Evaluation of substituted ebselen derivatives as potential trypanocidal agents

Article abstract of DOI:10.1016/j.bmcl.2016.12.021

Human African trypanosomiasis is a disease of sub-Saharan Africa, where millions are at risk for the illness. The disease, commonly referred to as African sleeping sickness, is caused by an infection by the eukaryotic pathogen, Trypanosoma brucei. Previously, a target-based high throughput screen revealed ebselen (EbSe), and its sulfur analog, EbS, to be potent in vitro inhibitors of the T. brucei hexokinase 1 (TbHK1). These molecules also exhibited potent trypanocidal activity in vivo. In this manuscript, we synthesized a series of sixteen EbSe and EbS derivatives bearing electron-withdrawing carboxylic acid and methyl ester functional groups, and evaluated the influence of these substituents on the biological efficacy of the parent scaffold. With the exception of one methyl ester derivative, these modifications ablated or blunted the potent TbHK1 inhibition of the parent scaffold. Nonetheless, a few of the methyl ester derivatives still exhibited trypanocidal effects with single-digit micromolar or high nanomolar EC₅₀values.

Full text of DOI:10.1016/j.bmcl.2016.12.021

Accepted Manuscript
Evaluation of substituted ebselen derivatives as potential trypanocidal agents
Heeren M. Gordhan, Stephen L. Patrick, Maria I. Swasy, Amber L. Hackler,
Mark Anayee, Jennifer E. Golden, James C. Morris, Daniel C. Whitehead
PII:
S0960-894X(16)31288-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.021
BMCL 24510
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 November 2016
5 December 2016
7 December 2016
Please cite this article as: Gordhan, H.M., Patrick, S.L., Swasy, M.I., Hackler, A.L., Anayee, M., Golden, J.E.,
Morris, J.C., Whitehead, D.C., Evaluation of substituted ebselen derivatives as potential trypanocidal agents,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.021
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Evaluation of substituted ebselen derivatives as potential trypanocidal agents
Heeren M. Gordhana; Stephen L. Patrickb; Maria I. Swasya; Amber L. Hacklerb;Mark
Anayeea; Jennifer E. Golden,c James C. Morrisb*; Daniel C. Whiteheada*
aDepartment of Chemistry, 467 Hunter Laboratories, Clemson University, Clemson, SC,
29634, USA
bEukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry,
249 Life Sciences Building, Clemson University, Clemson, SC, 29634, USA
cDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin,
777 Highland Ave., Madison, WI 53705-2222, USA
Abstract
Human African trypanosomiasis is a disease of sub-Saharan Africa, where
millions are at risk for the illness. The disease, commonly referred to as African sleeping
sickness, is caused by an infection by the eukaryotic pathogen, Trypanosoma brucei.
Previously, a target-based high throughput screen revealed ebselen (EbSe), and its
sulfur analog, EbS, to be potent in vitro inhibitors of the T. brucei hexokinase 1 (TbHK1).
1

These molecules also exhibited potent trypanocidal activity in vivo. In this manuscript,
we synthesized a series of sixteen EbSe and EbS derivatives bearing electron-
withdrawing carboxylic acid and methyl ester functional groups, and evaluated the
influence of these substituents on the biological efficacy of the parent scaffold. With the
exception of one methyl ester derivative, these modifications ablated or blunted the
potent TbHK1 inhibition of the parent scaffold. Nonetheless, a few of the methyl ester
derivatives still exhibited trypanocidal effects with single-digit micromolar or high
nanomolar EC50 values.
Keywords: ebselen; trypanosomes; hexokinases
2

Graphical Abstract
3

Human African trypanosomiasis (HAT), or African Sleeping Sickness, is caused
by the bloodstream form (BSF) of the eukaryotic pathogen Trypanosoma brucei and is
transmitted by the tsetse fly.1 Two subspecies of T. brucei cause human disease: T. b.
gambiense, responsible for West African sleeping sickness, and T. b. rhodesiense,
responsible for the more acute East African sleeping sickness.1-3 Although treatments for
HAT have existed for decades, these therapeutics are marked by serious adverse side-
effects, such as acute toxicity, neural disorders, and in some cases death.4 In other
cases, the therapeutics currently available are ineffective against the late stages of the
disease once the trypanosomes cross the blood-brain barrier.1,4
BSF trypanosomes generate energy currency, in the form of adenosine
triphosphate (ATP), through glycolysis, which occurs within a specialized organelle
called the glycosome.1,3,4 The first step of glycolysis involves the phosphorylation of
glucose by enzymes known as hexokinases (HKs).4 In trypanosomes, the HKs involved
in the first step of glycolysis are called TbHK1 and TbHK2, with the former having been
genetically and chemically validated for therapeutic design.5 Several known inhibitors of
mammalian hexokinases have demonstrated activity toward the inhibition of TbHK1 in
vitro and exhibit trypanocidal effects in whole cell assays.5 One such agent is
lonidamine exhibiting an IC50 value (half-maximal inhibitory concentration) of 850 M
toward hexokinases in recombinant TbHK1 (rTbHK1), expressed and purified as a
transgene in Escherichia coli or yeast.2
Through a high-throughput screening (HTS) effort conducted by some of us, two
related groups of novel inhibitors, the isobenzothiazolinone and isobenzoselenazolinone
classes, were discovered as inhibitors of TbHK1. One of six isobenzoselenazolinone
4

candidates was ebselen (EbSe, 1, Scheme 1), a drug that is non-toxic to humans and
has been evaluated in clinical trials in ischemic stroke patients.3,5 Ebselen demonstrated
high potency against rTbHK1 with an IC50 of 0.05 ± 0.03 M.3,5 Similarly, EbS (i.e. 2-
phenyl-1,2-benzisothiazol-3(2H)-one, 2, Scheme 1), which replaces the selenium in
ebselen with sulfur, had an IC50 value of 2.0 ± 0.5 M against rTbHK1.3,5 Subsequently,
we demonstrated that incubation of TbHK1 with EbSe and EbS inhibitors led to a five-
fold decrease in hexokinase activity that remained irreversible despite a 200–fold
dilution of enzyme and inhibitor.3 One hypothesis for the efficacy of EbSe toward the
inhibition of the trypanosome enzyme is the formation of selenyl-sulfide (Se-S) adducts
with Cys residues within the hexokinase, leading to the eventual oxidation of certain Cys
residues to the corresponding sulfonic acid.3 Indeed, we demonstrated that EbSe
induced the oxidation of two Cys residues in TbHK1, C327 and C369, resulting in
significantly reduced enzymatic activity.3
Scheme 1. EbSe (1) and EbS (2)
discovered through HTS and further
evaluated by Joice et al.
In this manuscript, we sought to expand upon this initial discovery by exploring
the synthesis of a series of EbSe/EbS derivatives modified with electron-withdrawing
carboxylic acid and methyl ester substituents. Our motivation to pursue these
derivatives was three-fold: 1.) we hypothesized that the incorporation of electron-
withdrawing substituents onto the scaffold might enhance the electrophilicity of the
chalcogen in the core scaffold, thus facilitating the covalent modification of TbHK1 and
5

enhance in vivo potency3, 2.) the carboxylic acid moiety, if tolerated, would offer a
convenient synthetic handle for further exploration of derivative structures, and 3.) the
carboxylic acid moiety would provide a simple handle for the incorporation of the drug
onto a peptidic targeting sequence in order to investigate a novel drug-delivery
strategy.6
Our study commenced with the synthesis of a series of EbSe/EbS derivatives
bearing a carboxymethyl moiety on the nitrogen atom of the heterocycle. The first EbS
derivative 6 was synthesized according to a one-pot protocol described by Dou, et al.11
Thus, the corresponding methyl ester 7 was isolated by alkylation of the nitrogen atom
of 3 in 38% yield (Scheme 2).7 Similarly, 3 was alkylated to give 5, followed by t-Bu
ester cleavage to give acid 6.
Scheme 2: Preparation of EbS derivatives 6 and 7.
The synthesis of the analogous EbSe derivative 13 (Scheme 3) initiated with the
diazotization of anthranilic acid 8.8-10 The diazonium salt 9 was treated with disodium
diselenide to provide diselenide 10, which was subsequently treated with thionyl
chloride under reflux to afford selenyl chloride 11. That compound was then acylated
with the methyl ester of glycine to afford 12 in a modest 20% yield.8,11 Hydrolysis of the
methyl ester in 12 with lithium hydroxide provided EbSe derivative 13 in 73% yield.7
6

Scheme 3: Preparation of EbSe derivatives 12 and 13.
Synthetic protocols for the second EbS and EbSe derivatives, bearing a
carboxylic acid at the ortho-position relative to the chalcogen, are shown in Scheme 4.
The symmetrical, commercially available, 2-bromo-1,3-dimethylbenzene 14 was
oxidized to the isophthalic acid 15 in 35% yield by action of potassium permanganate.12
Fischer esterification of 15 in the presence of methanol and sulfuric acid returned the
dimethyl ester 16 in 49% yield.12 The symmetrical dimethyl ester was then hydrolyzed
with sodium hydroxide to afford monoacid 17.13 Monoacid 17 was converted to the
corresponding amide 18 in 50% yield by a two-step process involving conversion to the
acid chloride followed by amidation with aniline. Amide 18 was then cyclized by a
convenient copper (I) iodide protocol in the presence of sulfur powder to afford 19 in
52% yield.14,15 The resulting EbS methyl ester was hydrolyzed with lithium hydroxide
monohydrate to provide carboxylic acid 20 in 77% yield.16 In a similar fashion, the EbSe
methyl ester 21 was obtained, albeit in a poor 12% yield, and was subsequently
hydrolyzed to afford 22 in 82% yield.12-16
7

Scheme 4. Synthesis of EbS derivatives 19 and 20 and EbSe derivatives 21 and 22.
Further varieties of EbS/EbSe derivatives were obtained by incorporating the
carboxylic acid functionality in either the para or meta positions of the N-phenyl ring.
The synthesis of these derivatives are depicted in general in Scheme 5. Iodobenzoic
Scheme 5. Synthesis of ester derivatives 26-29 and carboxylic acid derivatives 30-33.
acid 23 was converted to its corresponding acid chloride by action of thionyl chloride
followed by amidation with either the para or meta isomer of amino methylbenzoate to
afford amides 24 (89% yield) and 25 (62% yield), respectively. Amides 24 and 25 were
8

each reacted in the presence of either sulfur or selenium powder under the copper(I)
iodide/1,10-phenanthroline conditions described above.14,15 In this manner, methyl ester
analogs 26 (55% yield), 27 (65% yield), 28 (46% yield), and 29 (43% yield) were
prepared. This suite of methyl esters was then subjected to standard saponification
conditions16 to afford acid analogs 30 (79% yield), 31 (79% yield), 32 (84% yield), and
33 (91% yield).
Next, we evaluated the carboxylic acid and methyl ester derivatives of EbSe and
EbS against both TbHK1 in vitro and against BSF trypanosomes in whole-cell assays
(conducted in triplicate). This biological screening yielded four results: percent TbHK1
inhibition, IC50 values against TbHK1, percent BSF growth inhibition, and BSF LD50. The
derivatives were screened against TbHK1 and BSF T. brucei at a standard
concentration of 10 M. Enzyme activity of rTbHK1 was scored after incubation with the
drug with the percent inhibition of the enzyme being recorded. The reduction in
trypanosome growth in a cell culture, caused by incubation with the drug, is reflected by
the BSF growth inhibition value. BSF EC50 values refer to the dose of compound
necessary to reduce cell number by one-half in vitro and was pursued for those
compounds that demonstrated promising activity at 10 M. Figure 2 shows the
EbSe/EbS derivatives that were screened against TbHK1 and whole cell trypanosomes
(i.e. 6, 7, 12, 13, 19-22, and 26-33). The biological data resulting from the evaluation of
these molecules is assembled in Table 1.
Recall that percent TbHK1 inhibition (determined at 10 M drug concentration)
refers to the amount of rTbHK1 enzyme inhibition. Referring to Table 1, only a few of
the EbSe/EbSe derivatives exhibited any activity against TbHK1 in preliminary trials.
9

EbS ester 7 was weakly active against TbHK1 showing 12.8 ± 7.5% inhibition of the
enzyme. EbS derivative 26, bearing a para-methyl ester on the N-phenyl substituent
exhibited a 22.8 ± 3.8% inhibition of TbHK1, but its corresponding carboxylic acid 30
exhibited no activity. The most potent molecule in this assay was EbSe ester 28, which
demonstrated a 79.0 ± 2.8% TbHK1 inhibition. Thus, the IC50 value for TbHK1 inhibition
by EbSe ester 28 was determined to be 3.2 ± 0.01 M. Interestingly, positioning the
methyl ester in the meta position (i.e. 27) instead of para to the chalcogen completely
ablated the TbHK1 inhibitory activity observed for the latter compound. Finally, the only
free carboxylic acid that elicited only mild TbHK1 inhibition was EbSe acid 32, bearing a
carboxylic acid at the meta position on the N-aryl ring (18.5 ± 2.8% TbHK1 inhibition).
The relatively poor performance of the carboxylic acid derivatives as compared to the
corresponding methyl esters 26 and 28, may suggest that the anionic carboxylate may
stymie interactions with the enzyme.
The percent BSF growth inhibition (determined at 10 M) refers to the reduction
in trypanosome growth in a whole cell assay (conducted in triplicate) by the non-
conjugated EbSe/EbS analogues. Of the N-carboxymethylated EbSe/EbS derivatives,
EbS methyl ester 7 revealed a potent BSF growth inhibition of 98.3 ± 1.3%, and its
corresponding acid 6 effected a BSF growth inhibition of 77.6 ± 4.3%. Of the ortho-
substituted (i.e. relative to the chalcogen) analogues, the esters 19 and 21 exhibited
52.5 ± 4.9% and 55.6 ± 7.5% BSF growth inhibition, respectively. Further, the
corresponding acids 21 and 22 were ineffective toward promoting trypanosome growth
inhibition. The para-substituted N-aryl methyl ester 26 exhibited 66.6 ± 10.9% BSF
10

growth inhibition, while its corresponding acid 30 was ineffective against trypanosome
growth. The meta-substituted analogs were not effective at inhibiting trypanosome
growth. The para-substituted N-aryl methyl ester derivative of EbSe (i.e. 28) showed
38.6 ± 3.8% BSF growth inhibition, while its corresponding acid 32 was not active.
Similar to the meta-substituted N-aryl esters and carboxylic acids of EbS, the
corresponding meta-substituted EbSe ester 29 and acid 33 did not promote effective
growth inhibition against trypanosomes. Thus, the most active analog from the
collection of small molecule EbSe/EbS esters and carboxylic acids was N-
carboxymethylated ester of EbS (i.e. 7).
It is evident that the methyl ester derivatives (i.e. 7, 19, 21, 26, and 28) were
more effective at inhibiting the growth of BSF trypanosomes than their corresponding
carboxylic acid derivatives (with the notable exception of carboxylic acid 6). We
attribute the greater efficacy of the charge-neutral ester derivatives to their presumed
ability to more readily cross the cell membrane than their anionic carboxylic acid
counterparts.4,17 Further, it is curious that these molecules are relatively adept at
inhibiting BSF parasite growth despite their lackluster performance as in vitro TbHK1
inhibitors.
Four of the EbSe/EbS methyl esters yielded reasonably potent BSF EC50 values.
The N-carboxymethyl EbS acid 6 and ester 7 exhibited a BSF EC50 values of 7.24 ± 0.5
M and 0.28 ± 0.1 M, respectively. EbSe ester 28, with para N-phenyl
functionalization, revealed a higher BSF EC50 value of 13.0 ± 1.5 M. The para-
substituted N-phenyl EbS methyl ester 26 exhibited BSF EC50 value of 3.8 ± 0.1 M.
Table 1: Biological data on EbS and EbSe derivatives.a
11

STRUCTURE
TbHK1% Inhibition (10 M)
TbHK1 IC50 (M)
% BSF Growth Inhibition (10 M)
BSF EC50 (M)
1b
-
0.05 ± 0.03
-
2.9 ± 0.28
2b
-
2.0 ± 0.5
>10
-
0.03 ± 0.07
7.2 ± 0.5
0.28 ± 0.1
>10
0.87 ± 7.9%
12.8 ± 7.6%
0%
77.6 ± 4.3%
98.3 ± 1.3%
0%
6
7
>10
>10
12
0%
18.0 ± 0.4%
0%
>10
>10
0%
>10
~10
13
19
20
21
22
26
27
28
29
30
31
32
52.5 ± 4.9%
10.9 ± 10.9%
55.6 ± 7.5%
0%
>10
>10
2.7 ± 5.1%
0%
>10
~10
>10
>10
22.8 ± 3.8%
1.7 ± 0.3%
79.0 ± 2.8%
0%
>10
66.5 ± 10.9%
8.8 ± 22.1%
38.6 ± 3.8%
18.0 ± 20.8%
0%
3.8 ± 0.1
>10
>10
3.16 ± 0.06
>10
13.0 ± 1.5
>10
0%
>10
>10
0%
>10
15.8 ± 47.3%
12.0 ± 17.4%
>10
17.2 ± 14.6%
>10
>10
18.5 ± 2.8%
>10
0%
>10
33
aTrypanosome cultures were assayed in triplicate in a total volume of 40 L, and the DMSO vehicle was used as a negative
control. Percent inhibition was calculated by comparison to the growth of parasites grown with the DMSO controls from each plate.
EbS (10 M) was included in each assay as a positive control. Dose-response curves performed in triplicate for compounds that
elicited > 50% growth inhibition at 10 M were pursued in a 384-well plate format, with 50% effective concentrations (i.e. EC50)
determined using GraphPad Prism (version 6.0) software (GraphPad Software, Inc., La Jolla, CA). bdata taken from Ref. 5.
12

In summary, most of the carboxylic acid derivatives of 1 and 2 (i.e. 6, 13, 20, 22,
30, and 31) were ineffective against the TbHK1 enzyme in vitro, while EbSe acids 32
and 33 were modestly active, demonstrating ~17-18% inhibition. The corresponding
esters (7, 12, 19, 21, 26, and 27) showed improved activity against TbHK1 compared to
the acid derivatives, with EbSe ester 28 showing the greatest enzyme inhibition (ca.
79%).
With respect to growth inhibition of whole-cell BSF parasites, some of the ester
derivatives exhibited moderate to potent inhibitory activity while the corresponding
carboxylic acids (with the notable exception of 6) were poor inhibitors. Indeed, EbS
ester 7 demonstrated almost complete inhibition of BSF growth.
Ultimately, four of the sixteen derivatives prepared in this study exhibited similar
trypanocidal effects as compared to the parent compounds 1 and 2 (Scheme 6). The
most potent compound arising from our study was methyl ester 7 exhibiting an EC50
value of 0.28 ± 0.1 M, placing it in between EbS 2 (EC50 = 0.030 ± 0.067 M)5 and
EbSe 1 (EC50 = 2.9 ± 0.28 M)5 in terms of trypanocidal potency. Further, compounds
6 and 26 returned EC50 values below 10 M. It is particularly interesting that compound
28 was the only molecule in our study whose trypanocidal activity was harbingered by
potent in vitro TbHK1 inhibition (~80 % inhibition). Indeed, the most potent compound in
terms of trypanocidal activity (i.e. 7) arising from our study exhibited a relatively
unimpressive ~13% inhibition of TbHK1 in vitro. Thus, this study may suggest that the
potent trypanocidal activity of the parent molecules, EbSe (1) and EbS (2) may only be
partly explained by their potent TbHK1 inhibition.3,5 Rather, this class of molecules may
disrupt multiple vital functions within the BSF parasite in order elicit their potent
13

trypanocidal properties. Future studies in our laboratories will focus in part on
understanding the mechanism of action of his class of potent trypanocides.
Scheme 6. Most potent EbSe/EbS derivatives.
Acknowledgements
The authors would like to thank Clemson University and the Department of
Chemistry for providing funding. Work in the JCM laboratory was partially supported by
a Center for Biomedical Excellence (COBRE) grant of the National Institutes of Health
under award number P20GM109094 and by an award from the National Institute of
Allergy and Infectious Diseases of the National Institutes of Health (R21AI105656).
Additionally, the Clemson University Honors College and the Clemson University
Creative Inquiry Program are thanked for their support. The supplementary information
associated with this article can be found in the online version at [enter URL here].
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