In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium falciparum related to the efficacy of chalcones

Article abstract of DOI:10.1016/j.bmcl.2010.11.099

A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were found to be submicromolar inhibitors in vitro of drug-r

Full text of DOI:10.1016/j.bmcl.2010.11.099

Bioorganic & Medicinal Chemistry Letters 21 (2011) 786–789
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium
falciparum related to the efficacy of chalcones
Clare E. Gutteridge a, , Marshall M. Hoffman a, Apurba K. Bhattacharjee b, , Wilbur K. Milhous b,à
,
⇑
Lucia Gerena b
a Department of Chemistry, United States Naval Academy, Annapolis, MD 21402, USA
b Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same
mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were
found to be submicromolar inhibitors in vitro of drug-resistant Plasmodium falciparum, with the best
possessing activity comparable to chloroquine. Despite being developed from a lead that is a DHFR
inhibitor, these compounds do not mediate their antimalarial effects by inhibition of DHFR.
Published by Elsevier Ltd.
Received 5 October 2010
Revised 17 November 2010
Accepted 22 November 2010
Available online 26 November 2010
Keywords:
Plasmodium
Malaria
In vitro efficacy
Quinolinamine
Chalcone
Each year, more than 1 million people die from malaria.1 The
organism responsible for most of these deaths, Plasmodium
falciparum, has developed resistance to most available drugs.2,3
Thus, there is an urgent need for novel and affordable new prod-
ucts.4 Chalcones (1,3-diphenyl-2-propen-1-ones) and their carbo-
cyclic and heterocyclic analogs display a wide range of biological
activities including antiprotozoal, antibacterial, antifungal and
antiproliferative activity.5 Several groups have studied the relation-
ship between the structure of substituted chalcones and their anti-
malarial activity.6–14 Chalcones possessing submicromolar efficacy
against drug-resistant strains of P. falciparum in vitro have been
identified.6,15 The emerging SAR does not correlate with the SAR of
known antimalarial targets, implying that the mechanism by which
the chalcones act is distinct from established mechanisms.7–9 Both
reports that propose possible mechanisms for chalcone action
involve their modification of the erythrocyte membrane: either by
inhibiting the permeation pathways induced by the parasite in the
erythrocyte membrane,8 or by transformation of normal erythro-
cytes into echinocytes, which creates conditions unfavorable for
parasite growth.9 Several of these compounds have been tested in
animal models of malaria (Plasmodium yoelii- or Plasmodium
berghei-infected mice)6,12,14,16 and some possess significant in vivo
efficacy.12,14,16 However, in vitro and in vivo efficacies often do not
correlate.6,16 Although possibly due to the difference in Plasmodium
species employed between in vitro and in vivo assays, we suspect
that rapid compound metabolism also contributes because the
chalcones tested by us were all rapidly digested in vitro by micro-
some preparations.6
We therefore wished to identify a compound series structurally
distinct from the chalcones, but that mediates antimalarial activity
via the same mechanism. We aimed to achieve this through use of
a 3D-pharmacophore that we had developed using CATALYST soft-
ware which models the structural and electronic features required
by compounds to effect antimalarial activity via this novel
chalcone-mechanism.17 The pharmacophore was used in an in
silico screening of the collection of compounds at the Walter Reed
Army Institute of Research. It predicted that 6-(30-bromobenzyla-
mino)-2,4-quinazolinediamine (1) should mediate antimalarial
effects by a chalcone-like mechanism (Fig. 1).
Such quinazolinetriamines are known to exert potent antima-
larial activity by inhibition of dihydrofolate reductase (DHFR).18
Unfortunately, acquired drug resistance to DHFR inhibitors rapidly
develops.19 We, therefore, sought to design compounds instead
that exhibit antimalarial activity via a chalcone-like mechanism
and have minimal DHFR activity. Published data suggests that
the N1- and the 2-amino groups are important in mediating the
inhibition of DHFR by 6-benzylamino-quinazolinetriamines.20–22
⇑
Corresponding author. Tel.: +1 410 293 6638; fax: +1 410 293 2218.
E-mail address: gutterid@usna.edu (C.E. Gutteridge).
Present address: Department of Regulated Laboratories, Division of Regulated
Activities, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
à
Present address: Global Health Infectious Disease Research Program, College of
Public Health, University of South Florida, Tampa, FL 33612, USA.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.11.099

C. E. Gutteridge et al. / Bioorg. Med. Chem. Lett. 21 (2011) 786–789
787
2
N
NH2
a
7
NH.HCl
6
NH
3'
Br
N
N
O2N
N
H
N
H
1
4
R
2 HCl
(3)
NH2
NH2
b
(1)
(2a-f)
Figure 1. Structures of the in silico screening hit (1) and compounds designed from
it (2a–f).
c,d
N
N
O2N
O2N
Cl
e,f
N
N
H
N
g
R
H2N
NH2
2 HCl
NH2
(4)
R = H (2a), 3-Cl (2b), 4-Cl (2c),
3-Br (2d), 3-CF3 (2e), 3,4-Cl2 (2f)
Scheme 1. (a) KNO3 (aq), H2SO4 (aq); HCl, EtOH. (b) (KSO3)2NO, Na2CO3 (aq).
(c) m-CPBA. (d) p-TsCl, pyr. (e) (1) Ethanolamine. (f) H2, Pd/C. (g) R-C6H5CHO;
NaBH3CN; HCl, EtOH.
(2a–f) were prepared. The route commences with commercially
available 1,2,3,4-tetrahydroisoquinoline (3), and proceeds via
1,7-isoquinolinediamine (4).23,24 We have previously reported that
this sequence was used successfully to produce 1,7-isoquinolined-
iamine (4) on about a 1 mmol-scale.25 Subsequent efforts to scale-
up the synthesis by about 10-fold resulted in significantly reduced
yields in several of the steps. Significant optimization of the
synthesis would, therefore, be needed to produce the final com-
pounds in significant quantities. This finding, together with the
length of the synthetic sequence and the expense of the oxidant
employed in the second step, (KSO3)2NO, give us concern that it
may not be possible to bring down the production cost of the
isoquinolinamines sufficiently to be acceptable for their intended
use.
The in vitro antimalarial activities of the novel 1,7-diaminoiso-
quinolines (2a–f) were determined by measuring their ability to
inhibit [3H]-hypoxanthine uptake by P. falciparum.26 The results
against the mefloquine-resistant, chloroquine-sensitive D6 strain;
the Southeast Asian multi-drug resistant isolate TM91C235; and
the chloroquine-resistant, mefloquine-sensitive W2 strain; are
shown in Table 1. The compounds were found to inhibit parasite
Figure 2. The four structural features required for antimalarial activity via the
chalcone-mechanism, represented by the geometric shapes labeled according to
type, overlaid with the structure of compound 2d.
We proposed that 7-benzylamino-1-isoquinolinamines (2a–f),
which lack these functional groups, should inhibit DHFR to a con-
siderably lesser extent than do the 6-benzylamino-quinazolinetri-
amines (Fig. 1). Such isoquinolinamines are not described in the
literature, so have not previously been assessed as antimalarial
agents. Our pharmacophore model predicted that the bromo-
substituted 7-benzylamino-1-isoquinolinamines (2d) would be a
submicromolar inhibitor of P. falciparum. In Figure 2 the structure
of this isoquinolinamine is overlaid upon that of the pharmaco-
phore. The latter comprises the four structural features required
for inhibition via the chalcone-mechanism, and is shown by the
geometric shapes.17 The overlay shows that the isoquinolinamine
possesses all four of these structural features.
The seven-step synthetic sequence, shown in Scheme 1, was
developed by which the 7-benzylated 1,7-diaminoisoquinolines
Table 1
Analogs synthesized with in vitro efficaciesa against P. falciparum D6, W2 and TM91C235 and resistance indicesb,c calculated relative to D6
N
NH2
Br
N
N
N
H
N
H
R
2 HCl
NH2
NH2
(1)
(2a-f)
Compound number
R
P. falciparum D6
P. falciparum TM91C235
P. falciparum W2
Resistance indexb
TM91C235/D6
Resistance indexc
W2/D6
a
a
a
IC50
(l
M)
IC50
(lM)
IC50
(lM)
Chloroquine
Mefloquine
1
2a
2b
2c
2d
2e
2f
0.0134
0.0201
0.0712
0.0374
0.0208
0.486
0.158
0.205
0.142
0.111
0.0719
0.397
5.30
1.85
147
2.29
2.44
2.78
1.38
1.60
1.44
29.5
0.289
14.6
6.59
5.55
5.67
4.09
6.58
5.20
0.00601
0.00208
1.40
0.359
0.417
0.421
0.455
0.260
—
H
<0.000142
0.212
0.0648
0.0736
0.103
3-Cl
4-Cl
3-Br
3,4-DiCl
3-CF3
0.0691
0.0501
a
b
c
Inhibition of [3H]-hypoxanthine uptake by P. falciparum; values from one experiment.
IC50 against TM91C235/IC50 against D6.
IC50 against W2/IC50 against D6.

788
C. E. Gutteridge et al. / Bioorg. Med. Chem. Lett. 21 (2011) 786–789
IC50 (nM)
2000
D6
D6+FA
TM91C235
TM91C235+FA
W2
W2+FA
1500
1000
500
0
Pyrimethamine
Chloroquine
Compound 2b
Compound 2c
Figure 3. The effect of folinic acid, which can circumvent DHFR inhibition, on in vitro antimalarial efficacy. The IC50 of pyrimethamine (a DHFR inhibitor) increases
significantly in the presence of 1 mM folinic acid (+FA); in contrast, the IC50 of chloroquine (not a DHFR inhibitor) and of compounds 2b and 2c, are not significantly altered.
growth, generally at submicromolar concentrations. The best pos-
sess potencies comparable to chloroquine. The data suggest that
both the 3- and the 4-halo substituents on the benzyl group con-
tribute to potency. Potencies against the TM91C235 isolate on
average were twofold higher than against the D6 strain, with the
average for the W2 strain being almost sixfold higher. These results
suggest that 1,7-diaminoisoquinolines may not generally have
significant cross-resistance; but do display a modest reduction in
sensitivity, particularly to the 4-aminoquinoline, chloroquine.
In order to ascertain that the antimalarial properties of the
1,7-diaminoisoquinolines are not due to their inhibition of DHFR,
we tested two such compounds in an assay which employs folinic
acid to circumvent inhibition of parasite growth by inhibition of
DHFR.27 This method compares the IC50 for a test compound in
the presence, or the absence, of 1 mM folinic acid (+FA). When
the DHFR inhibitor pyrimethamine is tested with either pyrimeth-
amine-sensitive (D6), or pyrimethamine-resistant (TM91C235,
W2) parasites, the IC50 of pyrimethamine increases significantly
in the presence of 1 mM folinic acid, as shown in Figure 3. In con-
trast, folinic acid does not cause a significant change in the IC50 of
chloroquine, which is not a DHFR inhibitor. That folinic acid does
not cause a significant change in the IC50 of the 1,7-diaminoiso-
quinolines 2b or 2c suggests that they, like chloroquine but unlike
pyrimethamine, do not mediate their antimalarial effects by inhibi-
tion of DHFR.
U.S. Animal Welfare Act and other federal statutes and regulations
relating to animals and experiments involving animals and adheres
to principles stated in the Guide for the Care and Use of Laboratory
Animals, NRC Publication, 1996 edition. Material has been reviewed
by the Walter Reed Army Institute of Research. There is no objection
to its publication. The opinions or assertions contained herein are
the private views of the authors and are not to be construed as offi-
cial, or reflecting true views of the Department of the Army or the
Department of Defense.
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A 3D-pharmacophore model developed from the antimalarial
activities of a series of chalcones was used to design a series of
1,7-diaminoisoquinolinamines that are expected to mediate anti-
malarial activity by a novel chalcone-like mechanism. As predicted
by the model, substituted 1,7-diaminoisoquinolinamines were
found to be submicromolar inhibitors of drug-resistant P. falcipa-
rum, with the best possessing activity comparable to chloroquine.
Despite being developed from a lead that is a DHFR inhibitor,18
we have demonstrated that the 1,7-diaminoisoquinolinamines do
not mediate their antimalarial effects by inhibition of DHFR. Future
studies will include testing representative compounds against Pf
DHFR and in vivo efficacy testing.
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We are grateful for support of this work by the Military Infectious
Diseases Research Program; the Office of Naval Research; and the
United States Naval Academy, including from the Naval Academy
Research Council. Research was conducted in compliance with the
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26. In vitro efficacy was determined by a modified version of Desjardins’ method,
in which parasites were pre-exposed to test compound prior to measurement
of their [3H]-hypoxanthine uptake, as reported previously.15
27. The assay was a modification of the folic acid reversal assay method, using
normal plasma, described in Milhous, W. K.; Weatherly, N. F.; Bowdre, J. H.;
Desjardins, R. E. Antimicrob. Agents Chemother. 1985, 27, 525.