Structure-Based Design, Synthesis and Biological Evaluation of Bis-Tetrahydropyran Furan Acetogenin Mimics Targeting the Trypanosomatid F1 Component of ATP Synthase

Article abstract of DOI:10.1002/ejoc.201900541

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for the severely debilitating neglected Tropical diseases of African sleeping sickness, Chagas disease and leishmaniasis, respectively. As part of our ongoing programme exploring the potential of simplified analogues of the acetogenin chamuvarinin we identified the T. brucei FoF1-ATP synthase as a target of our earlier triazole analogue series. Using computational docking studies, we hypothesised that the central triazole heterocyclic spacer could be substituted for a central 2,5-substituted furan moiety, thus diversifying the chemical framework for the generation of compounds with greater potency and/or selectivity. Here we report the design, docking, synthesis and biological evaluation of new series of trypanocidal compounds and demonstrate their on-target inhibitory effects. Furthermore, the synthesis of furans by the modular coupling of alkyne- and aldehyde-THPs to bis-THP 1,4-alkyne diols followed by ruthenium/xantphos-catalysed heterocyclisation described here represents the most complex use of this method of heterocyclisation to date.

Full text of DOI:10.1002/ejoc.201900541

A Journal of
Accepted Article
Title: Structure-based design, synthesis and biological evaluation
of bis-tetrahydropyran furan acetogenin mimics targeting the
trypanosomatid F1 component of ATP synthase
Authors: Marija Zacharova, Lindsay Tulloch, Eoin Gould, Andrew
Fraser, Elizabeth King, Stefanie Menzies, Terry Smith, and
Gordon John Florence
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201900541
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
Structure-based design, synthesis and biological evaluation of bis-tetrahydropyran
furan acetogenin mimics targeting the trypanosomatid F1 component of ATP
synthase
Marija K. Zacharova, Lindsay B. Tulloch, Eoin R. Gould, Andrew L. Fraser, Elizabeth F.
King, Stefanie K. Menzies, Terry K. Smith* & Gordon J. Florence*
Dr M. K. Zacharova, Dr L. B. Tulloch, Dr E. R. Gould, Dr A. L. Fraser, Dr E. F. King, Dr S.
K. Menzies, Prof. T. K. Smith*, Dr G. J. Florence*
EaStCHEM School of Chemistry and School of Biology, Biomedical Science Research
Complex, University of St Andrews, North Haugh, St Andrews, KY16 9ST, UK
E-mail: gjf1@st-andrews.ac.uk and tks1@st-andrews.ac.uk
Web:
www.st-andrews.ac.uk/bsrc/research/?userid=gjf1
and
https://synergy.st-
andrews.ac.uk/tksmith/
Abstract: The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and
Leishmania spp. are responsible for the severely debilitating neglected Tropical diseases
of African sleeping sickness, Chagas disease and leishmaniasis, respectively. As part of
our ongoing programme exploring the potential of simplified analogues of the acetogenin
chamuvarinin we identified the T. brucei FoF1-ATP synthase as a target of our earlier
triazole analogue series. Using computational docking studies we hypothesized that the
central triazole heterocyclic spacer could be substituted for a central 2,5-substituted furan
moiety, thus diversifying the chemical framework for the generation of compounds with
greater potency and/or selectivity. Here we report the design, docking, synthesis and
biological evaluation of new series of trypanocidal compounds and demonstrate their on-
target inhibitory effects. Furthermore, the synthesis of furans by the modular coupling of
alkyne- and aldehyde-THPs to bis-THP 1,4-alkyne diols followed by ruthenium/xantphos-
catalysed heterocyclisation described here represents the most complex use of this
method of heterocyclisation to date.
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
Introduction
Infectious parasitic diseases are one of the major concerns in Developing countries. In
particular, the protozoan parasites of the kinetoplastid family are responsible for several
debilitating diseases, with Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.
being responsible for African sleeping sickness (human African trypanosomiasis, HAT),
Chagas disease (American trypanosomiasis), and leishmaniasis, respectively.[1–3] All three
diseases have a multi-thousand annual mortality rate, but have been inadequately
investigated, mainly due to the limited financial incentive in treating these conditions. The
existing treatments have severe side effects and are difficult to administer (usually
intravenously) given the challenging healthcare infrastructure in many affected countries.[4–
7] A number of novel candidate molecules have entered clinical trials, Fexinidazole[8,9] and
SCYX-7158[10] currently being in phase II/III trials. Nevertheless, emerging resistance
towards existing treatments[11–13] combined with the lack of preventive methods or
possibility for vaccination,[14–16] indicate an urgent need for novel, broad-spectrum and
selective trypanocidal treatments, particularly in the light of the ambitious target set by the
World Health Organisation (WHO) to eradicate HAT by 2020. [1]
Natural products are a valuable source of unexplored chemical frameworks for the
development of new drugs. Chamuvarinin (1), originally isolated from the roots of Uvaria
chamae, which has traditionally been used as a herbal remedy for HAT, and subsequently
synthesized in our laboratory,[17] has low micromolar trypanocidal activity (Figure 1).[18] We
have since described a series of bis-tetrahydropyran 1,4-triazole analogues based on the
chamuvarinin polyether core and assembled from a pool of readily accessible chiral
tetrahydropyran (THP) building blocks linked by different heterocyclic spacers including
1,4-triazoles of type 2 (Figure 1). Many of these simplified analogues displayed low
micromolar trypanocidal activities but more significantly excellent selectivities relative to
mammalian cell lines.[19–22]
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
OH
O
O
23
20
O
15
1
O
O
1: chamuvarinin
T. brucei (BSF) EC50 = 1.37± 0.08 µM
T. brucei (Pro) EC50 = 1.90 ± 0.11 µM
Simplified
architectures
R1
O
O
R2
Het
*
*
N
N
2: Previous
1,4-triazoles
Het
N
=
O
3: current study
2,5-substituted furan
* variation of stereochemistry
O
H2N
NH2
NH
NH
DB75
Figure 1. Trypanocidal chamuvarinin, simplified heterocyclic analogues and structure of
DB75.
Through a series of pull-down experiments we identified the T. brucei FoF1-ATP synthase
as a target for our lead triazole series.[23] FoF1 is a highly conserved mitochondrial protein
consisting of transmembrane (Fo) and matrix-facing (F1) moieties.[24,25] F1, which binds
our triazole inhibitors, is a heterohexamer consisting of alternating α- and β-subunits with
six nucleotide-binding sites at their interfaces, three of which are catalytic and primarily
constitute residues from the β-subunits, while the remaining three comprising mostly α-
subunit residues have a regulatory function. From our identification and verification of F1
as a target, we proposed that our compounds act as ADP mimics and bind at these
nucleotide-binding sites.
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We hypothesized that the triazole spacer unit in our earlier generation inhibitor series (of
type 2, Figure 1) could be substituted for a 2,5-subsitituted furan moiety, of type 3, and
that this alteration to the chemical framework would reduce conformational flexibility
across the heterocyclic array, while maintaining or offering greater potency and/or
selectivity. Such bis-tetrahydropyran 2,5-substituted furan compounds share a degree of
structural similarity with DB75 (Figure 1), a bis-phenyl 2,5-furan diamidine, which is also
reported to inhibit the trypanosomatid FoF1-ATP synthase.[26] Herein we undertook
computational docking of furans into the recently disclosed crystal structure of F1 from T.
brucei[25] to identify compounds with potential trypanocidal activities. We then undertook
the synthesis of a targeted library of stereochemically complex furan derivatives identified
as potential enzyme binders and assessed their biological potential in cytotoxicity assays
and F1 assays to confirm their on-target activities.
Results and Discussion
To confirm that AutoDock Vina had been set up correctly, and to estimate binding affinity
in kcal/mol, ADP and ATP were docked into the catalytic site (corresponding to the ATP-
bound conformation) of F1 from T. brucei.[25,27] Both compounds docked suitably close to
the position of ADP detected within the F1 crystal structure (Figure 2A-B), although the β-
phosphate was positioned where the γ-phosphate of ATP would normally reside, rather
than in its own pocket. This is likely a result of Mg2+ and water molecules in the crystal
structure, which help coordinate the β-phosphate within its phosphate-binding pocket,
being absent from the docking calculations (since inhibitors can often displace ions to
increase binding affinities). On this basis we were satisfied that our modeling methodology
had been setup appropriately.
We next docked our lead triazole 4, which we have established as an F1 inhibitor,[23] into
the same catalytic site of F1 from T. brucei. Triazole 4 docked in the same mode as that
predicted for bovine F1,[23] with the THP-Et side chain bound within the hydrophobic
adenine-binding pocket and the THP-OH interacting with Glu194, Arg195 and Ser’357 (the
prime denoting a residue from the α-subunit) within the γ-phosphate-binding pocket
(Figure 2C). This binding mode was predicted to have a similar affinity for the receptor as
the substrate and product (Table 1) despite possessing many fewer atoms capable of
forming hydrogen bonds than ADP/ATP. However, only the phosphates of ADP and ATP
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
actually form hydrogen bonds with the receptor; the adenine only forms hydrophobic
interactions (with Tyr350, Phe429, Val170 and Thr430) while the ribose acts as a
heterocyclic linker, thus triazole 4 is able to mimic the substrate(s).
The bis-tetrahydropyran furan 5 was designed as an analogue of our lead triazole 4 and
alternative substrate mimetic. Both 4 and 5 contain THP-Et and THP-OH moieties fused
by a heterocycle, but the different orientation of the furan linker could provide alternative
stereochemistry for further derivatization and improved inhibitor design. Docking of furan 5
revealed that it is likely to adopt a similar binding position to triazole 4 in the catalytic site,
with the THP-Et buried within the hydrophobic adenine-binding pocket and the free alcohol
forming hydrogen bonds with Glu194, Arg195 and Ser’357 in the γ-phosphate-binding
pocket (Figure 2D). Furthermore, the predicted affinity was similar to that of 4 (Table 1).
We postulated that the substitution of the Et group for a benzyl ether (OBn) at R2 would
lead to an affinity enhancement through the introduction of additional pi-stacking
interactions between Tyr350 and Phe429 in the adenine-binding pocket, as occurs with
ADP and ATP. Docking of 6 (Figure 2E) indicated that the OBn fits within the adenine-
binding pocket without disrupting the binding of the free alcohol within the γ-phosphate-
binding pocket and lead to a slight improvement in predicted binding affinity. To determine
whether different THP stereochemical permutations could optimize binding, syn-syn (furan
7), anti-syn (furan 8) and anti-anti (furan 9) isomers were also docked. All anchored the
OBn and OH moieties within the adenine- and γ-phosphate-binding pockets respectively
and through the conformational flexibility of the THP and furan framework to link together
(Figure 2E-H), resulting in similar docking scores.
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Table 1. Compound stereochemistries and docking scores.
Architecture
Dock affinity
Compound
Figure
R1
THP
THP
R2
(kcal/mol)
ADP
ATP
-
-
-
-
-9.5
-9.8
-9.0
-8.9
-9.7
-9.7
-9.1
-9.9
-9.6
-9.7
-8.7
-9.4
2A,B
-
-
-
-
-
Triazole 4
Furan 5
Furan 6
Furan 7
Furan 8
Furan 9
Furan 10
Furan 11 [a]
Furan 12
Furan 13
Et
Syn
Syn
Syn
Syn
Anti
Anti
Syn
Syn
Anti
Syn
Syn
Anti
Anti
Syn
Syn
Anti
Syn
Anti
Anti
Anti
OH
OH
OH
OH
OH
OH
OBn
OBn
OBn
OBn
2C
2D
2E
2F
2G
2H
2I
Et
OBn
OBn
OBn
OBn
OBn
OBn
OBn
Et
2J
2K
2L
[a] Furan 11 can also be represented as OBn-Anti-Syn-OBn.
To determine the importance of the free alcohol on inhibitor potency, the THP-OH was
replaced with THP-OBn and resulting bis-THP-OBn furans (furans 10, 11 and 12) were
docked into the catalytic site (Figure 2I-K). For each compound hydrogen bonding with
Glu194, Arg195 and Ser’357 in the γ-phosphate-binding pocket was completely lost, and
despite scoring well in docking, the bound conformations appeared unrealistic, with most
of the hydrophobic contacts appearing as intramolecular interactions rather than
interactions with the receptor.
As the presence of the OBn at R1 in addition to the OBn at R2 may make the molecules
too large to fit comfortably into the active site, we docked 13 with an OBn at R1 and a
smaller Et at R2 (Figure 2L). Due to the relative similarity of furan core architectures, 13
flipped within the binding site with the OBn, rather than the THP-Et, docking in the
adenine-binding pocket. The docked position of 13 was similar to that of furans 6-9 with
OBn at R1 instead of R2. This would suggest that the phenyl at R2 is likely to improve
inhibitor binding over the THP-Et motif of compounds 4 and 5. Given the promising
docking results for our furan compounds with free alcohols, we decided to synthesise
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10.1002/ejoc.201900541
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these select examples for biological evaluation, along with the bis-THP-OBn compounds
as negative controls.
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Figure 2. Docked positions of compounds within the T. brucei F1 catalytic site. A) Position of bound ADP coordinated with ions in the
crystal structure. B) Docked position of ADP in the absence of coordinating ions. The phosphates occupy a slightly different position, but
the ribose and adenine occupy the same positions as in the crystal structure. C-L colour scheme: yellow, syn-syn; green, syn-anti; cyan,
anti-syn; magenta, anti-anti. C-D) Docking of lead triazole 4 with Et at R1 and OH at R2 and furan analogue 5. The R2 OH forms
interactions with the phosphate-binding pocket. E-H) Furan 6-9 with OBn at R1 and OH at R2. The additional R1 OBn allows π-stacking in
the adenine-bonding pocket. I-K) Bis-OBn furans 10-12. Substitution of the R2 OH for OBn results in the loss of all H-bonds and potential
destabilisation of bound compound. L) Furan 13, with the OBn at R1, flips orientation indicating the phenyl is preferable in the adenine
pocket.
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Synthesis of 2,5-substituted furan inhibitors
The classical syntheses of furans commonly utilise 1,4-dicarbonyl precursors,[28,29] such
compounds are often challenging to access with limited capacity to vary precursor
substitution. Hence, multiple methodologies of heterocyclic ring formation have been
developed utilising dicarbonyl alternatives, such as allenes,[30–35] alkynyl epoxides,[35]
alkynyl ketones[36] or 1,4-alkyne diols.[37] The methodology introduced by Williams and co-
workers involving the ruthenium/xantphos-catalysed heterocyclisation of 1,4-alkyne
diols[38] avoids the isolation of challenging 1,4-diketone precursors which was particularly
attractive in our current system to access furans of type 14, given the sensitivity of α-
oxygenated carbonyl groups to epimerisation and the compatibility with our readily
accessible chiral THP building blocks of type 15 (Scheme 1).[17,18]
O
O
O
14
R1
R2
[Ru] cyclisation
OH
O
O
OH
sequential
alkyne addition
O
O
OH HO
15
R1
R2
syn and anti THP building blocks
R1/R2 = OBn, OTBDPS, Et
Scheme 1. Bis-tetrahydropyran 2,5-furan retrosynthesis.
Starting from our established THP building block syn-16 and anti-17,[17,19] Swern oxidation
followed by diastereoselective Carreira alkynylation[39–41] gave the corresponding propargyl
alcohols 18 and 19, respectively. While the stereochemical control of the alkynylation
reactions was inconsequential for the downstream synthesis, these conditions proved
more efficient compared with lithium TMS-acetylide addition to the respective
aldehydes.[42–45] The alkynylation products were then treated with potassium carbonate to
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
cleave the C-TMS group and protected as their respective TBS ethers in alkynes 20 and
21 (Scheme 2).
O
OH
16
OBn
a, b
OP1
O
P2
OBn
18: P1 = H, P2 = TMS (68%, >95 :5 dr)
20: P1 = TBS, P2 = H (80%)
c
O
OH
17
OBn
a, b
OP1
O
P2
OBn
19: P1 = H, P2 = TMS (64%, >95 :5 dr)
21: P1 = TBS, P2 = H (72%)
c
Scheme 2. Synthesis of THP-alkynes 20 and 21: a) (COCl)2, DMSO, CH2Cl2, -78 °C, 1 h;
then Et3N -78 °C to RT, 2 h; b) TMSCCH, Zn(OTf)2, (+)-N-methylephedrine, Et3N, PhMe,
70°C, 16 h ; c) (i) K2CO3, MeOH, RT 5h; (ii) TBSCl, ImH, DMAP, CH2Cl2, RT, 16 h. TBSCl
= t-Butyldimethylsilyl chloride, ImH = imidazole, DMAP = 4-dimethylaminopyridine.
With alkyne precursors 20 and 21 in hand along with the ethyl-terminated variant 22 (used
in our initial synthesis of 1),[17] we were in a position to begin the modular assembly of first
the key 1,4-alkyne diol framework followed by the Williams Ru-catalysed heterocyclisation
to access furan derivatives with both diastereomeric and terminal functionality variations,
as detailed in Scheme 3. Access to the intermediate 1,4-alkyne diol precursors was
achieved by n-BuLi deprotonation of the respective alkyne 20, 21 or 22 followed by
addition of the respective THP-aldehyde 23-26 to give precursors 27-35 (entries 1-9) after
silyl group cleavage in modest but workable yields over two steps. In the case of additions
to syn-THP aldehydes (entries 1,2 and 9) the products were formed with low
diastereoselectivity at the new carbinol centre which proved inconsequential in the
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subsequent cyclisation. In contrast the addition of alkynes to anti-THP aldehydes (entries
3-8) proceeded with high levels of Felkin-Anh selectivity (>95:<5 d.r. in all cases). The
intermediate alkyne 1,4-diols where then cyclized under the conditions described by
Williams and co-workers employing the Ru(PPh3)3(CO)H2/xantphos catalyst combination
to afford the 2,5-disubstituted furan derivatives 5-13, as detailed in Scheme 3. The yields
for this isomerization varied from low to modest, given the complexity of the 1,4-alkyne diol
precursors, in terms of α-oxygenated tetrahydropyran motifs flanking the central 1,4-alkyne
diol. However in all cases sufficient material was isolated to facilitate evaluation of
antitrypanosomatid activity.
O
OH
OTBS
O
O
a
O
O
O
O
H
b
O
OH
R1
R2
R1
R2
R1
R2
R1 = Et or OBn
R2 =OBn
R1 = Et or OBn
R2 =OBn or OH
R1 = Et or OBn
R2 = OBn or OH
or OTBDPS
Entry
Alkyne
OTBS
Aldehyde
Intermediate Alkyne Diol
Yield (%)
Furan
O
Yield (%)
O
O
OH
O
O
O
O
H
O
OH
R1
R2
R1
R2
R1
R2
20: R1 = OBn
20: R1 =OBn
1
2
23: R2 = OBn
24: R2 = OTBDPS
27: R1 = R2 = OBn
28: R1 =OBn R2 = OH
10: R1 = R2 = OBn
7: R1 =OBn R2 = OH
52
10
14
11a
O
OH
O
OTBS
O
O
O
O
O
H
O
OH
R1
R2
R1
R2
R1
R2
22: R1 = Et
18
39
34
49b
43
20
23
44
3
4
5
6
25: R2 = OBn
26: R2 = OTBDPS
25: R2 = OBn
26: R2 = OTBDPS
O
29: R1 = Et, R2 = OBn
30: R1 = Et R2 = OH
31: R1 = R2 = OBn
13: R1 = Et, R2 = OBn
5: R1 = Et R2 = OH
11: R1 = R2 = OBn
6: R1 = OBn R2 = OH
22: R1 = Et
20: R1 = OBn
20: R1 = OBn
32: R1 = OBn R2 = OH
OTBS
OH
O
O
O
O
O
O
H
O
OH
R1
R2
R1
R2
R1
R2
7
8
33: R1 = R2 = OBn
12: R1 = R2 = OBn
9: R1 =OBn R2 = OH
21: R1 = OBn
21: R1 =OBn
25: R2 = OBn
26: R2 = OTBDPS
O
58
37
36
32
34: R1 =OBn R2 = OH
OTBS
OH
O
O
O
O
O
O
H
O
OH
R1
21: R1 = OBn
R2
R1
R2
R1
R2
24: R2 = OTBDPS
9
35: R1 =OBn R2 = OH
7
8: R1 =OBn R2 = OH
36
Scheme 3. Alkyne coupling and ruthenium/xantphos-catalysed heterocyclisation to furans:
a) (i) Alkyne, n-BuLi, MTBE, -78 °C, 10 min; aldehyde, -78 to RT, 1 h; (ii) (±)-CSA,
MeOH/CH2Cl2 (1:1), RT, 15 h; (b) Ru(PPh3)3(CO)H2 (6 mol%), xantphos (6 mol%),
PhCO2H, PhMe, 120 °C, sealed tube, 15 h. aInitially co-isolated with aldehyde by-product,
upon reduction with NaBH4 in MeOH furan 7 was isolated in 11% yield over two steps; b32
was isolated in 49% yield after two cycles of (±)-CSA in MeOH/CH2Cl2 (1:1), see
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supporting information. MTBE = methyl t-butyltether, (±)-CSA = camphorsulfonic acid,
xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
Biological evaluation
Synthesised furans were tested in cytotoxicity assays against a panel of kinetoplastid cell
lines including bloodstream form (BSF) T. brucei, procyclic form (PF) T. brucei,
epimastigote form T. cruzi and promastigote form L. major, as well as human HeLa cells
for the determination of cell selectivity. EC50 values for 5, the furan analogue of our lead
triazole 4, were similar across all cell lines (Table 2, entries 1 and 2), ranging from 10.8–
28.9 µM, and were not dissimilar from 4. To confirm that 5 was acting on target, we
assessed the ability of the compound to inhibit F1 ATP production in digitonin-
permeabilised PF T. brucei, which contained permeable cell membranes but intact,
functional mitochondria. Like triazole 4, furan 5 ablated ATP production (Table 2, entry 2),
confirming that its trypanocidal activity is through F1 inhibition, supporting docking
predictions that both compounds would be similar. These data demonstrated that the
central 1,4-triazole heterocyclic spacer could indeed be substituted for a 2,5-furan moiety.
Table 2. Biological activities of compounds.
EC50 (µM)
T.cruzi
% ATP
inhibition
124 ± 24
163 ± 11
135 ± 13
148 ± 19
137 ± 17
134 ± 27
-33 ± 21
-22 ± 31
12 ± 6
Entry
Compound
SI [a]
T. brucei BSF
1.8 ± 0.1
T. brucei PF
8.4 ± 0.2
L.major
ND
HeLa
7.0 ± 1.0
11.3 ± 1.13
7.9 ± 0.3
34.4 ± 2.2
9.1 ± 1.4
7.9 ± 1.5
>500
1
2
Triazole 4
Furan 5
ND
4
1
12.2 ± 0.8
9.7 ± 0.7
10.8 ± 0.3
7.2 ± 0.2
28.9 ± 1.4
24.9 ± 1.1
30.8 ± 0.7
18.0 ± 1.0
21.3 ± 1.4
>500
17.6 ± 0.9
29.5 ± 1.8
14.3 ± 0.3
17.6 ± 0.6
25.4 ± 1.0
>500
3
Furan 6
1
4
Furan 7
10.9 ± 0.4
14.3 ± 0.6
9.6 ± 1.3
12.2 ± 0.4
5.0 ± 0.1
3
5
Furan 8
1
6
Furan 9
5.1 ± 0.3
1
7
Furan 10
Furan 11[b]
Furan 12
Furan 13
29.9 ± 3.1
23.2 ± 1.1
14.9 ± 0.6
44.0 ± 3.4
39.0 ± 1.2
70.3 ± 3.2
32.2 ± 2.1
77.3 ± 2.9
>17
>22
>34
>6
8
>500
>500
>500
9
>500
>500
>500
10
127 ± 10
>500
>250
ND
[a] SI (selectivity index) is the ratio of HeLa EC50 / BSF EC50 and measures how selective compounds are towards BSF T. brucei relative to human HeLa cell
line. [b] Furan 11 can also be represented as OBn-anti-syn-OBn.
Likewise, the F1 assay confirmed that OBn-substituted furans 6, 7, 8 and 9 also acted on
target, by eliminating F1 ATP production (Table 2, entries 3-6). Cytotoxicity assays
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revealed that all four compounds had similar potencies across all cell lines tested, ranging
from 5.0–34.4 µM, but disappointingly, no compound offered improved toxicity or
selectivity over the THP-Et analogue 5 (Table 2, entry 2), despite the introduction of π-
stacking interactions within the adenine-binding pocket.
It is possible that the
enhancement in binding through π-stacking does not outweigh the potential loss in affinity
created through increased inhibitor flexibility, and future studies will investigate the
removal of a THP to mitigate this.
Testing of the bis-THP-OBn furans 10, 11 and 12 against F1 revealed that, as expected,
they did not inhibit F1 ATP production (Table 2, entries 7-9), and compounds were
ineffective against T. cruzi, L. major and HeLa cells, failing to significantly inhibit cell
growth below 250 µM. This correlation between loss of activity against F1 and against
cells by furans 10-12 implies that in these cells, compounds 4-9 induce their cytotoxic
activities through F1 inhibition. Given that mammalian and T. brucei F1 are highly
similar[24,25] and that 4-9 are relatively non-specific towards trypanosomatid cells, the
design of trypanosomatid-specific F1 inhibitors of this type may prove challenging.
Remarkably, despite failing to inhibit T. brucei F1, 10, 11 and 12 still displayed activity
towards T. brucei, albeit with somewhat reduced potencies (Table 2, entries 7-9), providing
compounds with selectivities towards BSF T. brucei over human HeLa cells of >17, >22
and >34 respectively. This would suggest that these bis-THP-OBn compounds have an
alternative T. brucei-specific target and that in the design of potential F1-targetting
inhibitors, variants incapable of F1 inhibition have proven fruitful. Future compounds will
likely be designed for their inability to inhibit F1 in order to reduce their potent side effects.
Furan 13, which docks in a flipped orientation providing an anti-syn architecture, like
furans 8 and 11, but with the OH/OBn substituted for an Et group, also failed to
significantly inhibit the growth of HeLa, L. major and T. cruzi (Table 2, entry 10) and both
displayed cytotoxicity similar to 11. This further highlights the importance of hydrogen
bonding to the γ-phosphate-binding pocket for F1-binding and inhibition and may therefore
form a focal point for avoidance in future investigations.
Conclusions
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
Having previously identified the target of our original triazole inhibitor series as
mitochondrial F1 and established their hypothetical binding mode, furan analogues were
proposed to provide an alternative framework for future analogue derivatisation. We
therefore employed a structure-based approach to design, synthesise and biologically
evaluate compounds targeting the FoF1-ATP synthase. Computational docking indicated
that binding of furan analogues was feasible, so the compounds were synthesised by the
coupling of pairs of THP building blocks to access 1,4-alkyne diols, followed by
ruthenium/xantphos-catalysed heterocyclisation. This successful synthetic route is itself an
achievement given the complexity of the alkyne diol precursors, which to our knowledge,
are the most complex substrates thus described for this method of heterocyclisation.
Cytotoxicity assays confirmed that many of the synthesized furans were broadly cytotoxic
against a panel of kinetoplastid pathogens as well as human HeLa cells, while an F1 ATP
synthase assay confirmed that they acted on target at F1. Docking indicated that
substitution of the THP-Et (5) for THP-OBn (6-9) could potentially enhance inhibitor
binding through favourable π-stacking interactions with the receptor, and while all retained
F1-inhibitory properties, enhanced cytotoxicity was not evident. Inhibitors with terminal
hydroxylation appeared to form critical hydrogen bonding interactions. Upon substitution
with a benzyl ether moiety a loss of activity against most cell lines was observed along
with the loss of F1 inhibition, further indicating the on-target effects of previous analogues.
Unexpectedly, these di-benzoylated compounds 10, 11 and 12 retained modest activity
against T. brucei, with selectivities ranging from 17-34 fold, and therefore represent new
lead compounds for further development in finding new treatments for HAT.
Experimental Section
Compound synthesis and characterisation
Full details of compound synthesis and spectroscopic characterisation is available in the
supporting information.
Computational Studies
Compounds – bis-THP furan compounds of syn-syn, syn-anti, anti-syn and anti-anti
variants and 4 were drawn and converted to three-dimensional Protein Data Bank (PDB)
format structures and minimized using Schrødinger Suite 2012 update 2 (Maestro version
9.3.518) with Macromodel using the MMFF forcefield. PDB coordinates of ADP and ATP
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
were extracted directly from F1. Compounds were then converted to pdbqt format using
AutodockTools version 1.5.6.[46]
Protein receptor sites – the F1 crystal structures from T. brucei (6F5D.pdb)[25] was used for
docking with Autodock Vina.[27] The receptor was prepared exactly as described in the
user manual using Autodock tools.[46] A grid box of 17 x 15 x 23 Å was centred around the
catalytic site comprising chains B (α-subunit) and F (β-subunit).
Compound docking and evaluation – Ligand-macromolecule docking was carried out using
Autodock Vina. In the same directory files for the ligand and macromolecule, configuration
file, and the vina.exe file available for free online were placed. The top 10 binding modes
were output and visually assessed with Pymol (Schrødinger). Optimal binding mode and
corresponding AutoDock energy (Kcal/mol) for each compound were assessed.
Biological Evaluation
Compounds were screened against parasites (bloodstream form [BSF] T. brucei brucei
strain 427, procyclic [PF] form T. brucei strain 29-13, epimastigote T. cruzi strain CL
Brener, promastigote L. major strain Friedlin) and human HeLa cells.
General cell culture – BSF T. brucei were maintained between 103-106 cells/ml in full HMI-
11 (HMI-11 medium supplemented with 10% heat-inactivated foetal bovine serum (HI-
FBS, Gibco), 1 mM sodium pyruvate, 160 µM thymidine, 50 µM Bathcupromedisulfonic
acid, 1 mM hypoxanthine, 1.5 mM L-cysteine, 60 µM 1-thioglycerol, and 2.5 mg/L G418
(Calbiochem)) at 37 °C with 5% v/v CO2, as described previously.[47] PF T. brucei were
maintained between 105-107 cells/mL in full SDM-79 (SDM-79 medium supplemented with
10% HI-FBS, 23.8 mM sodium bicarbonate, 10 µM haemin, 12.5 mg/L G418 and 25 mg/L
hygromycin) at 28oC with 5% CO2.[48] T. cruzi were maintained between 106-107 cells/ml in
full RTH (RPMI 1640 medium supplemented with 10% HI-FBS, 16.53 mM HEPES pH 7.2,
4.2 g/L peptone (Fluka), 2 mM sodium glutamate, 100 mg/L streptomycin, 100 KU/L
penicillin, 30 µM haemin) at 28oC with 5% CO2. L. major were maintained between 105-
107 cells/ml in full M199 (M199 medium (Sigma) supplemented with 10% HI-FBS, 4.17 mM
sodium bicarbonate, 40 mM HEPES pH 7.4, 100 µM adenosine and 10 µM haemin) at
28oC with 5% CO2. HeLa cells were maintained in DMEM supplemented with 10% HI-FBS
at 37oC with 5% CO2.
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
Cytotoxicity assays – compounds were serially diluted across 96-well microtitre plates
containing 100 µl of relevant full growth medium in quadruplicate. The final concentration
of DMSO (used to solubilise compounds) never exceeded 1% (v/v). Cells were diluted to
2x required cell density in relevant full growth medium and 100 µl added to the 100 µl
serial dilution (total volume 200 µl per well), generating starting cell densities of 5 x 103/ml
(BSF T. brucei), 5 x 104/ml (PF T. brucei, promastigote L. major and HeLa) or 1 x 106/ml
(epimastigote T. cruzi). Cultures were incubated at the relevant temperature with 5% CO2
for 64 hours, then 10-20 µl of resazurin (1.1 mg/ml in PBS) was added and cultures were
incubated for a further 6 h at relevant temperature with 5% CO2 to allow cells to oxidise
resazurin to resorufin. Resorufin fluorescence was recorded with a Bio Tek FLX8000
spectrofluorimeter and Gen5 software, using an excitation of 545 ± 15 nm and emission of
590 nm. Curves were calculated using GraFit through the application of four-parameter
least squared non-linear regression and EC50 values were taken as the concentration of
drug at the inflection point.
FoF1 ATP synthase inhibition assay
Compounds were screened for their abilities to inhibit ATP production from succinate in
digitonin-permeabilised PF T. brucei as described previously.[23] Briefly, PF T. brucei were
washed in PBS, resuspended in isotonic SoTE buffer (20 mM Tris.HCl pH 7.5, 2 mM
EDTA pH 7.5, 600 mM sorbitol) and their plasma membranes permeabilised with 0.015%
(w/v) digitonin for 5 min on ice. Permeabilised cells were pelleted at 5000 g for 3 min at
4oC, resuspended in isotonic assay buffer (20 mM Tris.HCl pH 7.4, 15 mM KH2PO4, 10
mM MgSO4, 600 mM sorbitol, 2.5 mg/ml fatty acid-free BSA), and incubated with/without
inhibitor at 200 µM for 10 min at RT to allow inhibitors to take effect. Succinate (2.5 mM)
was added as substrate and incubated at RT for 10 min to allow uptake to the
mitochondrion. ATP production was initiated with the addition of 60 µM ADP, and after 30
min incubation at RT the reaction was terminated with the addition of 1.37% (final
concentration) perchloric acid, which also released generated ATP from the
mitochondrion. After neutralization with KOH, the degree of ATP production was
quantified using the CLSII bioluminescence assay kit (Roche) as directed, recording
bioluminescence using Spectramax Gemini XPD spectrofluorimeter (Molecular Devices)
and Softmax Pro software. Background luminescence was subtracted from each well and
ATP levels calculated relative to uninhibited controls.
Acknowledgments
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10.1002/ejoc.201900541
European Journal of Organic Chemistry
This work was funded by the Leverhulme Trust (G.J.F.), Wellcome Trust ISSF support
(G.J.F./T.K.S.) and the European Community’s Seventh Framework Programme under
grant agreement No. 602773 [Project KINDRED] (T.K.S.). We thank the EPSRC National
Mass Spectrometry Service Centre, Swansea, UK, for mass spectrometry services.
Keywords: Trypanosomatid • neglected tropical disease • drug design • FoF1-ATP
synthase • ruthenium/xantphos-catalysed heterocyclisation
Table of Contents
FULL PAPER
A series of 2,5-substituted furan
derivatives based on the tricyclic ether
core of the acetogenin, chamuvarinin,
display activity towards T. brucei, the
causative parasite of human African
Trypanosomiasis (HAT), by inhibition
of the F1 component of the ATP
synthase. Access to these privileged
scaffolds utilises a Ru-catalysed
heterocyclisation of alkyne 1,4-diol
precursors.
HAT Inhibitors
Marija K. Zacharova, Lindsay B. Tulloch,
Eoin R. Gould, Andrew L. Fraser,
Elizabeth F. King, Stefanie K. Menzies,
Terry K. Smith* & Gordon J. Florence*
Page No. – Page No.
Structure-based design, synthesis
and biological evaluation of bis-
tetrahydropyran furan acetogenin
mimics targeting the trypanosomatid
F1 component of ATP synthase
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