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O.M. Yvette et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
93.81 nM, respectively), when compared to CQ (IC50 = 300 nM).
These results showed that the AD-CQ conjugates exhibit marked
antimalarial activity. This was also confirmed by the selectivity
index (SI). The high SI values of the compounds (SI = 73–2825)
indicates greater selectivity towards the resistant parasite strain
K1 compared to the CHO cells and implies that the activity of the
conjugates is because of their antiplasmodial activity.52,53
When observing the RI, the aza-adamantanol conjugates (1–4)
in general showed better activity with lower resistance factors
compared to their imine adamantane (6–8) counterparts except
for 5. Evaluating the two series structurally in relation to their
activity, it was established that the presence of the hydroxyl
group may play a role in improved reversed CQ ability of the
aza-adamantanols. This may indicate that the hydroxyl group
could help or play a part in the binding of the compounds to
the PfCQRT and aid in blocking this channel or that the hybrid
with the OH is less susceptible towards efflux through this
channel.
Furthermore, the calculated pKa values of the tertiary adaman-
tane amine of the aza-adamantanol series (pKa: 8.3–9.2) were
closer to that of CQ (pKa = 10.2) compared to the imine-adaman-
tanes (pKa: 6.3–8.9). This increased pKa values of the aza-
adamantanols may have influenced their ability to accumulate
in the parasitic digestive vacuole and also could explain their
improved activity.54 It is worth mentioning that although the
imine-adamantane compounds showed significant activity
against the CQ sensitive strain, they had markedly reduced activ-
ity against the CQ resistant strain. This loss of activity may be
directly linked to the lower pKa values observed or other struc-
tural features making the imine-adamantane moiety a weaker
RA. Furthermore, imine containing compounds are known to
undergo hydrolysis in aqueous media, especially at lower pH val-
ues.55 However, with the imine-adamantane compounds the
imine is part of a six-membered ring in a lipophilic adamantine
polycyclic system which should provide enhanced stability of
the imine function against hydrolysis. Hydrocarbyl imines that
are lipophilic are relatively more stable than short chain and
hydrophilic hydrocarbyl imines towards hydrolysis.55 The
adamantane moiety and structurally related PCU’s have also been
shown to provide enhanced chemical stability and metabolic sta-
bility of conjugated molecules.56 In order to explore the stability
and if the loss of activity was due to degradation by imine hydrol-
ysis of the imine-adamantanes, 6 was selected as the representa-
tive imine-adamantane derivative and evaluated for chemical
stability under the same conditions as the pLDH antimalarial
assay procedure (see Supplementary material section 1.2.3). Com-
pound 6 was dissolved (1 mg/ml) in HEPES containing aqueous
buffer at the same pH (7.4) as the pLDH assay and at a pH within
a similar range as the parasites’ acidic digestive vacuole pH
(4.5)57 respectively. The solutions were then incubated at 37 °C
for 72 h to verify if the observed decreased activity in the anti-
malarial assays may be due to degradation. Results from this
study indicate that 6 remain relatively stable at pH 7.4, however
degradation was observed at pH 4.5 after 72 h. This indicates that
the imine-adamantanes will remain stable in the assay media
(aqueous HEPES-buffer, pH 7.4, 37 °C) and may undergo hydroly-
sis once inside the parasites’ acidic digestive vacuole. It is cur-
rently unclear if this observation may have influenced the pLDH
assay results, especially in the resistant strain, and further studies
are necessary to elaborate on these findings.
flexibility of the compounds in the PfCQRT protein increases and
may adopt an unfavourable conformation whereas the short chain
conjugates may adopt a more favourable conformation and thus
improves the efficacy of the compounds. From both series, the con-
jugates with alkyl linkers of 2–3 carbons showed optimum activity
for both NF54 and K1.
All the compounds tested showed reduced cross-resistance,
with a resistance index (RI) factor in the range 2.11–16 compared
to 38 for CQ. The aza-adamantanols generally had improved RI fac-
tors when compared to their imine-adamantane counterparts,
which indicates that the aza-adamantanol conjugates are better
RCQ compounds. The observed RI of the three most active com-
pounds (1, 2 and 5) was between 2.11 and 4.34. Furthermore, the
reversal effect of the adamantane moiety as a CQ resistance RA
compared to the structurally related PCU moiety used as a RA in
our previous study34 was compared. This was done by comparing
the RI of the AD-CQ conjugates with that of the PCU-CQ derivatives,
represented by PCU-CQ10 (see Fig. 1 and Table 1) identified as the
most promising RCQ agent in our previous work.34 To easily and
clearly compare the reversal effect, the reversal modification index
(RMI) was calculated (see Table 1). It revealed that PCU-CQ10 (with
a 2C linker) was 5.22 times better at retaining activity than CQ
whereas compound 1 (also with a 2C linker), was 18 times better
at retaining activity than CQ. In addition, six out of the eight AD-
CQ derivatives (1–5, 7; RMI: 5.3–18) showed improved RMIs com-
pared to PCU-CQ10 and the adamantane moieties, as used in the
AD-CQ conjugates, are thus more potent reversal agents in vitro
than the PCU moiety.
In conclusion, resistance to antimalarial drugs especially CQ
is a major setback in the use of chemotherapy to control malar-
ia. This created the need to identify and develop new improved
antimalarial agents. A total of eight non-toxic AD-CQ reversed
CQ derivatives were synthesized and compounds 1–6 exhibited
potent antimalarial activity in vitro superior to CQ against the
CQ resistant strain K1 and overcame P. falciparum resistance
to CQ. Compound 1 stood out as a potent conjugate (K1 IC50
= 98.92 nM) with the lowest resistance index (RI = 2.11) and
was identified as the most promising AD-CQ conjugate. Its abil-
ity to retain activity in the CQ resistant strain was 18-fold bet-
ter than that of CQ. The adamantane moiety, especially in the
aza-adamantanol series, was shown to be a significant P. falci-
parum CQ resistance reversal agent compared to the previously
used structurally related PCU moiety. Hence, the hybridization
of a CQ-like nucleus to adamantane moieties results in reversed
CQ molecules with improved antimalarial activity that could
overcome P. falciparum CQ resistance. The next step of this
study will be to carry out further in vitro and in vivo biological
and mechanistic studies to elaborate on the molecular mecha-
nism(s) involved in parasite-killing and reversal of the PfCQRT
efflux effect. Also, the role of the hydroxyl group on the activity
of the aza-adamantanol conjugates should be investigated fur-
ther to build on the structure activity relationships of the
compounds.
Acknowledgements
We are grateful to Prof Edith Antunes for assistance with NMR,
Mr Kippie for assistance with MS as well as the University of the
Western Cape and the National Research Foundation of South
Africa for financial support.
The IC50 values of the AD-CQ derivatives increased as the chain
length of the alkyl linker increased. This trend is significant in the
resistant strain K1 and concurs with literature in that chain length
changes has little influence on activity against CQ sensitive strains
but has a profound influence in CQ resistant strains.36,58 This can
be explained in that as the chain length increases the degree of
A. Supplementary data
Supplementary data associated with this article can be found, in