Synthesis and Structure-Activity Relationships of the Novel Antimalarials 5-Pyridinyl-4(1 H)-Pyridones

Article abstract of DOI:10.1021/acs.jmedchem.7b01256

Malaria is still one of the most prevalent parasitic infections in the world, with half of the world's population at risk for malaria. The effectiveness of current antimalarial therapies, even that of the most recent class of antimalarial drugs (artemisin

Full text of DOI:10.1021/acs.jmedchem.7b01256

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Synthesis and Structure-Activity Relationships
of Novel Antimalarial 5-Pyridinyl-4(1H)Pyridones
Jose Maria Bueno, Felix Calderon, Jesús Chicharro, Juan Carlos de la Rosa, Beatriz Diaz,
Jorge Fernandez, Jose M, Fiandor, María Teresa Fraile, Mercedes Garcia, Esperanza
Herreros, Adolfo García-Pérez, Milagros Lorenzo, Araceli Mallo, Margarita Puente, Anas
Saadeddin, Santiago Ferrer, Iñigo Angulo-Barturen, Jeremy N. Burrows, and María Luisa Leon
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01256 • Publication Date (Web): 28 Mar 2018
Downloaded from http://pubs.acs.org on March 28, 2018
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Journal of Medicinal Chemistry
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Synthesis and StructureꢀActivity Relationships of Novel
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Antimalarial 5ꢀPyridinylꢀ4(1H)ꢀPyridones.
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†
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José M. Bueno *, Félix Calderon , Jesús Chicharro , Juan C. De la Rosa , Beatriz Díaz , Jorge
Fernández†, José M. Fiandor†, María T. Fraile†, Mercedes García†, Esperanza Herreros†, Adolfo
García-Pérez†, Milagros Lorenzo†, Araceli Mallo†, Margarita Puente†, Anas Saadeddin†, Santiago
Ferrer †, Iñigo Angulo-Barturen†#, Jeremy N. Burrows‡, María L. León†*
†Tres Cantos Medicines Development Campus, Diseases of the Developing World. GlaxoSmithKline,
C/ Severo Ochoa, 2, 28760ꢀTres Cantos (Madrid, Spain)
‡Medicines for Malaria Venture, ICC, Route de PréꢀBois 20, PO Box 1826, 1215 Geneva, Switzerland
§
CHB Química
C/Quinto Centenario ꢀ54, Getafe 28905 (Madrid), Spain
KEYWORDS: malaria, pyridones, solubility, pyridine
Abstract
Malaria is still one of the most prevalent parasitic infection in the world with half of the world's
population at risk of malaria. The effectiveness of current antimalarial therapy is under continuous threat
through the spread of resistant Plasmodium strains, even to the most recent class of antimalarial drugs (ACT:
artemisininꢀcombinationꢀtherapies). As a consequence, there is still an urgent requirement for new
antimalarial drugs. We previously reported the identification of 4(1H)ꢀpyridones as a novel series with
potent antimalarial activity. The low solubility was identified as an issue to address. In this paper, we
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describe the synthesis and biological evaluation of 4(1H)ꢀpyridones with potent antimalarial activity in
vitro and in vivo and improved pharmacokinetic profile.Their main structural novelty is the presence of
polar moieties such as hydroxyl groups and the replacement of lipophilic phenyl rings by pyridine at the
lipophilic side chain.
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Introduction
Malaria continues to be a leading cause of death on the planet, with 50% of the population being
exposed to the risk of infection. More than 200 million new clinical cases are estimated annually that
leads to approximately 800,000 deaths each year primarily to children under five and expectant
mothers1.
Although the control of the disease still depends on chemotherapy, the spread of resistant
P.falciparum strains to most of the antimalarials currently in use, including chloroquine2, the limited
number of drugs currently available against malaria3 and the alarming evidences pointing to the
appearance of resistant or “less sensitive” P. falciparum strains to artemisinin derivatives4, the basic
components of the current gold standard combinations for the treatment of malaria (ACT), has fueled
the research effort to find novel drugs for the treatment of malaria5,6. In this way, the mitochondrial
respiratory chain of P. falciparum is an attractive target for chemotherapy due to its differences from the
analogous mammalian system7ꢀ10 and its essential caracter for the parasite. In the erythrocytic stages,
mitochondria are involved in several essential metabolic processes, which interesting enough do not
include ATP production and vary depending on parasitre species. Therefore, proteins involved in
mitochondrial physiology have the potental for antiparasitic drug discovery8.
The election of mitochondrial electron transport chain of P. falciparum as a putative target in
antimalarial drug discovery has been validated and has been succesful, as demonstrated with the
development of atovaquone (Figure 1), introduced into therapy in 1997 as a combination with proguanil.
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Journal of Medicinal Chemistry
The combination AtovaquoneꢀProguanil is currently used in treating multidrugꢀresistant malaria and for
prophylaxis in areas with chloroquine resistance11.
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Atovaquone inhibits the P. falciparum mitochondrial respiratory chain at the cytochrome bc1 level
(Complex III), demonstrating that it is a proven drug target in the prevention and treatment of malaria.
The bc1 complex is central to mitochondrial function in P. falciparum, and it is composed by two
binding sites, named Qo and Qi, for oxidation of Ubiquinol and reduction of Ubiquinone, respectively.
Most of the antimicrobial drugs, such as Atovaquone, target the Qo site12. Unfortunately, the rapid rise
of resistances, mainly caused by mutations at the binding site, has compromised the use of Atovaquone
as a first line antimalarial.
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In our efforts to find novel antimalarials, scientists at GSK have developed a Medicinal Chemistry
programme based on the 4(1H)ꢀpyridone core present at the known antiꢀcoccidial drug clopidol (Figure
1). Although initially it was presumed that 4(1H)ꢀpyridones were binding the Qo site at the cytochrome
bc1 , recent studies seem to point to Qi as the binding site for these compounds14.
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Clopidol displayed weak antimalarial activity in animal models15 and there is evidence indicating that
its anticoccidial action is related to mitochondrial impairment16. The strategy for increasing the
antimalarial activity of clopidol was the replacement of one of the chlorine atoms by different lipophilic
side chains. As a result, a series of potent 2,6ꢀdimethylꢀ4(1H)ꢀpyridones such as GW844520 and
GW308678, bearing que diphenylꢀether moiety, have been prepared (Figure 1). The novel compounds
have demonstrated to be superior to chloroquine in vitro against erythrocytic stages of the parasite and
in vivo in the P. yoelii mouse model17. Compared to clopidol, the most active 4(1H)ꢀPyridones prepared
showed >500 fold improvement in the in vitro activity (IC50 for inhibition of P. falciparum) and ca. 100
fold improvement in the in vivo efficacy (ED50 in a murine model of P. yoelii). In general, the most
potent compounds were characterized as class II compounds according to the Developability
Classification System18, with high permeability and low solubility in aqueous media. They display high
oral bioavailability in mouse and rat at low doses administered from solution formulations (50ꢀ100% at
doses <1 mg/Kg)19,20 and low oral bioavailability when administered as a solid form in suspension (4ꢀ
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20%)21, clearly suggesting that the absorption by oral route is hampered by low solubility and/or
dissolution rate of the solid sample. In this regard, it is expected that the physicochemical properties of
the ultimate solid dosage form and the particle size could be a key determinants of the preclinical and
clinical studies. In a continued effort to overcome the issues related to the poor solubility and oral
bioavailability, we have focused our attention on designing novel potent antimalarial 4(1H)ꢀpyridones
with improved solubility and physicochemical properties.
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Given the great influence of the lipophilic tail on the antimalarial activity of 4(1H)ꢀpyridones17, we
first tried to increase the solubility of diphenylꢀether derivatives by attaching polar groups and ionisable
tertiary amines and polar hydroxyl groups at the 4ꢀpyridone ring. Although the introduction of hydroxyl
groups, esters and tertiary amines at both positions C2 and C6 of the pyridone ring has led to a complete
loss of the antimalarial activity, the introduction of polar CH2OH groups exerted different effects on the
antimalarial activity depending on their position21. As shown in Figure 1, while the hydroxymethyl
group induces a significant drop in antimalarial activity in vitro in comparison with its non hydroxylated
analogue when it is located at position C2, it is possible to maintain high levels of activity by attaching
the hydroxymethyl group at position C6 of the 4ꢀpyridone ring, thus leading to potent and more polar
compounds with improved PK properties.
Within the strategies aimed at improving physicochemical properties and oral bioavailability, in
addition to the introduction of CH2OH groups at position C6, we have also explored the effect of
replacing phenyl rings by more polar heterocycles into the lipophilic side tail. In this paper, we report
the synthesis and biological evaluation of a new series of antimalarial 4(1H)ꢀpyridone derivatives
bearing substituted pyridine rings at position C5 (Figure 2). As shown, we have investigated the effect
of both flexible and rigid linkers between the aromatic rings (Ia-d) as well as the absence of any linker
between them (IIa,b).
In summary, according to BCS(18), antimalarial 4(1H)Pyridones have traditionally behave as Class II
compounds, displaying fair permeability and very low solubility in PBS and other aqueous media. For
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Journal of Medicinal Chemistry
this reason, since the very beginning of our Medicinal Chemistry Programme, we have tried to increase
the solubility by introducing polar/ionizable groups into the molecules. In this way, as commented
previously, while most of these changes have normally lead to a complete loss of in vitro antimalarial
activity, we have observed that the introduction of an OH group at position C6 maintain a good level of
antimalarial activity, thus offering new opportunities for both novel compounds or the synthesis of
appropriate prodrugs with improved solubility.
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Following this strategy, in this paper we describe the preparation of novel “hybrid” more polar
4(1H)ꢀpyridone derivatives that join the presence of pyridine rings at position C5 with the effect of the
polar hydroxymethyl groups at position C6 of the 4ꢀpyridone core (IIb) described previously. As
expected these structural change has led compounds with better physicochemical and PK profiles,
maintaining excellent antimalarial activity.
The novel compounds described in this paper have allowed us to wide the scope of antimalarial
4(1H)Pyridiones available, thus offering new opportunities to increase the current antimalarial pipeline.
Chemistry
In general, the 4(1H)ꢀpyridone core has been traditionally obtained by ammonolysis of the
corresponding 4ꢀpyranones. All the compounds described in this paper (except compounds of general
formulae Ib and IIb) have been obtained from key intermediate 4ꢀpyranone 6, which was prepared from
commercially available 5ꢀformylꢀ2ꢀmethoxypyridine 1, in a similar way as described previously for
other pyridones prepared by us17 (Scheme 1).
Alternatively, intermediate ketone 4 has been obtained in only one step via Stille CꢀC coupling by
reaction of 5ꢀbromoꢀ2ꢀmethoxypyridine 2 with isopropenyl acetate and tributyltin methoxide in the
presence of the tandem Palladium Acetate/Triꢀoꢀtolylphosphine (TOTP) as catalysts22. Condensation of
methyl ketone 4 with acetic anhydride was carried out by using either polyphosphoric acid (PPA)17,23 or
more conveniently the less viscous and commercially available Eaton’s reagent24 (mixture of
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phosphorous pentoxide in methanesulphonic acid), with advantages in ease of handling and aqueous
workꢀup.
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4ꢀpyranone 6, obtained by acidic hydrolysis of 5, was used directly in the synthesis of 6ꢀ
(benzyloxy)pyridine derivatives (Ia) as indicated in Scheme 2.
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Selective Oꢀbenzylation with a series of bencyl halides was accomplished in the presence of silver
carbonate25 to afford 4ꢀpyranones 7-13, which were subjected to ammonolysis under high pressure and
temperature to furnish the corresponding 4ꢀpyridones 14-20. Subsequent halogenation at position C3
with either Nꢀbromosuccinimide17,23 or with trichloroisocyanuric acid26, led to the corresponding 3ꢀ
bromo or 3ꢀchloro derivatives 21-27 and 28-34, respectively. The use of cheap and readily available
trichloroisocyanuric acid was found to provide a convenient substitute for other chlorinating agents such
as Nꢀchlorosuccinimide23, with advantages in shorter reaction times, atom economy and lower reaction
temperature, leading to higher yields of chlorinated material, particularly large scale preparations.
Phenyloxyꢀpyridinꢀ3ꢀyl derivative 40 was prepared following a completely different synthetic
approach, starting from commercially available 2,6ꢀdimethylꢀ4(1H)ꢀpyridone 38 (Scheme 3). Careful
monochlorination of 38 with a slight excess of Nꢀchlorosuccinimide at room temperature
(trichloroisocyanuric acid proved to be unsuccessful for the monochlorination step due to its high
reactivity), followed by iodination in basic solution afforded intermediate 39, which was used as a
suitable starting material for the introduction of the lipophilic side chain via Suzuki CꢀC coupling. The
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synthesis of the boronic acid 37 was performed from commercially available 2,5ꢀdibromopyridine 35 by
selective displacement of the more reactive 2ꢀbromine atom under Ullmann conditions and subsequent
lithiation and boronic acid formation under classical conditions to afford 37. Finally, reaction of 39 with
37 in the presence of a suitable Palladium catalyst led to phenyloxyꢀpyridinyl derivative 40.
With regard to the introduction of carbon substituents at position C6´of the pyridine ring (compounds
Ic,d and IIa, Scheme 4), while the activation of position C6´ hydroxyl group by reaction of 4ꢀpyranone
6 with either POBr3 or POCl3 led to complex mixtures of reaction, treatment with Nꢀphenylꢀ
trifluoromethaneꢀsulfonimide27 cleanly afforded the chemically stable and crystalline triflate 41 in high
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Journal of Medicinal Chemistry
yield and purity. Intermediate 41 has proved to be an ideal starting material for Sonogashira and Suzuki
coupling reactions that finally have led to final 4(1H)ꢀpyridones 47-49 (formula Ic), 54-55 (formula Id)
and 106-125 (formula IIa), respectively, according to the synthetic routes depicted in Scheme 4.
As mentioned previously, the 2,6ꢀdimethylꢀ4ꢀpyridone scaffold is accesible by condensation of the
appropriate 2ꢀpropanone derivatives with acetic anhydride in the presence of a dehydrating agent such
as polyphosphoric acid or Eaton´s reagent (Scheme 1). However, the presence of any substituent
different from methyl at positions C6 like in compounds of Formula IIb, precludes the use of the
symmetrical acetic anhydride, thus making their synthesis particularly challenging. Therefore, the
preparation of C6CH2OH derivatives of general formula IIb, has been tackled through a completely
different synthetic approach, described previously by us, starting from commercially available Kojic
acid21 (Scheme 5).
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The synthesis starts from benzyl derivative 130, available from Kojic acid28, and involves the
protection of the primary hydroxyl group as TBDMS ether, removal of benzyl group by hydrogenolysis
and subsequent triflylation of C3ꢀhydroxyl group to afford 133. It is noteworthy that in the absence of
the hindered TBDMS ether, migration of the reactive triflyl group from C3ꢀOTf to the vicinal C2ꢀ
CH2OH group29 takes place. Suzuki coupling between triflate 133 and the appropriate boronic acids 128
and 129 led to the corresponding 4ꢀpyranones 134, 135 which were subjected to ammonolysis with
concurrent loss of the TBDMS protective group, and further chlorination of the resulting 4ꢀpyridones
136, 137 to afford the target hydroxymethyl derivatives 138, 139.
Results and discussion
With regard to compounds bearing rigid linkers between the aromatic rings, from the data shown in
Table 1 it seems that, although compounds 47 and 48 are quite active, the presence of a rigid linker is
related to a lose of potency in comparison with their flexible analogs (compounds 22, 29 and 54). This
effect could be explained on the basis of the higher freedom degrees for flexible side chains, which
could help making more hydrophobic favored interactions, adopting the active site topology.
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On the other hand, it is noteworthy that the presence of two lipophilic groups such as CF3 in
compounds 25, 32 or Fluorine atoms in compounds 27, 34 led to a decrease of the antimalarial activity,
which is in contrast with the general correlation observed between lipophilicity and potency in other
antimalarial 4(1H)ꢀpyridones. This effect could also be explained on the basis of some clashing contacts
within the active site30
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In general, as shown previously in other pyridones17, the effect of the type of the halogen at position
C3 on the antimalarial activity is negligible as demonstrated the good correlation between the IC50
values of bromoderivatives 21-27 and chloroderivatives 28-34, with the only exception of 4ꢀFluoroꢀ
phenyl derivative 33 which displays slightly lower activity than its brominated counterpart 26.
In Table 2 the biological data for phenylꢀpyridinyl derivatives are reported. The main structural
feature of this large family of molecules is the rigidity of the lipophilic side tail caused by the absence of
any linker between the pyridine and the phenyl rings. From the data shown some observations on SAR
can be made.
Concerning substitution at the phenyl ring, it seems that the presence of lipophilic and electron
withdrawing groups such as CF3 or OCF3 is required to have high antimalarial activity, in a similar way
as observed in other pyridones previously reported17. The extreme sensitivity of the lipophilic tail to the
presence of polar moieties is also observed in the drop in the antimalarial activity displayed by
compounds 112-114 with less lipophilic substituents such as CN,SO2Me and the hydrophilic CH2OH
group. With regard to the effect of halogens (Cl and F), it is noteworthy that the antimalarial activity is
higher in compounds 109, 121 and 111, 123 bearing the halogen atom at position ortho (chlorine and
fluorine, respectively) in contrast with their isomers having the halogen atoms attached at positions meta
or para.
However, the most interesting effect observed in this series is the clear increase in the antimalarial
activity observed for diꢀsubstituted derivatives, in contrast with the series of flexible Oꢀbenzyl
derivatives.
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Journal of Medicinal Chemistry
With regard to physicochemical properties, in Table 3 is shown the lipophilicity in terms of
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Chromatographic Hydrophobicity Indexes (CHIlogD)32 at pH=2, 7.4 and 10.5, protein binding (HSA)33
and solubility in simulated biorelevant media (FaSSIF and FeSSIF)34 for a series of selected
compounds.
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As shown, benzyloxy derivatives 21 and 28 are lipophilic compounds, with similar CHIlogD7.4
values to other analogous pyridones having phenyl ring instead of pyridine21. Both compounds remained
unionized at pH=2 and pH=10.5 and highly protein bound (%HSA=97). Removal of the OCH2 linker
led to rigid, less lipophilic and less protein bound derivatives 106 and 116. Furthermore, from the
CHIlogD data obtained at different pH, it seems the conjugation of the phenyl ring contributes to
increase the basicity of the pyridine ring, as demonstrated the lower values of CHIogD obtained at pH=2
in comparison to neutral phenyl ring, which is in accordance with a decrease of the lipophilic character
of these molecules due to protonation of the pyridine ring in the acidic media. As expected, substitutions
by 2ꢀCl,4ꢀCF3 and 2ꢀF,4ꢀCF3 at phenyl ring in compounds 124 and 125 not only slightly increased the
lipophilicity, but also decrease the basicity of the pyridine in comparison to the monosubstituted
derivatives. This effect could be explained on the basis of the strong electronꢀwithdrawing effect exerted
by the 4ꢀtrifluoromethylꢀphenyl moiety into the pyridine ring35 and the steric effect exerted by the
substituents at the ortho position,that prevents the conjugation between the two aromatic rings.
As expected, C6ꢀCH2OH derivatives 138 and 139 are less lipophilic derivatives, with lower CHIlogD
values than their non hydroxylated counterparts 106 and 125 and higher solubilities, particularly in
FeSSIF. Furthermore, the introduction of the hydroxyl group decreases significantly the CHIlogD10.5
related to deprotonation of the 4(1H)ꢀpyridone ring, in comparison to the CHIlogD7.4, suggesting a
higher degree of ionization at pH=10.5 than the non hydroxylated derivatives.
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In general, the introduction of a more polar pyridine ring instead of phenyl does not significantly
increase the solubility profile of the compounds prepared at physiologically relevant media in
comparison to compounds described previously21. The extremely low solubility showed by the
antimalarial 4(1H)pyridones in aqueous media has been attributed not only to their relatively high
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lipophilicity but also to intrinsic structural features of the 4(1H)ꢀpyridone ring, in particular its marked
tendency to form strong hydrogen bonding networks. In this way, it has been reported in the literature
that clopidol and other 4(1H)pyridones36ꢀ38 adopt predominantly the 4ꢀketo tautomer form in the solid
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state, forming structures supported by strong hydrogen bonding networks. This is in accordance with the
high melting points (m.p.>250ºC) measured for some of the 4(1H)pyridones synthetized (data not
reported here).
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In Table 4 the main PK parameters measured for a series of selected antimalarial 4(1H)pyridones are
shown. In general, the oral bioavailabilities in mouse for compounds 106, 116, 118, 119, 124, 125 and
138, bearing substituted phenylꢀpyridinyl moieties attached to the pyridone ring are higher than that
observed for other more lipophilic phenyloxyphenylꢀ4(1H)pyridones described previously when
administered as a suspension in 1% Methyl Cellulose (~20%)20, 21. It is noteworthy the significant
increase in the oral bioavailability observed for the C6CH2OH derivative 138 (>100%) in comparison
with its non hydroxylated analog 116 (54.9%), which is in accordance with previous PK data reported
by us for the phenyloxyꢀphenyl series.
Concerning clearance and related halfꢀlives, it seems that they are mainly influenced by factors such
as the type of substituent attached to the pyridine ring and the relative position of the substituents at the
phenyl ring. With regard to the type of substituent, as mentioned previously, compounds having flexible
linkers such as 28, showed significantly shorter halfꢀlife (t1/2=5.3h) than its rigid counterpart 116
(t1/2=18.7), presumably due to the metabolic oxidation at the labile bencylic position and f urther
degradation, which could also explain its lower oral bioavailability. With regard to the substitution at
the pendant phenyl ring present at the end of the side chain, it seems that reducing metabolism by use of
carefully placed blocking groups is important, as demonstrated by the longer halfꢀlife displayed in para
substituted derivatives such as 118 (t1/2=23.4h) in comparison to its meta substituted isomer 119
(t1/2=5.1h).
The presence of a polar C6ꢀCH2OH group in compound 138 decreased the lipophilicity but increased
the clearance and shorten the halfꢀlife in comparison to its non hydroxylated and more lipophilic analog
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Journal of Medicinal Chemistry
116. This unusual behavior is in accordance with metabolic studies carried out for other 2,6ꢀdimethylꢀ
pyridones in which it has been observed that the hydroxylation at methyl groups is probably the main
Phase I metabolic pathway19, normally followed by conjugation with glucuronic acid (data not shown
here). This fact points to Phase II metabolism as the most likely route of elimination of the
4(1H)pyridones bearing CH2OH groups attached at position C6 of the pyridone ring.
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Table 5 shows the in vivo antimalarial efficacy determined in a mouse P. yoelii model39,40
,
administering test samples in four oral doses by gavage over four days. The in vivo data are expressed as
ED50 (mg/kg) values, representing the dose (estimated from the doseꢀresponse curve) for parasite
reduction of 50% relative to untreated controls.
As shown, with the only exception of compound 28, which displayed an ED50=16.3 mg/Kg, all the
pyridones tested have been highly efficacious in vivo, with ED50 values ≤ 1 mg/Kg. In general, it seems
that the compound exposure and duration of cover in vivo, along with the in vitro antimalarial potency
(PfIC50) correlates fairly well with the in vivo efficacy. Thus, the most efficacious compound (125)
displays high antimalarial activity in vitro, long halfꢀlive and high AUC. This observation is in
accordance with a study41 concerning the viability of the intraꢀerythrocytic forms of P. falciparum,
indicating that the effect of the inhibition of the Mitochondrial Electron Transport chain in P.
falciparum depends upon both the erythrocytic stage of the parasites and the duration of exposure. Thus
for example, as the ringꢀstage parasites appear to remain viable for up to 48h with an inhibited
Mitochondrial Electron Transport Chain, the drug needs to maintain inhibitory concentrations in plasma
for at least that length of time to achieve high levels of efficacy.
Conclusions
A new series of potent antimalarial 4(1H)pyridones in vitro and in vivo have been obtained by
introducing pyridine rings instead of phenyl into the lipophilic side chain attached to the 4(1H)ꢀpyridone
core. In general, the new compounds display improved pharmacokinetic profiles in comparison with the
more lipophilic phenoxyphenyl derivatives previously described by us whilst maintaining similar levels
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of antimalarial efficacy in vitro and in vivo. This improvement in the pharmacokinetic profile has been
particularly relevant in compounds that combine the effect of the pyridine ring with the presence of
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9
polar hydroxymethyl groups attached at position C6 of the 4(1H)ꢀpyridone ring, leading to a significant
increase of solubility in biorelevant media and oral bioavailability in mice. The results obtained
demonstrate that novel potent and more developable antimalarial 4(1H)pyridones can be prepared by
joining chemical transformations at both the very sensitive lipophilic tail and the 4(1H)ꢀpyridone
scaffold, thus opening new ways for the chemical exploration of this exciting family of antimalarials.
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Experimental section
Biology
Determination of activity in vitro against P.falciparum
The activity of the test compounds against P. falciparum in vitro was determined using a
modification of the semiꢀautomated microdilution technique of Desjardins.42 Further experimental
details can be found in the supporting information available.
Pharmacokinetics
Measurement of pharmacokinetic parameters in CD1 mice was carried out by using Specific
pathogenꢀfree female CD1 (Hsd:ICR) mice of 8 to 10 weeks of age were obtained from Harlan
Laboratories (Udine, Italy). The mice were maintained in the GlaxoSmithKline Laboratory Animal
Science animal facilities at Tres Cantos (Spain). Further experimental details are available in the
supplementary information provided.
In vivo efficacy in 4d P. yoelii murine model
The blood schizontocidal activity of test compounds was determined using a modified 4ꢀday
suppressive assay39 following the method previously described by us40. Further experimental details can
be found in the supplementary information available.
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Journal of Medicinal Chemistry
Physicochemical parameters
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8
Determination of CHIlogD, %HSA and equilibrium solubility in FaSSIF and FeSSIF have been
carried out following procedures reported in the literature32,33. Further experimental details are available
in the supplementary information provided.
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Chemistry
Starting materials were obtained from commercial suppliers and used without further purification
unless otherwise indicated. Flash chromatography was carried out using prepacked Isolute Flash or
Biotage silica gel columns as the stationary phase and analytical grade solvents as the eluent unless
otherwise stated. Preparative TLC purification was carried out in 20x20 cm plates Merck (silica gel 60,
F254, 2 mm), by using mixtures of analytical grade solvents as eluents. 1HꢀNMR spectra were
determined on a Varian Unity spectrometer (300 MHz). Chemical shifts are reported as δ values (ppm)
downfield from tetramethylsilane, used as an internal standard in the solvent indicated. Total ion
current traces were obtained for electrospray positive and negative ionization (ES+/ESꢀ) on a Waters
ZMD 2000. Analytical chromatographic conditions used for the LC/MS analysis were as follows. The
column was ACE, 4.6 mm _ 30 mm, and the stationary phase particle size was 3 ꢁm. Solvent A was an
aqueous solvent consisting of water + 0.1% formic acid. Solvent B was an organic solvent consisting of
acetonitrile + 0.1% formic acid. The composition of the solvent over 5 min is shown in Table 6.
Additional chromatographic parameters were as follows: flow rate, 1 mL/min; injection volume, 5 ꢁL;
column temperature, 30 _C; UV wavelength range, 220ꢀ330 nm.
Table 6. Analytical Chromatography: composition of eluent mixture over 5 min.
Time (min)
% solvent B
0
10
10
90
90
0.2
3.5
4
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4.01
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The purity of all tested compounds was >95% by using the analytical method described above. 1Hꢀ
NMR spectra of all the compounds listed not included here are available in the supplementary
information provided.
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2-Methoxy-5-[(2-nitroprop-1-en-1-yl]pyridine (3)
A solution of 5ꢀformylꢀ2ꢀmethoxypyridine 1 (14.56g, 0.1 mol) and butylamine (23.1 ml, 0.2 mol) in
dry toluene (90 ml) was heated to reflux in a flask equipped with a DeanꢀStark trap in order to remove
water. After heating for 3h, the mixture was concentrated to dryness and the residue dissolved in acetic
acid (45 ml). To the solution was added nitroethane (11.4 ml, 0.15 mol) and the mixture was heated to
100ºC. After 3h of heating the mixture was cooled to room temperature and poured onto 400 ml of a
mixture ice/water with vigorous stirring. The mixture was extracted with ethyl acetate (400 ml) and the
organic layer washed successively with water (2x300 ml), 10% NaHCO3 (2x300 ml), water (300 ml)
and brine (300 ml), then dried over MgSO4. Elimination of the solvent under vacuum gave a yellow
powder which was purified by column chromatography on silica gel, eluting with mixtures hexane/ethyl
1
acetate (v/v 20:1 and 10:1) to afford 16.65g of the title compound as a yellow powder (85% yield). Hꢀ
NMR(δ, ppm, CDCl3): 8.30(m, 1H); 8.02(bs, 1h)7.68(dd,1H); 6.83(d, 1H); 3.99(s, 3H); 2.47(s, 3H)
1-(6-Methoxypyridin-3-yl)acetone (4)
Method A:
In a 1L round bottom flask equipped with mechanical stirring were placed iron powder (55g, 1 mol)
and AcOH (220ml). A solution of Intermediate 3 (16.65g, 0.08 mol) in acetic acid (120ml) was added
dropwise over this suspension. The mixture was heated to reflux with vigorous stirring under nitrogen.
After 2 h of heating the mixture was cooled to room temperature and water (750ml) was added. The
mixture was extracted with dichloromethane (3x300ml) and the combined organic layers washed with
water (400 ml), saturated NaHCO3 (400 ml), water (400 ml) and brine (400 ml), then dried over MgSO4.
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Elimination of the solvent to dryness gave 12.5g of the title compound (93% yield) as a pale brown oil
which was used for the next step without further purification.
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Method B
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9
A solution of 5ꢀbromoꢀ2ꢀmethoxypyridine (16 ml, 23.2g, 0.12 mol) and dry toluene (625ml) was
deoxygenated by bubbling argon for 20 min. Triꢀoꢀtolylphosphine (2.99g, 0.009 mol), palladium acetate
(1.65g, 0.007 mol), isopropenyl acetate (20.3 ml, 0.18 mol) and tributyltin methoxide (49.6 ml, 0.17
mol) were added successively under argon atmosphere and the mixture heated to 80ºC. After 2.5 h the
mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness under
vacuum and treated with acetonitrile (800 ml). The solid precipitated was removed by filtration and the
filtrate washed with hexane (3x800 ml). The acetonitrile solution was dried over Na2SO4 and
concentrated to dryness. The residue was purified by column chromatography on silica gel, eluting with
mixtures hexane/ethyl acetate (v/v 9:1 and 3:1). 13.78g of the title compound were obtained as a yellow
oil (70% yield). 1HꢀNMR(δ, ppm, CDCl3): 7.97(bd, 1H); 7.41(dd,1H); 6.73(d, 1H); 3.92(s, 3H); 3.63(s,
2H); 2.19(s, 3H)
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3-(6-Methoxypyridin-3-yl)-2,6-dimethyl-4H-pyran-4-one (5)
Method A:
In a 1L round bottom flask equipped with mechanical stirring were placed polyphosphoric acid (117
g) and acetic anhydride (55 ml) and the thick mixture heated to 95ºC. To this mixture was added
dropwise a solution of Intermediate 4 (17.26g, 0.1 mol) in acetic anhydride (75 ml). The dark mixture
was heated at 95ºC under inert atmosphere for 1.5h. The mixture was cooled to room temperature and
water (700 ml) was added carefully (exotherm). The suspension obtained was takenꢀout the flask and
neutralized at 0ºC (ice/water bath) by adding solid NaOH and extracted with dichloromethane (3x300
ml). The organic layer was washed successively with 10% Na2CO3 (500 ml), water (500 ml) and brine
(500 ml), dried over MgSO4 and concentrated to dryness. The dark brown oily residue was purified by
column chromatography on silica gel, eluting with mixtures hexane/acetone (v/v 5:1 and 4:1). 15g of the
title compound were obtained as a orange powder (64% yield).
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Method B:
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To a mixture of Eaton´s reagent (4.5 ml) and acetic anhydride (3 ml) preꢀheated at 50ºC was added
dropwise a solution of Intermediate 4 (1g, 6 mmol) in acetic anhydride (3 ml). After heating overnight
the mixture was allowed to cool to room temperature, then water (50 ml) was added carefully. The
mixture was extracted with toluene (50 ml) and the aqueous layer was placed in an iceꢀwater bath, then
neutralized carefully with solid NaOH (3g). The resulting aqueous mixture was extracted with DCM
(2x50 ml) and the combined organic layers dried over MgSO4. Solvent was ecaporated and the crude
purified by silica gel column chromatography, eluting with a mixture Hexane/EtOAc v/v 3:1 to afford
0.8g of the title compound as a brown powder (58% yield).
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1HꢀNMR(δ, ppm, CDCl3): 8.01(bd, 1H); 7.51(dd, 1H); 6.81(d, 1H); 6.19(s, 1H); 3.95(s, 3H); 2.29(s,
3H); 2.23(s, 3H)
3-(6-Hydroxypyridin-3-yl)-2,6-dimethyl-4H-pyran-4-one (6)
A solution of Intermediate 5 (15g, 0.064 mol) in 6N Hydrochloric acid (330 ml) was heated at 110ºC
for 48h, then cooled to room temperature. The resulting solution was carefully neutralized at 0ºC
(ice/water bath) by slow addition of 6N NaOH. The aqueous solution was passed through a column of
resin DIAION HP20 (250ꢀ600°C) in order to eliminate inorganics and the organics were eluted with
methanol. Elimination of the solvent gave a brown powder which was dissolved in a mixture
dichloromethane/10% methanol and filtered through a pad of celite. Elimination of the solvent gave
13.4g of the title compound as a pale brown powder which was used for the next step without further
purification (95% yield). 1HꢀNMR(δ, ppm, CD3OD): 7.47(dd, 1H); 7.37(d, 1H); 6.59(d, 1H); 6.24(s,
1H); 2.34(s, 3H); 2.30(s, 3H)
2,6-dimethyl-5-(6-{[4-(trifluoromethoxy)benzyl]oxy}pyridin-3-yl)-4H-pyran-4-one (7)
To a solution of Intermediate 6 (0.326 g, 1.5 mmol) in dry toluene (15 ml) were added consecutively
4ꢀtrifluoromethoxybenzyl bromide (0.6 ml, 3 mmol) and silver carbonate (0.31 g, 1.12 mmol). The
mixture was heated at 60ºC in the dark for 30h, then cooled to room temperature. The remaining solids
were separated by filtration and the solid washed with ethyl acetate. The combined organics were
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Journal of Medicinal Chemistry
washed with 10% NaHCO3 (75 ml) and water (2x50 ml), then dried over Na2SO4. Elimination of the
solvent under vacuum gave an oil which was purified by column chromatography on silica gel, eluting
with mixtures hexane/ethyl acetate (v/v 2:1 and 1:1), to afford 0.423g (72% yield) of the title compound
as a pale brown powder. 1HꢀNMR(δ, ppm, DMSOꢀd6): 8.02(d, 1H); 7.61ꢀ7.58(m, 3H); 7.38(bd, 2H);
6.93(d, 1H); 6.21(s, 1H); 5.39(s, 2H); 2.27(s, 3H); 2.18(s, 3H)
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Compounds 8-13 were prepared in a similar manner to 7 by using the appropriates substituted benzyl
halides (spectroscopical data available in the supporting material).
2,6-Dimethyl-6'-{[4-(trifluoromethoxy)benzyl]oxy}-3,3'-bipyridin-4(1H)-one (14)
To a solution of Intermediate 7 (0.85g, 2.17 mmol) in ethanol (30 ml) was added commercial 30%
aqueous ammonia (110 ml) with stirring. The suspension thus obtained was placed into a steel reactor
and heated to 140ºC. After eight hours of heating, the reactor was allowed to cool to room temperature
overnight with mechanical stirring. The precipitate was filtered and washed with ethyl acetate. 0.495g of
the title compound were obtained as a pale brown powder after drying under vacuum (59% yield). 1Hꢀ
NMR(δ, ppm, DMSOꢀd6): 11.15(bs, 1H); 7.94(dd, 1H); 7.58(d, 2H); 7.54(dd,1H); 7.37(d, 2H); 6.88(d,
1H); 5.92(s, 1H); 5.38(s, 2H); 2.18(s, 3H); 2.08(s, 3H)
Compounds 15-20 were prepared in a similar manner to 14 from the appropriate 4ꢀpyranones
(spectroscopical data available in the supporting material).
5-Bromo-2,6-dimethyl-6'-{[4-(trifluoromethoxy)benzyl]oxy}-3,3'-bipyridin-4(1H)-one (21)
To a solution of Intermediate 14 (0.211g, 0.54 mmol) in a mixture dichloromethane/methanol v/v 2:1
(30 ml) was added portionwise Nꢀbromosuccinimide (0.106g, 0.6 mmol). The mixture was stirred at
room temperature under inert atmosphere for 1h. Removal of the solvent under vacuum gave a crude
white powder which was triturated with acetonitrile (20 ml) and filtered. The solid obtained was washed
with acetonitrile, then dried under vacuum to afford 0.217 g of the title compound as a white powder
(85% yield). 1HꢀNMR(δ, ppm, DMSOꢀd6): 11.15(bs, 1H); 7.97(bd, 1H); 7.62ꢀ7.55(m,3H); 7.37(d, 2H);
6.90(d, 1H); 5.39(s, 2H); 2.41(s, 3H); 2.1(s, 3H); [ES MS] m/z 470 (MH+)
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Compounds 22-27 were prepared in a similar manner to 21 from the appropriate 4(1H)pyridones
(spectroscopical data available in the supporting material).
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5-Chloro-2,6-dimethyl-6'-{[4-(trifluoromethoxy)benzyl]oxy}-3,3'-bipyridin-4(1H)-one (28)
To a cooled solution (0ꢀ4ºC, ice/water bath) of Intermediate 14 (0.276g, 0.71 mmol) in a mixture
dichloromethane/methanol v/v 2:1 (24 ml) was added portionwise under inert atmosphere
trichloroisocyanuric acid (67 mg, 0.29 mmol). The mixture was stirred at low temperature under inert
atmosphere for 1.5h, then diluted with a mixture dichloromethane/methanol v/v 2:1 (30 ml). The solid
precipitated was filtered and the filtrate was concentrated to dryness under vacuum to afford a solid
residue which was suspended in a mixture of aqueous 0.5N NaOH (30 ml) and methanol (10ml). After
stirring for 1h, the mixture was diluted by adding water (20 ml) and the remaining precipitate was
filtered and washed successively with water (3x10 ml), mixture methanol/water v/v 1:1 (2x10 ml) and
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acetonitrile (3x10ml). 0.193g of the title compound were obtained as a white powder after drying under
vacuum (64% yield). 1HꢀNMR(δ, ppm, DMSOꢀd6): 11.65(bs, 1H); 7.98(bd, 1H); 7.61ꢀ7.55(m,3H);
7.38(d, 2H); 6.91(d, 1H); 5.39(s, 2H); 2.37(s, 3H); 2.11(s,3H); [ES MS] m/z 425 (MH+)
Compounds 29-34 were prepared in a similar manner to 28 from the appropriate 4(1H)pyridones
(spectroscopical data available in the supporting material).
5-Bromo-2-({4-[(trifluoromethyl)oxy]phenyl}oxy)pyridine (36)
To a mixture of 4ꢀtrifluoromethoxyphenol (4.4ml, 0.033 mol) and sodium hydride (60% dispersion in
mineral oil, 1.35g, 0.033 mol) in dry DMF (3ml) was added a solution of 2,5ꢀdibromopyridine 35 (1g,
0.004 mol) in dry DMF (3mL). The resulting mixture was heated at 60ºC for 8h. Upon cooling, ethyl
acetate was added and the mixture was washed with NH4Cl 1N.The organic layer was dried over
Na2SO4 and the solvent removed by evaporation under reduced pressure. The crude product was
purified by column chromatography on silica gel (eluent: AcOEt/Hexane 1:10) to afford 1.08g of the
final compound as a colorless oil (80% yield). 1HꢀNMR(δ, ppm, CDCl3): 8.21 (d, 1H), 7.80 (dd, 1H),
7.25 (d, 2H), 7.14 (d, 2H), 6.87 (d, 1H).
[6-({4-[(Trifluoromethyl)oxy]phenyl}oxy)-3-pyridinyl]boronic acid (37)
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A solution of 36 (0.60g, 1.88 mmol) and triꢀisopropyl borate (0.54 mL, 2.33 mmol) in dry THF (6
1
2
mL) was added nꢀBuLi 1.6M (1.24mL, 1.98 mmol) at ꢀ78ºC. The reaction was allowed to warm to room
temperature and stirred for 8h, then quenched by addition of HCl 2N until pH=2. After stirring for 1h,
the aqueous phase was extracted with EtOAc, the organic phase was dried over Na2SO4, evaporated
under reduced pressure and the crude was washed with pentane to yield 0.330 g of the title compound as
a white solid (59% yield). 1H NMR (δ, ppm, CD3OD): 8.45 (d, 1H), 8.16 (dd, 1H), 7.41 (d, 2H), 7.25 (d,
2H), 7.03 (d, 1H)
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3-Chloro-5-iodo-2,6-dimethyl-4(1H)-pyridinone (39)
A suspension of 2,6ꢀdimethylꢀ4(1H)Pyridinone 38 (10g, 0.081 mol) and Nꢀchlorosuccinimide (14.3g,
0.107 mol) in a mixture of methanol (100 ml) and dichloromethane (250ml) as solvent was stirred under
inert atmosphere at room temperature overnight. The remaining precipitate was filtered and discarded,
then the filtrate was concentrated to dryness to afford a residue which was triturated with acetonitrile.
The insoluble material was filtered and washed with acetonitrile, then dried under vacuum to afford 8.6g
of material, which was added portionwise onto a solution of sodium hydroxide (2.2g, 0.07 mol) in water
(340ml). To the resulting solution was added iodine (13.8g, 0.054 mol) and the mixture stirred at room
temperature for 1h. The precipitate obtained was filteredꢀoff and washed with water and acetonitrile
(2x100 ml) to afford 11.5g of the title compound (50% yield). 1HꢀNMR(δ, ppm, CD3OD): 2.59(s, 3H);
2.45(s, 3H)
5-Chloro-2,6-dimethyl-6'-({4-[(trifluoromethyl)oxy]phenyl}oxy)-3,3'-bipyridin-4(1H)-one (40)
A mixture of boronic acid 37 (0.100 g, 0.33mmol), potassium carbonate (0.290g, 2.1mmol),
palladium acetate (0.008g, 0.03mmol) and 39 (0.211g, 0.7mmol) in DMF (2mL) was heated at 140ºC,
for 30 minutes under microwave irradiation. The reaction mixture was filtered through a pad of celite,
evaporated under reduced pressure and purified by column chromatography on silica gel (eluent
CH2Cl2/CH3OH, 10:1) to afford 0.087g of the title compound as a white solid (60% yield). 1H NMR (δ,
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ppm, DMSOꢀd6): 11.69 (s, 1H), 7.95 (d, 1H), 7.72 (dd, 1H), 7.42 (d, 2H), 7.30 (d, 2H), 7.10 (d, 1H),
2.38 (s, 3H), 2.12 (s, 3H); [ES MS] m/z 411 (MH+)
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2,6-Dimethyl-3-(6-trifluoromethanesulfonyloxypyridin-3-yl)-4H-pyran-4-one (41)
To a suspension of Intermediate 6 (4g, 0.018 mol) in dry N,Nꢀdimethylformamide (120 ml) was
added powdered potassium carbonate (7.62g, 0.07 mol). After stirring at room temperature for 2
minutes, solid Nꢀphenyltrifluoromethanesulfonimide (6.58g, 0.018 mol) was added portionwise. After
1h of stirring the suspension was diluted with ethyl acetate (300 ml) and washed with ammonium
chloride (3x250 ml). The solvent was removed to dryness under vacuum and the residue dissolved in
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diethyl ether (200 ml). The ethereal solution was washed with 1N NaOH (4x200 ml) and brine (200 ml),
dried over Na2SO4 and concentrated to dryness to afford 5g of the title compound as a pale yellow
powder (78% yield), which was used for the next step without further purification. 1HꢀNMR(δ, ppm,
CDCl3): 8.24(d, 1H); 7.86(dd, 1H); 7.23(d, 1H); 6.23(s, 1H); 2.32(s, 3H); 2.26(s,3H)
2,6-Dimethyl-3-(6-{[4-(trifluoromethyl)phenyl]ethynyl}-3-pyridinyl)-4H-pyran-4-one (42)
To a solution of Intermediate 41 (0.25g, 0.72 mmol) in a mixture of dry N,Nꢀdimethylformamide (5
ml) and triethylamine (2 ml) were added consecutively CuI (0.051g, 0.27 mmol)), (PPh3)2PdCl2(0.050
g, 0.07 mmol) and PPh3 (0.111g, 0.42 mmol). The mixture was deoxygenated by bubbling argon and 4ꢀ
ethynylꢀα,α,αꢀtrifluorotoluene (0.23 ml, 1.35 mmol) was added, then heated to reflux for 1h. The
mixture was cooled to room temperature, diluted with ethyl acetate and washed with 1N NH4Cl. The
solvent was evaporated and the resulting crude was dissolved in diethyl ether. The ethereal solution was
washed with 1N HCl until no product was detected in the organic layer. The acidic aqueous solution
was basified by adding 2N NaOH and extracted with tertꢀbutylꢀmethylꢀethyl. Elimination of the solvent
gave 0.14g of the title compound (52% yield). 1HꢀNMR(δ, ppm, CD3OD): 8.47(s,1H); 7.79(m, 6H);
6.30(s, 1H); 2.37(s, 3H); 2.29(s,3H)
Compounds 43 and 44 were prepared in a similar manner to 42 by using the appropriate ethynyl
derivatives (spectroscopical data available in the supporting material).
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Journal of Medicinal Chemistry
2,6-Dimethyl-6'-{[4-(trifluoromethyl)phenyl]ethynyl}-3,3'-bipyridin-4(1H)-one (45)
To a solution of Intermediate 42 (0.07g, 0.19 mmol) in MeOH (1 ml) was added commercial 30%
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aqueous ammonia (3ml) and the suspension heated to 140ºC for 1h under microwave radiation. Upon
cooling, the solid precipitated was filtrated and washed with acetonitrile. 0.035g of the title compound
were obtained (50% yield). 1HꢀNMR(δ, ppm, CD3OD): 8.45(m,1H); 7.75(m, 6H); 6.30(s, 1H); 2.36(s,
3H); 2.23(s, 3H)
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Compound 46 was prepared in a similar manner to 45 from the appropriate 4ꢀpyranone 43
(spectroscopical data available in the supporting material).
5-Bromo-2,6-dimethyl-6'-{[4-(trifluoromethyl)phenyl]ethynyl}-3,3'-bipyridin-4(1H)-one (47)
To a solution of Intermediate 45 (0.012 g, 0.032 mmol) in a mixture of dichloromethane/methanol
v/v 5:1 (6 ml) was added portionwise at room temperature Nꢀbromosuccinimide (0.0064g, 0.035 mmol).
The suspension was stirred at room temperature for 30 min, then concentrated to dryness under vacuum.
The residue thus obtained was triturated with acetonitrile and filtered. The solid was washed with
acetonitrile and dried under vacuum. 0.010 g of the title compound were obtained as a white powder
(69% yield). 1HꢀNMR(δ, ppm, CD3OD): 8.47(m,1H); 7.77(m, 6H); 2.56(s, 3H); 2.23(s, 3H); [ES MS]
m/z 447 (MH+)
5-Chloro-2,6-dimethyl-6'-{[4-(trifluoromethyl)phenyl]ethynyl}-3,3'-bipyridin-4(1H)-one (48)
To a solution of Intermediate 45 (0.027g, 0.073 mmol) in a mixture of dichloromethane/methanol v/v
5:1 (6 ml) was added portionwise at room temperature trichloroisocyanuric acid (0.0068g, 0.029 mmol).
The suspension was stirred at room temperature for 30 min, then concentrated to dryness under vacuum.
The residue thus obtained was purified by preparative TLC chromatography, eluting with a mixture
dichloromethane/5% methanol. 0.015 g of the title compound were obtained (50% yield).1HꢀNMR(δ,
ppm, CD3OD): 8.47(m,1H); 7.77(m, 6H); 2.51(s, 3H); 2.23(s, 3H); [ES MS] m/z 401 (MH+)
Compound 49 was prepared in a similar way described for 48 from 4(1H)ꢀpyridone 46
(spectroscopical data available in the supporting material).
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2,6-Dimethyl-3-(6-{2-[4-(trifluoromethyl)phenyl]ethyl}-3-pyridinyl)-4H-pyran-4-one (50)
A solution of Intermediate 42 (0.2g, 0.54 mmol) in a mixture of ethyl acetate/methanol v/v 3:1 (20
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ml) was deoxygenated by bubbling argon for 5 minutes, then Palladium 10% w/w on activated charcoal
(0.050 g) and 1H HCl (0.541 ml) were added. The mixture was hydrogenated at 30 psi for 4 hours. The
catalyst was removed by filtration and the solvent evaporated to dryness under vacuum to afford 0.168g
of the expected compound (83% yield). 1HꢀNMR(δ, ppm, CDCl3): 8.41(m,1H); 7.70ꢀ7.64(m, 1H); 7.56ꢀ
7.52(m, 2H); 7.33(d, 2H); 7.17(d, 1H); 6.21(m, 2H); 3.14(m, 4H); 2.30(s, 3H); 2.23(s,3H)
Compound 51 was prepared in a similar manner to 50 from compound 43(spectroscopical data
available in the supporting material).
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2,6-Dimethyl-6'-{2-[4-(trifluoromethyl)phenyl]ethyl}-3,3'-bipyridin-4(1H)-one (52)
To a solution of Intermediate 50 (0.164g, 0.44 mmol) in MeOH (1 ml) was added commercial 30%
aqueous ammonia (3ml) and the suspension heated to 145ºC for 45 minutes under microwave radiation.
Upon cooling, the solid precipitated was filtrated and washed with acetonitrile. 0.092g of the title
compound were obtained (56% yield). 1HꢀNMR(δ, ppm, DMSOꢀd6): 11.18(bs, 1H); 8.28(m, 1H);
7.63(d, 2H); 7.51ꢀ7.46(m, 3H); 7.25(d, 1H); 5.93(s, 1H); 3.08(m, 4H); 2.19(s, 3H); 2.06(s, 3H).
Compound 53 was synthetized in a similar way to 52 from the intermediate 51 (spectroscopical data
available in the supporting material).
5-Chloro-2,6-dimethyl-6'-{2-[4-(trifluoromethyl)phenyl]ethyl}-3,3'-bipyridin-4(1H)-one (54)
To a solution of Intermediate 52 (0.038g, 0.1 mmol) in a mixture dichloromethane/methanol v/v 2:1
(6 ml) was added portionwise Trichloroisocyanuric acid (9.3 mg, 0.04 mmol). After 30 min of stirring at
room temperature the solvent was removed under vacuum and the residue purified by preparative TLC
chromatography, eluting with a mixture dichloromethane/10% methanol. 0.025g of the title compound
were obtained (61% yield). 1HꢀNMR(δ, ppm, CD3OD): 8.34(m,1H); 7.60(dd, 1H); 7.47(m, 2H); 7.30(d,
1H); 3.14(m,4H); 2.50(s,3H); 2.17(m, 3H); [ES MS] m/z 405 (MHꢀ)
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Journal of Medicinal Chemistry
Compound 55 was prepared in a similar manner to 54 from the appropriate 4(1H)ꢀpyridone 53
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(spectroscopical data available in the supporting material).
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2,6-dimethyl-3-(6-{4-[(trifluoromethyl)oxy]phenyl}-3-pyridinyl)-4H-pyran-4-one (56).
To a solution of Intermediate 41 (2.27 g, 6.49 mmol) in a mixture of toluene (120 ml) and ethanol
(60 ml) were added successively (PPh3)2PdCl2(0.456 g, 0.39 mmol) and 4ꢀtrifluoromethoxyphenyl
boronic acid (1.6g, 7.76 mmol) and the resulting suspension was deoxygenated by bubbling argon for 5
minutes. Saturated NaHCO3 (23 ml) was added dropwise and the mixture heated to 95ºC for 2.5 h. The
mixture was cooled to room temperature and the mixture concentrated to dryness under vacuum. The
oily residue thus obtained was dissolved in tertꢀbutylꢀmethyl ether (400 ml) and washed with 1N NaOH
(2x250ml). The organic layer was washed with 1N HCl (7x100 ml) until no remaining material was
detected in the organic layer. The aqueous layer was carefully basified by slow addition of 6N NaOH
(80 ml) and 2N NaOH (150ml), then extracted with tertꢀbutylꢀmethyl ether (2x300 ml). The combined
organic layers were washed with brine (500 ml), dried over Na2SO4 and concentrated to dryness to
afford the title compound (2g) as a yellowish powder (85% yield). 1HꢀNMR (δ, ppm, CDCl3): δ 8.54(m,
1H); 8.06ꢀ8.01(m, 2H); 7.79ꢀ7ꢀ72(m, 2H); 7.32(bd, 2H); 6.24(s, 1H); 2.32(s, 3H); 2.29(s, 3H).
Compounds 57-79 were prepared in a similar manner to 56, by coupling between triflate 41 and the
appropriate boronic acids (spectroscopical data available in the supporting material).
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2,6-dimethyl-3-(6-{4-[(2,2,2-trifluoroethyl)oxy]phenyl}-3-pyridinyl)-4H-pyran-4-one (80).
In a sealed tube, to a mixture of intermediate 71 (0.1 g, 0.34 mmol) and 2ꢀiodoꢀ1,1,1ꢀtrifluoroethane
(0.12 ml, 0.1.19 mmol) dissolved in dry DMF (1.40 ml) cesium carbonate (0.22 g, 0.68 mmol) was
added under argon atmosphere and the mixture was heated to 50ºC overnight. The reaction was
quenched with water and extracted with EtOAc. The aqueous layer was extracted with EtOAc (2x15 ml)
and the combined organic layers were washed with 1N NH4Cl and dried over Na2SO4. Column
chromatography (eluent: CH2Cl2/EtOAc) of the crude afforded the title compound (0.067g) as a yellow
solid (52% yield). 1HꢀNMR (δ, ppm, CD3OD): δ 8.50(m, 1H); 8.12ꢀ8.05(m,2H); 7.94ꢀ7.91(m,1H); 7.81ꢀ
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7.77(m,1H); 7.36ꢀ7.12(m, 2H); 6.30(s, 1H); 4.87(m, 2H); 2.38(s, 3H); 2.30(s, 3H); [ES MS] m/z 376
(MH+).
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3-(6-{4-[(difluoromethyl)oxy]phenyl}-3-pyridinyl)-2,6-dimethyl-4H-pyran-4-one (81).
Intermediate 72 (0.036 g, 0.118 mmol) was placed in a teflon round botton flask and
diethylaminosulfur trifluoride (DAST) (1.0 ml, 5.0 mmol) was added dropwise at 0ºC under argon
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atmosphere. The mixture was stirred at room temperature overnight. Then reaction was cooled in an ice
bath and carefully quenched with water. The mixture was diluted with CH2Cl2 (15 mL) and the layers
partitioned. The aqueous layer was extracted with CH2Cl2 (3x15 ml) and the combined organic layers
washed with 10% NaHCO3 (1x15mL) and brine, dried over Na2SO4, filtered and concentrated under
vacuo. The crude was purified by column chromatography (eluent: MeOH/CH2Cl2) to afford the title
compound (0.104 g) as a brown solid (32% yield). 1HꢀNMR (δ, CDCl3): δ 7.61ꢀ8.55(m, 1H); 8.11(d,
2H); 7.84ꢀ7.78(m,2H); 7.64ꢀ7.61(m,2H); 6.71(m, 1H); 6.24(m, 1H); 4.87(m, 2H; 2.32(s, 3H); 2.30(s,
3H); [ES MS] m/z 328 (MH+).
2,6-dimethyl-6´-{4-[(trifluoromethyl)oxy]phenyl}-3,3´-bipyridin-4(1H)-one (82).
To a solution of Intermediate 56 (2.115 g, 5.87 mmol) in ethanol (13 ml) was added commercial 30%
aqueous ammonia (44 ml, 610 mmol) with stirring. The suspension thus obtained was placed into a steel
reactor and heated to 140ºC. After eight hours of heating, the reactor was allowed to cool to room
temperature overnight. The precipitate was filtered and washed successively with ethyl acetate and
dichloromethane to give compound 82 (1.38 g, 65%) as a pale brown powder after drying under
vacuum. 1HꢀNMR (δ, DMSOꢀd6): δ 11.24(bs, 1H); 8.48(m,1H); 8.23(m, 2H); 7.99(d, 1H); 7.73(dd,1H);
7.48(m, 2H); 5.97(s, 1H); 2.21(s, 3H); 2.14(s, 3H).
Compounds 83-105 were prepared by a similar method to that described for compound 82 from the
corresponding 4ꢀpyranones (spectroscopical data available in the supporting material).
5-Bromo-2,6-dimethyl-6'-{4-[(trifluoromethyl)oxy]phenyl}-3,3'-bipyridin-4(1H)-one (106).
To a solution of intermediate 82 (0.064 g, 0.18 mmol) in a mixture of dichloromethane/methanol v/v
1:1 (2 ml) was added portionwise at room temperature Nꢀbromosuccinimide (0.026 g, 0.15 mmol). The
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Journal of Medicinal Chemistry
suspension was stirred at room temperature for 30 min, then concentrated to dryness under vacuum. The
residue thus obtained was triturated with ethyl acetate and filtered. The solid was washed with ethyl
acetate and dried under vacuum. 0.062g of compound 106 was obtained as a white powder (78% yield).
1HꢀNMR (δ, DMSOꢀd6): δ 11.7ꢀ11.6(bs, 1H); 8.49(m,1H); 8.27ꢀ8.22(m, 2H); 8.03(d, 1H); 7.75(dd,1H);
7.48(m, 2H); 2.44(s, 3H); 2.15(s, 3H); [ES MS] m/z 439 (MH+).
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Compounds 107-115 were prepared in a similar manner to compound 106 from the corresponding
4(1H)ꢀpyridone (spectroscopical data available in the supporting material).
5-Chloro-2,6-dimethyl-6'-{4-[(trifluoromethyl)oxy]phenyl}-3,3'-bipyridin-4(1H)-one (116).
To a cooled solution (0ꢀ4ºC, ice/water bath) of 82 (1.39 g, 3.86 mmol) in a mixture of
dichloromethane/methanol v/v 2:1 (120 ml) was added portionwise at room temperature
trichloroisocyanuric acid (0.365 g, 1.57 mmol) and stirring continued for 1h. The mixture was filtered
and the precipitate washed with a mixture dichloromethane/methanol v/v 2:1. The filtrate was
evaporated and the solid thus obtained suspended in aqueous 0.5N NaOH (60 ml) and diluted with
methanol (20 ml) and water (140 ml) and the precipitate formed was isolated by filtration. The solid was
washed successively with water (3x20 ml) and acetonitrile (3x20 ml). 1,1g of compound 116 were
obtained as a white powder after drying under vacuum (72% yield). 1HꢀNMR (δ, ppm, DMSOꢀd6): δ
11.7ꢀ11.6(bs, 1H); 8.50(m,1H); 8.24(m, 2H); 8.02(d, 1H); 7.75(m,1H); 7.48(m, 2H); 2.39(s, 3H); 2.15(s,
3H); [ES MS] m/z 393 (MH+).
Compounds 117-125 were prepared by a similar way followed to achieve compound 116 starting
from the corresponding 4(1H)pyridones (spectroscopical data available in the supporting material).
5-bromo-2-{4-[(trifluoromethyl)oxy]phenyl}pyridine (126)
A mixture of 2,5ꢀdibromopyridine 35 (5.00g, 21.11 mmol), cesium carbonate (7.56g, 23.22 mmol)
and 4ꢀ(trifluoromethoxy)benzeneboronic acid (4.13g, 20.05 mmol) in Tol/EtOH 10/1 (165 ml) was
stirred and deoxygenated with argon for 15 min. (PPh3)2PdCl2 (0.741 g, 1.055 mmol) was added over
this mixture and the reaction was heated at 80 ºC for 1h. The reaction was quenched with water and
extracted with AcOEt. The organic layer was washed with brine, dried over sodium sulfate and the
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solvent evaporated under vacuum. The residue obtained was purified by flash column chromatography
(silica gel, 15:1 Hex:AcOEt) to afford 5.8 g of the title compound (90% yield). 1H NMR (δ, ppm,
DMSOꢀd6 ): 8.80 (d, 1H); 8.25ꢀ8.10 (m, 3H); 7.99 (d, 1H); 7.48 (d, 2H).; [ES MS] m/z: 318 [M+H]+.
5-bromo-2-[2-fluoro-4-(trifluoromethyl)phenyl]pyridine (127)
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(trifluoromethyl)phenyl]boronic acid (3g, 14.4 mmol) and 2M Na2CO3 (21.6 ml, 43.3 mmol) in 108 ml
of toluene and 36 ml of ethanol (3 : 1), were added Pd(PPh3)4 (0.83g, 0.7 mmol) under nitrogen. The
mixture was heated at 80ºC overnight. The suspension obtained was diluted with 100 ml of AcOEt and
washed with water (3x50 ml). The resulting organic layer was dried over anhydrous MgSO4 and
evaporated to afford 4.1g of crude material which was used for the next step without any further
purification (83% yield). 1H NMR (δ, ppm, DMSOꢀd6 ) 8.89 (d, 1 H); 8.22 (ddd, 1 H); 8.13 (t, 1 H); 7.8ꢀ
7.7 (m, 2 H); 7.73 (m, 1 H).
(6-{4-[(trifluoromethyl)oxy]phenyl}-3-pyridinyl)boronic acid (128)
Compound 128 was prepared following an analogous procedure to that used for compound 129
starting from bromoderivative 126. 1HꢀNMR (δ, ppm, DMSOꢀd6): 8.96 (s, 1H); 8.38 (s,2H); 8.29ꢀ8.16
(m, 3H); 7.97 (d, 1H); 7.47 (d, 2H); [ES MS] m/z: 284 [M+H]
{6-[2-fluoro-4-(trifluoromethyl)phenyl]-3-pyridinyl}boronic acid (129)
A solution of crude 127 (3.75g, 11.72 mmol) in 1,4ꢀdioxane (60mL) was deoxygenated with argon,
then bis(pinacolato)ꢀdiboron (3.57g, 14.06 mmol), (PPh3)2PdCl2 (0.164 g, 0.234 mmol) and potassium
acetate (3.45g, 35.1 mmol) were added. The mixture was heated to 100 ºC for approx 5 h then allowed
to cool to room temperature with stirring overnight, filtered through celite, and concentrated to dryness.
The crude was suspended in a 120 ml of a mixture acetone/water v/v 1:1 and ammonium acetate (2.0 g,
26.4 mmol) was added followed by NaIO4 (5.6g, 26.4 mmol). The mixture was stirred at room
temperature overnight. The organic solvent was evaporated under reduced pressure and 2N NaOH (30
mL) was added. The mixture was stirred at room temperature for 15 min then extracted with CH2Cl2 (2x
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Journal of Medicinal Chemistry
60 mL). The aqueous layer was cooled to 0 ºC, and 2N HCl was added dropꢀwise until pH was adjusted
to 5. The white solid precipitated was filtered and dried under vacuum to afford 1.2g of the title
compound (36% yield). 1HꢀNMR (δ, ppm, DMSOꢀd6): 9.03 (s, 1H); 8.26ꢀ8.15 (m, 2H); 7.85ꢀ7.82 (m,
2H); 7.74ꢀ7.71 (d, 1H).
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3-Benzyloxy-6-methyl-2-(O-tert-butyldimethylsilyl)hydroxymethyl-pyran-4(1H)-one (131)
In a 2L jacketed reactor were introduced 4ꢀpyranone 130 (66.65 g, 0.27 mol, Ref. 26) and dry N,Nꢀ
dimethylformamide (500 mL). Imidazole (55.28g, 0.81 mol) and a solution of tertꢀbutyldimethylsilyl
chloride (48.94g, 0.32 mol) in dry N,Nꢀdimethylformamide (50 mL) were added. After stirring for 3 h,
ethyl acetate (900 mL) and 1N NH4Cl (700 mL) were added. The two layers were partitioned and the
organic layer washed with 1N NH4Cl (2x900 mL) and brine (900 mL), dried over Na2SO4, filtered and
concentrated to dryness under vacuum to give 100.68 g of the title compound as a pale yellow oil (100%
yield). 1HꢀNMR(δ, ppm, CDCl3): 7.35(m, 5H); 6.20(s,1H); 5.16(s, 2H); 4.39(s, 2H); 2.26(s, 3H); 0.87(s,
9H); 0.04 (s, 6H)
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3-Hydroxy-6-methyl-2-(O-tert-butyldimethylsilyl)hydroxymethyl-pyran-4(1H)-one (132)
Over a solution of 131 (23.71g, 65 mmol) in ethyl acetate (600 mL) under N2 atmosphere was added
palladium 10% w/w on activated charcoal (700.9 mg). The mixture was hydrogenated at 30 psi for 3
hours, after that time the catalyst was removed by filtration. The solvent was evaporated to dryness to
afford 16.72 g of the title compound (94% yield). 1HꢀNMR (δ, ppm, CDCl3): 6.43(bd, 1H); 6.23(s,1H);
4.70(s, 2H); 2.32(s, 3H); 0.91 (s, 9H); 0.12 (s, 6H)
6-Methyl-2-(O-tert-butyldimethylsilyl)hydroxymethyl-3-trifluoromethanesulfonyloxy-pyran-
4(1H)-one (133)
In a 500 mL round bottom flask was introduced compound 132 (16.72g, 61 mmol) in dry N,Nꢀ
dimethylformamide (170 mL) and the solution stirred under N2 atmosphere for 10 min. Then Nꢀ
phenyltrifluoromethanesulfonimide (23.85g, 66 mmol) and powdered potassium carbonate (10.68g, 77
mmol) were added in portionwise. After stirring for 1.5 h, potassium carbonate was removed by
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Journal of Medicinal Chemistry
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filtration and washed with tertꢀbutylꢀmethylꢀether (300 mL). The filtrate was diluted with 100 mL of
tertꢀbutylꢀmethylꢀether and washed with 1N NH4Cl (2x400mL). The aqueous layer was extracted with
tertꢀbutylꢀmethylꢀether (200 mL). The organic layers were washed with Na2CO3 (3x400 mL) and brine
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(400 mL), dried over Na2SO4, filtered and concentrated in vacuo to give 24.7 g of the title compound as
a pale orange solid (100% yield). 1HꢀNMR (δ, ppm, CDCl3): 6.29(s, 1H); 4.6624(s, 2H); 2.34(s, 3H);
0.92 (s, 9H); 0.13 (s, 6H)
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2-({[(1,1-dimethylethyl)(dimethyl)silyl]oxy}methyl)-6-methyl-3-(6-{4-
[(trifluoromethyl)oxy]phenyl}-3-pyridinyl)-4H-pyran-4-one (134)
To a deoxygenated solution of boronic acid 128 (0.736g, 2.60 mmol) and triflate 133 (1.361g, 3.38
mmol) in a mixture of anhydrous ethanol (21 ml) and anhydrous toluene (7 ml) were added solid
Na2CO3 (1.103g, 10.4 mmol) and (PPh3)2PdCl2 (0.183g, 0.260 mmol). The reaction mixture was flushed
with argon for 10 min, then heated to 80 °C for 2h. Additional boronic acid 128 (0.45g, 1.6 mmol) and
(PPh3)2PdCl2(0.183g, 0.26 mmol) were added and the mixture stirred at 80ºC for 1h. The reaction
mixture was filtered, solvent removed by evaporation and the residue obtained was purified by flash
chromatography on silica gel (Hex:AcOEt 0ꢀ50%) to afford 0.850 g of the title compound (66% yield).
1HꢀNMR (δ, ppm, CD3OD): 8.58ꢀ8.51(m, 1H); 8.17ꢀ8.07(m, 2H); 8.00ꢀ7.91(m, 1H); 7.88ꢀ7.79(m, 1H);
7.45ꢀ7.35(m, 2H); 6.38ꢀ6.32(m, 1H); 4.53ꢀ4.46(m, 2H); 2.44ꢀ2.36(m, 3H); 0.91ꢀ0.83(m, 9H); 0.08ꢀ
0.01(m, 6H); [ES MS] m/z: 492 [M+H]+.
2-({[(1,1-dimethylethyl)(dimethyl)silyl]oxy}methyl)-3-{6-[2-fluoro-4-(trifluoromethyl)phenyl]-
3-pyridinyl}-6-methyl-4H-pyran-4-one (135)
Compound 135 was prepared following an analogous procedure to that used for compound 134,
starting from boronic acid 129.
1HꢀNMR (δ, ppm, CD3OD) 8.58(s, 1 H), 8.08(t, 1 H), 7.9ꢀ7.8(m, 2 H), 7.59(m, 2 H), 6.31(s, 1 H),
4.46 (s, 2 H), 2.36(s, 3 H), 0.87(s, 9 H), 0.05(s, 6 H); [ES MS] m/z: 494 [M+H]+.
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Journal of Medicinal Chemistry
2-(hydroxymethyl)-6-methyl-6'-{4-[(trifluoromethyl)oxy]phenyl}-3,3'-bipyridin-4(1H)-one
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In a 20 mL vessel microwave was introduced compound 134 (0.5g, 1.017 mmol) dissolved in ethanol
(8ml). Ammonia (32% in water, 5.78 ml, 85 mmol) was added and the reaction vessel sealed and heated
in a microwave oven to 140 ºC for 50 min. After cooling, reaction was evaporated to dryness. The solid
residue was triturated with diethyl ether, filtered and washed with diethyl ether, dried under vacuum to
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afford 355 mg of the title compound (94% yield). H NMR (δ, ppm, DMSOꢀd6): 8.48(dd, 1H); 8.21(d,
2H); 7.93(dd, 1H); 7.72(dd, 1H); 7.46(d, 2H); 5.97 (s, 1H); 4.17(s, 2H); 2.18(s, 3H); [ES MS] m/z: 377
[M+H]+.
6'-[2-fluoro-4-(trifluoromethyl)phenyl]-2-(hydroxymethyl)-6-methyl-3,3'-bipyridin-4(1H)-one
(137)
Compound 137 was prepared following an analogous procedure to that used for compound 136,
starting from compound 135. 1H NMR(δ, ppm, DMSOꢀd6) 11.22(s, NH); 8.58(s, 1 H); 8.21(t, 1 H); 7.8ꢀ
7.7(m, 4 H); 6.03(s, 1 H); 5.61(s, OH); 4.27(s, 2 H); 2.27(s, 3 H).
5-chloro-2-(hydroxymethyl)-6-methyl-6'-{4-[(trifluoromethyl)oxy]phenyl}-3,3'-bipyridin-4(1H)-
one (138)
To a stirred solution of compound 136 (4.85g, 12.89 mmol) in a mixture of dichloromethane (150
ml) and methanol (75 ml) were added at 0ºC under nitrogen trichloroisocyanuric acid (1.225g, 5.27
mmol). The reaction mixture was stirred at 0 °C for 1h, then filtered and concentrated, The residue was
stirred with 5% Na2CO3 solution for 1h, filtered and washed with H2O, this solid was dried at vacuum
overnight. The solid was triturated with AcOEt to afford 4.24 g of the title compound (80% yield).
1H NMR (δ, ppm, DMSOꢀd6): 11.69(bs, 1H); 8.54ꢀ8.50(m, 1H); 8.25(d, 2H); 8.03(d, 1H); 7.78(dd, 1H);
7.49(d, 2H); 5.67(s, 1H); 4.28(s, 2H); 2.49(s, 3H); [ES MS] m/z: 411 [M+H]+
5-chloro-6'-[2-fluoro-4-(trifluoromethyl)phenyl]-2-(hydroxymethyl)-6-methyl-3,3'-bipyridin-
4(1H)-one (139)
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