Development of Selective Steroid Inhibitors for the Glucose-6-phosphate Dehydrogenase from Trypanosoma cruzi

Article abstract of DOI:10.1021/acsmedchemlett.0c00106

Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socioeconomic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt the identification of novel, efficient, and affordable drug candidates. To address this issue, one strategy could be probing the susceptibility of Trypanosoma parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have been described as highly potent but nonselective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17 compounds were shown to display moderate to high selectivity for T. cruzi over the human G6PDH. In addition, three compounds were effective in killing intracellular T. cruzi forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data around G6PDH and further supports this target for treating Chagas disease.

Full text of DOI:10.1021/acsmedchemlett.0c00106

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Letter
Development of selective steroid inhibitors for the glucose-6-
phosphate dehydrogenase from Trypanosoma cruzi
Fabrício Fredo Naciuk, Jéssica do Nascimento Faria, Amanda
Gonçalves Eufrásio, Artur Torres Cordeiro, and Marjorie Bruder
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2020
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Development of selective steroid inhibitors for the glucose-6-
phosphate dehydrogenase from Trypanosoma cruzi
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Fabrício Fredo Naciuk, Jéssica do Nascimento Faria, Amanda Gonçalves Eufrásio, Artur Torres
Cordeiro* and Marjorie Bruder*
Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas-SP, Brazil
KEYWORDS: Chagas disease, Trypanosoma cruzi, G6PDH, selective inhibitors, steroids
ABSTRACT: Chagas disease is a parasitic infection affecting millions of people across Latin America, imposing a dramatic socio-
economic burden. Despite the availability of drugs, nifurtimox and benznidazole, lack of efficacy and incidence of side-effects prompt
the identification of novel, efficient and affordable drug candidates. To address this issue, one strategy could be probing the
susceptibility of Trypanosoma parasites toward NADP-dependent enzyme inhibitors. Recently, steroids of the androstane group have
been described as highly potent but non-selective inhibitors of parasitic glucose-6-phosphate dehydrogenase (G6PDH). In order to
promote selectivity, we have synthesized and evaluated 26 steroid derivatives of epiandrosterone in enzymatic assays, whereby 17
compounds were shown to display moderate to high selectivity for T. cruzi over the human G6PDH. In addition, three compounds
were effective in killing intracellular T. cruzi forms infecting rat cardiomyocytes. Altogether, this study provides new SAR data
around G6PDH, and further supports this target for treating Chagas disease.
Discovered in the early 20th century by the Brazilian
physician Carlos Chagas, Chagas disease is attributed to the
infection by the flagellate protozoan Trypanosoma cruzi (T.
cruzi). According to the World Health Organization, it is
estimated that about 6 to 7 million people worldwide are at risk
of infection by T. cruzi.1 Currently there are two licensed drugs
for the treatment of Chagas disease: nifurtimox (NFX) and
benznidazole (BNZ). Both have serious side effects such as
neurological and digestive disorders, hypersensitivity in the
form of rashes, in addition to having low efficacy and efficiency
during the chronic stage.2,3 Given this current scenario, it is
necessary to develop more effective and efficient drugs for the
treatment of all stages of infection. The enzyme glucose-6-
phosphate dehydrogenase (G6PDH) is the first of the oxidative
branch of the pentose-phosphate pathway (PPP). This pathway
is key to cellular metabolism because it is a major producer of
Steroids such as dehydroepiandrostone (DHEA, 1) and
epiandrosterone (EA, 2) have long been known for inhibiting
human glucose-6-phosphate dehydrogenase (HsG6PDH).8
Recently, HsG6PDH inhibition by DHEA in erythrocytes was
shown to promote resistance towards infection by Plasmodium
falciparum, the causative agent of malaria.9 DHEA is further
believed to act as an immunomodulating agent by increasing the
host’s immunity toward infections by T. cruzi.10–12
Epiandrosterone (EA) analogues have been studied in the
context of their potential anticancer activity in phenotypic
assays, with modifications occurring mainly at the C3 or the
C16/17 of the androstane core (Figure 1).13,14 Hamilton and co-
workers, in particular, have developed a series of EA-
derivatized inhibitors of HsG6PDH, allowing for the
identification of key features that promote or disfavor binding
to this enzyme.15 16-Bromoepiandrosterone (BrEA, 3), which
is a potent inhibitor of HsG6PDH, was also shown to strongly
inhibit T. cruzi G6PDH (TcG6PDH),16 and its parasiticidal
effects have been reported for Taenia and Entamoeba.17 In
particular, the safety of BrEA (also registered as HE2000) has
been confirmed in clinical trials in HIV and malaria patients
while its immune-boosting properties were assessed.18–20
Although the mode of inhibition (uncompetitive mechanism)
has long been elucidated,21 the exact mode of binding of steroid
dihydronicotinamide
adenine
dinucleotide
phosphate
(NADPH), a molecule necessary for biosynthetic reductions
and important in the maintenance of cellular redox homeostasis,
by counteracting the deleterious effects of oxygen radicals.4 The
PPP is especially important for parasites, since mammalian
hosts use oxidative burst as the first line of defense against
infection via the formation of reactive oxygen species, which
are counterbalanced in the parasite by NADPH-dependent
reactions. This assertion is corroborated by in vitro experiments
performed in T. cruzi by Maugeri and co-workers, which points
out the increased flow through the PPP in response to oxidative
stress.5 G6PDH is of central importance for PPP, since it often
presents a high control coefficient, being considered a potential
target for the development of drugs.6 In addition, the genetic
validation of this enzyme as a target was demonstrated in T.
brucei by Cordeiro and co-workers through the partial depletion
of this enzyme using tetracycline inducible RNA interference.7
inhibitors to the enzyme remains unknown, despite
a
combination of computational and biochemical efforts
suggesting it lies near the binding site of its substrate, G6P, and
co-factor, NADP.22,23 So far, attempts by Mercaldi and co-
workers have yielded the crystal structure of TcG6PDH
obtained in a ternary complex with the substrate G6P and
reduced cofactor NADPH, however the desired quaternary
complex with a steroid inhibitor, for instance, has not yet been
achieved.24 Despite BrEA displaying high affinity for the
parasitic enzyme TcG6PDH, it is only twice less active against
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analogue 32. In order to introduce polar groups similar to the
the human homologous enzyme. Likewise, already known 16-
halogenated analogues (4, 6 and 7) have also proven strong
inhibitors of HsG6PDH, which could potentially hamper their
use for the treatment of this parasitic disease, on the basis of
undesired drug-host interactions.
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heterocycle-containing compounds 19-24, we focused on
analogues of derivatives 22 and 24, where the carbonyl/sulfonyl
group and the methylene adjacent to the nitrogen atom would
be inverted, producing amide 33 and sulfonamide 34,
respectively. From a synthetic point of view, these new
compounds would be formed by an SN2 or a Michael addition-
type O-alkylation of the corresponding -bromoacetamide (35)
and vinyl sulfone (36) derivatives, which were prepared by
known procedures.28,29 Treating ketal 25 with n-butyllithium,
compound 35 and tetrabutylammonium iodide (TBAI) gave the
desired ether (37) in modest yield,30 and further deprotection of
the ketal delivered the target compound (33). For the alkylation
of 25 with vinyl sulfone 36, sodium hydroxide and
tetrabutylammonium bromide (TBAB) were used, giving the
desired ether (38) in 66% yield.31 Likewise, removal of the ketal
group yielded the proposed compound (34). Pleasingly, we
were able to introduce the 16-bromine atom under the usual
conditions, delivering target compounds 39 and 40.
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Figure 1. Endogenous steroid hormone DHEA (1) and
synthetic analogues (2-7).
According to Hamilton and co-workers, the presence of an
H-bond donor at C3 substituents is essential for human
G6PDH inhibition,15 but this has not yet been reported for
TcG6PDH. Thus, the derivatization of the C3-OH group
would allow for the removal of the key H-bonding feature,
while introducing chemical diversity to the androstane core,
potentially yielding TcG6PDH selective inhibitors. As such, we
herein describe the synthesis and biochemical evaluation of a
series of C3-O-modified analogues of EA and BrEA,
composed of carboxylic and sulfonic esters, as well as ethers,
where aliphatic and heterocyclic groups are introduced. To the
best of our knowledge, these compounds have not been
described in the literature in the context of G6PDH inhibition
and would thus provide the scientific community with novel
data in this field.
Finally, we designed a series of compounds deprived of
hydrolyzable groups, where the attachment to the steroid and
the introduction of heterocycles would be facilitated by using
the reactivity of an epoxide (Scheme 1C). Seeking to produce
both epimers of the resulting secondary alcohols, we used the
adequate glycidyl tosylate surrogates (R)-41 and (S)-42,
obtained from the commercially available (S)-(+)-glycidol (43)
and (R)-(-)-glycidol (44), respectively. Substitution of the
tosylate group by the C3-alcohol of 25 occurred best using an
excess of sodium hydride at room temperature, despite the
modest yields observed for the glycidyl ethers 45 and 46. Next,
the desired target compounds 47-50 were achieved by
aminolysis of the epoxides with either morpholine or N-
methylpiperazine under gentle heating, subsequent treatment of
the crude reactions mixtures with aqueous hydrochloric acid to
remove the ketal group, and purification by recrystallization.
The first set of compounds (8-24) was composed of
carboxylic and sulfonic esters of EA and BrEA25 containing
either lipophilic or heterocyclic moieties. Compounds 8-15
represented esters of increasing size, as well as the simple
methyl sulfonic esters, and were prepared by reacting steroids 2
and 3 with the desired acyl or sulfonyl chlorides (Scheme 1A).
Intermediates 16-18 were synthesized using the same
conditions with the appropriate reagent, and further treated with
either morpholine or N-methylpiperazine to yield heterocyclic
steroid analogues 19-24. Although the 16-brominated
equivalents of compounds 16-18 (Scheme 1A, X = Br) were
successfully synthesized (data not shown), their reaction with
the two heterocyclic amines led to partial elimination of the
bromine atom, and attempts to brominate compounds 20 and 24
with copper(II) bromide in methanol at reflux led to the
elimination of the carboxylic or sulfone ester moieties at C3.
All 26 target compounds were evaluated in biochemical
assays against Tc and HsG6PDH to calculate the selectivity of
the inhibitors and perform a structure-activity relationship
(SAR) analysis (Supporting Information, Table S1). Reference
compounds DHEA (1), EA (2), and their C3-deoxygenated and
16-halogenated analogues (3-7) were also assayed. The
potency of the inhibitors towards both enzymes are illustrated
in Figure 2, facilitating the SAR analysis.
As mentioned in the introduction, all reference compounds
(1-7)* are HsG6PDH-selective (Figure 2, above the gray dotted
line), to the exception of BrEA (3), which is, however, only
twice more potent for TcG6PDH. These compounds all have in
common the free C3-alcohol (1-4) or its absence (5-7), further
supporting our strategy to produce C3-derivatized steroids to
favor the inhibition of TcG6PDH. Indeed, most of the
synthesized EA-analogues are found below the dotted gray line.
Special attention should be given to the ester derivatives of
BrEA, (12-14), in the dotted green triangle, which showed
highest potency (0.15 < IC50 (µM) < 0.38) and selectivity
toward TcG6PDH, especially compound 14 for which no
activity was detected for HsG6PDH even at the highest assayed
concentration of 80 µM. Further, it appears that increasing the
bulkiness of the ester group contributes to increased selectivity,
as can be noted by comparing the acetyl (12, 160-fold) and the
isobutyryl (14, > 400-fold) esters of BrEA. This same trend is
observed for the non-halogenated analogues (8-10), in the
dotted orange rectangle, which display moderate selectivity,
Because of the potential lability of esters in vivo, we sought
to produce C3-ether derivatives, thus demanding the use of
harsher conditions to perform O-alkylations at C3. As a
consequence, the protection of the C17-ketone as a ketal (25)
was required and was performed under conditions described by
Hitchin and co-workers (Scheme 1B).26 We chose the simple
methyl ethers of EA and BrEA, as well as the equivalent of
compounds 10 and 14, that is, the isobutyl ethers. Ketal (25)
was thus treated with sodium hydride in methanol under reflux,
followed by the addition of methyl iodide to form the desired
methyl ether (26).27 Deprotection of the ketal group under acidic
condition delivered the target methyl ether of EA (27), and
further treatment with copper(II) bromide generated the 16-
brominated analogue 28. In a similar fashion, isobutyl tosylate
(29) was reacted with ketal 25 and sodium hydride in the
presence of sodium iodide to yield the desired isobutyl ether
(30). After deprotection of the ketal, ketone 31 was treated with
copper(II) bromide, thus producing its 16-brominated
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due to lower potencies for TcG6PDH (IC50 > 3 µM; pIC50 <
5.5, Figure 2).
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Scheme 1. Synthesis of EA (2) and BrEA (3) derivatives comprising: A) aliphatic and heterocycle-containing esters; B)
aliphatic and heterocycle-containing ethers; C) non-hydrolysable moieties - target compounds are highlighted in blue
rectangles.
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Methyl sulfones 11 and 15 showed weak (2-fold) and moderate
(6-fold) selectivity, respectively.
highlighting structural features unique to TcG6PDH,32 the exact
mechanism underlying high selectivities for 16BrEA-O-
acetylated inhibitors, for instance, still cannot be rationalized.
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More polar EA ester analogues containing morpholine and N-
methylpiperazine moieties (19-22) were weak inhibitors (IC50
= 10 µM), while the heterocyclic sulfone esters (23-24)
performed better (IC50 = ~5 µM), only 23 showing moderate
selectivity. In the C3-ether series, 16-brominated ethers 28
and 32 achieved high selectivities (50-fold range), while
morpholine-containing analogues 39 and 40 showed moderate
selectivity (5 to 10-fold). Non-halogenated methyl ether 27
achieved some selectivity toward TcG6PDH, while
unexpectedly, isobutyl ether 31 proved 3-fold HsG6PDH-
selective, but also a weak inhibitor. Compounds 47-50 showed
almost no activity (IC50 > 40 µM) toward either of the G6PDH
homologues, noting that they all contain a hydrogen-bond
donating group in their propanolamine moiety. Similarly,
compound 33, which bears a hydrogen-bond acceptor (amide)
at the same position, was also one of the weakest compounds of
the series, although the addition of the 16-bromine somewhat
restored potency toward TcG6PDH, as shown with compound
39. Compound 34 was also a weak inhibitor (IC50 = 20 µM)
and non-selective.
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Next, we evaluated the efficacy of TcG6PDH inhibitors (with
pIC50_TC > 5) in killing intracellular T. cruzi (Y strain)
amastigotes infecting H9C2 rat cardiomyocytes by an image-
based cellular assay. Such multiparametric assay enables to
assess compound efficacy against parasites and toxicity towards
host cells, simultaneously. Initially, compounds were assayed
at the single concentration of 20 µM. At this concentration, no
compounds were able to reduce infection without showing
significant host cell toxicity (Figure 3), as being the case for
compounds 3, 6, 12, 13, 15, 32, 39 and 40, all derived from
BrEA. Non-halogenated steroids 2, 8, 9, 10, 11, 23, 24 and 27,
on the other hand, were not cytotoxic towards host cells, but
neither were they effective against T. cruzi. These observations
reinforce the idea that bromine addition at C-16 is important for
potent TcG6PDH inhibition but may also be responsible for the
pronounced toxicity in cell assays. Concerning fluorine-
derivatives 4 and 7, and BrEA derivative 14, which were not
active in the cell assays despite showing potent enzyme
inhibition, one ought to remind that several factors such as
solubility and membrane permeability (which needs to occur
twice to reach intracellular parasites), as well as metabolization
can impact on the general bioavailability of drug candidates.
Complex cellular pharmacokinetics have also been suggested
by Khare and co-workers for the intracellular T. cruzi forms in
particular, while assessing proteasome inhibitors of
kinetoplastids.34
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Finally, looking at the distribution of the compounds
according to their calculated partition coefficient (cLogP),
compounds with values higher than 3.5 tend to make the more
potent inhibitors, while polar compounds (cLogP < 3.5) are
poor inhibitors (Figure 2). Clearly, the most potent inhibitors
owe their high affinities to the presence of the 16-bromine
atom.
Figure 3. Anti-T.cruzi intracellular assay at single
concentration of 20 µM. Black and gray bars represent mean
and standard deviation of normalized total host and infected
cells, respectively. DMSO values were used for data
normalization.
Due to the toxicity observed for a set of compounds at 20 µM,
these were selected to be retested at lower concentrations in a
dose-response assay format for EC50 and selectivity index
(S.I.) determination. The results are presented in Table 1.
Table 1: EC50 values (µM) for host cells and intracellular
T. cruzi forms.
Figure 2. SAR analysis of EA (2) and BrEA (3) derivatives
against the parasitic and human G6PDHs. Affinities are shown
as pIC50 values, where IC50 is expressed in µM (pIC50 = -
log(IC50.10-6). The green dotted triangle represents highly Tc-
selective compounds, defined by an IC50_Hs/IC50_Tc ratio >
40. Moderate Tc-selectivity (5 < ratio < 30) is represented by a
dotted orange rectangle. The light green filled rectangle covers
BrEA derivatives, whereas the light orange filled rectangle
represents EA derivatives. The chart was generated with the
DataWarrior software (Osiris).33
Sample
BNZ
40
EC50_T. cruzi
2.2 ± 0.4
2.4 ± 0.4
2.4 ± 0.3
3.2 ± 1
EC50_host
> 40
S.I.
> 18
3.5
3.1
1.8
2.4
2.9
2.0
1.7
3.2
8.5 ± 1.9
7.5 ± 0.6
5.6 ± 1.6
8.4 ± 1.8
12.3 ± 1.8
13 ± 2
15
39
6
3.5 ± 0.4
4.2 ± 0.5
6.4 ± 2
32
3
In summary, our strategy to remove a H-bond donating
feature at epiandrosterone’s C3 substituents, which is key for
HsG6PDH binding, proved successful for producing TcG6PDH
selective inhibitors. Despite a recent comparative study
12
8.5 ± 1.5
16.7 ± 4.2
14.7 ± 4.2
54 ± 3.3
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S.I. = selectivity index calculated as EC50_host/EC50_T. cruzi.
Mean and standard deviation for 3 independent experiments.
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At first glance, the Tc/HsG6PDH selectivity and potencies
drug candidates.36 Altogether, these are encouraging results
showing for the first time that uncompetitive G6PDH inhibitors
are able to kill intracellular T. cruzi amastigotes, the parasite
form associated with the chronic stage of Chagas disease.
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observed in enzymatic assays were not reflected in cell assays.
On one hand, highly Tc-selective compounds 32, 12 and 13
showed higher EC50 values for host cells than compounds with
no or low Tc-selectivity such as 40, 15, 39 and 6 (Table 1),
which indicates that a certain degree of selectivity in cell assays
was achieved, at least in terms of toxicity. However, it is the
least Tc-selective compounds, which despite having lower
potencies for the parasitic enzyme, showed the highest
efficacies toward T. cruzi in cell assays. On the other hand,
BrEA (3), which is only twice more potent for TcG6PDH, still
has a higher EC50_host value, and is thus less toxic than its
moderately selective congeners. One could propose that
possible metabolic pathways could be involved. In fact, BrEA
possesses a free C3-OH group which could be prone to
sulfatation, as is the case in vivo for DHEA, which renders it
inactive toward HsG6PDH.35 In a similar fashion, compounds
12 and 13, which are highly Tc-selective but show lower
efficacies could also easily be metabolized. Being esters, they
could indeed suffer hydrolysis by esterases and further undergo
conjugation. As such, these transformations occurring in the
host cells’ cytosol would lower the concentration of inhibitor
entering the parasites, which would account for their higher
EC50 values in T. cruzi. Conversely, this could explain why
non-hydrolyzable ethers 39 and 40 were more active in cell
assays. Still, this is merely speculative, since T. cruzi
pharmacokinetics are reported to be quite complex.34
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Nevertheless, we were pleased to observe that compounds 40,
15 and 32 showed EC50 values for intracellular T. cruzi
equivalent to BNZ and at least three times higher for host cells
(Table 1 and Figure 4).
Figure 4. Dose-response curves for normalized host (black) and
infected cells (gray) for BNZ, compounds 32, 15 and 40. Plots
prepared with GraphPad Prism.
Inspection of cell assay images for these compounds acquired
at 10 µM (Figure 5), the concentration closest to the EC50 for
host cells, shows a significative reduction in the number of
infected cells and infection ratio, equivalent to BNZ. Despite
the observed decrease in total host cells, a significative
reduction in parasitemia was clearly observed in the remaining
cells, supporting the notion that the parasites are more
susceptible to the compounds’ action than the host cells.
Figure 5. Image analysis of anti-T. cruzi intracellular assay of
compounds 32, 15 and 40 (10 µM). Mean and standard
deviation for total host cells (A), infected cells (B) and infection
ratio (C) per image (N=15). (D) Representative images (left)
and zoom from blue boxes (right) for DMSO, BNZ and
compounds 32, 15 and 40. Host cell nuclei are colored in yellow
and T. cruzi amastigotes appear as white spots in the cytoplasm
of infected cells (indicated by blue arrows).
In conclusion, for the first time, selective inhibitors of the
parasitic G6PDH enzyme, with respect to the human isoform,
have been described. Our data shows that esters of the C3-OH
group of epiandrosterone (2) abolish activity towards the human
enzyme, while maintaining high affinity for TcG6PDH,
especially in the presence of the 16-bromine atom, as
supported by consistent SAR analyses. On assessing the
efficacy of these compounds in cell-based assays, however,
most of the halogenated compounds proved cytotoxic at 20 µM.
On performing dose-response experiments, isobutyl ether 32,
sulfone 15 and sulfonamide 40 displayed EC50 values close to
BNZ’s, but with narrow therapeutic windows. New chemical
scaffolds are thus pursued by our group in order to achieve safer
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures for the synthesis of compounds 3-42 and
45-50; experimental procedures and data for the biological
evaluation of the target compounds - enzymatic and cell-based
assays (docx). 1H and 13C NMR spectra as ANNEX (docx).
AUTHOR INFORMATION
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Effect and Mechanism of Inhibiting Glucose-6-Phosphate
Corresponding Author
Dehydrogenase Activity on the Proliferation of Plasmodium
Falciparum. Biochim. Biophys. Acta - Mol. Cell Res. 2017, 1864
(5), 771–781.
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* E-mail : marjorie.bruder@lnbio.cnpem.br.
* E-mail : artur.cordeiro@lnbio.cnpem.br.
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Santos, C. D.; Toldo, M. P. A.; Santello, F. H.; Filipin, M. D. V.;
Brazão, V.; do Prado Júnior, J. C. Dehydroepiandrosterone
Increases Resistance to Experimental Infection by Trypanosoma
Cruzi. Vet. Parasitol. 2008, 153 (3–4), 238–243.
Domingues Santos, C.; Loria, R. M.; Rodrigues Oliveira, L. G.;
Collins Kuehn, C.; Alonso Toldo, M. P.; Albuquerque, S.; do
Prado Júnior, J. C. Effects of Dehydroepiandrosterone-Sulfate
(DHEA-S) and Benznidazole Treatments during Acute Infection
of Two Different Trypanosoma Cruzi Strains. Immunobiology
2010, 215 (12), 980–986.
Author Contributions
F.F.N.‡ and M.B. performed the synthesis; J.N.F.‡ produced the
purified enzymes and performed the enzymatic assays; A.G.E.
performed the cell-assays; A.T.C. designed and supervised the
biological assays; M.B. planned the target compounds; A.T.C. and
M.B. jointly designed the study and analyzed the results. The
manuscript was written through contributions of all authors. ‡These
authors contributed equally.
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Kuehn, C. C.; Oliveira, L. G. R.; Santos, C. D.; Augusto, M. B.;
Toldo, M. P. A.; Do Prado, J. C. Prior and Concomitant
Dehydroepiandrosterone Treatment Affects Immunologic
Response of Cultured Macrophages Infected with Trypanosoma
Cruzi in Vitro? Vet. Parasitol. 2011, 177 (3–4), 242–246.
Romero-Hernández, L. L.; Merino-Montiel, P.; Meza-Reyes, S.;
Vega-Baez, J. L.; López, Ó.; Padrón, J. M.; Montiel-Smith, S.
Synthesis of Unprecedented Steroidal Spiro Heterocycles as
Potential Antiproliferative Drugs. Eur. J. Med. Chem. 2018, 143,
21–32.
Cui, H. W.; Peng, S.; Gu, X. Z.; Chen, H.; He, Y.; Gao, W.; Lv,
F.; Wang, J. H.; Wang, Y.; Xie, J.; Liu, M. Y.; Yi, Z.; Qiu, W. W.
Synthesis and Biological Evaluation of D-Ring Fused 1,2,3-
Thiadiazole Dehydroepiandrosterone Derivatives as Antitumor
Agents. Eur. J. Med. Chem. 2016, 111, 126–137.
Hamilton, N. M.; Dawson, M.; Fairweather, E. E.; Hamilton, N.
S.; Hitchin, J. R.; James, D. I.; Jones, S. D.; Jordan, A. M.; Lyons,
A. J.; Small, H. F.; Thomson, G. J.; Waddell, I. D.; Ogilvie, D. J.
Novel Steroid Inhibitors of Glucose 6-Phosphate Dehydrogenase.
J. Med. Chem. 2012, 55 (9), 4431–4445.
Cordeiro, A. T.; Thiemann, O. H. 16-Bromoepiandrosterone, an
Activator of the Mammalian Immune System, Inhibits Glucose 6-
Phosphate Dehydrogenase from Trypanosoma Cruzi and Is Toxic
to These Parasites Grown in Culture. Bioorganic Med. Chem.
2010, 18 (13), 4762–4768.
Funding Sources
This research was funded by the São Paulo Research Foundation
[FAPESP grants for M.B. (2013/16534-4), and A.T.C.
(2016/14271-4 and 2018/22202-8)] and by the Coordination for the
Improvement of Higher Education Personnel (CAPES 7775/2014-
98). The Brazilian Ministry of Science, Technology, Innovation
and Communication sponsored F.F.N. (PCI-DB 313424/2015-2).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors wish to thank Prof. Ronaldo Pilli and the Chemistry
Institute / UNICAMP for their support, as well as the Nuclear
Magnetic Resonance Laboratory at LNBio/CNPEM.
ABBREVIATIONS
NADP, nicotinamide adenine dinucleotide phosphate; G6PDH,
glucose-6-phosphate dehydrogenase; NFX, nifurtimox; BNZ,
benznidazole; PPP, pentose-phosphate pathway; NADPH,
dihydronicotinamide adenine dinucleotide phosphate; DHEA,
dehydroepiandrosterone; EA, epiandrosterone; BrEA, 16-
bromoepiandrosterone; DMSO, dimethyl sulfoxide; HIV, human
immunodeficiency virus.
Carrero, J. C.; Cervantes-Rebolledo, C.; Vargas-Villavicencio, J.
A.; Hernández-Bello, R.; Dowding, C.; Frincke, J.; Reading, C.;
Morales-Montor,
J.
Parasiticidal
Effect
of
16α-
Bromoepiandrosterone (EpiBr) in Amoebiasis and Cysticercosis.
Microbes Infect. 2010, 12 (8–9), 677–682.
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Allylations of Functionalized α-Haloalkylphenyl Sulfones.
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ACS Medicinal Chemistry Letters
A
O
O
O
O
1
2
3
4
5
X
X
O
Y
O
O
O
O
O
N
O
N
O
6
MeN
O
8 (X = H, Y = C, 96%)
7
8
9
10
11
12
13
14
15
16
16 (X = H, 61%)
O
11 (X = H, Y = S(O), 99%)
12 (X = Br, Y = C, 74%)
15 (X = Br, Y = S(O), 90%)
19 (97%)
20 (72%)
R1
Cl
X
or
O
S
NH
O
O
O
O
O
(X = NMe, O)
(2 eq.)
O
R2
Cl
X
X
X
O
O
O
O
MeN
O
Et3N, CH2Cl2
0 ºC-rt, 16 h
CH2Cl2
rt, 16 h
Br
N
N
O
O
O
HO
17
O
18
19
9* (X = H, 58%)
*(R1 = Me(CH)2))
13 (X = Br, 57%)
2 (EA, X = H)
3 (BrEA, X = Br, 76%)
CuBr2, MeOH
17* (X = H, 70%)
*(BrCH2C(O)Br used)
21 (87%)
22 (98%)
reflux, 16 h
EA (2)
Br
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
O
O
O
O
X
X
O
O
O
O
S
O
O
O
O
O
S
S
O
N
O
N
MeN
O
18* (X = H, 61%)
*(R2 = Cl(CH2)2)
10 (X = H, 77%)
14 (X = Br, 72%)
23 (93%)
24 (99%)
36
B
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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57
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60
O
O
O
O
ethyleneglycol
pTSA
CH(OMe)3
O
NaH (10 eq.)
MeI (8 eq.)
X
5% aq. H2SO4
CH2Cl2
rt, 16 h
Dioxane
rt, 5 h
THF
reflux, 16 h
MeO
MeO
HO
CuBr2, MeOH
reflux, 16 h
26 (77%)
27 (X = H, 77%)
28 (X = Br, 71%)
25 (87%)
O
O
25
NaH (10 eq.)
NaI (1.0 eq.)
O
TsCl
Et3N, DMAP
aq. HCl (2M)
Acetone
X
OH
OTs
THF
reflux, 16 h
CH2Cl2
rt, 16 h
THF/MeOH
rt, 2 h
29 (81%)
(10 eq.)
O
O
CuBr2, MeOH
reflux, 16 h
30 (60%)
31 (X = H, 88%)
32 (X = Br, 75%)
O
O
O
25
nBuLi
TBAI
O
O
aq. HCl (2M)
Acetone
NH
X
N
Br
Br
O
O
Et3N (1 eq.)
CH2Cl2
0 ºC, 1 h
THF anh.
0 ºC-rt, 20 h
THF/MeOH
rt, 2 h
O
O
N
N
O
O
35 (74%)
O
O
37 (42%)
33 (X = H, 78%)
39 (X = Br, 74%)
CuBr2, MeOH
reflux, 16 h
O
O
O
25
NaOH
TBAB
O
O
aq. HCl (2M)
Acetone
NH
X
Cl
Cl
N
S
S
O
S
O
O
S
O
O
O
Toluene
rt, 20 h
Et3N (2 eq.)
CH2Cl2
0 ºC, 1 h
O
THF/MeOH
rt, 2 h
O
N
O
N
O
36 (71%)
O
O
38 (66%)
34 (X = H, 83%)
40 (X = Br, 74%)
CuBr2, MeOH
reflux, 16 h
C
X
NH
O
O
O
O
O
O
41 or 42
NaH (8 eq.)
(X = NMe, O)
(5 eq.)
S
O
O
25
DMF anh.
0 ºC-rt, 20 h
MeOH
50 ºC, 1.5 h
41 (85%)
*
N
O
O
*
O
X
OH
Et3N
CH2Cl2
0 ºC-rt, 20 h
45 (*(R), 46%)
46 (*(S), 41%)
OH
O
43
O
O
O
O
S
Cl
N
O
N
O
O
OH
MeN
OH
aq. HCl (2M)
Acetone
Et3N
CH2Cl2
0 ºC-rt, 20 h
OH
47 (35%)
48 (43%)
O
THF/MeOH
rt, 2 h
44
O
O
O
S
O
O
N
O
N
O
O
O
OH
MeN
OH
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42 (85%)
49 (64%)
50 (72%)

O
O
O
16α
1A7CS Medicinal Chemistry Letters
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X
3β
1
2
H
H
HO
HO
3
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DHEA (1)
EA (2, X = H)
BrEA (3, X = Br)
FEA (4, X = F)
dEA (5, X = H)
dBrEA (6, X = Br)
dFEA (7, X = F)

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X
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