Synthesis and evaluation of a novel series of indoloisoquinolines as small molecule anti-malarial leads

Article abstract of DOI:10.1016/j.bmcl.2011.12.071

A group of novel synthetic indoloisoquinolines was prepared and its potential as a novel series of smallmolecule anti-malarial leads was assessed. The structure-activity relationship on variation of three distinct regions of chemical space was investigate

Full text of DOI:10.1016/j.bmcl.2011.12.071

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1770–1773
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a novel series of indoloisoquinolines as small
molecule anti-malarial leads
Paul Horrocks a,b, Samantha Fallon c, Laura Denman a, Oliver Devine a, Liam J. Duffy b,c, Anthony Harper d,
Emma-Louise Meredith d, Sandra Hasenkamp b, Adam Sidaway d, Daniel Monnery a, Theresa R. Phillips c,
Steven M. Allin c,
⇑
a School of Medicine, Keele University, Staffs. ST5 5BG, UK
b Institute for Science and Technology in Medicine, Keele University, Staffs. ST5 5BG, UK
c School of Physical and Geographical Sciences and Institute for the Environment, Physical Sciences and Mathematics, Keele University, Staffs. ST5 5BG, UK
d School of Life Sciences, Keele University, Staffs. ST5 5BG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of novel synthetic indoloisoquinolines was prepared and its potential as a novel series of small-
molecule anti-malarial leads was assessed. The structure–activity relationship on variation of three dis-
tinct regions of chemical space was investigated. A lead compound was generated with an activity close
to that observed for a known anti-malarial natural product, dihydrousambarensine, that shares the indo-
loisoquinoline template structure.
Received 2 November 2011
Revised 12 December 2011
Accepted 13 December 2011
Available online 21 December 2011
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Indoloisoquinoline
Anti-malarial
Natural product
Malaria
Dihydrousambarensine
Malaria continues to pose a significant global health and socio-
economic burden on those regions where it is endemic. Whilst sub-
stantial investment in the delivery of front-line artemisinin-based
combination therapies and use of insecticide impregnated bednets
have seen a fall in the mortality attributed to malaria over recent
years, data from 2009 show that this disease still imposes a signif-
icant impact both in terms of morbidity (ꢀ255 million cases) and
mortality (ꢀ781,000 deaths) annually.1 A major challenge is the
narrow drug discovery pipeline, a problem exacerbated by recent
reports of artemisinin treatment failure in South East Asia.2,3 In re-
cent years, however, high throughput screening of small-molecule
libraries as well as natural products derived from plants and marine
organisms have sought to seed this pipeline with diverse and novel
chemical entities.4–6 Indole alkaloids isolated from various Strych-
nos species of plants have been demonstrated to show in vitro
selectivity and activity against both chloroquine resistant (CQR)
and sensitive (CQS) isolates of Plasmodium falciparum, the aetiolog-
ical agent of the most severe form of human malaria.7,8 Of these
compounds characterized, dihydrousambarensine, 1, emerged as
an interesting candidate showing altered levels of activity between
CQR and CQS isolates, with a clear preferred activity against CQR
isolates (0.85 lM vs 0.03 l
M for CQS and CQR, respectively).8 Over
recent years our research group has become expert in the stereose-
lective synthesis of indole alkaloid targets and we have recently re-
ported the asymmetric synthesis of alkaloids including harmicine 2,
deplancheine 3 and 12b-epidevinylantirhine 4 (Fig. 1).9 Given the
structural similarity of dihydrousambarensine, 1, to these com-
pounds we have initiated a research program to investigate the
structure–activity relationship (SAR) on the indoloisoquinoline
template 5. Examination of a collection of heterocyclic compounds
originating from our historical research efforts identified 10 com-
pounds that shared some structural similarity to dihydrousamba-
rensine. Of these, the indoloisoquinoline derivative 6a (Fig. 2) was
identified as a potential lead compound for this investigation
(IC50 of 20 lM, Table 1). Based on this structure we decided to
undertake an initial investigation of three areas of chemical space
(Fig. 2) with the aim of gaining an increased appreciation of the
resulting activity that could be realised by simple chemical modifi-
cation of the readily available indoloisoquinoline core 5a, namely:
(i) the indole N-substituent (R); (ii) the hydroxymethyl O-substitu-
ent (R1); (iii) alkenic substitution on or around the lactam ring (R2).
Compound 5a was prepared by our previously reported route
from L
-tryptophan,10 and utilized as a starting point for further
structural diversity in our investigation. Symmetrical alkylation
of the N- and O-groups was readily achieved by the general proce-
dure outlined in Scheme 1 to yield compounds 7a–c.
⇑
Corresponding author. Tel.: +44 1782 724371.
E-mail address: s.m.allin@chem.keele.ac.uk (S.M. Allin).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.071

P. Horrocks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1770–1773
1771
OR1
O
OH
N
O
i) NaH in DMF
N
N
H
N
N
H
N
N
H
ii) alkyl halide
Yields 14-90%
H
N
N
H
H
N
R
H
N
H
H
N
H
N
H
H
2
1
3
7a-c
5a
OH
O
O
O
OR1
O
O
N
O
N
N
N
N
H
N
N
H
H
H
H
N
H
N
R
H
4
5
6a
OH
6b
8a-c
Figure 1. The indoloisoquinoline template and anti-malarial leads.
Scheme 1. Synthesis of indoloisoquinoline targets.
OR1
O
OR1
O
OR1
O
O
(ii)
O
N
N
N
N
N
R
N
R
H
H
N
N
H
H
R2
R2
R2
(iii)
R
(i)
6a
L-series
D-series
Figure 3. Alternative template structures of the lead indoloisoquinoline compounds.
Figure 2. Initial lead compound and proposed sites of chemical modification on the
indoloisoquinoline template.
Anti-malarial activity was determined using an adaptation of
the lactate dehydrogenase (LDH) assay.12,13 Assays to determine
the 50% inhibitory concentration (IC50) were carried out using
intraerythrocytic cultures of P. falciparum lines Dd2 (chloroquine
resistant) and 3D7 (chloroquine sensitive). It is immediately appar-
ent from Table 1 that the compounds tested in this screen show a
significant increase in antiplasmodial activity (p = 0.015) on mov-
We were mindful of the fact that the absolute stereochemistry
present within the indoloisoquinoline core of the anti-malarial nat-
ural product dihydrousambarensine, 1, did not match that of our
initial series of compounds and, due to the known potential differ-
ences in activity of enantiomeric series of biologically active com-
pounds, we also prepared the analogous series of diastereoisomeric
compounds, 8a–c, by following the same series of alkylation reac-
tions from the diastereoisomeric template 5b, itself produced from
ing from the
the importance of absolute stereochemistry in this indoloisoquin-
oline template. Indeed the more active -series of compounds
L-tryptophan to the D-tryptophan series, highlighting
the D
-enantiomer of the original tryptophan precursor (Fig. 3).10
D
Unsymmetrically substituted compounds 9–17 (Table 1) were pre-
pared by a modified procedure in which the appropriate precursor
(5a or 5b) underwent stepwise derivatization. Initial alkylation
using NaH in DMF with 1.5 equiv of the alkyl halide gave the
N-alkyl product followed (if required) by a second alkylation on
the hydroxymethyl oxygen atom (using NaH in DMF with 2 equiv
of the appropriate alkyl halide). We have also prepared and inves-
tigated the enantiomer 6b of our lead compound from the initial
screen, 6a. The synthesis of 6b followed the previously reported
has the same absolute stereochemistry at the ring junction within
the heterocyclic core as the natural product that formed the basis
of this study (dihydrousambarensine, 1) and also shares the rela-
tionship between stereochemistry and activity with other natural
products from the Frédérich study.8As a result we therefore
decided to continue our own study with compounds derived from
the D-tryptophan enantiomer.
Themes that emerge from this study include the apparent pref-
erence for a benzyl substituent on the indole nitrogen atom (R),
with a methyl (as in 11b) or allyl (as in 12b) substituent at R1 lead-
ing to the highest levels of activity in this screen (both have an IC50
synthesis of 6a albeit starting from the D-enantiomer of the original
tryptophan precursor.11 It is worth noting at this point that com-
pounds 5, 7–17 lacked the presence of the vinyl substituent (R2)
on the lactam ring.
value of 1.3 l
M, Table 1). An increase in steric bulk at R1 on moving
Table 1
Structure–activity relationships of indoloisoquinoline derivatives14
Compound
L-series
R
R1
R2
IC50
(
lM) Dd2
Compound
D
-series
R
R1
R2
IC50 (lM)Dd2
5a
6a
7a
7b
7c
H
H
H
vinyl
H
H
H
71
20
30
16
12
5b
6b
8a
8b
8c
H
H
H
Vinyl
H
H
H
56
13
29
13
3.5
5
Benzyl
Methyl
Allyl
Benzyl
Methyl
Allyl
Benzyl
Methyl
Allyl
Benzyl
Benzyl
Methyl
Allyl
Benzyl
H
Benzyl
Benzyl
9
Benzyl
H
10
11b
12b
13
14
15
16
17
Allyl
Benzyl
Benzyl
Benzyl
H
H
H
H
H
H
H
H
H
41
1.3
1.3
2.8
19
9
11a
12a
Benzyl
Benzyl
Methyl
Allyl
H
H
32
35
Methyl
Allyl
Propyl
Cyclohexylmethyl
Allyl
Benzyl
Cyclohexylmethyl
Cyclobutylmethyl
4-Pyridyl methyl
Allyl
Allyl
7
2.1

1772
P. Horrocks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1770–1773
OR1
N
N
R
H
Figure 4. Tertiary amine derivatives 18 and 19.
from an allyl (in 12b) to either a propyl group, as in 13, or to a
cyclohexyl ring, 14, gave a fall in activity. The propyl moiety may
be viewed as being less planar, and consequently more degrees
of freedom, than allyl as a result of the introduction of the double
bond in the latter. The fully saturated cyclohexyl moiety would be
expected to show conformational preference for a chair rather than
the planarity present in the original aromatic ring of 12b. The dif-
ference in activity between 8c (R1 = benzyl) and 14 (R1 = cyclo-
hexylmethyl) is significant and again points to the preference for
some degree of planarity in the ring component, and also possibly
an increase in electron density, within the R1 substituent.
Variation of the ring moiety within the R grouping in 12b leads
to significant falls in activity with compounds 15 and 16. Each of
these analogues contains an all-carbon ring structure at R, but nei-
ther alternative group (cyclohexylmethyl nor cyclobutylmethyl)
shares the electronic or steric properties of the aromatic ring con-
tained within the original benzyl substituent at R in 12b.
Compound 17 retains almost the same level of activity as 12b
on introduction of an alternative nitrogen-bearing pyridylmethyl
substituent at R. Indeed, one would expect that the steric and elec-
tronic properties of the pyridylmethyl group would closely mimic
that of the benzyl substituent at R in 12b, albeit with an increase in
the overall basicity of pyridyl analogue, 17. In this current series of
compounds an increase in the basic nature of the compounds does
not seem to lead to a corresponding increase in activity, a fact sup-
ported by the work on compounds 18 and 19 (Fig. 4), discussed
below.
Comparing compound 18 with its amide analogue 11b (Table 1)
we observe a slight decrease in activity on removal of the lactam
functionality. Compound 19, bearing an allyl substituent at R1,
shows comparable activity (IC50 1.5
lM) to its amide parent 12b
(IC50 1.3 M). Removal of the lactam carbonyl group to generate
l
tertiary amines does not therefore lead to an apparent increase
in antiplasmodial activity in this series.
Based on the observed increase in activity of 12b over 8c we
decided to prepare compound 20 (Fig. 5), incorporating the O-allyl,
N-benzyl substitution pattern of 12b but now introducing the vinyl
group present at R2 in the original lead compound 6a. Compound 20
was prepared by a route that is analogous to the preparation of
compound 6b, albeit starting from the N-benzyl, O-allyl compound,
12b. Compound 20 was found to have an IC50 of 1.1 lM, and is our
most active compound to date. It is interesting to compare this
activity with that of compounds 6a and 6b (Table 1) which share
the presence of the vinyl substituent at R2. Simply replacing the
O-benzyl substituent of 6b by O-allyl (in 20) leads to a considerable
increase in activity. Although close to the margin for error, com-
pound 20, containing the vinyl substituent at R2, is more active than
its precursor 12b and the original vinyl-containing leads 6a and 6b.
Finally, compounds 21 and 22(Fig. 6) were prepared by typical
selenoxide-induced unsaturation chemistry in order to investigate
an alternative modification of the lactam ring, effecting a change in
ring conformation on introduction of a conjugated double bond
into this ring.
On comparing these results to those of the ‘parent’ compounds
12b and 8c there is no significant change in activity of their unsat-
urated analogues, 21 and 22 respectively (Table 3).
In this study we set out to investigate the structure–activity
relationships of a range of synthetic indoloisoquinolines sharing
some structural similarities to the known antiplasmodial natural
product dihydrousambarensine, 1. Through a series of rational
structural modifications we have developed our compound series
With the results of the pyridyl analogue 17 in hand we decided
to explore an alternative mode to introduce a higher level of basi-
city to the series. Tertiary amine analogues 18 and 19 (Table 2)
were prepared by amide group reduction of compounds 11b and
12b respectively (using lithium aluminium hydride in dry THF).
Table 2
Activity of tertiary amine analogues
from initial activities of around 70
lM to our current lead com-
pound, 20, with an IC50 of 1.1 M. This level of inhibition is compa-
l
Compound Yield (%)
R
R1
IC50 (lM) Dd2
rable, if not better, than those previously reported for monoindole
alkaloid moieties isolated from Strychnos spp.8 Of note is the fact
that, like most indole alkaloids, the inhibitory effects reported here
for the CQR isolate Dd2 were not significantly different from those
determined from the CQS isolate 3D7 (see Supplementary data).
18 (45%)
19 (12)
Benzyl
Benzyl
Me
Allyl
3.3
1.5
O
O
N
Table 3
Activity of unsaturated lactam derivatives
N
H
Compound Yield (%)
R
R1
IC50 (lM) Dd2
20
Figure 5. Most active synthetic indoloisoquinoline.
21 (25%)
22 (40)
Benzyl
Benzyl
Allyl
Benzyl
1.7
3.6
OR1
O
N
N
H
R
Figure 6. Unsaturated indoloisoquinolines 21 and 22.

P. Horrocks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1770–1773
1773
We have identified the following general structural properties
Supplementary data
as having an effect on antiplasmodial activity in the synthetic
indoloisoquinolines:
Supplementary data (representative experimental conditions
and characterization data for the synthesis of compounds 8a, 9,
11b, 12b, 19,20, and 21) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.12.071.
(i) absolute chirality of the template: compounds derived from
D-tryptophan are preferred;
(ii) the steric bulk and a degree of planarity within R is impor-
tant, with the presence of an aromatic ring being preferred;
(iii) introduction of allyl at R1 can lead to an increase in activity;
(iv) an increase in basicity at R, or within the lactam ring, does
not lead to an increase in activity;
References and notes
1. World Malaria Report 2010, World Health Organization.
2. Fidock, D. Nature 2010, 465, 297.
3. Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong, M.;
Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.;
Day, N. P.; Lindegardh, N.; Socheat, D.; White, N. J. N. Eng. J. Med. 2009, 361, 455.
4. Gamo, F. J.; Sanz, L. M.; Vidal, J.; de Cozar, C.; Alvarez, E.; Lavandera, J. L.;
Vanderwall, D. E.; Green, D. V.; Kumar, V.; Hasan, S.; Brown, J. R.; Peishoff, C. E.;
Cardon, L. R.; Garcia-Bustos, J. F. Nature 2010, 465, 305.
(v) introducing unsaturation to the lactam ring does not lead to
an increase in activity;
(vi) introduction of substitution, currently a vinyl group, at R2
can lead to an increase in activity.
5. Oliveira, A. B.; Dolabela, M. F.; Braga, F. C.; Jácome, R. L.; Varotti, F. P.; Póvoa, M.
M. An. Acad. Bras. Cienc. 2009, 81, 715.
That chirality of the template, and the size and nature of substi-
tutions at the R and R1 position, correlates with the antiplasmodial
activity in this compound series suggesting an interaction with a
specific parasite target. Compounds 8c and 20 when added to
6. Batista, R.; Silva, Ade. J., Jr.; de Oliveira, A. B. Molecules 2009, 14, 3037.
7. Frédérich, M.; Tits, M.; Angenot, L. Trans. R. Soc. Trop. Med. Hyg. 2008, 102, 11.
8. Frédérich, M.; Jacquier, M. J.; Thépenier, P.; De Mol, P.; Tits, M.; Philippe, G.;
Delaude, C.; Angenot, L.; Zèches-Hanrot, M. J. Nat. Prod. 2002, 65, 1381.
9. Allin, S. M.; Thomas, C. I.; Doyle, K.; Elsegood, M. R. J. J. Org. Chem. 2005, 70, 357;
Allin, S. M.; Khera, J. S.; Witherington, J.; Elsegood, M. R. J. Tetrahedron Lett.
2006, 47, 5737; (c) Allin, S. M.; Gaskell, S. N.; Elsegood, M. R. J.; Martin, W. P.
Tetrahedron Lett. 2007, 48, 5669.
10. Allin, S. M.; Thomas, C. I.; Allard, J. E.; Doyle, K.; Elsegood, M. R. J. Tetrahedron
Lett. 2004, 45, 7103.
11. Allin, S. M.; Khera, J. S.; Thomas, C. I.; Witherington, J.; Doyle, K.; Elsegood, M. R.
J.; Edgar, M. E. Tetrahedron Lett. 2006, 47, 1961.
12. Makler, M. T.; Hinrichs, D. J. Am. J. Trop. Med. Hyg. 1993, 48, 205.
13. Intraerythrocytic cultures of P. falciparum were maintained in standard
continuous culture.14 Cultures were maintained at 37 °C in a 1% O2: 3% CO2:
96% N2 environment. When required, cultures were synchronised to ring stages
using the sorbitol lysis technique.15All compounds were solubilised in 100%
dimethyl sulphoxide (DMSO) to a 100 mM stock (stored at À20 °C), with dilution
to appropriate concentration made in complete P. falciparum cell culture
P. falciparum culture at 5 lM appear to induce major morphological
alterations in the trophozoite stage of parasite development (data
not shown), although this provides only very provisional insights
to a potential target for the indoloisoquinoline target. Introduction
of a vinyl substituent and the resulting levels of biological activity
in compound 20 tantalizingly suggest that additional exploration
of the R2 position is desirable, with the aim of extending the indolo-
isoquinoline core to produce analogues that more closely mimic the
structural properties found in this region of chemical space within
the bisindole alkaloid, dihydrousambarensine, 1. Such modifica-
tions, and more detailed analysis of the temporal and spatial effect
of these compounds on parasite growth and development, will form
the basis of future studies in our group and our results will be re-
ported in due course.
medium immediately prior to use. Assays were carried out in
microtitre plate using an initial 200 l of 2% haematocrit 1% ring stage culture
and five-fold dilutions of drug (800 M–51.2 nM). 100% growth was established
from cultures where no drug was added, and 0% from cultures subjected to a
supralethal dose (100 nM) of artemether. Following 48 h of incubation, 25 l of
resuspended culture were transferred to a fresh 96-well microtitre plate and
100 l of Malstat reagent (0.1 M Tris pH9.1, 0.2 M sodium lactate, 10
acetylpyridine adenine dinucleotide (APAD), 0.2% Triton X-100) and 25
NBT/PES (16
g mlÀ1nitrobluetetrazolium, 0.239 M phenazineethosulphate)
a 96-well
l
l
l
Acknowledgments
l
lM
ll of
Financial and facilities support was provided by Keele Univer-
sity (studentship to SF), including Innovation Keele (IK) funding,
and from the North Staffordshire Medical Institute. We also
acknowledge bursary support to AS and OD from the Nuffield
Foundation and Wellcome Trust, respectively. Paul Horrocks is a
BBSRC New Investigator. EPSRC is gratefully acknowledged for pro-
vision of financial support to LJD (EP/F026552/1) and for access to
the EPSRC Mass Spectrometry Service Centre, Swansea.
l
added and mixed by pipetting. After 1hr incubation in the dark at room
temperature, the absorbance at 650 nm was measured using a Scientific Biotek
EL800 plate reader. The mean % growth of at least n = 6 assays was plotted using a
log dose–response curve and the IC50 extrapolated using GraphIT (v3 Erithacus
Software).
14. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
15. Lambros, C.; Vanderberg, J. P. J. Parasitol. 1979, 65, 418.