Non-natural Acetogenin Analogues as Potent Trypanosoma brucei Inhibitors

Article abstract of DOI:10.1002/cmdc.201402272

Neglected tropical diseases remain a serious global health concern. Here, a series of novel bis-tetrahydropyran 1,4-triazole analogues based on the framework of chamuvarinin, a polyketide natural product isolated from the annonaceae plant species are deta

Full text of DOI:10.1002/cmdc.201402272

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DOI: 10.1002/cmdc.201402272
Non-natural Acetogenin Analogues as Potent
Trypanosoma brucei Inhibitors
Gordon J. Florence,* Andrew L. Fraser, Eoin R. Gould, Elizabeth F. B. King,
Stefanie K. Menzies, Joanne C. Morris, Lindsay B. Tulloch, and Terry K. Smith*[a]
Neglected tropical diseases remain a serious global health con-
cern. Here, a series of novel bis-tetrahydropyran 1,4-triazole an-
alogues based on the framework of chamuvarinin, a polyketide
natural product isolated from the annonaceae plant species
are detailed. The analogues synthesized display low micromo-
lar trypanocidal activities towards both bloodstream and insect
forms of Trypanosoma brucei, the causative agent of African
sleeping sickness, also known as Human African Trypanosomia-
sis (HAT). A divergent synthetic strategy was adopted for the
synthesis of the key tetrahydropyran intermediates to enable
rapid access to diastereochemical variation either side of the
1,4-triazole core. The resulting diastereomeric analogues dis-
played varying degrees of trypanocidal activity and selectivity
in structure–activity relationship studies. Together, the biologi-
cal potency and calculated lipophilicity values indicate that
while there is room for improvement, these derivatives may
represent a promising novel class of anti-HAT agents.
Introduction
Neglected tropical diseases are
a continuing health concern in
developing countries due to the
lack of effective prevention
methods
and
therapeutic
agents.[1] One of these prevalent
neglected tropical diseases,
which has been slowly attract-
ing attention over the past few
years, is African sleeping sick-
ness or Human African Trypano-
somiasis (HAT), caused by the
protozoan parasite Trypanosoma
brucei that is transmitted by the
bite of the Tsetse fly. HAT is
a major health concern in sub-
Saharan
Africa
threatening
more than 60 million people.
The annual mortality rate of
HAT estimated by the World
Health Organization (WHO)
Figure 1. Current drugs for treatment of Human African trypanosomiasis (HAT).
stands at approximately 8000
with less than 30000 new cases
per year based on recent re-
and dependent upon the disease stage of HAT infection—the
lymphatic first stage or the second neurological stage when
the T. brucei parasite has crossed the blood–brain barrier. Four
drugs are used in the treatment of HAT (Figure 1), namely sura-
min (1) and pentamidine (2) for stage 1 HAT, and melarsoprol
(3) and eflornithine (4) for stage 2.
These drugs are difficult to administer to patients, often re-
quiring lengthy infusion rates; they have varying degrees of
human toxicity and resistance is becoming a significant prob-
lem.[3] In an effort to decrease the costs associated with these
ports.[2] At present, the treatment for HAT is extremely limited
[a] Dr. G. J. Florence, A. L. Fraser, Dr. E. R. Gould, E. F. B. King, S. K. Menzies,
Dr. J. C. Morris, Dr. L. B. Tulloch, Prof. T. K. Smith
EaStCHEM School of Chemistry, Biomedical Sciences Research Complex
University of St Andrews, North Haugh, St Andrews, KY16 9ST (UK)
E-mail: gjf1@st-andrews.ac.uk
tks1@st-andrews.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402272.
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drugs, improve the logistics of
their
administration,
and
combat drug resistance (a
common problem associated
with parasitic diseases), a nifurti-
mox–eflornithine combination
therapy (NECT; nifurtimox 5 is
used clinically to treat Chagas
disease) has been recently intro-
duced for stage 2 HAT by the
WHO.[4] This combination thera-
py maintains the efficacy of
Figure 2. Rationale for simplified analogue design.
eflornithine at lower dosages,
while decreasing the toxic side
effects associated with eflornithine monotherapy. Despite the
success of NECT, there is a lack of new effective therapeutic
agents, and the onset drug resistance remains a serious threat
to current therapies,[5] highlighting the urgent demand for the
development of new drug-like molecules and their clinical im-
plementation as effective HAT therapies.
(IC50 =0.8 nm).[8] In 2011, we reported the total synthesis of
chamuvarinin (6); we showed that 6 and a series of synthetic
derivatives exhibited low micromolar activities towards both
the blood-stream and procyclic forms of T. brucei.[9] These en-
couraging preliminary results and the limited investigations
into trypanocidal activity of the acetogenins[10] prompted our
initial interest in designing simplified analogues of 6, which
retain the important structural features of the acetogenin
family of natural products. In an effort to decrease structural
complexity, it was hypothesized that a 1,4-triazole motif could
form an effective spatial mimic of the central C20ꢀC23 tetrahy-
drofuran (THF) motif found in chamuvarinin (6). The synthesis
The acetogenins are a class of polyketide natural products
isolated from the annonaceae plant species found in the tropi-
cal regions of West Africa and South America.[6,7] Chamuvarinin
(6; Figure 2) was isolated in 2004 by Laurens et al. from the
roots of the bush banana plant Uvaria chamae and showed
significant cytotoxicity against the KB 3-1 cervix cancer cell line
Scheme 1. Synthesis of azides 11 a–c, 12 and 13 and alkynes 16a–b and 17a–b. Reagents and conditions: a) CH2CHCH2CH2MgBr, CuI, THF, ꢀ408C!RT, 2 h;
b) m-CPBA, CH2Cl2, 08C!RT, 3 h; then (ꢁ)-CSA (20 mol%), RT, 2.5–18 h; c) PPh3, iPr2NEt, DIAD, DPPA, 08C!RT, 16 h; d) (COCl)2, DMSO, CH2Cl2, Et3N, ꢀ788C!
RT, 1 h; e) dimethyl diazo-2-oxopropylphosphonate, K2CO3, MeOH, RT, 16 h; f) CBr4, PPh3, CH2Cl2, ꢀ788C, 45 min; g) nBuLi, THF, ꢀ788C, 1.5 h. Abbreviations:
(ꢁ)-camphorsulfonic acid [(ꢁ)-CSA]; 3-chloroperbenzoic acid (m-CPBA); diisopropyl azodicarboxylate (DIAD); dimethyl sulfoxide (DMSO); diphenyl phosphoryl
azide (DPPA).
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and incorporation of THF motifs are notoriously difficult, hence
the C16–19 THF ring system of 6 was substituted with the
readily accessible and manageable tetrahydropyran (THP)
motif (7 in Figure 2). The heterocyclic spacer was envisioned to
arise from the “click” reaction of two THP subunits, whilst the
butenolide moiety could be appended to the ether core in an
analogous manner to 6 by a suitable length alkyl spacer.
ic acid (m-CPBA) epoxidation and subsequent in situ acid-
mediated ring closure to provide a readily separable mixture of
diastereomeric alcohols 8a and 9a. This divergent approach
provided an excellent opportunity to introduce structural varia-
tion within the THP scaffolds. With the alcohols in hand, both
diastereomers could be independently converted to the azide
by Mitsunobu reaction (diphenylphosphoryl azide (DPPA), dii-
sopropyl azodicarboxylate (DIAD), N,N-diisopropylethylamine,
triphenylphosphine) of alcohols 8a and 9a to give 11 a and
12.[12] The enantiomeric syn azide (13) could be accessed in an
analogous manner from (R)-epoxyoctane.[11] For the alkyne sub-
units, syn-8a and anti-9a alcohols were oxidized under Swern
conditions to afford aldehydes 14a and 15a in excellent
yields, and subsequent exposure of 14a to Ohira–Bestmann
homologation[13] gave exclusively syn-16a in 72% yield. Due to
epimerization of aldehyde 15a under the mildly basic condi-
Results and Discussion
Chemistry
As outlined in Scheme 1, intermediate THP alcohols 8a and 9a
could be readily assembled in three steps from (S)-epoxyoc-
tane (10a)[11] via copper(I)-promoted ring opening of 10a with
homoallyl magnesium bromide followed by 3-chloroperbenzo-
Scheme 2. Synthesis of triazole analogues 18a–d, 19a,b, 20a,b, 21a,b and 22. Reagents and conditions: a) CuSO4·5H2O, Na ascorbate, H2O, tBuOH, RT, 16 h.
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tions, an alternative two-step
Corey–Fuchs
homologation[14]
approach was adopted, giving
17a in 70% yield. The incorpo-
ration of terminal oxygenation
as a functional handle for ex-
tended elaboration was ach-
ieved by synthesis of the corre-
sponding benzylated and silylat-
ed series of azides 11 b,c and al-
kynes 16b and 17b from epox-
ides 10b and 10c.
The central 1,4-triazole scaf-
fold was conveniently installed
by application of the copper(I)-
catalyzed Huisgen 1,3-dipolar
cycloaddition
(Scheme 2).[15]
Thus, diastereomeric analogues
of the alkyl, benzylated and sily-
lated bis-THP triazole motifs
were synthesized by treatment
of the corresponding azides
11 a–c, 12, 13 and alkynes
16a,b, 17a,b under “click” reac-
tion conditions (copper(II) sul-
Scheme 4. Synthesis of triazoles 31 and 32. Reagents and conditions: a) 1H-mercaptophenyltetrazole, PPh3, DIAD,
08C, 3 h; b) (NH4)6Mo7O24·4H2O, H2O2, EtOH, 08C!RT, 16 h; c) NaHMDS, THF, ꢀ788C; then 30, THF ꢀ78!ꢀ208C,
1.5 h; d) TsNHNH2, NaOAc, DME, H2O, 1008C, 3 h. Abbreviations: diisopropyl azodicarboxylate (DIAD); dimethoxy-
ethane (DME); sodium hexamethyldisilylazide (NaHMDS).
fate, sodium ascorbate) to afford triazole products 18a–d,
19a,b, 20a,b, 21a,b and 22 in good yields.
Benzylated 18b-c, 19b, 20b, 21b and 22 analogues, out-
lined in Scheme 3, were deprotected under atmospheric hy-
drogenolysis (20% Pd(OH)2/C, H2) to the corresponding alco-
hols 23–28 (entries 1–6; Scheme 3) in excellent yields.
Having previously established structure–activity relationship
(SAR) data for the advanced analogues of chamuvarinin, it was
observed that incorporation of the butenolide side chain im-
pacted positively on the trypanocidal activity. Thus, installation
of the butenolide motif to successfully mimic chamuvarinin
was accomplished in four steps from lead alcohol 23, outlined
in Scheme 4. Alcohol 23 was manipulated to sulfone 29 via
a two-step Mitsunobu reaction and catalytic MoVI[7] oxidation
protocol.[16] Subsequent Julia–Kocienski olefination[16] with al-
dehyde 30[9,18] and diimide reduction[19] (para-toluenesulfonyl
hydrazide, sodium acetate) provided advanced analogue 31.
To ascertain whether the triazole analogue would bind in a di-
rectional manner to the unknown protein target, analogue 32
was prepared in an analogous manner to 31 from alcohol 28
via sulfone 33.
Having successfully installed the butenolide motif and in an
effort to further extend the SAR data, bis-directional analogue
34, outlined in Scheme 5, was synthesized in six steps from ad-
vanced triazole 18d. Silyl deprotection of 18d to alcohol 35,
followed by a Mitsunobu/oxidation protocol, gave sulfone 36.
Julia–Kocienski olefination of sulfone 36 with 30 and subse-
quent diimide reduction gave triazole 37. Benzyl deprotection
of 37 with boron trichloride furnished alcohol 34 in 23% yield.
Diol analogue 38 was accessed in excellent yield from alcohol
35 under atmospheric hydrogenolysis.
Structure–activity relationship summary
The primary mode of action for acetogenins in mammalian
cells is inhibition of complex I of the mitochondrial electron
transport chain (ETC).[20] In contrast, bloodstream (BSF) T. brucei
lacks functional complexes I, III and IV and instead relies on al-
Scheme 3. Synthesis of alcohols 23–28. Reagents and conditions: a) 20%
Pd(OH)2/C, H2 (1 atm), EtOH, RT, 1.5 h.
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and 72.4ꢁ3.9 mm and SI values
of 23 and 0.8, respectively. The
anti–anti analogue (25; entry 3;
Table 1) was still devoid of para-
sitic activity, despite transforma-
tion to the free hydroxy. Gratify-
ingly, introducing the free hy-
droxy to analogue 23 (entry 9;
Table 1) led to a slight increase
in T. brucei inhibition, and 23
has shown comparable levels of
parasitic activity to that of cha-
muvarinin (6): EC50 values of
1.80ꢁ0.10 and 1.4ꢁ0.1 mm, re-
spectively.
On the basis of 23, bis-direc-
tional analogue 35 (entry 10;
Table 1) was tested in the hope
of further improving the activity.
Unfortunately, 35 was nine
times less potent than 23, and
subsequent removal of the
benzyl group to give diol 38
(entry 11; Table 1) resulted in
significantly diminished activi-
ties. Comparison of the biologi-
cal data for the ethyl, benzyl
Scheme 5. Synthesis of advanced alcohol 34. Reagents and conditions: a) TBAF, THF, 08C!RT, 4 h; b) 1H-mercapto-
phenyltetrazole, PPh3, DIAD, 08C, 16 h; c) (NH4)6Mo7O24·4H2O, H2O2, EtOH, 08C!RT, 16 h; d) NaHMDS, THF, ꢀ788C;
then 30, THF ꢀ78!ꢀ208C, 3 h; e) TsNHNH2, NaOAc, DME, H2O, 1008C, 3 h; f) BCl3·SMe2, CH2Cl2, ꢀ788C!RT, 16 h;
g) 20% Pd(OH)2/C, H2 (1 atm), EtOH, RT, 1.5 h. Abbreviations: diisopropyl azodicarboxylate (DIAD); dimethoxy-
ethane (DME); sodium hexamethyldisilylazide (NaHMDS); tetrabutylammonium fluoride (TBAF).
ternative oxidases (TAO, AOX2) to enable mitochondrial respi-
ration, while the procyclic (Pro) form expresses both a fully
functional ETC and the alternative oxidases.[21] Thus in order to
assess whether the triazole-based analogues share a common
mitochondrial target and identify key structural features re-
quired for parasitic inhibition, screening against the BSF and
Pro forms of T. brucei and the mammalian HeLa cell line[22] was
performed (Table 1). From the data, it was evident that the ste-
reochemistry surrounding the THP ring systems and the func-
tionalization of the terminal motifs impacted on the T. brucei
inhibition profile. In comparison to chamuvarinin (6), the initial
set of diastereomeric alkyl triazoles 20a, 21a and 18a (en-
tries 1, 4, 7; Table 1) were more than 17 times less potent, indi-
cating that stereochemistry has little effect on the inhibition
profile. Surprisingly, analogue 19a (entry 12; Table 1) displayed
good levels of T. brucei inhibition with an EC50 value of 7.6ꢁ
0.3 mm and moderate levels of parasite selectivity (SI=21.9).
Introduction of terminal oxygenation as a handle for further
manipulation to one side of the alkyl side chain, analogues
21b, 18b, 19b and 22 (entries 5, 8, 13, 15; Table 1), resulted in
good micromolar activities with EC50 values below 10 mm. Inter-
estingly, the anti–anti analogue 20b (entry 2; Table 1) was
devoid of HeLa and T. brucei activity. By comparison, analogue
21b (entry 5; Table 1) displayed good levels of parasite inhibi-
tion and selectivity, with a selectivity index (SI) value of 23. For
analogues 24 and 27 (entries 14 and 16; Table 1), removal of
the benzyl group resulted in approximately twofold loss in par-
asite activity. Removal of the benzyl group from analogue 21b
(entry 5; Table 1) to give 26 (entry 6; Table 1) led to a significant
loss in parasitic activity and selectivity: EC50 values of 3.1ꢁ0.1
and alcohol triazole series implies that the benzyl group may
be potentially interacting with residues at a protein target site,
which an alcohol group cannot accommodate. Alternatively, or
as well, the benzyl group may be able to readily insert itself
into the lipophilic membrane, whereas the hydrophilic alcohol
incurs a greater penalty for membrane insertion.
The synthesis of triazole analogues with terminal oxygena-
tion has provided a functional handle for further elaboration
(Table 2). The non-natural chamuvarinin-like analogues previ-
ously reported revealed that introduction of the butenolide
side chain resulted in a greater than fivefold increase in
T. brucei activity. Encouraged by the potent activity of lead al-
cohol 23 (entry 1; Table 2), it was decided to incorporate the
butenolide moiety based on this structure. Analogues 31 and
32 (entries 2 and 3; Table 2) clearly highlight the importance of
the spatial orientation of the pendent butenolide side chain.
While 31 was essentially inactive, 32 displayed low micromolar
selective activities against both BSF and Pro forms of T. brucei,
with EC50 values of 3.2ꢁ0.1 and 5.7ꢁ0.6 mm, respectively and
a selectivity index (BSF/HeLa) of 15.8. This suggests that al-
though the structures are only subtly different at face value,
their binding/interaction is highly specific and indicative of
a protein target, rather than the biophysical properties of the
compounds.
On the basis of the SAR data, incorporation of both the bu-
tenolide moiety and a benzyl-protected alcohol in place of the
alkyl side chain indicated that benzyl analogue 37 (entry 4,
Table 2) had similar activity to 32. Removal of the benzyl group
to reveal the free hydroxy analogue (34; entry 5, Table 2), was
found to diminish activity and was nine-times less active than
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to estimate the drug-likeness
(LipE>5) of a compound. The
calculated LipE values for tria-
zoles 23, 32, 34, 37 and chamu-
varinin 6 fall in the range +0.6
to ꢀ3.7 (Table 3), which al-
though below the optimal
value are in line with values
often displayed by natural prod-
ucts and their derivatives.[24]
Furthermore the LipE values of
our designed triazole analogues
are comparable with bioactive
compounds that cross the
blood–brain barrier,[23,24] which
when coupled with their toxici-
ty towards T. brucei, provides
the exciting prospect of devel-
oping an effective dual pronged
treatment for both stage 1 and
2 HAT if selectivity profiles can
be further improved.
Table 2. Biological profiles for analogues 23, 31-32, 34 and 37.
Entry
Compd
EC50 [mm]
SI
BSF
Pro
HeLa
7.0ꢁ1.0
1
2
3
4
5
23
31
32
37
34
1.8ꢁ0.1
0.8ꢁ0.1
3.9
–
15.8
0.4
0.3
>500
>1000
>1000
3.2ꢁ0.1
5.2ꢁ0.3
28.5ꢁ4.6
5.7ꢁ0.6
19.2ꢁ0.8
5.6ꢁ0.6
50.8ꢁ3.7
2.1ꢁ0.4
8.3ꢁ1.1
Molecular modelling
[a] EC50 values were determined against bloodstream (BSF) and procyclic (Pro) T. brucei, or in mammalian HeLa
cells. Data represent the meanꢁSD of n=3 independent experiments performed in triplicate. The selectivity
index (SI) refers to BSF parasitic inhibition versus mammalian HeLa inhibition.
Our initial rationale for the
design and synthesis of simpli-
fied triazole analogues was
based on the molecular model-
ling of the central tricyclic core
32, with EC50 values of 28.5ꢁ4.6 and 3.2ꢁ0.1 mm, respectively.
of chamuvarinin (6), as outlined in Figure 3a, indicating that 6
adopts a “U-shape” conformation.[25] We hypothesized that
a five-membered heterocyclic spacer (Figure 3b,c) would act as
an effective spatial mimic for the central THF core of 6 and
thus retain potent biological activity. Modelling highlights that
the lowest energy conformation of the syn-syn bis-THP rings is
predicted to act as a better mimic of this “U-shaped” confor-
mation than the corresponding anti-anti, and this would
appear to be broadly borne out by the biological results.[25]
The encouraging trypanocidal activity of the triazole com-
This may indicate that a non-hydrophilic arm on the opposite
side of the butenolide moiety is required for maximum pro-
tein/analogue/lipid interaction.
Lipophilic efficiency
A theoretical comparison of the triazole analogues molecular
properties was conducted in an effort to highlight the lipophi-
licity of these compounds and provide an indication as to
whether they would be suitable drug candidates. As outlined
in Table 3, lead compound alcohol 23 (entry 1) possesses a suit-
able calculated lipophilicity (clogP) value of 5.163, has a molec-
ular weight of less than 500, and has a suitable number of hy-
drogen-bond donors and acceptors, adhering to Lipinski’s rule
of five. Introduction of the butenolide moiety on one end of
the THP ring system and an alkyl side chain on the opposite
side in analogue 32 (entry 2, Table 3), led to an increased
clogP value of 9. Exchange of the hydrophobic alkyl side chain
with either a benzyl group (37; entry 4, Table 3) or a free hy-
droxy group (34; entry 3, Table 3) decreased the clogP values
while increasing the potential for hydrogen-bond donation.
Chamuvarinin itself (6; entry 5, Table 3) is calculated to have
a high clogP value of 9, in line with the observed ability of
acetogenin family members to cross the blood–brain barrier[23]
The lipophilic efficiency (LipE) is a combination of the calcu-
lated lipophilicity (clogP) and the potency (pEC50) of analogues
Table 3. Theoretical data of molecular properties for triazole analogues 6,
23, 32, 34 and 37.[a]
Entry Compd
EC50
pEC50 clogP LipE MW
H bond
[mm]
donors acceptors
1
2
3
4
5
23
32
34
37
6
1.8ꢁ0.1 5.75 5.163
0.59 407.6
1
0
1
1
1
6
7
8
8
6
3.2ꢁ0.1 5.49 9.056 ꢀ3.57 571.8
28.5ꢁ4.6 4.55 7.336 ꢀ2.79 559.8
5.2ꢁ0.3 5.29 8.935 ꢀ3.65 649.9
1.4ꢁ0.1 5.86 9.208 ꢀ3.35 604.9
[a] EC50 values were determined against bloodstream (BSF) T. brucei; data
represent the meanꢁSD of n=3 independent experiments performed in
triplicate. Theoretical molecular properties: (clogP); (LipE); molecular
weight (MW); number of H bond donors and acceptors, were calculated
using Molinspiration Cheminformatics calculation of molecular properties
and bioactivity scores (http://www.molinspiration.com/cgi-bin/proper-
ties).
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Acknowledgements
This work was funded by The
Royal Society (UK), the Lever-
hulme Trust (UK), the UK Engi-
neering and Physical Sciences Re-
search Council (EPSRC) (G.J.F.)
and the Wellcome Trust (UK)
(T.K.S., WT 093228). The authors
thank the EPSRC National Mass
Spectrometry
Service
Centre
(Swansea, UK) for mass spec-
trometry services.
Keywords: acetogenin · Human
African Trypanosomiasis (HAT) ·
Figure 3. Molecular modelling of the lowest energy conformations for a) chamuvarinin framework; b) syn-syn tria-
zole core; and c) anti-anti triazole core.
neglected diseases
·
natural
product analogues
·
stereochemistry · Trypanosoma
brucei
pounds discussed in this paper has now formed the basis of
further design iterations and lead structure optimization. The
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procyclic forms of T. brucei. Analogue 23 displayed comparable
levels of T. brucei inhibition to that of natural product chamu-
varinin (6), with EC50 values of 1.8ꢁ0.1 and 1.4ꢁ0.1 mm, re-
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