cyano (22) derivatives were significantly more active
than robenidine. 22 in particular had activity in the same range
as the early hit compound 16, so the ortho (20) and meta (21)
analogues were prepared to explore the position effects of the
nitrile group. This series was expected to show a similar pattern
to the trifluoromethoxy compounds, and so it came as quite a
surprise when the 3-CN analogue 21 was found to have IC50
Cytotoxicity in Hep2G did not track proportionally with
antimalarial activity in any of the four tested P. falciparum
strains. Overall, substitution at the 3 (meta) position resulted
in increased cytotoxicity relative to the 2 (ortho) and 4 (para)
positions, though the cyano substituent was shown to be an
exception to this trend. Several of the aminoguanidines,
including the active compound 21, had no measurable effect
on Hep2G activity at concentrations as high as 200 μM. Most
aminoguanidines in the series had HepG2 IC50 values above 10
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μM, and nearly all active aminoguanidines had an in vitro
therapeutic index of over 1000-fold (this value cannot be
calculated for those aminoguanidines having no antimalarial
activity). It is noteworthy that 1, with the greatest
antiplasmodial activity among compounds in this series,
exhibits an IVTI value of 2000, which is indicative of its
highly selective antiparasitic action.
Aminoguanidine Activity Is Concentration Depend-
ent but Not Time Dependent. Many drugs have activity
dependent on their exposure time in addition to concentration,
and antimalarial potency is frequently stage-specific. To
determine whether the aminoguanidines acted by a time-
dependent mechanism against malaria parasites, the SYBR
Green activity assay was adapted to include additional
incubation intervals. Two potent aminoguanidines, 16 and
21, were incubated with P. falciparum Dd2 infected
erythrocytes for 48, 72, or 96 h (Figure 3). Notably, this
concentration rather than by lengthening drug incubation time.
Additional experiments are planned to explore for possible
stage-specific activity of the most active compounds in this
series however our results combine to suggest that the
molecules are not acting in a manner consistent with a
“delayed death” phenotype as shown for other drugs such as
doxycycline and azithromycin.45
Lead Aminoguanidines Have High in Vivo Efficacy in
a Mouse Model of Malaria. Aminoguanidines exhibiting
high in vitro antimalarial potency and robenidine were assessed
for in vivo efficacy in a murine model of malaria (Plasmodium
yoelii, Table 5). In this modified 4-day Peters test,41 mice were
a
Table 5. In Vivo ED50 and ED90 of Lead Aminoguanidines
P. yoelii ED50
(mg/kg/day)
P. yoelii ED90
(mg/kg/day)
P. yoelii NRD
(mg/kg/day)
compound
robenidine
1.6
1.2
2.7
7.1
0.25
4.9
3.7
7.7
9.4
0.28
>25
12.5
>25
>25
>25
16
22
21
1
a
In vivo activity values were determined from a modified 4-day Peters
test. Compounds were administered by oral gavage up to 25 mg/kg/
day, near the solubility limit of the PEG delivery vehicle. NRD =
nonrecrudescence dose (cure dose).
inoculated with parasites from a donor mouse (day 0), and
then dosed orally with drugs on each of the subsequent 4 days
(days 1−4). On day 5 of the experiment, the parasitemia for
each mouse was determined microscopically by examining
methanol-fixed and Giemsa-stained blood smears. The ED50
represents the interpolated dose of a compound at which
parasitemia was suppressed to one-half that of untreated
controls. Similarly, the ED90 represents the dose at which
parasitemia is suppressed 10-fold. Mice were considered cured
of their infection if no parasites were detected in the blood 30
days from the first drug administration, and the non-
recrudescence dose (NRD) represents the lowest dose to
achieve a cure.
Robenidine, 16, 22, and 21 were evaluated for in vivo
antimalarial efficacy. That robenidine exhibited respectable in
vivo antimalarial activity was somewhat surprising given that it
is poorly absorbed and known to accumulate in the
gastrointestinal tract.44 The early hit compound 16 (the 4-
OCF3 analogue, ED50 1.2 mg/kg/day) showed improved in
vivo activity over robenidine (ED50 = 1.6 mg/kg/day), while
22 (the 4-CN analogue, ED50 = 2.7 mg/kg/day) did not.
Unexpectedly, 21 (the 3-CN analogue, ED50 = 7.1 mg/kg/day)
was 6-fold less efficacious than 16 in vivo, despite being 6-fold
more active than 16 in vitro. For comparative purposes,
consider that the ED50 of chloroquine in this system is 1.5 mg/
kg/day.42
Figure 3. Inhibition of P. falciparum Dd2 growth by 16 (green) and
21 (purple) at 48 h (dotted curves), 72 h (dashed curves), and 96 h
(solid curves) time points.
assay measures drug-treated parasite growth during the
incubation time relative to untreated parasites. A shorter
incubation time allows for less parasite growth for all drug
conditions, producing data with proportionally greater
variability or “noise”. Conversely, longer incubation times
reduce noise in the resulting data.
IC50 values for both 16 and 21 were somewhat higher at 48
h than at longer drug incubation times (Table 4), though this
Table 4. P. falciparum Dd2 IC50 of 16 and 21 vs Drug
a
Incubation Length
IC50 Dd2 (nM)
compound
48 h
72 h
96 h
16
21
410
31
195
21
192
22
a
See Table 1 legend and Methods.
may be the result of noise in the data associated with the short
incubation time. This finding may also stem from the use of
asynchronous parasite cultures, wherein one life-cycle stage
may be more impacted than another (16 in particular appears
to exhibit a biphasic concentration−response curve, indicative
of stage specificity). IC50 values for both compounds were
virtually identical between 72-h and 96-h incubation time
points. These results indicate that a 72-h incubation may be
required to attain full in vitro activity, but that no additional
benefit is construed with longer incubation times. Amino-
guanidine activity appears to be driven primarily by drug
The only aminoguanidine which produced a cure in this
model was 16 with NRD of 12.5 mg/kg/day, while other
compounds were not curative (including compound 1
described below). It is important to note that failure to
produce a cure in this model and by this dosing regimen is not
predictive of clinical failure. Indeed, several approved clinical
drugs such as chloroquine are not curative in this model at any
dose level.
1 is a Highly Potent Antimalarial in Vitro and in Vivo.
The discrepancy between the in vitro success (Table 2) and in
E
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vivo mediocrity (Table 5) of 21 remained a mystery which we
later explored. From previous in vivo SAR studies on other
scaffolds, we had noted that aryl groups without protective
substitutions at the para positions were biologically unstable.
While 16 was substituted at the para position, 21 was not,
potentially leaving this position vulnerable to hepatic micro-
somal degradation.
To interrogate this hypothesis, an analogue of 21 was
prepared with an additional para-fluoro substituent (1, 3-CN,
4-F). Given the low activity of 4 (4-F), this substitution was
expected to have little effect on in vitro potency while
potentially improving upon the in vivo activity of 21.
Unexpectedly, 1 had excellent in vitro activity, becoming the
new series lead in potency. 1 had a single-digit nanomolar IC50
value against D6 (4 nM), IC50 values in the low double digits
for the drug-resistant strains, and an in vitro therapeutic index
of 2000-fold.
The in vivo efficacy of 1 was even more pronounced with an
ED50 value of 0.25 mg/kg/day, 5-fold lower than its nearest
competitor 16. The single atom difference between 21 (3-CN,
4-H) and 1 (3-CN, 4-F) resulted in a nearly 30-fold
improvement in in vivo efficacy.
Murine Microsomal Stability of Aminoguanidines
Correlates with in Vivo Activity. To investigate the
relationship between the aminoguanidines’ in vivo efficacy
and their metabolic properties, the murine microsomal stability
of a selection of aminoguanidines was evaluated (Table 6).
combination of high intrinsic potency and biological stability.
Although speculative at this time, it is also possible that the
nitrogen atoms in the aminoguanidine bridge of 1 exhibit
diminished basicity (i.e., ionizability) due to the presence of
two strongly electron withdrawing groups (F and CN) at the
para and meta positions of the flanking aromatic rings, which
may in turn enhance oral bioavailability.
Further exploration of the aminoguanidine scaffold is
certainly warranted, as is developing more knowledge of its
antimalarial properties, including the mode of action. Assessing
the in vivo activity of these compounds in humanized mice may
refine predictions of clinical success. Assessing the compounds
against synchronous parasites will elucidate potential stage-
specific potency effects. Beyond the blood stage, evaluating the
aminoguanidines against other stages of the malaria life cycle
such as the liver stage will provide valuable information useful
for their potential development for use in humans. Systemati-
cally exploring the mechanism of action, including chemical
biology techniques and resistance studies, can further guide
SAR for this chemotype, and we are currently engaged in this
work. Potential synergy with other antimalarial compounds
such as artemisinin, atovaquone, and ELQ-300 will also be
assessed.
As we look for new entries in the antimalarial pipeline, it
may be useful to reexamine drugs and chemotypes effective in
related parasites and pathogens. This appears to be the case
with robenidine, a drug discovered in 1970 but which has not
been methodically explored in malaria using a medicinal
chemistry approach until this point. Though robenidine itself
has reasonably good antimalarial activity in vitro and in vivo, it
did not take long to improve upon this activity in both settings.
In this study, a second look at an old drug efficiently produced
a new and promising chemical lead.
a
Table 6. Murine Microsomal Stability of Aminoguanidines
murine microsomal stability, t1/2
predicted Clint
(mL/min/kg)
compound
(min)
ketanserin
robenidine
16
21
1
14.47
158.65
172.16
58.18
377.20
34.40
31.70
93.80
29.32
METHODS
■
General Chemistry: Materials and Instruments. All
solvents, starting materials, and reagents were acquired from
commercial sources (Sigma-Aldrich and Combi-Blocks).
Robenidine was obtained from Santa Cruz Biotechnology
(Santa Cruz, California). All materials were used without
further purification. 1H and 13C NMR spectra were taken on a
Bruker 400 MHz instrument, and chemical shifts are reported
relative to TMS (0.0 ppm). Fluorescence measurements were
recorded using a Molecular Devices Spectramax iD3 equipped
with Softmax Pro 7 software. Final compounds were measured
to be >95% pure by high performance liquid chromatography
(HPLC) using an Agilent Technologies 1260 Infinity II system
(unless otherwise noted). High-resolution mass spectrometry
(HRMS) using electrospray ionization was performed by the
Portland State University BioAnalytical Mass Spectrometry
Facility. Melting points were measured using a Stanford
Research Systems OptiMelt Automated Melting Point System
(model MPA100).
186.15
a
Data from ChemPartner Co. Ltd., Shanghai, P.R. China. See
Methods for full details.
Robenidine, 16, 21, 1, and the control compound ketanserin
were incubated with pooled murine liver microsomes and
monitored for degradation by LC/MS/MS for 1 h. The
concentration vs time plot for each compound was used to
determine its biological half-life (t1/2) and predicted intrinsic
clearance (Clint).
For all of the aminoguanidines evaluated, murine micro-
somal stability correlated with in vivo efficacy. Robenidine, 16,
and 1 were all biologically stable with half-lives above 150 min
(with the same rank order for in vivo activity and stability). 21
was metabolically unstable in the presence of murine
microsomes, with a half-life of only 58.18 min.
This data supports the notion that 1 is more efficacious in
vivo than 21 due in part to improved stability. Substituting the
4-position H for F resulted in a threefold increase in metabolic
stability. Presumably the prolonged presence of 1 in the
bloodstream contributes to its excellent performance in vivo.
1: 2,2′-Bis[(3-cyano-4-fluorophenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 3-
cyano-4-fluorobenzaldehyde (1.31 g, 8.8 mmol, 2.2 equiv) and
1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 1.54 g, 99%. 1H NMR
(400 MHz, DMSO-d6) δ 12.56 (s, 2H), 8.76 (s, 2H), 8.64 (d,
CONCLUSIONS
■
1 is a robenidine derivative with excellent in vitro potency,
virtually no cross-resistance in multidrug-resistant strains, and a
high in vitro therapeutic index. In a murine model of malaria, 1
displayed robust in vivo antimalarial activity propelled by a
F
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J = 5.63 Hz, 2H), 8.46 (s, 2H), 8.33−8.32 (m, 2H), 7.68 (t, J =
8.92, 2H). 13C NMR (400 MHz, DMSO-d6) δ 164.98, 162.40,
153.57, 146.25, 136.94, 135.95, 133.22, 131.61, 131.57, 117.73,
117.53, 114.22, 101.56, 101.40. HRMS found 352.1111, M+H.
MP = 317−319 °C.
(1.23 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 g, 4 mmol, 1 equiv) in ethanol (5 mL) was
refluxed for 16 h. Diethyl ether (10 mL) was added, and the
product carbonimidic dihydrazide crashed out of solution as a
white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.48 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 12.39
(s, 2H), 8.69 (s, 2H), 8.43 (s, 2H), 8.17 (s, 2H), 7.83 (d, J =
6.49 Hz, 2H), 7.59−7.50 (m, 2H). 13C NMR (400 MHz,
DMSO-d6) δ 153.40, 147.93, 135.91, 134.27, 131.11. 130.92,
127.71, 127.06. HRMS found 334.0619, M+H. MP = 268−270
°C.
7: 2,2′-Bis[(2-bromophenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 2-bromobenzaldehyde
(1.23 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 g, 4 mmol, 1 equiv) in ethanol (5 mL) was
refluxed for 16 h. Diethyl ether (10 mL) was added, and the
product carbonimidic dihydrazide crashed out of solution as a
white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.83 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 12.52
(s, 2H), 8.80 (s, 2H), 8.66 (s, 2H), 8.39 (d, J = 7.64 Hz, 2H),
7.73 (d, J = 7.94, 2H), 7.51 (t, J = 7.43 Hz, 2H), 7.43 (t, J =
7.56, 2H). 13C NMR (400 MHz, DMSO-d6) δ 153.21, 133.66,
133.00, 132.53, 128.87, 128.44, 124.44. HRMS found
423.9586, M+H. MP = 279−281 °C.
2: 2,2′-Bis[(2-fluorophenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 2-fluorobenzaldehyde
(1.1 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
1
Yield: 1.34 g, 99%. H NMR (400 MHz, DMSO-d6) δ 12.54
(s, 2H), 8.72 (s, 2H), 8.64 (s, 2H), 8.36 (dt, J = 7.9, 1.72, 2H),
7.59−7.53 (m, 2H), 7.34 (t, J = 7.9 Hz, 4H). 13C NMR (400
MHz, DMSO-d6) δ 162.21, 159.71, 152.74, 141.62, 132.93,
127.31, 124.75, 120.82, 116.07, 115.86. HRMS found
302.1206, M+H. MP = 293−295 °C.
3: 2,2′-Bis[(3-fluorophenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 3-fluorobenzaldehyde
(1.09 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
8: 2,2′-Bis[(3-bromophenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 3-bromobenzaldehyde
(1.63 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
1
Yield: 1.31 g, 97%. H NMR (400 MHz, DMSO-d6) δ 12.56
(s, 2H), 8.69 (s, 2H), 8.48 (s, 2H), 7.98 (d, J = 10.3 Hz, 2H),
7.70(d, 7.6 Hz, 2H), 7.53 (q, J = 7.3, 2H), 7.33 (t, J = 8.3, 2H).
13C NMR (400 MHz, DMSO-d6) δ 164.14, 161.72, 153.47,
148.02, 136.37, 136.28, 131.34, 131,26, 125.37, 118.15, 117.94,
113.89, 113.66. HRMS found 302.1206, M+H. MP = 281−283
°C.
1
Yield: 1.80 g, 98%. H NMR (400 MHz, DMSO-d6) δ 12.28
4: 2,2′-Bis[(4-fluorophenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 4-fluorobenzaldehyde
(1.09 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
(s, 2H), 8.66 (s, 2H), 8.39 (s, 2H), 8.29 (t, J = 3.08 Hz, 2H),
7.87 (d, J = 7.80, 2H), 7.68 (dd, J = 8.00, 2.79, 2H), 7.45 (t, J =
7.86, 2H). 13C NMR (400 MHz, DMSO-d6) δ 136.13, 133.80,
131.34, 129.95, 128.05, 122.83. HRMS found 423.9589, M+H.
MP = 258−260 °C.
9: 2,2′-Bis[(4-bromophenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 4-bromobenzaldehyde
(1.63 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 g, 4 mmol, 1 equiv) in ethanol (5 mL) was
refluxed for 16 h. Diethyl ether (10 mL) was added, and the
product carbonimidic dihydrazide crashed out of solution as a
white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
1
Yield: 1.33 g, 99%. H NMR (400 MHz, DMSO-d6) δ 12.32
(s, 2H), 8.56 (s, 2H), 8.44 (s, 2H), 8.03 (t, J = 6.5 Hz, 4H),
7.34 (t, J = 8.2, 4H). 13C NMR (400 MHz, DMSO-d6) δ
165.24, 162.77, 153.41, 148.14, 130.71, 130.62, 130.48, 130.45,
116.40, 116.19. HRMS found 302.1205, M+H. MP = 295−297
°C.
1
Yield: 1.73 g, 94%. H NMR (400 MHz, DMSO-d6) δ 12.32
5: 2,2′-Bis[(2-chlorophenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 2-chlorobenzaldehyde
(1.23 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 g, 4 mmol, 1 equiv) in ethanol (5 mL) was
refluxed for 16 h. Diethyl ether (10 mL) was added, and the
product carbonimidic dihydrazide crashed out of solution as a
white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
(s, 2H), 8.61 (s, 2H), 8.41 (s, 2H), 7.91 (d, J = 8.24 Hz, 4H),
7.70 (d, J = 8.21, 4H). 13C NMR (400 MHz, DMSO-d6) δ
153.32, 148.29, 133.05, 132.23, 130.23, 124.70. HRMS found
423.9587, M+H. MP = 295−297 °C.
10: 2,2′-Bis[(4-methylphenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 4-methylbenzalde-
hyde (1.06 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
1
Yield: 1.42 g, 96%. H NMR (400 MHz, DMSO-d6) δ 12.62
(s, 2H), 8.88 (s, 2H), 8.69 (s, 2H), 8.43 (d, J = 7.15 Hz, 2H),
7.58−7.46 (m, 6H). 13C NMR (400 MHz, DMSO-d6) δ
153.19, 145.53, 134.06, 132.77, 131.03, 130.39, 128.51, 127.95.
HRMS found 334.0620, M+H. MP = 295−297 °C.
6: 2,2′-Bis[(3-chlorophenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 3-chlorobenzaldehyde
1
Yield: 1.11 g, 84%. H NMR (400 MHz, DMSO-d6) δ 12.18
(s, 2H), 8.46 (s, 2H), 8.39 (s, 2H), 7.83 (d, J = 7.8 Hz, 4H),
G
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7.30 (d, J = 7.8 Hz, 4H), 2.37 (s, 6H). 13C NMR (400 MHz,
DMSO-d6) δ 152.08, 148.22, 140.13, 130.00, 128.74, 127.24,
20.51. HRMS found 294.1708, M+H. MP = 246−248 °C.
11: 2,2′-Bis[(4-methoxyphenyl)methylene]carbonimidic
Dihydrazide Hydrochloride. A solution of 4-methoxybenzal-
dehyde (1.2 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
1
methanol as a hydrochloride salt. Yield: 660 mg, 70%. H
NMR (400 MHz, DMSO-d6) δ 12.43, (s, 2H), 8.71 (s, 2H),
8.47 (s, 2H), 8.09 (s, 2H), 7.93 (d, J = 7.6 Hz, 2H), 7.63 (t, J =
7.9, 2H), 7.49 (d, J = 8.3 Hz, 2H). 13C NMR (400 MHz,
DMSO-d6) δ 153.48, 149.29, 147.93, 136.22, 131.31, 128.06,
123.45, 121.84, 120.13. HRMS found 434.1039, M+H. MP =
241−243 °C.
16: 2,2′-Bis[(4-trifluoromethoxyphenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
trifluoromethoxybenzaldehyde (1.67 g, 8.8 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 1.03 g, 55%. 1H NMR
(400 MHz, DMSO-d6) δ 12.24 (s, 2H), 8.61 (s, 2H), 8.45 (s,
2H), 8.10 (d, J = 8.9 Hz, 4H), 7.50 (d, J = 8.1 Hz, 4H). 13C
NMR (400 MHz, DMSO-d6) δ 150.26, 133.07, 130.34,
121.70. HRMS found 434.1041, M+H. MP = 278−280 °C.
17: 2,2′-Bis[(4-trifluoromethoxyphenyl)ethylidene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
trifluoromethoxyacetophenone (1.79 g, 8.8 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 1.88 g, 95%. 1H NMR
(400 MHz, DMSO-d6) δ 11.91 (s, 2H), 8.78 (s, 2H), 8.19 (d,
J = 8.8 Hz, 4H), 7.44 (d, J = 8.1, 4H), 2.46 (s, 6H). 13C NMR
(400 MHz, DMSO-d6) δ 154.66, 149.85, 136.38, 129.60,
121.79, 121.17, 119.24. HRMS found 462.1352, M+H. MP =
323−325 °C.
18: 2,2′-Bis[(4-difluoromethoxyphenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
difluoromethoxybenzaldehyde (757 mg, 4.4 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (250 g, 2 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 0.63 g, 73%. 1H NMR
(400 MHz, DMSO-d6) δ 12.28 (s, 2H), 8.56 (s, 2H), 8.43 (s,
2H), 8.02 (d, J = 8.6, 4H), 7.38 (t, J = 74, 2H), 7.29 (d, J = 8.6
Hz, 4H). 13C NMR (400 MHz, DMSO-d6) δ 153.17, 130.67,
130.22, 119.01, 116.58. HRMS found 398.1231, M+H. MP =
245−247 °C.
1
Yield: 1.37 g, 94%. H NMR (400 MHz, DMSO-d6) δ 12.00
(s, 2H), 8.37 (s, 2H), 8.35 (s, 2H), 7.88 (d, J = 9.1 Hz, 4H),
7.04 (d, J = 9.1 Hz, 4H), 3.83 (s, 6H). 13C NMR (400 MHz,
DMSO-d6) δ 181.34, 152.55, 129.56, 125.90, 114.22, 55.38.
HRMS found 326.1608, M+H. MP = 218−220 °C.
12: 2,2′-Bis[(4-trifluoromethylphenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of
trifluoromethylbenzaldehyde (1.53 g, 8.8 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
1
methanol as a hydrochloride salt. Yield: 1.75 g, 100%. H
NMR (400 MHz, DMSO-d6) δ 12.55 (s, 2H), 8.78 (s, 2H),
8.55 (s, 2H), 8.19 (d, J = 7.8 Hz, 4H), 7.85 (d, J = 8.1, 4H).
13C NMR (400 MHz, DMSO-d6) δ 153.12, 147.44, 137.21,
130.44, 130.12, 128.51, 125.53, 125.39, 122.68. HRMS found
402.1140, M+H. MP = 273−275 °C.
13: 2,2′-Bis[(4-trifluoromethylphenyl)ethylidene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
trifluoromethylacetophenone (1.65 g, 8.8 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 1.78 g, 96%. 1H NMR
(400 MHz, DMSO-d6) δ 12.04 (s, 2H), 8.89 (s, 2H), 8.28 (d,
J = 7.6 Hz, 4H), 7.81 (d, J = 8.4, 4H), 2.5 (s, 6H). 13C NMR
(400 MHz, DMSO-d6) δ 154.75, 141.00, 128.26, 125.63,
123.28. HRMS found 430.1455, M+H. MP = 334−336 °C.
14: 2,2′-Bis[(2-trifluoromethoxyphenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 2-
trifluoromethoxybenzaldehyde (1.67 g, 8.8 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
1
19: 2,2′-Bis[(4-difluoromethoxyphenyl)ethylidene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
difluoromethoxyacetophenone (818 mg, 4.4 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (250 mg, 2 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 1.87 g, 100%. H
NMR (400 MHz, DMSO-d6) δ 12.54 (s, 2H), 8.75 (s, 2H),
8.71 (s, 2H), 8.48 (d, J = 7.8 Hz, 2H), 7.67−7.50 (m, 6H). 13
C
NMR (400 MHz, DMSO-d6) δ 152.21, 146.36, 131.98,
127.28, 127.14, 125.67, 121.03, 120.74, 118.19. HRMS found
434.1039, M+H. MP = 194−196 °C.
15: 2,2′-Bis[(3-trifluoromethoxyphenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 3-
trifluoromethoxybenzaldehyde (835 mg, 4.4 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (250 mg, 2 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
1
methanol as a hydrochloride salt. Yield: 990 mg, 100%. H
NMR (400 MHz, DMSO-d6) δ 11.78 (s, 2H), 8.72 (s, 2H),
8.13 (d, J = 8.8 Hz, 4H), 7.36 (t, J = 73.56, 2H), 7.24 (d, J =
8.6, 2H). 13C NMR (400 MHz, DMSO-d6) δ 154.53, 152.90,
H
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152.66, 133.97, 129.39, 119.19, 118.54, 116.62, 114.06, 15.33.
HRMS found 426.1538, M+H. MP = 273−275 °C.
carbonimidic dihydrazide crashed out of solution as a white
solid. The product was filtered, washed with diethyl ether, and
recrystallized from methanol as a hydrochloride salt. Yield:
1.19 g, 99%. 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 2H),
8.53 (s, 2H), 8.44 (s, 2H), 7.96−7.94 (m, 4H), 7.50−7.49 (m,
6H). 13C NMR (400 MHz, DMSO-d6) δ 133.27, 130.77,
128.73, 127.86. HRMS found 266.1398, M+H. MP = 245−247
°C.
25: 2,2′-Bis[(4-phenol)methylene]carbonimidic Dihydra-
zide Hydrochloride. A solution of 4-hydroxybenzaldehyde (1.1
g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine hydro-
chloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL) was
refluxed for 16 h. Diethyl ether (10 mL) was added, and the
product carbonimidic dihydrazide crashed out of solution as a
white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.33 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 11.95
(s, 2H), 10.15 (s, 2H), 8.28 (s, 2H), 7.75 (d, J = 8.1 Hz, 4H),
6.87 (d, J = 8.3, 4H). 13C NMR (400 MHz, DMSO-d6) δ
160.56, 130.15, 124.76, 116.10. HRMS found 298.1296, M+H.
MP = 185−187 °C.
26: 2,2′-Bis[(4-diphenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 4-phenylbenzaldehyde
(1.60 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.81 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 12.32
(s, 2H), 8.59 (s, 2H), 8.49 (s, 2H), 8.05 (d, J = 7.3 Hz, 2H),
7.81 (d, J = 7.3 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.51 (t, J =
7.3 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H). 13C NMR (400 MHz,
DMSO-d6) δ 153.28, 142.71, 139.72, 132.89, 129.52, 128.99,
128.51, 127.42, 127.28. HRMS found 418.2022, M+H. MP =
286−288 °C.
27: 2,2′-Bis[(4-nitrophenyl)methylene]carbonimidic Dihy-
drazide Hydrochloride. A solution of 4-nitrobenzaldehyde
(1.33 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.56 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 12.68
(s, 2H), 8.82 (s, 2H), 8.57 (s, 2H), 8.33 (d, J = 8.3 Hz, 4H),
8.24 (d, J = 8.3 Hz, 4H). 13C NMR (400 MHz, DMSO-d6) δ
153.79, 148.78, 147.27, 139.98, 129.35, 124.35. HRMS found
356.1098, M+H. MP = 285−287 °C.
20: 2,2′-Bis[(2-cyanophenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 2-cyanobenzaldehyde
(1.15 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
1
Yield: 1.23 g, 88%. H NMR (400 MHz, DMSO-d6) δ 12.88
(s, 2H), 8.8 (s, 2H), 8.74 (s, 2H), 8.52 (d, J = 7.8 Hz, 2H),
7.96 (d, J = 7.8 Hz, 2H), 7.84 (t, J = 7.7 Hz, 2H), 7.68 (t, J =
7.7, 2H), 7.34 (t, J = 50.59, 2H). 13C NMR (400 MHz,
DMSO-d6) δ 145.01. 136.32, 133.91, 133.88, 131.56, 127.55,
117.67, 111.51, 56.48, 19.03. HRMS found 316.1301, M+H.
MP = 213-215 °C.
21: 2,2′-Bis[(3-cyanophenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 3-cyanobenzaldehyde
(1.15 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.40 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 12.44
(s, 2H), 8.74 (s, 2H), 8.56 (s, 2H), 8.46 (s, 2H), 8.22 (d, J =
8.1 Hz, 2H), 7.95 (d, J = 7.8 Hz, 2H), 7.71 (t, J = 7.6 Hz, 2H).
13C NMR (400 MHz, DMSO-d6) δ 153.60, 147.27, 135.10,
134.21, 133.25, 131.24, 130.50, 118.95, 112.49. HRMS found
316.1300, M+H. MP = 278−280 °C.
22: 2,2′-Bis[(4-cyanophenyl)methylene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 4-cyanobenzaldehyde
(1.15 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 1.40 g, 100%. 1H NMR (400 MHz, DMSO-d6) δ 12.69
(s, 2H), 8.79 (s, 2H), 8.52 (s, 2H), 8.17 (d, J = 8.2 Hz, 4H),
7.97 (d, J = 8.3 Hz, 4H). 13C NMR (400 MHz, DMSO-d6) δ
153.64, 147.70, 138.14, 133.11, 128.92, 119.09, 113.05. HRMS
found 316.1303, M+H. MP = 303−305 °C.
23: 2,2′-Bis[(3-cyanophenyl)ethylidene]carbonimidic Di-
hydrazide Hydrochloride. A solution of 3-cyanoacetophenone
(1.28 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguanidine
hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5 mL)
was refluxed for 16 h. Diethyl ether (10 mL) was added, and
the product carbonimidic dihydrazide crashed out of solution
as a white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
28: 2,2′-Bis[(4-dimethylaminophenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of
N,N-dimethylaminobenzaldehyde (1.31 g, 8.8 mmol, 2.2
equiv) and 1,3-diaminoguanidine hydrochloride (500 mg, 4
mmol, 1 equiv) in ethanol (5 mL) was refluxed for 16 h.
Diethyl ether (10 mL) was added, and the product
carbonimidic dihydrazide crashed out of solution as a white
solid. The product was filtered, washed with diethyl ether, and
recrystallized from methanol as a hydrochloride salt. Yield: 90
mg, 6%. This compound was chemically unstable in
chromatography solvents but was greater than 80% pure
when used in in vitro assays. 1H NMR (400 MHz, DMSO-d6)
δ 11.74 (s, 2H), 8.22 (s, 2H), 8.14 (s, 2H), 7.71 (d, J = 7.8 Hz,
4H), 6.75 (d, J = 7.8 Hz, 4H), 3.00 (s, 6H). 13C NMR (400
1
Yield: 1.40 g, 92%. H NMR (400 MHz, DMSO-d6) δ 11.89
(s, 2H), 8.90 (s, 2H), 8.63 (t, J = 1.44 Hz, 2H), 8.33 (dt, J =
7.75, 1.39, 2H), 7.92 (dt, J = 7.76, 2.44, 2H), 7.67 (t, J = 7.88,
2H), 2.48 (s, 6H). 13C NMR (400 MHz, DMSO-d6) δ 154.70,
152.16, 138.21, 133.59, 132.06, 131.04, 130.09, 119.21, 112.16,
15.29. HRMS found 344.1612, M+H. MP = 304−306 °C.
24: 2,2′-Bis(phenylmethylene)carbonimidic Dihydrazide
Hydrochloride. A solution of benzaldehyde (933 mg, 8.8
mmol, 2.2 equiv) and 1,3-diaminoguanidine hydrochloride
(500 mg, 4 mmol, 1 equiv) in ethanol (5 mL) was refluxed for
16 h. Diethyl ether (10 mL) was added, and the product
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MHz, DMSO-d6) δ 152.50, 152.35, 129.69, 129.41, 121.22,
120.96, 112.01. HRMS found 352.2237, M+H. MP = 132−134
°C.
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
1
29: 2,2′-Bis[(phenyl-4-carbonate)methylene]carbonimidic
Dihydrazide Hydrochloride. A solution of 4-formylphenylcar-
bonate (1.32 g, 8.8 mmol, 2.2 equiv) and 1,3-diaminoguani-
dine hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5
mL) was refluxed for 16 h. Diethyl ether (10 mL) was added,
and the product carbonimidic dihydrazide crashed out of
solution as a white solid. The product was filtered, washed with
diethyl ether, and recrystallized from methanol as a hydro-
chloride salt. Yield: 600 mg, 39%. 1H NMR (400 MHz,
DMSO-d6) δ 13.13 (s, 2H), 12,57 (s, 2H), 8,70 (s, 2H), 8.52
(s, 2H), 8.11 (d, J = 7.7 Hz, 4H), 8.03 (d, J = 7.7 Hz, 4H). 13C
NMR (400 MHz, DMSO-d6) δ 193.48, 167.31, 167.02,
153.47, 148.46. 139.35, 137.72, 136.12, 132.79, 130.39, 130.03,
128.40. HRMS found 354.1195, M+H. MP = 336−338 °C.
30: 2,2′-Bis[(4-carbonamidophenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
carbonamidobenzaldehyde (655 mg, 4.4 mmol, 2.2 equiv) and
1,3-diaminoguanidine hydrochloride (250 mg, 2 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 850 mg, 90%. H
NMR (400 MHz, DMSO-d6) δ 11.84 (s, 2H), 8.26 (s, 4H),
7.77 (d, J = 8.1 Hz, 4H), 7.01 (d, J = 8.1 Hz, 4H), 3.75 (s,
8H), 3.24 (s, 8H). 13C NMR (400 MHz, DMSO-d6) δ 153.09,
152.75, 149.18, 129.58, 123.87, 114.53, 66.40, 47.78. HRMS
found 436.2449, M+H. MP = 280−282 °C.
34: 2,2′-Bis[(3-tetrazole-1-phenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 3-
tetrazole-1-benzaldehyde (766 mg, 4.4 mmol, 2.2 equiv) and
1,3-diaminoguanidine hydrochloride (250 g, 2 mmol, 1 equiv)
in ethanol (5 mL) was refluxed for 16 h. Diethyl ether (10 mL)
was added, and the product carbonimidic dihydrazide crashed
out of solution as a white solid. The product was filtered,
washed with diethyl ether, and recrystallized from methanol as
a hydrochloride salt. Yield: 870 mg, 100%. This compound was
not sufficiently soluble in chromatography solvents to obtain a
1
quantitative purity by HPLC. H NMR (400 MHz, DMSO-
d6) δ 8.84 (s, 2H), 8.75 (s, 2H), 8.66 (s, 2H), 8.20 (t, J = 9.0
Hz, 4H), 7.76 (t, J = 7.78, 2H). 13C NMR (400 MHz, DMSO-
d6) δ 152.27, 147.44, 133.85, 129.83, 129.27, 128.30, 125.70,
124.25. HRMS found 402.1641, M+H. MP = 289−291 °C.
35: 2,2′-Bis[(4-tetrazole-1-phenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
tetrazole-1-benzaldehyde (1.53 g, 8.8 mmol, 2.2 equiv) and
1,3-diaminoguanidine hydrochloride (500 mg, 4 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
methanol as a hydrochloride salt. Yield: 1.75 g, 100%. This
compound was not sufficiently soluble in chromatography
1
methanol as a hydrochloride salt. Yield: 770 mg, 100%. H
NMR (400 MHz, DMSO-d6) δ 12.43 (s, 2H), 8.85 (s, 2H),
8.50 (s, 2H), 8.13 (s, 2H), 8.04 (d, J = 7.8 Hz, 4H), 7.98 (d, J
= 7.8 Hz, 4H), 7.50 (s, 2H). 13C NMR (400 MHz, DMSO-d6)
δ 167.70, 153.41, 148.59, 136.32, 136.26, 128.30, 128.15.
HRMS found 352.5100, M+H. MP = 309−311 °C.
31: 2,2′-Bis[(4-sulphonamidophenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
sulphonamidobenzaldehyde (814 mg, 4.4 mmol, 2.2 equiv)
and 1,3-diaminoguanidine hydrochloride (250 mg, 2 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
(10 mL) was added, and the product carbonimidic dihydrazide
crashed out of solution as a white solid. The product was
filtered, washed with diethyl ether, and recrystallized from
1
solvents to obtain a quantitative purity by HPLC. H NMR
(400 MHz, DMSO-d6) δ 8.70 (s, 2H), 8.52 (s, 2H), 8.20 (s,
8H), 3.47−3.42 (m, 2H), 1.06 (t, J = 7.0 Hz, 2H). 13C NMR
(400 MHz, DMSO-d6) δ 153.39, 148.45, 136.23, 129.19,
127.77. HRMS found 402.1641, M+H. MP = 277−279 °C.
36: 2,2′-Bis{[4-(2,2-difluoro-2,3-dioxazolo)]phenyl}-
methylene]carbonimidic Dihydrazide Hydrochloride. A
solution of 4-(2,2-difluoro-2,3-dioxazolo)benzaldehyde (820
mg, 4.4 mmol, 2.2 equiv) and 1,3-diaminoguanidine hydro-
chloride (250 mg, 2 mmol, 1 equiv) in ethanol (5 mL) was
refluxed for 16 h. Diethyl ether (10 mL) was added, and the
product carbonimidic dihydrazide crashed out of solution as a
white solid. The product was filtered, washed with diethyl
ether, and recrystallized from methanol as a hydrochloride salt.
Yield: 800 mg, 87%. 1H NMR (400 MHz, DMSO-d6) δ 12.58
(s, 2H), 8.66 (s, 2H), 8.60 (s, 2H), 8.04 (d, J = 7.74 Hz, 2H),
7.52 (dd, J = 7.97, 0.93, 2H), 7.32 (t, J = 8.12, 2H). 13C NMR
(400 MHz, DMSO-d6) δ 142.55, 140.90, 140.27, 130.60,
123.91, 120.71, 116.23, 111.11. HRMS found 426.0814, M+H.
MP = 276−278 °C.
37: 2,2′-Bis[(2,4-dimethoxy-5-chlorophenyl)ethylidene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 2,4-
dimethoxy-5-chloroacetophenone (655 mg, 4.4 mmol, 2.2
equiv) and 1,3-diaminoguanidine hydrochloride (250 mg, 2
mmol, 1 equiv) in ethanol (5 mL) was refluxed for 16 h.
Diethyl ether (10 mL) was added, and the product
carbonimidic dihydrazide crashed out of solution as a white
solid. The product was filtered, washed with diethyl ether, and
recrystallized from methanol as a hydrochloride salt. Yield: 400
1
methanol as a hydrochloride salt. Yield: 920 mg, 100%. H
NMR (400 MHz, DMSO-d6) δ 12.41, (s, 2H), 8.69 (s, 2H),
8.50 (s, 2H), 8.14 (6, J = 7.6 Hz, 4H), 7.91 (d, J = 7.6, 4H),
7.49 (s, 4H). 13C NMR (400 MHz, DMSO-d6) δ 153.49,
146.00, 136.81, 128.72, 125.43. HRMS found 424.0852, M+H.
MP = 285−287 °C.
32: 2,2′-Bis[(4-propynoxyphenyl)methylene]carbonimidic
Dihydrazide Hydrochloride. A solution of 4-propynoxyben-
zaldehyde (704 mg, 4.4 mmol, 2.2 equiv) and 1,3-
diaminoguanidine hydrochloride (250 g, 2 mmol, 1 equiv) in
ethanol (5 mL) was refluxed for 16 h. Diethyl ether (10 mL)
was added, and the product carbonimidic dihydrazide crashed
out of solution as a white solid. The product was filtered,
washed with diethyl ether, and recrystallized from methanol as
a hydrochloride salt. Yield: 0.82 g, 100%. 1H NMR (400 MHz,
DMSO-d6) δ 12.19 (s, 2H), 8.42 (s, 2H), 8.37 (s, 2H), 7.91
(d, J = 8.4 Hz, 4H). 13C NMR (400 MHz, DMSO-d6) δ
159.67, 153.09, 129.93, 127.06, 115.56, 79.38, 79.06, 56.09.
HRMS found 374.1606, M+H. MP = 217−219 °C.
33: 2,2′-Bis[(4-morpholinophenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of 4-
morpholinobenzaldehyde (840 mg, 4.4 mmol, 2.2 equiv) and
1,3-diaminoguanidine hydrochloride (250 mg, 2 mmol, 1
equiv) in ethanol (5 mL) was refluxed for 16 h. Diethyl ether
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1
mg, 39%. H NMR (400 MHz, DMSO-d6) δ 11.64 (s, 2H),
commercial malaria drugs atovaquone and chloroquine were
used as control drugs. Test plates were incubated for 72 h at 37
°C in an environmental chamber with a controlled low oxygen
atmosphere (5% O2, 5% CO2, 90% N2). After the incubation
period, 100 μL SYBR Green I dye-detergent solution was
added to each well, and the plates were incubated at ambient
temperature and atmosphere in the dark for at least 1 h.
Fluorescence was read at 497 nm excitation and 520 nm
emission bands using a Spectramax iD3 plate reader.
Fluorescence readings were normalized with respect to the
untreated control wells representing normal parasite growth
and plotted against the logarithm of drug concentration. An
IC50 was determined for each compound by fitting this data to
a variable slope nonlinear regression curve using Graphpad
Prism software (v. 8).
8.55 (s, 2H), 7.68 (s, 2H), 6.85 (s, 2H), 3.94 (s, 6H), 3.91 (s,
6H), 2.31 (s, 6H). 13C NMR (400 MHz, DMSO-d6) δ 157.97,
156.84, 154.64, 154.53, 130.97, 130.65, 120.76, 112.75, 98.42,
98.30, 57.21, 56.96, 56.79, 32.07, 19.31. HRMS found
482.1350, M+H. MP = 229−231 °C.
38: 2-(Phenylmethylene)-2′-[(2-Fluorophenyl)methylene]-
carbonimidic Dihydrazide Hydrochloride. A solution of
benzaldehyde (466 mg, 4.4 mmol, 1.1 equiv), 2-fluorobenzal-
dehyde (546 mg, 4.4 mmol, 1.1 equiv), and 1,3-diaminogua-
nidine hydrochloride (500 mg, 4 mmol, 1 equiv) in ethanol (5
mL) was refluxed for 16 h. Diethyl ether (10 mL) was added,
and the product carbonimidic dihydrazide crashed out of
solution as a white solid. The product was filtered, washed with
diethyl ether, and recrystallized from methanol as a hydro-
chloride salt. This product was isolated as a mixture also
containing 24 and 2. 38 was the dominant product accounting
for greater than 50% of the total material in the mixture. Yield:
1.27 g mixture, 50%. 1H NMR (400 MHz, DMSO-d6) δ 12.30
(s, 3H), 8.69 (s, 1H), 8.62 (s, 1H), 8.57 (s, 1H), 8.53 (s, 1H),
8.44 (s, 1H), 8.35 (t, J = 6.5 Hz, 1H), 7.96−7.94 (m, 2H), 7.56
(q, J = 8.1 Hz, 1H), 7.51−7.49 (m, 2H), 7.34 (t, J = 8.1, 2H).
13C NMR (400 MHz, DMSO-d6) δ 133.27, 130.78, 128.74,
127.89, 127.28, 124.80, 115.88. HRMS found 284.1301, M+H.
MP = 281−283 °C.
General Biology: Parasite Culture and Drug Sensi-
tivity. The following parasite strains were used in this study
and obtained through BEI Resources, NIAID, NIH. Plasmo-
dium falciparum, Strain D6 (MRA-285, originally from Sierra
Leone, has modest resistance to mefloquine).46 Strain Dd2
(MRA-150, originated from Indochina; derived from W2-mef
and is resistant to chloroquine, pyrimethamine, and
mefloquine). P. falciparum strain Tm90−C2B (Thailand;
resistant to mefloquine, chloroquine, atovaquone, and
pyrimethamine) was obtained directly from the Division of
Experimental Therapeutics of Walter Reed Army Institute of
Research (WRAIR) in Silver Spring, Maryland, United
States.39 Strain SB1-A6 (MRA-1002, Sierra Leone was derived
from D6 clone and is resistant to Atovaquone and ELQ-300.40
P. falciparum parasites were thawed from frozen stock and
cultured in suspended human erythrocytes (Lampire Biological
Laboratories, Pipersville, PA) not more than 28 days old at 2%
hematocrit. The culture medium used was RPMI-1640,
supplemented with 25 mM HEPES buffer, 25 mg/L
gentamicin sulfate, 45 mg/L hypoxanthine, 10 mM glucose,
2 mM glutamine, and 0.5% Albumax II (complete medium).43
Cultures were maintained in a standard low oxygen
atmosphere (5% O2, 5% CO2, 90% N2) in an environmental
chamber and incubated at 37 °C. Cultures were subpassaged
every 3−4 days into a fresh culture flask containing complete
media and erythrocytes.
HepG2 Cytotoxicity Assay. Compounds were prepared as
10 mM stock solutions in DMSO. Human hepatocarcinoma
(HepG2) cells were maintained in culture at 37 °C in a
humidified 5% CO2 atmosphere in RPMI-1640 medium
containing 10% fetal bovine serum. HepG2 cells were added
to each well of flat bottom 96-well tissue culture plates at an
initial density of 2 × 104 cells per well and an initial volume of
160 μL complete medium per well. After an overnight
incubation at 37 °C to adhere the cells to the culture plates,
40 μL drug solutions in complete medium were applied to
each well at a final concentration range of 0 to 200 μM across
each plate. Drugs were tested in triplicate or quadruplicate.
The cells were incubated for 24 h at 37 °C and 5% CO2 with
the drug solutions, which were then aspirated and replaced
with 200 μL per well of complete medium for an additional 24
h incubation under the same conditions. To each well was
added 20 μL of resazurin (Alamar Blue) in PBS buffer to a final
concentration of 10 μM, and the plates were incubated for 3 h.
Fluorescence was measured at 560 nm excitation and 590 nm
emission bands using a Spectramax iD3 plate reader.
Fluorescence readings were normalized with respect to the
untreated control wells and plotted against the logarithm of
drug concentration. An IC50 was determined for each
compound by fitting this data to a variable slope nonlinear
regression curve using Graphpad Prism software (v. 8).
In Vivo Efficacy against Murine Malaria. The in vivo
ED50 and ED90 of selected aminoguanidines was measured
using a modified 4-day Peters test. Female CF1 mice from
Charles River Laboratories were inoculated intravenously with
approximately 2.5−5.0 × 104 parasitized erythrocytes (murine
malaria P. yoelii, Kenya strain MR4 MRA-428) from a donor
mouse (experiment day zero). On the following 4 days
(experiment days 1−4), solutions of the test compounds in
PEG-300 (or PEG-300 only for control mice) were
administered by oral gavage once daily. Aminoguanidines
were initially assessed at 2.5, 5, and 10 mg/kg/day, and
experiments were repeated to adjust the dose range as needed
to obtain an interpolated ED50 and ED90 value (1 required a
dosing down to 0.1 mg/kg/day to attain this result).
Experiments were repeated with doses up to 25 mg/kg/day
to obtain a nonrecrudescence dose, though only 16 was found
to produce a cure in this model. Parasitemia of each mouse was
determined by microscopic examination of Giemsa stained
blood smears on day 5. ED50 and ED90 values were assessed by
generating dose−response curves relative to untreated controls
using Graphpad Prism (v. 8). Mice were considered cured of
malarial infection if they maintained 0% parasitemia at
experiment day 30. The procedures involved, together with
IC50 Determination by the Fluorescence-Based SYBR
Green Assay. The aminoguanidine series was assessed for in
vitro antiplasmodial activity using the fluorescence-based SYBR
Green assay described previously by Smilkstein and co-
workers.38 Compounds were evaluated in quadruplicate in
flat-bottomed Costar clear 96-well plates (Model #3585). A 2-
fold serial dilution of each compound was performed across
the columns of the test plates starting with 20 μM and ending
with a final untreated column to serve as control wells.
Asynchronous parasite infected erythrocytes in growth media
were added to each well for a total volume of 100 μL, final
hematocrit of 2%, and initial parasitemia of 0.2%. The
K
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all matters relating to the care, handling, and housing of the
animals used in this study, were approved by the Portland VA
Medical Center Institutional Animal Care and Use Committee.
Murine Microsomal Stability. Metabolic stability studies
of selected aminoguanidines were performed at ChemPartner,
Shanghai, China. Compounds were incubated at 37 °C and 1
μM concentration in murine liver microsomes (Corning) for 1
h at a protein concentration of 0.5 mg/mL in potassium
phosphate buffer at pH 7.4 containing 1.0 mM EDTA. The
metabolic reaction was initiated by NADPH and quenched
with ice-cold acetonitrile at 15 min increments up to 1 h. The
progress of compound metabolism was followed by LC-MS/
MS (ESI positive ion, LC-MS/MS-034(API-6500+) using a
C18 stationary phase (ACQUITY UPLC BEH C18 (2.1 × 50
mm, 1.7 μm)) and a MeOH/water mobile phase containing
0.25% FA and 1 mM NH4OAc. Imipramine or Osalmid were
used as internal standards, and ketanserin was used as a
metabolically unstable control compound. Concentration
versus time data for each compound were fitted to an
exponential decay function to determine the first-order rate
constant for substrate depletion, which was then used to
calculate the degradation half-life (t1/2) and predicted intrinsic
clearance value (Clint) from an assumed murine hepatic blood
flow of 90 mL/min/kg.
ACKNOWLEDGMENTS
■
This project was supported with funds from the United States
Department of Veterans Affairs, Veterans Health Adminis-
tration, Office of Research and Development Program Award
i01 BX003312 (M.K.R.). M.K.R. is a recipient of a VA
Research Career Scientist Award (14S-RCS001). Research
reported in this publication was also supported by the United
States National Institutes of Health under Awards
R01AI100569 and R01AI141412 (M.K.R.), the United States
Department of Defense Peer Reviewed Medical Research
Program (Log # PR181134) (M.K.R.), and the National
Institute of Allergy and Infectious Diseases under Award
T32AI007472 (A.K.). Murine metabolic stability studies were
performed by ChemPartner, Shanghai, P.R. China. HRMS was
performed at the Portland State University BioAnalytical Mass
Spectrometry Facility by Rozalia Dodean, MS.
DEDICATION
■
This manuscript is dedicated to Dr. Jonathan Vennerstrom in
honor of his 65th birthday and for his outstanding
contributions to the field of antimalarial drug research and
development.
REFERENCES
■
(1) World Malaria Report 2020. World Health Organization:
Geneva, 2020.
ASSOCIATED CONTENT
■
(2) Dondorp, A. M., Nosten, F., Yi, P., Das, D., Phyo, A. P., Tarning,
J., Lwin, K. M., Ariey, F., Hanpithakpong, W., Lee, S. J., Ringwald, P.,
Silamut, K., Imwong, M., Chotivanich, K., Lim, P., Herdman, T., An,
S. S., Yeung, S., Singhasivanon, P., Day, N. P., Lindegardh, N.,
Socheat, D., and White, N. J. (2009) Artemisinin resistance in
Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455−467.
(3) Hooft van Huijsduijnen, R., and Wells, T. N. (2018) The
antimalarial pipeline. Curr. Opin. Pharmacol. 42, 1−6.
(4) Kantor, S., Kennett, R. L., Jr., Waletzky, E., and Tomcufcik, A. S.
(1970) 1,3-Bis(p-chlorobenzylideneamino)guanidine hydrochloride
(robenzidene): new poultry anticoccidial agent. Science 168, 373−374.
(5) Reid, W. M., Kowalski, L. M., Taylor, E. M., and Johnson, J.
(1970) Efficacy evaluations of robenzidene for control of coccidiosis
in chickens. Avian Dis. 14, 788−796.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00001.
Calculated error values for antiplasmodial IC50 and
cytotoxicity assays (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Alina Krollenbrock − Department of Chemical Physiology
and Biochemistry, Oregon Health & Science University,
Portland, Oregon 97239, United States; VA Medical Center,
Portland, Oregon 97239, United States; orcid.org/0000-
0003-3460-0808; Email: krollenb@ohsu.edu
Michael K. Riscoe − VA Medical Center, Portland, Oregon
97239, United States; Department of Molecular Microbiology
and Immunology, Oregon Health & Science University,
Portland, Oregon 97239, United States; orcid.org/0000-
0002-1343-5279; Email: riscoem@ohsu.edu
(6) Ryley, J. F., and Wilson, R. G. (1971) Studies on the mode of
action of the coccidiostat robenidene. Z. Parasitenkd. 37, 85−93.
(7) Ryley, J. F., and Wilson, R. G. (1972) The development of
Eimeria brunetti in tissue culture. J. Parasitol. 58, 660−663.
(8) Ryley, J. F., and Wilson, R. G. (1972) Comparative studies with
anticoccidials and three species of chicken coccidia in vivo and in vitro.
J. Parasitol. 58, 664−668.
(9) Kennett, R. L., Kantor, S., and Gallo, A. (1974) Efficacy studies
with robenidine, a new type of anticoccidial, in the diet. Poult. Sci. 53,
978−986.
Authors
(10) Wong, D. T., Horng, J. S., and Wilkinson, J. R. (1972)
Robenzidene, an inhibitor of oxidative phosphorylation. Biochem.
Biophys. Res. Commun. 46, 621−627.
Yuexin Li − VA Medical Center, Portland, Oregon 97239,
United States
Jane Xu Kelly − VA Medical Center, Portland, Oregon 97239,
United States; orcid.org/0000-0001-8871-0207
(11) Berger, H., Wang, G. T., Shor, A. L., Gale, G. O., and Simkins,
K. L. (1974) Safety evaluation of robenidine in the feed of broiler
chickens. Poult. Sci. 53, 1013−1015.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.1c00001
(12) Long, P. L., and Millard, B. J. (1978) Studies on Eimeria grenieri
in the guinea fowl (Numida meleagris). Parasitology 76, 1−9.
(13) Peeters, J. E., Halen, P., and Meulemans, G. (1979) Efficacy of
robenidine in the prevention of rabbit coccidiosis. Br. Vet. J. 135,
349−354.
Notes
The authors declare the following competing financial
interest(s): Coauthors M.R. and A.K. have filed an invention
disclosure relating to selected compounds described in this
paper to the United States Department of Veterans Affairs and
to Oregon Health & Science University as required by law.
(14) Wilson, R. J., Denny, P. W., Preiser, P. R., Rangachari, K.,
Roberts, K., Roy, A., Whyte, A., Strath, M., Moore, D. J., Moore, P.
W., and Williamson, D. H. (1996) Complete gene map of the plastid-
like DNA of the malaria parasite Plasmodium falciparum. J. Mol. Biol.
261, 155−172.
L
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pubs.acs.org/journal/aidcbc
Article
(15) Ito, S., Tsunoda, K., and Nishikawa, H. (1974) Effects of
robenidine and some other drugs on mice experimentally infected
with toxoplasma. Natl. Inst Anim Health Q (Tokyo) 14, 129−136.
(16) Krylov, M. V., Sokolov, A. N., Zaionts, V. I., Korovitskaia, L. A.,
and Zakharova, N. A. (1976) Khimotsid for treatment and prevention
of toxoplasmosis. Veterinariia 11, 69−71.
J., and McCluskey, A. (2018) Gram-Positive and Gram-Negative
Antibiotic Activity of Asymmetric and Monomeric Robenidine
Analogues. ChemMedChem 13, 2573−2580.
(34) Russell, C. C., Stevens, A., Young, K. A., Baker, J. R.,
McCluskey, S. N., Khazandi, M., Pi, H., Ogunniyi, A., Page, S. W.,
Trott, D. J., and McCluskey, A. (2019) Discovery of 4,6-bis(2-((E)-
benzylidene)hydrazinyl)pyrimidin-2-Amine with Antibiotic Activity.
ChemistryOpen 8, 896−907.
(35) Abraham, R. J., Abraham, S., Stevens, A. J., Page, S. W.,
McCluskey, A., Trott, D. J., and O’Handley, R. M. (2019)
Aminoguanidines: New leads for treatment of Giardia duodenalis
infection. Int. J. Parasitol.: Drugs Drug Resist. 10, 38−44.
(36) Khazandi, M., Pi, H., Chan, W. Y., Ogunniyi, A. D., Sim, J. X. F.,
Venter, H., Garg, S., Page, S. W., Hill, P. B., McCluskey, A., and Trott,
D. J. (2019) In vitro Antimicrobial Activity of Robenidine,
Ethylenediaminetetraacetic Acid and Polymyxin B Nonapeptide
Against Important Human and Veterinary Pathogens. Front. Microbiol.
10, 837.
(37) Das Gupta, R., Krause-Ihle, T., Bergmann, B., Muller, I. B.,
Khomutov, A. R., Muller, S., Walter, R. D., and Luersen, K. (2005) 3-
Aminooxy-1-aminopropane and derivatives have an antiproliferative
effect on cultured Plasmodium falciparum by decreasing intracellular
polyamine concentrations. Antimicrob. Agents Chemother. 49, 2857−
2864.
(17) Bakhtari, S. K., and Jira, J. (1988) Chemotherapy of
experimental toxoplasmosis with special reference to robenidine.
Folia Parasitol (Praha) 35, 193−198.
(18) Ricketts, A. P., and Pfefferkorn, E. R. (1993) Toxoplasma
gondii: susceptibility and development of resistance to anticoccidial
drugs in vitro. Antimicrob. Agents Chemother. 37, 2358−2363.
(19) Mukhopadhyay, R., Kapoor, P., and Madhubala, R. (1996)
Antileishmanial effect of a potent S-adenosylmethionine decarbox-
ylase inhibitor: CGP 40215A. Pharmacol. Res. 33, 67−70.
(20) Yao, J. M., Zhang, H. B., Liu, C. S., Tao, Y., and Yin, M. (2015)
Inhibitory effects of 19 antiprotozoal drugs and antibiotics on Babesia
microti infection in BALB/c mice. J. Infect. Dev. Countries 9, 1004−
1010.
(21) Huang, Q., Cao, J., Zhou, Y., Huang, J., Gong, H., Zhang, H.,
Zhu, X. Q., and Zhou, J. (2017) Babesia microti Aldo-keto Reductase-
Like Protein Involved in Antioxidant and Anti-parasite Response.
Front. Microbiol. 8, 2006.
(22) Bacchi, C. J., Brun, R., Croft, S. L., Alicea, K., and Buhler, Y.
(1996) In vivo trypanocidal activities of new S-adenosylmethionine
decarboxylase inhibitors. Antimicrob. Agents Chemother. 40, 1448−
1453.
(23) Brun, R., Burri, C., and Gichuki, C. W. (2001) The story of
CGP 40 215: studies on its efficacy and pharmacokinetics in African
green monkey infected with Trypanosoma brucei rhodesiense. Trop.
Med. Int. Health 6, 362−368.
(24) Beswick, T. C., Willert, E. K., and Phillips, M. A. (2006)
Mechanisms of allosteric regulation of Trypanosoma cruzi S-
adenosylmethionine decarboxylase. Biochemistry 45, 7797−7807.
(25) Dec, M., Puchalski, A., Stepien-Pysniak, D., Marek, A., and
Urban-Chmiel, R. (2020) Susceptibility of chicken Lactobacillus
bacteria to coccidiostats. J. Vet. Med. Sci. 82, 333−336.
(26) Stanley, V. G., Woldesenbet, S., Gray, C., and Hinton, A., Jr.
(1996) Sensitivity of Escherichia coli O157:H7 strain 932 to selected
anticoccidial drugs in broiler chicks. Poult. Sci. 75, 42−46.
(27) Ogbunude, P. O., and Asiri, S. A. (2001) In vitro effect of
diamidines on intracellular polyamines of Acanthamoeba polyphaga.
Drugs Exp Clin Res. 27, 127−133.
(28) Molnar, K., and Ostoros, G. (2007) Efficacy of some
anticoccidial drugs for treating coccidial enteritis of the common
carp caused by Goussia carpelli (Apicomplexa: Eimeriidae). Acta Vet.
Hung. 55, 67−76.
(29) Hansen, M., Krogh, K. A., Brandt, A., Christensen, J. H., and
Halling-Sorensen, B. (2009) Fate and antibacterial potency of
anticoccidial drugs and their main abiotic degradation products.
Environ. Pollut. 157, 474−480.
(38) Smilkstein, M., Sriwilaijaroen, N., Kelly, J. X., Wilairat, P., and
Riscoe, M. (2004) Simple and inexpensive fluorescence-based
technique for high-throughput antimalarial drug screening. Anti-
microb. Agents Chemother. 48, 1803−1806.
(39) Nilsen, A., LaCrue, A. N., White, K. L., Forquer, I. P., Cross, R.
M., Marfurt, J., Mather, M. W., Delves, M. J., Shackleford, D. M.,
Saenz, F. E., Morrisey, J. M., Steuten, J., Mutka, T., Li, Y., Wirjanata,
G., Ryan, E., Duffy, S., Kelly, J. X., Sebayang, B. F., Zeeman, A. M.,
and Riscoe, M. K. (2013) Quinolone-3-diarylethers: a new class of
antimalarial drug. Science Translational Medicine 5, 177ra37.
(40) Smilkstein, M. J., Forquer, I., Kanazawa, A., Kelly, J. X., Winter,
R. W., Hinrichs, D. J., Kramer, D. M., and Riscoe, M. K. (2008) A
drug-selected Plasmodium falciparum lacking the need for conven-
tional electron transport. Mol. Biochem. Parasitol. 159, 64−68.
(41) Peters, W., Fleck, S. L., Robinson, B. L., Stewart, L. B., and
Jefford, C. W. (2002) The chemotherapy of rodent malaria. LX. The
importance of formulation in evaluating the blood schizontocidal
activity of some endoperoxide antimalarials. Ann. Trop. Med. Parasitol.
96, 559−573.
(42) Pou, S., Winter, R. W., Nilsen, A., Kelly, J. X., Li, Y., Doggett, J.
S., Riscoe, E. W., Wegmann, K. W., Hinrichs, D. J., and Riscoe, M. K.
(2012) Sontochin as a guide to the development of drugs against
chloroquine-resistant malaria. Antimicrob. Agents Chemother. 56,
3475−3480.
(43) Trager, W., and Jensen, J. B. (1976) Human malaria parasites in
continuous culture. Science 193, 673−675.
(44) Zulalian, J., and Gatterdam, P. E. (1973) Absorption, excretion,
and metabolism of robenz, robenidine hydrochloride (1,3-bis(p-
chlorobenzylideneamino)guanidine hydrochloride), in the rat. J. Agric.
Food Chem. 21, 794−797.
(45) Dahl, E. L., and Rosenthal, P. J. (2007) Multiple antibiotics
exert delayed effects against the Plasmodium falciparum apicoplast.
Antimicrob. Agents Chemother. 51, 3485−3490.
(46) Oduola, A. M., Weatherly, N. F., Bowdre, J. H., and Desjardins,
R. E. (1988) Plasmodium falciparum: cloning by single-erythrocyte
micromanipulation and heterogeneity in vitro. Exp. Parasitol. 66, 86−
95.
(30) Claes, Z., Jonkhout, M., Crespillo-Casado, A., and Bollen, M.
(2019) The antibiotic robenidine exhibits guanabenz-like cytopro-
tective properties by a mechanism independent of protein
phosphatase PP1:PPP1R15A. J. Biol. Chem. 294, 13478−13486.
(31) Abraham, R. J., Stevens, A. J., Young, K. A., Russell, C., Qvist,
A., Khazandi, M., Wong, H. S., Abraham, S., Ogunniyi, A. D., Page, S.
W., O’Handley, R., McCluskey, A., and Trott, D. J. (2016)
Robenidine Analogues as Gram-Positive Antibacterial Agents. J.
Med. Chem. 59, 2126−2138.
(32) Ogunniyi, A. D., Khazandi, M., Stevens, A. J., Sims, S. K., Page,
S. W., Garg, S., Venter, H., Powell, A., White, K., Petrovski, K. R.,
Laven-Law, G., Totoli, E. G., Salgado, H. R., Pi, H., Coombs, G. W.,
Shinabarger, D. L., Turnidge, J. D., Paton, J. C., McCluskey, A., and
Trott, D. J. (2017) Evaluation of robenidine analog NCL195 as a
novel broad-spectrum antibacterial agent. PLoS One 12,
No. e0183457.
(33) Russell, C. C., Stevens, A., Pi, H., Khazandi, M., Ogunniyi, A.
D., Young, K. A., Baker, J. R., McCluskey, S. N., Page, S. W., Trott, D.
M
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ACS Infect. Dis. XXXX, XXX, XXX−XXX