Antimalarial activity of novel 4-cyano-3-methylisoquinoline inhibitors against: Plasmodium falciparum: Design, synthesis and biological evaluation

Article abstract of DOI:10.1039/c5ob02517f

Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum parasites. Following each cycle of intracellular development and replication, parasites activate a cellular program to egress from their current host cell and invade a new one. The orchestration of this process critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number of central kinases. Parasite kinases are emerging as novel antimalarial targets as they have diverged sufficiently from their mammalian counterparts to allow selectable therapeutic action. Parasite protein kinase A (PfPKA) is highly expressed late in the cell cycle of the parasite blood stage and has been shown to phosphorylate a critical invasion protein, Apical Membrane Antigen 1. This enzyme could therefore be a valuable drug target so we have repurposed a substituted 4-cyano-3-methylisoquinoline that has been shown to inhibit rat PKA with the goal of targeting PfPKA. We synthesised a novel series of compounds and, although many potently inhibit the growth of chloroquine sensitive and resistant strains of P. falciparum, they were found to have minimal activity against PfPKA, indicating that they likely have another target important to parasite cytokinesis and invasion.

Full text of DOI:10.1039/c5ob02517f

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Antimalarial activity of novel 4-cyano-3-
methylisoquinoline inhibitors against
Plasmodium falciparum: design, synthesis
and biological evaluation†
Cite this: DOI: 10.1039/c5ob02517f
a
b,c
a
a
Melissa J. Buskes,‡ Katherine L. Harvey,‡ Benjamin J. Richards, Robabeh Kalhor,
a
a
d
b
Rebecca M. Christoff, Chamodi K. Gardhi, Dene R. Littler, Elliott D. Cope,
Boris Prinz, Greta E. Weiss, Nathan J. O’Brien, Brendan S. Crabb,
Leslie W. Deady, Paul R. Gilson and Belinda M. Abbott*
b
b
a
b,c,d
a
b,d
a
Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum para-
sites. Following each cycle of intracellular development and replication, parasites activate a cellular
program to egress from their current host cell and invade a new one. The orchestration of this process
critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number
of central kinases. Parasite kinases are emerging as novel antimalarial targets as they have diverged
sufficiently from their mammalian counterparts to allow selectable therapeutic action. Parasite protein
kinase A (PfPKA) is highly expressed late in the cell cycle of the parasite blood stage and has been shown
to phosphorylate a critical invasion protein, Apical Membrane Antigen 1. This enzyme could therefore be a
valuable drug target so we have repurposed a substituted 4-cyano-3-methylisoquinoline that has been
shown to inhibit rat PKA with the goal of targeting PfPKA. We synthesised a novel series of compounds
and, although many potently inhibit the growth of chloroquine sensitive and resistant strains of
P. falciparum, they were found to have minimal activity against PfPKA, indicating that they likely have
another target important to parasite cytokinesis and invasion.
Received 9th December 2015,
Accepted 31st March 2016
DOI: 10.1039/c5ob02517f
www.rsc.org/obc
Introduction
Malaria represents a major global health challenge causing
widespread morbidity and mortality and placing a huge
a
Department of Chemistry and Physics, La Trobe Institute for Molecular Science,
La Trobe University, Melbourne, Victoria 3086, Australia.
medical, social and economic burden upon resource-poor
populations. Nearly half of the world’s population is at risk of
infection and the World Health Organisation estimates that
over 500 000 deaths and nearly 200 million clinical cases
E-mail: b.abbott@latrobe.edu.au; Tel: +61 3 94792520
b
Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria 3004,
Australia
c
Department of Microbiology and Immunology, Peter Doherty Institute, University of
1
arise each year. Although these figures indicate there has
Melbourne, Melbourne, Victoria 3010, Australia
d
been progress in recent years in reducing the burden of
disease much remains to be done to achieve eventual elimin-
Monash University, Melbourne, Victoria 3800, Australia
Electronic supplementary information (ESI) available: Copies of the H and
1
†
1
3
C NMR spectra of compounds 9–50. Counter screen of compounds 4, 12, 13, ation and eradication. Despite significant efforts to reduce the
2
3 and 25. Video S1. Live cell image sequence of a Plasmodium falciparum schi-
disease’s impact, major obstacles to overcome are a lack of an
zont-stage parasite treated with 10 µM compound 25 showing egressing mero-
effective vaccine and increasing resistance to available thera-
pies. New antimalarial therapeutics are therefore urgently
needed but their design requires the identification and charac-
zoites and their attempts to invade nearby erythrocytes. Some invasions were
successful (white arrows) whilst some failed to penetrate (pink arrow) or could
penetrate but not reseal their erythrocyte host (red arrow). Video S2. Live cell
image sequence of a Plasmodium falciparum schizont-stage parasite treated with terisation of novel potential drug targets.
2,3
1
0 µM compound 25 showing egressing merozoites and their attempts to invade
Malaria parasites have a complex life cycle that encom-
passes a succession of developmental stages across different
cells types and host species, and a detailed understanding of
the molecular mechanisms that underpin key events will be
nearby erythrocytes. Some invasions were successful (white arrows) whilst some
failed to penetrate (pink, yellow, orange and green arrows) or could penetrate
but not reseal their erythrocyte host (red and blue arrows). See DOI: 10.1039/
c5ob02517f
4
‡
These authors contributed equally.
essential to inform the development of therapeutic strategies.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Proliferation, and specifically invasion, requires the co-
ordinated modulation of specific gene sets and strict control
of the cellular machinery. Malaria parasites regulate these pro-
cesses by several means including phospho-signaling, a univer-
5
–7
sally described intracellular regulatory mechanism.
Several
malarial protein kinases are now known to be essential for
parasite growth, replication and dissemination and these have
arguably diverged sufficiently from their host counterparts to
represent selectable therapeutic targets and a route to the next
8
,9
generation of antimalarials.
The cyclic AMP-dependent protein kinase (PKA) of
P. falciparum, the causative agent of the most lethal form of
human malaria, represents an attractive target for the develop-
1
0,11
ment of drugs against this species.
In most organisms
PKA exists as a heterotetramer comprising of two regulatory
Fig. 1 Structures of commercially available PKA inhibitors H89 and
subunits that bind and inhibit the activity of two catalytic sub- KT5720.
units, but PfPKA has only a single regulatory and catalytic
subunit. Binding of two cyclic AMP (cAMP) molecules to the
5
1 kDa regulatory subunit causes disassociation and activation
Two commercially available PKA inhibitors, N-[2-[[3-(4-
of the 40 kDa catalytic subunit that phosphorylates substrates
using ATP as a phosphate donor.
1
1–13
bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfon-
Despite its relatively
1
9,20
amide (H89)
and (5R,6S,8S)-hexyl 6-hydroxy-5-methyl-13-
high sequence homology with that of Homo sapiens (45.8%
identity; 77.2% similarity), the catalytic subunit of PfPKA dis-
plays distinctiveness in ATP anchoring and mechanisms of
substrate recognition and phosphorylation. Selective targeting
of these aspects would allow the tailoring of compounds that
selectively inhibit PfPKA with minimal cross inhibition of
human PKA and consequent toxicity to the host.
oxo-6,7,8,13,14,15-hexahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methano-
dibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacene-6-carboxylate
2
1
(
KT5720) (Fig. 1), have been used in over 2000 studies to
investigate PKA activity, but widespread non-specific effects
2
2–24
have been identified.
A series of 4-cyano-3-methyl-
isoquinoline derivatives, 1–4 (Fig. 2), have also been reported
as selective inhibitors of the catalytic subunit of PKA from rat
Several studies using stimulants and inhibitors of PKA
activity and modulation of gene expression have now high-
lighted the indispensability of PfPKA for parasite
2
5
liver, acting competitively with ATP as a substrate. The
unsubstituted compound, 2, is commercially available and has
been utilised as a chemical biology tool in a number of mam-
1
1,12,14,15
16
growth.
A recent phospho-proteome study identified
2
6,27
malian systems.
Preliminary study and testing of com-
4
25 schizont phosphoproteins that display PKA phosphoryl-
pound 4 and a small set of analogues against P. falciparum
indicated sufficient activity to warrant further investigation.
ation motifs, indicating that this kinase likely has multiple
important functions during the replicative phase of the
asexual blood stage cell cycle. The role of PfPKA is becoming
increasingly well characterised in invasion of erythrocytes via
5
6
This report describes the preparation of a library of 4-cyano-
-methylisoquinoline analogues, based upon the structure of
3
1
7
compound 4, with a view to studying their structure–activity
relationships as inhibitors of PfPKA.
phosphorylation of Apical Membrane Antigen 1 (PfAMA1).
PfAMA1 is a membrane spanning ligand with a pivotal role in
erythrocyte invasion, namely formation of a ‘moving junction’;
a circumferential membrane junction that bridges the host–
parasite interface and is thought to serve as a stable anchoring
structure upon which the parasite can apply traction as it
invades (reviewed in ref. 18). Within the parasite cytoplasm is
Results and discussion
Chemical synthesis
the C-terminal domain of PfAMA1, which is phosphorylated at Synthesis of a library of 4-cyano-3-methylisoquinolines was
serine S610 by PfPKA. Mutation of PfAMA1S610 renders para- undertaken principally through the key intermediate
sites unable to efficiently invade and thus it appears that phos- N-(4-cyano-3-methyl-1H-isochromen-1-ylidene)acetamide
6,
phorylation of PfAMA1S610 by PfPKA is a central event in obtained by acetylation of commercially available 2-cyano-
1
7
28
establishing infection of erythrocytes. Other substrates of phenylacetonitrile 5 (Scheme 1).
PfPKA have also been identified in recent times, namely To investigate the role of the alkyl substituent at the 3-posi-
myosin A (PfMyoA), glideosome associated protein GAP45 tion, 5 was reacted with two other anhydrides in the
PfGAP45) and calcium-dependent protein kinase 1 (PfCDPK1), presence of sodium acetate. Residual amounts of the acid pro-
(
1
6
which all function during erythrocyte invasion. Thus inhi- duced as a byproduct in such an acylation reaction can
bition of PfPKA will likely disrupt multiple key processes result in the rearrangement of the acylated intermediate into
2
9
throughout erythrocyte invasion to halt the parasites’ infection the corresponding isocoumarin. As a consequence, ana-
cycles.
logues 7 and 8 were not isolated but were immediately coupled
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 2 Structures of known 4-cyano-3-methylisoquinoline inhibitors of rat liver PKA.
1
Scheme 1 Synthesis of 1-, 3- and 4-substituted isoquinolines. Reagents and conditions: (a) (R CO)
2
O, NaOAc, reflux, 2 h; (b) For 11; aq. NH
3
, 1,4-
2
9
2
dioxane, reflux, 1 h; (c) For 4, 9–10 and 12–25; R H, acetone, <25 °C, 0.5 h; (d) 15, 1 : 1 H
f) For 28; NaOBu, n-BuOH, reflux, 1 h.
2 4 2
SO /H O, reflux, 65 h; (e) For 27; KOEt, EtOH, reflux, 1 h;
(
with n-butylamine to produce the target compounds 9 and 10 isolation. For water soluble amines, addition of ice-water
with 3-propyl and 3-pentyl substituents respectively. resulted in precipitation of the solid product. However, for the
The reaction of acetamide 6 with amine nucleophiles has more hydrophobic alkylamines, such treatment resulted in an
previously been described through a mechanism of ring oily mix of the product and excess amine. Careful addition of
opening and subsequent cyclisation to form the desired dilute acid to the mixture was able to preferentially solubilise
3
0
29
isoquinoline, including the lead compound 4 and 11.
the amine so that the crude product could be separated.
Modification of the 1-position was explored through the syn- Chromatography or recrystallisation then completed the purifi-
thesis of compounds 12–25 by treatment of the 3-methyl com- cation process. A number of variations on this theme were
pound 6 in acetone at low temperature (<25 °C) with an required for the various amines, and the details are given in
extensive variety of linear and branched alkylamines, as well as the Experimental section.
aralkylamines.
Acid hydrolysis of 15 was carried out to afford the
It was found that a large excess of amine was needed to corresponding carboxamide 26, to provide insight into the
force the complex mechanism to favor formation of the target importance of the nitrile at the 4-position. Introduction of a
isoquinolines. This resulted in some difficulties in the product 1-alkoxy moiety was also undertaken by the reaction of 6 with
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
ethoxide and butoxide in the corresponding anhydrous alcohols,
Derivatisation at the 7-position was further explored by
29
to give the known 1-ethoxy compound 27 and 1-butoxy com- nitration of the lead compound 4, which, as for bromination,
56
pound 28, the latter providing a direct analogue of the lead gave largely the 7-nitro isomer 37 (Scheme 3) and a trace
compound 4. amount of the 5-nitro isomer. Palladium-catalysed hydrogen-
Electrophilic bromination of the benzo ring of compound 4 ation produced the desired amino analogue 38 in high yield.
resulted in preferential formation of 7-bromo-1-butylamino-4- Reaction of 38 with acetyl chloride gave the acetamido com-
cyano-3-methylisoquinoline 31 (Scheme 2), with <10% of the pound 39. Treatment of 38 with isocyanuric acid, produced
5
-isomer. Carefully raising the pH to ca. 5 during aqueous in situ from sodium cyanate in 80% aqueous acetic acid,
workup resulted in predominant precipitation of the 7-isomer, afforded the urea analogue 40, along with a substantial
and recrystallisation completed the purification. The orien- amount of 39 as byproduct. Separation was readily achieved as
tation of the 7-bromo compound was established from NMR only the latter was soluble in dichloromethane.
3
experiments. In particular, there was strong JCH coupling in
The 7-amino compound 38 was also reacted with 4-chloro-
the HMBC spectrum between C4 (at a characteristic δ benzaldehyde in ethanol to generate the imine 41, which was
8.9 ppm) and H5, an ortho-coupled doublet at δ 7.63 ppm. isolated and then reduced with sodium borohydride to the
This doublet therefore required the bromine to be at position 7-(4-chlorobenzyl)amino compound 42 in a moderate yield. It
8
3
7
(
.
Similarly,
δ 155.4 ppm) and H8, a meta-coupled doublet (δ 8.62 ppm). PfPKA as a series of 7-benzylamino-1-isoquinolinamine inhibi-
Brominated compounds 29 and 30 were also prepared without tors had previously shown submicromolar inhibition against
JCH coupling was observed between C1 was of interest to explore the activity of this substituent against
3
1
any further derivatisation.
P. falciparum, with an unknown mechanism of action. The
Suzuki–Miyaura couplings of 31 with phenylboronic acid amino group of 38 was also indirectly converted into an ether.
and meta-methoxyphenylboronic acid, in the presence of Iodination of an intermediate diazonium salt gave the desired
caesium fluoride and a catalytic amount of bis(diphenyl- product 43, which was reacted with ethanol in the presence of
phosphino)ferrocene]dichloropalladium(II) in a 4 : 1 mixture of caesium carbonate, copper iodide and 1,10-phenanthroline to
3
2
acetonitrile and water, gave 32 and 33, respectively. Buchwald– give the ethoxy ether 44.
Hartwig amination of 31, using potassium carbonate, tris- Synthesis of analogues exploring the 5-position of the iso-
dibenzylideneacetone)dipalladium(0) and XPhos® in tert- quinoline exploited the previously prepared 7-bromo isomer
butanol, with aniline, n-butylamine and benzylamine gave the 31. Nitration of 31 gave the 7-bromo-5-nitro compound 45
(
corresponding products 34–36.
(Scheme 4). Suzuki–Miyaura coupling, as for 31, gave the
-nitro-7-phenyl analogue 46. Palladium-catalysed hydrogen-
5
ation of 45 in the presence of triethylamine reduced the nitro
group and removed the bromine to afford the 5-amino com-
pound 47 in high yield. Acetylation, as before, gave 48. Imine
4
4
9 was generated by the reaction of 4-chlorobenzaldehyde with
7, following methodology used for the 7-isomer 41 but, in
this instance, reflux was required. This sluggishness was most
likely due to the steric hindrance by the nitrile at the peri
4-position. Reduction of imine 49 by sodium borohydride
afforded the 5-(4-chlorobenzyl)amino compound 50.
Inhibitory potency of 4-cyano-3-methylisoquinolines against
parasite growth
Biological testing of the compounds was carried out on
asexual blood stages of Plasmodium falciparum chloroquine
sensitive 3D7 and resistant W2mef strains. W2mef parasites
possess mutations in the chloroquine transporter, which
confer greater resistance to the antimalarial, and an extra copy
of the multi-drug resistance transporter, which boosts efflux of
3
3–35
antimalarial drugs.
Testing was carried out using a
Malstat assay to measure parasite lactate dehydrogenase levels,
3
6
which correlate with parasite proliferation. Assay results
from the isoquinolines with modifications at the 1-, 3-, and
4
-positions are shown in Table 1.
It was observed that extension of the 3-methyl group of 4
Scheme 2 Synthesis of 7-substituted isoquinolines via brominated ana-
logue. Reagents and conditions: (a) H
2
SO
, 4 : 1 CH
3
, XPhos®, t-BuOH, reflux, 17–18 h.
4
, NBS, 0 °C, 0.5 h; then 0 °C →
1
resulted in a moderate improvement in potency of both 9 and
10, with the 3-propyl and 3-pentyl substituents respectively.
rt, 2 h; (b) R -B(OH)
2
, CsF, Pd(dppf)Cl
2
3
CN/H O, 90 °C, 24 h; (c)
2
1
R NH
2
, K
2
CO
3
, Pd
2
(dba)
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
Scheme 3 Synthesis of 7-substituted isoquinolines via nitrated analogue. Reagents and conditions: (a) H
2
SO
4
, KNO
3
, 0 °C, 0.5 h; (b) 10% Pd/C (8%
w/w), H
EtOH, rt, 2 h; (f) NaBH
10 °C, 18 h.
2
, EtOH, rt, 0.5 h; (c) For 39; AcCl, Et
3
N, THF, 0 °C, 5 min; then 0 °C → rt, 2 h; (d) For 40; NaOCN, 5 : 1 AcOH/H CHO,
3
, concd. HCl, −5 °C, 1 h; KI, H O, −5 °C → rt, 4 h; (h) CuI, 1,10-phenanthroline, CsCO , EtOH,
2
O, rt, 16 h; (e) 4-ClC
6
H
4
4
, EtOH, rt, 16 h; (g) NaNO
2
2
1
Scheme 4 Synthesis of 5-substituted isoquinolines. Reagents and conditions: (a) H
CH CN/H O, 90 °C, 24 h; (c) 10% Pd/C (8% w/w), H , Et N, EtOH, rt, 2 h; (d) AcCl, Et
EtOH, reflux, 24 h; (f) NaBH , EtOH, rt, 16 h.
2
SO , 4 : 1
4
, KNO
3
, 0 °C, 1 h; (b) PhB(OH)
2
, CsF, Pd(dppf)Cl
2
3
2
2
3
3
N, THF, 0 °C, 5 min; then 0 °C → rt, 4 h; (e) 4-ClC H CHO,
6 4
4
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
a
Table 1 Inhibition of malarial parasite 3D7 and W2mef proliferation by 1-, 3- and 4-substituted isoquinoline analogues
3
D7
W2mef
IC50 (µM) ± SD
1
3
4
R
R
R
IC50 (µM) ± SD
4
9
1
CH
3
CN
CN
CN
34 ± 10
33 ± 8.7
11 ± 1.1
6.7 ± 0.4
(CH
2
)
2
CH
3
3
13 ± 3.6
6.0 ± 0.3
0
(CH
2
)
4
CH
1
1
1
2
CH
3
CN
CN
34 ± 12
36 ± 10
CH
3
3
3
5.7 ± 2.6
2.4 ± 2.6
1
1
3
4
CH
CH
CN
CN
16 (n = 1)
8.8 (n = 1)
27 ± 12
>50
1
1
1
1
5
6
7
8
CH
CH
CH
CH
3
3
3
3
CN
CN
CN
CN
>50
28 ± 5
>50
>50
14 ± 4.3
10 ± 5.7
17 ± 2.7
14 ± 8.0
1
2
9
0
CH
3
3
CN
CN
2.7 ± 0.8
15 ± 0.7
2.5 ± 1.2
2.8 ± 2.3
CH
2
2
1
2
CH
3
3
CN
CN
40 ± 17
>50
CH
9.7 ± 3.3
6.7 ± 1.4
2
2
3
4
CH
3
3
CN
CN
0.6 ± 0.4
10 ± 3.1
1.0 ± 0.6
9.4 ± 1.4
CH
25
CH
3
CN
1.0 ± 0.5
0.7 ± 0.8
26
27
28
CH
CH
CH
3
3
3
CONH
2
>50
32 ± 4.2
>50
CN
>50
CN
36 ± 4.0
16 ± 0.1
a
Data represents the mean ± SD for two or three independent experiments unless otherwise specified.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
Removal of the alkyl group to amino compound 11 retained of the 4-nitrile functional group of 15 to the corresponding
similar activity comparable to the lead compound 4. Extending amide of 26 did not result in any improvement of inhibitory
the length of the 1-alkylamino chain was found to be favor- activity against the parasites.
able, with a lower IC50 observed for the 1-hexylamino analogue
The results of the testing of the 7-substituted isoquinoline
2. This effect was reduced for the largest 1-octyl extension of analogues in the Malstat assay are presented in Table 2. Bromi-
3 against 3D7 parasites, but not for the W2mef parasites. nation at the 7-position usually resulted in moderate inhibitors
Branching of the 1-alkyl substituents was also explored, of parasite growth as observed for analogues 29 and 31 but not
with a mixture of structure–activity results. At best, inhibition for compound 30.
similar to the lead compound 4 was observed for compounds Introduction of an unsubstituted phenyl ring by Suzuki–
4, 15 and 16 or no inhibition at all, depending on the parasite Miyaura coupling was found to be detrimental to the activity of
1
1
1
tested. The branched butylamino groups of 17 and 18, in com-
parison with lead compound 4, gave only a modest improve-
ment of inhibition over the lead compound 4 but the (±)-1-((4-
methyl-2-pentyl)amino) analogue 19 obtained an order of mag-
nitude improvement in potency with an IC50 of 2.7 ± 0.8 µM
against 3D7 parasites and 2.5 ± 1.2 µM against W2mef para-
sites. It was noted that 17, 19 and 20 contained a chiral stereo-
centre, however the inhibitory activity of these compound was
not sufficient to warrant separation and testing of each of the
enantiomers. The (±)-1-((5-diethylamino)2-pentyl)amino) sub-
stituent of the known malarial treatment chloroquine gave
compound 20, which was found to be a better inhibitor of
W2mef than 3D7 parasites.
The introduction of the 1-morpholino moiety for com-
pound 21 was found to reduce growth inhibitory activity.
However, we were delighted to observe that alpha-methylation
of the 1-benzylamino functional group resulted a significant
improvement in parasite growth inhibition, in particular the
S-enantiomer 23 with an IC50 of 0.6 ± 0.4 µM against 3D7 para-
sites and 1.0 ± 0.6 µM against W2mef parasites. This result,
together with the potency of 19 and, to a lesser extent, 20,
suggested that inhibitory activity was favoured by to the pres-
ence of an alpha-methyl group. Attempts to replicate this low
micromolar potency without requiring a stereocentre were
made, with some inhibition observed for the biphenyl com-
pound 24 but, very pleasingly, the achiral 1-ethylpropylamino
analogue 25 was found to have an IC50 of 1.0 ± 0.5 µM against
Table 2 Inhibition of malarial parasite 3D7 and W2mef proliferation by
a
7-substituted isoquinoline analogues
3
D7
W2mef
1
7
R
R
IC50 (µM) ± SD IC50 (µM) ± SD
2
9
0
15 ± 2.3
>50
11 ± 2.8
>50
3
3
3
3
1
2
3
12 ± 4.8
>50
2.5 ± 2.1
>50
5.7 ± 0.1
15 ± 0.2
3
3
3
3
3
4
4
5
6
7
8
0
5.9 ± 1.5
18 ± 2.3
18 ± 3.3
5.5 ± 0.7
31 ± 6.9
9.7 ± 13
5.0 ± 1.2
24 ± 6.8
21 ± 2.0
4.3 ± 1.8
15 ± 3.3
6.1 ± 1.4
3
D7 parasites and 0.7 ± 0.8 µM against W2mef parasites. The
large standard deviations observed for some compounds
resulted from the fact that the IC50 modestly differed between
assay repeats (n = 2 or 3 unless otherwise stated). This could
be due the compounds acting during narrow replicative phase
of the 48 h cell cycle which the parasites would be expected to
pass through once or twice during the 72 h assay period. Para-
sites that attempted to pass though replication twice could be
more adversely affected than those that passed through once.
Although reasonable attempts were made to synchronise the
parasites similarly between repeats so that they would only
replicate once, in some repeats there might have been a
greater proportion that attempted to replicate twice.
The importance of the 1-alkylamino functionality to the
activity of this family of compounds was also demonstrated by
the substitution of a 1-alkoxy functional group, with modest to
poor inhibition observed for the 1-butoxy analogue 28 when
compared the lead compound 4 and the 1-ethoxy analogue 27
4
1
27 ± 3.8
5.4 ± 3.6
29 ± 12
>50
42
5.2 ± 2.0
16 ± 6.7
44
a
Data represents the mean ± SD for two or three independent
not producing any inhibition of the assay at 50 µM. Reduction experiments unless otherwise specified.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
the compound 32, but the meta-methoxyphenyl substituent of 33
While 7-nitration resulted in compound 37, which was a
and the extended 7-anilino compound 34 showed good inhibitory good inhibitor of parasite growth, reduction to the 7-amino
activity. Neither the 7-butylamino or 7-benzylamino compounds, analogue returned compound 38 which had only similar
35 and 36 respectively, were particularly potent. However, the potency to that of the lead compound 4. Conversion of the
addition of a p-chloro substituent to the benzylamine functional 7-amino functional group to the 7-urea substituent of 40
group resulted in the p-chlorobenzylamino compound 42 which resulted in a moderately potent analogue. The 7-ethoxy ana-
was observed to lead to a good improvement in activity, which logue 44 was found to be comparable to the lead compound 4
was not observed for the corresponding imine precursor 41, against the parasites, with some improvement in inhibitory
suggesting the potential for a hydrogen bonding interaction.
activity against the W2mef strain.
Finally, results from the Malstat assays conducted using
the 5-substituted analogues synthesised are shown in Table 3.
Nitration of the 7-bromo analogue 31 resulted in compound
Table 3 Inhibition of malarial parasite 3D7 and W2mef proliferation by
a
5-substituted isoquinoline analogues
4
2
5 which was found to be equipotent with inhibitors 23 and
5 with an IC50 of 0.7 ± 0.2 against 3D7 parasites and 0.6 ± 0.4
against W2mef parasites. Suzuki coupling of 45 with
phenylboronic acid gave analogue 46, which was observed to
have only moderate potency. Reduction of the 5-nitro
compound 45 to the 5-amino compound 47 improved potency
only modestly compared to 4, which again was completely
removed by acetylation to analogue 48. An improvement of
inhibition was observed for both imine compound 49 and the
p-chlorobenzylamino analogue 50, compared to 7-substituted
analogues of 41 and 42.
Overall, it was observed that an increase in the 1-aminoalkyl
chain length or the introduction of a bulkier substituent gen-
erally resulted in an increase in compound potency. The pres-
ence of alpha-branching on the alkyl chain was determined to
be the most crucial structural change resulting in a much
greater increase in activity over the lead compound 4. Substi-
tution of the 1-amino moiety by 1-ethoxy or the morpholino
functional group removed all activity. Extension of the
3
D7
W2mef
IC50 (µM) ± SD
7
5
R
R
IC50 (µM) ± SD
4
4
4
4
5
6
7
8
0.7 ± 0.2
0.6 ± 0.4
4.4 ± 1.9
23 ± 14
>50
13 ± 8.8
13 ± 1.8
>50
4
5
9
0
9.2 ± 4.1
4.2 ± 1.7
3-methyl group was also found to be acceptable, while hydro-
1.2 (n = 1)
1.8 (n = 1)
lysis of the nitrile substituent was not tolerated. Analogues
were generally better tolerated at the 5-position over the 7-posi-
tion of the compound, with most changes increasing potency
moderately. A summary of the structure–activity relationships
is shown in Fig. 3.
a
Data represents the mean ± SD for two or three independent
experiments unless otherwise specified.
Fig. 3 Structure–activity relationships summary of analogues of 4-cyano-3-methylisoquinoline 4.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
Compounds 23 and 25 were among the most potent growth PfAMA1WT and PfAMA1S610only substrates from lysates
inhibitors tested and represented a significant improvement supplemented with cAMP while H89 strongly inhibited cAMP-
over the lead compound 4. Both are drug-like compounds dependent phosphorylation (Fig. 4B). This indicates that
which comply with Lipinki’s rule of 5, which predicts poor the ELISA method incorporating parasite lysates supple-
absorption or permeation is more likely with molecular mented with cAMP could be used to quantitatively and
weights of more than 500 Daltons, more than 5 hydrogen specifically measure PfPKA activity with the specific AMA1
bond donors, more than 10 hydrogen bond acceptors and a tail phospho-S610 antibody achieving a great signal to noise
3
7
calculated log P of greater than 5. Good oral bioavailability is ratio.
also predicted for these compounds which all possess less
Next we attempted to determine the effect of the most
than 10 rotatable bonds and total polar surface area (tPSA) of growth inhibitory 4-cyano-3-methylisoquinolines on PfPKA
2
38
less than 140 Å . These compounds, like most that were tested activity. Compounds 4, 12, 13, 23, 25 and 45 were titrated into
here, had similar levels of growth inhibition against 3D7 and the ELISA-based phosphorylation assays along with H89 as a
W2mef P. falciparum strains, indicating that neither of the positive control. While H89 clearly inhibited phosphorylation
chloroquine resistance mechanisms increased the tolerance for of PfAMA1 at S610, none of the 4-cyano-3-methylisoquinolines
the compounds, boding well for potential therapeutic use.
had a significant effect on the phosphorylation levels even at
Before progressing to PfPKA inhibition assays we ensured concentrations as high as 100 µM, indicating that they do not
that the most potent compounds were not inhibiting the target PfPKA (Fig. 5).
Malstat reaction and thereby giving the false impression they
were reducing parasites growth.
performed on fresh 3D7 parasite culture to which 4, 12, 13, 23
A counter screen was
Invasion inhibitory capacity of 4-cyano-3-methylisoquinolines
and 25 had been added to 100 µM. Relative to a no drug The lack of inhibition by the 4-cyano-3-methylisoquinoline
control, the compounds only weakly inhibited the screen compounds on PfPKA was surprising given that the original
2
6
reaction by 5–10%, much less than the ∼100% inhibition compound 4 was found to be active against rat PKA. Given
evident in the growth assays (ESI†). The weak counter screen that the 4-cyano-3-methylisoquinolines are thought to compe-
inhibition was probably due to the DMSO carrier.
titively inhibit ATP binding, we hypothesised that these com-
pounds might target an enzyme with an ATP binding pocket.
Many signaling enzymes function late in the cell cycle as they
are utilised extensively to coordinate egress and invasion,
Specificity of 4-cyano-3-methylisoquinolines for PfPKA
An ELISA-based assay was developed to measure PfPKA activity therefore the effect of these compounds upon erythrocyte
via phosphorylation of a recombinant version of the 56 amino egress and invasion was examined. Assays to quantify egress
acid, C-terminal cytoplasmic tail of PfAMA1 expressed as a and invasion inhibition were performed using transgenic para-
fusion protein with glutathione S transferase (GST) (Fig. 4A). sites that express and secrete nanoluciferase (NanoLuc) into
3
9,40
The GST-AMA1 fusion proteins were used to coat the wells of the parasitophorous vacuole (PV).
The most growth inhibi-
ELISA plates for subsequent phosphorylation. To obtain a tory compounds, 4, 12, 13, 23, 25 and 45, were added to pre-
source of PfPKA we attempted to express a His-6 tagged version invasion cultures and incubated for 4 hours over which time
of the catalytic domain of PfPKA that was codon optimised for the parasites attempted to egress and invade new erythrocytes.
expression in Escherichia coli but the protein was insoluble and After the 4 hour incubation period, parasite media containing
therefore likely inactive. Instead we used lysates of late stage NanoLuc released from the ruptured host cell and PV was har-
intraerythrocytic P. falciparum that contained active PfPKA. vested for analysis of egress inhibition. To quantify invasion
Because the lysate also contained other active kinases, we used inhibition, the 4-cyano-3-methylisoquinolines were washed out
a panel of mutant PfAMA1 C-terminal fusion proteins to derive after the 4 hour invasion window and the cultures were treated
specific PfPKA activity. In addition to the wildtype AMA1 tail, a to prevent further invasions, thereby only including parasites
PfAMA1S610only tail was utilised where S610 is the PfPKA target that invaded during drug treatment. These parasites were
1
7
site and all other phosphorylatable residues are mutated, allowed to grow for a further 24 hours until NanoLuc
allowing only phosphorylation by PfPKA on this substrate. The expression was high enough to measure. To quantify the levels
inverse of this tail was also generated where only S610 is of NanoLuc in the egress media or in the invaded parasite
mutated allowing this substrate to accept phosphorylation cultures, NanoGlo substrate was added and relative light units
from multiple kinases except for PfPKA (Fig. 4A). A commercial (RLU) were measured. The RLUs were normalised against
phospho-serine antibody was used to detect any serine phos- those for egress or invasion in untreated cultures (100%) and
phorylation of the AMA1 tail as well as an antibody raised in uninfected cultures (0%) and inhibition curves were
against PfAMA1 tail peptide phosphorylated at S610 to specifi- plotted. All of the compounds poorly inhibited egress except at
cally detect only PfPKA-specific (S610) phosphorylation the highest concentrations (Fig. 6). In comparison, the
(Fig. 4B). To validate this assay as being quantitative for PfPKA compounds were much more potent at inhibiting invasion
activity, the tail substrates were also used with or without with compound 45 being the most effective. This indicates
added cAMP and the PKA inhibitor H89. As expected, S610 that the target of the 4-cyano-3-methylisoquinolines probably
phosphorylation was maximally detected on only the functions during the invasion stage of the life cycle and may
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 4 An ELISA-based method can be used to measure PfPKA activity against the S610 residue of PfAMA1 expressed as a GST-AMA1 tail fusion
protein. A) Diagram of a panel of GST-AMA1 tail fusion proteins showing how they were modified by PfPKA and other kinases. B) Immobilised recom-
binant PfAMA1WT, PfAMA1S610A and PfAMA1S610only fusion proteins were phosphorylated with parasite lysates supplemented with cAMP and H89 to
trigger and inhibit PfPKA activity, respectively. Following the reactions, an ELISA was performed to detect phosphorylation of S610 (the PfPKA site)
using a commercial phosphoserine antibody and a specific antibody against a PfAMA1 peptide phosphorylated at S610. As expected, maximal
PfPKA-specific signals resulted from phosphorylation of the PfAMA1WT and PfAMA1S610only substrates by parasite lysates supplemented with cAMP.
be another kinase(s) that phosphorylates invasion proteins microscopy was performed on late-stage W2mef parasites as
similar to PfAMA1.
they egressed and invaded new erythrocytes. The basic steps of
To more precisely observe the phenotype of parasites egress and invasion are as follows. Before rupture and release
treated with the 4-cyano-3-methylisoquinolines, live cell video of daughter parasites, the infected erythrocyte which is
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
the pore further opening up the erythrocyte and ejecting more
4
2
merozoites. Erythrocyte invasion is a three step process
beginning with the pre-invasion phase where the merozoite
4
3,44
contacts and deforms the erythrocyte surface.
This phase
lasts about 10 seconds during which the merozoite reorien-
tates its penetrative apical end onto the erythrocyte surface.
Then the active invasion phase lasts about 10 seconds during
4
4
which a vacuole forms around the invading merozoite.
During the final post-invasion phase, the internalised mero-
zoite and erythrocyte become separated when scission of the
membranous isthmus connecting the vacuole and erythrocyte
membranes occurs. Precisely when this happens is difficult to
visualise because 30 seconds after invasion the erythrocyte
surface becomes spiculated and the merozoite inside is
4
4
difficult to see. Over several minutes the erythrocyte recovers
its normal biconcave shape by which time the merozoite has
differentiated into an amoeboid ring. Due to the amount of
effort and compound required for this method, achiral com-
pound 25 was selected for testing at a concentration of 10 µM,
Fig. 5 Effect of the 4-cyano-3-methylisoquinolines on PfPKA activity.
The compounds were titrated into the ELISA-based phosphorylation
assay for detection of PfPKA activity via PfAMA1S610only phosphorylation
but were found to have no effect at their growth inhibitory concen- which conferred about 70% invasion inhibition in the
trations, indicating that they do not target PfPKA.
NanoLuc assay. Of 14 schizonts treated with 25 that ruptured,
eight appeared normal with each liberating 12–24 merozoites.
Six ruptures were aberrant and released a single incompletely
normally flattened rounds up into a sphere and the hemozoin divided schizont (Fig. 7). From the eight normal ruptures,
4
1
becomes highly condensed. Egress begins with the sudden 30 merozoites were observed to contact and deform their sur-
ejection of a few merozoites from a pore in the erythrocyte rounding erythrocytes and of these 15 invaded normally and
surface followed by curling back of the membranous edges of progressed to form intra-erythrocytic ring-stage parasites
Fig. 6 Inhibition of parasite egress and invasion by 4-cyano-3-methylisoquinolines. The most growth inhibitory compounds were tested for their
egress and invasion inhibition capacity to determine whether this stage of the life cycle is affected and when the drug target(s) may function. The
compounds were titrated into NanoLuc-expressing late stage 3D7 cultures for 4 hours covering a period of egress and invasion. The levels of
NanoLuc released into the media during this period were a marker for parasite egress and the dose–response curves indicate the compounds did
not substantially inhibit egress except at higher concentrations. To measure invasion inhibition the compounds were removed after 4 hours and the
parasites were grown for 24 hours to increase the NanoLuc signal in the parasites. Lysis of the whole cultures and measurement of NanoLuc levels
indicated that the compounds were much more effective against invasion than egress. The data has been normalised against untreated cultures
(
100%) and uninfected cultures (0%). Assays were conducted in triplicate and error bars denote one standard deviation.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 7 Live cell microscopy panels and a diagram of invasion in the presence of compound 25. At 10 µM, 25 blocked cytokinesis as indicated in the
live cell micrograph (time points in seconds are shown). Compound 25 blocked multiple points of the invasion phase after cytokinesis including
active invasion and resealing of the host cell.
(Videos S1 and S2,† white arrows). The remaining merozoites released but were unable to invade properly. Known phos-
however failed to form viable ring-stage parasites and exhibi- phorylation targets involved in cytokinesis and formation of
ted three general kinds of abnormal behavior (Fig. 7). The the inner membrane complex such as Rab11A could be func-
1
6,45
most upstream blockage occurred in about half the unsuccess- tionally compromised.
ful merozoites where they deformed their erythrocyte but did
not cleanly invade and instead seemed to embed themselves
into the erythrocyte surface (Videos S1 and S2†). This triggered
echinocytosis of the erythrocyte and as it recovered its normal
Screening for potential kinase targets of 4-cyano-3-
methylisoquinoline inhibitors
shape several minutes later the merozoites or by now differen- Given that the lead compound 4 is thought to have competitive
tiated ring-stage parasites, remained attached to the outside ATP-binding activity against rat PKA, we reasoned that the syn-
(Videos S1 and S2†). Other aberrant merozoites appeared to thesised compounds might exhibit a similar mode of action
enter the erythrocytes normally but later appeared to exit at albeit against a different parasite kinase. To identify potential
the invasion point either as a merozoite or ring. Some invad- kinase targets of the compounds, we introduced compound 25
ing merozoites remained internal and formed rings but soon into a commercial human kinase screen (MRC PPU Inter-
triggered lysis of their host erythrocyte. This indicates that the national Centre for Kinase Profiling, University of Dundee,
target of compound 25 has a role in cytokinesis, invasion and United Kingdom) and tested a panel of available kinases with
resealing. The observed defects suggests that active invasion homologs in P. falciparum, where inhibited human kinases
powered by the actomyosin invasion motor is not functioning might indicate a parasite target given the homology. Aurora B
correctly. Proteins within this motor such as GAP45 are known kinase, glycogen synthase kinase 3-beta, cyclin-dependent
to be phosphorylated by kinases such as PKA, PKB, PKG and kinase 2, cyclin-dependent kinase 9 and cyclin-dependent
1
6
CDPK1. A notable surprise from the live cell imaging was the kinase like were found to have reduced activity in the presence
egress of whole undivided schizonts. It seems unlikely that of 10 µM 25 (29%, 38%, 32%, 54% and 53% respectively)
these were immature schizonts that had not yet formed mero- (Table 4). In support of our PfPKA assays was the observation
zoites and had been stimulated to prematurely egress because that 25 did not inhibit Human PKA indicating that modifi-
there was no increase in egress following the addition of com- cation of 4 to 25 had ablated activity against mammalian PKA.
pound 25 according to the NanoLuc assay. It is possible that
These kinases (PfARK2, PfGSK3, PfPK5, PfCLK1 and PfCRK1) in
when compound 25 was added to schizonts before they had P. falciparum are known to have roles in mitosis, cell cycle regu-
46–48,57
undergone cytokinesis, the process was arrested but the egress lation and transcription, among others.
Specifically, aurora
program continued unimpeded. If cytokinesis was complete or kinase B, which was inhibited to the greatest extent, is required
nearly so when compound 25 was added, merozoites were for chromosome condensation and segregation as well as for cyto-
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
a
Table 4 Inhibition of human kinases
Hs kinase tested
Pf Kinase homolog
Activity after treatment (%)
Function
PKA (AGC kinase)
PKB (AGC kinase)
PKC (AGC-related kinase)
PDK1 (AGC-related kinase)
Aurora A (aurora kinase)
Aurora B (aurora kinase)
GSK3β (glycogen synthase kinase)
CK1 (casein kinase)
PKA (PFI1685w)
PKB (PFL2250c)
PKC (PF11_0464)
Putative (PF11_0227)
ARK1 (PFF0260w)
ARK2 (PFC0385c)
GSK3 (PFC0525c)
CK1 (PF11_0377)
CK2 (PF11_0096)
PK5 (MAL13P1.279)
CRK1 (PFD0865c)
CLK1 (PF14_0431)
MAP2 (PF11_0147)
PK2 (PFL1885c)
92 ± 7
122 ± 22
96 ± 24
78 ± 31
87 ± 5
29 ± 2
38 ± 6
117 ± 16
87 ± 1
32 ± 3
54 ± 5
53 ± 2
Mitosis
Multiple
CK2 (casein kinase)
CDK2-Cyclin A (cyclin-dependent kinase)
CDK9-Cyclin T1 (cyclin-dependent kinase)
CLK2 (cyclin-dependent kinase like)
ERK1 (mitogen-activated protein knase)
CAMK1 (calcium/calmodulin-dependent kinase)
NEK2a (NIMA kinase)
Cell cycle
Cell cycle
Transcription
133 ± 11
98 ± 1
84 ± 19
96 ± 1
NEK1 (PFL1370)
PK4 (PFF1370w)
TLK1 (PFB0520w)
EIF2aK3 (eukaryotic translation initiation factor 2a kinase)
MEKK1 (tyrosine kinase like)
99 ± 4
a
Data represents the mean % activity remaining of assay duplicates ± SD.
kinesis in eukaryotes. PfARK2 being a target of the 4-cyano-3- will be a particularly important consideration since compound
methylisoquinoline inhibitors may explain the abnormal pheno- 25 has shown some inhibition of four human kinases.
type observed where parasites do not complete division before
49
egressing in the presence of 25. In future studies, these com-
pounds will be tested against recombinant forms of these para-
site-specific kinases to elucidate the target of the inhibitors.
Experimental
Chemistry
All commercial materials were used as received without further
purification. Glassware used in moisture sensitive reactions
was oven dried and cooled under nitrogen prior to use and
Conclusion
The large evolutionary distance between mammals and Plas- non-aqueous reactions performed under an atmosphere of
modium and the subsequent divergence in sequence identity nitrogen. All reactions were carried out at room temperature,
between orthologous enzymes such as kinases, makes it feas- unless otherwise noted. Purification of solvents and reagents,
ible to repurpose mammalian enzyme inhibitors towards if required, was carried out by procedures described by Chai
5
0
greater activity against their Plasmodium orthologs at little and Armarego. Melting points were determined on a Reichert
expense to the original mammalian enzyme. For this reason, ‘Thermopan’ microscope hot stage apparatus as uncorrected
the synthesis and structure–activity relationships of a library of values. All organic extracts were dried over magnesium sulfate
synthesised 4-cyano-3-methylisoquinolines and related ana- unless otherwise stated. All reactions were monitored by Thin
logues have been explored. Low micromolar inhibition of para- Layer Chromatography (TLC) unless otherwise noted. Analyti-
site growth was observed against both 3D7 and chloroquine- cal TLC was performed on Merck Kieselgel 60 F254 aluminium
resistant W2mef strains of Plasmodium falciparum for com- backed plates and visualised using a 254 nm UV lamp. Flash
pounds 23 (IC50 0.6 ± 0.4 µM against 3D7 parasites and IC50 chromatography was performed on silica gel (Davisil® LC60 Å
5
1
1
.0 ± 0.6 µM against W2mef parasites) and 25 (IC50 1.0 ± 0.5 40–63 micron) according to the method of Still et al. Low-
µM against 3D7 parasites and IC50 0.7 ± 0.8 µM against resolution electrospray ionisation (ESI) mass spectra were
W2mef parasites) but, surprisingly, the compounds appeared to recorded on a Bruker Daltronics Esquire 6000 Ion Trap mass
have no effect on their supposed target of PfPKA. Importantly, spectrometer in methanol or acetonitrile (0.1% formic acid) at
however, the compounds inhibited parasite cytokinesis and 300 °C, a 40 eV cone voltage, with a scan rate of 5500 m/z/s.
erythrocyte invasion at multiple steps indicating that the target, High-resolution ESI mass spectra were recorded on an Agilent
like PfPKA, functions during invasion and is possibly another 6224 TOF LC/MS coupled to an Agilent 1290 Infinity LC. All
kinase. These compounds provide much needed new tools to data was acquired and reference mass corrected via a dual-
provide insight into the mechanisms by which malaria parasites spray ESI source. Analytical Reverse-Phase High Performance
establish infection, particularly if their molecular targets can be Liquid Chromatography (RP-HPLC) was performed on a Shi-
identified. To pave the way for the optimisation of 4-cyano-3- madzu LC-20AB Prominence Liquid Chromatography system
methylisoquinolines as potential antimalarial agents they fitted with a Phenomenex® Jupiter C18 300 Å column
should be tested for toxicity against cultured human cells. This (250 mm × 4.6 mm, 10 µm). Using either Method A: 10 → 90%
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
ACN in 0.1% TFA or Method B: 10 → 95% ACN in 10 mM 156.1, 136.3, 131.5, 126.3, 124.7, 121.5, 118.7, 115.3, 91.9, 41.5,
NH HCO over 25 minutes with a flow rate of 1 mL min−
1
,
31.7, 24.4, 20.4, 14.0. LRMS-ESI (m/z): 240.1 (M + H) .
monitored at 254 nm. Semi-preparative reverse phase HPLC RP-HPLC: t 12.3 min (Method A).
was performed on a Beckman system (125 Solvent Module and 1-(Butylamino)-3-propylisoquinoline-4-carbonitrile (9). 2-Cyano-
66 Detector) fitted with a Phenomenex® Jupiter C18 300 Å phenylacetonitrile 5 (0.50 g, 3.5 mmol) was treated according
+
4
3
R
1
column (250 mm × 10.0 mm, 10 µm) at a flow rate of 5 to General procedure A, except freshly distilled butanoic anhy-
mL min− , monitored at 254 nm. NMR spectra were recorded dride (3.2 mL) was used. The resulting butyramide intermedi-
1
on either a Bruker AV-300, AV-400 and AV-500 spectrometer at ate 7 (0.93 g, 3.3 mmol) was coupled with n-butylamine
1
3
00.19, 400.13 and 500.02 MHz respectively, for H nuclei and (11 mL, 0.11 mol) according to General procedure B1, except
1
3
at 75.78, 100.62 and 125.74 MHz respectively, for C nuclei at the oil that separated was extracted with dichloromethane (×3).
00 K. For H NMR the residual CDCl3 peak (7.26 ppm) or The combined organics were washed with brine (×2), dried, fil-
1
3
DMSO-d peak (2.50 ppm) were used as internal standards. tered and concentrated in vacuo. The crude solid was purified
6
1
3
Similarly, C NMR spectra were referenced to the residual by flash chromatography (5% ethyl acetate in hexanes) and
solvent; the central peak of the CDCl ‘triplet’ (77.16 ppm) or subsequent purification by semi-preparative reverse phase
3
6
DMSO-d ‘heptet’ (39.52 ppm). Chemical shifts were reported HPLC (10 → 90% acetonitrile in 0.1% trifluoroacetic acid over
as δ values in parts per million (ppm). The following abbrevi- 120 min) provided compound 9 (10 mg, 1% over two steps) as
1
ations have been used upon reporting spectral data: s, singlet; a transparent solid, mp 117–119 °C. H NMR (400 MHz,
3
d, doublet; t, triplet; q, quartet; m, multiplet; app, apparent; CDCl ): δ 7.97 (d, J = 8.0 Hz, 1H), 7.73–7.68 (m, 2H), 7.50–7.46
and br, broad. Coupling constants (J) are reported in Hz to the (m, 1H), 5.72 (br s, 1H), 3.72–3.67 (m, 2H), 2.97–2.94 (m, 2H),
nearest 0.5 Hz.
1.91–1.82 (m, 2H), 1.75–1.67 (m, 2H), 1.52–1.42 (m, 2H),
1
3
3
1.04–0.97 (m, 6H). C NMR (100 MHz, CDCl ): δ 163.1, 156.1,
General procedure A: preparation of substituted amides
1
36.2, 131.2, 126.1, 124.7, 121.5, 118.6, 115.3, 91.7, 41.3, 39.3,
+
N-(4-Cyano-3-methyl-1H-isochromen-1-ylidene)acetamide 31.5, 22.3, 20.2, 13.9, 13.8. LRMS-ESI (m/z): 268.2 (M + H) .
+
+
(
6). Prepared according to the original method of Gabriel and HRMS-ESI (m/z): (M + H) calcd for C17
H
22
N
3
, 268.1808;
5
2
52
Neumann.
(Synthesis
reported,
correct
structure found, 268.1808. RP-HPLC: t
R
15.5 min (Method A).
2
9
reported ). A mixture of 2-cyanophenylacetonitrile 5 (5.0 g,
1-(Butylamino)-3-pentylisoquinoline-4-carbonitrile
(10).
3
5 mmol) and anhydrous sodium acetate (2.5 g) in distilled 2-Cyanophenylacetonitrile 5 (0.50 g, 3.5 mmol) was treated
acetic anhydride (25 mL) was heated under reflux for 2 h. The according to General procedure A, except freshly distilled hexa-
resulting mixture was poured onto ice and stirred for 0.25 h. noic anhydride (4.4 mL) was used. The resulting hexanamide
The solid that separated was filtered, washed with water and intermediate 8 (0.96 g, 2.8 mmol) was coupled with n-butyl-
dried to give acetamide 6 (7.2 g, 91%) as a brown solid. The amine (9.5 mL, 97 mmol) according to General procedure B1,
1
obtained material was sufficiently pure (>95% by H-NMR) for except the oil that separated was extracted with ethyl acetate
further reaction. A portion was recrystallised from carbon tetra- (×3). The combined organics were washed with brine (×2),
chloride to afford the amide as a light brown solid, mp dried, filtered and concentrated in vacuo. The crude solid was
5
2
1
1
8
1
35–136 °C (lit. 137–138 °C). H NMR (400 MHz, CDCl ): δ purified by flash chromatography (10% ethyl acetate in
3
.13 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.58 (d, J = 8.0 Hz, hexanes) to provide compound 10 (60 mg, 7% over two steps)
H), 7.53–7.49 (m, 1H), 2.50 (s, 3H), 2.31 (s, 3H). C NMR as a white solid, mp 114–115 °C. H NMR (400 MHz, CDCl ): δ
3
13
1
(100 MHz, CDCl ): δ 182.7, 163.7, 145.9, 134.6, 130.0, 129.7, 7.96 (d, J = 8.0 Hz, 1H), 7.73–7.66 (m, 2H), 7.49–7.45 (m, 1H),
3
1
28.2, 123.5, 119.8, 114.3, 92.7, 25.9, 19.5.
5.74 (br s, 1H), 3.72–3.67 (m, 2H), 2.97 (t, J = 7.5 Hz, 2H),
1
1
3
.87–1.79 (m, 2H), 1.75–1.67 (m, 2H), 1.51–1.43 (m, 2H),
General procedure B1: preparation of 1-substituted 4-cyano-3-
methylisoquinoline compounds via aminolysis
.41–1.37 (m, 4H), 0.99 (t, J = 7.5 Hz, 3H), 0.90 (t, J = 7.5 Hz,
13
3
H). C NMR (100 MHz, CDCl ): δ 163.4, 156.1, 136.3, 131.3,
1
-(Butylamino)-3-methylisoquinoline-4-carbonitrile (4). Pre- 126.1, 124.7, 121.4, 118.6, 115.3, 91.5, 41.3, 37.3, 31.6, 31.4,
2
9
+
pared according to the method reported by Deady and Quazi.
A solution of acetamide 6 (0.50 g, 2.2 mmol) in acetone HRMS-ESI (m/z): (M + H) calcd for C19
7.4 mL) was added dropwise to a stirring solution of n-butyl- found, 296.2123. RP-HPLC: t
R
amine (7.4 mL, 75 mmol) in acetone (7.4 mL). After 0.5 h,
ensuring the temperature remained <25 °C, the mixture was according to the method of Deady and Quazi. A solution of
poured onto ice and the resulting solid that separated was acetamide 6 (0.20 g, 0.88 mmol) and ammonia (10 mL, 30%
filtered, washed with water, dried and recrystallised from aqueous, d 0.89) in dioxane (10 mL) was heated under reflux
carbon tetrachloride to afford the title compound 4 (0.41 g, for 1 h. Water was added and the solid that separated was fil-
8%) as a pink solid, mp 135–137 °C (lit. 136–138 °C). tered, washed with water and recrystallised from ethanol to
H NMR (400 MHz, CDCl ): δ 7.95 (dd, J = 9.0, 1.0 Hz, 1H), afford amine 11 (0.15 g, 93%) as a light pink solid, mp
28.7, 22.6, 20.3, 14.0, 13.9. LRMS-ESI (m/z): 296.2 (M + H) .
+
+
H
26
N
3
, 296.2121;
23.6 min (Method B).
1-Amino-3-methylisoquinoline-4-carbonitrile (11). Prepared
(
2
9
2
9
7
1
3
2
9
1
7
.72–7.68 (m, 2H), 7.50–7.46 (m, 1H), 5.65 (br s, 1H), 3.71–3.67 273–274 °C (lit. 273–274 °C). H NMR (400 MHz, DMSO-d
6
):
(
1
m, 2H), 2.70 (s, 3H), 1.75–1.68 (m, 2H), 1.53–1.43 (m, 2H), δ 8.29 (d, J = 8.5 Hz, 1H), 7.88 (br s, 2H), 7.81–7.77 (m, 1H),
.00 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, CDCl ): δ 159.6, 7.73 (dd, J = 8.5, 1.0 Hz, 1H), 7.56–7.52 (m, 1H), 2.54 (s, 3H).
3
1
3
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
1
3
+
+
C NMR (100 MHz, DMSO-d
6
): δ 159.3, 158.9, 135.8, 132.3, H) calcd for C14
H
16
N
3
, 226.1339; found, 226.1339. RP-HPLC:
1
1
26.0, 124.7, 122.8, 118.2, 114.5, 89.5, 23.4. LRMS-ESI (m/z): tR 10.2 min (Method A).
+
84.0 (M + H) . RP-HPLC: t
R
8.5 min (Method A).
1-(2-Methylpropylamino)-3-methylisoquinoline-4-carbonitrile
(
2
15). Acetamide 6 (0.50 g, 2.2 mmol) was coupled with
-methylpropylamine (7.5 mL, 75 mmol) according to General
General procedure B2: preparation of 1-substituted 4-cyano-3-
methylisoquinoline compounds via aminolysis using an acidic
workup
procedure B1. The crude product was recrystallised from
toluene to afford the title compound 15 (0.30 g, 57%) as a light
1
1
-(Hexylamino)-3-methylisoquinoline-4-carbonitrile (12). To pink solid, mp 147–149 °C. H NMR (300 MHz, CDCl ): δ 7.95
3
a stirring solution of n-hexylamine (4.0 mL, 30 mmol) in (d, J = 8.0 Hz, 1H), 7.74–7.67 (m, 2H), 7.51–7.45 (m, 1H), 5.78
acetone (4.0 mL), solution of acetamide (0.20 g, (br s, 1H), 3.53 (dd, J = 7.0, 5.5 Hz, 2H), 2.69 (s, 3H), 2.11–1.97
.88 mmol) in acetone (4.0 mL) was added dropwise ensuring (m, 1H), 1.04 (d, J = 6.5 Hz, 6H). C NMR (75 MHz, CDCl ): δ
a
6
1
3
0
3
the reaction temperature was maintained <25 °C. After stirring 159.6, 156.2, 136.4, 131.6, 126.3, 124.8, 121.5, 118.7, 115.3,
+
at room temperature for 0.5 h, the mixture was poured onto 92.0, 49.1, 28.5, 24.4, 20.5. LRMS-ESI (m/z): 240.0 (M + H) .
+
+
ice and the oil separated was extracted with dichloromethane HRMS-ESI (m/z): (M + H) calcd for C H N , 240.1495;
1
5
18 3
(×3). The combined organics were washed with brine (×2), found, 240.1496. RP-HPLC: t
R
11.8 min (Method A).
dried, filtered and concentrated in vacuo to give a brown oil 3-Methyl-1-(2,2-dimethylpropanamino)isoquinoline-4-carbo-
residue. Water was added and the mixture neutralised with 3 M nitrile (16). Acetamide 6 (0.30 g, 1.3 mmol) was coupled with
hydrochloric acid or until a solid precipitated and the mixture 2,2-dimethyl-1-propanamine (5.3 mL, 45 mmol) according to
was subsequently kept overnight at 4 °C. The crude product General procedure B1, except the oil that separated was
was purified by flash chromatography (10% ethyl acetate in extracted with dichloromethane (×3). The combined organics
hexanes) and subsequently recrystallised from toluene/pet- were washed with brine (×2), dried, filtered and concentrated
roleum ether (bp 80–110 °C) to give the title compound 12 in vacuo. The crude solid was recrystallised from toluene to
1
(
(
86 mg, 36%) as a white solid, mp 113–115 °C. H NMR give compound 16 (0.18 g, 54%) as a white solid, mp
1
300 MHz, CDCl
3
): δ 7.94 (d, J = 8.5 Hz, 1H), 7.74–7.66 (m, 2H), 176–178 °C. H NMR (300 MHz, CDCl
3
): δ 7.94 (d, J = 8.0 Hz,
7
3
3
1
2
.50–7.45 (m, 1H), 5.72 (br s, 1H), 3.71–3.64 (m, 2H), 2.70 (s, 1H), 7.75–7.67 (m, 2H), 7.52–7.46 (m, 1H), 5.79 (br s, 1H), 3.57
1
3
H), 1.77–1.67 (m, 2H), 1.47–1.33 (m, 6H), 0.90 (t, J = 7.0 Hz, (d, J = 6.0 Hz, 2H), 2.69 (s, 3H), 1.04 (s, 9H). C NMR (75 MHz,
1
3
H). C NMR (75 MHz, CDCl
3 3
): δ 159.6, 156.0, 136.3, 131.6, CDCl ): δ 159.5, 156.4, 136.4, 131.6, 126.4, 124.8, 121.3, 118.7,
26.3, 124.7, 121.6, 118.7, 115.3, 91.9, 41.8, 31.7, 29.5, 26.9, 115.3, 91.9, 52.4, 32.4, 27.7, 24.3. LRMS-ESI (m/z): 254.1 (M +
+
+
+
+
4.3, 22.7, 14.2. LRMS-ESI (m/z): 268.1 (M + H) . HRMS-ESI H) . HRMS-ESI (m/z): (M + H) calcd for C H N , 254.1652;
1
6
20 3
+
+
(
m/z): (M + H) calcd for C17
RP-HPLC: t 14.8 min (Method A).
-Methyl-1-(octylamino)isoquinoline-4-carbonitrile
22 3 R
H N , 268.1808; found, 268.1809. found, 254.1652. RP-HPLC: t 13.8 min (Method A).
R
(±)-3-Methyl-1-((2-methylbutyl)amino)isoquinoline-4-carbo-
3
(13). nitrile (17). Acetamide 6 (0.30 g, 1.3 mmol) was coupled with
Acetamide 6 (0.50 g, 2.2 mmol) was coupled with n-octylamine (±)-2-methylbutylamine (5.3 mL, 45 mmol) according to
12 mL, 75 mmol) according to General procedure B2. The General procedure B2. The crude product was recrystallised
crude product was recrystallised from ethanol to afford the from toluene to afford the title compound 17 (0.13 g, 39%) as
(
1
title compound 13 (99 mg, 15%) as a light pink solid, mp an off-white solid, mp 130–132 °C. H NMR (400 MHz, CDCl
3
):
): δ 8.32 (d, J = 8.5 δ 7.96 (dd, J = 8.0, 1.5 Hz, 1H), 7.72–7.68 (m, 2H), 7.51–7.46
Hz, 1H), 8.29 (br t, J = 5.5 Hz, 1H), 7.80–7.72 (m, 2H), (m, 1H), 5.72 (br s, 1H), 3.69–3.47 (m, 2H), 2.70 (s, 3H),
.57–7.53 (m, 1H), 3.58–3.53 (m, 2H), 2.56 (s, 3H), 1.67–1.61 1.85–1.75 (m, 1H), 1.56–1.47 (m, 1H), 1.34–1.23 (m, 1H), 1.02
1
1
06–109 °C. H NMR (400 MHz, DMSO-d
6
7
(
1
3
13
m, 2H), 1.35–1.24 (m, 10H), 0.84 (t, J = 7.0 Hz, 3H). C NMR (d, J = 7.0 Hz, 3H), 0.97 (t, J = 7.0 Hz, 3H). C NMR (100 MHz,
(
100 MHz, DMSO-d ): δ 158.9, 156.3, 135.3, 131.9, 126.1, 123.7, CDCl ): δ 159.6, 156.2, 136.4, 131.6, 126.3, 124.8, 121.4, 118.7,
6
3
1
2
22.9, 118.4, 115.1, 89.1, 40.8, 31.2, 28.7, 28.6, 28.3, 26.5, 23.9, 115.3, 91.9, 47.3, 35.0, 27.4, 24.4, 17.7, 11.5. LRMS-ESI (m/z):
+
+
+
+
20 3
2.1, 13.9. LRMS-ESI (m/z): 296.2 (M + H) . HRMS-ESI (m/z): 254.0 (M + H) . HRMS-ESI (m/z): (M + H) calcd for C16H N ,
+
+
(M + H) calcd for C H N , 296.2121; found, 296.2122. 254.1652; found, 254.1652. RP-HPLC: t 13.4 min (Method A).
19 26 3 R
RP-HPLC: t
R
17.3 min (Method A).
1-(Isopentylamino)-3-methylisoquinoline-4-carbonitrile (18).
1
-(2-Propylamino)-3-methylisoquinoline-4-carbonitrile (14). Acetamide 6 (0.20 g, 0.88 mmol) was coupled with isopentyl-
Acetamide 6 (2.0 g, 8.9 mmol) was coupled with 2-propylamine amine (3.5 mL, 30 mmol) according to General procedure B2.
26 mL, 0.30 mol) according to General procedure B1. The Purification by flash chromatography (10% ethyl acetate in
(
crude product was recrystallised from carbon tetrachloride to hexanes) and subsequent recrystallisation from toluene/
afford the title compound 14 (1.4 g, 70%) as a pale brown petroleum ether (bp 80–110 °C) gave the title compound 18
1
1
solid, mp 203–206 °C. H NMR (300 MHz, DMSO-d
6
): δ 8.40 (d, (32 mg, 14%) as a light pink solid, mp 149–151 °C. H NMR
): δ 8.32 (d, J = 8.5 Hz, 1H), 8.27 (br t, J =
.58–7.52 (m, 1H), 4.63–4.52 (m, 1H), 2.56 (s, 3H), 1.27 (d, J = 5.5 Hz, 1H), 7.81–7.72 (m, 2H), 7.57–7.53 (m, 1H), 3.62–3.57
J = 8.0 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 7.83–7.71 (m, 2H), (400 MHz, DMSO-d
6
7
6
1
2
1
3
.5 Hz, 6H). C NMR (75 MHz, DMSO-d
35.4, 131.9, 126.0, 123.9, 122.9, 118.4, 115.1, 89.0, 42.3, 24.0, 0.94 (d, J = 6.5 Hz, 6H). C NMR (100 MHz, DMSO-d ):
2.0. LRMS-ESI (m/z): 226.1 (M + H) . HRMS-ESI (m/z): (M + δ 159.0, 156.3, 135.3, 131.9, 126.1, 123.7, 123.0, 118.4, 115.2,
6
): δ 159.0, 155.6, (m, 2H), 2.56 (s, 3H), 1.71–1.61 (m, 1H), 1.58–1.52 (m, 2H),
1
3
6
+
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
8
9.0, 39.1 (obscured by DMSO peak), 37.5, 25.4, 24.0, 22.5.
(R)-3-Methyl-1-((1-phenylethyl)amino)isoquinoline-4-carbo-
nitrile (22). Acetamide 6 (0.26 g, 1.2 mmol) was coupled with
(R)-(+)-1-phenylethylamine (5.0 mL, 39 mmol) according to
General procedure B2. The crude product was recrystallised
+
+
LRMS-ESI (m/z): 254.1 (M + H) . HRMS-ESI (m/z): (M + H)
calcd for C16
1
+
20 3 R
H N , 254.1652; found, 254.1653. RP-HPLC: t
3.5 min (Method A).
(
±)-3-Methyl-1-((4-methyl-2-pentyl)amino)isoquinoline-4- from ethanol to afford the title compound 22 (0.14 g, 42%) as
1
carbonitrile (19). Acetamide 6 (85 mg, 0.38 mmol) was a lime green solid, mp 152–154 °C. H NMR (400 MHz, DMSO-
coupled with (±)-4-methyl-2-pentylamine (1.8 mL, 13 mmol) ): δ 8.55 (d, J = 8.5 Hz, 1H), 8.52 (d, J = 7.5 Hz, 1H), 7.83–7.79
d
6
according to General procedure B2. Purification by flash (m, 1H), 7.74 (dd, J = 8.0, 1.0 Hz, 1H), 7.61–7.57 (m, 1H),
chromatography (10% ethyl acetate in hexanes) provided the 7.47–7.44 (m, 2H), 7.32–7.28 (m, 2H), 7.22–7.18 (m, 1H),
1
3
title compound 19 (22 mg, 22%) as a pale yellow semi-solid, 5.71–5.63 (m, 1H), 2.51 (s, 3H), 1.60 (d, J = 7.0 Hz, 3H).
C
1
mp 78–83 °C. H NMR (400 MHz, CDCl ): δ 7.94 (dd, J = 8.5, NMR (100 MHz, DMSO-d ): δ 158.7, 155.4, 144.7, 135.4, 132.1,
3
6
1
7
1
3
1
9
2
2
.0 Hz, 1H), 7.71–7.67 (m, 2H), 7.49–7.45 (m, 1H), 5.40 (d, J = 128.2, 126.6, 126.3, 126.2, 124.0, 122.9, 118.2, 115.0, 89.7, 49.5,
.5 Hz, 1H), 4.69–4.62 (m, 1H), 2.69 (s, 3H), 1.76–1.65 (m, 1H), 23.9, 21.9. LRMS-ESI (m/z): 288.1 (M + H) . HRMS-ESI (m/z):
+
+
+
.61–1.37 (m, 2H), 1.29 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 5.5 Hz, (M + H) calcd for C H N , 288.1495; found, 288.1495.
1
9
18 3
1
3
H), 0.95 (d, J = 5.5 Hz, 3H). C NMR (100 MHz, CDCl
59.7, 155.5, 136.5, 131.5, 126.2, 124.7, 121.5, 118.8, 115.2,
1.6, 46.9, 45.2, 25.4, 24.4, 23.0, 22.9, 21.5. LRMS-ESI (m/z): nitrile (23). Acetamide 6 (0.50 g, 2.2 mmol) was coupled with
3
): δ RP-HPLC: t
R
16.8 min (Method A).
(S)-3-Methyl-1-((1-phenylethyl)amino)isoquinoline-4-carbo-
+
+
+
68.1 (M + H) . HRMS-ESI (m/z): (M + H) calcd for C17
H
22
N
3
,
(S)-(−)-1-phenylethylamine (9.7 mL, 75 mmol) according to
68.1808; found, 268.1808. RP-HPLC: t 14.9 min (Method A).
R
General procedure B2, except after pH adjustment, the super-
(±)-1-((5-(Diethylamino)2-pentyl)amino)-3-methylisoquinoline- natant was decanted off and the residual solid was suspended
4
-carbonitrile (20). Acetamide 6 (0.20 g, 0.88 mmol) was in water. The solid was extracted with dichloromethane (×3),
coupled with (±)-5-diethylamino-2-pentylamine (5.8 mL, washed with brine (×2), dried, filtered and concentrated in
0 mmol) according to General procedure B1, except the reac- vacuo to give a brown oil residue. The residue was recrystallised
tion mixture was not poured onto ice. The reaction mixture from ethanol to give the title compound 23 (0.34 g, 54%) as a
3
1
6
was concentrated in vacuo and the excess amine was distilled pink solid, mp 151–154 °C. H NMR (300 MHz, DMSO-d ): δ
off to give a brown residue. The crude residue was dissolved in 8.54 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 7.82–7.71 (m,
ethanol, to which a drop of phosphoric acid was added, fol- 2H), 7.61–7.55 (m, 1H), 7.47–7.44 (m, 2H), 7.33–7.28 (m, 2H),
lowed by diethyl ether (2 mL) and the solvent subsequently 7.22–7.17 (m, 1H), 5.71–5.62 (m, 1H), 2.51 (s, 3H), 1.60 (d, J =
1
3
decanted. Following the addition of water (5 mL) the suspen- 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d ): δ 158.6, 155.4,
6
sion was basified with 25% sodium hydroxide and the whole 144.7, 135.4, 132.0, 128.1, 126.5, 126.3, 126.1, 124.0, 122.9,
extracted with dichloromethane (×3). The combined organics 118.1, 115.0, 89.7, 49.5, 23.8, 21.9. LRMS-ESI (m/z): 288.1 (M +
+
+
+
were washed with brine (×2), dried, filtered and concentrated H) . HRMS-ESI (m/z): (M + H) calcd for C H N , 288.1495;
1
9
18 3
in vacuo to afford compound 20 (54 mg, 19%) as a green oil, found, 288.1495. RP-HPLC: t
mp 31–35 °C. H NMR (400 MHz, CDCl ): δ 7.89 (d, J = 8.5 Hz,
3
R
16.8 min (Method A).
1-(Benzhydrylamino)-3-methylisoquinoline-4-carbonitrile
H), 7.84 (d, J = 8.5 Hz, 1H), 7.68–7.64 (m, 1H), 7.47–7.42 (m, (24). Acetamide 6 (0.18 g, 0.80 mmol) was coupled with benz-
1
1
1
2
H), 6.21 (d, J = 8.0 Hz, 1H), 4.57–4.50 (m, 1H), 2.66 (s, 3H), hydrylamine (4.7 mL, 27 mmol) according to General
.58 (q, J = 7.0 Hz, 4H), 2.51 (td, J = 7.0, 2.5 Hz, 2H), 1.80–1.58 procedure B1. Upon pouring the reaction mixture onto ice the
1
3
(m, 4H), 1.30 (d, J = 6.5 Hz, 3H), 1.03 (t, J = 7.0 Hz, 6H).
C
supernatant was decanted off. The crude mixture was boiled in
NMR (100 MHz, CDCl
3
): δ 159.6, 155.8, 136.4, 131.4, 126.1, ethanol and the off-white solid that separated upon cooling
1
2
24.5, 122.1, 118.9, 115.4, 91.4, 52.8, 47.0, 46.8, 34.7, 24.4, was filtered. The crude product was purified by flash
+
3.5, 20.9, 11.1. LRMS-ESI (m/z): 325.2 (M + H) . HRMS-ESI (m/z): chromatography (2.5–5% ethyl acetate in hexanes) and sub-
2
+
+
4
(
M + 2H) calcd for C20
RP-HPLC: t 9.45 min (Method A).
-Methyl-1-morpholinoisoquinoline-4-carbonitrile
Acetamide 6 (0.30 g, 1.3 mmol) was coupled with morpholine Hz, 1H), 7.85–7.76 (m, 2H), 7.62–7.57 (m, 1H), 7.42–7.33 (m,
29
H N
, 163.1230; found, 163.1227. sequent recrystallisation from ethanol provided compound 24
1
R
(35 mg, 13%) as a white solid, mp 194–196 °C. H NMR
3
(21). (400 MHz, DMSO-d ): δ 8.91 (d, J = 8.5 Hz, 1H), 8.67 (d, J = 8.5
6
(
3.9 mL, 45 mmol) according to General procedure B2. The 8H), 7.29–7.25 (m, 2H), 6.98 (d, J = 8.5 Hz, 1H), 2.55 (s, 3H).
1
3
crude product was recrystallised from toluene/petroleum ether
C NMR (100 MHz, DMSO-d ): δ 158.5, 155.5, 142.0, 135.5,
6
(
bp 80–110 °C) to afford the title compound 21 (95 mg, 28%) 132.2, 128.3, 127.9, 127.0, 126.3, 124.3, 123.0, 118.0, 115.1,
1
+
as a deep orange solid, mp 158–161 °C. H NMR (400 MHz, 90.5, 57.3, 23.9. LRMS-ESI (m/z): 350.1 (M + H) . HRMS-ESI
CDCl ): δ 8.03 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), (m/z): (M + H) calcd for C H N , 350.1652; found, 350.1655.
+
+
3
24 20 3
7
3
R
.76–7.70 (m, 1H), 7.54–7.48 (m, 1H), 3.92 (t, J = 5.0 Hz, 4H), RP-HPLC: t 21.2 min (Method A).
1
3
.64 (t, J = 5.0 Hz, 4H), 2.75 (s, 3H). C NMR (100 MHz,
3-Methyl-1-(1-ethylpropylamino)isoquinoline-4-carbonitrile
CDCl ): δ 161.8, 157.5, 138.0, 131.7, 126.3, 126.0, 124.7, 117.8, (25). Acetamide 6 (1.0 g, 4.4 mmol) was coupled with 1-ethyl-
3
+
1
17.7, 96.8, 67.0, 51.5, 24.1. LRMS-ESI (m/z): 254.0 (M + H) . propylamine (18 mL, 0.15 mol) according to General procedure
HRMS-ESI (m/z): (M + H) calcd for C15
+
+
H
16
N
3
O , 254.1288; B2. Purification by flash chromatography (10% ethyl acetate in
found, 254.1292. RP-HPLC: t 15.8 min (Method A).
hexanes) and subsequent recrystallisation from toluene/
R
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
petroleum ether (bp 80–110 °C) afforded the title compound 8.5 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.81–7.76 (m, 1H),
5 (0.89 g, 79%) as a light brown solid, mp 102–104 °C. 7.59–7.55 (m, 1H), 4.57 (t, J = 6.5 Hz, 2H), 2.77 (s, 3H),
2
1
H NMR (400 MHz, CDCl
3
): δ 7.95 (d, J = 8.0 Hz, 1H), 7.73–7.67 1.91–1.84 (m, 2H), 1.59–1.53 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H).
1
3
(m, 2H), 7.49–7.45 (m, 1H), 5.42 (br d, J = 8.0 Hz, 1H),
3
C NMR (100 MHz, CDCl ): δ 162.2, 158.1, 137.2, 132.5, 127.3,
4
7
1
1
.46–4.38 (m, 1H), 2.68 (s, 3H), 1.80–1.54 (m, 4H), 0.96 (t, J = 124.9, 123.7, 117.5, 117.4, 97.2, 67.2, 31.1, 24.0, 19.5, 14.0.
1
3
+
+
.5 Hz, 6H). C NMR (100 MHz, CDCl
31.5, 126.2, 124.8, 121.4, 118.8, 115.2, 91.6, 53.4, 27.2, 24.4, calcd for C15
3
): δ 159.7, 156.1, 136.5, LRMS-ESI (m/z): 241.1 (M + H) . HRMS-ESI (m/z): (M + H)
+
H
17
N
2
O , 241.1335; found, 241.1335. RP-HPLC: t
R
+
0.4. LRMS-ESI (m/z): 254.1 (M + H) . HRMS-ESI (m/z): 23.6 min (Method A).
+
+
(M + H) calcd for C16
H
20
N
3
, 254.1652; found, 254.1651.
General procedure C: preparation of 7-bromo-4-cyano-3-
methylisoquinoline compounds via bromination using
N-bromosuccinimide
RP-HPLC: t
R
12.9 min (Method A).
1
-(2-Methylpropylamino)-3-methylisoquinoline-4-carboxamide
26). A solution of 15 (0.10 g, 0.42 mmol) in 1 : 1 concentrated
sulfuric acid/water (4 mL) was heated under reflux for 65 h.
(
1-Amino-7-bromo-3-methylisoquinoline-4-carbonitrile (29).
2
9
The solution was then cooled, poured onto ice and adjusted to Isoquinoline 11 (0.10 g, 0.55 mmol) was added portion wise
pH 9 with 25% sodium hydroxide. The whole was extracted to ice cold concentrated sulfuric acid (2 mL). Once the solid
with dichloromethane (×3), washed with brine (×2), dried, was dissolved, N-bromosuccinimide (0.12 g, 0.66 mmol) was
filtered and concentrated in vacuo. Purification by flash added in portions and the reaction mixture stirred at 0 °C for
chromatography (15 → 80% ethyl acetate in hexanes) afforded 0.5 h, then allowed to come to room temperature and stirred
amide 26 (27 mg, 25%) as a light brown solid, mp 157–159 °C. for a further 2 h. The reaction mixture was poured onto ice,
1
H NMR (400 MHz, DMSO-d ): δ 8.24 (d, J = 8.5 Hz, 1H), 7.72 adjusted to pH 5 with 25% sodium hydroxide and the solid
6
(br s, 1H), 7.65–7.57 (m, 2H), 7.47 (br s, 1H), 7.44–7.39 (m, that separated was filtered and washed with water. The crude
2
1
1
1
2
H), 3.32 (obscured by H O peak), 2.36 (s, 3H), 2.12–2.02 (m, product was purified by flash chromatography (70% ethyl
1
3
H), 0.93 (d, J = 6.5 Hz, 6H). C NMR (100 MHz, DMSO-d ): δ acetate in hexanes) to afford compound 29 (82 mg, 57%) as a
6
1
70.7, 154.5, 145.4, 134.4, 129.7, 124.4, 123.9, 122.8, 116.8, light brown solid, mp 255–260 °C (dec). H NMR (400 MHz,
15.4, 48.4, 27.2, 22.8, 20.5. LRMS-ESI (m/z): 258.0 (M + H) . DMSO-d
+
6
): δ 8.65 (d, J = 2.0 Hz, 1H), 8.21 (br s, 2H), 7.98 (dd,
+
+
13
HRMS-ESI (m/z): (M + H) calcd for C H N O , 258.1601; J = 9.0, 2.0 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 2.54 (s, 3H).
C
1
5
20 3
found, 258.1600. RP-HPLC: t
-Ethoxy-3-methylisoquinoline-4-carbonitrile
amide 6 (0.20 g, 0.88 mmol) and potassium ethoxide (0.19 g, (M[ Br] + H) , 264.0 (M[ Br] + H) . HRMS-ESI (m/z): (M[ Br]
R
7.53 min (Method A).
NMR (100 MHz, DMSO-d
6
): δ 159.7, 156.0, 137.3, 133.9, 127.9,
1
(27). Acet- 125.7, 120.3, 116.8, 116.1, 91.5, 21.0. LRMS-ESI (m/z): 262.0
7
9
+
81
+
79
+
+
2
.2 mmol) was dissolved in anhydrous ethanol (25 mL) and + H) calcd for C11
the reaction was heated under reflux for 1 h. The mixture was HPLC: t 10.2 min (Method A).
then concentrated in vacuo, water added (25 mL), neutralised 7-Bromo-1-(2-propylamino)-3-methylisoquinoline-4-carbo-
9 3
H BrN , 261.9974; found, 261.9977. RP–
R
with 3 M hydrochloric acid and the whole extracted with nitrile (30). Isoquinoline 14 (0.30 g, 1.3 mmol) was brominated
chloroform (×3). The combined organic extracts were washed according to General procedure C, except was further kept
with brine (×2), dried, filtered and concentrated in vacuo. The overnight at 4 °C prior to filtering. The crude product recrystal-
crude solid was recrystallised from ethanol to give ethoxide 27 lised from methanol to afford compound 30 (0.14 g, 35%) as a
2
9
1
(
13 mg, 7%) as an orange solid, mp 113–115 °C (lit.
6
brown solid, mp 177–179 °C. H NMR (400 MHz, DMSO-d ): δ
1
1
1
7
2
14–116 °C). H NMR (400 MHz, CDCl ): δ 8.27 (ddd, J = 8.0, 8.72 (d, J = 2.0 Hz, 1H), 8.09 (br d, J = 7.0 Hz, 1H), 7.92 (dd, J =
.5, 0.5 Hz, 1H), 7.99 (ddd app dt, J = 8.0, 1.0 Hz, 1H), 9.0, 2.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 4.59–4.51 (m, 1H),
.81–7.76 (m, 1H), 7.59–7.57 (m, 1H), 4.63 (q, J = 7.0 Hz, 2H), 2.55 (s, 3H), 1.26 (d, J = 6.5 Hz, 6H). C NMR (100 MHz,
.77 (s, 3H), 1.51 (t, J = 7.0 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 159.5, 154.6, 134.8, 134.3, 126.3, 125.2, 118.8,
3
1
3
1
3
6
CDCl
3
): δ 162.1, 158.0, 137.2, 132.5, 127.2, 124.9, 123.7, 117.4, 118.0, 116.4, 88.8, 42.5, 24.0, 21.9. LRMS-ESI (m/z): 303.9 (M
+
79
+
81
+
79
1
17.4, 97.2, 63.2, 24.0, 14.6. LRMS-ESI (m/z): 213.0 (M + H) . [ Br] + H) , 305.9 (M[ Br] + H) . HRMS-ESI (m/z): (M[ Br] +
+
+
RP-HPLC: t 20.9 min (Method A).
H) calcd for C H BrN , 304.0444; found, 304.0444.
14 15 3
R
1
R
-Butoxy-3-methylisoquinoline-4-carbonitrile (28). Sodium RP-HPLC: t 16.0 min (Method A).
in anhydrous n-butanol (25 mL), under nitrogen, was heated to 7-Bromo-1-(butylamino)-3-methylisoquinoline-4-carbonitrile
complete dissolution and then allowed to cool to room temp- (31). Isoquinoline 4 (2.0 g, 8.4 mmol) was brominated accord-
erature. To this acetamide 6 (0.20 g, 0.88 mmol) was added ing to General procedure C. The crude product was recrystal-
and the reaction heated under reflux for 1 h. The mixture was lised from methanol to afford compound 31 (1.9 g, 70%) as a
1
then concentrated in vacuo, water added (25 mL), neutralised beige solid, mp 176–178 °C. H NMR (400 MHz, DMSO-d ): δ
6
with 3 M hydrochloric acid and the whole extracted with 8.62 (d, J = 2.0 Hz, 1H), 8.34 (br t, J = 5.0 Hz, 1H), 7.89 (dd, J =
dichloromethane (×3). The combined organic extracts were 9.0, 2.0 Hz, 1H), 7.63 (d, J = 9.0 Hz, 1H), 3.55–3.51 (m, 2H),
washed with brine (×2), dried, filtered and concentrated in 2.53 (s, 3H), 1.66–1.59 (m, 2H), 1.41–1.32 (m, 2H), 0.92 (t, J =
1
3
vacuo. The crude solid obtained was recrystallised from 7.0 Hz, 3H). C NMR (100 MHz, DMSO-d
6
): δ 159.5, 155.4,
n-butanol to give the butoxide 28 (55 mg, 26%) as an off-white 134.7, 134.2, 126.2, 125.2, 118.8, 117.9, 116.4, 88.9, 40.7, 30.4,
1
79
+
solid, mp 80–83 °C. H NMR (400 MHz, CDCl ): δ 8.26 (d, J = 24.0, 19.7, 13.7. LRMS-ESI (m/z): 318.0 (M[ Br] + H) , 320.0 (M
3
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
8
1
+
79
+
[
Br] + H) . HRMS-ESI (m/z): (M[ Br] + H) calcd for then allowed to cool, water was added and the whole extracted
+
C H BrN , 318.0600; found, 318.0600. RP-HPLC: tR with dichloromethane (×2). The combined organic extracts
1
5
17
3
1
7.4 min (Method A).
were washed with brine (×2), dried, filtered and concentrated
in vacuo to give a brown oil. The crude oil was purified by flash
chromatography (10% ethyl acetate in hexanes) to provide
General procedure D: preparation of 7-substituted 4-cyano-3-
methylisoquinoline via boronic acid coupling
compound 34 (38 mg, 36%) as a yellow powder, mp
1
1
-(Butylamino)-3-methyl-7-phenylisoquinoline-4-carbonitrile 240–245 °C. H NMR (400 MHz, CDCl
3
): δ 7.84 (d, J = 9.0 Hz,
(
32). To a solution of 31 (0.20 g, 0.63 mmol) in 4 : 1 1H), 7.41 (dd, J = 9.0, 2.0 Hz, 1H), 7.37–7.32 (m, 2H), 7.28 (d,
acetonitrile : water (50 mL), phenylboronic acid (0.12 g, J = 2.0 Hz, 1H), 7.16–7.13 (m, 2H), 7.06–7.02 (m, 1H), 5.93 (s,
.95 mmol) and caesium fluoride (0.29 g, 1.9 mmol) were 1H), 5.38 (s, 1H), 3.67–3.62 (m, 2H), 2.67 (s, 3H), 1.71–1.63 (m,
0
1
3
added and the reaction mixture degassed under nitrogen. To 2H), 1.49–1.40 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H). C NMR
this 1,1′ bis(diphenylphosphino)ferrocene]dichloropalladium (100 MHz, CDCl ): δ 156.8, 154.9, 142.2, 142.0, 130.7, 129.7,
II) (23 mg, 5 mol%) was added and the reaction mixture was 126.0 124.1, 122.3, 118.8, 118.7, 116.3, 106.0, 91.7, 41.4, 31.5,
3
(
+
heated to 90 °C for 24 h. The mixture was then cooled, brine 23.9, 20.3, 13.9. LRMS-ESI (m/z): 331.2 (M + H) . HRMS-ESI (m/
+
+
was added and the whole extracted with ethyl acetate (×2). The z): (M + H) calcd for C21
organic extracts were dried, filtered and concentrated in vacuo RP-HPLC: t 21.1 min (Method B).
to give a brown solid. The crude solid was purified by flash 1,7-Bis(butylamino)-3-methylisoquinoline-4-carbonitrile (35). A
23 4
H N , 331.1917; found, 331.1921.
R
chromatography (10% ethyl acetate in hexanes) to provide mixture of 31 (0.20 g, 0.63 mmol), n-butylamine (75 µL,
compound 32 (81 mg, 41%) as a white solid, mp 193–196 °C. 0.76 mmol) and potassium carbonate (0.19 g, 1.4 mmol) in
1
H NMR (500 MHz, CDCl ): δ 7.99 (dd, J = 8.5, 0.5 Hz, 1H), anhydrous tert-butanol (20 mL) was degassed under nitrogen.
3
7
(
3
.91 (dd, J = 8.5, 2.0 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.66–7.63 To this mixture 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-
m, 2H), 7.51–7.47 (m, 2H), 7.42–7.39 (m, 1H), 5.84 (br s, 1H), biphenyl (60 mg, 20 mol%) and tris(dibenzylideneacetone)
.73–3.69 (m, 2H), 2.71 (s, 3H), 1.76–1.70 (m, 2H), 1.52–1.45 dipalladium(0) (58 mg, 10 mol%) were added and the reaction
1
3
(m, 2H), 1.00 (t, J = 7.0 Hz, 3H). C NMR (125 MHz, CDCl
3
): δ heated under reflux for 18 h. The mixture was then allowed to
59.4, 156.1, 140.1, 139.4, 135.3, 130.9, 129.1, 127.9, 127.3, cool, water was added and the whole extracted with ethyl
25.1, 119.6, 118.6, 115.5, 91.6, 41.5, 31.6, 24.2, 20.3, 13.9. acetate (×2). The combined organic extracts were washed with
1
1
+
+
LRMS-ESI (m/z): 316.2 (M + H) . HRMS-ESI (m/z): (M + H)
calcd for C21
brine (×2), dried, filtered and concentrated in vacuo. The crude
+
H
22
N
3
, 316.1808; found, 316.1809. RP-HPLC: t
R
solid was purified by flash chromatography (10% ethyl acetate
2
2.7 min (Method B).
in hexanes) to provide compound 35 (34 mg, 17%) as a yellow
1
1
-(Butylamino)-7-(3-methoxyphenyl)-3-methylisoquinoline-4- solid, mp 110–120 °C. H NMR (500 MHz, CDCl
3
): δ 7.74 (d, J =
carbonitrile (33). Compound 31 (0.10 g, 0.32 mmol) was 9.0 Hz, 1H), 7.05 (dd, J = 9.0, 2.0 Hz, 1H), 6.52 (d, J = 2.0 Hz,
coupled to 3-methoxyphenylboronic acid (72 mg, 0.47 mmol) 1H), 5.35 (br s, 1H), 3.69–3.65 (m, 2H), 3.19 (t, J = 7.0 Hz, 2H),
according to General procedure D. The crude solid was puri- 2.65 (s, 3H), 1.74–1.65 (m, 4H), 1.52–1.43 (m, 4H), 1.00 (t, J =
1
3
fied by flash chromatography (10% ethyl acetate in hexanes) 7.5 Hz, 6H). NH not observed. C NMR (100 MHz, CDCl
3
): δ
and subsequently recrystallised from petroleum ether (bp 155.0, 154.9, 147.1, 128.4, 125.7, 121.5, 119.3, 116.8, 98.9, 91.8,
8
0–110 °C) to provide compound 33 (39 mg, 36%) as a white 43.8, 41.5, 31.8, 31.5, 23.9, 20.43, 20.45, 14.05, 14.04.
1
+
+
solid, mp 161–166 °C. H NMR (400 MHz, CDCl
3
): δ 7.98 (d, J = LRMS-ESI (m/z): 311.2 (M + H) . HRMS-ESI (m/z): (M + H)
+
9
.0 Hz, 1H), 7.90 (dd, J = 9.0, 1.5 Hz, 1H), 7.83 (d, J = 1.5 Hz, calcd for C H N , 311.2230; found, 311.2227. RP-HPLC: t
1
9
27
4
R
1
H), 7.41 (dd app t, J = 8.0 Hz, 1H), 7.23 (ddd, J = 8.0, 2.0, 1.0 11.0 min (Method A).
Hz, 1H), 7.17 (dd app t, J = 2.0 Hz, 1H), 6.95 (ddd, J = 8.0, 2.0,
7-(Benzylamino)-1-(butylamino)-3-methylisoquinoline-4-carbo-
1
2
7
1
1
.0 Hz, 1H), 5.80 (br s, 1H), 3.89 (s, 3H), 3.75–3.68 (m, 2H), nitrile (36). A mixture of 31 (0.10 g, 0.30 mmol), benzylamine
.71 (s, 3H), 1.77–1.69 (m, 2H), 1.53–1.43 (m, 2H), 1.00 (t, J = (41 µL, 0.38 mmol) and potassium carbonate (96 mg,
1
3
3
.5 Hz, 3H). C NMR (100 MHz, CDCl ): δ 160.2, 159.4, 156.0, 0.69 mmol) in anhydrous tert-butanol (10 mL) was degassed
41.6, 139.2, 135.4, 130.9, 130.1, 125.1, 119.8, 119.6, 118.6, under nitrogen. To this mixture 2-dicyclohexylphosphino-
15.4, 113.5, 112.9, 91.7, 55.5, 41.5, 31.6, 24.2, 20.3, 13.9. 2′,4′,6′-triisopropylbiphenyl (30 mg, 20 mol%) and tris(dibenzy-
+
+
LRMS-ESI (m/z): 346.2 (M + H) . HRMS-ESI (m/z): (M + H)
calcd for C H N O , 346.1914; found, 346.1916. RP-HPLC: t
1
lideneacetone)dipalladium(0) (29 mg, 10 mol%) were added
and the reaction heated under reflux for 17 h. The mixture was
then allowed to cool, water was added and the whole extracted
+
2
2
24
3
R
6.9 min (Method A).
1
-(Butylamino)-3-methyl-7-(phenylamino)isoquinoline-4- with ethyl acetate (×2). The combined organic extracts were
carbonitrile (34). A mixture of 31 (0.10 g, 0.32 mmol), aniline washed with brine (×2), dried, filtered and concentrated in
35 µL, 0.38 mmol) and potassium carbonate (96 mg, vacuo. The crude solid was purified by flash chromatography
.69 mmol) in anhydrous tert-butanol (10 mL) was degassed (10% ethyl acetate in hexanes) to provide compound 36
(
0
1
under nitrogen. To this mixture 2-dicyclohexylphosphino- (60 mg, 55%) as a deep red solid, mp 179–181 °C. H NMR
′,4′,6′-triisopropylbiphenyl (15 mg, 10 mol%) and tris(dibenzyl- (500 MHz, CDCl ): δ 7.75 (d, J = 9.0 Hz, 1H), 7.41–7.36 (m, 4H),
2
3
ideneacetone)dipalladium(0) (14 mg, 5 mol%) were added and 7.33–7.29 (m, 1H), 7.09 (dd, J = 9.0, 2.5 Hz, 1H), 6.56 (d, J = 2.5
the reaction heated under reflux for 17 h. The mixture was Hz, 1H), 5.24 (br s, 1H), 4.43 (s, 2H), 3.64–3.61 (m, 2H), 2.64 (s,
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
3
3
1
1
H), 1.69–1.63 (m, 2H), 1.47–1.39 (m, 2H), 0.98 (t, J = 7.5 Hz, 2.0 Hz, 1H), 8.14 (br t, J = 5.5 Hz, 1H), 7.76 (dd, J = 9.0, 2.0 Hz,
1
3
H). NH not observed. C NMR (100 MHz, CDCl ): δ 155.2, 1H), 7.70 (d, J = 9.0 Hz, 1H), 3.57–3.52 (m, 2H), 2.54 (s, 3H),
3
54.8, 146.5, 138.4, 128.9, 128.6, 127.6, 127.5, 125.7, 121.4, 2.10 (s, 3H), 1.66–1.59 (m, 2H), 1.40–1.31 (m, 2H), 0.92 (t, J =
1
3
6
19.1, 116.6, 99.4, 91.7, 48.4, 41.3, 31.6, 23.8, 20.2, 13.9. 7.5 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 168.4, 157.4,
+
+
LRMS-ESI (m/z): 345.2 (M + H) . HRMS-ESI (m/z): (M + H)
156.0, 137.0, 131.5, 125.8, 123.6, 118.5, 115.5, 113.2, 88.9, 40.6,
+
+
calcd for C22
0.7 min (Method B).
-(Butylamino)-3-methyl-7-nitroisoquinoline-4-carbonitrile found, 297.1711. RP-HPLC: t 10.7 min (Method A).
H
25
N
4
, 345.2074; found, 345.2070. RP-HPLC: t
R
30.6, 23.79, 23.78, 19.7, 13.8. LRMS-ESI (m/z): 297.2 (M + H) .
+
+
2
21 4
HRMS-ESI (m/z): (M + H) calcd for C17H N O , 297.1710;
1
R
(37). Isoquinoline 4 (0.30 g, 1.3 mmol) was added portion
1-(1-(Butylamino)-4-cyano-3-methylisoquinolin-7-yl)urea (40).
wise to ice cooled concentrated sulfuric acid (6 mL). Once the To a solution of amine 38 (50 mg, 0.20 mmol) in acetic acid
solid was dissolved potassium nitrate (0.19 g, 1.9 mmol) was (5 mL) was added sodium cyanate (25 mg, 0.38 mmol) in water
added in portions and the reaction mixture stirred at 0 °C for (1 mL) and the mixture allowed to stir at room temperature for
0
.5 h. The reaction mixture was poured onto ice and the solid 16 h. The reaction was poured onto ice and the solution neu-
that separated was filtered, washed with water and recrystal- tralised with saturated sodium hydrogen carbonate. The solid
lised from ethanol to provide compound 37 (0.27 g, 76%) as a that separated was filtered, stirred in dichloromethane (5 mL)
1
bright orange solid, mp 214–216 °C. H NMR (400 MHz, and subsequently filtered. The crude product was recrystallised
DMSO-d ): δ 9.38 (d, J = 2.0 Hz, 1H), 8.99 (br t, J = 5.5 Hz, 1H), from ethanol to afford urea 40 (18 mg, 31%) as a white solid,
6
1
8
.47 (dd, J = 9.0, 2.0 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 3.60–3.55 mp 290 °C (dec.). H NMR (400 MHz, DMSO-d
6
): δ 8.75 (br s,
(
m, 2H), 2.59 (s, 3H), 1.69–1.62 (m, 2H), 1.43–1.33 (m, 2H), 1H), 8.14 (d, J = 2.0 Hz, 1H), 8.05 (br t, J = 5.5 Hz, 1H), 7.81
1
3
0
1
3
.94 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 163.1, (dd, J = 9.0, 2.0 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 6.03 (br s, 2H),
6
57.1, 144.8, 139.6, 125.7, 124.7, 121.1, 117.6, 114.2, 89.2, 40.9, 3.56–3.51 (m, 2H), 2.52 (s, 3H), 1.66–1.59 (m, 2H), 1.41–1.31
+
13
0.3, 24.3, 19.7, 13.7. LRMS-ESI (m/z): 285.1 (M + H) . (m, 2H), 0.93 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO-
+
+
HRMS-ESI (m/z): (M + H) calcd for C H N O , 285.1346; d6): δ 156.5, 155.9, 155.8, 138.7, 130.2, 125.0, 123.5, 118.6,
1
5
17 4 2
found, 285.1346. RP-HPLC: t
R
20.0 min (Method A).
115.8, 110.9, 88.9, 40.5, 30.6, 23.7, 19.7, 13.8. LRMS-ESI (m/z):
+
+
7
-Amino-1-(butylamino)-3-methylisoquinoline-4-carbonitrile
298.2 (M + H) . HRMS-ESI (m/z): (M + H) calcd for
+
(38). A suspension of 37 (1.0 g, 3.5 mmol) and ethanol C H N O , 298.1662; found, 298.1665. RP-HPLC: t 9.85 min
1
6
20
5
R
(40 mL) in the presence of 10% palladium on charcoal (8% (Method A).
w/w, 80 mg) was hydrogenated at atmospheric pressure at
1-(Butylamino)-7-((4-chlorobenzylidene)amino)-3-methyl-
room temperature for 0.5 h. The reaction mixture was filtered isoquinoline-4-carbonitrile (41). A suspension of amine 38
through a pad of Celite®, the filter cake rinsed with hot (0.10 g, 0.39 mmol) in ethanol (10 mL) was heated to complete
ethanol and the solvent was removed in vacuo. The crude dissolution and then allowed to cool to room temperature. To
product was recrystallised from toluene to afford amine 38 this was added 4-chlorobenzaldehyde (61 mg, 0.43 mmol) and
1
(
(
9
1
(
0.73 g, 82%) as a dull mauve solid, mp 180–182 °C. H NMR the reaction was stirred for 2 h. Upon reaction completion, as
1
400 MHz, DMSO-d
6
): δ 7.71 (br t, J = 5.5 Hz, 1H), 7.47 (d, J = indicated by H-NMR, the mixture was cooled on ice and the
.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.16 (dd, J = 9.0, 2.0 Hz, solid that separated was filtered and washed with cold
H), 5.45 (br s, 2H), 3.53–3.49 (m, 2H), 2.48 (s, 3H), 1.65–1.58 ethanol. The crude product was recrystallised from toluene to
13
m, 2H), 1.40–1.31 (m, 2H), 0.92 (t, J = 7.0 Hz, 3H). C NMR give imine 41 (70 mg, 47%) as a brown solid, mp 177–179 °C.
1
(
100 MHz, DMSO-d ): δ 155.0, 153.6, 147.4, 126.3, 123.9, 122.0,
H NMR (400 MHz, DMSO-d ): δ 8.77 (s, 1H), 8.30 (br t, J = 5.5
6
6
1
19.0, 117.0, 103.3, 89.2, 40.4, 30.7, 23.4, 19.7, 13.8. LRMS-ESI Hz, 1H), 8.24 (d, J = 1.0 Hz, 1H), 8.00 (d, J = 8.5 Hz, 2H),
+
+
(
m/z): 255.1 (M + H) . HRMS-ESI (m/z): (M + H) calcd for 7.81–7.76 (m, 2H), 7.63 (d, J = 8.5 Hz, 2H), 3.60–3.55 (m, 2H),
+
C H N , 255.1604; found, 255.1605. RP-HPLC: t 10.5 min 2.57 (s, 3H), 1.69–1.61 (m, 2H), 1.43–1.33 (m, 2H), 0.93 (t, J =
1
5
19
4
R
1
3
(Method A).
6
7.5 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 160.3, 158.5,
N-(1-(Butylamino)-4-cyano-3-methylisoquinolin-7-yl)acetamide 156.4, 148.9, 136.3, 134.7, 133.7, 130.3, 129.1, 126.0, 124.2,
39). To a solution of amine 38 (50 mg, 0.20 mmol) in an- 118.4, 115.8, 115.6, 89.1, 40.6, 30.6, 23.9, 19.7, 13.8. LRMS-ESI
(
3
5
+
37
+
hydrous tetrahydrofuran (5 mL) at 0 °C was added triethyl- (m/z): 377.2 (M[ Cl] + H) , 379.2 (M[ Cl] + H) . HRMS-ESI (m/
3
5
+
+
amine (41 µL, 0.30 mmol) and acetyl chloride (21 µL, z): (M[ Cl] + H) calcd for C22
H
22ClN
4
, 377.1528; found,
0
.30 mmol) and the mixture was stirred for 5 min. The reac- 377.1519. RP-HPLC: t 10.5 min (Method A).
R
tion mixture was warmed to room temperature and stirred for
1-(Butylamino)-7-((4-chlorobenzyl)amino)-3-methylisoquinoline-
a further 2 h. The mixture was concentrated in vacuo, water 4-carbonitrile (42). To a suspension of sodium borohydride
was added and the whole extracted with ethyl acetate (×3). The (60 mg, 1.6 mmol) in ethanol (5 mL) was added imine 41
combined organic extracts were washed with brine (×2), dried, (60 mg, 0.16 mmol) and the mixture was stirred at room temp-
filtered and concentrated in vacuo. The crude product was pur- erature for 16 h. Water was added and the solid that separated
ified by flash chromatography (50% ethyl acetate in hexanes) was filtered and washed with water. The crude solid was recrys-
and subsequently recrystallised from toluene to afford acet- tallised in toluene to give compound 42 (34 mg, 56%) as a
1
amido 39 (42 mg, 72%) as off-white needles, mp 240–242 °C. plum solid, mp 180–182 °C. H NMR (400 MHz, DMSO-d
6
): δ
1
H NMR (400 MHz, DMSO-d ): δ 10.19 (br s, 1H), 8.45 (d, J = 7.76 (br t, J = 5.0 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.44 (d, J =
6
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
9
1
=
2
.0 Hz, 2H), 7.38 (d, J = 9.0 Hz, 2H), 7.24 (dd, J = 9.0, 2.0 Hz, mixture stirred at 0 °C for 1 h. The reaction mixture was
H), 7.16 (d, J = 2.0 Hz, 1H), 6.66 (br t, J = 6.0 Hz, 1H) 4.40 (d, J poured on ice and the solid that separated was filtered,
6.0 Hz, 2H), 3.55–3.50 (m, 2H), 2.48 (s, 3H), 1.65–1.57 (m, washed with water and recrystallised from methanol to provide
13
H), 1.39–1.30 (m, 2H), 0.92 (t, J = 7.5 Hz, 3H). C NMR compound 45 (0.36 g, 79%) as a deep red solid, mp
1
(100 MHz, DMSO-d ): δ 155.0, 153.8, 147.2, 138.6, 131.4, 129.5, 211–213 °C. H NMR (400 MHz, DMSO-d ): δ 8.91 (d, J = 2.0
6 6
1
3
3
28.2, 126.6, 123.9, 122.2, 118.9, 116.8, 99.8, 89.2, 45.7, 40.4, Hz, 1H), 8.77 (br t, J = 5.5 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H),
3
5
+
0.8, 23.5, 19.8, 13.8. LRMS-ESI (m/z): 379.2 (M[ Cl] + H) , 3.60–3.55 (m, 2H), 2.58 (s, 3H), 1.68–1.61 (m, 2H), 1.42–1.33
3
7
+
35
+
13
81.2 (M[ Cl] + H) . HRMS-ESI (m/z): (M[ Cl] + H) calcd for (m, 2H), 0.93 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO-
+
C
(
22
H
24ClN
Method A).
-(Butylamino)-7-iodo-3-methylisoquinoline-4-carbonitrile (M[ Br] + H) , 365.1 (M[ Br] + H) . HRMS-ESI (m/z): (M[ Br]
4
, 379.1684; found, 379.1685. RP-HPLC: t
R
16.0 min
d
6
): δ 163.8, 155.2, 145.9, 130.3, 129.7, 124.4, 117.6, 117.0,
115.7, 83.6, 41.0, 30.2, 24.6, 19.7, 13.7. LRMS-ESI (m/z): 363.1
7
9
+
81
+
79
1
+
+
(
43). To a suspension of amine 38 (0.10 g, 0.39 mmol) in con- + H) calcd for C15
20.7 min (Method A).
1-(Butylamino)-3-methyl-5-nitro-7-phenylisoquinoline-4-carbo-
4 2
H16BrN O , 363.0451; found, 363.0453.
centrated hydrochloric acid (4 mL), at −5° C, was added RP-HPLC: t
sodium nitrite (54 mg, 0.79 mmol) in water (2.5 mL) dropwise
R
over 5 min and the reaction mixture was stirred for 1 h. The nitrile (46). Compound 45 (0.20 g, 0.55 mmol) was coupled to
resulting solution was added dropwise to a solution of potass- phenylboronic acid (0.10 g, 0.83 mmol) according to General
ium iodide (1.6 g, 9.8 mmol) in water (8 mL) and the reaction procedure D. The crude solid was purified by flash chromato-
mixture stirred at −5° C for 0.25 h and for a further 4 h at graphy (10% ethyl acetate in hexanes) to provide compound 46
1
room temperature. The reaction mixture was diluted with (54 mg, 27%) as a yellow solid, mp 240–245 °C. H NMR
water and extracted with ethyl acetate (×3). The combined (400 MHz, DMSO-d ): δ 8.92–8.90 (m, 2H), 8.60 (d, J = 1.5 Hz,
6
organic extracts were washed with 2.5 M sodium hydroxide 1H), 7.94–7.92 (m, 2H), 7.59–7.55 (m, 2H), 7.50–7.46 (m, 1H),
(
×2), 10% sodium thiosulfate (×2), 2.5 M sodium hydroxide, 3.66–3.61 (m, 2H), 2.61 (s, 3H), 1.72–1.64 (m, 2H), 1.45–1.35
1
3
water, brine, dried, filtered and concentrated in vacuo to (m, 2H), 0.95 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO-
provide compound 43 (0.10 g, 70%) as a brown solid, mp ): δ 163.2, 156.2, 146.1, 137.2, 136.8, 129.1, 128.7, 127.1,
): δ 8.78 (d, J = 1.5 125.4, 124.8, 124.4, 116.9, 116.1, 83.6, 40.9, 30.4, 24.5, 19.7,
d
6
1
2
04–208 °C. H NMR (400 MHz, DMSO-d
6
+
Hz, 1H), 8.37 (br t, J = 5.0 Hz, 1H), 8.05 (dd, J = 9.0, 1.5 Hz, 13.8. LRMS-ESI (m/z): 361.1 (M + H) . HRMS-ESI (m/z): (M +
1
+
+
H), 7.51 (d, J = 9.0 Hz, 1H), 3.57–3.52 (m, 2H), 2.54 (s, 3H), H) calcd for
.67–1.59 (m, 2H), 1.42–1.32 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H). RP-HPLC: t 21.3 min (Method B).
5-Amino-1-(butylamino)-3-methylisoquinoline-4-carbonitrile
32.1, 124.9, 117.9, 116.7, 91.3, 88.9, 40.7, 30.4, 24.0, 19.8, (47). A suspension of 45 (0.52 g, 1.4 mmol), triethylamine
21 21 4 2
C H N O , 361.1659; found, 361.1664.
1
R
1
3
C NMR (100 MHz, DMSO-d ): δ 159.4, 155.0, 140.0, 134.4,
6
1
1
(
+
3.8. LRMS-ESI (m/z): 366.0 (M + H) . RP-HPLC: t
R
22.3 min (0.20 mL, 1.4 mmol) and ethanol (20 mL) in the presence of
10% palladium on charcoal (8% w/w, 40 mg) was hydrogenated
Method B).
1
-(Butylamino)-7-ethoxy-3-methylisoquinoline-4-carbonitrile
at atmospheric pressure at room temperature for 2 h. The reac-
(44). To a suspension of 43 (0.10 g, 0.27 mmol), caesium car- tion mixture was filtered through a pad of Celite®, the filter
bonate (0.18 g, 0.55 mmol) and 1,10-phenanthroline (9.9 mg, cake was rinsed with hot ethanol and the solvent was removed
0 mol%) in ethanol (4.4 mL) was added copper(I) iodide in vacuo. The crude residue was dissolved in ethyl acetate,
5.2 mg, 10 mol%) and the mixture heated to 110 °C in a washed with water (×2), brine (×2), dried, filtered and concen-
2
(
pressure tube for 18 h. The reaction mixture was allowed to trated in vacuo. The crude mixture was recrystallised from
cool, filtered through a silica pad and solvent removed in petroleum ether (bp 80–110 °C)/toluene to afford amine 47
1
vacuo to give a brown solid. The solid was purified by flash (0.31 g, 85%) as a maroon solid, mp 151–154 °C. H NMR
chromatography (10% ethyl acetate in hexanes) to provide (400 MHz, DMSO-d ): δ 8.01 (br t, J = 5.0 Hz, 1H), 7.50 (d, J =
6
compound 44 (34 mg, 44%) as a white solid, mp 150–161 °C. 8.0 Hz, 1H), 7.24 (dd app t, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz,
1
3
H NMR (500 MHz, CDCl ): δ 7.86 (d, J = 9.0 Hz, 1H), 7.34 (dd, 1H), 5.65 (s, 2H), 3.55–3.50 (m, 2H), 2.54 (s, 3H), 1.65–1.57 (m,
1
3
J = 9.0, 2.5 Hz, 1H), 6.98 (d, J = 2.5 Hz, 1H), 5.47 (br s, 1H), 2H), 1.39–1.30 (m, 2H), 0.92 (t, J = 7.0 Hz, 3H). C NMR
4
.14 (q, J = 7.0 Hz, 2H), 3.69–3.65 (m, 2H), 2.67 (s, 3H), (100 MHz, DMSO-d
6
): δ 159.0, 156.7, 143.0, 126.8, 122.1, 122.0,
1
.74–1.68 (m, 2H), 1.51–1.43 (m, 5H), 1.00 (t, J = 7.5 Hz, 3H). 116.6, 115.8, 111.7, 85.0, 40.6, 30.7, 24.2, 19.8, 13.8. LRMS-ESI
1
3
+
+
C NMR (125 MHz, CDCl ): δ 157.4, 156.9, 155.2, 130.7, 126.2, (m/z): 255.1 (M + H) . HRMS-ESI (m/z): (M + H) calcd for
3
+
1
1
22.1, 118.8, 116.1, 103.0, 91.6, 64.1, 41.4, 31.6, 23.9, 20.3,
4.8, 13.9. LRMS-ESI (m/z): 284.1 (M + H) . HRMS-ESI (m/z): (Method A).
C
15
H
19
N
4
R
, 255.1604; found, 255.1604. RP-HPLC: t 10.4 min
+
+
+
(M + H) calcd for C H N O , 284.1757; found, 284.1760.
N-(1-(Butylamino)-4-cyano-3-methylisoquinolin-5-yl)acetamide
(48). To a solution of amine 47 (50 mg, 0.20 mmol) in an-
1
7
22 3
R
RP-HPLC: t 14.0 min (Method A).
7
-Bromo-1-(butylamino)-3-methyl-5-nitroisoquinoline-4- hydrous tetrahydrofuran (5 mL) at 0 °C was added triethyl-
carbonitrile (45). Compound 31 (0.40 g, 1.3 mmol) was added amine (55 µL, 0.40 mmol) and acetyl chloride (28 µL,
portion wise to ice cold concentrated sulfuric acid (8 mL). 0.40 mmol) and the mixture was stirred for 5 min. The reac-
Once the solid was dissolved, potassium nitrate (0.19 g, tion mixture was warmed to room temperature and stirred for
1
.9 mmol) was added in small portions and the reaction a further 4 h. The mixture was concentrated in vacuo, water
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
was added and the whole extracted with ethyl acetate (×3). The Malstat assays for growth inhibition
combined organic extracts were washed with brine (×2), dried,
filtered and concentrated in vacuo. The crude product was
P. falciparum 3D7 and W2mef parasites were cultured accord-
ing to the procedure described by Trager et al. in RPMI-
53
recrystallised from ethanol to afford acetamido 48 (45 mg,
HEPES media supplemented with L-glutamine (Sigma) and
Albumax II (Invitrogen). 100 µL of P. falciparum ring-stage cul-
tures were seeded into 96-well microplates at 0.3% parasitaemia
and 2% haematocrit. Compounds were serially diluted in
DMSO at 500× working concentration and were added to cul-
tures with a final concentration of 0.2% DMSO. The growth
assays were performed in triplicate for 1.5 cell cycles (37 °C,
1
7
7%) as a pink solid, mp 245–248 °C. H NMR (400 MHz,
DMSO-d
6
): δ 9.88 (s, 1H), 8.27 (br t, J = 5.5 Hz, 1H), 8.22 (dd,
J = 8.0, 1.0 Hz, 1H), 7.53–7.45 (m, 2H), 3.59–3.54 (m, 2H), 2.56
(
(
1
8
s, 3H), 2.07 (s, 3H), 1.67–1.60 (m, 2H), 1.42–1.32 (m, 2H), 0.93
13
t, J = 7.0 Hz, 3H). C NMR (100 MHz, DMSO-d
6
): δ 169.7,
60.9, 156.3, 132.7, 131.9, 131.6, 125.7, 122.2, 119.9, 116.4,
6.5, 40.6, 30.6, 24.3, 22.9, 19.7, 13.8. LRMS-ESI (m/z): 297.2
7
3
0
2 h). The cultures were then lysed by a freeze–thaw cycle and
+
+
+
(
2
M + H) . HRMS-ESI (m/z): (M + H) calcd for C17
97.1710; found, 297.1710. RP-HPLC: t 8.58 min (Method A).
-(Butylamino)-5-((4-chlorobenzylidene)amino)-3-methyl-
isoquinoline-4-carbonitrile (49). A suspension of amine 47
50 mg, 0.20 mmol) in ethanol (5 mL) was heated to complete
21 4
H N O ,
0 µL was mixed with 75 µL Malstat reagent (0.1 M Tris pH 8.5),
R
−1
−1
.2 g mL lactic acid, 0.2% v/v Triton X-100 and 1 mg mL
1
−1
acetylpyridine adenine dinucleotide [Sigma], 0.01 mg ml phe-
nozine ethosulfate [Sigma] and 0.2 mg mL nitro blue tetr-
−1
(
azolium [Sigma]. Once the no drug control wells had developed
a purple color the absorbance was measured at 650 nm in a
spectrophotometer. Absorbance values were plotted in Prism 6
dissolution and then allowed to cool to room temperature. To
this was added 4-chlorobenzaldehyde (41 mg, 0.30 mmol) and
the reaction refluxed for 24 h. Upon reaction completion, as
(
GraphPad) and normalised against untreated and uninfected
1
indicated by H-NMR, the mixture was cooled on ice and the
samples to generate drug curves and IC50 values.
solid that separated was filtered and washed with cold
ethanol. The crude product was recrystallised from toluene/
petroleum ether (bp 80–110 °C) to give imine 49 (29 mg, 39%)
Expression of recombinant PfPKA
1
Synthetic cDNA encoding the Plasmodium falciparum 3D7 PKA
catalytic subunit (CAA11945) was cloned into pET-28b based
expression vector. Two different constructs were trialled con-
sisting of residues 1–342 and 14–342. Recombinant protein
was expressed in E. coli BL21(DE3) cells grown for 4 hours at
37 °C in Luria Broth containing 34 μM Kanamycin before
being induced for 4–22 hours at 18, 22 or 37 °C with 0.1 mM
Isopropyl thiogalactopyranoside. Cells were harvested and
resuspended in buffer A (20 mM HEPES pH 7.5, 250 mM NaCl,
as a brown solid, mp 176–177.5 °C. H NMR (400 MHz,
DMSO-d
6
): δ 8.63 (s, 1H), 8.23 (br t, J = 5.5 Hz, 1H), 8.18 (dd,
J = 8.0, 1.0 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz,
2
1
1
H), 7.54 (dd app t, J = 8.0 Hz, 1H), 7.40 (dd, J = 8.0, 1.0 Hz,
H), 3.60–3.55 (m, 2H), 2.59 (s, 3H), 1.69–1.61 (m, 2H),
1
3
.43–1.33 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H). C NMR (100 MHz,
): δ 160.9, 159.7, 156.1, 146.1, 136.3, 134.6, 131.3,
30.1, 128.8, 126.4, 121.1, 120.5, 119.4, 116.1, 86.9, 40.6, 30.6,
DMSO-d
1
2
6
3
5
+
4.4, 19.7, 13.8. LRMS-ESI (m/z): 377.3 (M[ Cl] + H) , 379.2
−
1
3
7
+
35
+
1 mM TCEP) and lysed using sonication and 1 mg mL Lyso-
zyme and centrifuged at 15 000g for 30 minutes. Samples of
the re-solubilised pellet and supernatant where run on
SDS-PAGE gels.
(M[ Cl] + H) . HRMS-ESI (m/z): (M[ Cl] + H) calcd for
+
C
(
22
H
22ClN
Method B).
-(Butylamino)-5-((4-chlorobenzyl)amino)-3-methylisoquinoline-
-carbonitrile (50). To a suspension of sodium borohydride
4 R
, 377.1528; found, 377.1533. RP-HPLC: t 20.8 min
1
4
In vitro phosphorylation assays
(
(
0.10 g, 2.7 mmol) in ethanol (10 mL) was added imine 49
0.10 g, 0.27 mmol) and the mixture was stirred at room temp-
Nunc MaxiSorp 96 well ELISA plates were coated with 1 µg ml−
of the GST-PfAMA1 substrate. Phosphorylation reactions were
performed in the wells of the plate in kinase activity buffer
1
erature for 16 h. Water was added and the solid that separated
was filtered and washed with water. Purification by flash
chromatography (15% → 100% ethyl acetate in hexanes) and
subsequent recrystallisation from petroleum ether (bp
(
2 2
20 mM Tris, 20 mM MgCl , 2 mM MnCl , 1 mM DTT, Roche
PhosSTOP) with 2% parasite extract, 100 µM ATP and 2 µM
cAMP and 10 µM H89 as indicated (37 °C, 30 min). Inhibitors
were titrated at a final concentration of 0.1% DMSO. Following
the reaction the wells were washed with TBS and probed with a
rabbit anti phospho-PfAMA1S610, and then washed again with
TBS and probed with goat anti rabbit-HRP (Abcam). To
develop the assay ABTS was added to the wells for 30 min and
the reaction was stopped with 1% SDS before reading at
8
0–110 °C)/toluene provided compound 50 (34 mg, 34%) as a
1
pale green solid, mp 142–144 °C. H NMR (400 MHz, DMSO-
): δ 8.05 (br t, J = 6.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.49 (d,
d
6
J = 9.0 Hz, 2H), 7.40 (d, J = 9.0 Hz, 2H), 7.28 (dd app t, J = 8.0
Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.24 (br t, J = 5.5 Hz, 1H), 4.43
(
(
(
1
3
3
d, J = 5.5 Hz, 2H), 3.57–3.52 (m, 2H), 2.58 (s, 3H), 1.65–1.58
13
m, 2H), 1.40–1.31 (m, 2H), 0.92 (t, J = 7.5 Hz, 3H). C NMR
100 MHz, DMSO-d ): δ 159.5, 156.5, 142.2, 138.0, 131.4, 129.2,
28.3, 126.7, 123.8, 121.6, 116.3, 112.0, 111.6, 85.1, 46.8, 40.6,
405 nm. Absorbance values were plotted in Prism
6
6
(
GraphPad).
3
5
+
0.7, 24.3, 19.7, 13.8. LRMS-ESI (m/z): 379.3 (M[ Cl] + H) ,
3
7
+
35
+
Luciferase assays for egress and invasion inhibition
81.0 (M[ Cl] + H) . HRMS-ESI (m/z): (M[ Cl] + H) calcd for
+
C
22
H
24ClN
, 379.1684; found, 379.1685. RP-HPLC: t 23.0 min P. falciparum 3D7 parasites transfected with an exported
4 R
3
9
(Method B).
NanoLuc fusion protein were cultured as above. The com-
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
pounds were serially diluted as above and added to synchro- undergraduate chemistry students who contributed to this
nised late-stage parasite cultures (46–48 h post invasion) and project, Banin Rasouli, Dylan Smith, Nikolaj Villadsen, Kristo-
were seeded into 96-well microplates at 5% parasitaemia and fer Cupic and Bethany Davey (Department of Chemistry and
2
% haematocrit. Compounds were titrated in triplicate and Physics, La Trobe Institute for Molecular Science, La Trobe
incubated over egress and invasion (37 °C, 4 h). Samples of University). We also acknowledge the contribution made by Dr
culture supernatant were collected to determine the amount of Jason Dang (Monash Institute of Pharmaceutical Sciences,
luciferase released during egress. Samples were treated with Monash University) for HRMS analysis and the MRC PPU
5
% sorbitol in water to lyse any unruptured schzionts and International Centre for Kinase Profiling (University of
were then washed in 3× in 2 volumes of culture medium to Dundee, United Kingdom) for the human kinase screening.
wash out the PKA inhibitors. The parasites were then returned This work was supported by grants from the National Health
to culture (37 °C, 24 h) and samples were collected again to and Medical Research Council (NHMRC) of Australia. The
determine the amount of luciferase carried over during inva- authors gratefully acknowledge the contribution of the Victor-
sion. From the egress media and the 24 h old cultures, a small ian Operational Infrastructure Support Program, Australia
aliquot was removed (5–10 µL) and added to 0.6× sample received by the Burnet Institute and Monash Micro Imaging.
volume PBS, 0.4× sample volume Luciferase Cell Culture Lysis
5
X Reagent (Promega) and 0.01× sample volume NanoGlo
Luciferase Assay Substrate (Promega). Relative light units
References
(
RLU) emitted from the reaction were measured in a FLUOstar
Omega Luminometer (BMG Labtech) with the gain reduced by
0% for the sample with the highest signal to prevent satur-
1 World Malaria Report 2014. World Health Organization,
1
2014.
http://www.who.int/malaria/publications/world_
ation. Relative light units were plotted in Prism 6 (GraphPad)
and normalised against untreated and uninfected samples to
generate drug curves and IC50 values.
malaria_report_2014/en/ (accessed Dec 8, 2015).
2 D. Horn and M. T. Duraisingh, Annu. Rev. Pharmacol.
Toxicol., 2014, 54, 71–94.
3
D. Leroy, B. Campo, X. C. Ding, J. N. Burrows and
S. Cherbuin, Trends Parasitol., 2014, 30, 478–490.
Video microscopy
P. falciparum W2mef strain asexual blood stage parasites were
grown to the late schizont stage. They were diluted from 4% to
4 N. J. White, S. Pukrittayakamee, T. T. Hien, M. A. Faiz,
O. A. Mokuolu and A. M. Dondorp, Lancet, 2014, 383, 723–735.
5 M. E. Francia and B. Striepen, Nat. Rev. Microbiol., 2014,
12, 125–136.
6 A. F. Cowman and B. S. Crabb, Cell, 2006, 124, 755–766.
7 L. Solyakov, J. Halbert, M. M. Alam, J. P. Semblat, D. Dorin-
Semblat, L. Reininger, A. R. Bottrill, S. Mistry, A. Abdi,
C. Fennell, Z. Holland, C. Demarta, Y. Bouza, A. Sicard,
M. P. Nivez, S. Eschenlauer, T. Lama, D. C. Thomas,
P. Sharma, S. Agarwal, S. Kern, G. Pradel, M. Graciotti,
A. B. Tobin and C. Doerig, Nat. Commun., 2011, 2, 1–12.
0
.16% hematocrit in RPMI media to which the PfPKA inhibi-
tors had been added and were allow to settle on the bottom of
a 35 mm Fluorodish (World Precision Instruments). The
dishes were mounted in a humidified gas chamber (90% N2,
1
2 2
% O , and 5% CO ) at 37 °C on a Zeiss AxioObserver Z1 fluo-
rescence microscope and time-lapse videos were recorded with
an AxioCam MRm camera usually at 4 frames per second.
Human kinase screen
8
B. Hardy, R. Affentranger, A. Contini, H. Gutierrez de
Teran, J. Spitzner, R. Papoian, W. L. Seibel, S. Nelson,
J. Wiseman, S. D. Bryant, I. Lucet and C. Doerig, in Protein
Phosphorylation in Parasites Novel Targets for Antiparasitic
Intervention, ed. C. Doerig, G. Späth and M. Wiese, Wiley-
VCH Verlag, Germany, 2014, pp. 375–416.
Compound 25 was tested at 10 mM against a panel of available
human kinases with homologs in P. falciparum by MRC PPU
International Centre for Kinase Profiling (University of
Dundee, United Kingdom). The principal method utilised is a
3
3
54,55
radioactive filter binding assay using
P ATP.
The
enzyme–substrate mixture is incubated for 5 min at rt followed
3
3
9 B. Morahan and J. Garcia-Bustos, Mol. Biochem. Parasitol.,
014, 193, 23–32.
by the addition of P ATP to begin the reaction. The assay is
halted by orthophosphoric acid and the components harvested
onto a P81 filter plate then air-dried. Scintillation counting is
undertaken using a Topcount NXT. A mean percentage activity
with standard deviation for all duplicates is calculated.
2
10 A. Sudo, K. Kato, K. Kobayashi, Y. Tohya and H. Akashi,
Mol. Biochem. Parasitol., 2008, 160, 138–142.
1
1 C. Syin, D. Parzy, F. Traincard, I. Boccaccio, M. B. Joshi,
D. T. Lin, X.-M. Yang, K. Assemat, C. Doerig and
G. Langsley, Eur. J. Biochem., 2001, 268, 4842–4849.
2 A. Merckx, M. P. Nivez, G. Bouyer, P. Alano, G. Langsley,
K. Deitsch, S. Thomas, C. Doerig and S. Egee, PLoS Pathog.,
2008, 4, 1–11.
1
Acknowledgements
M.B. acknowledges the support of the CRC for Biomarker
Translation, funded by the Australian Government, in the pro- 13 G. M. Fimia and P. Sassone-Corsi, J. Cell Sci., 2001, 114,
vision of her postgraduate scholarship. K.H. is the recipient of 1971–1972.
an Australian Postgraduate Award from the University of Mel- 14 F. H. Beraldo, F. M. Almeida, A. M. da Silva and
bourne, Australia. The authors also wish to acknowledge the
C. R. Garcia, J. Cell Biol., 2005, 170, 551–557.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
1
5 N. Wurtz, J. Desplans and D. Parzy, Parasitol. Res., 2009, 39 M. F. Azevedo, C. Q. Nie, B. Elsworth, S. C. Charnaud,
1
05, 1691–1699.
P. R. Sanders, B. S. Crabb and P. R. Gilson, PLoS One, 2014,
9, 1–12.
1
6 E. Lasonder, J. L. Green, G. Camarda, H. Talabani,
A. A. Holder, G. Langsley and P. Alano, J. Proteome Res., 40 M. P. Hall, J. Unch, B. F. Binkowski, M. P. Valley,
2
012, 11, 5323–5337.
B. L. Butler, M. G. Wood, P. Otto, K. Zimmerman,
G. Vidugiris, T. Machleidt, M. B. Robers, H. A. Benink,
C. T. Eggers, M. R. Slater, P. L. Meisenheimer,
D. H. Klaubert, F. Fan, L. P. Encell and K. V. Wood, ACS
Chem. Biol., 2012, 7, 1848–1857.
1
7 K. Leykauf, M. Treeck, P. R. Gilson, T. Nebl, T. Braulke,
A. F. Cowman, T. W. Gilberger and B. S. Crabb, PLoS
Pathog., 2010, 6, 1–11.
8 K. L. Harvey, A. Yap, P. R. Gilson, A. F. Cowman and
B. S. Crabb, Int. J. Parasitol., 2014, 44, 853–857.
1
1
41 A. J. Crick, T. Tiffert, S. M. Shah, J. Kotar, V. L. Lew and
P. Cicuta, Biophys. J., 2013, 104, 997–1005.
9 T. Chijiwa, A. Mishima, M. Hagiwara, M. Sano, K. Hayashi,
T. Inoue, K. Naito, T. Toshioka and H. Hidaka, J. Biol. 42 M. Abkarian, G. Massiera, L. Berry, M. Roques and
Chem., 1990, 265, 5267–5272. C. Braun-Breton, Blood, 2011, 117, 4118–4124.
0 R. A. Engh, A. Girod, V. Kinzel, R. Huber and 43 J. A. Dvorak, L. H. Miller, W. C. Whitehouse and
D. Bossemeyer, J. Biol. Chem., 1996, 271, 26157–26164. T. Shiroishi, Science, 1975, 187, 748–750.
1 H. Kase, K. Iwahashi, S. Nakanishi, Y. Matsuda, K. Yamada, 44 P. R. Gilson and B. S. Crabb, Int. J. Parasitol., 2009, 39, 91–
2
2
M. Takahashi, C. Murakata, A. Sato and M. Kaneko,
Biochem. Biophys. Res. Commun., 1987, 142, 436–440.
2 A. J. Murray, Sci. Signaling, 2008, 1, 1–6.
96.
45 C. Agop-Nersesian, B. Naissant, F. Ben Rached,
M. Rauch, A. Kretzschmar, S. Thiberge, S. Menard,
D. J. P. Ferguson, M. Meissner and G. Langsley, PLoS
Pathog., 2009, 5, 1–15.
2
2
3 A. Lochner and J. A. Moolman, Cardiovasc. Drug Rev., 2006,
2
4, 261–274.
2
2
2
2
2
2
3
3
4 S. P. Davies, H. Reddy, M. Caivano and P. Cohen, Biochem. 46 R. Rangarajan, A. Beia, N. Henrya, M. Madametb,
J., 2000, 351, 95–105.
5 Z. X. Lu, N. H. Quazi, L. W. Deady and G. M. Polya, Biol.
Chem. Hoppe-Seyler, 1996, 377, 373–384.
6 F. L. Smith, R. R. Javed, P. A. Smith, W. L. Dewey and
B. H. Gabra, Pharmacol. Res., 2006, 54, 474–480.
7 J. M.-Y. Bobalova and N. Violeta, Eur. J. Pharmacol., 2006,
D. Parzyb, M.-P. Nivezc, C. Doerigc and A. Sultana, Exp.
Parasitol., 2006, 112, 202–207.
47 R. Graeser, B. Wernli, R. M. Franklin and B. Kappes, Mol.
Biochem. Parasitol., 1996, 82, 37–49.
48 E. Droucheaua, A. Primota, V. Thomasa, D. Matteib,
M. Knockaerta, C. Richardsonc, P. Sallicandrod, P. Alanod,
A. Jafarshade, B. Barattea, C. Kunickf, D. Parzyg, L. Pearlc,
C. Doerige and L. Meijer, Biochim. Biophys. Acta, Proteins
Proteomics, 2004, 1697, 181–196.
5
6, 128–132.
8 L. W. Deady and S. M. Devine, J. Heterocycl. Chem., 2004,
1, 549–555.
4
9 L. W. Deady and N. H. Quazi, Synth. Commun., 1995, 25, 49 L. Reininger, J. M. Wilkes, H. Bourgade, D. Miranda-Saave-
3
09–320.
0 L. W. Deady, P. M. Loria and N. H. Quazi, Aust. J. Chem.,
996, 49, 485–488.
dra and C. Doerig, Mol. Microbiol., 2011, 79, 205–
221.
50 W. L. F. Armarego and C. L. L. Chai, Purification of Labora-
tory Chemicals, Butterworth-Heinemann, Great Britain, 5th
edn, 2003.
1
1 C. E. Gutteridge, M. M. Hoffman, A. K. Bhattacharjee,
W. K. Milhous and L. Gerena, Bioorg. Med. Chem. Lett.,
2
011, 21, 786–789.
51 W. C. Still, M. Kahn and A. Mitra, J. Org. Chem., 1978, 43,
2923–2925.
52 S. Gabriel and A. Neumann, Ber. Dtsch. Chem. Ges., 1892,
25, 3563–3573.
3
3
3
3
2 M. Wolter, G. Nordmann, G. E. Job and S. L. Buchwald,
Org. Lett., 2002, 4, 973–976.
3 M. B. Reed, K. J. Saliba, S. R. Caruana, K. Kirk and
A. F. Cowman, Nature, 2000, 403, 906–909.
53 W. Trager and J. B. Jensen, Science, 1976, 193, 673–675.
4 A. B. Sidhu, D. Verdier-Pinard and D. A. Fidock, Science, 54 C. J. Hastie, H. J. McLauchlan and P. Cohen, Nat. Protoc.,
002, 298, 210–213. 2006, 1, 968–971.
5 T. E. Wellems, L. J. Panton, I. Y. Gluzman, V. E. do Rosario, 55 J. Bain, L. Plater, M. Elliott, N. Shpiro, C. J. Hastie,
2
R. W. Gwadz, A. Walker-Jonah and D. J. Krogstad, Nature,
990, 345, 253–255.
H. McLauchlan, I. Klevernic, J. S. Arthur, D. R. Alessi and
P. Cohen, Biochem. J., 2007, 408, 297–315.
1
3
3
3
6 M. T. Makler and D. J. Hinrichs, Am. J. Trop Med. Hyg., 56 M. J. Buskes, K. L. Harvey, B. Prinz, B. S. Crabb,
1
993, 48, 205–210.
P. R. Gilson, D. J. D. Wilson and B. M. Abbott, Bioorg.
Med. Chem., 2016, DOI: 10.1016/j.bmc.2016.03.048, in
press.
7 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney,
Adv. Drug Delivery Rev., 2001, 46, 3–26.
8 D. F. Veber, S. R. Johnson, H. Y. Cheng, B. R. Smith, 57 S. Agarwal, S. Kern, J. Halbert, J. M. Przyborski,
K. W. Ward and K. D. Kopple, J. Med. Chem., 2002, 45,
615–2623.
S. Baumeister, T. Dandekar, C. Doerig and G. Pradel, J. Cell
Biochem., 2011, 112, 1295–1310.
2
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.