Aminopurine and aminoquinazoline scaffolds for development of potential dengue virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.10.008

Previous efforts led to dicarboxamide derivatives like 1.3, comprising either an imidazole, pyrazine or fenyl ring as the central scaffold, with many congeners displaying strong inhibitory effects against dengue virus (DENV) in cell-based assays. Followin

Full text of DOI:10.1016/j.ejmech.2016.10.008

European Journal of Medicinal Chemistry 126 (2017) 101e109
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Aminopurine and aminoquinazoline scaffolds for development of
potential dengue virus inhibitors*
Akkaladevi Venkatesham a, Milind Saudi a, Suzanne Kaptein b, Johan Neyts b,
,
*
Jef Rozenski a, Mathy Froeyen a, Arthur Van Aerschot a
a KU Leuven, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49, 3000 Leuven, Belgium
b KU Leuven, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Herestraat 49, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous efforts led to dicarboxamide derivatives like 1.3, comprising either an imidazole, pyrazine or
fenyl ring as the central scaffold, with many congeners displaying strong inhibitory effects against
dengue virus (DENV) in cell-based assays. Following up on some literature reports, the rationale was
borne out to preserve the pending groups, now attached to either a 2,6-diaminopurine or 2,4-
diaminoquinazoline scaffold. Synthetic efforts turned out less straightforward than expected, but yiel-
ded some new derivatives with low micromolar anti-DENV activity, albeit not devoid of cellular toxicity.
Received 13 July 2016
Received in revised form
3 October 2016
Accepted 4 October 2016
Available online 5 October 2016
The purine 14 proved the most potent compound for this series with an EC50 of 1.9
mM and a selectivity
Keywords:
Flavivirus inhibitors
Dengue virus
index of 58, while the quinazoline 18a displayed an EC50 of 2.6 M with SI of only 2.
m
© 2016 Elsevier Masson SAS. All rights reserved.
Diaminopurines
Diaminoquinazolines
NS5 polymerase
1. Introduction
Although DENV is much more prevalent and wide-spread,
currently there still is neither any vaccine nor any antiviral ther-
apy available for DENV [6,7]. Indeed, dengue vaccine development
is not straightforward, as an immunogenic response is needed
against all four serotypes of DENV. When a “vaccinated” person
becomes infected with a serotype against which this patient is not
(or insufficiently) protected, an aggravated form of the disease will
develop, due to an incompletely understood mechanism, antibody-
dependent enhancement (ADE) with increased viral loads [8].
However, a tetravalent dengue vaccine under development by
Sanofi-Pasteur recently was registered in Brazil [9]. This should
avoid the ADE complication, but meanwhile in addition, a fifth
serotype was reported [10] further complicating vaccine
development.
Regarding antiviral therapy, the intensive efforts of many
research groups led to a large variety of possible targets and a host
of compounds have been reported to be able inhibiting DENV
growth in a laboratory setting. Thus recently, a series of 2-aroyl-3-
arylquinoline was reported to strongly inhibit DENV2 RNA
expression without significant cell cytotoxicity [11]. However,
many more reports are appearing each year and an excellent review
of Klein et al. tries to cover all aspects on the medicinal chemistry of
dengue virus [12], while another review of Soliman et al. specif-
ically discusses the potential of targeting non-structural proteins
The dengue virus (DENV), member of the Flaviviridae, is a very
common viral infection transmitted by mosquito bites [2]. Dengue
incidence and prevalence are rising considerably in endemic areas
of the tropical and subtropical regions. It is estimated that
approximately 390 million infections occur each year [2] and cases
continue to rise worldwide [3]. Indeed, global warming and
increasing international contacts and travelling make up the per-
fect breeding ground for an exponential increase in Dengue as well
as other previously exotic virus outbreaks carried by arthropods.
The most recent newcomer is the Zika virus with outbreaks in
Central and South America reaching pandemic levels especially in
Brazil. This virus is likewise member of the Flaviviridae and related
to dengue, yellow fever and West Nile viruses [4]. Like dengue it
spreads through mosquito bites, and is expected to spread further
over both American continents. The symptoms are usually mild, but
infection of pregnant women leads to severe birth defects and poor
pregnancy outcomes [5].
*
This is part 5 in a series on dengue virus inhibitors, part 4 being reference [1].
* Corresponding author.
E-mail address: Arthur.Vanaerschot@rega.kuleuven.be (A. Van Aerschot).
http://dx.doi.org/10.1016/j.ejmech.2016.10.008
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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A. Venkatesham et al. / European Journal of Medicinal Chemistry 126 (2017) 101e109
and especially proteases for combating neglected diseases as
caused by arboviruses with in particular Dengue virus [13]. In
addition it was recently determined that host molecular chaper-
ones like Hsp70 are required for viral entry, RNA replication and
virion production and allosteric inhibitors of Hsp70 potently inhibit
DENV replication [14]. We likewise recently reported on tritylated
nucleosides [15e17] as well as imidazole and pyrazine dicarbox-
amides [1,18] as potential inhibitors of both Dengue and Yellow
fever virus, but have not been able to uncover the mechanism of
action of the latter although we suspect the RNA polymerase.
Bisaryl amide compounds likewise have been reported as weak
inhibitors of influenza virus, supposedly interacting with APO-
BEC3G, an RNA editing enzyme [19]. In addition, DENV NS5 RdRp
inhibitors binding in its palm subdomain were uncovered recently
using an X-ray based fragment screening methodology, resulting in
low micromolar EC50 values in cell-based assays [20]. Finally and
alternatively, the vector of dengue fever, Aedes aegypti, could be
targeted in an effort to control the spread of dengue virus [21,22].
imidazole (2.1) and the phthalic acid series (2.2), inspired us to
envisage compound series like 3.1 or 3.2 for their potential antiviral
activity, as shown below (Fig. 3).
2. Results and discussion
2.1. Synthetic procedures
The 2,6-dichloropurine and 2,4-dichloroquinazoline scaffolds
are the logic precursors for synthesis of the compound series 3.1
and 3.2. Both share a 2,4-dichloropyrimidine ring allowing selec-
tive reaction to introduce different aniline substituents. Coupling of
two different anilines can be done selectively by optimization of the
temperature around 60 ꢀC and 90 ꢀC, respectively. Both steps pro-
ceed via
a straightforward SNAr mechanism on the 2,6-
dichloropyrimidine ring. Hereto, either a preformed heterocycle
substituted aniline could be used, or a concluding Suzuki reaction
on a brominated aniline can afford the target compounds 3.1 and
3.2.
1.1. Rationale: building on previous leads
Corroborating on these plans, somewhat unexpectedly 2,6-
dichloropurine 4 displayed no nucleophilic substitution at C6 us-
ing 2-bromo-toluidine (5) under different reaction conditions [28]
(either in Et3N, n-BuOH at 100 ꢀC for 12 h, or in Et3N, amyl alcohol,
100e110 ꢀC for 16 h or in presence of DIPEA in acetonitrile,
80e90 ꢀC for 18 h) (Scheme 1). Likewise, nucleophilic substitution
at the C4 position of 2,4-dichloroquinazoline [29] (7) to obtain 8
proved not possible using 5 in either amyl alcohol or acetonitrile at
elevated temperatures. This lack of reactivity could be attributed to
the use of the sterically hindered electron-poor aniline [30] 5. Ac-
cording to the literature [31], however, the pKa value of a tri-
fluoroacetyl (TFA) protected aniline approaches the pKa for phenol
(9.9 and 10.0 respectively). Activation of the sterically hindered
electron-poor aniline nitrogen by a TFA group therefore will lower
the pKa of the anilide 9 increasing its nucleophilicity under basic
conditions. The corresponding 9 was easily obtained in 90% yield by
treatment of 5 with triflic anhydride and Et3N in DCM at rt for 3 h.
In parallel, reaction of 2,6-dimethylquinolin-4-ol (10) with hydra-
zine afforded the envisaged pyrazole containing aniline 11 as
described previously [32].
However, reaction of 2,6-dichloropurine (4) with 9 failed like-
wise to afford the desired substitution, and hence 4 was first pro-
tected on the imidazole nitrogen with 4-methoxybenzyl chloride
(PMBCl) (Scheme 2). This resulted in a 2:1 mixture of the 9- and 7-
PMB regioisomers [33] 12a and 12b respectively, which were
conveniently separated by column chromatography.
Nucleophilic aromatic substitution at the C6-position of 12a
with the acylated aniline 9 was accomplished using K2CO3 in 1,2-
dimethoxyethane (DME) at reflux for 24 h to obtain 13 in 48%
yield. Various other reaction conditions were less successful, with
decreased yields using NaH or Cs2CO3 in DME, while no reaction
took place with K2CO3 in an aprotic solvent like DMF, even at
elevated temperature. Deprotection of the PMB group using TFA in
DCM at rt for 5 h afforded 6 in 51% yield. In contrast with 5, the
Among our previously synthesized heterocyclic compounds,
analogue 1.2 exhibited the most potent anti-DENV activity with an
EC50 ¼ 0.5
initial lead (1.1) having an imidazole dicarboxamide central scaffold
displayed an EC50 ¼ 2.5 M. In addition, evaluation of the congeners
with a pyrazine central scaffold lead to compound 1.3 being the
mM and a selectivity index (SI) of above 235 while the
m
most potent congener for this series with again an EC50 ¼ 0.5
mM
and SI of over 235. In addition, the latter compound upon removal
of the methyl moiety of the benzene ring at the right, displayed
strong YFV inhibition with EC50 ¼ 0.4
mM (Fig. 1). Unfortunately, we
have been unable to generate resistant viruses and no target so far
could be pinpointed.
Searching for structurally analogous compounds, it has been
reported in literature that a series of anthranilic acid derivatives are
potent inhibitors of the hepatitis C NS5B polymerase. One of the
compounds (1.4, Fig. 2) of this series displayed an IC50 of 17 nM and
very low cellular toxicity affording selectivity indexes over 7000
using the MTS cell proliferation assay [23]. The Novartis corporate
compound archive also led to the identification of N-sulfonylan-
thranilic acid 1.5 which inhibited DENV RdRp with an IC50 of 0.7 mM
[24]. In addition, scientists at NITD identified compound 1.6 as one
of their inhibitors which displayed an average EC50 of 119 nM
against dengue virus serotype 2 in a human cell line [25]. In another
series of compounds, 2,4-diaminoquinazoline derivative 1.7 was
observed to display both the highest antiviral potency
(EC50 ¼ 2.8 nM, SI > 1000) and an excellent pharmacokinetic profile
against DENV [26]. Finally, a series of substituted quinazoline-2,4-
diamines (1.8) has been shown to display interesting anti-
Leishmania activities with favorable physicochemical properties
[27].
Combination of the structural information in these reports with
our previous lead structures for DENV inhibition within the
Fig. 1. Structures of the most active anti-DENV compounds from our previous work.

A. Venkatesham et al. / European Journal of Medicinal Chemistry 126 (2017) 101e109
103
Fig. 2. Compounds with biological activity and sharing some structural aspects with our series or displaying antimicrobial activity.
BINAP, Cs2CO3, Toluene, reflux for 7 h) lead to degradation. Micro-
wave conditions [35] (toluidine, EtOH, 30 min at 150 ꢀC) furnished a
low yield reaction (20%), but optimal results finally were obtained
upon heating in IPA at 100 ꢀC in a sealed tube [36] affording 66%
and 46% of 18a and 18b, respectively.
Introduction of amines via substitution of chlorine at the C2-
position of purines on many occasions in the past has shown to
be problematic, especially with an electron donating nitrogen
already present at C6. Ciszewski et al. carefully analyzed the
problem and found slight acidic catalysis using TMSCl to be optimal
[37]. However, upon treatment of 15 with low molecular % amounts
of TMSCl in refluxing n-butanol for 12 h, the starting material had
disappeared and a brown solid was filtered, but the desired product
could not be detected. Preserving the PMB group at N9 as in 14,
substitution at C2-position likewise proved unsuccessful under
various Buchwald-Hartwig [38] conditions with toluidine (17a)
(either (Pd(OAc)2, BINAP, Cs2CO3, toluene, reflux; Pd2(dba)3, BINAP,
Cs2CO3, dioxane, MW at 150 ꢀC for 15 min; or Pd(OAc)2, BINAP,
KOtBu, toluene, reflux) (Scheme 2). Possibly, complexation of the
catalyst by interaction with the pyrazole moiety causes poor reac-
tivity of 14 towards palladium catalyzed cross-coupling reactions.
Unfortunately, heating in IPA which was successful for the quino-
lone congeners, likewise did not afford the desired compound. The
Fig. 3. New envisaged structures for DENV inhibition based on aminopurine or amino-
quinazoline scaffolds.
ꢀ
work on 2,6,9-trisubstituted purines of Houze et al. [38] with prior
hindered aniline 11 displayed adequate nucleophilicity and 37% of
14 was obtained on refluxing 12a and 11 in DME for 24 h, followed
by deprotection to afford 15 in moderate yield. Direct substitution
of 4 with 11 to provide 15 proved feasible as well on small scale in
low yield (50 mg batch), albeit the reaction proved not reproducible
(Scheme 2).
functionalization of the C2 position starting from a C2-amine like 2-
amino-6-chloropurine could have solved our issue but was not
attempted further.
2.2. Assessment of antiviral activity
As for purines, nucleophilic aromatic substitution of the qui-
nazoline ring is directed preferably to the C4-position displaying
higher reactivity. Hence, the quinazoline 7 reacted swiftly with 9 or
11 using K2CO3 in DME at reflux conditions to afford 8 and 16 in 88%
and 61%, respectively (Scheme 3). Further nucleophilic substitution
at C2 of the quinazoline 16 in presence of conc. HCl under reflux in
acetone [29] proved unsuccessful due to degradation of the starting
material. Also Buchwald coupling conditions (Pd2(dba)3 [34],
Our previous work entailing imidazole-4,5-and pyrazine-2,3-
dicarboxamides targeting dengue virus (see chemical structures
1.1e1.3) resulted in several compounds endowed with sub-
micromolar inhibitory acitivities and high selectivity indices.
Combined with literature reports on some quinazoline derivatives
displaying strong dengue inhibitory properties (structures 1.6e1.8),
we sought to combine some features of both series in either a

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A. Venkatesham et al. / European Journal of Medicinal Chemistry 126 (2017) 101e109
Scheme 1. i) triflic anhydride, Et3N, DCM, rt, 3 h (90%); (ii) NH2NH2/NH2NH2. 2HCl, ethylene glycol, 200 ꢀC, sand bath, 5 h (75%).
Scheme 2. i) PMBCl, K2CO3, DMF, rt., 24 h (12a: 55%; 12b: 26%); ii) K2CO3, DME, reflux, 24 h (48% of 13 (along with 25% recovery of 12a) and 37% of 14; iii) TFA, DCM, rt, 5 h (51%); iv)
K2CO3, DME, reflux, 24 h (35% of 15).
quinazoline or purine functionalized scaffold (see Fig. 3). The
resulting compounds were evaluated for their inhibitory properties
against DENV (serotype 2) in Vero-B cells. Following completion of
our work presented here, Vincetti et al. [39] disclosed a series of
double substituted purine compounds with some of them dis-
playing micromolar inhibitory effects, targeting the dengue virus
NS5 polymerase at an allosteric pocket. Best results for this series
were obtained for the 2,6-diaminopurine congener 1.9 with an EC50
of 5.3 mm and having strong resemblance to our target compounds.
Hence, the results for compounds 1.3 and 1.9 are included here in
Table 1 as reference compounds.
Only for the diaminoquinazoline series the double modified
target compounds 18a and 18b comprising the 3-methyl-1H-
pyrazol-5-yl-phenyl moiety were attained. To our satisfaction we
noted the strong inhibitory effect on denge virus proliferation
(2.6 mm and 6.7 mm, respectively, based on RNA viral load deter-
mination), as was hoped for based on our starting rationale.
However, both compounds are about 10 times less active compared
to the lead dicarboxamide 1.3 (EC50 0.5 mm) [1] being at the same
time much more toxic and devoid of selectivity. The mono-
aminated quinazolines 8 and 16 proved only twice less inhibitory,
but displayed lower toxicity, endowing these congeners with a
selectivity index (SI) of around 10. In the purine series, the inter-
mediate N9 or N7-methoxybenzyl protected derivatives 12a,b
already displayed inhibitory activity concomitant with toxicity in
the lower micromolar range, while the mono-aminated purines 6

A. Venkatesham et al. / European Journal of Medicinal Chemistry 126 (2017) 101e109
105
the DENV-3 structure should be a good model for the experimental
inhibition measurements obtained with DENV-2 [40].
The ligands were positioned in cavity B simulating induced fit
docking by introducing flexibility in some protein residues defining
cavity B (Leu326, Leu327, Lys329, Thr858, Trp859, Asn862, and
Ile863). Due to high flexibility of the shallow cavity multiple
binding modes for the different analogues are observed having a
binding energy difference of less than 0.2 kcal/mol for different
conformations. Fig. 4 shows a docking pose in cavity B for several of
the new reported compounds as well as for the reference com-
pound 1.9 (structure 16i in the respective reference) [39]. Selection
of this conformation is based on the docking score (in the top 5 out
of 50) and maximal hydrophobic contact with the residues Leu327,
Lys329, Trp859 and Ile863, which were proven before to be
important for NS3-NS5 interaction by mutational analysis.
Remarkably, our docking result for this reference compound is
different from the one reported by Vincetti, with 3 hydrogen bonds
instead of one but absence of the reported cation-pi interaction
with Lys329 which was not observed in our study. However, the
inhibitor shows hydrophobic contact with Lys329, Trp859 and
Ile863, 3 out of 4 residues important for viral replication (no contact
with Leu327, using DENV3 numbering). This deviation is not sur-
prising, as often different docking positions are found when using
different docking programs [42] Using a similar procedure allowed
to dock the other compounds, all binding cavity B and displaying
different hydrogen bonds and hydrophobic contacts. All hydro-
phobic contacts with the key amino acids are given in the caption
for Fig. 4. The nice hydrophobic fit for 14 (panel D) is noteworthy.
Compounds 18a and 18b (panels E and F) likewise display good
interaction. The docking of compound 14 is different when
compared to orientation of compounds 15 and 18 despite the fact
they have a similar scaffold. This likewise is not remarkable, as
small changes in substituents may result in a completely different
binding mode [43]. Nevertheless, all these docking experiments
may help to explain the experimental findings. From the obtained
results it seems plausible that binding may interfere with NS3-NS5
protein interaction and concomitant initiation of RNA synthesis
[39] but only a qualitative interpretation can be provided.
Scheme 3. i) K2CO3, DME, reflux, 6e12 h (88% for 8 and 61% for 16); ii) IPA, 100 ꢀC,
4e5 h (66% for 18a and 46% of 18b).
Table 1
Dengue inhibitory activity and cytotoxicity of the various obtained compounds.
compound
MW
MTS/PMS CC50
(
mm)
RT-qPCR EC50
(m
m)
SI
12a
12b
13
6
14
15
8
16
18a
18b
1.3
1.9
308
308
457
337
459
339
347
349
420
424
426
256
32.4
12.3
>109a
>148
>109a
>147
>144a
97
5.8
8.7
>117
168
17.6
9.9
7.2
33.5
1.9
28.9
10.9
9.2
2.6
6.7
0.5
5.3
2
1
15
4
58
5
13
11
2
1
>235
32
a
Deviating CC50 on visual inspection of cell viability: 63
mm (8).
m
m (13); 10
m
m (14);
32
3. Conclusion
Based on our previous findings for dicarboxamide derivatives
like 1.3 endowed with strong inhibitory effects on dengue virus,
and on the biological activities of 2,4-diaminoquinazoline struc-
tures as reported in literature, efforts were undertaken to combine
both features based on either a central quinazoline or purine het-
erocyclic scaffold.
Accurate activity data were obtained using a virus yield reduc-
tion assay determining the viral RNA load by real-time quantitative
RT-PCR. As hoped for, the double modified target compounds 18a
and 18b displayed low micromolar inhibitory effects albeit with
low selectivity. The pyrazolyl containing monocarboxylated pre-
cursor purine 14 unexpectedly proved to be the most active of the
and 15 displayed only marginal activity around 30 mm with an SI of
about 5. Finally, their precursors 13 and 14 still functionalized with
the methoxybenzyl moiety, proved most active and selective of this
series. The pyrazolyl containing purine 14 herein showed most
promising with an EC50 of 1.9 mm and an SI of 109. Visual inspection
for cell abnormalities (in contrast to inhibition of cell metabolism as
determined in the MTS cell viability assay) however tempered our
enthusiasm and afforded a reduced SI of only 5. In comparison, the
diaminated purine 1.9 displayed inhibitory effects at 5 mm with an
SI of 32 [39].
2.3. Molecular docking on dengue virus NS5 polymerase
present series with an EC50 of 1.9 mM and an SI of 109. Based on
literature results, analogous molecular docking studies for our
compounds confirmed the NS5 polymerase to be the probable
target, highlighting the potential of bis-aminated purine and qui-
nazoline scaffolds for further study as flavivirus inhibitors.
Vincetti et al. described a novel series of dengue virus inhibitors
targeting an allosteric pocket coined “cavity B” on DENV-NS5 [39]
with their purine derivative 1.9 (16i in the original publication)
interacting with crucial residues within this cavity. With our
compounds displaying structural similarity to the reported com-
pounds, a similar modeling study was carried out. Hereto, the
envisaged compounds were docked in cavity B [40] of the dengue
virus DENV-3 RNA-dependent RNA polymerase (DENV-3 RdRp NS5
pdb code 2j7u [41] being part of the NS3-NS5 protein interface. This
cavity is conserved in DENV RdRps so our docking models based on
4. Materials and methods
4.1. Chemistry
General methods are largely as described before [18] and can be
found in the supplementary section. Chromatographic purifications

106
A. Venkatesham et al. / European Journal of Medicinal Chemistry 126 (2017) 101e109
Fig. 4. Docking pose for different compounds in cavity B of DENV3 RdRp with residue numbering according to DENV-3 with panel A: 1.3 from Ref. [1] (Leu327, Lys329, Trp859 and
Ile863); panel B: reference compound 1.9 [39] (hydrophobic contacts with Lys329, Trp859, Ile863); panel C: 15 (Leu327, Lys329, Trp859, Ile863); panel D: 14 (Lys329); panel E: 18a
(Trp859 and Ile863); panel F: 18b (Lys329, Trp859 and Ile863).
on silica gel were carried out via slow methanol gradients in
dichloromethane, unless indicated otherwise.
evaporated. The product was purified by flash chromatography on
silica gel to give the title compounds 12a and 12b as white powders.
2, 6-Dichloro-9-(4-methoxybenzyl)-9H-purine (12a): Yield:
2.71 g (55%); 1H NMR (300 MHz, CDCl3)
d
: 8.04 (s, 1H), 7.30 (d,
J ¼ 8.5 Hz, 2H), 6.92 (d, J ¼ 8.5 Hz, 2H), 5.36 (s, 2H, PhCH2), 3.82 (s,
3H, OMe); 13C NMR (75 MHz, CDCl3)
168.6, 159.8, 152.8, 151.5,
4.1.1. N-(2-Bromo-4-methylphenyl)-2,2,2-trifluoroacetamide (9)
Trifluoroacetic anhydride (6.38 mL, 45.16 mmol) was added
dropwise to a stirring solution of aniline 5 (5.50 g, 31.1 mmol) and
Et3N (10.5 mL, 75.3 mmol) in DCM (100 mL) that was kept at 0 ꢀC
with the aid of an ice bath. After 15 min the solution was allowed to
warm to room temperature (rt.) and was stirred for 3 h more,
cooled again to 0 ꢀC and quenched with saturated aq. NaHCO3 so-
lution. The aqueous layer was extracted with DCM (3 ꢁ 100 mL).
The organic layer was separated and dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure, and the residue
was purified by column chromatography on silica gel to give 7.3 g
(90%) of the title compound 9 as a white solid.
d
145.1, 130.4, 129.4, 125.6, 114.4, 55.0, 47.4; calcd.: 331.0124; found:
331.0124.
2, 6-Dichloro-7-(4-methoxybenzyl)-7H-purine (12b): Yield:
1.3 g (26%); 1H NMR (300 MHz, CDCl3)
d
: 8.19 (s, 1H), 7.14 (d,
J ¼ 8.7 Hz, 2H), 6.90 (d, J ¼ 8.7 Hz, 2H), 5.59 (s, 2H, PhCH2), 3.80 (s,
3H, OMe); 13C NMR (75 MHz, CDCl3)
: 163.4, 159.8, 152.9, 149.9,
d
143.6, 128.6, 125.4, 121.4, 114.5, 55.1, 50.3; HRMS for C13H10Cl2N4O1
([MþNa]þ) calcd.: 331.0124; found: 331.0122.
1H NMR (300 MHz, CDCl3)
J ¼ 8.4 Hz, 1H), 7.42 (s, 1H), 7.18 (d, J ¼ 8.4 Hz, 1H), 2.34 (s, 3H, CH3);
13C NMR (75 MHz, CDCl3)
d: 8.36 (brs, 1H, NH), 8.15 (d,
4.1.3. N-(2-Bromo-4-methylphenyl)-2-chloro-9-(4-
methoxybenzyl)-9H-purin-6-amine (13)
d
: 154.24 (q, J ¼ 37.6 Hz), 137.3, 132.5,
To a solution of compound 12a (60 mg, 0.19 mmol) and 9 (65 mg,
0.23 mmol) in DME (5 mL) was added anhydrous K2CO3 (80 mg,
0.58 mmol) at rt. and under N2. The reaction mixture was slowly
heated to 60e70 ꢀC. Further work-up was as carried out for the
synthesis of 8 using 10 mL of EtOAc, and purification by flash
chromatography on silica gel gave 43 mg (48%) of the title com-
pound 13 as a white solid while recovering some of the starting
material 12a (15 mg, 25%).
130.3, 129.0, 121.6, 115.0 (q, J ¼ 288.8 Hz), 113.8, 20.3; HRMS for
C9H8BrF3NO ([MþH]þ) calcd.: 281.9736; found: 281.9742.
4.1.2. N-Alkylation of 2,6-dichloropurine (12a, 12b)
A mixture of 2,6-dichloropurine 4 (3 g, 15.9 mmol) and anhy-
drous K2CO3 (6.57 g, 47.6 mmol) in dry DMF (100 mL) was stirred at
rt. under N2 atmosphere for 30 min p-Methoxybenzyl chloride
(4.3 mL, 31.7 mmol) was slowly added to the reaction mixture at rt.
and progress of the reaction was monitored by TLC. After stirring for
24 h, the reaction mixture was filtered and evaporated in vacuo. The
mixture was diluted with EtOAc (100 mL) and washed with H2O
(100 mL) and the aqueous layer was back extracted twice with
100 mL of EtOAc. The organic phase was dried with Na2SO4 and
1H NMR (300 MHz, CDCl3)
d
: 8.47 (d, J ¼ 8.4 Hz, 1H), 8.06 (s, 1H),
7.76 (s, 1H), 7.43 (s, 1H), 7.31e7.25 (m, 2H), 7.22 (d, J ¼ 8.4 Hz, 1H),
6.91 (d, J ¼ 8.4 Hz, 2H), 5.30 (s, 2H, PhCH2), 3.82 (s, 3H, OMe), 2.35
(s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d: 159.5, 151.8, 150.6, 140.7,
134.7, 132.8, 32.4, 129.2, 128.7, 126.7, 121.8, 119.2, 114.2, 113.9, 55.0,
46.7, 20.2; HRMS for C20H18BrClN5O ([MþH]þ) calcd.: 458.0378;

A. Venkatesham et al. / European Journal of Medicinal Chemistry 126 (2017) 101e109
107
found: 458.0378.
aniline 9 (5.08 g, 18.09 mmol) in dimethoxyethane (DME, 50 mL)
was added anhydrous K2CO3 (6.23 g, 45.2 mmol) at rt. under N2. The
reaction mixture was slowly heated to 60e70 ꢀC. Progress of the
reaction was monitored by TLC and after 6 h, the mixture was
cooled to rt. The solvent was removed in vacuo and the mixture was
diluted with EtOAc (50 mL) and washed with H2O (50 mL) and the
aqueous layer was back extracted twice with 50 mL of EtOAc. The
organic phase was dried with Na2SO4 and evaporated. The product
was purified by flash chromatography on silica gel to give 4.7 g
(88%) of the title compound 8 as a white solid.
4.1.4. 2-Chloro-9-(4-methoxybenzyl)-N-(4-methyl-2-(3-methyl-
1H-pyrazol-5-yl)phenyl)-9H-purin-6-amine (14)
Following the procedure for 13, to a mixture of compounds 12a
(0.600 g, 1.95 mmol) and 11 (0.437 g, 2.33 mmol) dissolved in DME
(60 mL) was added anhydrous K2CO3 (0.800 g, 5.85 mmol) at rt.
under N2. Progress of the reaction was monitored by TLC and after
24 h the solvent was evaporated in vacuo. The mixture was diluted
with EtOAc (60 mL) and washed with H2O (60 mL) and the aqueous
layers were back extracted twice with EtOAc. The organic phase
was dried with Na2SO4 and evaporated and the product was puri-
fied by on silica gel to afford the desired product 14 (0.33 g, 37%)
along with partial recovery of the unreacted mixture of 12a and 11.
1H NMR (600 MHz, CDCl3)
d
8.57 (d, J ¼ 8.5 Hz, 1H), 8.16 (brs, 1H,
NH), 7.96e7.77 (m, 3H), 7.59 (dtd, J ¼ 8.5, 6.8 and 1.7 Hz, 1H),
7.49e7.38 (m, 1H), 7.23 (dd, J ¼ 8.5, 1.7 Hz, 1H). 2.35 (s, 3H, CH3); 13
C
NMR (150 MHz, CDCl3) d: 157.7, 156.7, 151.0, 135.4, 133.6, 132.4,
1H NMR (600 MHz, DMSO)
d: 13.14 (s, 1H, NH), 12.16 (s, 1H, NH),
132.3, 128.9, 128.0, 126.8, 122.2, 120.1, 114.3, 113.4, 20.3; HRMS for
8.65 (d, J ¼ 8.5 Hz, 1H), 8.42 (s, 1H), 7.58 (s, 1H), 7.29 (d, J ¼ 8.7 Hz,
2H), 7.18 (d, J ¼ 8.5 Hz, 1H), 6.92 (d, J ¼ 8.7 Hz, 2H), 6.59 (s, 1H), 5.34
(s, 2H, PhCH2), 3.72 (s, 3H, OCH3), 2.34 (s, 3H, CH3), 2.30 (s, 3H,
C
15H12BrClN3 ([MþH]þ) calcd.: 347.9898; found: 347.9900.
4.1.8. 2-Chloro-N-(4-methyl-2-(3-methyl-1H-pyrazol-5-yl)phenyl)
quinazolin-4-amine (16)
CH3); 13C NMR (150 MHz, DMSO)
d: 159.0, 152.5, 152.1, 150.7, 150.4,
142.7, 139.8, 133.5, 132.0, 129.1, 128.6, 128.4, 128.3, 121.4, 120.7,
119.5, 114.2, 102.6, 55.2, 46.5, 20.6, 10.4; HRMS for C24H23ClN7O
([MþH]þ) calcd.: 460.16470; found: 460.1652.
To a solution of 7 (0.500 g, 2.5 mmol) and 11 (0.563 g, 3.0 mmol)
in DME (50 mL) was added anhydrous K2CO3 (1.04 g, 7.5 mmol) at rt.
under N2 atmosphere. The reaction mixture was slowly heated to
60e70 ꢀC for 24 h. Further work-up was as carried out for the
synthesis of 8, and purification on silica gel afforded 0.545 g (62%)
of the title compound 16 as a pale yellow solid.
4.1.5. N-(2-Bromo-4-methylphenyl)-2-chloro-9H-purin-6-amine
(6)
To a stirred solution of compound 13 (30 mg, 0.065 mmol) in
1H NMR (500 MHz, DMSO)
d: 13.7 (brs, 1H, NH), 12.7 (brs, 1H,
DCM (5 mL) was added TFA (40
m
L, 0.65 mmol) at 0 ꢀC under N2
NH), 7.98 (d, J ¼ 7.9 Hz, 1H), 7.87 (t, J ¼ 7.9 Hz, 1H), 7.79 (s, 1H), 7.55
(d, J ¼ 8.2 Hz,1H), 7.42 (t, J ¼ 6.9 Hz, 2H), 7.34 (d, J ¼ 6.9 Hz,1H), 6.46
(s, 1H), 2.45 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR (125 MHz,
atmosphere. The reaction mixture was slowly heated to 60 ꢀC and
progress of the reaction was monitored by TLC. After 5 h, the
mixture was cooled to 0 ꢀC and neutralized with saturated aqueous
K2CO3. The aqueous layer was extracted with DCM (3 ꢁ 10 mL). The
organic layer was separated and dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure and the residue
was purified by column chromatography on silica gel to afford
11 mg (51%) of the title compound 6 as a white solid.
DMSO)
d 159.4, 152.4, 147.8, 140.5, 139.2, 137.7, 136.2, 131.1, 130.3,
129.9, 129.5, 127.7, 125.6, 125.0, 116.7, 115.3, 102.7, 21.0, 10.3; HRMS
for C19H17ClN5 ([MþH]þ) calcd.: 350.1166; found 350.1165.
4.1.9. N4-(4-Methyl-2-(3-methyl-1H-pyrazol-5-yl)phenyl)-N2-(p-
tolyl)quinazoline-2,4-diamine (18a)
1H NMR (600 MHz, DMSO)
d: 9.59 (s, 1H, NH), 8.20 (s, 1H), 7.54
A stirred solution of 16 (65 mg, 0.186 mmol) and 17a (23 mg,
0.22 mmol) in 5 mL of isopropanol was heated to 100 ꢀC in a closed
tube. After 4 h, a pale yellow solid was filtered and the solid was
washed with a minimum amount of cold isopropanol, and dried in
vacuo to afford (52 mg, 66%) of the title compound 18a as a pale
yellow solid.
(d, J ¼ 1.1 Hz, 1H), 7.53 (d, J ¼ 8.1 Hz, 1H), 7.24 (ddd, J ¼ 8.1 Hz, 1.8Hz
and 0.6 Hz), 3.66 (brs,1H, NH), 2.34 (s, 3H, CH3); 13C NMR (150 MHz,
DMSO)
d 153.6, 152.8, 152.2, 141.8, 137.4, 134.0, 133.0, 128.9, 128.0,
120.2, 117.3, 114.2, 20.2; HRMS for C12H10BrClN5 ([MþH]þ) calcd.:
337.9803; found: 337.9797.
1H NMR (600 MHz, DMSO)
d: 13.28 (brs, 1H, NH), 12.84 (brs, 1H,
4.1.6. 2-Chloro-N-(4-methyl-2-(3-methyl-1H-pyrazol-5-yl)
phenyl)-9H-purin-6-amine(15)
NH), 10.46 (brs, 1H, NH), 8.41 (d, J ¼ 8.2 Hz, 1H), 8.28 (brs, 1H), 7.89
(t, J ¼ 7.6 Hz, 1H), 7.64 (d, J ¼ 8.7 Hz, 2H), 7.56 (t, J ¼ 7.6 Hz, 1H), 7.36
(d, J ¼ 7.9 Hz, 2H), 7.21 (d, J ¼ 7.3 Hz, 2H), 6.99 (s, 1H), 6.56 (s, 1H),
2.35 (s, 2 ꢁ 3H, 2 ꢁ CH3); 2.30 (s, 3H, CH3); 13C NMR (150 MHz,
Analogous to the procedure for 13, to a mixture of compound 4
(50 mg, 0.26 mmol) and 11 (60 mg, 0.32 mmol) dissolved in DME
(5 mL) was added anh. K2CO3 (109 mg, 0.79 mmol) at rt. under N2.
After reaction for 24 h, the solvent was evaporated in vacuo, and the
mixture was partitioned between EtOAc (10 mL) and H2O (10 mL).
The aqueous phase was back extracted twice with EtOAc. The
organic phase was dried with Na2SO4 and evaporated. The product
was purified by flash chromatography on silica gel to give 32 mg
(35%) of the title compound 15 as a white solid.
DMSO)
d 158.4, 152.0, 149.7, 140.3, 139.6, 135.4, 135.0, 133.9, 132.1,
129.5, 128.4, 128.2, 125.4, 123.9, 123.8, 123.8, 123.6, 118.0, 111.3,
102.9, 20.7 (2 ꢁ C), 10.5; HRMS for C26H25N6 ([MþH]þ) calcd.:
421.2135; found: 421.2132.
4.1.10. N2-(4-fluorophenyl)-N4-(4-methyl-2-(3-methyl-1H-
pyrazol-5-yl)phenyl)-quinazoline-2,4-diamine (18b)
Alternatively, deprotection of 14 with TFA as described for
compound 6 afforded 15.
A stirred solution of 16 (100 mg, 0.286 mmol) and 17b (38 mg,
0.344 mmol) in 5 mL of isopropanol was heated in a closed vessel
for 6 h at 100 ꢀC. The precipitate formed was filtered and the solid
was washed with a minimum amount of cold isopropanol and dried
under vacuum to afford 56 mg (46%) of the title compound 18b as a
pale yellow solid.
1H NMR (600 MHz, DMSO)
d: 13.36 (s, 1H, NH), 13.14 (s, 1H, NH),
12.10 (s, 1H, NH), 8.68 (d, J ¼ 8.5 Hz, 1H), 8.26 (s, 1H), 7.57 (s, 1H),
7.17 (dd, J ¼ 8.5,1.4 Hz,1H), 6.57 (s,1H), 2.33 (s, 3H, CH3), 2.30 (s, 3H,
CH3); 13C NMR (150 MHz, DMSO)
d: 152.2, 152.0, 151.2, 150.7, 140.9,
139.7, 133.7, 131.8, 128.4, 128.3, 121.2, 120.5, 119.3, 102.5, 20.5, 10.4;
1H NMR (600 MHz, DMSO)
d 13.27 (brs, 1H, NH), 12.80 (s, 1H,
HRMS for C16H15ClN7 ([MþH]þ) calcd.: 340.1072; found: 340.1071.
NH), 10.45 (s, 1H, NH), 8.45 (d, J ¼ 8.1 Hz, 1H), 8.30 (brs, 1H), 7.91 (t,
J ¼ 7.6 Hz, 1H), 7.67 (d, J ¼ 6.1 Hz, 2H), 7.61e7.49 (m, 3H), 7.26 (s,
4.1.7. N-(2-Bromo-4-methylphenyl)-2-chloroquinazolin-4-amine
2H), 7.05 (s, 1H), 6.60 (s, 1H), 2.37 (s, 3H, CH3), 2.30 (s, 3H, CH3); 13
C
(8)
NMR (150 MHz, DMSO)
d 160.6, 159.0, 158.6, 152.4, 152.3, 149.9,
To a solution of compound 7 (3 g, 15.07 mmol) and the protected
140.0, 135.4, 135.0, 133.0, 132.1, 128.4, 128.2, 125.9, 125.4, 123.9,

108
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25H22FN6 ([MþH]þ) calcd.: 425.1884; found: 425.1875.
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presence and in absence of virus. Afterwards, the 50% effective
concentrations (EC50) and the 50% cytotoxic concentrations (CC50),
which are defined as the compound concentration that is required
to inhibit the virus-induced cytopathogenic effect (CPE), are
calculated based on microscopic scoring data. Indeed, besides
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wells were checked microscopically for minor signs of virus-
induced CPE or alterations of host cell morphology caused by the
compound, and these results sometimes deviate from the MTS/PMS
method [44]. More accurate activity data are obtained with a virus
yield reduction assay, which determines the viral RNA load by real-
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Using AutodockTools [45] a box of 60 ꢁ 60 ꢁ 60 units of 0.375 Å
was centered on atom CB of the Lys329 sidechain. Polar hydrogens
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Docking using autodock 4.2 was performed 100 times for every
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Milind Saudi was beneficiary of a scholarship of the Erasmus
Mundus External Cooperation Window (EMCW lot 13). Mass
spectrometry was made possible by the support of the Hercules
Foundation of the Flemish Government (grant 20100225e7). The
antiviral work was supported by EU FP7 project SILVER (contract no
HEALTH-F3-2010-260644). We are indebted to Luc Baudemprez for
providing NMR spectra and to C. Biernaux for final typesetting.
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