Optimization of 2-Anilino 4-Amino Substituted Quinazolines into Potent Antimalarial Agents with Oral in Vivo Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b01673

Novel antimalarial therapeutics that target multiple stages of the parasite lifecycle are urgently required to tackle the emerging problem of resistance with current drugs. Here, we describe the optimization of the 2-anilino quinazoline class as antimalarial agents. The class, identified from publicly available antimalarial screening data, was optimized to generate lead compounds that possess potent antimalarial activity against P. falciparum parasites comparable to the known antimalarials, chloroquine and mefloquine. During the optimization process, we defined the functionality necessary for activity and improved in vitro metabolism and solubility. The resultant lead compounds possess potent activity against a multidrug resistant strain of P. falciparum and arrest parasites at the ring phase of the asexual stage and also gametocytogensis. Finally, we show that the lead compounds are orally efficacious in a 4 day murine model of malaria disease burden.

Full text of DOI:10.1021/acs.jmedchem.6b01673

Subscriber access provided by University of Newcastle, Australia
Article
Optimization of 2-anilino 4-amino substituted quinazolines
into potent antimalarial agents with oral in vivo activity
Paul R Gilson, Cyrus Tan, Kate E. Jarman, Kym N. Lowes, Joan M. Curtis, William Nguyen,
Adrian E. Di Rago, Hayley E. Bullen, Boris Prinz, Sandra Duffy, Jonathan B. Baell, Craig A Hutton,
Helene Jousset Sabroux, Brendan S. Crabb, Vicky M. Avery, Alan F. Cowman, and Brad E. Sleebs
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01673 • Publication Date (Web): 12 Jan 2017
Downloaded from http://pubs.acs.org on January 12, 2017
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Optimization of 2-Anilino 4-Amino Substituted
Quinazolines into Potent Antimalarial Agents with Oral in
Vivo Activity
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Paul R. Gilson, Cyrus Tan, Kate E. Jarman, Kym N. Lowes, Joan M. Curtis,
†
,‡
†
⊥,
⊥,
William Nguyen, Adrian E. Di Rago, Hayley E. Bullen, Boris Prinz, Sandra
§
€
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†,‡
Duffy, Jonathan B. Baell, Craig A. Hutton, Helene Jousset Subrox, Brendan S.
⊥,#, ‡
§
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†,‡,*
Crabb,
Vicky M. Avery, Alan F. Cowman, and Brad E. Sleebs.
†
The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia
Department of Medical Biology, The University of Melbourne, Parkville 3010, Australia
‡
€
¥
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia.
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of
Melbourne, Parkville, VIC 3010, Australia
§
Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland
4
111, Australia
⊥Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, 3004, Australia.
#
Monash University, Clayton, Victoria, 3800, Australia.
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*
Correspondence to:
Brad E. Sleebs
The Walter and Eliza Hall Institute of Medical Research
G Royal Parade, Parkville 3052, Victoria, Australia
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Phone: 61 3 9345 2718
Email: sleebs@wehi.edu.au
KEYWORDS
Malaria, Plasmodium, antimalarial, quinazoline.
ABSTRACT
Novel antimalarial therapeutics that target multiple stages of the parasite lifecycle are urgently
required to tackle the emerging problem of resistance with current drugs. Here we describe the
optimization of the 2ꢀanilino quinazoline class as antimalarial agents. The class, identified from
publicly available antimalarial screening data, was optimized to generate lead compounds that possess
potent antimalarial activity against P. falciparum parasites comparable to the known antimalarials,
chloroquine and mefloquine. During the optimization process we defined the functionality necessary
for activity and improved in vitro metabolism and solubility. The resultant lead compounds possess
potent activity against a multiꢀdrug resistant strain of P. falciparum and arrest parasites at the ring
phase of the asexual stage and also gametocytogensis. Finally, we show that the lead compounds are
orally efficacious in a 4 day murine model of malaria disease burden.
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INTRODUCTION
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Malaria is a lifeꢀthreatening disease caused by infection with protozoan parasites of the genus
Plasmodium. Each year Plasmodium parasites cause over two hundred million infections and over
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38,000 deaths, predominantly children. The two most lethal forms of malaria are caused by infection
with either P. falciparum, which is hyperꢀendemic in Africa and the most deadly parasite, or P. vivax,
responsible for recrudescent infection via activation of dormant liverꢀstage hypnozoites that reꢀ
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establish the clinical bloodꢀstage of infection.
Preventive interventions against malaria, such as bed nets, insecticide spraying, removal of stagnant
water from living areas and improved access to therapies has considerably reduced the global incidence
of malaria since the turn of the century; however, malaria is still an enormous problem throughout the
world, and particularly in Africa. While antimalarial agents, such as chloroquine, have been used
successfully to treat millions of malaria infections, the emergence and spread of chloroquine resistance
necessitated the introduction of combination therapies of mefloquine, atovaquone, and artemisinin
3
analogues. Despite this, resistance to artemisininꢀbased combination therapies has now been reported
4
along the ThaiꢀCambodian border and has potential to spread. The continual emergence of resistant
plasmodial strains of malaria highlights the urgent need for the development of new antimalarial
therapies against novel targets.
To combat the onset of resistance, the WHO has stipulated that new classes of drugs should possess
activity against multiple stages of the parasite’s lifecycle. To catalyse the identification of these agents,
Medicines for Malaria Venture (MMV) recently encouraged industry and academic medical research
institutes to conduct phenotypic high throughput screens (HTS) using their internal and proprietary
compound libraries to identify compounds that not only reduced asexual stage parasite viability but
also acted on liver and sexual blood stage parasites. Over the last 10 years several high throughput
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screens have been undertaken by various organisations and the data from these screens have been
made publicly available to the research community.
We used this resource to identify a small molecule starting point that demonstrated activity across
multiple stages of the parasites lifecycle. In identifying this starting point, we also took into
consideration other important attributes such as activity against parasite lines with resistance to known
antimalarials, cytotoxicity profile, chemical liabilities and synthetic tractability. Synthetic ease was not
only important with respect to medicinal chemistry, but also important when considering the WHO
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recommendation that the cost of one antimalarial treatment is less than USD$1. Taking these criteria
into consideration, a survey of the HTS datasets identified the 2ꢀanilino quinazoline as a promising
starting point.
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A subꢀstructure analysis across multiple screening data sets revealed several analogues possessing
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the 2ꢀanilino quinazoline scaffold, with examples also included as part of the MMV Malaria Box. A
summary of the number of hit analogues in each screening set and the activity of these analogues are
shown in Table S1. These analogues possess modest activity against asexual P. falciparum 3D7
parasites (EC ~0.1 to 2 ꢀM) and the multiꢀdrug resistant parasite lines, K1, DD2 and W2 (EC ~0.1
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to 2 ꢀM). Further, this class has demonstrated modest activity against P. falciparum NF54 early
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stage IꢀIII gametocytes (EC ~1.5 ꢀM) and examples that possessed P. yoelii liver stage schizonts
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(EC ~0.1 ꢀM to 4 ꢀM), while possessing a modest mammalian cell cytotoxicity window (EC >2
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50
ꢀM).
Structurally related scaffolds have also been reported to possess antimalarial activity. A 2,4ꢀ
unsubstituted diaminoquinazoline scaffold was reported to target Plasmodium dihydrofolate reductase
9
, 11
(
DHFR) and possess potent antimalarial activity (Figure 1).
More recently, Chibale et al. have
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described the optimization of the 2,4ꢀdiaminothienopyrimidine series and Sambandamurthy et al. on
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the development of a 2ꢀaminopyridyl pyrimidine scaffold that have yielded compounds with potent
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antimalarial activity. Interestingly, the human methyl transferase inhibitor, BIXꢀ01294, a 2,4ꢀ
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substituted diaminoquinazoline was also found to be a potent antimalarial compound (Figure 1). The
ꢀanilino quinazoline scaffold although structurally related to these other classes was sufficiently
structurally divergent to warrant further optimization. Only one other group 35 years ago has evaluated
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the 2ꢀanilino quinazoline scaffold for potential antimalarial activity. In this study they show several
analogues possess varying degrees of efficacy in a P. berghei mouse model of disease burden, but did
not present any in vitro data or physicochemical data to facilitate optimization of this class, which we
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have now readdressed in the study undertaken here.
Herein, we describe the optimization of the 2ꢀanilino quinazoline scaffold to generate a series of
compounds with potent activity primarily against the asexual stage of P. falciparum. Compounds with
these properties were then evaluated against P. falciparum gametocytes and multiꢀdrug resistant strains,
and were further assessed in asexual stage growth arrest and rate of kill assays. Physicochemical
parameters were also assessed in parallel and assisted in the design and selection of analogues for
further evaluation in a mouse model of malaria disease burden.
RESULTS AND DISCUSSION
The parasite activity of the analogues identified in the HT screening data sets, varied from one
research lab to another (Table S1). An attempt to build an early structure activity relationship (SAR)
proved challenging due to the variation in compound activity between data sets which was influenced
by different screening technologies, assay conditions and strain of parasite. Another challenge was that
the difference in functionality between analogues was highly varied and therefore difficult to
distinguish trends in early SAR. Thus, we initially sought to generate analogues inꢀhouse and acquire a
small set of closely related analogues to determine which functionality was important for activity
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against P. falciparum asexual blood stages. We then optimized the activity of these scaffolds
primarily based on P. falciparum asexual stage activity while monitoring mammalian cell cytotoxicity.
To test the activity of compounds against asexual stage parasites, we utilized a Plasmodium lactate
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dehydrogenase (pLDH) assay previously described. Briefly, human erythrocytes infected with ringꢀ
stage P. falciparum were treated with compounds or vehicle controls and incubated for 72 h. At the end
of this period, pLDH activity (monitored by the consumption of 3ꢀacetylpyridineꢀadenine dinucleotide
(
APADH), an NADH analogue) was determined as a measure of parasitemia. The human cellular
cytotoxicity of compounds was monitored by using a HepG2 growth inhibition assay using Cell Titreꢀ
Glo.
Chemistry
The synthetic route undertaken to generate the 2ꢀanilino 4ꢀsubstituted quinazolines involves two
generally high yielding S Ar reactions using inexpensive building blocks (Scheme 1). The first S Ar
N
N
reaction involves reaction of an amine that gives exclusively the 4ꢀsubstituted regioisomer in moderate
to high yields across a range of different substrates. The second S Ar reaction required the addition of
N
a strong acid to adequately activate the 2ꢀposition of the quinazoline for nucleophilic addition by the
aniline. Using these conditions and microwave irradiation the 2ꢀanilino substituted products were
obtained in acceptable yields.
Structure and activity relationship
We first investigated whether the substitution at the 4ꢀposition was important for activity, while
retaining an aniline substituent in the 2ꢀposition. It was observed (Table 1) that disubstitution of the 4ꢀ
nitrogen (examples 2, 3, and 8) was detrimental to activity (7.6 ꢀM, 1.8 ꢀM and 418 nM respectively)
compared to the monoꢀalkyl amino compounds 1 (124 nM) and 4 (329 nM). However, the compound
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possessing an Nꢀmethyl substituent at the 4ꢀposition (analogue 7) had comparable activity (124 nM) to
analogue 3 with a larger Nꢀ(3ꢀhydroxypropyl) substituent. This data suggested that the 4ꢀNH group was
required for binding to its cellular target, and that substitution on the 4ꢀamino group is likely oriented
towards solvent space. A number of screening hits (as seen in Table S1) possessed either a
furfurylamino or tetrahydrofurfurylamino group in the 4ꢀposition. To investigate whether these groups
in the 4ꢀposition were important for activity, several analogues were generated with this functionality.
As a general observation, these groups had no effect on activity (Table 1), and therefore could be used
interchangeably for SAR purposes.
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We next investigated the effect of substitution on the 2ꢀaniline moiety on the activity against
erythrocytic stage parasites, while maintaining either the furfurylamino and tetrahydrofurfurylamino
groups in the 4ꢀposition. It was shown that the metaꢀmethoxy compound 23 and related paraꢀmethoxy
compound 1 both possessed EC50 values of 230 nM and 124 nM respectively, however the orthoꢀ
methoxy analogue 24 possessed reduced activity (526 nM) (Table 1). This was further exemplified
with orthoꢀsubstituted dimethoxy analogues 25 and 26, which were observed to have weaker activity
(1.6 and 1.3 ꢀM) compared to the 3,4ꢀmethylenedioxy analogues 22 and 5 (169 and 114 nM
respectively). Larger polar substituents in the 4ꢀposition, such as an ester (17), 1ꢀhydroxyethyl (18),
amide (19 and 20), or an acetamide (21) were not well tolerated (574, 702, 691, 569, and 274 nM
respectively) compared to 1 (124 nM).
We noted that the methoxy analogues carried potential metabolic liabilities particularly in the 4ꢀ
position of the aniline. Metabolism studies (Table S2) identified that the 4ꢀmethoxy substituent on the
aniline is Oꢀdemethylated and subsequently oxidized to the quinone functionality that is potentially
susceptible to the addition of nucleophiles and a site for glucuronidation. To address this liability, we
then replaced the 4ꢀmethoxy substituent with methyl and halogen substituents. It was observed that the
4
ꢀhalogen analogues 10-12 (125, 112, and 113 nM respectively) and the 4ꢀmethyl analogue 9 (109 nM)
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all retained potency compared to the 4ꢀmethoxy analogue 1 (124 nM) (Table 1). Notably, the 4ꢀhalogen
analogues also maintained a selectivity window of approximately 50ꢀfold or greater with a slightly
better selectivity profile to the methoxy analogues 1 and 23. Consistent with the decrease in activity of
other ortho- substituted 2ꢀanilino analogues, the ortho-fluoroaniline (16) also had decreased
antimalarial activity (317 nM). However, analogues with either metaꢀchloro or metaꢀfluoro substitution
on the aniline (14 and 15 respectively) exhibited activity (134 and 144 nM) comparable to the paraꢀ
fluoro analogue 12 (112 nM).
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The furfurylamino and tetrahydrofurfurylamino groups present in many of the screening hits (Table S1)
and compounds detailed in Table 1, have been previously identified as liabilities in liver microsome
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studies.
The identification of metabolites from liver microsome studies corroborated this
observation (Table S2). In the first instance, to block this metabolic liability the 4ꢀfurfurylamino and 4ꢀ
tetrahydrofurfurylamino groups were replaced with a 4ꢀbenzylamino group. The activity (77 and 264
nM) of 4ꢀbenzylamino analogues 29 and 27 with either 4ꢀmethoxy or 4ꢀfluoro substitution on the 2ꢀ
anilino moiety (Table 2) demonstrated that the benzyl group was tolerated compared to their furfuryl
counterparts, 1 and 12 (124 and 112 nM). Furthermore, it suggested that the 4ꢀposition could be used as
a site at which to attach functionalities that could improve the overall physicochemical properties of the
scaffold, given that physicochemical analysis revealed that 27 possesses high cLogP (Table 2) and
limited aqueous solubility (Table 5).
The incorporation of the 4ꢀhalogen substitution on the aniline prevented Oꢀdemethylation, but
2
1
presented its own metabolic liability. Metabolism identification studies revealed that the 4ꢀhalogen
substituent (analogues 36 and 35) was susceptible to hydroꢀdehalogenation (Table S2). A known
method in literature to suppress hydro–dehalogenation from occurring is to incorporate a 3ꢀhalogen
2
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substituent next to the 4ꢀhalogen. Several analogues 31-33 with 3,4ꢀdihalogen substitution were
generated and possessed similar antimalarial activity (56 to 121 nM) compared to the 4ꢀfluoro analogue
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7 (77 nM) (Table 2). Notably, the 3ꢀchloroꢀ4ꢀfluoro analogue 32 was similarly potent (56 nM) to other
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diꢀhalogen analogues 31 and 33 (98 and 121 nM respectively). It was also observed that replacing the
aniline with 4ꢀtrifluoromethyl pyridinꢀ3ꢀyl amine (34) retained activity (102 nM) compared to the
trifluoromethylaniline orthologue 30 (105 nM). This results suggests that the aniline moiety can be
replaced with a pyridine and provides a future opportunity to overcome metabolism issues and adverse
toxicity of reactive metabolites associated with anilines. Data from the metabolite study undertaken on
compounds 53-56 (see below) corroborated that hydroꢀdehalogenation was suppressed by the 3,4ꢀ
dihalogen substitution. However, inserting a benzyl group in the 4ꢀposition and substituting halogens
on the 2ꢀaniline (compounds 29-33) to improve metabolism, impacted cLogP and in turn LipE values
were unsatisfactorily low (Table 2).
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To improve the LipE, solubility, and target selectivity of the series, we then incorporated a heteroatom
replacement into the 5, 7 or 8 positions of the quinazoline ring, and also replaced the quinazoline
system with similar heteroaromatic systems. Several analogues 37-42 were synthesized to evaluate
whether these changes would be tolerated with respect to asexual antimalarial activity. Analogues 37,
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8, 40-42 in this assay showed reduced potency (539 nM, 636 nM, 866 nM, 1.3 ꢀM and 7.7 ꢀM
respectively) compared to the comparator compound 29 (77 nM) (Table 3). Compound 39 was the
exception, possessing similar activity (57 nM) to 29 (77 nM). Compound 39 can form an
intramolecular hydrogen bond between the 5ꢀheteoatom and the 4ꢀNH group, potentially masking the
4ꢀNH from its binding partner and affecting the intrinsic lipophilicity. However, methylation of the 4ꢀN
position reduces antimalarial activity, and therefore it is unclear whether the intramolecular hydrogen
bond contributes to biological activity. Nevertheless, the pyridyl scaffold present in compound 39
offered an avenue to improve LipE and thus was further utilized later in the optimisation of the series.
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,7ꢀDimethoxy analogues 35 and 36 were also prepared to improve LipE, and this substitution
preserved activity (57 and 41 nM respectively) (Table 3) compared to 29 (77 nM) (Table 2). However,
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Oꢀdemethylation of both 6 and 7 methoxy groups was shown to be responsible for high intrinsic
clearance in liver microsome studies (Table S2). Therefore, this substitution was not pursued further.
In an ongoing effort to improve physicochemical properties of the series we next looked at replacing
the hydrophobic aliphatic substitution in the 4ꢀposition with polar substituents. This change was
orchestrated to more specifically reduce cLogP and improve solubility. From earlier SAR (Figure 2), it
was evident that the substitution in the 4ꢀposition was not important for modulating antimalarial
activity, suggesting that this moiety of the molecule not involved in binding to its cellular target, but
instead oriented towards solvent space. This provided an avenue to install groups known to assist in
improving solubility. To test this hypothesis, we generated several analogues 47-52 with polar
functionality in the 4ꢀposition while retaining the 4ꢀfluoroaniline in the 2ꢀposition and evaluated their
asexual antimalarial activity (Table 4). Compared to 29 (77 nM), 47, 49 and 50 had similar activity
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(104, 118 and 77 nM), while 48, 51 and 52 had a marginal improvement in activity (42, 64 and 51 nM).
Notably, 47-52 had an improved selectivity window of 250ꢀfold or greater and improved LipE
compared to the progenitor compound 29.
We then combined the functionality from the 2 and 4 positions that best modulated antimalarial
potency to generate analogues 53-56 that had a combination of 2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀethylamino
or 4ꢀaminoꢀ1ꢀmethylpiperidinyl groups in the 4ꢀposition and either 4ꢀchloroꢀ3ꢀfluoroanilino or 3ꢀ
chloroꢀ4ꢀfluoroanilino groups in the 2ꢀposition of the quinazoline (Table 4). The same substitution
patterns were also installed on the most promising heteroaryl quinazoline system, pyrido[3,2ꢀ
d]pyrimidine (derived from compound 29), to generate analogues 43-46 (Table 4). The evaluation of
these compounds, 43-46 and 53-56, against the asexual stage of P. falciparum parasites showed that the
pyrido[3,2ꢀd]pyrimidine analogues (43-46) all possessed EC values greater than 110 nM (Table 4).
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~250ꢀfold, whereas the 4ꢀaminoꢀ1ꢀmethylpiperidinyl analogues 55 and 56 ~150ꢀfold (Table 4). Given
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that 53-56 possessed the most potent activity against P. falciparum parasites in vitro, we further
assessed the physicochemical properties and parasite activity of these analogues.
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The solubility of analogues 53-56 at both pH 2 and pH 6.5, as measured by nephelometry, was
enhanced compared to compounds that did not possess 2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀethylamino or 4ꢀ
aminoꢀ1ꢀmethylpiperidinyl functionality in the 4ꢀposition (Table 5). The stability of 53-56 in the
presence of both human liver and mouse microsomes was also enhanced as shown by the improvement
in the hepatic extraction ratio and predicted intrinsic clearance values compared to the earlier analogue
27 (Table 5). Notably, an across species difference in the liver microsome study was observed, where
compounds were generally more susceptible to degradation in the presence of mouse liver microsomes
than human. Interestingly, the 4ꢀaminoꢀ1ꢀmethylpiperidinyl 55 and 56 were more stable than their 2ꢀ
(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀethylamino counterparts 53 and 54 in the presence of mouse liver
microsomes.
The major metabolites identified in the microsome studies for analogues 53-56 were Nꢀdemethylation
of the methylpiperidine or piperazine, and monoꢀoxygenation, presumably at the 6ꢀposition of the
quinazoline (Table S2). In these studies, a minor metabolite with a mass consistent with hydro
defluorination was also observed for the 3ꢀchloroꢀ4ꢀfluoro anilino analogues 53 and 55. It is known that
23
a 4ꢀfluorine substituent is more susceptible to hydro dehalogenation than a 4ꢀchloro, which is
consistent with the differences observed in this metabolism study between 53/55 and 54/56.
Nevertheless, the hydro defluorination was suppressed in the cases of the dihalo analogues 53-56
compared to that of monoꢀfluoro analogue 36 (Table S2). It is currently not known whether the
metabolites of compounds 53-56 possess antimalarial activity, so blocking these sites of metabolism
may not be beneficial for in vivo antimalarial activity. The blood plasma concentrations of the
metabolites are also unknown, and therefore will be investigated in the future development of the 2ꢀ
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anilino quinazoline series. Further efforts to block these metabolic events and improve on the
physicochemistry of the series, particularly concentrating LipE, will be the focus of future
optimization.
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Evaluation against a multi-drug resistant strain of P. falciparum
Several hit compounds from previous studies have been shown to be effective against different drugꢀ
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resistant strains of P. falciparum (Table S1). We evaluated a selection of compounds from our series
against the chloroquine and mefloquine resistant W2mef strain of P. falciparum using the LDH assay
format described above. This strain expresses a mutant PfCRT that confers chloroquine resistance, as
well as amplified levels of the digestive vacuole drug transport channel, Pꢀglycoprotein homologue 1
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(
Pgh1), encoded by the pfmdrl gene that confers resistance to mefloquine. From the data presented in
Table 6, W2mef parasites showed sensitivity towards the selected compounds that were similar to the
chloroquineꢀ and mefloquineꢀsensitive 3D7 parasite strain. Notably, compounds 53-56, that harbor
basic functionality with pKa values similar to the functionality found on mefloquine and chloroquine,
were active against the W2mef line, suggesting they are not substrates of PfCRT or the Pgh1 drug
transport channel.
Determination of the rate of kill and the stage of arrest in asexual stage P. falciparum parasites
To align the 2ꢀanilino quinazoline series with target product profile criteria suggested by world
1
health governing bodies, it is important to define the stage at which these compounds impact asexual
lifecycle progression, and also the rate at which this series arrests P. falciparum parasites in vitro
(Figure 3 and Figure 4). To determine the stage of arrest, synchronous ring (>95%) or trophozoite stage
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D7 parasites (>90%), were treated with compound at a concentration 10 times their EC . At 0, 24 and
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48 h, blood smears were prepared with Giemsa staining and the morphology and parasite form was
quantified. The data from this study is summarized in Figure 3 and shows that four compounds, namely
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3-56, generally arrest the majority of parasites at the ring stage of asexual stage development,
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commonly leading to parasite death. It was also observed that when compared to DMSOꢀtreated
controls, treatment with any one of compounds 53-56 at the ring stage, only one third of parasites
progressed to the trophozoite stage, and virtually none completed schizogony or advanced into the next
cell cycle. Conversely, a proportion of parasites that were treated with one of 53-56 at the trophozoite
stage were able to complete schizogony and enter the next lifecycle, but were then arrested at the next
ring stage. It is interesting to note that compounds 53-56 behave similarly to artemisinin, the current
gold standard antimalarial treatment, in that they also arrest parasite growth at the ring stage of the
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parasite's asexual lifecycle (Figure 3).
We next performed a compound washout experiment to confirm the stage of asexual arrest and rate of
action of 53 and 54, as 55 and 56 performed so similarly in the stage arrest experiments. In this study,
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synchronous P. falciparum 3D7 ring stage parasites expressing nanoluciferase were treated with a
dilution series of the compound (maximum concentration 500 times the EC ) at time point 0, and then
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the compound was washed out at 3, 6 and 24 h time points (Figure 4). All parasites were allowed to
grow for a total of 24 h and the parasite viability was subsequently quantified by measuring
bioluminescence. EC values were then calculated for each pulse point. The data presented in Figure 4
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demonstrates that the EC50 for 53 and 54 gradually declines after 6 h suggesting the compounds
progressively arrest parasite growth over 24 h in a concentration dependent manner. The fact the drugs
reduce ringꢀstage growth over the first several hours of treatment supports the findings in Figure 3.
Chloroquine acted over a similar timeframe, but chloroquine was significantly more potent at the
timepoints measured than 53 and 54. Whereas artemisinin appears to maximally reduce parasite growth
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between 3 h and 6 h, indicating that it acts more quickly and at lower concentrations than 53 and 54.
Lead compound evaluation against sexual stage P. falciparum gametocytes
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In the sexual stage of the parasites lifecycle, a small number of the asexual forms commit to the
sexual development of the parasite through a process termed gametocytogenesis. After commitment,
which occurs over a single interꢀerythrocytic replication cycle, ring stage parasites are present which
although not distinguishable from asexual blood stage parasites initially, continue through a
developmental differentiation pathway through five morphological recognizable stages (I to V)
ultimately culminating in the presence of both male and female gametocytes that can be transmitted
from the blood of the host to the mosquito during a blood meal. The ability for an antimalarial agent to
block transmission of the malaria parasite from the human host to the mosquito is critical in the
worldwide effort to prevent malaria transmission. World governing bodies and MMV have suggested
several target product profiles that encompass agents that kill gametocytes and could potentially act as
transmission blocking agents.
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To determine if the lead compounds 53-56 from the 2ꢀanilino quinazoline series were suitable to fit
this profile, these compounds were evaluated for their ability to prevent gametocyte development and
reduce viability at three stages of gametocyte development, ring stage, early stage (IꢀIII) or late stage
(
IVꢀV). A previously described high content imaging assay using a highly synchronous culture of P.
ꢀpfs16ꢀLUCꢀGFP
falciparum NF54
parasites that have been treated with Nꢀacetyl glucosamine at day 0 of
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gametocyte development to prevent asexual growth and replication was employed. Compound effects
on gametocyte development and viability after 72 h incubation were determined using automated
image analysis of confocal microscopic images whereby GFP was used to define gametocyte
morphology and MitoTracker Red (MTR CMꢀH2XRos) was sued to define parasite viability.
The results of this study demonstrate that compounds 53-56 are most potent against ring stage
gametocytes (179 ꢀ 223 nM), and less so against early stage gametocytes (326 ꢀ 554 nM) and weakly
active against late stage gametocytes (>2 ꢀM) (Table 7). A comparison of this data with the asexual
stage data shows that parasites arrest at both the early stages of asexual and sexual stage development.
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These compounds are also 10ꢀfold less potent against ring stage gametocytes and 15 to 25ꢀfold less
potent against early stage gametocytes compared with activity against early asexual stage parasites (26
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35 nM). Some reasons postulated for this difference are that the cellular target(s) of 53-56 are not
expressed or are not as essential for gametocytogenesis as they are for the asexual blood stage. Another
hypothesis is that 53-56 acts on two or more cellular targets essential for asexual stage progression, but
only have modest affinity for a target(s) that is only expressed during gametocyte development. To find
a more definitive reason for these differences in stage specific potency, we are now attempting to
identify the cellular target of the 2ꢀanilino quinazoline series.
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Evaluation of compounds in a mouse model of malaria disease burden
Overall the physical properties installed in analogues 53-56, along with their potent asexual
antimalarial activity, suggested they were suitably placed for evaluation in a mouse model of malaria
disease burden. A Peters 4 day mouse model was employed to determine the primary in vivo
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antimalarial activity of compounds 53-56. In this model, mice were infected with P. berghei ANKA
parasites on day 0. Compounds were then administered either by intraperitoneal injection (i.p.) or oral
gavage (p.o.) at 20 mg/kg, 4 hours after infection and then on day 1, 2 and 3. On the fourth day blood
smears were taken and parasitemia evaluated. A summary of these studies is shown in Figure 5 (panels
A and B).
The mouse blood plasma exposure levels of compounds 53-56 in this mouse model were also
monitored at 2, 5 and 20 h after administration of treatment at day 0. The results for each compound
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3-56 are shown in Figure 5 (panels C and D) and Table S3. The blood plasma exposure levels
observed with compounds 53-56 in this model by either i.p. or p.o administration over 20 h were
significantly higher than the concentrations required to suppress parasite growth in vitro, consistent
with the in vivo efficacy observed.
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In the Peter’s 4ꢀday mouse model, compounds that possess a 4ꢀamino ethyl piperazine substituent,
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3 and 54, reduced parasitemia by >98% when administered intraperitoneally (Figure 5A) and by
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90% when administered orally (Figure 5B). Compounds 53 and 54 reduced parasitemia to a similar
extent, even though 53 had a greater blood plasma exposure than 54 when dosing either by i.p. or p.o.
The mean levels of parasitemia in mice treated intraperitoneally with compounds that possess the 4ꢀ
amino piperidine, 55 and 56, were higher than 53 and 54 (Figure 5A). A likely explanation for this
trend is that compounds 55 and 56 had lower blood plasma exposure levels in comparison to 53 and 54
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(
Figure 5C). When dosed by oral gavage, compound 55 reduced parasitemia to similar levels to 53 and
54, in contrast the mean parasitemia value for compound 56 was higher (Figure 5B). Reflecting this
result, compound 56 demonstrated the lowest blood plasma exposure level in comparison to
compounds 53-55 (Figure 5D). In summary of the mouse model, the structural differences versus the
performance between compounds 53-56 could not be definitively concluded (dosing either i.p. or p.o.)
because the statistical test between compounds was not significant. However, the data between the
vehicle control and compounds 53-56, was statistically significant. Collectively this preliminary in vivo
data shows that the lead compounds in the anilino quinazoline series are orally efficacious, but require
further optimization for complete suppression of parasitemia at lower doses of compound.
CONCLUSIONS
In this report, we have described the optimization of the 2ꢀanilino quinazoline series to generate lead
compounds that have potent antimalarial activity against asexual stage parasites. Furthermore, these
compounds are shown here to have comparable in vitro activity to chloroquine and mefloquine. The 2ꢀ
anilino quinazoline series also has a similar activity profile against the W2mef multiꢀdrug resistant
parasite line compared to 3D7 P. falciparum parasites. The lead compounds, however, are 10ꢀfold
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weaker against ring stage gametocytes, and less potent against early stage and late stage gametocytes.
The 2ꢀanilino quinazoline series was shown to be moderately fast acting and exerts its greatest effect at
the ring phase in the asexual stage and at ring stage of gametocytogenesis.
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The remaining challenges for the next phase of optimization of this series will include monitoring of
cardiotoxicity risk (hERG) and other mammalian targets that are commonly associated with adverse
safety risks; particularly given the lipophilicity and basic functionality the series currently possesses
and that this scaffold is often considered as promiscuous. To identify potential offꢀtarget promiscuity, a
subꢀstructure search of literature was performed and revealed several compound series that possess the
2
8ꢀ30
4ꢀamino 2ꢀanilinoquinazoline substructure that inhibit, with varying potencies, human kinases,
31ꢀ33
34ꢀ36
ATPases
and Gꢀprotein coupled receptors.
As a consequence, offꢀtarget activity, particularly
those associated with safety risks, will be monitored in the future development of this compound class.
The potential metabolic liability and toxicity associated with the 2ꢀaniline moiety also needs to be
addressed. It is foreseen that this liability will likely be tackled by replacing the 2ꢀanilino group with
pyridyl functionality, given that the 2ꢀpyridyl analogue 34 was shown to have equivalent potency and
improved cLogP compared to the parent 2ꢀaniline compound 30. Improving the LipE of the series is
also a priority. Inserting heteroatoms into the scaffold, such as that seen in 34, along with substituting
different polar aliphatic functionality in the 4ꢀposition, as seen in 51, is an obvious avenue to improve
LipE in further development of the 2ꢀanilino quinazoline series. Although the physicochemical
properties, namely solubility and metabolism, of the lead compounds 53 and 54, are superior to the
screening starting points, additional effort is required to optimize these properties.
In closing, we have optimized a set of publicly available screening hits to compounds that are able to
arrest the growth and development of asexual stage parasites, with comparable activity to chloroquine,
and can suppress 99.8% and 95% of parasitemia in a mouse model when dosed at 20 mg/kg
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intraperitoneally and orally and respectively. It is foreseen that with further optimization, this
chemotype will join the current arsenal of new agents progressing towards and through the clinic to
assist in treating and eliminating this devastating disease.
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EXPERIMENTAL SECTION
Asexual Stage Parasite Viability Assay. P. falciparum 3D7 and W2mef parasites were cultured
37
according to the procedure described by Jensen et al. in RPMIꢀHEPES media supplemented with Lꢀ
glutamine and Albumax II. 100 µL of P. falciparum ringꢀstage cultures were seeded into 96ꢀwell
microplates at 0.3% parasitemia and 2% hematocrit. Compounds were serially diluted in DMSO at the
appropriate working concentration and were added to cultures with a final concentration of 0.2%
DMSO. The growth assays were performed in triplicate for 1.5 cell cycles (37°C, 72 h). The cultures
18
were then lysed by a freezeꢀthaw cycle and 30 µL was mixed with 75 µL od modified Malstat reagent
(0.1 M Tris pH 8.5, 0.2 g/mL lactic acid, 0.2% v/v Triton Xꢀ100 and 1 mg/mL acetylpyridine adenine
dinucleotide), 0.01 mg/mL phenazine ethosulfate and 0.2 mg/mL nitro blue tetrazolium. 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 using a 4 parameter log dose, nonꢀlinear regression
analysis, with sigmoidal dose response (variable slope) curve fit in GraphPad Prism (ver 6.05) and
normalized against untreated and uninfected samples to generate drug curves and EC50 values.
Chloroquine (EC of 23 nM for 3D7 and an EC of 250 nM for W2mef) and Artemisinin (EC of 8
5
0
50
50
nM for 3D7 and an EC of 6 nM for W2mef) were used as control compounds.
5
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HepG2 Viability Assay. HepG2 cells were cultured in Dulbeccos modified eagles medium (DMEM)
supplemented with 10% fetal calf serum (FCS), in a humidified incubator at 37˚C and 5% CO . Ten
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point compound titration assays were performed by treating cells (1 x 10 ) for 48 h in 384 well tissue
culture treated plates (Greiner). Cytotoxicity was determined using Cell Titer Glo (Promega) and
calculated as a percentage using DMSO as a positive growth control and 10 µM Bortezomib as a
negative growth control. EC50 values were calculated using a 4 parameter log dose, nonꢀlinear
regression analysis, with sigmoidal dose response (variable slope) curve fit using Graph Pad Prism (ver
6.05). 0 and 100 constraint parameters were used for curve fitting. Etoposide was used as a control
compound and was determined to have an EC of 15.9 ꢀΜ, compared to the literature value EC of
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30.2 ꢀΜ that had an incubation time of 48 h using an MTT assay to determine cell viability. For a
comparison, the following known antimalarials were evaluated in the HepG2 viability assay;
chloroquine EC >40 ꢀM; artemisinin EC >40 ꢀM; atovaquone EC 23.2 ꢀM; mefloquine EC
50
50
50
50
1
1.6 ꢀM.
2
6
Rate and Stage of Asexual Parasite Arrest Study. 3D7 parasites ± nanoluciferase were
maintained as synchronized cultures by frequent sorbitol treatment of ring stages. Prior to commencing
experiments, Giemsaꢀstained smears the parasites were counted to ensure the stage required was over
9
0% pure. To determine stage of arrest, following synchronization (rings) or 24 h later (trophozoites),
parasites were treated with 10 times EC of each compound. Parasites were smeared immediately prior
50
to addition of compound (time point 0) and again after 24 and 48 h and subsequently fixed in methanol
30 sec) and stained by incubation with 10% Giemsa solution (5 min). 1000 cells were counted per
(
treatment, per time point for three independent biological replicates.
To determine the rate of parasite arrest, sorbitol synchronized parasites were diluted to 1%
hematocrit and 100 µL was added to the wells of a 96ꢀwell plate. Compounds were added in a twoꢀfold
dilution series in DMSO with the highest concentration being 500 times the EC of each compound
50
and final DMSO concentration at 0.2%. After specific periods of drug incubation (3, 6 or 24 h), treated
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cells were washed four times by removing 80 µL of media and replacing it with 200 µl fresh media.
Following washing, parasites were incubated at 37˚C for the remainder of the 24 h growth period. To
measure parasite viability, parasites were reꢀsuspended and 10 µL of each well was added to a 96ꢀwell
Costar luminescence plate. Cells were subsequently lysed by addition of 90 µL lysis buffer (10 mM tris
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phosphoric acid, 5 mM Ka EDTA, 0.2% NP40, 5 mM DTT). To measure bioluminescence, 5 µL of
2
NanoGloꢀcontaining buffer (20 µL NanoGlo substrate per ml of buffer (10 mM tris phosphoric acid,
1
32 mM NaCl, 5 mM Ka EDTA, 5 mM DTT, 0.5 mM TrisꢀHCl, pH 7.5)) was injected into each well,
2
prior to shaking (700 pm/30 sec). Relative light units were measured with a ClARIOstar multimode
plate reader (BMG Labtech) and data was subsequently analyzed using GraphPad PRISM software.
Sexual Stage High Content Imaging Assay. Sexual stage activity of compounds was evaluated
5
ꢀpfs16ꢀLUCꢀGFP
according to the method of Duffy et al. Briefly, NF54
transgenic parasites expressing GFP
linked to luciferase under the control of the early gametocyte specific pfs16 promoter, Pfs16 were
maintained in asexual culture. At day ꢀ3 and ꢀ2 of the induction of gametocytogenesis, magnet purified
3
9
trophozoites were placed under nutritional stress in order to induce gametocytogenesis. At day 0 of
gametocyte development, Nꢀacetyl glucosamine (NAG) (50 mM) was added to the parasite culture to
prevent asexual parasite replication. Gametocytes were then obtained after continuous culture at the
39
appropriate time points, ring stage (day 0), early stage (day 2) and late stage (day 8). Titrated
compound diluted in 4% DMSO was transferred into 384ꢀwell imaging plates. The independent
gametocyte cultures at the appropriate stage were dispensed into the compound containing imaging
plates which were then sealed and incubated for 72 h in standard incubation conditions of 5% CO , 5%
2
O and 60% humidity at 37°C. After 72 h incubation, 5 µL of MitoTracker Red CMH2XRos (MTR) in
2
phosphate buffered saline (PBS) was added to each well and plates were resealed with membranes and
incubated overnight. The following day image acquisition and analysis was undertaken on the Opera
QEHS microꢀplate confocal imaging system. Single images were taken for each well and the GFP
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intensity was measured with an exposure time of 400 msec (488 nm), then to ascertain the fluorescence
intensity of the viability stain, MTR, measurements were made for 600 msec (532 nm). All images, for
each well, were analyzed using an Acapella based script which relates the MTR fluorescent signal and
the GFP designated object quantifying viable stage dependent parasite morphology. Gametocyte
viability after a 72 h incubation was calculated as a percentage of the positive (5 µM puromycin) and
negative (0.4% DMSO) controls contained in each assay plate. EC values were calculated using a 4
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parameter log dose, nonꢀlinear regression analysis, with sigmoidal dose response (variable slope) curve
fit using GraphPad Prism (ver 4.0). No constraints were used in the curve fit. Chloroquine, Artesunate,
Pyronaridine were used as control compounds and their EC values are indicated in Table 7 and are
5
0
5
directly comparable to EC values cited in Duffy et al.
50
Solubility Determination using Nephelometry. Compound in DMSO was spiked into either pH
6
.5 phosphate buffer or 0.01 M HCl (approx pH 2.0) with the final DMSO concentration being 1%.
4
0
Samples were then analyzed via nephelometry to determine a solubility range.
In Vitro Metabolism using Mouse and Human Liver Microsomes. Metabolic stability was
o
assessed by incubating test compounds individually (1 ꢁM) at 37 C with either mouse or human liver
microsomes. The metabolic reaction was initiated by the addition of an NADPH regenerating system
and quenched at various time points over the incubation period by the addition of acetonitrile. The
relative loss of parent compound and formation of metabolic products was monitored by LCꢀMS. Test
compound concentration versus time data was fitted to an exponential decay function to determine the
firstꢀorder rate constant for substrate depletion. In cases where clear deviation from firstꢀorder kinetics
was evident, only the initial linear portion of the profile was utilized to determine the degradation rate
constant (k). Each substrate depletion rate constant was then used to calculate an in vitro intrinsic
clearance value (CLint, in vitro) according to the equation, CL , in vitro = k/microsomal protein
int
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content (0.4 mg protein/mL); t1/2 = ln(2)/k; E = CL /Q + CL . The scaling parameters determined
H
int
int
42
in literature were used in the aforementioned calculations.
2
7
Mouse Model of Malaria Disease Burden. The method by Fidock et al. was followed to
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undertake the Peter’s 4 day mouse model to evaluate the ability of compounds to suppress malaria
7
infection. Briefly, male Swiss black mice were infected with P. berghei parasites (2 x 10 parasites) by
tail vein injection. For oral administration, an aqueous vehicle containing 0.5% (wt/vol)
hydroxypropylꢀmethylcellulose (HPMC), 0.4% (vol/vol) Tween 80, and 0.5% (vol/vol) benzyl alcohol
(HPMCꢀSV) was used. For intraperitoneal administration, a formulation consisting of a 10%
DMSO/90% 5% Solutol HSꢀ15 in 0.9% saline vehicle was used. Compounds 53-56 were administered
at 20 mg/kg. Chloroquine administered at 10 mg/kg was used as a positive control. Formulated drugs
and the vehicle control were then injected either by i.p. or p.o. four hours after infection (day 0) and
then on day 1, 2 and 3. On the fourth day blood smears were taken and parasitemia evaluated.
Microscopic counts of blood films from each mouse were exported into GraphPad Prism (ver 6.05) and
expressed as percentages of parasitemia.
Bioanalysis of blood plasma samples from mouse model. To determine blood plasma exposure of
compounds, plasma samples were taken from the compound treated parasite infected mice at 2, 5 and
2
0 h post administration either by i.p. or p.o. Blood samples were collected into EDTA containing
tubes then snap frozen. Samples were thawed and spun down at 13,000 rpm for 10 min and the
supernatant removed and used for analysis. Plasma standards were freshly prepared from a stock
solution (DMSO) for each analytical run with each set of standards comprising at least seven different
analyte concentrations. Plasma standards were then prepared by spiking blank mouse plasma (50 ꢁL)
with solution standards (10 ꢁL) and the internal standard (IS) diazepam (10 ꢁL, 6.25 ꢁg/mL in 50%
acetonitrile/water). Plasma samples from the study were similarly prepared except that blank
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acetonitrile (10 ꢁL) was added instead of the solution standard. Protein precipitation for plasma
standards and samples was carried out by the addition of acetonitrile (120 ꢁL), vortexing and
centrifugation (10,000 rpm) for 3 minutes in a microcentrifuge. The supernatant was subsequently
separated and 3 ꢁL injected directly onto the column for LCꢀMS analysis. Quantitation of samples was
performed using a Waters Micromass Quattro Premier coupled to a Waters Acquity UPLC coupled to a
positive electrospray ionization multipleꢀreaction monitoring mode detector. Column used was a
Supelco Ascentis Express RP Amide column (50 x 2.1 mm, 2.7 ꢁm); LC conditions used: Gradient
cycle time: 4 min; Injection vol: 3 ꢁL; Flow rate: 0.4 mL/min; Mobile phase Acetonitrileꢀwater
gradient with 0.05% formic acid.
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General Chemistry Procedures. Analytical thinꢀlayer chromatography was performed on Merck
2
54
silica gel 60F aluminumꢀbacked plates, and visualized by fluorescence quenching under UV light or
by KMnO staining. Flash chromatography was performed with silica gel 60 (particle size 0.040ꢀ0.063
4
1
ꢀm). NMR spectra were recorded on a Bruker Avance DRX 300 with the solvents indicated ( H NMR
at 300 MHz). Chemical shifts are reported in ppm on the δ scale and referenced to the appropriate
solvent peak. HRMS were acquired by Jason Dang at the Monash Institute of Pharmaceutical Sciences
Spectrometry Facility using an Agilent 1290 infinity 6224 TOF LCMS. Column used was RRHT 2.1 x
5
0 mm 1.8 µm C18. Gradient was applied over the 5 min with the flow rate of 0.5 mL/min. For MS:
o
Gas temperature was 325 C; drying gas 11 L/min; nebulizer 45 psig and the fragmentor 125V. LCMS
were recorded on a Waters ZQ 3100 using a 2996 Diode Array Detector. LCMS conditions used to
assess purity of compounds were as follows, column: XBridge TM C18 5 µm 4.6 x 100 mm, injection
volume 10 µL, gradient: 10ꢀ100% B over 10 min (solvent A: water 0.1% formic acid; solvent B: AcCN
0
.1% formic acid), flow rate: 1.5 mL/min, detection: 100ꢀ600 nm. All final compounds were analyzed
using high performance liquid chromatography/ultraviolet/evaporative light scattering detection
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coupled to mass spectrometry. Unless otherwise noted, all compounds were found to be >95% pure by
this method.
The following compounds were purchased commercially and used without further purification, 2-11,
14-25 and 28.
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General Procedure A. N4-(Furan-2-ylmethyl)-N2-(4-methoxyphenyl)quinazoline-2,4-diamine (1).
A mixture of N4ꢀ(furanꢀ2ꢀylmethyl)ꢀ2ꢀchloroquinazolinꢀ4ꢀamine 57 (50 mg, 0.19 mmol), TFA (60 ꢀL,
0
.80 mmol) and 4ꢀmethoxyaniline (47 mg, 0.39 mmol) in iPrOH (3 mL) was stirred under microwave
o
irradiation (200 W) at 120 C for 15 min. The mixture was partitioned between ethyl acetate (10 mL)
and 10% sodium hydrogen carbonate solution (10 mL). The layers were then separated. The organic
layer was then washed with brine solution (1 x 5 mL). The organic layer was dried (magnesium sulfate)
and the organic layer was concentrated in vacuo to obtain an oil. The oil was subjected to column
1
chromatography eluting 100% DCM to 10% MeOH/DCM to obtain 1 as a solid (44 mg, 68%). H
NMR (d ꢀDMSO): δ 8.85 (s, 1H), 8.47 (br s, 1H), 8.05 (d, J 8.4 Hz, 1H), 7.75ꢀ7.73 (m, 1H), 7.59ꢀ7.53
6
(m, 2H), 7.34 (d, J 8.4 Hz, 1H), 7.11 (t, J 7.8 Hz, 1H), 6.82 (d, J 8.4 Hz, 2H), 6.37ꢀ6.32 (m, 2H), 4.73
+
(
d, J 6.0 Hz, 2H), 3.68 (s, 3H). MS, m/z = 347 [M + H] . HRMS found: (M + H) 347.1506;
C H N O requires (M + H), 347.1508.
20
18
4
2
N2-(4-Fluorophenyl)-N4-(furan-2-ylmethyl)quinazoline-2,4-diamine (12). General procedure A
was followed using 57 (100 mg, 0.31 mmol) and 4ꢀfluoroaniline (60 ꢀL, 0.63 mmol) to give the title
1
compound 12 as a white solid (75 mg). H NMR (d ꢀDMSO): δ 9.11 (s, 1H), 8.56 (t, J 5.4 Hz, 1H),
6
8
.12ꢀ8.08 (m, 1H), 7.92ꢀ7.86 (m, 2H), 7.62ꢀ7.57 (m, 2H), 7.42ꢀ7.39 (m, 1H), 7.02ꢀ7.05 (m, 3H), 6.41ꢀ
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+
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.35 (m, 2H), 4.77 (d, J 5.7 Hz, 2H). MS, m/z = 335 [M + H] . HRMS found: (M + H) 335.1305;
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C H FN O requires (M + H), 335.1308.
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N4-Benzyl-N2-(4-methoxyphenyl)quinazoline-2,4-diamine (27). General procedure A was followed
using 58 (100 mg, 0.37 mmol) and 4ꢀmethoxyaniline (91 mg, 0.74 mmol) to obtain 27 as a solid (50
1
mg, 38%). H NMR (d ꢀDMSO): δ 10.01 (br s, 1H), 8.30 (d, J 7.8 Hz, 1H), 7.79ꢀ7.76 (m, 1H), 7.55 (d,
6
J 7.8 Hz, 1H), 7.45 (d, J 7.8 Hz, 2H), 7.43ꢀ7.41 (m, 1H), 7.35ꢀ7.24 (m, 5H), 7.09 (d, J 7.8 Hz, 2H),
+
6
3
.93ꢀ6.91 (m, 1H), 4.75ꢀ4.72 (m, 2H), 3.57 (s, 3H). MS, m/z = 357 [M + H] . HRMS found: (M + H)
57.1714; C H N O requires (M + H), 357.1715.
22 20
4
N4-Benzyl-N2-(4-fluorophenyl)quinazoline-2,4-diamine (29). General procedure A was followed
using 58 (50 mg, 0.19 mmol) and 4ꢀfluoroaniline (18 ꢀL, 0.19 mmol) to obtain 29 as a solid (40 mg,
1
60%). H NMR (MeOD): δ 7.95 (d, J 7.8 Hz, 1H), 7.58ꢀ7.56 (m, 1H), 7.51ꢀ7.49 (m, 2H), 7.41 (d, J 8.4
+
Hz, 1H), 7.35ꢀ7.16 (m, 6H), 6.92ꢀ6.89 (m, 2H), 4.80 (m, 2H). MS, m/z = 345 [M + H] . HRMS found:
(
M + H) 345.1512; C H N F requires (M + H), 345.1515.
21 17 4
N4-Benzyl-N2-[4-(trifluoromethyl)phenyl]quinazoline-2,4-diamine (30). General procedure A was
followed using 58 (50 mg, 0.19 mmol) and 4ꢀtrifluoromethylaniline (60 mg, 0.37 mmol) to obtain 30 as
1
a solid (45 mg, 62%). H NMR (d ꢀDMSO): δ 10.64 (bs, 1H), 10.40 (bs, 1H), 8.46ꢀ8.43 (m, 1H), 7.90ꢀ
6
+
7
.50 (m, 7H), 7.38ꢀ7.27 (m, 5H), 4.82 (d, J 5.9 Hz, 2H). MS, m/z = 395 [M + H] . HRMS found: (M +
H) 395.1484; C H F N requires (M + H), 395.1484.
22 17
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N4-Benzyl-N2-(4-bromo-3-chlorophenyl)quinazoline-2,4-diamine (31). General procedure A was
followed using 58 (60 mg, 0.22 mmol) and 4ꢀbromoꢀ3ꢀchloroaniline (77 mg, 0.44 mmol) to obtain 31
1
as a solid (30 mg, 37%). HꢀNMR (d ꢀDMSO): δ 9.40 (s, 1H), 8.81ꢀ8.76 (m, 1H), 8.34 (d, J 2.4 Hz,
6
1
H), 8.19ꢀ8.16 (m, 1HH), 7.71ꢀ7.21 (m, 9H), 4.83 (d, J 5.7 Hz, 2H). MS, m/z (%) = 441 (100) [M +
0
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
81 35
H] , 439 (90), 443 (30), 442 (30). HRMS found: (M + H) 441.0306; C H Br ClN requires (M +
21
16
4
H), 441.0305.
N4-Benzyl-N2-(3-chloro-4-fluorophenyl)quinazoline-2,4-diamine (32). General procedure A was
followed using 58 (50 mg, 0.19 mmol) and 4ꢀfluoroꢀ3ꢀchloroaniline (54 mg, 0.37 mmol) to obtain 32 as
1
a solid (65 mg, 93%). H NMR (d ꢀDMSO): δ 10.47 (br s, 1H), 10.38 (br s, 1H), 8.45ꢀ8.42 (m, 1H),
6
7
.86ꢀ7.83 (m, 2H), 7.63ꢀ7.60 (m, 1H), 7.53ꢀ7.26 (m, 8H), 4.77 (d, J 5.9 Hz, 2H). MS, m/z (%) = 379
+
35
16
(100) [M + H] , 381 (30). HRMS found: (M + H) 379.1123; C H ClFN requires (M + H),
2
1
4
3
79.1126.
N4-Benzyl-N2-(4-chloro-3-fluorophenyl)quinazoline-2,4-diamine (33). General procedure A was
followed using 58 (50 mg, 0.19 mmol) and 4ꢀchloroꢀ3ꢀfluoroaniline (54 mg, 0.37 mmol) to obtain 33 as
1
a solid (65 mg, 93%). H NMR (d ꢀDMSO): δ 9.42 (s, 1H), 8.81ꢀ8.76 (m, 1H), 8.19ꢀ8.15 (m, 2H),
6
+
7
.67ꢀ7.21 (m, 10H), 4.83 (d, J 5.6 Hz, 2H). MS, m/z (%) = 379 (100) [M + H] , 381 (30). HRMS
3
16
5
found: (M + H) 379.1125; C H ClFN requires (M + H), 379.1126.
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N4-Benzyl-N2-[6-(trifluoromethyl)pyridin-3-yl]quinazoline-2,4-diamine (34). General procedure A
was followed using 58 (60 mg, 0.22 mmol) and 6ꢀ(trifluoromethyl)pyridinꢀ3ꢀamine (72 mg, 0.44
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Journal of Medicinal Chemistry
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2
1
mmol) to obtain 34 as a solid (85 mg, 96%). H NMR (CDCl ): δ 8.89 (s, 1H), 8.61ꢀ8.58 (m, 1H), 7.69ꢀ
3
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
7
.60 (m, 4H), 7.43ꢀ7.28 (m, 5H), 5.99 (br s, 1H), 4.87 (d, J 5.4 Hz, 2H). MS, m/z = 396 [M + H] .
HRMS found: (M + H) 396.1438; C H F N requires (M + H), 396.1436.
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3
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N2-(4-Fluorophenyl)-6,7-dimethoxy-N4-(oxolan-2-ylmethyl)quinazoline-2,4-diamine (35). General
procedure A was followed using 59 (100 mg, 0.31 mmol) and 4ꢀfluoroaniline (59 ꢀL, 0.62 mmol) to
1
obtain 35 as a solid (90 mg, 73%). H NMR (d ꢀDMSO): δ 8.81 (s, 1H), 7.92ꢀ7.87 (m, 3H), 7.53 (s,
6
1
H), 7.08ꢀ7.01 (m, 2H), 6.81 (s, 1H), 4.17ꢀ4.10 (m, 1H), 3.84ꢀ3.81 (m, 7H), 3.67ꢀ3.53 (m, 3H), 1.95ꢀ
+
1.77 (m, 3H), 1.67ꢀ1.58 (m, 1H). MS, m/z = 399 [M + H] . HRMS found: (M + H) 399.1834;
C H FN O requires (M + H), 399.1832.
21
23
4
3
N4-Benzyl-N2-(4-fluorophenyl)-6,7-dimethoxyquinazoline-2,4-diamine (36). General procedure A
was followed using 60 (800 mg, 2.43 mmol) and 4ꢀfluoroaniline (461 ꢀL, 4.85 mmol) to obtain 36 as a
1
solid (850 mg, 87%). H NMR (d ꢀDMSO): δ 8.83 (s, 1H), 8.36ꢀ8.32 (m, 1H), 7.87ꢀ7.81 (m, 2H), 7.58
6
(s, 1H), 7.42ꢀ7.22 (m, 5H), 7.05ꢀ6.99 (m, 2H), 6.85 (s, 1H), 4.80 (d, J 5.8 Hz, 2H), 3.88 (s, 3H), 3.84
+
(
s, 3H). MS, m/z = 405 [M + H] . HRMS found: (M + H) 405.1727; C H FN O requires (M + H),
23
22
4
2
4
05.1727.
N4-Benzyl-N2-(4-fluorophenyl)pyrido[3,4-d]pyrimidine-2,4-diamine (37). General procedure A
was followed using 61 (50 mg, 0.19 mmol) and 4ꢀfluoroaniline (41 mg, 0.38 mmol) to obtain 37 as a
1
solid (40 mg, 63%). H NMR (CDCl ): δ 9.02 (s, 1H), 8.35 (d, J 5.5 Hz, 1H), 7.71ꢀ7.66 (m, 2H), 7.43ꢀ
3
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1
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1
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2
2
2
2
2
2
2
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3
3
3
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5
5
6
+
7
.30 (m, 6H), 7.08ꢀ7.01 (m, 3H), 5.93 (br s, 1H), 4.84 (d, J 5.5 Hz, 2H). MS, m/z = 346 [M + H] .
HRMS found: (M + H) 346.1465; C H FN requires (M + H), 346.1468.
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5
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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7
8
9
0
1
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7
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9
0
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9
0
1
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4
5
6
7
8
9
0
N4-Benzyl-N2-(4-fluorophenyl)pyrido[2,3-d]pyrimidine-2,4-diamine (38). General procedure A
was followed using 62 (50 mg, 0.19 mmol) and 4ꢀfluoroaniline (41 mg, 0.38 mmol) to obtain 38 as a
1
solid (40 mg, 63%). H NMR (CDCl ): δ 8.89ꢀ8.86 (m, 1H), 7.92 (dd, J 8.0 and 1.9 Hz, 1H), 7.75ꢀ7.70
3
(m, 2H), 7.42ꢀ7.35 (m, 5H), 7.14ꢀ6.99 (m, 4H), 5.95 (br s, 1H), 4.84 (d, J 5.4 Hz, 2H). MS, m/z = 346
+
[
M + H] . HRMS found: (M + H) 346.1464; C H FN requires (M + H), 346.1468.
2
0
16
5
N4-Benzyl-N2-(4-fluorophenyl)pyrido[3,2-d]pyrimidine-2,4-diamine (39). General procedure A
was followed using 63 (50 mg, 0.19 mmol) and 4ꢀfluoroaniline (41 mg, 0.38 mmol) to obtain 39 as a
1
solid (40 mg, 63%). H NMR (CDCl ): δ 8.39ꢀ8.37 (m, 1H), 7.80 (dd, J 8.5 and 1.5 Hz, 1H), 7.67ꢀ62
3
+
(m, 2H), 7.51ꢀ7.30 (m, 7H), 7.08ꢀ6.97 (m, 3H), 4.80 (d, J 5.9 Hz, 2H). MS, m/z = 346 [M + H] .
HRMS found: (M + H) 346.1466; C H FN requires (M + H), 346.1468.
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16
5
N4-Benzyl-N2-(4-fluorophenyl)thieno[3,2-d]pyrimidine-2,4-diamine (40). General procedure A was
followed using 64 (50 mg, 0.18 mmol) and 4ꢀfluoroaniline (40 mg, 0.36 mmol) to obtain 40 as a solid
1
(
35 mg, 55%). H NMR (CDCl ): δ 7.64 (d, J 5.3 Hz, 1H), 7.59ꢀ7.54 (m, 2H), 7.40ꢀ7.30 (m, 5H), 7.22
3
+
(
d, J 5.3 Hz, 1H), 7.02ꢀ6.96 (m, 3H), 5.65 (br s, 1H), 4.84 (d, J 5.6 Hz, 2H). MS, m/z = 351 [M + H] .
HRMS found: (M + H) 351.1081; C H FN S requires (M + H), 351.1080.
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15
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N6-Benzyl-N2-(4-fluorophenyl)-7-methyl-7H-purine-2,6-diamine (41). General procedure A was
followed using 65 (50 mg, 0.18 mmol) and 4ꢀfluoroaniline (41 mg, 0.36 mmol) to obtain 41 as an oil
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
40 mg, 63%). 1H NMR (CDCl ): δ 7.77 (s, 1H), 7.62ꢀ7.56 (m, 1H), 7.39ꢀ7.33 (m, 5H), 7.10ꢀ7.04 (m,
3
+
1
H), 6.95ꢀ6.85 (m, 2H), 4.83ꢀ4.79 (m, 2H), 4.02ꢀ3.96 (m, 3H). MS, m/z = 349 [M + H] . HRMS found:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(M + H) 349.1578; C H FN requires (M + H), 349.1577.
19 17 6
N4-Benzyl-N6-(4-fluorophenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
(42).
General procedure A was followed using 66 (50 mg, 0.18 mmol) and 4ꢀfluoroaniline (41 mg, 0.36
1
mmol) to obtain 42 as an oil (45 mg, 71%). H NMR (CDCl ): δ 7.68 (s, 1H), 7.62ꢀ7.57 (m, 2H), 5.37ꢀ
3
7
.25 (m, 5H), 7.07 (br s, 1H), 7.01ꢀ6.95 (m, 2H), 5.65 (br s, 1H), 4.77 (d, J 5.7 Hz, 2H), 3.90 (s, 3H).
+
MS, m/z = 349 [M + H] . HRMS found: (M + H) 349.1577; C H FN requires (M + H), 349.1577.
19
17
6
N2-(3-Chloro-4-fluorophenyl)-N4-(2-(4-methylpiperazin-1-yl)ethyl)pyrido[3,2-d]pyrimidine-2,4-
diamine (43). General procedure A was followed using 67 (50 mg, 0.16 mmol) and 3ꢀchloroꢀ4ꢀfluoroꢀ
1
aniline (47 mg, 0.32 mmol) to obtain 43 as a solid (45 mg, 66%). H NMR (CDCl ): δ 8.44 (d, J 4.2
3
and 1.5 Hz, 1H), 8.08 (d, J 6.7 and 2.7 Hz, 1H), 7.83ꢀ7.79 (m, 2H), 7.53ꢀ7.48 (m, 1H), 7.44ꢀ7.38 (m,
1
8
H), 7.30 (br s, 1H), 7.07 (t, J 8.8 Hz, 1H), 3.70 (q, J 6.3 Hz, 2H), 2.73 (t, J 6.3 Hz, 2H), 2.60ꢀ2.50 (m,
+
H), 2.33 (s, 1H). MS, m/z (%) = 416 [M + H] (100), 418 (30). HRMS found: (M + H) 416.1766;
35
C H ClFN requires (M + H), 416.1766.
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23
7
N2-(4-Chloro-3-fluorophenyl)-N4-(2-(4-methylpiperazin-1-yl)ethyl)pyrido[3,2-d]pyrimidine-2,4-
diamine (44). General procedure A was followed using 68 (50 mg, 0.16 mmol) and 4ꢀchloroꢀ3ꢀfluoroꢀ
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