Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases

Article abstract of DOI:10.1021/acs.jmedchem.5b00108

This study describes the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug resistance. Starting from known c-Src inhibitors

Full text of DOI:10.1021/acs.jmedchem.5b00108

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Article
Discovery of multi-target antivirals acting on both the dengue
virus NS5-NS3 interaction and the host Src/Fyn kinases
Paolo Vincetti, Fabiana Caporuscio, Suzanne Keptein, Antimo Gioiello, Valentina Mancino,
Youichi Suzuki, Naoki Yamamoto, Emmanuele Crespan, andrea Lossani, Giovanni Maga, Giulio
Rastelli, Daniele Castagnolo, Johan Neyts, Pieter LEYSSEN, Gabriele Costantino, and Marco Radi
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 03 Jun 2015
Downloaded from http://pubs.acs.org on June 3, 2015
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Discovery of multiꢀtarget antivirals acting on both
the dengue virus NS5ꢀNS3 interaction and the host
Src/Fyn kinases
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a
b
g
c
c
Paolo Vincetti, Fabiana Caporuscio, Suzanne Kaptein, Antimo Gioiello, Valentina Mancino,
d
d
e
e
e
Youichi Suzuki, Naoki Yamamoto, Emmanuele Crespan, Andrea Lossani, Giovanni Maga,
b
f
g
g
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Giulio Rastelli, Daniele Castagnolo, Johan Neyts, Pieter Leyssen, Gabriele Costantino and
a,
Marco Radi *
a
P4T Group, Dipartimento di Farmacia, Università degli Studi di Parma, Viale delle Scienze,
27/A, 43124 Parma, Italy.
b
Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via
Campi 183, 41125 Modena, Italy.
c
Laboratory of Medicinal and Advanced Synthetic Chemistry (Lab MASC), Dipartimento di
Scienze Farmaceutiche, Università degli Studi di Perugia, Via del Liceo 1, Iꢀ06123 Perugia,
Italy.
d
Department of Microbiology, Yong Loo Lin School of Medicine, National University of
Singapore, Center for Translational Medicine, 14 Medical Drive, #15ꢀ02, Level 15, Singapore
117599, Singapore
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Istituto di Genetica Molecolare, IGMꢀCNR, Via Abbiategrasso 207, 27100 Pavia, Italy
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Department of Applied Sciences, Northumbria University Newcastle, Ellison Place, NE1 8ST
Newcastle upon Tyne, United Kingdom
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Laboratory of Virology and Experimental Chemotherapy, Rega Institute for Medical Research,
KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
Abstract
This study describe the discovery of novel dengue virus inhibitors targeting both a crucial viral
proteinꢀprotein interaction and an essential host cell factor as a strategy to reduce the emergence
of drugꢀresistance. Starting from known cꢀSrc inhibitors, a virtual screening was performed to
identify molecules able to interact with a recently discovered allosteric pocket on the dengue
virus NS5 polymerase. The selection of cheapꢀtoꢀproduce scaffolds and the exploration of the
biologically relevant chemical space around them suggested promising candidates for chemical
synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus
replication at low micromolar concentrations with no significant toxicity to the host cell. Among
the identified antivirals, compound 16i proved to be ten times more potent than ribavirin, showed
a better selectivity index and represents the firstꢀinꢀclass DENVꢀNS5 allosteric inhibitor able to
target both the virus NS5ꢀNS3 interaction and the host kinases cꢀSrc/Fyn.
Introduction
Dengue fever (DF) or breakꢀbone fever is the most widespread arthropodꢀborne disease in the
world and, each year, accounts for more than 50ꢀ100 million patients, 99% of all reported cases
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of viral hemorrhagic fever, and around 20.000 deaths. The causative agent is the dengue virus
(
DENV), which belongs to the Flaviviridae family and is divided into five serotypes (DENV 1ꢀ
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). DENV is predominantly transmitted through the bite of infected Aedes aegyptii, although
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other mosquito species can be carrier of the virus as well. Considering the expanding
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geographical distribution of both the virus and the mosquito vector, the increasing frequency of
epidemics, and the fact that multiple serotypes are coꢀcirculating in many regions, WHO has
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classified dengue as a major international public health concern. While infection with a single
serotype is often mild and believed to induce lifeꢀlong immunity to that serotype, crossꢀ
protection to other serotypes only lasts for a few weeks and subsequent infection with another
serotype has been associated with the clinically more severe dengue hemorrhagic fever (DHF)
and dengue shock syndrome (DSS). Although vaccines are already available for different
flaviviruses (e.g., yellow fever virus, tickꢀborne encephalitis, and Japanese encephalitis virus),
the development of a vaccine for DENV proves to be very cumbersome because of the existence
of multiple serotypes and the underlying mechanism that leads to DHF/DSS (i.e., antibodyꢀ
dependent enhancement of infection). Past clinical trials with tetravalent vaccines have not
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delivered their promises and a few are still ongoing. Moreover, a drug for the treatment or
prevention of DENV infection is not yet available and the management of patients is limited to
symptomatic treatment and supportive care. Therefore, the development of new smallꢀmolecule
antivirals against DENV is of significant interest.
A direct correlation has been demonstrated between a high DENV viral load in the blood and
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the development of the more severe, lifeꢀthreatening form of the disease. Thus, a drug that
would reduce the viral load is expected to have a significant impact on the number of patients
that will progress to DHF/DSS. Furthermore, a lower viraemia should also result in a decrease in
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the number of uninfected mosquitoes that become carrier after feeding on a viraemic patient, and
thus would also slow down transmission. Therefore, an efficient and safe drug, delivered early in
the course of the disease or even taken prophylactically, will not only save lives but will also
curb epidemics.
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Conventional antiviral drugs are typically designed to directly inhibit virus replication by
targeting viral proteins. Among the ten known DENV proteins, the atomic structures of the
capsid, envelope, NS3 (protease domain, helicase domain, and fullꢀlength NS3), NS5
(methyltransferase and RNAꢀdependent RNA polymerase RdRp) have been solved and have
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provided a solid basis for the development of such inhibitors (Figure 1).
However, because RNA viruses are known to mutate quite rapidly, viral protein alterations that
induce resistance to drugs are expected to emerge, thus jeopardizing the longꢀterm clinical use of
such compounds. Different approaches can be pursued to increase the barrier to resistance: (i) the
implementation of combination treatments with drugs that target different viral proteins, (ii) the
development of compounds that target host cell factors essential for viral replication but
dispensable for host cell survival, and (iii) the design of multiꢀtarget inhibitors that interfere with
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two (or more) targets at the same time, preferably a viral and a host cell factor. Drugs developed
according to the latter two strategies may also offer a therapeutic option in an outbreak setting of
viral pathogens of unknown etiology, or combat those that depend on the same host factor for
replication and against which no drug has been developed so far. Even though the targeting of
host cell proteins is considered a risky approach in the context of antiviral drug development,
many, if not most of the drugs that are on the market do target host cell proteins and the risk of
side effects is wellꢀaccepted. Because viruses heavily rely on host cell proteins for their
replication, many efforts are ongoing to identify proteins that can be targeted with a minimal
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effect on host cell wellbeing. In this context, pursuing the development of drugs with a dual
mechanism of action is expected to allow treatment with a significantly lower dose, which will
reduce the risk of side effects while retaining antiviral efficacy. Furthermore, the use of a single
multiꢀtarget drug would simplify ADMEꢀTOX and PK studies, and the risk of drugꢀdrug
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interactions may be avoided.
Host cell kinases have been proven to be essential for DENV assembly and secretion. In
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particular, cꢀSrc has been identified as one of the kinases that is required for DENV replication.
Src kinase inhibitors Dasatinib and Saracatinib (AZD0530) have been shown to inhibit virion
assembly of DENV 1ꢀ4, thus validating the Src family of tyrosine kinases as potential drug
targets and their inhibitors as promising lead therapeutics for the development of a treatment for
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dengue virus infection (Figure 1).
Viral polymerases have been extensively studied and proven to be suitable targets for the
development of antiviral drugs. Despite the fact that most nucleoside polymerase inhibitors are
characterized by low selectivity and severe side effects, recent mutagenesis studies pointed
towards an allosteric pocket on DENVꢀNS5 (hereafter named Cavity B) that could be exploited
for the development of safer nonꢀnucleoside inhibitors. In fact, mutation of few residues within
this allosteric pocket (Leu328, Lys330, Trp859, Ile863) has proven to interfere with the initiation
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of RNA synthesis and with the formation of the functional NS5ꢀNS3 complex. To the best of
our knowledge, no inhibitor targeting this site on the DENV NS5 protein has been reported so
far. However, compounds targeting a similar site on the thumb domain of HCV NS5B
polymerase have recently been shown to have a promising antiviral profile and are currently in
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clinical trial for the treatment of HCVꢀinfected patients.
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In this study, we aimed to explore the possibility to develop multiꢀtarget compounds acting on
both the host cꢀSrc kinase and the allosteric site on the viral NS5 polymerase as a complementary
strategy to block DENV replication and escape from the insurgence of drugꢀresistance. In fact,
although drugs targeting host cell kinases will not constitute a selective pressure on the virus to
select host mutants, in a few cases, prolonged treatment with kinase inhibitors may result in the
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selection of alternative pathways that could theoretically be exploited for viral replication. As a
consequence, the allosteric inhibition of a viral protein by the same multiꢀtarget molecule may
overcome the potential selection of an alternative kinase pathway while making it more difficult
for the virus to develop an allosteric mutation. The known host kinase involved in DENV
replication (cꢀSrc) and the only druggable allosteric pocket on a DENV protein (cavity B on
NS5) were thus chosen as ideal targets of a multiꢀtarget DENV inhibitor.
Considering the fact that the majority of DENV patients live in underꢀprivileged regions, our
effort was specifically aimed at the identification of antiviral compounds that would be cheap to
produce/optimize by selecting appropriate, easyꢀtoꢀsynthesize chemical scaffolds.
Results and discussion
The identification of costꢀeffective dual NS5/Src inhibitors was accomplished by combining a
structureꢀbased virtual screening on the allosteric site of NS5 and a virtual library generation,
starting from a library of known Src active scaffolds. Structures of compounds with known
bioactivity data against tyrosineꢀprotein kinase Src were obtained from databases of bioactive
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molecules (ChEMBL, Binding DB ) and from an internal collection of kinase inhibitors. Only
compounds with an IC ≤100 µM were retained. While this activity cutoff may also include
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weak Src inhibitors, the higher number of compounds may increase the chance of identifying
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NS5ꢀbinding scaffolds that could be cheap to synthesize and easily modified in a virtual library
expansion to reach the optimal binding efficiency. Moreover, the synergistic effect of a dual
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inhibitor may lead to an improved activity profile. The Glide Standard Precision (SP) docking
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protocol and Autodock Vina were used to dock Src ligands (about 3.000 compounds) to the
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allosteric pocket of the DENV NS5 RdRp. This pocket corresponds to the aboveꢀmentioned
cavity B and is made up by residues Leu327, Leu328, Lys330, Thr858, Trp859, Asn862, Ile863,
and Ala866 (the corresponding numbering of the used DENVꢀ3 RdRp crystal structure is
Leu326, Leu327, Lys329, Thr858, Trp859, Asn862, Ile863, and Ala866). Unless specified
otherwise, DENVꢀ2 RdRp numbering will be used hereafter. Leu328, Trp859, and Ile863 are
highly conserved among flaviviruses. Moreover, alanine scanning experiments have shown that
L327A, W859A, and I863A mutations remarkably reduce the de novo RNA synthesis, while the
K330A mutation inhibits the NS3ꢀNS5 interaction. On the basis of this mutagenesis analysis, it
was hypothesized that compounds that are able to bind to this cavity could act as allosteric NS5
inhibitors or NS3ꢀNS5 proteinꢀprotein interaction inhibitors. Despite the challenge of targeting
proteinꢀprotein interfaces with smallꢀmolecules, stateꢀofꢀtheꢀart structureꢀbased design methods
have proven to be successful in the identification of smallꢀmolecule proteinꢀprotein interaction
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modulators. To select scaffolds for chemical synthesis, the most recurrent scaffolds were
identified by clustering compounds on the basis of the Tanimoto similarity with the Canvas
Similarity and Clustering tool available in the Schrödinger Suite 2011. Clusters were analyzed by
considering first of all the synthetic accessibility of each scaffold. Binding modes within the NS5
allosteric pocket and docking scores further aided in the selection. Finally, compounds 4a and
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3a,b were selected from Autodock Vina docking results, while compounds 16a-c were selected
on the basis of Glide docking results (see Table 1). Starting from the identified scaffolds, the
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biologically relevant chemical space was sampled by docking to the NS5 allosteric site a virtual
library designed by the chemical expansion of the three identified scaffolds (4, 13, and 16).
Based on the synthetic approaches reported for the preparation of these scaffolds (Schemes 1ꢀ4),
a virtual library of synthetically accessible derivatives was designed using the software SmiLib
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v2.0. In particular, a series of building blocks available in our stockroom were combined to
generate ~10,000 virtual compounds that were docked to cavity B using the same docking
protocols/parameters previously applied to the ChEMBL/Binding DB compounds (see the
experimental section for details). Compounds were selected by considering (i) binding modes
within the NS5 allosteric pocket and (ii) docking scores. The set of compounds listed in Table 1
was thus selected and freshly synthesized for evaluation in a virusꢀcellꢀbased assay.
2ꢀPyridone derivatives were synthesized by a twoꢀstep procedure by reacting 4ꢀhydroxyꢀ6ꢀ
methylꢀ2Hꢀpyranꢀ2ꢀone (1) with the amines 2a-d in refluxing water, followed by alkylation of
the C4 hydroxy group with substituted benzylbromides to give the desired compounds 4a-e
(Scheme 1). For the synthesis of the triazole derivatives 6a,b, the approach described above
proved to be unsuccessful either using the required triazoloꢀamines directly or by trying to build
the triazole ring in two steps through the propargyl derivative 5. In the latter case, the reaction of
2ꢀpyranone 1 with propargylamine in refluxing water gave the side product 7 with no trace of the
desired derivative 5. It is likely that compound 7 derives from the nucleophilic attack of 5 in the
enol form to the triple bond. This was indeed avoided by blocking the phenolic OH by Oꢀ
alkylation as reported in Scheme 2. As an alternative strategy to prepare compounds 6a,b, 2ꢀ
pyranone 1 was first converted into the corresponding pyridinꢀ2ꢀone by reaction with
concentrated ammonia, followed by C4 Oꢀalkylation to give the intermediate 8a (Scheme 2). The
latter intermediate was selectively Nꢀpropargylated by treatment with NaH/LiBr in a mixture of
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DMF and DME to give the desired compound 9a in good yields. Final compounds 6a,b were
quickly obtained by reacting 9a with substituted benzylbromides under microwaveꢀassisted
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Clickꢀchemistry conditions.
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Quinolineꢀ3ꢀcarbonitrile derivatives 13a-c were synthesized following a previously reported
procedure (Scheme 3): the tandem condensation/cyclization reaction of anilines 10a,b with ethyl
2ꢀcyanoꢀ3ꢀethoxyacrylate under microwave irradiation led to 4ꢀquinolones 11a,b in good yields
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with no need of any chromatographic purification. The C4ꢀchlorination of 11a,b with
phosphorus oxychloride gave 4ꢀchloroquinolines 12a,b that were directly submitted to a
nucleophilic substitution with selected aniline derivatives to afford the final compounds 13a-c.
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Compounds 13a,b have been previously reported as potent cꢀSrc inhibitors. Purine derivatives
16a-m were easily obtained by developing a fast and practical twoꢀstep microwaveꢀassisted
protocol starting from commercially available 2,6ꢀdichloro purine 14 (Scheme 4). In the first
step, regioselective C4 nucleophilic substitution with different anilines in the presence of
triethylamine led to the monosubstitued intermediates 15a-d that were finally submitted to a C2
nucleophilic substitution with a wide range of different amines to give final compounds 16a-m
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(see Table 1). Compounds 16a-c were previously reported as potent cꢀSrc inhibitors. The
antiviral activity of all synthesized compounds was evaluated in either a CPE reduction assay or
a virus yield reduction assay (for details on the assays, see the experimental section). In both
assays, DENV serotype 2 was used and the assay was performed in a clone of Vero (African
Green Monkey kidney) cells that has been selected for high susceptibility to dengue virus
infection. The EC values (the concentration at which 50% inhibition of virusꢀinduced CPE or
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viral RNA replication in infected, treated cells is observed), as well as CC50 values (the
concentration at which 50% reduction of metabolic activity in uninfected, treated cells is
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observed) were determined. Microscopic evaluationꢀbased eligibility criteria were also applied in
the identification of promising “active compounds”: (i) no alteration of normal cell morphology
in treated, uninfected cells and (ii) a significant reduction of virusꢀinduced CPE or viral RNA
replication. Moreover, the selectivity index (defined as CC /EC ) should at least be greater than
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10 for a compound to be even considered as a selective antiviral. Results are reported in Table
in comparison with ribavirin, which was used as a reference compound. While 2ꢀpyridones
4a-e; 6a,b) and quinolineꢀ3ꢀcarbonitrile derivatives (13a-c) were characterized by a high
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cellular toxicity, the purine derivatives (16a-m) showed promising antiviral activity. Known Srcꢀ
inhibitors 16a-c showed no antiviral activity, and no or little toxicity (only for 16c). Purines
bearing a 4ꢀacetylaniline moiety at the C6 position (16d-f) were characterized by a high
micromolar activity and a low selectivity index independent of the C2 substituent. Purines
bearing a 3ꢀhydroxyaniline moiety at C6 showed the most interesting antiꢀDENV profile, with
the only exception of the C2ꢀbenzyl derivative 16g: compounds 16h-m showed better EC50
values than the broadꢀspectrum inhibitor ribavirin and a promising selectivity index. A typical
doseꢀresponse curve is depicted in Figure 4 for the best antiꢀDENV compound 16i. All the
synthesized compounds were then tested in a recently developed AlphaScreen assay to evaluate
24
their ability to block the formation of the functional NS3/NS5 complex. A preliminary
AlphaScreen assay was performed with 500 µM compound: only purine derivatives (16a-m)
2
5
showed inhibition of the NS5ꢀNS3 interaction at this high concentration. The previously
unpublished purines 16d-m were subsequently tested at lower concentrations (50 µM for 16d-m
and 25 µM for 16e,f): compound 16i emerged as the only hit able to inhibits the NS3ꢀNS5
interaction by 33% at 50 µM and represents the firstꢀinꢀclass inhibitor with such a mechanism of
action (Figure 2). Our previous study on the establishment of an AlphaScreenꢀbased NS3ꢀNS5
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interaction assay has demonstrated that 25 ꢀ 100 nM of recombinant NS3 and NS5 is needed to
obtain a significant level of luminescent interaction signals with a robust Z' factor, and also
showed that an excess amount of competitor molecules was required to block the NS3ꢀNS5
interaction in the AlphaScreen assay. Since NS3ꢀNS5 protein concentrations in DENVꢀinfected
cells are considered to be lower than those used in the in vitro AlphaScreen assay, it is
reasonable to assume that the concentration of small molecules that is required to disrupt the in
vitro NS3ꢀNS5 interaction may be lower than that used in the AlphaScreen assay and, hence, its
contribution to the potency of the antiviral activity may be underestimated. Although 16i was not
very potent in inhibiting the NS3ꢀNS5 proteinꢀprotein interaction, possible underestimation in
the AlphaScreen assay and the lack of inhibitors endowed with this novel mechanism of action
for comparison purposes, make this compound a promising candidate for further
optimization/investigation. In addition, the AlphaScreen assay can only indicate allosteric
compounds that disrupt the NS3ꢀNS5 interaction by interfering with the Lys330 functions, as
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suggested by previous mutagenesis studies, while allosteric antivirals that block the initiation
of RNA synthesis cannot be detected. Nonetheless, the in vitro antiviral effect of DENV
replication may be the result of its action on multiple targets, as per our design strategy.
Consequently, the most active inhibitors 16h-m and selected compounds from all synthesized
chemical classes (4, 6, 13 and 16) were subsequently analyzed for their activity on kinases
known to be involved in DENV replication. Interestingly, after the start of this work, the Yang
group reported that Fyn kinase has a more pronounced role (compared to Src) in the replication
of DENV: Fyn inhibition by treatment with Dasatinib and Saracatinib (nanomolar inhibitors of
3
3,34
both Fyn and Src)
was proposed as the main mechanism responsible for their antiꢀDENV
activity (EC around 1.0 and 6.0 µM, respectively) without excluding the possibility that these
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compounds may affect processes beyond RNA replication. A mutation in the viral NS4B
26
protein (T108I) was in fact selected after repeated treatments with Dasatinib. Although cꢀSrc
inhibitors showed low micromolar ID50s in our experimental conditions (see Table 2, entries 4ꢀ5,
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ꢀ9), a few general considerations can be drawn: i) 2ꢀpyridone derivatives 4 and 6 were inactive
against cꢀSrc/Fyn and highly cytotoxic in cellꢀbased assays; ii) known cꢀSrc inhibitors 13a,b
were also active against Fyn while 13c was completely inactive. All these quinolone derivatives
are however highly cytotoxic in cellꢀbased assays; iii) all purine derivatives tested showed a
similar range of activity against cꢀSrc and Fyn but only compounds 16d-m were able to inhibit
DENV replication. Overall, these data indicate that the dual cꢀSrc/Fyn inhibition may not be a
sufficient prerequisite for the identification of an effective DENV inhibitor and many different
factors, still unidentified and difficult to predict, may play a key complementary role in the
replication of dengue virus. At antiviral concentrations, Dasatinib and Saracatinib are in fact
reported to inhibit many different kinases and further experiments are needed to clearly
understand the mechanism of action of these drugs that exhibit an antiviral potency comparable
to that of our compounds. At the present however, we cannot exclude that our compounds (but
also Dasatinib/Saracatinib) could potentially exploit a different mechanism of action, as the one
reported for the cꢀSrc inhibitor herbimycin A, which inhibits HCV replication by blocking the
36
interaction between cꢀSrc and the viral proteins NS5/NS3.
Finally, the binding mode of the dual compound 16i was further confirmed with Induced Fit
Docking (IFD), i.e., a docking protocol that takes into account protein flexibility, thus allowing
the retrieval of optimized binding modes. Protein flexibility, which always plays a pivotal role in
all proteinꢀligand recognition events, may be particularly important in the case of the solventꢀ
17
exposed cavity B that is part of a proteinꢀprotein interface. Compound 16i was characterized by
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a good steric and electronic complementarity with the cavity B of DENV polymerase (interacting
with six out of the eight conserved residues that line cavity B) and was the topꢀscoring hit in
terms of Glide SP ligand efficiency among the tested compounds (Figure 3). The 6ꢀamine NH of
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6i makes a hydrogen bond with the backbone carbonyl oxygen of Leu327 (DENVꢀ3 Leu326),
while the ligand hydroxyl group forms a hydrogen bond with the sideꢀchain carboxylate of
Asp333 (DENVꢀ3 Asp332). Moreover, 16i makes favorable hydrophobic contacts with Leu327
(DENVꢀ3 Leu326), Trp859, Ile863, Ala866, and the alkyl chains of Lys330 (DENVꢀ3 Lys329)
and Asn862. The binding mode is further stabilized by a cationꢀpi interaction between the purine
ring and the side chain of Lys330 (DENVꢀ3 Lys329). As is evident from the aboveꢀdescribed
binding mode, 16i interacts with Lys330, Trp859, and Ile863, i.e., three out of the four residues
that have been shown to be critical for viral replication. Notably, the ability of 16i to disrupt the
NS3ꢀNS5 interaction may be ascribed to the hydrophobic contacts and the cationꢀpi interaction
with Lys330 that is involved in the recognition of NS3.
Conclusions
In summary, we have described a multidisciplinary approach for the discovery of dengue virus
inhibitors that act on the viral RdRp (NS5) as well as on host cell kinases previously shown to be
involved in DENV replication (cꢀSrc, Fyn). Docking of known cꢀSrc active scaffolds to the
allosteric pocket of DENV NS5 RdRp allowed the selection of three cheapꢀtoꢀproduce chemical
scaffolds that were subsequently expanded in a virtual library of highly substituted derivatives to
identify the most promising compounds for chemical synthesis. The synthesized compounds
were evaluated for their: (i) antiviral activity in a virusꢀcellꢀbased assay for DENVꢀ2, (ii) ability
to block the NS3ꢀNS5 interaction, and (iii) activity on cꢀSrc and Fyn kinases. Purine derivatives
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6h-m inhibited DENVꢀ2 replication and kinase activity at low micromolar concentrations, with
an antiviral profile comparable to that of Dasatinib and Saracatinib. Notably, compound 16i was
discovered as the first multiꢀtarget antiviral blocking the formation of the viral NS3ꢀNS5
complex and inhibiting the activity of host cꢀSrc/Fyn kinases. This compound can therefore be
considered as a promising starting point for further optimization and development.
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Experimental section
Molecular modeling
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Glide docking. ChEMBL and Binding DB databases were searched for tyrosineꢀprotein
kinase Src inhibitors. Collected inhibitors from the two databases were merged and only
27
compounds whose IC50 was ≤ 100 µM were retained. The Schrödinger Suite 2011 Virtual
Screening Workflow (VSW) tool was used to i) remove duplicates and compounds with a
molecular weight higher than 600 g/mol; ii) generate stereoisomers and retain up to 4
stereoisomers for compounds with unspecified stereocenters; and iii) generate tautomers and
ionization states at a pH range of 6ꢀ8 using Epik and opting for the removal of highꢀenergy
tautomers and ionization states. Virtual library compounds were prepared using the same
protocol. The dengue virus DENVꢀ3 RNAꢀdependent RNA polymerase (RdRp) catalytic domain
structure was downloaded from the Protein Data Bank (PDB code 2J7U). The structure was then
processed with the Schrödinger Suite 2011 Protein Preparation Wizard (PPW) tool.
Crystallization additives and water molecules were removed, while the magnesium and zinc ions
were retained. PPW automatically adjusted the ionization and tautomerization state of the protein
at a neutral pH, set the orientation of any misoriented groups (Asn, Gln, and His residues) as well
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as charges and atom types for metal atoms, and optimized the hydrogen bond network.
Moreover, side chains that were missing, even if away from the region of interest, were added
and optimized by running a Prime structure refinement job through the PPW graphical interface.
Finally, the protein structure was refined to relieve steric clashes with a restrained minimization
with the OPLS2005 force field till a final RMSD of 0.30 Å with respect to the input protein
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coordinates. Docking was performed with Glide 57114 (Schrödinger Suite 2011). The protein
structure, prepared as described above, was used to build the energy grid. The enclosing box was
centered at the centroid of residues Leu326, Leu327, Lys329, Thr858, Trp859, Asn862, Ile863,
and Ala866 (the numbering of amino acids is based on DENVꢀ3 RdRp), which define cavity B
28
as reported by Malet et al. Box dimensions and ligand diameter midpoint box sides were set to
30 Å × 30 Å × 30 Å and 10 Å ×10 Å × 10 Å, respectively. The Glide Standard Precision (SP)
docking protocol was used. Ligands were docked flexibly, the sampling of ring conformations
and nitrogen inversions was included, and nonꢀplanar amide conformations were penalized. Epik
state penalties were added to docking scores. All SP parameters were set to their default values.
Docking results were filtered to remove compounds that did not contact residues Leu326,
Leu327, Lys329, Thr858, Trp859, Asn862, Ile863, and Ala866 by using the Schrödinger Suite
2011 Pose Filter tool with a contact maximum distance of 3.5 Å. Remaining compounds were
clustered with the Canvas Similarity and Clustering tool that is available through the Schrödinger
Suite 2011 Maestro graphical interface by using the MolPrint2D fingerprints, the Tanimoto
similarity metric, and the Average linkage method. Virtual library compounds were docked with
the same Glide SP docking protocol. Binding modes of purine derivatives were reꢀgenerated with
2
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Induced Fit Docking. An initial Glide SP docking of the ligand was performed by using a
softened potential, i.e., a van der Waals radius scaling factor of 0.50 for receptor atoms with a
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partial atomic charge (absolute value) less than 0.25 and 0.50 for ligand atoms with a partial
atomic charge (absolute value) less than 0.15. A maximum number of fifty poses were saved and
submitted to the subsequent Prime sideꢀchain orientation prediction of residues within a shell of
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Å around the ligand. After the Prime minimization of the selected residues and the ligand for
each pose, a Glide SP reꢀdocking of each proteinꢀligand complex structure within 30 kcal/mol of
the lowest energy structure was performed. Finally, the binding energy (IFDScore) for each
output pose was calculated.
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Autodock Vina docking. Docking studies were performed with Autodock Vina through
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PyRx, while PyMol was used to visualize the results. Ligands were prepared by first
generating an energy minimized 3D structure in Openbabel and then processed with Autodock
Tools 1.5.4 to assign Gasteiger charges, merge nonpolar hydrogens, and set torsional bonds.
Docking runs were performed within a 30 Å × 30 Å × 30 Å cubic box (grid spacing = 0.375 Å)
surrounding the above described allosteric pocket. A search exhaustiveness of 8 was used and
output modes were ranked according to the binding affinity.
Chemistry
General. All commercially available chemicals were purchased from both Sigma ꢀ Aldrich
and Alfa Aesar and, unless otherwise noted, used without any previous purification. Solvents
used for workꢀup and purification procedures were of technical grade. TLC was carried out using
SigmaꢀAldrich TLC plates (silica gel on Al foils, SUPELCO Analytical). Where indicated,
products were purified by silica gel flash chromatography on columns packed with Merck
1
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Geduran Si 60 (40ꢀ63 µm). H and C NMR spectra were recorded on BRUKER AVANCE 300
MHz and BRUKER AVANCE 400 MHz spectrometers. Chemical shifts (δ scale) are reported in
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parts per million relative to TMS. HꢀNMR spectra are reported in this order: multiplicity and
number of protons; signals were characterized as: s (singlet), d (doublet), dd (doublet of
doublets), ddd (doublet of doublet of doublets), t (triplet), m (multiplet), bs (broad signal). ESIꢀ
mass spectra were recorded on an API 150EX apparatus and are reported in the form of (m/z).
Elemental analyses were performed on a PerkinꢀElmer PE 2004 elemental analyzer, and the data
for C, H, and N were within 0.4% of the theoretical values. Melting points were taken using a
Gallenkamp melting point apparatus and were uncorrected. All target compounds possessed a
purity of ≥95% as verified by elemental analyses by comparison with the theoretical values.
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Microwave Irradiation Experiments. Microwave reactions were conducted using a CEM
Discover Synthesis Unit (CEM Corp., Matthews, NC). The machine consists of a continuous
focused microwave power delivery system with an operatorꢀselectable power output from 0 to
3
00 W. The temperature inside the reaction vessel was monitored using a calibrated infrared
temperature control mounted under the reaction vessel. All experiments were performed using a
stirring option whereby the reaction mixtures were stirred by means of a rotating magnetic plate
located below the floor of the microwave cavity and a Teflonꢀcoated magnetic stir bar in the
vessel.
General Procedures for the Synthesis of intermediates 3a-d: To an aqueous suspension (16
mL) of 4ꢀhydroxyꢀ6ꢀmethylꢀ2ꢀpyrone (1) (500 mg, 3.96 mmol) the proper amine (3.96 mmol)
(
2a-d) was added dropwise. The resulting reaction mixture was heated at reflux for 2ꢀ7 hours,
then cooled down to room temperature. A precipitate was obtained and separated by filtration
over a Buchner funnel. The solid was purified by flash chromatography using the proper eluent.
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a: CHCl /MeOH 99/1ꢀ97/3; 3c: CHCl /MeOH 99/1; 3d: CHCl /MeOH 98/2. Compound 3b
3
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was used in the following step without any further purification.
1-(4-chlorobenzyl)-4-hydroxy-6-methylpyridin-2(1H)-one (3a). Yield: 55%. MS (ESI) [Mꢀ
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ꢀ
1
H] : 248.1 m/z; HꢀNMR (DMSOꢀd 300 MHz): δ 2.16 (s, 3H), 5.16 (bs, 2H), 5.59 (d, 1H, J =
6
3.58 Hz), 5.80 (dd, 1H, J = 2.64, 0.81 Hz), 7.12 (d, 2H, J = 8.64 Hz), 7.39 (d, 2H, J = 8.58 Hz),
1
0.50 (bs, 1H).
4-hydroxy-1-(4-methoxybenzyl)-6-methylpyridin-2(1H)-one (3b). Yield: 34%. MS (ESI)
+
+ +
1
[M+H] : 246.5 m/z; [M+Na ] : 268.4 m/z; HꢀNMR (DMSOꢀd 300 MHz) δ 2.17 (s, 3H), 3.71
6
(s, 3H), 5.10 (bs, 2H), 5.58 (d, 1H, J = 2.61 Hz), 5.77 (dd, 1H, J = 2.62, 0.76 Hz), 6.88 (d, 2H, J
=
8.76 Hz), δ= 7.04 (d, 2H, J = 8.82 Hz), 10.48 (bs;1H).
4-hydroxy-6-methyl-1-(4-(trifluoromethyl)benzyl)pyridin-2(1H)-one (3c). Yield: 40%. MS
+
+ +
1
(ESI) [M+H] : 284.1 m/z; [M+Na ] : 306.2 m/z; HꢀNMR (DMSOꢀd 300 MHz): δ 2.16 (s, 3H),
6
5
.27 (bs, 2H), 5.62 (d, 1H, J = 2,61 Hz), 5.84 (dd, 1H, J = 2.64, 0.81 Hz), 7.30 (d, 2H, J = 7.98
Hz), 7.69 (d, 2H, J = 8.07 Hz), 10.59 (bs, 1H).
-([1,1'-biphenyl]-4-ylmethyl)-4-hydroxy-6-methylpyridin-2(1H)-one (3d). Yield: 50%.
1
+
1
MS (ESI) [M+H] : 292.5 m/z; HꢀNMR (DMSOꢀd 300 MHz): δ 2.21 (s, 3H), 5.23 (bs, 2H),
6
5
.62 (d, 1H, J = 2.43 Hz), 5.82 (d, 1H, J = 1.98 Hz), 7.19 (d, 2H, J = 8.13 Hz), 7.35 (t, 1H, J =
.27 Hz), 7.45 (t, 2H, J = 7.48 Hz), 7.62 (m, 4H), 10.55 (bs, 1H).
7
General Procedures for the Synthesis of Compounds 4a-e: In a threeꢀway necked roundꢀ
bottom flask, intermediates 3a-d (0.706 mmol) and K CO (196 mg, 1.41 mmol) were stirred at
2
3
room temperature for 15 minutes in dry DMF (7 mL). The proper benzylbromides (0.85 mmol),
diluted in dry DMF (3 mL), were added dropwise to the reaction mixture. The resulting
suspension was stirred at room temperature for 2 hours, after which H O and ethyl acetate were
2
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added to the reaction mixture. The organic phase was washed with an aqueous solution of LiCl
(
5%), brine, dried over Na SO and concentrated under vacuum. The crude was purified by flash
2 4
chromatography using the proper eluent. 4a: petroleum ether/ethylacetate 8/2ꢀ1/1; 4b: petroleum
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ether/ethylacetate 8/2ꢀ0/10; 4c and 4d: CHCl /MeOH 99/1; 4e: petroleum
3
ether/ethylacetate/MeOH 73/20/2ꢀ70/20/5.
1-(4-chlorobenzyl)-4-((3-fluorobenzyl)oxy)-6-methylpyridin-2(1H)-one (4a). Yield: 55%.
+
1
Mp 99ꢀ100 °C. MS (ESI) [M+H] : 358.00 m/z. HꢀNMR (CDCl 300 MHz): δ 2.20 (s, 3H), 4.99
3
1
3
(s, 2H), 5.22 (s, 2H), 5.86 (d, 1H, J = 1.47 Hz), 5.96 (d, 1H, J = 2.04 Hz), 7.82 (m, 8H). Cꢀ
NMR (CDCl 75 MHz): δ 19.95, 45.51, 68.57, 95.31, 101.19, 113.84, 114.74, 122.45, 127.38,
3
1
28.38, 129.80, 132.55, 134.88, 137.47, 145.93, 160.79, 164.36, 165.80. Anal. (C H ClFNO )
20 17 2
C, H, N.
4-((3-fluorobenzyl)oxy)-1-(4-methoxybenzyl)-6-methylpyridin-2(1H)-one (4b). Yield:
+
+
1
4
2%. Mp 102ꢀ103 °C. MS (ESI) [M+H] : 354.3 m/z, [M+Na] :376.3 m/z. HꢀNMR (CDCl 400
3
MHz): δ 2.23 (s, 3H), 3.76 (s, 3H), 4.99 (s, 2H), 5.21 (s, 2H), 5.84 (s, 1H), 5.96 (s, 1H), 6.83 (d,
H, J = 8 Hz), 7.03 (t, 1H, J = 8.4 Hz), 7.11 (d, 2H, J = 8.4 Hz), 7.15 (d, 2H, J = 8 Hz), 7.35 (bq,
2
1
3
1H, J = 6.4 Hz ). CꢀNMR (CDCl 100 MHz): δ 20.53, 46.08, 55.27, 69.03, 95.88, 101.39,
3
1
14.16, 114.36, 115.24, 122.9, 127.84, 128.90, 130.28, 138.08, 146.69, 158.83, 161.75, 164.20,
65.29, 166.13. Anal. (C H FNO ) C, H, N.
1
2
1
20
3
4-((2,4-difluorobenzyl)oxy)-6-methyl-1-(4-(trifluoromethyl)benzyl)pyridin-2(1H)-one
+
1
(
4c). Yield: 55%. Mp 136ꢀ137 °C. MS (APCI) [M+H] : 410.0 m/z. HꢀNMR (CDCl 300 MHz):
3
δ 2.22 (s, 3H), 5.03 (s, 2H), 5.34 (s, 2H), 5.86 (dd, 1H; J = 2.73, 0.78 Hz), 6.03 (d, 1H, J = 2.73
Hz), 6.91 (m, 2H), 7.28 (d, 2H, J = 8.01 Hz), 7.43 (dt, 1H, J = 8.4 6.66 6.66 Hz), 7.58 (d, 2H, J =
,
,
13
8
.1 Hz). CꢀNMR (CDCl 75 MHz): δ 19.95, 45.51, 54.70, 68.46, 95.27, 100.89, 113.56,
3
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2
2
2
2
2
2
2
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3
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6
1
13.59, 113.78, 114.64, 122.41, 127.25, 128.33, 129.75, 137.56, 146.24, 158.25, 160.74, 164.38,
1
65.64. Anal. (C H F NO ) C, H, N.
21 16
5
2
1-([1,1'-biphenyl]-4-ylmethyl)-4-((2,4-difluorobenzyl)oxy)-6-methylpyridin-2(1H)-one
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
+
+
(4d). Yield: 50%. Mp 134ꢀ135 °C (with decomposition). MS (ESI) [M+H] :418.3 m/z, [M+Na] :
1
440.4 m/z. HꢀNMR (CDCl 400 MHz): δ 2.28 (s, 3H), 5.04 (s, 2H), 5.34 (s, 2H), 5.86 (s, 1H),
3
1
3
6
.05 (d, 1H, J = 2 Hz), 6.91 (m, 2H), 7.41 (m, 10H). CꢀNMR (CDCl 100 MHz): δ 20.60,
3
4
6.40, 63.35, 63.26, 95.73, 101.45, 104.16, 111.58, 118.78, 126.85, 127.04, 127.32, 127.53,
128.78, 131.15, 135.84, 140.5, 146.64, 160.8, 163.29, 164.48, 165.28. Anal. (C H F NO ) C,
2
6
21
2
2
H, N.
-((2,4-difluorobenzyl)oxy)-1-(4-methoxybenzyl)-6-methylpyridin-2(1H)-one (4e). Yield:
4
+
1
34%. Mp 121 °C. MS (ESI) [M+H] : 372.1 m/z. HꢀNMR (CDCl 400 MHz): δ 2.24 (s, 3H),
3
3.78 (s, 3H), 5.01 (s, 2H), 5.22 (s, 2H), 5.81 (d, 1H, J = 2.16 Hz), 6.00 (d, 1H, J = 2.44 Hz), 6.84
1
3
(d, 2H, J = 8.48 Hz), 6.90 (m, 2H), 7.12 (d, 2H, J = 8.48 Hz), 7.42 (q, 1H, J = 8.28 Hz). Cꢀ
NMR (CDCl 100 MHz): δ 20.52, 46.07, 55.27, 63.26, 95.70, 101.27, 104.12, 111.56, 114.14,
3
118.80, 127.84, 128.90, 131.11, 146.69, 158.82, 160.75, 163.24, 165.25, 166.07. Anal.
(C H F NO ) C, H, N.
2
1
19
2
3
5-methyl-2-methylene-2,3-dihydro-7H-oxazolo[3,2-a]pyridin-7-one (7). To an aqueous
suspension (16 mL) of 4ꢀhydroxyꢀ6ꢀmethylꢀ2ꢀpyrone (1) (500 mg, 3.96 mmol) propargylamine
(253 µL, 3.96 mmol) was added portionwise in three subsequent additions every 60 minutes. The
resulting mixture was refluxed for 3 hours, then cooled down to room temperature; a precipitate
was obtained and separated by filtration over a Buchner funnel. The solid was purified by flash
+
chromatography using (CHCl /MeOH 95/5ꢀ9/1). Yield: 50%. MS (APCI) [M+H] : 164.4 m/z.
3
1
HꢀNMR (DMSOꢀd 300 MHz): δ 2.20 (s, 3H), 4.63 (dt, 1H, J = 2.18, 2.22, 3.45 Hz), 4.88 (dt,
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H, J = 2.55, 2.55, 3.45 Hz), 4.97 (t, 2H, J = 2.37, 2.43 Hz), 5.56 (d, 1H, J = 2.19 Hz), 5.82 (dd,
1
3
H, J = 2.13, 2.16 Hz). CꢀNMR (CDCl 75 MHz): δ 17.43, 47.55, 86.95, 91.33, 113.83,
3
43.40, 152.42, 157.99, 179.33.
10
11
12
13
14
15
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58
59
60
Synthesis of 4-hydroxy-6-methylpyridin-2(1H)-one. 4ꢀhydroxyꢀ6ꢀmethylꢀ2ꢀpyrone (5g, 39.7
mmol) and concentrated ammonia (35 mL) were heated at reflux for 4 hours. After cooling down
to room temperature, the reaction mixture was concentrated under vacuum until a brown
precipitate was obtained. The solid was separated by filtration over a Buchner funnel and washed
+
1
with H O. Yield: 90%. MS (APCI) [M+H] : 126.3 m/z. HꢀNMR (DMSOꢀd 300 MHz): δ 2.08
2
6
(
s, 3H), 5.35 (s, 1H), 5.61 (s, 1H), 10.53 (bs, 1H), 11.03 (bs, 1H).
Synthesis of 4-((2,4-difluorobenzyl)oxy)-6-methylpyridin-2(1H)-one (8a). 4ꢀhydroxyꢀ6ꢀ
methylpyridinꢀ2(1H)ꢀone (500 mg, 3.99 mmol) and K CO (552 mg, 3.99 mmol) were stirred at
2
3
room temperature for 15 minutes in dry DMF (20 mL) until the suspension turned green. 1ꢀ
bromomethyl)ꢀ2,4ꢀdifluorobenzene (513 µL, 3.99 mmol), in dry DMF (10 mL), was added
(
dropwise to the reaction mixture. The resulting suspension was stirred at room temperature for 6
hours and then H O and ethyl acetate were added. The organic phase was washed with an
2
aqueous solution of LiCl (5%), brine, dried over Na SO and concentrated under vacuum. The
2
4
crude was purified by flash chromatography using chloroform/methanol (99/1ꢀ95/5) as eluent.
ꢀ
Yield: 50%. MS (APCI) [MꢀH] :
1
HꢀNMR (DMSOꢀd 400 MHz): δ 2.09 (s, 3H), 5.03 (s, 2H), 5.69 (s, 1H), 5.73 (s, 1H), 7.13 (t,
6
1
H, J = 8.26, 8.26 Hz), 7.30 (t,1H; J = 8.9, 8.9 Hz), δ= 7.60 (q, 1H; J = 8.04, 8.04, 8.04 Hz), δ=
1.16 (bs, 1H).
1
Synthesis of 4-((2,4-difluorobenzyl)oxy)-6-methyl-1-(prop-2-yn-1-yl)pyridin-2(1H)-one
(
9a). Compound 8a (200 mg, 0.80 mmol) was suspended in a mixture of dry DME and dry DMF
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(
4/1 in volume). The suspension was cooled to 0 °C, NaH (60% dispersion in mineral oil) (35
mg, 0.86 mmol) was added and the reaction mixture was stirred at room temperature for 15
minutes. LiCl (67 mg, 1.6 mmol) was added and the mixture was stirred for 15 minutes. Then,
propargyl bromide (177 µL, 1.6 mmol) was added and the reaction mixture was stirred at 65 °C
0
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5
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8
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0
for 26 hours, after which H O and ethyl acetate were added. The organic phase was washed with
2
an aqueous solution of LiCl (5%), brine, dried over Na SO and concentrated under vacuum. The
2
4
crude was purified by flash chromatography using ethyl acetate/methanol (98/2ꢀ9/1) as eluent.
+
1
Yield: 73%. MS (APCI) [M+H] :290.3 m/z. HꢀNMR (CDCl 300 MHz): δ 2.25 (t, 1H, J = 2.50,
3
2
.50 Hz), 2.47 (s, 3H), 4.83 (d, 2H, J = 2.52 Hz), 4.99 (s, 2H), 5.84 (dd, 1H, J = 2.76, 0.84 Hz),
.90 (m, 2H), δ= 7.40 (m, 1H).
6
General Procedures for the Synthesis of Compounds 6a-b: In a microwave tube, sodium
azide (5 mg, 0.076 mmol) and the opportune benzylbromide (0.076 mmol) were suspended in
mixture 1/1 of H O/tꢀBuOH (3 mL) and stirred at room temperature for 10 minutes. Sodium
2
ascorbate (1.34 mg, 0.0076 mmol) and CuSO (0.121 mg, 0.00076 mmol) were added to the
4
tube, the mixture was stirred at room temperature for 10 minutes, 9a (22 mg, 0.076 mmol) was
then added and the tube was heated at 125 °C for 10 minutes in a microwave oven (max µW
power input: 250 W; ramp time: 1 minute; power max: off; maximum pressure: 180 psi). At the
end of the irradiation, the tube was cooled down to room temperature and NaOH 0.1 M was
added till pH = 14. The solid formed at the bottom of the tube was separated by filtration over a
Hirsch funnel and washed with NaOH 0.1 M, H O and petroleum ether.
2
1-((1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-4-((2,4-difluorobenzyl)oxy)-6-
methylpyridin-2(1H)-one (6a). Yield: 70%. Mp 188ꢀ189 °C (decomposition: 180 °C). MS
+
+
1
(ESI) [M+H] :501.2 m/z, [M+Na] : 523.2 m/z. HꢀNMR (DMSOꢀd 400 MHz): δ 2.44 (s, 3H),
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.04 (s, 2H), 5.13 (s, 2H), 5.56 (s, 2H), 5.86 (bs, 1H), 5.91 (d, 1H), 7.13 (m, 1H), 7.32 (m, 3H),
1
3
.57 (m, 3H), 8.08 (s, 1H). CꢀNMR (DMSOꢀd 100 MHz): δ 20.42, 38.69, 52.35, 63.66, 95.22,
6
00.42, 104.85, 112.23, 119.70, 122.25, 124.44, 127.63, 131.41, 132.95, 139.05, 143.94, 147.73,
59.76, 162.02, 162.55, 163.80, 164.32, 166.24. Anal. (C H BrF N O ) C, H, N.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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55
56
57
58
59
60
2
3
19
2
4
2
4-((2,4-difluorobenzyl)oxy)-6-methyl-1-((1-(3-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-
yl)methyl)pyridin-2(1H)-one (6b). Yield: 61%. Mp162ꢀ163 °C (decomposition: 160 °C). MS
+
+
1
(
ESI) [M+H] :491.4 m/z, [M+Na] : 513.4 m/z. H ꢀNMR (DMSOꢀd6 400 MHz): δ 2.45 (s, 3H),
5.04 (s, 2H), 5.14 (s, 2H), 5.67 (s, 2H), 5.86 (d, 1H, J = 3.6 Hz), 5.92 (d, 1H, J = 2.92 Hz), 7.13
13
(
m, 1H), 7.31 (m, 1H), 7.60 (m, 3H), 7.70 (bs, 2H), 8.12 (s, 1H). CꢀNMR (DMSOꢀd 400
6
MHz): δ 20.39, 38.70, 52.45, 63.67, 95.20, 100.40, 104.63, 112.26, 119.77, 123.11, 124.53,
125.17, 125.41, 125.82, 129.66, 130.42, 132.70, 132.94, 137.85, 143.98, 147.72, 159.99, 161.77,
162.47, 163.80, 164.23, 166.24. Anal. (C H F N O ) C, H, N.
24 19 5 4 2
General Procedure for the Synthesis of 11a-b: Ethyl 2ꢀcyanoꢀ3ꢀethoxyacrylate (275 mg,
.62 mmol) and the proper aniline (10a,b) (1.62 mmol) were heated at 120 °C for 5 minutes in a
1
microwave oven (max µW power input: 250 W; ramp time: 1 minute; reaction time: 5 minutes;
power max: off; maxim pressure: 190 psi). The solid formed at the bottom of the tube was
separated by filtration, suspended in 5 mL of diphenylether and irradiated at 230 °C for 7
minutes (max µW power input: 250 W; ramp time: 3 minutes; reaction time: 7 minutes; power
max: off; maxim pressure: 180 psi). At the end of the irradiation, petroleum ether was added to
the reaction mixture and the solid obtained was filtered over a Buchner funnel, washed
thoroughly with petroleum ether and used in the following step without any further purification.
General Procedure for the Synthesis of 12a-b: Under nitrogen atmosphere intermediates
1
1a-b (0.99 mmol) and freshly distilled POCl (5 mL) were heated at reflux for 2 hours. The
3
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reaction mixture was cooled down to room temperature and volatile residues were removed
under vacuum. The solid obtained was cooled down to 0 °C and then CH Cl , H O, and solid
2
2
2
K CO were added until pH reached 11. The aqueous phase was extracted twice with CH Cl and
2
3
2
2
0
1
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8
9
0
the combined organic phases were dried over Na SO , filtered and dried under vacuum.
2
4
Intermediates 12a-b were used in the next step without any further purification. Quantitative
yield.
General Procedure for the Synthesis of 13a-b: 2,4ꢀDichloroaniline (65 mg, 0.40 mmol) was
added to a suspension of NaH (60% dispersion in mineral oil) (16 mg, 0.4 mmol) in dry DMF (5
mL). The reaction mixture was stirred at room temperature for 1 hour, followed by the addition
of intermediate 12 or 12b (0.2 mmol). The mixture was heated at reflux for 2 hours after which
H O and ethyl acetate were added and the reaction mixture was cooled down to room
2
temperature. The organic phase was washed with an aqueous solution of LiCl (5% w/w), brine,
dried over Na SO and concentrated under vacuum. The crude was purified by flash
2
4
chromatography: CH Cl /acetone (100/0ꢀ98/2) for 13a; petroleum ether/ethyl acetate (7/3) for
2
2
13b.
4-((2,4-dichlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (13a). Yield: 64%. Mp
+
1
1
94ꢀ195 °C. MS (APCI) [M+H] : 374.08 m/z, HꢀNMR (CDCl 400 MHz): δ 3.99 (s, 3H), 6.98
3
(d, 1H, J = 8.64 Hz), 7.17 (dd, 1H, J = 9.3, 2.44 Hz), 7.21 (dd, 1H, J = 8.7, 2.0 Hz), 7.44 (d, 1H,
1
3
J = 2.44 Hz), 7.53 (d, 1H, J = 2.2 Hz), 7.71 (d, 1H, J = 9.28 Hz), 8.76 (s, 1H). CꢀNMR (CDCl3
100 MHz): δ 56.04, 56.42, 94.74, 101.13, 109.13, 115.06, 116.30, 122.12, 126.44, 127.60,
1
29.66, 129.87, 136.67, 147.18, 147.68, 147.82, 149.67, 150.14, 154.50. Anal. (C H Cl N O)
17 11
2
3
C, H, N.
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-((2,4-dichlorophenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile (13b). Yield: 57%.
+
1
Mp 243ꢀ244 °C. MS (ESI) [M+H] :374.4 m/z. HꢀNMR (CDCl 400 MHz): δ 3.79 (s, 3H), 4.06
3
(
s, 3H), 6.78 (bs, 1H), 6.84 (d, 1H, J = 8.67 Hz), 6.89 (s, 1H), 7.17 (dd, 1H, J = 8.64, 2.28 Hz),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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36
37
38
39
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42
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44
45
46
47
48
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50
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52
53
54
55
56
57
58
59
60
13
7
.43 (s, 1H), 7.52 (d, 1H, J = 2.28 Hz), 8.71 (s, 1H). CꢀNMR (CDCl 100 MHz: δ 44.52, 47.38,
3
53.88, 56.08, 85.68, 109.09, 113.37, 116.18, 117.71, 118.01, 127.30, 131.29, 149.38, 151.24,
52.10, 154.38, 162.34. Anal. (C H Cl N O ) C, H, N.
1
1
8
13
2
3
2
Synthesis
of
7-methoxy-4-((4-(4-methylpiperazin-1-yl)phenyl)amino)quinoline-3-
carbonitrile (13c). 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)aniline (350 mg, 1.82 mmol) was added to a
suspension of NaH (60% dispersion in mineral oil) (73 mg, 1.82 mmol) in dry DMF (11 mL).
The reaction mixture was stirred at room temperature for 1 hour, after which intermediate 12a
was added and the mixture was heated at reflux for 2 hours. Next, the reaction mixture was
cooled to room temperature, NaOH 1 M was added until pH 11 was reached. The obtained grey
solid was separated by filtration over a Buchner funnel, washed with H O, dried and purified by
2
flash chromatography using acetone/MeOH/NEt (90/9/1) as eluent. Yield: 35%. Mp 231ꢀ232 °C
3
+
1
(
decomposition: 225 °C). MS (ESI) [M+H] :374.4 m/z. HꢀNMR (DMSOꢀd 400 MHz): δ 2.50
6
(s, 3H), 2.86 (bs, 4H), 3.30 (bs, 4H), 3.92 (s, 3H), 6.99 (d, 2H, J = 8.76 Hz), 7.18 (d, 2H, J = 8.64
1
3
Hz), 7.23 (dd, 1H, J = 9.22, 2.4 Hz), 8.41 (d, 1H, J = 10.28 Hz), 8.42 (s, 1H), 9.63 (s, 1H). Cꢀ
NMR (DMSOꢀd 100 MHz): δ 44.52, 47.38, 53.88, 56.08, 85.68, 109.09, 113.37, 116.18,
6
117.71, 118.01, 127.30, 131.29, 149.38, 151.24, 152.10, 154.38, 162.34. Anal. (C H N O) C,
2
2
23
5
H, N.
General Procedure for the Synthesis of 15a-d: In a microwave tube 2,6ꢀdichloroꢀ9Hꢀpurine
4 (100 mg, 0.53 mmol) and the proper aniline (2.64 mmol) were suspended in nꢀBuOH (3 mL).
1
NEt (265 µL, 1.90 mmol) was added and the tube was heated at 70 °C for 10 minutes (max µW
3
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power input: 300 W; ramp time: 1 minute; reaction time: 10 minutes; power max: off; maximum
pressure: 260 psi). At the end of the irradiation, nꢀBuOH was evaporated under vacuum. The
solid obtained was isolated by filtration over a Buchner funnel and washed with nꢀhexane and
cold (4 °C) ethyl acetate.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
4-((2-chloro-9H-purin-6-yl)amino)benzenesulfonamide (15a). Yield: 75%. HꢀNMR
(
DMSOꢀd , 200 MHz), δ 7.01 (t, 1H, J = 7.28 Hz), 7.29 (t, 2H, J = 7.40 Hz), 7.78 (d, 2H, J =
6
7.63 Hz), 8.25 (s, 1H), 10.16 (bs, 1H).
1
2-chloro-N-phenyl-9H-purin-6-amine (15b). Yield: 61%. HꢀNMR (DMSOꢀd , 200 MHz), δ
6
7.26 (s, 2H), 7.73 (d, 2H, J = 8.83 Hz), 7.96 (d, 2H, J = 8.78 Hz), 8.44 (s, 1H), 10.75 (bs, 1H).
1
1
-(4-((2-chloro-9H-purin-6-yl)amino)phenyl)ethan-1-one (15c). Yield: 74%. HꢀNMR
(DMSOꢀd , 200 MHz), 2.53 (s, 3H), 7.97 (d, 2H, J = 8.61 Hz), 8.04 (d, 2H, J = 8.74 Hz), 8.62 (s,
6
1
H), 10.94 (bs, 1H).
-((2-chloro-9H-purin-6-yl)amino)phenol (15d). Yield: 81%. MS (ESI) [MꢀH] :260.2 m/z.
HꢀNMR (DMSOꢀd 300 MHz), δ 6.50 (dd, 1H; J = 8.01, 1.42 Hz), 7.12 (t, 1H; J = 8.04 Hz),
ꢀ
3
1
6
7.25 (d, 1H; J = 8.19 Hz), 7.34 (bs, 1H), 8.29 (s, 1H), 9.36 (bs, 1H), 10.04 (s, 1H), 13.37 (bs,
1H).
General Procedure for the Synthesis of 16a-l: In a microwave tube intermediates 15a-d (50
mg, 0.19 mmol) and the opportune amine (0.47 mmol) were suspended in nꢀBuOH (1.5 mL).
Trifluoroacetic acid (14.63 µL, 0.19 mmol) was added and the tube was heated in the microwave
in two consecutive steps: first at 170 °C for 10 minutes and then at 150 °C for 10 minutes
(STEPꢀ1: max µW power input: 300 W; ramp time: 1 minute; reaction time: 10 minutes; power
max: off; maximum pressure: 260 psi; STEPꢀ2: max µW power input: 300 W; reaction time: 10
minutes; power max: off; maximum pressure: 260 psi).
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-((2-(4-(2-hydroxyethyl)piperazin-1-yl)-9H-purin-6-yl)amino)benzenesulfonamide (16a).
1
Yield: 54%. Mp > 250 °C (decomposition). HꢀNMR (DMSOꢀd , 400 MHz): δ 2.42 (t, 2H, J =
6
6
.12 Hz), 2.45ꢀ2.52 (m, 4H), 3.53 (d, 2H, J = 3.92 Hz), 3.68 (s, 4H), 4.43 (bs, 1H), 7.17 (bs, 2H),
.73 (d, 2H, J = 8.6Hz), 7.91 (s, 1H), 8.04 (d, 2H, J = 8.48 Hz), 9.88 (bs, 1H), 12.53 (bs, 1H).
10
11
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
1
3
CꢀNMR (DMSOꢀd , 100.6 MHz): δ 44.90, 53.52 (2 x), 58.96 (2 x), 60.80, 114.13, 119.71 (2 x),
6
1
26.74 (2 x), 137.00, 137.94, 143.69, 151.46, 153.37, 158.87. Anal. (C H N O S) C, H, N.
1
7
22
8
3
2-(4-(6-(phenylamino)-9H-purin-2-yl)piperazin-1-yl)ethan-1-ol (16b). Yield: 44%. Mp >
1
250 °C (decomposition). HꢀNMR (DMSOꢀd , 200 MHz): δ 2.23ꢀ243 (m, 6H), 3.44 (s, 2H), 3.60
6
(s, 4H), 4.40 (bs, 1H), 6.91 (t, 1H, J = 7.03 Hz), 7.23 (t, 2H, J = 7.72 Hz), 7.80 (m, 3H), 9.41 (bs,
1
3
1
H). CꢀNMR (DMSOꢀd , 100.6 MHz): δ 44.99 (2 x), 53.69 (2 x), 59.09, 60.95, 114.01, 120.70
6
(2 x), 122.43, 128.91 (2 x), 137.46, 140.63, 152.02, 153.01, 159.10. Anal. (C H N O) C, H, N.
1
7
21
7
4-((2-morpholino-9H-purin-6-yl)amino)benzenesulfonamide (16c). Yield: 61%. Mp > 250
1
°
C (decomposition). HꢀNMR (DMSOꢀd , 200 MHz): δ 3.50 (s, 8H), 7.14 (s, 2H), 7.69 (d, 2H, J
6
1
3
=
8.77 Hz), 7.90 (s, 1H), 8.00 (d, 2H, J = 8.83 Hz), 9.90 (bs, 1H). CꢀNMR (DMSOꢀd , 100.6
6
MHz): δ 45.35 (2 x), 66.43 (2 x), 117.00, 119.74 (2 x), 126.73 (2 x), 136.99, 138.08, 143.56,
151.43, 153.19, 158.90. Anal. (C H N O S) C, H, N.
1
5
17
7
3
1-(4-((2-(isopentylamino)-9H-purin-6-yl)amino)phenyl)ethan-1-one (16d). Yield: 66%. Mp
1
>
250 °C (decomposition). HꢀNMR (DMSOꢀd , 200 MHz): δ 0.79ꢀ0.86 (m, 6H), 1.38ꢀ1.50 (m,
6
1
3
2H), 1.57ꢀ1.63 (m, 1H), 2.48 (s, 3H), 2.70ꢀ2.78 (m, 2H), 7.97ꢀ8.10 (m, 4H), 8.62 (s, 1H). Cꢀ
NMR (DMSOꢀd , 100.6 MHz): δ 22.74 (2 x), 25.30, 26.06, 35.74, 41.32, 112.83, 120.18 (2 x),
6
1
29.32 (2 x), 131.11, 142.07, 143.83, 151.64, 152.08, 154.22, 196.80. Anal. (C H N O) C, H,
18 22 6
N.
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1
-(4-((2-(methylamino)-9H-purin-6-yl)amino)phenyl)ethan-1-one (16e). Yield: 47%. Mp >
1
2
2
1
50 °C (decomposition). HꢀNMR (DMSOꢀd , 200 MHz): δ 2.53 (s, 3H), 3.06 (s, 3H), 7.94 (d,
6
13
H, J = 11.72 Hz), 8.04 (d, 2H, J = 8.76 Hz), 8.62 (s, 1H), 10.94 (bs, 1H). CꢀNMR (DMSOꢀd ,
6
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
00.6 MHz): δ 25.99, 26.35, 115.30, 119.24 (2 x), 129.22 (2 x), 131.33, 141.57, 143.24, 150.18,
152.26, 153.22, 196.39. Anal. (C H N O) C, H, N.
1
4
14
6
1-(4-((2-((2-hydroxyethyl)amino)-9H-purin-6-yl)amino)phenyl)ethan-1-one (16f). Yield:
1
4
2%. Mp > 250 °C (decomposition). HꢀNMR (DMSOꢀd , 200 MHz): δ 2.58 (s, 3H), 3.26 (t, 2H,
6
J = 8.20 Hz), 3.50 (t, 2H, J = 1.80 Hz), 7.97 (d, 2H, J = 8.64 Hz), 8.05 (d, 2H, J = 9.24 Hz), 8.68
13
(
s, 1H), 10.96 (bs, 1H). CꢀNMR (DMSOꢀd , 100.6 MHz): δ 26.05, 39.95, 62.22, 114.22,
6
1
19.90 (2 x), 128.53 (2 x), 130.99, 140.11, 142.33, 152.24, 154.83, 156.53. Anal. (C H N O )
15 16 6 2
C, H, N.
3-((2-(benzylamino)-9H-purin-6-yl)amino)phenol (16g). Yield: 49%. Mp 267ꢀ268 °C (with
+
+
1
decomposition). MS (ESI) [M+H] : 333.3m/z, [M+Na] : 355.3 m/z. HꢀNMR (DMSOꢀd 300
6
MHz): δ 4.52 (d, 2H; J = 6.21 Hz), 6.39 (dd, 1H; J = 7.83, 1.11 Hz), 7.01 (m, 2H), 7.18 (t, 1H; J
=
7.08 Hz), 7.28 (t, 2H; J = 7.35 Hz), 7.43 (bs, 2H), 7.49 (d, 2H; J = 7.14 Hz), 7.80 (s, 1H), 8.35
1
3
(bs, 1H), 9.21 (bs, 1H), 13.37 (bs, 1H). CꢀNMR (DMSOꢀd 75 MHz): δ 45.02, 107.65, 109.37,
6
1
11.52, 126.73, 127.45, 128.50, 129.23, 136.91, 141.70, 141.83, 152.11, 157.79, 159.47. Anal.
(
(
C H N O) C, H, N.
1
8
16
6
3-((2-(isopentylamino)-9H-purin-6-yl)amino)phenol (16h). Yield: 70%. Mp 223ꢀ224 °C
+
+
1
with decomposition). MS (ESI): [M+H] : 313.2 m/z, [M+Na] : 335.4 m/z. HꢀNMR (DMSOꢀd
6
3
00 MHz): δ 0.90 (d, 6H; J = 6.6 Hz), 1.46 (dd, 2H; J = 14.7, 7.08 Hz), 1.67 (e, 1H; J = 6.68
Hz), 3.30 (dd, 2H; J = 14.5, 7.3 Hz), 6.37 (m, 2H), 7.02 (t, 1H, J = 8.08 Hz), 7.43 (s, 1H), 7.50
13
(
d, 1H, J = 8.37 Hz), 7.78 (s, 1H), 8.14 (s, 1H), 9.10 (s, 1H), 9.18 (s, 1H), 12.31 (bs, 1H). Cꢀ
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NMR (DMSOꢀd 75 MHz): δ 23.06, 25.92, 39 41, 107.60, 109.33, 111.42, 129.25, 141.94,
6
1
57.78, 159.59, 163.54. Anal. (C H N O) C, H, N.
16 20 6
3-((2-(methylamino)-9H-purin-6-yl)amino)phenol (16i). Yield: 45%. Mp 293 °C (with
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60
ꢀ
1
decomposition). MS (ESI) [MꢀH] : 255.3. HꢀNMR (DMSOꢀd 300 MHz): δ 2.81 (d, 3H, J =
6
4.32 Hz), 6.39 (d, 1H; J = 8.16 Hz), 6.5 (d, 1H; J = 4.1 Hz), 7.03 (t, 1H; J = 7.92 Hz), 7.45 (d,
1
3
1
H, J = 8.22 Hz), 7.50 (s, 1H), 8.36 (bs, 1H), 9.22 (s, 1H), 12.10 (bs, 1H). CꢀNMR (DMSOꢀd
6
7
5 MHz): δ 28.99, 107.65, 109.32, 111.35, 129.24, 136.98, 141.98, 151.94, 153.97, 157.86,
160.19. Anal. (C H N6O) C, H, N.
1
2
12
3
-((2-((2-hydroxyethyl)amino)-9H-purin-6-yl)amino)phenol (16l). Yield: 69%. Mp 252ꢀ253
+
+
1
°
C (with decomposition). MS (ESI) [M+H] : 287.3 m/z, [M+Na] : 309.4 m/z. HꢀNMR (DMSOꢀ
d6 300 MHz): δ 3.35 (d, 2H, J = 2.85 Hz), 3.58 (d, 2H, J = 2.85 Hz), 4.72 (t, 1H, J = 5.3 Hz),
.27 (t, 1H, J = 5.5 Hz), 6.39 (dd, 1H, J = 8.01, 1.89 Hz), 7.04 (t; 1H, J = 8.02 Hz), 7.45 (d, 1H,
6
1
3
J = 8.31 Hz), 7.50 (bs, 1H), 7.80 (s, 1H), 9.17 (s, 1H), 12.38 (bs, 1H). CꢀNMR (DMSOꢀd 75
6
MHz) δ 44.51, 60.61, 107.68, 109.34, 111.49, 114.06, 129.30, 136.43, 141.89, 152.44, 157.74,
1
59.56, 161.66. Anal. (C H N O ) C, H, N.
13 14 6 2
3
-((2-((2-(1H-imidazol-4-yl)ethyl)amino)-9H-purin-6-yl)amino)phenol (16m).
In
a
microwave tube intermediate 15d (50 mg, 0.19 mmol) and histamine dihydrochloride (88 mg,
0.49 mmol) were suspended in nꢀBuOH (3.0 mL). Et N (173 µL, 1.24 mmol) was then added
3
and the tube was heated in the microwave at 130 °C for 10 minutes (max µW power input: 300
W; ramp time: 1 minute; reaction time: 10 minutes; power max: off; maximum pressure: 260
psi). At the end of the irradiation, nꢀBuOH was evaporated under vacuum and the solid obtained
was purified by flash chromatography using ethyl acetate/MeOH + NEt (8/2+3%) as eluent.
3
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