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M. Ghavami et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Instead a reductive protocol21 was applied to give 1r in 27% yield
over two steps. When this protocol was applied to the synthesis
of acetylene-containing 1s, reduction of the C„C triple bond was
observed. Suspecting that borane formed in the reaction was
responsible for this outcome, cyclohexene was added as a trap.
This modification proved successful and 1s was isolated in 33%
yield over 2 steps.
To assess antimalarial activity of these compounds, the well-
established SYBR Green method22 was used to measure growth
inhibition of erythrocytic Plasmodium falciparum 3D7, a CQ- and
MQ-sensitive parasite line, over one replication cycle (Table 1).23
Atovaquone and WR99210 were selected as positive controls,
and as expected, they very potently inhibited growth (IC50 values
of 0.6 and 0.12 nM, respectively). MQ inhibited growth with an
IC50 value of 9.1 nM, and the non-piperidine MQ analogs featured
IC50 values ranging from 4 to 22,000 nM. To benchmark the
growth inhibition data obtained for 3D7 strain parasites, in Table 1
we also list published10 data on the W2 strain, which like 3D7 is
MQ-sensitive. Although these data are derived from a different
assay, i.e. measurement of [3H]hypoxanthine incorporation,24
and from a different calculated parameter (IC90 vs IC50), others
have shown that SYBR Green- and [3H]hypoxanthine-derived
IC50 values on a single strain can be very closely correlated.23
Where comparisons can be made, compounds that were potent
(IC50 < 10 nM) against 3D7 strain in the SYBR Green assay (MQ,
1c, 1f, 1i, 1j, 1m), also potently inhibited [3H]hypoxanthine incor-
poration in W2 strain.
Likewise compounds that are weakly potent against 3D7 strain
in the SYBR Green assay (1d, 1e, 1k, 1o) are weak growth inhibitors
of W2 strain in the [3H]hypoxanthine incorporation assay. A
log–log plot of our measured 3D7 strain IC50 values versus
published W2 strain IC90 values is shown in Figure 2. In view of
the fact that these data were obtained using two different parasite
lines, two different growth inhibition assays, and reflect two differ-
ent parameters the correlation seen in Figure 2 is surprisingly good
and recapitulates the previously reported structure–activity
relationships,3b namely: i) non-piperidine MQ analogs lacking an
pendant amine (3) are much less potent than those that do
(1c–1s); ii) phenethyl-substitution (1m) confers good potency,
but anilino- (1k) does not; iii) a pendant 1° alcohol reduces
Figure 1. Selected quinoline antimalarial drugs and drug candidates.
the asexual replication cycle. In contrast CQ was found to only
weakly inhibit endocytosis, but inhibited vesicle trafficking.20b
Thus it has been proposed that the antimalarial action of MQ
derives from inhibition of hemoglobin endocytosis.7c,20a
As a test of this hypothesis, in this work we compare the anti-
malarial potency (growth inhibition, SYBR Green) of MQ and 18
non-piperidine analogs to their potency to inhibit amino acid
efflux from Plasmodium falciparum-infected erythrocytes. We have
recently shown that amino acid efflux provides a reliable surrogate
measure for hemoglobin endocytosis and subsequent catabolism.12
The required non-piperidine MQ analogs 1c–q (12 known, 6 new,
all racemic) were prepared from the commercial epoxide 2 and
the required amines by heating in ethanol in a sealed tube at
130 °C for 2–24 h (Scheme 1). In general our yields with conven-
tional heating were in the 70–99% range, similar to the yields
reported by other authors using microwave heating.3b Only in
the cases of 1k, 1l, 1n, 1o & 1s were yields below 60% observed.
Reaction with cyanamide under these conditions however gave
compound 3, due to reaction with the solvent. Diamine
derivatives 1r–s were prepared by ring-opening 2 with the
indicated phthalimide-protected 1° amines, and removal of the
phthalimide group. Interestingly the standard hydrazine
deprotection protocol was not successful, perhaps due to
unwanted reaction with the electron-deficient quinoline ring.
potency (cf. 1e
& 1c); and iv) within the mono- and
dialkylamine-substituted compounds an intricate relationship
exists between structure and potency (cf. 1d & 1c, 1g & 1d, 1n &
1m).
With P. falciparum growth inhibition activities of MQ, 1c–s and
3 established, their effects on hemoglobin uptake and catabolism
was assayed using a published procedure for quantitation of leu-
cine (Leu) efflux from intact P. falciparum 3D7-infected erythro-
cytes into the culture medium.12 In this assay, parasites are
cultured in medium lacking the amino acids Leu and Val, and con-
taining the internal standard D-norvaline. These modifications do
not affect parasite growth.12 After defined time periods, condi-
tioned culture medium is recovered and the Leu concentration is
determined by UPLC (AccQ-Tag). The effect of MQ (160 nM) on
Leu efflux is shown in Figure 3.
As can be seen, DMSO-treated P. falciparum 3D7-infected ery-
throcytes efflux Leu steadily from 0 to 240 min. However, for
infected erythrocytes treated with 160 nM MQ, Leu efflux substan-
tially slows after 60 min, resulting in a 4 h Leu concentration
roughly 1/8 of the DMSO-treated erythrocytes. Several lines of evi-
dence indicate that Leu efflux serves as a reliable surrogate mea-
sure of rates of hemoglobin uptake and catabolism: (i) amino
acid efflux from uninfected erythrocytes is negligible; (ii) relative
efflux rates of amino acids correspond well to their abundance in
hemoglobin; and (iii) administration of a protease inhibitor greatly
suppresses amino acid efflux.12 Thus MQ (160 nM) dramatically
Scheme 1. Synthesis of racemic MQ analogs 1c-s and ethanol opening product 3.
See Table 1 for the definition of R1 and R2. Reagents and conditions: (i) R1R2NH (2–
7 equiv), EtOH, sealed tube, 130 °C, 1–24 h. (ii) BuNH(CH2)3NPhth (2 equiv), EtOH,
sealed tube, 130 °C, 2 h. (iii) NaBH4 (10 equiv), i-PrOH/H2O, rt, 22 h; HOAc (1 equiv)
80 °C, 5 h. (iv) HC„C(CH2)2NH-(CH2)3NPhth (2 equiv), EtOH, sealed tube, 130 °C,
2 h. (v) NaBH4 (10 equiv), cyclohexene (10 equiv), i-PrOH/H2O, rt, 22 h; HOAc
(1 equiv) 80 °C, 5 h. (vi) NH2CN (2 equiv), EtOH, sealed tube, 130 °C, 2 h.
Ghavami, Maryam
