1-Benzyl-3-aryl-2-thiohydantoin Derivatives as New Anti-Trypanosoma brucei Agents: SAR and in Vivo Efficacy

Article abstract of DOI:10.1021/acsmedchemlett.7b00230

A high throughput screening and subsequent hit validation identified compound 1 as an inhibitor of Trypanosoma brucei parasite growth. Extensive structure-activity relationship optimization based on antiparasitic activity led to the highly potent compounds, 1-(4-fluorobenzyl)-3-(4-dimethylamino-3-chlorophenyl)-2-thiohydantoin (68) and 1-(2-chloro-4-fluorobenzyl)-3-(4-dimethylamino-3-methoxyphenyl)-2-thiohydantoin (76), with a T. brucei EC₅₀ of 3 and 2 nM, respectively. This represents >100-fold improvement in potency compared to compound 1. In vivo efficacy experiments of 68 and 76 in an acute mouse model of Human African Trypanosomiasis showed a 100% cure rate after 4 days of oral treatment at 50 mg/kg twice per day.

Full text of DOI:10.1021/acsmedchemlett.7b00230

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Letter
1-Benzyl-3-aryl-2-thiohydantoin derivatives as new anti-
Trypanosoma brucei agents: SAR and in-vivo efficacy
Andriy Buchynskyy, J. Robert Gillespie, Zackary Michel Herbst,
Renae M. Ranade, Frederick S. Buckner, and Michael H. Gelb
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00230 • Publication Date (Web): 10 Jul 2017
Downloaded from http://pubs.acs.org on July 18, 2017
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-Benzyl-3-aryl-2-thiohydantoin derivatives as new anti-
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Trypanosoma brucei agents: SAR and in-vivo efficacy
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Andriy Buchynskyy, J. Robert Gillespie, Zackary M. Herbst, Ranae M. Ranade, Frederick S.
b*
a*
Buckner, Michael H. Gelb
a
Department of Chemistry, University of Washington, Seattle, WA 98195, USA
b
Department of Medicine, University of Washington, Seattle, WA 98109, USA.
KEYWORDS: Human African Trypanosomiasis, ‘‘Sleeping Sickness”, Trypanosoma brucei inhibitor, Thiohydantoins, Hit-
to-lead optimization.
ABSTRACT: A high throughput screening and subsequent hit validation identified compound 1 as an inhibitor of Trypanosoma
brucei parasite growth. Extensive SAR optimization based on antiparasitic activity led to the highly potent compounds, 1ꢀ(4ꢀ
fluorobenzyl)ꢀ3ꢀ(4ꢀdimethylaminoꢀ3ꢀchlorophenyl)ꢀ2ꢀthiohydantoin (68) and 1ꢀ(2ꢀchloroꢀ4ꢀfluorobenzyl)ꢀ3ꢀ(4ꢀdimethylaminoꢀ3ꢀ
methoxyphenyl)ꢀ2ꢀthiohydantoin (76), with a T. brucei EC50 of 3 nM and 2 nM, respectively. This represents >100ꢀfold improveꢀ
ment in potency compared to compound 1. In vivo efficacy experiments of 68 and 76 in an acute mouse model of Human African
Trypanosomiasis showed a 100 % cure rate after 4 days of oral treatment at 50 mg/kg twice per day.
1
1,12
13
HAT (Human African Trypanosomiasis) or sleeping sickness is
one of the most neglected diseases of human, caused by parasites
of Trypanosoma brucei species, and spread through the bite of
infected tsetse flies. Most of the morbidity is due to the late neuroꢀ
logical stage when parasites cross the bloodꢀbrain barrier. Without
treatment, the disease is invariably fatal. The WHO approved
treatment for lateꢀstage HAT is nifurtimoxꢀeflornithine combinaꢀ
tion therapy (NECT) which requires intravenous administration,
antimicrobials,
antiꢀulcer and antiꢀinflammatories, as well as
14
herbicides. Enzalutamide is an FDA approved anticancer drug
with a 2ꢀthiohydantoin pharmacophore and is an androgenic reꢀ
ceptor antagonist. Despite their use in multiple disease indicaꢀ
tions, 2ꢀthiohydantoins should not be regarded as promiscuous
inhibitors, but rather as a scaffold with a distinct intermolecular
interaction profile that can be usefully exploited for drug develꢀ
1
5
16
opment.
1
skilled staff and inpatient monitoring. A new drug, effective for
Different synthetic methods to prepare 2ꢀthiohydantoin and its
derivatives have been described. The most commonly used methꢀ
ods are the treatment of αꢀamino acids with acetic anhydride folꢀ
lowed by ammonium thiocyanate or the coupling reaction beꢀ
tween αꢀamino acid derivatives and isothiocyanate.
late stage disease, that is nontoxic and orally administered, is
urgently needed. Moreover, this drug should be affordable by
national health systems in the diseaseꢀendemic countries located
in subꢀSaharan Africa.
17
18,19
We have
In the course of a high throughput phenotypic screen for comꢀ
pounds that inhibit T. brucei growth in vitro, we discovered the
substituted 2ꢀthiohydantoin (1) (Figure 1).
used the isothiocyanate route to prepare our thiohydantoin library,
as it is the most suitable for our purposes. Nꢀbenzyl substituted
glycine esters and isothioocyanates were prepared as reagents for
the synthesis of 1,3ꢀdisubstituted 2ꢀthiohydantions (Scheme 1, 2).
Depending on the commercial availability of starting materials
and chemical tractability, Nꢀsubstituted glycine esters were preꢀ
pared by reductive amination of aromatic aldehydes with glycine
esters (Scheme 1) or by nucleophilic substitution of bromoacetic
esters with benzyl amines (Scheme 2). For synthesis of isothiocyꢀ
anates, not only the commercial availability and chemical tractaꢀ
bility, but also structural features, such as the presence of a haloꢀ
gen in the aromatic ring, determined the synthetic route. The 3ꢀ
chloroꢀ4ꢀN,Nꢀdimethylphenyl isothiocyanate (7) was prepared by
aromatic nucleophilic substitution of fluorine with dimethylamine,
reduction of the nitroꢀgroup in the presence of halogens (chlorine)
with tin chloride, and finally the conversion of the aminoꢀgroup to
the isothiocyanate by treatment with thiophosgene in a heterogeꢀ
neous mixture (Scheme 1).
2
,3
Figure 1. Screening hit, a 2ꢀthiohydantoin (compound 1)
Compounds with the 2ꢀthiohydantoin moiety have been investiꢀ
Scheme 1. Representative synthesis of 2-thiohydantoin
derivative 68
4
a
gated for a variety of applications, including hypolipidemics,
5
6
7
8ꢀ10
anticarcinogenics, antimutagenics, antithyroidals, antivirals,
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2
Compound
R
R
T.brucei
EC50 (nM)
a
Reagents and conditions: (a) Et N, mol. sieves 3Å, NaCNBH ,
3
3
1
4ꢀOMe
4ꢀOMe
3ꢀCl
3ꢀCl
H
346
CHCl , rt, yield 48%; (b) dimethylamine HCl salt, K CO ,
DMSO, 80ºC, yield 95%; (c) SnCl , EtOAc/EtOH, 80ºC, yield
6
3
2
3
14
15
>10000
>20000
>10000
>10000
>10000
>20000
>10000
>10000
>10000
>10000
>10000
>10000
1500
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4%; (d) CSCl , DCMꢀaq.NaHCO , 4ºCꢀrt, yield 84%; (e) EtOH,
2
3
H
rt, yield 70%.
1
1
6
7
H
H
The preparation of 3ꢀmethoxyꢀ4ꢀN,Nꢀdimethylphenyl isothiocyaꢀ
nate (13) started with reductive alkylation of 2ꢀmethoxyꢀ4ꢀ
nitroaniline, followed by reduction of the nitroꢀgroup with hydroꢀ
gen/palladium catalyst and conversation of the aminoꢀgroup to the
isothiocyanate (Scheme 2). The final condensation and subseꢀ
quent cyclization reaction of the Nꢀsubstituted glycine ester with
isothiocyanates was done in ethanol at room temperature. The
corresponding 2ꢀthiohydantoin derivatives were precipitated from
the reaction mixture or purified by column chromatography.
3ꢀOMe
4ꢀCl
H
18
19
H
2ꢀCl
H
2
2
2
0
1
2
5ꢀCl
2ꢀOMe
3ꢀCl
3ꢀCl
2ꢀF
H
4ꢀCF
4ꢀF
3
23
24
5ꢀCl
4ꢀF
Scheme 2. Representative synthesis of 2-thiohydantoin
derivative 76
a
2
2
2
5
6
7
3ꢀCF
4ꢀN(Me)
4ꢀisoPr
4ꢀOH
3
5ꢀCF
H
3
2
H
>10000
>10000
>10000
>10000
637
28
H
2
3
3
9
0
1
4ꢀC(O)Me
4ꢀCOOH
H
H
4ꢀOMe
2ꢀOMe
3ꢀOMe
3ꢀOMe
3ꢀCl
a
_
Reagents and conditions: (a) THF, 4 ºC rt, yield 62% ; (b) paraꢀ
formaldehyde, AcOH, NaCNBH , yield 94%; (c) Pd/C, H ,
32
4ꢀOMe
161
3
2
3
3
4ꢀOMe, 5ꢀOMe
>10000
16
MeOH, yield 60%; (d) CSCl , DCMꢀaq.NaHCO , 4 ºC –rt, yield
9
2
3
34
4
ꢀN(Me)2
4%; (e) EtOH, rt, yield 84%.
3
5
4ꢀN(Me)
2
3ꢀF
125
Hit compound 1 showed subꢀmicromolar activity in T. brucei
growth inhibition assays, low CYP inhibition activity, and no
detected cytotoxicity on the mammalian CRLꢀ8155 or Hep G2
cell lines. Compound 1 displayed moderately rapid degradation in
the presence of murine microsomes (Figure 1). Improving T.
brucei activity and microsomal stability were the first prerequiꢀ
sites for advancing this scaffold to efficacy testing in mouse modꢀ
els of T. brucei infection.
36
37
4ꢀN(Me)2
4ꢀOMe
3ꢀOMe
3ꢀN(Me)
13
431
2
a
Concentration of compound required to inhibit growth by 50%
EC ) in T. brucei brucei strain BF427. Pentamidine isethionate
was included as a control for all of the T.brucei EC50 assays with
average ± SEM: 1.15 ± 0.09 nM (n=32)
The breakthrough came with the 4ꢀN,Nꢀdimethylamino analog
26), with an EC50 of 1500 nM, 5 times less active than 1, but at
(
50
We started SAR investigation of this scaffold by modification of
the 3ꢀchloroꢀ4ꢀmethoxyphenyl moiety. Replacing 3ꢀCl and/or 4ꢀ
OMe with hydrogen(s) led to inactive compounds 14, 15, and 16
(Table 1). Changing the substitution position of chlorine or methꢀ
oxyꢀgroup in monoꢀderivatives 17, 18, 19 did not show any posiꢀ
tive effects on the compound’s activity. The relative position of
chlorine and methoxy group was important for the activity as seen
by the inactive 5ꢀchloroꢀ2ꢀmethoxy analog (20). The results indiꢀ
cated that both substituents had synergetic effects on activity, and
their positions in the ring were important. When the methoxy
group in 1 was replaced with the trifluoromethyl 21 or the fluoro
22 group the potency decreased. The combination of chlorine and
fluorine groups in compound 23 as well as monoꢀfluoroꢀ
substitution in the compound 24 phenyl ring led to inactive comꢀ
pounds. Bis–trifluoromethyl derivative 25 was also inactive.
(
least 6 times more potent than 4ꢀmethoxy analogue (14), which
indicated a higher influence on activity from the N,Nꢀ
dimethylamino group compared to the methoxy group. 4ꢀIsoꢀ
propyl 27, 4ꢀphenol 28, 4ꢀacetyl 29 and 4ꢀcarboxylic 30 derivaꢀ
tives were all inactive. Substitution of chlorine in 1 with a methꢀ
oxy group increased the potency of the 3,4ꢀdimethoxy derivative
31 twofold, while 2,4ꢀdimethoxy derivative 32 showed a decrease
in antiꢀtrypanosomal activity (EC50 of 161 nM and 637 nM reꢀ
spectively), confirming the preferable substitution position as the
paraꢀmeta combination. Addition of the third methoxy group at
position 5 to compound 31 was detrimental to activity, as the
corresponding compound 33 was completely inactive. By combinꢀ
ing the best groups and positions mentioned above, we syntheꢀ
sized the 4ꢀN,Nꢀdimentylaminoꢀ3ꢀchloro derivative 34 with T.
brucei EC50 = 16 nM, 4ꢀN,NꢀDimethylaminoꢀ3ꢀmethoxy analog
36 with EC50 = 13 nM, and 4ꢀN,NꢀDimethylaminoꢀ3ꢀfluoro anaꢀ
log 35 with EC50 = 125 nM. Switching the substitution position of
Table 1. In vitro anti-trypanosomal evaluation of N-
benzyl-2-thiohydantoins derivatives (SAR R -R )
1
2
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dimethylamino and methoxyꢀgroup in 36, led to a 30ꢀfold drop in
potency 6ꢀfold compared to 39 while addition at ortho position
1
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9
1
1
1
1
1
1
1
1
1
1
2
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2
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2
2
2
2
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activity in compound 37, which provided further support of the
importance of paraꢀmeta positions for activity.
(48) retained the activity. The relative position of both fluorines
was important as seen by inactive 2,5ꢀdifluoro analog (49). Introꢀ
ducing a larger (compared with H, or F) substituent like cyano
Table 2. In vitro anti-trypanosomal evaluation of 3-chloro-
(
50) at meta position eliminated the activity. The ortho position
4-(dimethylamino)phenyl-2-thiohydantoin
(SAR of R -R )
derivatives
in compound 39 tolerated changes to the size and nature of the
substituents, with 4ꢀfluoroꢀ2ꢀchloro derivative (51) as the most
active analog with EC50 = 46 nM and 4ꢀfluoroꢀ2ꢀmethoxy (52), 4ꢀ
fluoroꢀ2ꢀnitro (53), 4ꢀfluoroꢀ2ꢀtrifluoromethyl (54) analogs conꢀ
sidered for further optimization to increase potency and stability.
3
4
0
1
2
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0
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3
4
5
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7
8
9
0
3
4
Compound
R
R
T.brucei
EC50
a
(
nM)
346
853
52
1
H
4ꢀCl
H
H
38
3
4
4
9
0
1
4ꢀF
H
3ꢀF
H
968
4ꢀMe
4ꢀOMe
4ꢀCF3
4ꢀCN
4ꢀC(O)OEt
3ꢀpyridyl
4ꢀF
H
1553
>20000
>20000
17818
>20000
3000
313
42
H
4
4
4
3
4
5
H
H
Figure 2. 2ꢀThiohydantoin ring modifications
H
To determine the importance of the 2ꢀthiohydantoin core, we
modified the 2ꢀthiohydantoin cycle by conversion to hydantoin (S
to O change) (55) or substitution of the glycine αꢀhydrogen with
ethoxy (56), methyl (57), or carbonyl (58) groups. All changes to
the 2ꢀthiohydantoin core were highly detrimental for antiꢀ
trypanozomal activity. Methylation of the αꢀbenzyl position of 39
resulted in loss of activity for compound 59. It was determined
that the 2ꢀthiohydantoin cycle should remain unmodified in furꢀ
ther optimization. All compounds with a chiral center were preꢀ
pared as racemates.
46
H
4
4
4
5
7
8
9
0
3ꢀF
2ꢀF
2ꢀF
3ꢀCN
2ꢀCl
2ꢀOMe
2ꢀNO2
4ꢀF
63
5ꢀF
9260
>20000
46
4ꢀF
51
4ꢀF
5
5
5
2
3
4
4ꢀF
191
4ꢀF
350
Based on the SAR described above, the next step was to combine
the substituents with the highest influence on activity into one
molecule in order to optimize the compounds antitrypanosomal
properties. We chose more than one pair of substituents (four for
the phenyl ring and seven for the benzyl ring) in order to have a
large pool to select the best compounds from.
4ꢀF
2ꢀCF3
924
a
Concentration of compounds required to inhibit growth by 50%
(EC ) of T. brucei brucei strain BF427. Pentamidine isethionate
5
0
was included as a control for all of the T. brucei EC50 assays with
average ± SEM: 1.15 ± 0.09 nM (n=32)
The next region that has been explored was the substitution on the
Table 3. In vitro activity and cytotoxicity for select 2-
thiohydantoins.
benzyl
ring
in
3ꢀchloroꢀ4ꢀ(dimethylamino)phenylꢀ2ꢀ
thiohydantoin, starting with introduction of substituents at posiꢀ
tion 4. The 4ꢀchloro derivative (38) was less potent than unsubstiꢀ
tuted benzyl compound 1. Compounds with activating or deacꢀ
tivating groups such as 4ꢀmethyl (41), 4ꢀmethoxy (42), 4ꢀtrifluoro
(
43), 4ꢀcyano (44), 4ꢀethylcarboxylate (45) and 3ꢀpyridyl anaꢀ
logue (46) had low or no activity. Improvement of antiparasitic
potency was observed with the 4ꢀfluoro derivative (39), which is
seven times more potent against T. brucei parasites (EC50 = 52
nM) relative to 1. Changing the position of the fluorine from para
to meta (compound 40) decreased potency 18ꢀfold. Based on theꢀ
se results, we speculate that there is not enough space in the bindꢀ
ing pocket for a large substituent near para position and/or there
is specific hydrogen bonding to the fluorine at para position.
Nevertheless, we kept the 4ꢀfluorine substitution in the benzyl
ring and added substituents at ortho and meta positions. The addiꢀ
tion of the second fluorine at meta position (47) decreased the
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1
2
3
4
values below 50 nM and showed no cytotoxicity up to 50 µM.
The most active compounds 68 and 76, with EC50 of 3 and 2
Cmpd
R
R
R
R
T.brucei Cytotoxycity
EC50a CC
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
50
3
b
nM, respectively, were assessed for plasma protein binding,
solubility, and metabolic stability against pooled mouse liver
microsomes. They were also tested in mice for oral pharmacokiꢀ
netics and brain concentrations (at 60 min postꢀdose) to assess
the brain to plasma ratios. (Table 4).
(
nM)
14
49
33
107
115
555
37
2
(x10 nM)
36.8
>50
>50
>50
>50
>50
>50
>50
39
6
6
0
1
OMe
OMe
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
2ꢀCl
H
62
2ꢀF
Table 4. Activity, CYP inhibition, metabolic stability, oral
pharmacokinetics, brain penetration, protein binding and
solubility for compounds 68 and 76.
6
6
6
6
6
6
6
7
3
4
5
6
7
8
9
0
2ꢀOMe
3ꢀF
2ꢀNO2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2ꢀCF3
Compound
68
3.2
76
1.9
a
N(Me)2
Cl
2ꢀCl
T. brucei EC50 (nM)
b
H
2ꢀF
3
CYP3A4 IC50 (nM)
12798
15
7760
c
4
>50
12.6
>50
>50
>50
>50
>50
>50
>50
>50
39.6
>50
>50
>50
>50
>50
>50
Microsome T1/2 (min)
11
d
2ꢀOMe
3ꢀF
11
13
19
22
15
28
2
2.1 ± 0.1
1376 ± 187
Oral PK Cmax (µM)
2.5 ± 0.3
750 ± 130
71
Oral PK AUCd
(min*umol/L)
7
7
7
2
3
4
2ꢀNO2
2ꢀCF3
2ꢀCl
2ꢀF
e
Brain/plasma ratio
0.74 ± 0.30
88.8 ± 1.5
2.1
1.68 ± 0.35
96.3 ± 0.5
4.1
f
Protein binding (%)
N(Me)2
F
g
Solubility at pH 7.4 (µM)
75
a
7
7
7
7
6
7
8
9
N(Me)2 OMe
2ꢀCl
H
Concentration of compound required to inhibit growth by 50%
(EC ) of T. brucei brucei strain BF427. Pentamidine isethionate
50
7
was included as a control for all of the T.brucei EC5 assays with
0
2ꢀF
6
b
average ± SEM= 1.15 ± 0.09 nM (n=32). Concentration to inꢀ
2ꢀOMe
3ꢀF
11
28
44
28
9
hibit CYP3A4 by 50%. Ketoconazole was run as a control with a
value of 29.6 nM, which aligned with other assays (25.62 ± 3.48
80
c
nM (n=29)). Compound halfꢀlife time in mouse microsomes.
8
8
8
1
2
3
2ꢀNO2
2ꢀCF3
2ꢀCl
H
Testosterone (T1/2= 3.97 minutes) and dextromethorphan HBr
(T1/2= 9.51 minutes) were included as controls and fell into acꢀ
ceptable ranges. average ± SEM of testosterone 4.6 ± 0.6 min
PYR
Cl
d
(n=47) and dextromethorphan HBr 10.2 ± 0.9 min (n=51) Avꢀ
84
27
10
erage values ± SEM of three mice each given a single dose at 50
e
mg/kg by oral gavage. Average values ± SEM of three mice
8
5
2ꢀF
f
each given a single ip dose at 5 mg/kg. Protein binding was
a
Concentration of compound required to inhibit growth by 50%
EC ) of T. brucei brucei strain BF427. Pentamidine isethionate
was included as a control for all of the T.brucei EC50 assays with
assessed for each compound in triplicate at 2 ꢁM solution in 50%
mouse plasma. Propranolol was included as a binding control with
(
5
0
g
91.7 ± 1.0 % total binding. Solubility was assessed in phosphate
b
average ± SEM: 1.15 ± 0.09 nM (n=32). Concentration of comꢀ
buffered saline.
pound required to inhibit growth by 50% (CC ) of human lymꢀ
phoblasts (CRLꢀ8155). Quinacrine was included as a control for
all CRLꢀ8155 assays, with an average ± SEM: 2.34 ± 0.31 µM
5
0
With rather moderate metabolic stability, high brain plasma ratios,
a reasonable PK profile and low protein binding, compounds 68
and 76 were selected for in vivo mouse efficacy experiments. In a
model of the acute phase of HAT, five mice were infected with T.
brucei rhodesiense at day 0 and were given the test compounds
for 4 days, beginning 2 days postꢀinfection. Compounds 68 and
76 attained 5/5 cures as determined by the absence of detectable
parasitemia in any of the treated mice for 60 days postinfection
(Figure 3). All mice that received only vehicle showed high paraꢀ
sitemia on the last day of dosing, with all concentrations >1.5 ×
(n=5)
For substituents R and R on the phenyl group, we selected 4ꢀ
1
2
methoxy and 3ꢀmethoxy groups (60-66) and three 4ꢀ
dimethylamino pairs: 4ꢀdimethylamino and 3ꢀchloro (67-73), 4ꢀ
dimethylamino and 3ꢀfluoro (74-75), and 4ꢀdimethylamino and 3ꢀ
methoxy (76-82) groups (Table 3). 4ꢀPyrrolidinoꢀ3ꢀchloro anaꢀ
logues (83-85) were prepared with the aims discussed below. As
for substituents R and R at the benzyl moiety we selected the
7
10 parasites/mL of blood, and were euthanized.
3
4
seven most active 4ꢀfluoro analogues: 4ꢀfluoro and 2ꢀchloro, 4ꢀ
fluoro with H at R , 4ꢀfluoro and 2ꢀfluoro, 4ꢀfluoro and 2ꢀ
4
methoxy, 4ꢀfluoro and 3ꢀfluoro, 4ꢀfluoro and 2ꢀnitro, and 4ꢀfluoro
and 2ꢀtrifluoromethyl groups (Table 3). The influence of each pair
of substituents on the antiꢀT. brucei activity can be evaluated as
1
2
follows (R and R ): (4ꢀN(CH ) and 3ꢀCl) ≥ (4ꢀN(CH ) and 3ꢀ
3
2
3 2
OMe) > (4ꢀPyrrolidino and 3ꢀCl) > (4ꢀN(CH ) and 3ꢀF) ≥ (4ꢀ
3 2
3
4
OMe and 3ꢀOMe). The R and R substituents are in the order : (4ꢀ
F and 2ꢀCl) > (4ꢀF and H) ≥ (4ꢀF and 2ꢀF) > (4ꢀF and 2ꢀOMe) >
(
4ꢀF and 3ꢀF) > (4ꢀF and 2ꢀCF ) ≥ (4ꢀF and 2ꢀNO ) and ordered in
3
2
Table 3 accordingly. Out of 26 compounds, 23 had T. brucei EC50
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Funding Sources
1
2
3
4
5
6
7
8
9
This work was supported by the National Instituted of Health
Grant 5R01AI106850).
(
Notes
The authors declare no competing financial interest.
All rodent experiments were done in compliance with the Univerꢀ
sity of Washington Institutional Animal Care and Use Committee
(IACUC) approved protocol.
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11
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60
ACKNOWLEDGMENT
Authors would like to thank Uyen T. T. Nguyen for her contribuꢀ
tions in collecting cytotoxicity data and Martin Sadilek for recordꢀ
ing high resolution mass spectra.
Figure 3. Mouse efficacy model of acute T. brucei infection. All
mice were infected with T. b. rhodesiense STIB900 on day 0.
Groups of five mice each were treated with compound 68 and
compound 76 (50 mg/kg by oral gavage b.i.d.) or vehicle from
day 2−5 (grayꢀshaded area). Mice were monitored for parasitemia
in tail blood samples through day 60 postꢀinfection
ABBREVIATIONS
HAT, Human African Trypanosomiasis; SAR, Structure–Activity
Relationship; WHO, World Health Organization; PYR, Pyrroliꢀ
dine; SEM, Standard Error of the Mean.
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The 100% cure rate in the murine model demonstrates that in vitro
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thiohydantion core with the highest improvement in antiparasitic
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ACS Publications website.
Biochemical assays, pharmacokinetic and in vivo experiments,
experimental procedures for the synthesis of compounds 68 and
76, general experimental procedure and characterization of comꢀ
pounds 1, 14-85 (PDF)
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(1)
(68)
(76)
EC50 (T.brucei): 346 nM
EC50 (T.brucei): 3.2 nM
EC50 (T.brucei): 1.9 nM
Efficacy (acute HAT mice model):
Efficacy (acute HAT mice model):
1
00% cure rate
100% cure rate
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