Histone lysine methyltransferase structure activity relationships that allow for segregation of G9a inhibition and anti-Plasmodium activity

Article abstract of DOI:10.1039/c7md00052a

Plasmodium falciparum HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and represent exciting new anti-malarial epigenetic targets. Using an inhibitor series derived from the diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly promising antimalarial activity, including irreversible asexual cycle blood stage-independent cytotoxic activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will need to address host versus parasite selectivity, where inhibitory activity against human G9a is removed from the lead compounds, while maintaining potent anti-Plasmodium activity. Herein, we report an extensive study of the SAR of this series against both G9a and P. falciparum. We have identified key SAR features which demonstrate that high parasite vs. G9a selectivity can be achieved by selecting appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered that potent G9a inhibitors can be identified by employing a 6-carbon 'Nle mimic' at position 7. Together, this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences. Based on this, we believe this scaffold to have clear potential for development into a novel anti-malarial therapeutic.

Full text of DOI:10.1039/c7md00052a

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Chen, A. Lubin, G. Lueg, F. Lu, A. J. P. White, N. Malmquist, M. Vedadi, A. Scherf and M. Fuchter, Med.
Chem. Commun., 2017, DOI: 10.1039/C7MD00052A.
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Identification of diaminoquinazoline histone lysine methyltransferase
structure activity relationships that allow for segregation of human G9a
†
inhibition and anti-Plasmodium activity
a
b
a
a
c
Sandeep Sundriyal , Patty B. Chen , Alexandra S. Lubin , Gregor A. Lueg , Fengling Li , Andrew J. P.
a
b
c,d
b
a*
White , Nicholas A. Malmquist , Masoud Vedadi , Artur Scherf , Matthew J. Fuchter
a
Department of Chemistry, Imperial College London, London SW7 2AZ (UK)
b
Unité Biologie des Interactions HôteꢀParasite, Département de Parasites et Insectes Vecteurs, Institut
Pasteur, Paris 75015, (France)
b
CNRS ERL 9195, Paris 75015, France
b
INSERM Unit U1201, Paris 75015, France
c
Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7, Canada
d
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, M5S 1A8, Canada
CORRESPONDING AUTHOR
*
Eꢀmail: m.fuchter@imperial.ac.uk; Fax: +44 (0)2075945805; Tel: +44 (0)2075945815
†
The authors declare no competing interests.
1
ABBREVIATIONS
1
PfHKMT, Plasmodium histone lysine methyltransferase; ACTs, Artemisininꢀbased combination
therapies; PfHDAC, Plasmodium histone deacetylases; TPSA, total polar surface area; ICW, InꢀCell
Western assay; PRC2, polycomb repressive complex 2; ECSD, enzymeꢀcoupled SAH detection assay;
CLOT, chemiluminescenceꢀbased oxygen tunnelling assay
1

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ABSTRACT
Plasmodium HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and
represent exciting new antiꢀmalarial epigenetic targets. Using an inhibitor series derived from the
diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly
promising antimalarial activity, including irreversible asexual cycle blood stageꢀindependent cytotoxic
activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to
reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will
need to address host versus parasite selectivity; where inhibitory activity against human G9a is removed
from the lead compounds, while maintaining potent antiꢀPlasmodium activity. Herein, we report an
extensive study of the SAR of this series against both G9a and P. falciparum. We have identified key
SAR features which demonstrate that high parasite vs G9a selectivity can be achieved by selecting
appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered
that potent G9a inhibitors can be identified by employing a 6ꢀcarbon ‘Nle mimic’ at position 7. Together,
this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets
and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences.
Based on this, we believe this scaffold to have clear potential for development into a novel antiꢀmalarial
therapeutic.
KEYWORDS
Histone lysine methyltransferase, G9a, malaria, diaminoquinazoline, epigenetics, BIX01294
2

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INTRODUCTION
Despite a global reduction in malaria incidence and mortality rate over the past 15 years, this disease
1
remains a major health burden, with over 200 million new cases reported in 2015 . The majority of the
malaria related deaths occur in subꢀSaharan Africa, particularly amongst children under five and pregnant
women. The emergence of multiꢀdrug resistant strains of Plasmodium, the malaria causing parasite, have
2
, 3
rendered many of the conventional antimalarial drugs ineffective
. Artemisininꢀbased combination
4
therapies (ACTs) have, in part, addressed this issue and are widely used as an effective treatment .
5, 6
However, recent reports of artemisinin resistant Plasmodium strains along the ThaiꢀCambodian border
is of significant concern. Thus, there is an increasing demand for the discovery of novel classes of
antimalarial drugs, with distinct modes of action, in order to tackle multiꢀdrug resistant Plasmodium
7
parasites .
8,
Epigenetic regulation has been shown to affect gene expression throughout the life cycle of Plasmodium
9
and thus, modulation of epigenetic targets in the malarial parasite presents a novel approach for
antimalarial drug discovery. Indeed, inhibitors of malarial histone deacetylases (PfHDACs) have been
shown to possess parasite killing activity and are currently being explored as a new class of antimalarial
1
0ꢀ14
drugs
. However, the poor selectivity, unfavourable pharmacokinetics and toxicity issues associated
with the hydroxamic acidꢀbased HDAC inhibitors poses a major challenge to their clinical development
15
.
Thus other potential epigenetic targets in Plasmodium are of significant interest.
Histone lysine methyltransferases (HKMTs) act as vital components of epigenetic regulation by serving
as ‘writers’ that install methyl marks on histones and other proteins. Among various human HKMTs, G9a
(
EHMT2) is a wellꢀstudied enzyme, which catalyses the addition of one or two methyl groups to lysine 9
16
of histone H3 (H3K9me1and H3K9me2) . Like most of the other HKMTs, the active site of G9a resides
in the SET (suppressor of variegation 3−9, enhancer of zeste and trithorax) domain, where the substrate
peptide binds and is, in turn, methylated by an Sꢀadenosyl methionine (SAM) cofactor. G9a has been
shown to play key role in various physiological and pathophysiological processes such as mental health
3

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17
18, 19
20ꢀ22
,
cocaine addiction
, differentiation and cancer
and thus together with other HKMTs, it is under
2
3ꢀ26
investigation in context of drug discovery
.
Plasmodium HKMTs (PfHKMTs) play key role in controlling Plasmodium gene expression through
9
epigenetic pathways . Computational analysis predicts the presence of ten SET domain containing
2
7
PfHKMTs , six of which were found to be essential in the asexual bloodꢀstages of the parasite and thus
28, 29
may represent good drug targets
. Moreover, knockout of PfSET2 (now renamed PfSETvs) was found
to reverse the silencing of the var gene family, which is centrally involved in the immune evasion
8
, 28
mechanism by which Plasmodium avoids the host antibody response
. Despite this potential,
production of enzymatically active PfHKMTs has proved to be challenging, with only a few successful
29, 30
reports in the literature
, thus hindering the prospect of PfHKMT inhibitor discovery.
We have recently reported our initial attempts to validate the PfHKMTs as a novel approach for
31ꢀ34
antimalarial therapy
. In the absence of the full complement of purified essential PfHKMTs – required
for targetꢀbased hit discovery and SAR ꢀ we used a phenotypicallyꢀled approach; examining the activity
of an established HKMT chemotype for antimalarial activity. Specifically, a focused library of inhibitors
exemplifying the diaminoquinazoline HKMT chemotype was explored. Diaminoquinazoline HKMT
activity was initially identified through a high throughput screen, with BIX01294 (1, Fig. 1) identified as
35
an inhibitor of human G9a . While a number of medicinal chemistry studies ꢀ most notably those of Jin
and coꢀworkers ꢀ have been reported that improve the activity, selectivity, cell permeability and in vivo
36ꢀ41
activity of G9a probes derived from 1
, it is becoming increasingly apparent that the HKMT activity
of this chemotype is not limited to G9a. Indeed, by modifying the amino side chains of this scaffold,
4
2, 43
26
diaminoquinazoline inhibitors have been reported exhibiting human SETD8
Fig. 1). Given this broad HKMT activity, it would seem that ‘repurposing’ the diaminoquinazoline
scaffold as inhibitors of the homologous PfHKMTs (for a comparison of the homology of select P.
and EZH2 activity
(
3
1
falciparum SET domains to human proteins, see Table S1 in Malmquist et al ) is a valid approach to
progress these exciting new drug targets.
4

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Figure 1: Representative examples of G9a (1ꢀ6), SET8 (7) and dual G9a/EZH2 (8, 10) inhibitors.
In our initial studies, 1 (Fig. 1) and a related analogue TM2ꢀ115 (60, Table 3) were found to exhibit rapid
and irreversible asexual cycle blood stageꢀindependent cytotoxic activity at nM concentrations,
comparable potency against resistant strains (including artemisinin) and clinical isolates of P. falciparum
and P. vivax, and oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum
31, 32
infection
. Highly promising effects were also observed for other life cycle stages, with mature
3
2
gametocyte progression to gamete formation inhibited at submicromolar concentrations , and the
34
unprecedented ability to reactivate dormant liver stages (hypnozoites) in a novel in vitro model system .
A doseꢀdependent reduction in H3K4 methylation and, to a lesser extent, H3K9 methylation was observed
3
1
in parasites upon treatment (Western analysis), suggesting onꢀtarget PfHKMT activity , and that the
broad ranging effects of these compounds is likely due to their target. A preliminary SAR study on our
diaminoquinazoline series revealed that some pharmacophoric features might be conserved for both
parasiteꢀkilling and G9a inhibition, thereby suggesting potential similarities between G9a and the yet
3
3
unidentified PfHKMT target(s) responsible for the antiꢀparasitic activity . However, future development
of this series will need to address host versus parasite selectivity; where inhibitory activity against human
G9a is removed from the lead compounds, while maintaining potent antiꢀPlasmodium activity. Hence, we
5

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set out to identify regions around the scaffold that can be fineꢀtuned to improve the parasiteꢀkilling to G9a
inhibition ratio. Herein, we report an extensive study of the SAR of this series against both G9a and P.
falciparum. To provide a more complete picture of the underlying SAR, some of the analogues included
3
3
(
mainly in the ESI), and their antiꢀPlasmodium activities, were previously reported by us. However, G9a
inhibition for such analogues is reported here for the first time. Such cases are clearly marked in Tables 1ꢀ
. Important and previously unidentified trends are determined for activity against both targets and,
4
critically, we elucidate features of this scaffold that allow for high parasite versus host selectivity. We
believe this study further cements the potential of this scaffold as a candidate for development into greatly
needed novel therapies to control malaria.
CHEMISTRY
Molecules 8ꢀ59 (Table 1 and 2) were synthesized following a wellꢀestablished twoꢀstep synthetic
36ꢀ38, 41
procedure (Scheme 1)
. Hence, 6,7ꢀdimethoxyꢀ2,4ꢀdichloroquinazoline was treated with various
commercially available or synthesized (see Electronic Supplementary Information) Nꢀsubstitutedꢀ4ꢀ
piperidylamines, in order to obtain 4ꢀsubstituted quinazoline derivatives. These intermediates were
subsequently heated with a second amine nucleophile to access the desired diaminoquinazolines
analogues.
Scheme 1 Synthesis of diaminoquinazolines in Table 1 and 2. Reagents and conditions: (a) various
amines, Et N (or DIEA), THF (or DMF), RT, 18ꢀ24 h; (b) various amines (5ꢀ10 eqv), microwave, toluene
3
(
or neat), 130ꢀ185 °C, 30ꢀ50 min or iꢀPrOH, 4 M HCl/dioxane, microwave, 160 °C, 15 min.
6

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Scheme 2 Synthesis of diaminoquinazolines in Table 3. Reagents and conditions: (i) Benzyl bromide,
K CO , DMF, 0 °C – RT, 18 h; (ii) HNO , Ac O, RT, 18 h; (iii) Fe, NH Cl, iPrOH/H O, reflux, 18 h; (iv)
2
3
3
2
4
2
a) NaOCN, H O/AcOH, RT, 18 h; b) NaOH, MeOH, reflux, 6 h; (v) POCl , DIEA, acetonitrile, reflux, 6
2
3
h; (vi) various amines, Et N (or DIEA), THF (or DMF), RT, 18ꢀ24 h; (vii) various amines (5ꢀ10 eqv),
3
o
o
microwave, toluene (or neat), 130ꢀ185 C, 30ꢀ50 min or iꢀPrOH, 4 M HCl/dioxane, MW, 160 C, 15 min;
(
viii) TFA, reflux, 3 h; ix) K CO (or Cs CO ), DMF, alkyl halides, 3ꢀ18 h 80 °C or PPh , DIAD, THF, 20
2 3 2 3 3
h, RT
Analogues with benzyl or alkoxy substituents at position 7 (Table 3) were synthesized following a
36ꢀ38
previously described methodology
, with slight modifications, as shown in Scheme 2. The phenolic
oxygen of the commercially available 4ꢀhydroxyꢀ3ꢀmethoxybenzoate (92) was first benzylated to give 93.
Nitration of 93 gave nitro compound 94 which was further reduced to obtain aniline 95. Conversion of 95
into a urea intermediate, followed by baseꢀmediated ring closure yielded quinazolinedione 96 that was
subsequently heated with phosphorous oxychloride to obtain the 2,4ꢀdichloroquinazoline derivative 97.
7

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Subsequent displacement of the chloride atoms from position 4 and 2, analogously to that described in
Scheme 1, yielded final compounds 60ꢀ63 possessing 7ꢀOBn substituents. Alternatively, 98 was
debenzylated under acidic conditions to yield intermediates 99 possessing a free phenol group at position
7. The phenol oxygen was either alkylated using a selection of alkyl halides or else treated with primary
alcohols under Mitsunobu conditions to give substituted 2ꢀchloroquinazolines 100. Finally, substitution of
the chloride at position 2 of 100 with a second amine yielded the desired analogues 68ꢀ70 and 73ꢀ84. The
synthesis of analogues 64ꢀ67, 71, 72 (Table 3) has been reported earlier by Jin et al. using similar
36ꢀ38
synthetic scheme
.
For the synthesis of diaminoisoquinoline and diaminoquinoline analogues 85ꢀ91 (Table 4) a route
analogous to the synthesis of diaminoquinazolines was envisaged, where the desired amines could be
installed late stage. This strategy required the synthesis of the corresponding 1,3ꢀdichloroꢀ6,7ꢀ
dimethoxyisoquinoline (107) and 2,4ꢀdichloroꢀ6,7ꢀdimethoxyquinoline (110) intermediates, as shown in
Scheme 3 and 4, respectively.
Following a reported procedure, 5,6ꢀdimethoxyꢀ1ꢀindanone (101) was treated with butylnitrite under
4
4, 45
acidic conditions to yield ketoꢀoxime 102 (Scheme 3)
. Compound 102 was tosylated and ringꢀopened
4
6
in a one pot
procedure to give the benzoic acid derivative 103 that was esterified to obtain 104.
Subsequent, addition of methoxide to the nitrile moiety of 104, followed by cyclisation resulted in the
isoquinolinꢀ1ꢀone derivative 105 that was further hydrolysed under acidic conditions to obtain
dihydroisoquinolineꢀ1,3ꢀdione (106). Attempts to convert 106 to the key intermediate 1,3ꢀdichloroꢀ6,7ꢀ
dimethoxyisoquinoline (107) using widelyꢀused chlorinating reagents, such as phosphorous oxychloride,
phosphorous pentachloride or thionyl chloride, all failed with little/no conversion of the starting material.
4
7
Finally, the use of dichlorophenylphosphine oxide at high temperature was identified as a suitable
method to obtain 107 in good yield.
8

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Scheme 3: Synthesis of isoquinoline analouges in Table 5. Reagents and conditions: (i) butyl nitirite,
HCl (cat.), MeOH, 40 °C, 15 min; (ii) a) NaOH, 50 °C, TosCl b) 80 °C, 15 min; (iii) K CO , MeI, DMF,
2
3
2
h; (iv) NaH, MeOH (anhyd), 80 °C, 3 h; (v) 3 M HCl, MeOH, 100 °C, 1 h; (vi) PhP(O)Cl , 160 °C, 3 h,
2
sealed tube; (vii) 180 °C, 1,2ꢀdichlorobenzene, 1ꢀbenzylꢀ4ꢀpiperidylamine, MW, 2 h; (viii) (SPhos)
palladium(II) phenethylamine chloride, KꢀtOBu, various cyclic amines THF, 90 °C, 5ꢀ8 h.
It has been previously reported that conversion of 1,3ꢀdichloroisoquinoline (with no methoxy groups in
48
position 6 and 7) to diaminoisoquinolines is possible under thermal conditions, albeit in low yield
.
Notably, the first substitution reaction (with ammonia or a primary amine) was reported to occur at the
o
o
more reactive position 1 at 100 C, while temperatures up to 220 C were used for the second substitution
reaction (using a secondary amine) at position 3. However, in case of 107, which contains methoxy
groups in positions 6 and 7, a higher temperature was required for the first substitution reaction. Hence,
o
treatment of 107 with 1ꢀbenzylꢀ4ꢀpiperidylamine at 180 C resulted in the substituted isoquinoline
analogue 108. Prolonged reaction time or the use of higher temperatures resulted in significant
degradation of the starting material. Attempts to substitute position 3 of 108 with either ammonia or a
o
secondary amine at temperatures of up to 220 C proved fruitless, resulting in either no conversion, or
degradation of 108 into a complex an intractable mixture. Given this, a BuchwaldꢀHartwig CꢀN coupling
reaction was investigated as an alternative approach. While a comparable coupling reaction to mediate Cꢀ
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N bond formation at position 3 was not found in the literature, one example was identified which
described the coupling between an aromatic amine and position 1 of a 1ꢀchloroꢀ3ꢀaminoisoquinoline
4
6
using palladium acetate and BINAP under microwave irradiation . Based on the precedent of successful
4
9ꢀ51
coupling between heteroaromatic halides and cyclic secondary amines using PdꢀPEPPSIꢀiPr
, we
initially surveyed this catalyst for coupling of 108 and cyclic amines. However, low conversion and an
intractable mixture of components was obtained under the standard coupling conditions. Hence,
dialkylbiaryl phosphane based palladium catalysts were employed which are known for their high
5
2
53
54
stability and broad applicability especially for arylchloride substrates . The use of a SPhos based
palladium catalyst in particular resulted in excellent reaction between 108 and a variety of cyclic amines
(
Scheme 3). Through this method, sufficient quantities of analogues 85ꢀ87 were obtained for the current
study. 2D NMR (NOESY) of the final compounds was employed to confirm the regiochemistry of amine
substitution (see Electronic Supplementary Information).
For the synthesis of 6,7ꢀdimethoxyꢀ2,4ꢀdiaminoquinoline analogues (88ꢀ91) we modified our earlier
41
55, 56
adopted synthetic methodology , which contained a number of nonꢀoptimal synthetic steps
. Thus,
the key intermediate, dimethoxyꢀ2,4ꢀdichloroquinoline (110), was synthesized by heating 3,4ꢀ
dimethoxyaniline (109) and malonic acid with phosphoryl chloride, following a reported procedure
57
(
Scheme 4) . Compound 110 was subsequently heated with a variety of secondary amines to give the
desired 2ꢀsubstituted isomers 111a-c, together with minor amounts of the 4ꢀsubstituted regioisomer and
,4ꢀdisubstituted products. These minor sideꢀproducts were readily removed by column chromatography.
2
The conditions reported for this reaction in Scheme 4 were found to be optimum in order to obtain the
maximum yield of the desired isomers, often together with recovery of the unreacted starting material
110. The final structures of the regioisomers were confirmed by either 2D NMR (NOESY) and/or single
Xꢀray crystallography (for 111a, see Figure SF1 Electronic Supplementary Information). Finally, as was
developed for the isoquinolines, the 2ꢀsubstituted quinolines (111a-c) were subjected to a Buchwaldꢀ
Hartwig amination reaction, using either 1ꢀbenzylꢀ4ꢀpiperidylamine or 1ꢀmethylꢀ4ꢀpiperidylamine under
1
0

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palladium catalysis, in order to obtain the desired analogues 88ꢀ91. While the BuchwaldꢀHartwig
coupling steps in both Scheme 3 and 4 proved suitable to provide sufficient material for SAR analysis, we
note that the methods are not optimized to maximize product yield. Hence there is scope to screen other
catalysts, bases and solvents to further improve this synthetic methodology towards diaminoquinolines
and diaminoisoquinolines – scaffolds poorly represented in the literature.
Scheme 4: Synthesis of quinoline analogues in Table 4. Reagents and conditions: (i) malonic acid,
POCl , reflux, 3 h; (ii) THF, DIEA, various amines, 120 °C, MW, 48 h; (iii) PdꢀPEPPSIꢀiPr, LiꢀtOBu (1
3
M in THF), 1ꢀbenzylꢀ4ꢀpiperidylamine or 1ꢀmethylꢀ4ꢀpiperidylamine, THF, 100 °C, 18 h.
RESULTS AND DISCUSSION
For the library of compounds synthesised, we measured and compared parasite killing effects with G9a
inhibition. For select compounds, we also used a cellꢀviability counter screen to evaluate potential nonꢀ
specific host toxicity using a HepG2 hepatoma cell line. We divide our discussion into key regions of
SAR for this series.
Quinazoline SAR: Position 2
Initially, we explored the effect that the amine at position 2 had on parasiteꢀkilling and G9a inhibition,
when 1ꢀbenzylꢀ4ꢀpiperidylamine was present at position 4 (Table 1). Previously, we have shown that
decreasing the ring size at position 2, as in the case of compound 8, or removal of the basic nitrogen from
1
1

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Table 1: SAR at position 2
3
3
Nꢀmethyl homopiperazine (9 and 10) does not affect the parasiteꢀkilling activity . However, analogues 9
and 10, lacking a basic nitrogen group in the ring, were found to be 4ꢀ5 fold less potent against G9a
3
6
compared to their counterparts 1 and 2, which is in accordance with an earlier report . The protonated Nꢀ
methylhomopiperazine is known to foster a hydrogen bond (Hꢀbond) with the Asp1074 residue in the
substrate binding pocket of the G9a (Fig. 2) thereby contributing to overall binding strength. However,
this loss of binding contribution can be compensated by a ‘lysine mimic’ group at position 7, as
evidenced by the high reported potencies of analogues such as 2, which contains a cyclohexyl ring at
3
9
position 2 . A variety of substituted rings that lacked such a basic nitrogen (e.g. 9ꢀ16 and Table ST1
Electronic Supplementary Information,) exhibited poorer G9a inhibition than 1, whereas parasiteꢀkilling
activity was less effected. In particular, bulky cyclic amines at position 2, such as in 14ꢀ16, were found to
be suitable for maintaining high parasiteꢀkilling vs G9a inhibition, especially in the absence of a ‘lysine
38
mimic’ . Interestingly, substitution with a primary acyclic amine instead of a cyclic amine at position 2
(
17) resulted in poor potency against Plasmodium while maintaining good G9a inhibition. Together, these
results suggest that the substituent at position 2 can have a significant effect on both parasiteꢀkilling and
G9a inhibition. Importantly, removal of the basic centre from position 2, together with the use of bulky
substituents, can be used to improve the parasite to G9a inhibition ratio.
1
2

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Pf3D7
HepG2
IC50
G9a
R2
IC50
nM)
G9a/Pf3D7
HepG2/Pf3D7 clogP TPSA
*
IC (nM)
5
0
*
(
(nM)
1
67
a
43
1.6
5.6
4800
5500
3800
111.6
305.5
131
3.86 65.99
3.48 65.99
5.10 62.75
b
c
BIX01294
(110 /290 )
a
8
9
18
29
101
a
332
11.4
a
a
a
10
11
12
23
76
38
472
1326
576
20.5
17.4
15.2
4700
5400
201.7
71.0
4.71 62.75
4.96 62.75
4.34 71.98
10100
265.8
a
a
a
a
1
1
1
3
4
5
37
67
26
72
506
13.7
5900
3600
2600
6100
159.5
53.7
100
4.25 65.99
5.21 62.75
4.44 78.88
~10000
3190
~149.3
122.7
1
6
7
>10000
>138.9
84.7
6.11 62.75
4.09 74.78
1
>1000
33
123
ND
<0.1
ND
ND
ND
ND
ND
Pyrimethamine
1
3

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Chloroquine
8
ND
ND
ND
ND
a
b
c
33
Parasiteꢀkilling activity reported earlier
36, 37
IC reported using enzymeꢀcoupled SAH detection (ECSD) assay
5
0
36, 37
IC reported using chemiluminescenceꢀbased oxygen tunnelling (CLOT) assay
5
0
*
ND = not determined; IC determination experiments were performed either in duplicates or triplicates
50
Quinazoline SAR: Position 4
Next we examined the effect of the substituent at position 4 of the diaminoquinazoline scaffold in
conjunction with a variety of substituents at position 2. According to the crystal structure of 1 bound to
58
GLP , a close homologue of G9a, the benzyl moiety of the 1ꢀbenzylꢀ4ꢀpiperidylamine is solvent exposed
and thus, its removal or modification should not have considerable effect on G9a inhibitory activity. This
3
6, 37
fact is clearly apparent in previously described SAR studies of quinazoline G9a inhibitors
. Indeed,
analogues with methyl (18-20), fluoro (21-23) or methoxy (24) substitution on the phenyl ring of the 1ꢀ
benzylꢀ4ꢀpiperidylamine displayed good G9a IC values, ranging from 78ꢀ169 nM (Table 2). However,
5
0
once again the effect of a basic nitrogen within the substituent at position 2 was found to be key to potent
G9a inhibition, where analogues 25ꢀ29 ꢀ possessing a piperidine in place of Nꢀmethylpiperazine ꢀ
consistently exhibited lower potency against G9a (IC = 690 to 1214 nM). In contrast, all these analogues
5
0
displayed good to moderate (IC < 150 nM) parasiteꢀkilling activity, (except 24 and 28) irrespective of
50
the position 2 substituent. Together these results suggest that the benzyl group of 1ꢀbenzylꢀ4ꢀ
piperidylamine can be modified without losing significant activity against either G9a or Plasmodium, thus
providing a handle for tuning the physicochemical parameters of compounds for lead optimization.
Table 2 SAR at position 4
1
4

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Pf3D7 G9a
HepG2
ID
R4
R2
IC50
IC50 G9a/Pf3D7 IC50 HepG2/Pf3D7 clogP TPSA
*
*
(
nM) (nM)
(nM)
3100
1
1
8
9
74
69
116
78
1.6
1.1
41.9
47.8
3.78 65.99
3.78 65.99
3300
4100
2
0
80
169
2.1
51.2
3.78 65.99
2
2
1
2
NT
112
132
ꢀ
ND
ND
ND
ND
3.61 65.99
3.61 65.99
148
0.9
2
3
137
116
0.8
ND
ND
3.61 65.99
2
2
2
4
5
6
237
77
128
869
690
0.5
11.3
9.0
ND
ND
36.4
35.1
3.48 75.22
5.02 62.75
5.02 62.75
2800
2700
77
1
5

MedChemComm
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2
7
78
~1000
~12.8
2900
37.2
5.02 62.75
2
2
3
3
3
3
8
9
0
1
2
3
191
NT
1214
~1000
~1000
~1000
344
6.4
ꢀ
ND
ND
ND
ND
53.3
35.4
ND
67
4.85 62.75
4.85 62.75
3.48 65.99
4.71 62.75
3.09 65.99
4.32 62.75
a
197
178
330
~5.1
~5.6
1.0
8.8
10500
6300
ND
a
a
a
a
94
93
830
6300
3
3
4
5
~10000 ~107.5
5600
ND
60.2
ND
4.06 78.88
4.08 95.95
a
2
47 >10000
>40.5
a
3
69
ND
ND
4.35 79.82
3.55 79.82
3
3
6
7
>10000
>27.1
>9.3
a
107
>1000
17800
166.3
b
3
30 /
a
3
8
>300
ꢀ
ND
ND
2.30 65.99
c
230
1
6

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b
6
80 /
a
3
4
9
>300
ꢀ
ND
ND
1.90 65.99
3.14 62.75
c
2
00
a
0
1
2
37
591
16.0
14200
383.8
a
4
4
56
>1000
>17.9
ꢀ
5500
98.2
2.88 78.88
3.00 62.75
b
9
10 /
a
28
>10000
>357
c
6
500
b
1
100 /
a
4
4
4
4
4
3
4
5
6
7
34
ꢀ
>10000
ND
>294
ND
ND
ND
ND
2.22 62.75
4.25 65.99
3.86 65.99
5.09 62.75
4.72 71.98
c
9
00
174
130
144
47
10
25
0.06
0.19
2.0
ND
295
185
ND
3.9
ND
4
4
5
8
9
0
205
222
108
55
49
0.27
0.22
ꢀ
ND
ND
ND
ND
ND
ND
4.00 65.99
3.61 65.99
4.85 62.75
NT
1
7

MedChemComm
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5
5
5
5
1
2
3
4
470
55
0.12
ND
ND
ND
ND
ND
ND
ND
ND
3.07 65.99
2.68 65.99
3.55 71.98
3.92 62.75
280
126
71
123
NT
319
0.44
ꢀ
4.5
5
5
5
5
5
6
7
8
215
107
6438
3877
29.9
36.2
ND
ND
ND
ND
ND
ND
ND
ND
3.14 62.75
3.53 62.75
3.14 62.75
4.38 59.51
a
570 >10000
>17.5
>62.1
a
161 >10000
a
5
9
77
>10000 >129.9
12200
158.4
4.87 59.51
a
b
c
33
Parasiteꢀkilling activity reported earlier
36, 37
IC reported using enzymeꢀcoupled SAH detection (ECSD) assay
50
36, 37
IC reported using chemiluminescenceꢀbased oxygen tunnelling (CLOT) assay
50
*
ND = not determined; NT = not tested; IC determination experiments were performed either in duplicates
50
or triplicates
1
8

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Interestingly, analogues with 1ꢀbenzylꢀ3ꢀpiperidylamine (30, 31) or 1ꢀbenzylꢀ3ꢀpyrrolidinylamine (32ꢀ34)
at position 4, were less potent against G9a compared to compounds bearing a 1ꢀbenzylꢀ4ꢀpiperidylamine
in this position, for example 8 vs 30 and 32, 10 vs 31 and 33, and 15 vs 34 (see also Table ST1,
Electronic Supplementary Information). The protonated basic ring nitrogen of the piperidylamine moiety
3
7
is expected to form an Hꢀbond with the backbone carbonyl of Asp1078 in G9a (Fig. 2) . However, this
interaction is dependent on the conformation of the sixꢀmembered piperidylamine, which might present
3
7
39
the protonated nitrogen NꢀH either towards (PDB 3K5K) or away (PDB 3RJW) from Asp1078. In
analogues 30ꢀ34, the corresponding basic nitrogen is positioned differently within the rings and thus
likely disrupts the Hꢀbond with Asp1078. Such analogues have not been previously reported in the G9a
inhibitor literature and therefore present a new aspect of SAR for this series. However, it should be noted
5
9
that indolylamine based analogues such as 5 (Aꢀ366, Fig. 1) possess potent G9a activity despite the
absence of a basic centre in the corresponding position; suggesting that maintenance of an interaction
with Asp1078 is not critical for potent G9a inhibition per se, but can be compensated by other
interactions; particularly those provided by a ‘lysine mimic’. In general, 30ꢀ34 retained good (93 nM) to
moderate (330 nM) activity against Pf3D7, depending on the substituent at position 2. This suggests that
an equivalent Hꢀbond interaction is either maintained despite the different orientation in the PfHKMT
target(s) or is not required for activity. The importance of this basic centre was further explored by testing
analogues 35ꢀ37 with an acylated piperidylamine nitrogen. All such analogues were found to be
completely inactive against G9a, while a few (35ꢀ37, see also, Table ST1 Electronic Supplementary
Information) retained moderate parasiteꢀkilling activity, depending on the nature of the substituent at
position 2.
1
9

MedChemComm
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Figure 2: Twoꢀdimensional diagram depicting key interactions of UNC0224 (66) in G9a substrate
binding pocket (PDB ID 3K5K).
Replacing 1ꢀbenzylꢀ4ꢀpiperidylamine with 1ꢀmethylꢀ4ꢀpiperidylamine at position 4 in 1 or 8, thus
yielding 38 and 39 respectively, is known to retain G9a activity due to the solvent exposed nature of the
36, 37
terminal substituent group (vide supra)
. However, these compounds were both found to have low
Pf3D7 activity. Both 38 and 39 have a calculated topological surface area (TPSA) identical to 1, but have
a significantly lower clogP. Previously, diaminoquinazoline analogues possessing lower clogP values
were found to have poor G9a activity in the cell based assays, suggesting poor permeability across cell
38
membranes . In accordance with this, the poor parasiteꢀkilling activity exhibited by 38 and 39 may be
due to their limited cellular uptake, rather than associated to a targetꢀbased effect. Indeed, analogues 40
and 41, with increased clogP were found to recover the parasiteꢀkilling activity and displayed Pf3D7 IC50
values comparable to 1. Conversely however, analogues 40 and 41 were weak inhibitors of G9a, either
due to the absence of a basic centre (as in 40) or the presence of a bulky substituent (as in 41) at position
3
6, 37
2
(vide supra). Remarkably, 42 and 43, previously reported to be weak inhibitors of G9a
, displayed
excellent parasiteꢀkilling activity. To further investigate the effect of increased clogP on parasiteꢀkilling
2
0

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activity we synthesized analogues having cyclohexylmethyl (44ꢀ47) or cyclohexyl (48ꢀ50) Nꢀcapping
groups on the 4ꢀsubstituent. These lipophilic rings have previously been reported to improve the cellular
3
8
permeability of G9a inhibitors bearing a ‘lysine mimic’ at position 7 . However, the parasiteꢀkilling
activity of most of these analogues was less potent than 1, suggesting no direct correlation between clogP
and the antiꢀPlasmodium activity. Broadly, analogues lacking a basic nitrogen in the 2ꢀsubstituent ring
exhibited better potency against Plasmodium compared to the analogues containing Nꢀmethylpiperazine
(
45 and 49) and Nꢀmethylhomopiperazine (44 and 48) at this position. A similar trend in parasiteꢀkilling
activity was observed for molecules containing a 1ꢀisopropylꢀ4ꢀpiperidylamine, cyclohexylamine or
aniline at position 4 (51ꢀ59): Analogues bearing a piperidine derivative at position 2 (such as 53, 54, 58
and 59) exhibited better antiꢀPlasmodium activity than comparable compounds with a piperazine or
homopiperazine substituent in this position (e.g. 51, 52, 55 and 57). As observed earlier, the trend for the
G9a inhibition was opposite to the antiꢀPlasmodium activity in this regard: analogues with a piperazine or
homopiperazine in the position 2, such as 44, 45, 48, 49, 51 and 52, were found to be more potent against
G9a compared to those without such substituents (such as 46, 47, 54). Analogues having either a
cyclohexylamine (55 and 56) or aniline (57-59) at position 4 were found to be devoid of G9a inhibition,
while maintaining good to moderate (77ꢀ570 nM) parasiteꢀkilling activity, the precise potency of which
depended on the substituent at position 2 (see also Table ST1 in Electronic Supplementary Information).
This SAR can once again be justified through the necessity of a hydrogen bond with Asp 1078 for G9a
inhibition, but not for antiꢀparasitic activity. Substitution of other amines such as isopropylamine, 1ꢀ
methylꢀ3ꢀpyrrolidinylamine or tetrahydroꢀ2Hꢀpyranꢀ4ꢀylamine at position 4 (Table ST1, Electronic
Supplementary Information) resulted in poor antiꢀPlasmodium and G9a inhibition.
In summary, modification of the benzyl moiety of 1ꢀbenzylꢀ4ꢀpiperidylamine at position 4 or replacing it
with a range of other groups can be tolerated to retain both G9a and Pf3D7 activity, while other amine at
position 4 are detrimental to G9a activity. This is presumably due to the loss of the Hꢀbond with
Asp1078. Pf3D7 activity does not follow this trend however and many a large variety of amines at
2
1

MedChemComm
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position 4 are tolerated. In particular, an aromatic amine at this position can be used to achieve high
selectivity in favour of antiꢀPlasmodium activity.
We note that methylation of the 4ꢀamino group, or its substitution with an oxygen or sulphur, was found
to dramatically decrease the parasiteꢀkilling activity of this series (Table ST2, Electronic Supplementary
33
Information) . For G9a inhibition, such a structural alteration eliminates a hydrogen donor interaction
3
7
with Asp1083 (Fig. 2), and is thereby known to abolish G9a inhibition ; which was further confirmed by
the G9a inhibition data obtained by us (Table ST2, Electronic Supplementary Information). This
highlights the importance of a hydrogen bond donor at position 4 for both antiꢀPlasmodium and G9a
inhibition, thus making it an indispensable SAR feature for both targets. We note however that once
again, a suitable ‘lysine mimic’ can potentially compensate for this effect, in terms of G9a potency (cf 5)
59
.
Quinazoline SAR: Position 7
As already stated, installation of a ‘lysine mimic’ at position 7 has been found to be central to the
36ꢀ40, 59
development of potent G9a inhibitors
. Interestingly, analogues devoid of the methoxy groups at
position 6 and 7 were found to maintain moderate parasiteꢀkilling activity, but exhibited no G9a
41,
inhibition (Table ST3, Electronic Supplementary Information) as also reported earlier by us and others
60
.
This difference may indicate the inherent differences in the respective lysine binding channels of the
G9a and putative PfHKMT target(s), since the methoxy groups are known to occupy regions close to the
3
6, 37
lysine binding channel in G9a (vide infra)
reported 60 (Table 3), a positional isomer of 1, to be equally efficacious against Plasmodium
Interestingly, such ‘swapping’ of the terminal benzyl and methyl groups between positions 4 and 7 (1 vs
0) resulted in a complete loss of G9a activity. Analogues 61 and 62 also maintained this selectivity for
. Focusing specifically on position 7, our previous study
31, 32
.
6
the parasite, exhibiting virtually no G9a inhibition. However, analogue 63 with a primary acyclic amine at
position 2 lost activity against Plasmodium. This is in accordance with the similar effect observed above
2
2

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for analogue 17 (Table 1), more broadly suggesting a primary amine at position 2 to be deleterious to
Table 3: SAR at position 7
parasite activity.
2
3

MedChemComm
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Pf3D7
G9a
IC50
HepG2
R
R7
R2
IC50
G9a/Pf3D7 IC50 HepG2/Pf3D7 SlogP TPSA
(nM)
*
*
(
nM)
(nM)
6
0
a
ꢀ
Me
ꢀBn
ꢀBn
ꢀBn
ꢀBn
ꢀBn
ꢀBn
43
>1000
~10000
>50000
23.2
122
4700
2900
2900
110.1
35.4
50.9
3.86 65.99
5.05 65.99
6.28 62.75
TM2ꢀ115
6
6
1
2
82
57
877.2
6
6
3
4
ꢀMe
ꢀMe
ꢀMe
>1000 >1000
ꢀ
ꢀ
ꢀ
ND
ND
ND
ND
ND
ND
4.09 74.78
3.54 69.23
3.15 69.23
a
b
c
>2000 25 /20
a
b
6
6
5
6
>2000
8
a
b
c
ꢀMe
>2000 43 /57
ꢀ
ND
ND
2.62 69.23
(
UNC0224)
a
b
c
6
6
6
7
7
7
7
8
9
0
1
2
ꢀMe
ꢀiPr
ꢀBn
ꢀBn
ꢀMe
ꢀMe
>2000 110 /120
ꢀ
0.004
>0.005
0.018
ꢀ
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.23 69.23
3.93 69.23
4.19 69.23
3.80 69.23
3.09 75.22
4.10 65.99
1120
632
4
< 3
449
8
a
c
319
>10000
a
b
150
3400 /
ꢀ
2
4

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c
5200
73
74
75
76
77
78
79
80
ꢀBn
ꢀBn
ꢀBn
ꢀBn
ꢀBn
ꢀBn
ꢀBn
ꢀiPr
268
235
248
242
231
173
62
211
39
0.79
0.17
0.16
4.2
ND
ND
ND
ND
ND
ND
ND
ND
46.8
71.4
6.60 65.99
5.82 65.99
6.04 74.78
6.21 65.99
5.43 65.99
5.04 65.99
4.65 65.99
3.85 65.99
39
ND
1018
114
~1000
683
843
ND
0.5
ND
~5.8
11.0
15.0
ND
2900
4000
56
8
8
8
8
1
2
3
4
ꢀBn
ꢀiPr
ꢀBn
ꢀBn
61
37
5344
2034
223
87.6
55.0
1.4
1800
2400
ND
29.5
64.9
ND
4.89 65.99
4.10 65.99
4.60 65.99
4.99 65.99
162
241
575
2.4
ND
ND
a
b
c
33
Parasiteꢀkilling activity reported earlier
36, 37
IC reported using enzymeꢀcoupled SAH detection (ECSD) assay
5
0
36, 37
IC reported using chemiluminescenceꢀbased oxygen tunnelling (CLOT) assay
50
2
5

MedChemComm
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*
ND = not determined; IC determination experiments were performed either in duplicates or triplicates
50
Analogues with linear 7ꢀaminoalkoxy substituents (or ‘lysine mimics’) such as 64ꢀ67 (Table 3) exhibit
potent activity against G9a due to the additional interactions in the lysine binding channel of this enzyme
3
6ꢀ40, 59
(
Fig. 2)
. However, all these analogues were found to lack antiꢀPlasmodium activity as reported by
3
3
us previously (see also Table ST4, Electronic Supplementary Information) . We previously rationalized
the poor parasiteꢀkilling activities of these analogues as attributed to their low clogP and/or high TPSA
and hence poor cellular permeability. However, when considering such factors more broadly, it is
apparent that the calculated physicochemical parameters for 64 and 65 are comparable to 1. Indeed, both
these analogues have been reported to possess moderate G9a activity in cells as measured by the
reduction of H3K9me2 levels in an InꢀCell Western (ICW) assay, suggesting sufficient cellular
3
8
permeability of these compounds, at least under the assay concentrations employed . Hence, we
synthesized analogues 68ꢀ70 with a higher clogP while retaining the basic ‘lysine mimic’ group. All three
analogues exhibited potent G9a activity, as expected, but showed only slight improvement in parasiteꢀ
killing activity. In contrast, replacement of the basic ‘lysine mimic’ with less polar ether (71) or neutral
hydrocarbon (72) chain further improved the parasiteꢀkilling activity while it significantly reduced the
G9a potency of these analogues. Comparison of 72 and 69 is particularly interesting as both analogues
have very similar clogP and TPSA values, but the former is ~ 4 fold more potent against Plasmodium
3
7
while reported to be considerably less potent against G9a . Together, this data suggests that unlike G9a,
the lysine binding channel in the PfHKMT target(s) is better able to accommodate the hydrophobic
benzyl or hydrocarbon chains. Indeed, computational analysis of lysine binding channels of various
HKMTs suggests that they have diverse hotspot profiles that can be exploited for designing selective
61
inhibitors .
A number of recent reports suggest that for cases (in cancer) where the substrate histone lysine residues
are mutated to either methionine (Met) or a hydrocarbon based residue such as norleucine (Nle) (e.g.
2
6

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H3K9M or H3K27M), these mutant histones act as potent inhibitors of the respective HKMTs: such as
6
2ꢀ68
G9a inhibition by H3K9M or polycomb repressive complex 2 (PRC2) inhibition by H3K27M
. Very
recently Judge et al. have reported peptide inhibitors of SETD8 (a H4K20 methylase) by replacing the
lysine (K20) residue of the substrate peptide to Nle/Met (H4K20Nle/Met) and other hydrophobic residues
69
.
Indeed, coꢀcrystallized structures of such mutated peptide substrates with both human
PRC2/G9a/SETD8 show Met and Nle to occupy the lysine binding channel in a manner similar to lysine
67ꢀ69
in the nonꢀmutated substrates
. This data clearly suggests that ‘lysine mimic’ groups which interact
with the lysine binding channels of HKMTs need not be limited to polar and basic sidechains. Given this,
and given the fact that hydrocarbon chains were tolerated at position 7 for antiꢀparasite activity, analogues
containing ‘Met or Nle mimics’ at position 7 were investigated. However, poor inhibition of G9a by 72
suggested that the length and nature of the ‘Nle mimic’ in context of diaminoquinazoline inhibitory
scaffolds was not directly analogous to that in H3 peptides. Analogues 73ꢀ77 with a 6ꢀ8 carbon linear
‘
Nle mimic’ all displayed very similar moderate IC values (~231ꢀ268 nM) against the parasite. On the
50
other hand, analogues with the linear or branched ‘Nle mimic’ containing 4ꢀ5 carbons (78ꢀ82) showed
good parasiteꢀkilling activities, with 82 exhibiting an IC50 value (37 nM) comparable to 1. The G9a
inhibitory SAR was found to be different however: a linear chain of 6 carbons was found to be optimum
for G9a inhibition as evidenced by the low G9a IC50 values of 74, 75, and 77. Indeed, G9a inhibition
seemed to be intolerant of chain length variations, as increasing (73, 76) or decreasing (78ꢀ82) the chain
length led to a negative effect on inhibitor potency. To the best of our knowledge, analogues 74, 75 and
7
7 represent the first examples of nonꢀpeptidic small molecule G9a inhibitors possessing a hydrophobic
Nle mimic’ at position 7. Analogues 83 and 84 representing a ‘Met mimic’ showed only moderate
parasiteꢀkilling activities and poor G9a inhibition. This is in accordance with the data reported for peptide
‘
6
4
based inhibitors where Met mutation was found to be less effective than Nle mutation . However,
assessment of analogues possessing varying length ‘Met mimics’ and different position 2 substituents
would need to be tested in order to draw firm conclusions over the SAR surrounding ‘Met mimics’.
2
7

MedChemComm
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SAR for the central heterocyclic ring
Finally, we examined the role of the central fused aromatic scaffold. We have previously synthesized and
41
surveyed other fused heteroaromatic scaffolds, related to diaminoquinazolines, against G9a
.
Specifically, we examined cases where the fused benzenoid ring was replaced by a thiophene, furan,
imidazole or a cyclopentane ring (Table ST5, Electronic Supplementary Information) All these analogues
were found to be inactive against both G9a and Plasmodium suggesting both biological activities to be
restricted to the fused six membered ring scaffold. Next, we tested the importance of pyrimidine ring
nitrogens of the quinazoline scaffold, by comparing analogues based on diaminoquinolines and
diaminoisoquinolines (Table 4). The protonated Nꢀ1 nitrogen of quinazoline interacts strongly with
Asp1088 (Fig. 2) in the G9a pocket, while the Nꢀ3 nitrogen does not appear to form any specific
interaction. Accordingly, isoquinoline analogues (85-87) lacking a basic centre at position 1 displayed
poor activities against both G9a and the parasite while the quinoline compounds 88ꢀ91 were found to be
potent against both targets. In particular, 88 displayed ~5 fold increase in potency against G9a than the
4
1
parental analogue 1; a fact that we have rationalised previously . This data highlights this SAR feature
to clearly be conserved between G9a and the potential PfHKMT target(s) of this compound series.
Table 4: SAR of pyrimidine ring of quinazoline core
Pf3D7
G9a
IC50
HepG2
IC50
ID
R
X Y
R2
IC50
G9a/Pf3D7
HepG2/Pf3D7 SlogP TPSA
*
*
(
nM)
955
(nM)
(nM)
85
ꢀBn C N
NT
ꢀ
ND
ND
4.08
53.1
2
8

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DOI: 10.1039/C7MD00052A
8
6
7
ꢀBn C N
ꢀBn C N
1087 >50000
>46.00
>18.3
ND
ND
ND
ND
4.94 59.09
5.32 49.86
8
1367 >25000
8
8
9
ꢀBn N C
ꢀBn N C
97
46
13
21
0.13
0.46
5600
3400
57.7
73.9
4.47
4.08
53.1
53.1
8
9
0
1
ꢀBn N C
ꢀMe N C
49
119
30
2.4
3600
73.5
66.5
4.94 59.09
9
203
0.15
13500
2.51
53.1
*
ND = not determined; NT = not tested; IC determination experiments were performed either in duplicates
5
0
or triplicates
HepG2 cytotoxicity
Earlier, we screened several diaminoquinazoline analogues in a cellꢀviability assay to evaluate potential
3
3
host toxicity using a HepG2 hepatoma cell line . The HepG2 cell line is routinely used to measure
7
0ꢀ73
72
toxicity
and displays slightly more sensitivity compared to the other commonly used cell lines . Of
the select analogues examined in this assay, most display high antiꢀHepG2 IC values. No correlation is
5
0
observed between the antiꢀPlasmodium and antiꢀHepG2 activities. For example, analogues 12, 40, 42 and
3 exhibit potent parasiteꢀkilling activity but are amongst the least toxic compounds studied, with antiꢀ
4
7
4
HepG2 IC50 > 10 µM. While lipophility can give rise to nonꢀspecific cellular toxicity , we did not
observe any significant correlation between the cLogP and antiꢀHepG2 activity. Finally, no clear
correlation is apparent between G9a inhibition and antiꢀHepG2 activity. For example, BIX01294 (1) has a
2
9