Efficacy of a series of alpha-pyrone derivatives against Leishmania (L.) infantum and Trypanosoma cruzi

Article abstract of DOI:10.1016/j.ejmech.2017.08.055

The neglected tropical diseases Chagas disease and leishmaniasis affect together more than 20 million people living mainly in developing countries. The mainstay of treatment is chemotherapy, however the drugs of choice, which include benznidazole and milt

Full text of DOI:10.1016/j.ejmech.2017.08.055

Accepted Manuscript
Efficacy of a series of alpha-pyrone derivatives against Leishmania (L.) infantum and
Trypanosoma cruzi
Andre Gustavo Tempone, Daiane Dias Ferreira, Marta Lopes Lima, Thais Alves
Costa Silva, Samanta E.T. Borborema, Juliana Quero Reimão, Mariana K. Galuppo,
Juliana Mariotti Guerra, Angelie J. Russell, Graham M. Wynne, Roy Y.L. Lai, Melissa
M. Cadelis, Brent R. Copp
PII:
S0223-5234(17)30666-9
10.1016/j.ejmech.2017.08.055
EJMECH 9699
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 June 2017
Revised Date: 23 August 2017
Accepted Date: 23 August 2017
Please cite this article as: A.G. Tempone, D.D. Ferreira, M.L. Lima, T.A. Costa Silva, S.E.T.
Borborema, J.Q. Reimão, M.K. Galuppo, J.M. Guerra, A.J. Russell, G.M. Wynne, R.Y.L. Lai,
M.M. Cadelis, B.R. Copp, Efficacy of a series of alpha-pyrone derivatives against Leishmania (L.)
infantum and Trypanosoma cruzi, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.055.
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ACCEPTED MANUSCRIPT
Leishmania
Trypanosoma cruzi

ACCEPTED MANUSCRIPT
Efficacy of a series of alpha-pyrone derivatives against Leishmania (L.) infantum
and Trypanosoma cruzi
Andre Gustavo Tempone1*, Daiane Dias Ferreira1, Marta Lopes Lima1, Thais Alves
Costa Silva1, Samanta E. T. Borborema1, Juliana Quero Reimão2, Mariana K.
Galuppo1, Juliana Mariotti Guerra1, Angelie J. Russell4,5, Graham M. Wynne4, Roy Y. L.
Lai3, Melissa M. Cadelis3 and Brent R. Copp3*
1
Centre for Parasitology and Mycology, Instituto Adolfo Lutz, Avenida Dr. Arnaldo, 351, 8º
andar. Cerqueira César, CEP 01246-902 – São Paulo/SP, Brazil.
2
Faculty of Medicine of Jundiai, Rua Francisco Telles, 250, Vila Arens, CEP 13202-550 –
Jundiai/SP, Brazil
3 School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand.
4
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield
Road, Oxford OX1 3TA, UK
5 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
*Corresponding authors. Brent R. Copp (b.copp@auckland.ac.nz) and Andre G.
Tempone (atempone@usp.br)
Keywords: alpha-pyrones, Leishmania, Trypanosoma cruzi, SAR, phenotypic
screening
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Abstract
The neglected tropical diseases Chagas disease and leishmaniasis affect together
more than 20 million people living mainly in developing countries. The mainstay of
treatment is chemotherapy, however the drugs of choice, which include benznidazole
and miltefosine, are toxic and have numerous side effects. Safe and effective therapies
are urgently needed. Marine alpha-pyrones have been previously identified as
scaffolds with potential antiprotozoan activities. In this work, using a phenotypic screen,
twenty-seven examples of 3-substituted 4-hydroxy-6-methyl alpha-pyrones were
synthesized and their antiparasitic efficacy evaluated against Leishmania (L.) infantum
and Trypanosoma cruzi in order to evaluate structure-activity relationships within the
series. The mechanism of action and the in vivo efficacy of the most selective
compound against T. cruzi were evaluated using different techniques. In vitro data
indicated that compounds 8, 15, 25, 26 and 28 presented IC50 values in the range
between 13 to 53 µM against L. infantum intracellular amastigotes. Among them,
hexanoyl substituted pyrone 8 was the most selective and potent, with a Selectivity
Index (SI) >14. Fifteen of the alpha-pyrones were effective against T. cruzi
trypomastigotes, with 3-undecanoyl (11) and 3-tetradecanoyl (12) substituted pyrones
being the most potent against trypomastigotes, with IC50 values of 1 and 2 µM,
respectively, and SI higher than 70. Using flow cytometry and fluorescent-based
assays, pyrone 12 was found to induce hyperpolarization of the mitochondrial
membrane potential of T. cruzi, without affecting plasma membrane permeability. An
experimental acute phase-murine model, demonstrated that in vivo dosing of 12 (30
mg/kg/day; 5 days), had no efficacy at the first parasitemia onset of T. cruzi, but
reduced the second onset by 55% (p<0.05), suggesting a delayed action in BALB/c
mice. Additionally, a histopathology study demonstrated no toxic effects to the treated
mice. The finding that several 3-substituted alpha-pyrones have in vitro efficacy against
both L. infantum and T. cruzi, and that one analogue exhibited moderate and non-toxic
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in vivo efficacy against T. cruzi is encouraging, and suggests that this compound class
should be explored as long-term treatments in experimental Chagas disease.
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1. Introduction
Neglected diseases affect mainly developing countries and are usually
associated with poverty conditions.[1] Visceral Leishmaniasis (VL) is a fatal protozoan
disease transmitted by the bite of infected female sand flies. About 350 million people
are at risk and 12 million people are affected worldwide. It is estimated that 500,000
new cases of VL occur annually, with more than 90% of cases concentrated in six
countries, namely Bangladesh, Brazil, India, Nepal and Sudan and South Sudan.[1]
Considering the lack of vaccines, therapeutic treatment with drugs represents the main
intervention. Such drugs include pentavalent antimony and amphotericin B and the oral
drug miltefosine in India. However, their use is limited by the need for prolonged
administration of toxic drugs, hospitalization and the occurrence of moderate to severe
adverse effects. More recently, efforts to discover classes of antileishmanial whole
organism growth inhibitors and leishmanial target inhibitors have been reported.[2–5]
Trypanosoma cruzi is the etiologic agent of Chagas disease, an important
cause of mortality and morbidity in Latin America. About 30% of patients progress to
chagasic cardiomyopathy, a debilitating heart condition associated with conduction
disturbances and heart failure, leading to sudden death. There are about 9.4 million
people living with Chagas disease especially in Latin America and the US, leading to
untreatable heart disease and cardiomyopathy.[6] A recent study evaluated the clinical
efficacy of benznidazole (BZ), the only available drug; despite the reduction of the
parasitemia, the study concluded that BZ induced no significant improvement in cardiac
clinical outcomes. According to the Drugs for Neglected Diseases initiative (DNDi), in
the next five years, 200,000 people living with Chagas disease will die from heart
disease and related complications.[7] The DNDi also pointed out “We urgently need to
redouble our efforts to identify and treat young people who are still in the early stages
of their illness, but ultimately we need to find better treatments and new cures”.[8]
The pyrone ring system is present in a diverse array of natural products isolated
from a wide variety of sources, collected from both marine and terrestrial environments.
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Of the two pyrone isomers, alpha (2)-pyrone-bearing natural products[9] are more
common than their gamma (4)-pyrone counterparts.[10] Both alpha- and gamma-
pyrones have been reported to exhibit biological properties including antitumor,
antimicrobial and anti-HIV activities.[9–11] There are only a few reports describing the
evaluation of the pyrone class of natural products for activity towards infectious
diseases such as leishmaniasis and Chagas disease. For example, gamma-pyrones 1
and 2, isolated from the seeds of Podolepsis hieracioides (Asteraceae), exhibit in vitro
antileishmanial activity in the micromolar range while kojic acid (3), isolated from
Aspergillus fungi, demonstrates in vitro and in vivo antileishmanial efficacy (Figure
1).[12,13] With regard to alpha-pyrones, styryl pyrone,11-methoxyyangonin (4) exhibits
activity towards Trypanosoma brucei rhodesiense (IC50 31 µM) and Plasmodium
falciparum (IC50 3.0 µM) while the marine microbial pyrones pseudopyronine A (5) and
B (6) were found to be growth inhibitors of Trypanosoma brucei rhodesiense (IC50
13.09 and 12.46 µg/mL, respectively), Leishmania (L.) donovani (IC50 2.63 and 1.38
µg/mL, respectively) and Plasmodium falciparum (IC50 14.89 and 14.2 µg/mL,
respectively).[14,15]
Figure 1. Structures of alpha and gamma pyrone natural products (1–6).
Structure-activity relationship studies were carried out on this series with a set
of twenty-seven alpha-pyrone derivatives related to the pseudopyronines being
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synthesized and their antiparasitic efficacy evaluated against L. infantum and T. cruzi.
The efficacy of the most selective compound against T. cruzi was investigated in an
acute phase in vivo murine model. Additionally, using flow cytometry analysis and
fluorescent-based assays, the lethal action of the most selective compound in T. cruzi
parasites was evaluated.
2. CHEMISTRY
Target alpha-pyrones were chosen to explore the effects of chain length, steric
bulk and lipophilicity on bioactivity. To this end, the pyrones were prepared by
electrophilic substitution of 4-hydroxy-6-methyl-2-pyrone (7) with a variety of acyl
chlorides in TFA and heated under reflux for 5 hours (Scheme 1). Solvent removal
under reduced pressure followed by purification of the crude product by silica gel
column chromatography afforded alkanoyl pyrones 8–15 in yields ranging from 43% to
63%. Subsequently, reaction of alkanoyl pyrones 8–15 with sodium cyanoborohydride
and HCl in THF for 2–5 hours afforded alkyl pyrones 16–23 in yields ranging from 63%
to 76% after purification.
Scheme 1. Synthesis of alkanoyl and alkyl pyrones (8–23).
Reagents and conditions: a) Acyl chloride (2.1 eq.), TFA, reflux, 5 h, 53% (8), 48% (9), 59% (10), 52%
(11), 49% (12), 43% (13), 62% (14), 53% (15); b) sodium cyanoborohydride (2.5 eq.), HCl, THF, 2–5 h,
76% (16), 75% (17), 70% (18), 71% (19), 63% (20), 75% (21), 68% (22), 70% (23).
Further variation at the pyrone C-3 position was then explored by the
introduction of amido or thioamido substituents. To achieve this, pyrone 7 was reacted
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with the appropriate isocyanate or isothiocyanate and TEA in toluene at 120 ºC for 6
hours (Scheme 2). Purification of the crude product by column chromatography
afforded amido pyrones 24–32 and thioamido pyrones 33 and 34 in yields ranging from
6% to 70%.
Scheme 2. Synthesis of amido and thioamido pyrones (24–34).
Reagents and conditions: Isocyanate/isothiocyanate (5 eq.), TEA (0.25 eq.), toluene, 120 ºC, 6 h, 67%
(24), 70% (25), 53% (26), 42% (27), 46% (28), 37% (29), 34% (30), 25% (31), 50% (32), 45% (33), 6%
(34).
The synthesis and characterization of alpha-pyrones 8–11, 13, 14, 17 and 18
1
have been previously reported and in all cases the H NMR spectroscopic and mass
spectrometric data from the current study were in agreement with literature values.[16–
20] Novel compounds 12, 15, 16 and 19–34 were characterized utilizing a combination
of 1D and 2D NMR and HRMS.
3. RESULTS
3.1. In vitro Efficacy and Cytotoxicity
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Among the twenty seven synthesized alpha-pyrones, eighteen compounds
demonstrated activity against Leishmania and T. cruzi parasites (Table 1). The in vitro
data indicated that compounds 8, 15, 25, 26 and 28 presented activity in the range of
13 to 53 µM against Leishmania intracellular amastigotes. Among them, hexanoyl
analogue 8 was the most selective and potent, with an IC50 value of 13 µM and a
Selectivity Index (SI) higher than 14. Miltefosine was used as standard drug and
resulted in an IC50 value of 16.2 µM against the promastigotes and 7.4 µM against the
intracellular amastigotes. Fifteen of the tested alpha-pyrones were effective against T.
cruzi trypomastigotes, with IC50 values in the range of 1 to 90 µM. Compounds 11 and
12 were the most potent against trypomastigotes, with IC50 values of 1.3 and 2.6 µM,
respectively, and with SI values >70. The most selective compound (12) was tested
against intracellular amastigotes of T. cruzi, but demonstrated limited selectivity,
eliminating 14% ±3 of amastigotes at 25 µM. Benznidazole was used as standard drug
and resulted in an IC50 value of 16.4 µM against the trypomastigotes. The series
demonstrated low mammalian cytotoxicity (NCTC); among the 27 compounds, 10
(37%) presented no mammalian cytotoxicity to the highest tested concentration (200
µM), and only compounds 20 and 21 presented 50% Cytotoxic Concentration (CC50)
values below 50 µM (Table 1).
Table 1. Antiparasitic and mammalian cytotoxicity of alpha-pyrone derivatives.
Nº
IC50 (µM)a
(CI 95%)b
Selectivity Index
L. inf proc
L. inf amad
T. cruzie
NCTC
L. inf
T.
amad
cruzie
8
9
NAf
NA
13.8
(10.32 – 18.39)
NA
>200
>14.5
−
−
NA
NA
25.7
(22.6 – 29.3)
140.1
(106.7 –
184.0)
5.4
10
NA
17.5
134.1
−
7.6
(13.4 – 22.8)
(107.4 –
167.4)
11
12
13
NA
NA
NA
NA
NA
NA
1.3
(0.6 – 2.5)
2.6
(1.0 – 6.6)
66.3
90.3
(59.7 – 136.5)
>200
−
−
−
71.1
>77.5
2.5
164.5
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(48.0 – 91.7)
(114.2 –
237.0)
>200
14
15
NA
NA
NA
NA
−
−
39.2
(20.7 – 74.4)
NA
4.4
(2.7 – 7.12)
NA
117.2
(90.1 – 152.4)
>200
2.9
26.8
16
17
NA
NA
−
−
−
3.6
NA
40.0
144.7
(36.1 – 44.3)
(123.9 –
168.8)
18
19
20
21
22
23
117.7
(88.9 – 155.7)
137.5
(119.7 – 157.9)
NA
NA
NA
NA
NA
NA
NA
NA
56.0
(41.9 – 74.9)
57.2
(44.4 – 73.6)
24.1
(17.4 – 33.4)
38.2
−
−
−
−
−
−
−
16.1
(13.4 – 19.4)
4.9
(3.3 – 7.3)
26.6
3.5
4.9
1.4
NA
NA
NA
(16.2 – 43.4)
NA
(32.2 – 45.4)
>200
32.2
109.1
3.4
(26.3 – 39.5) (71.5 – 166.4)
24
25
NA
NA
NA
52.8
NA
NA
> 200
> 200
–
5.0
–
–
(34.7 – 80.4)
(182.5 –
389.2)
178.3
26
27
28
29
NA
NA
NA
NA
46.8
NA
3.8
–
–
(31.8 – 69.1)
(115.3 –
275.6)
142.9
(110.8 –
184.2)
195.5
(168.8 –
226.4)
186.6
NA
81.4
(75.8 – 87.4)
1.7
2.7
2.4
54.0
(42.7 – 68.2)
72.6
(56.8 – 92.7)
3.6
–
NA
78.1
(64.4 – 94.6)
(148.8 –
234.1)
> 200
> 200
> 200
30
31
32
33
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
–
–
–
–
–
–
–
–
>100
> 200
34
NA
–
NA
–
90.5
(60.4 – 135.5)
176.7
(153.3 –
203.6)
>200
–
1.9
BZ
16.2
>36
(12.9 – 20.3)
MT
16.2
(14.6 – 16.9)
7.4
(6.7 – 7.6)
241.4
(236.7 –
254.1)
32.6
a 50% inhibitory concentration; b 95% Confidence interval ; c Leishmania infantum promastigotes; d
Leishmania Infantum amastigotes; e Trypanosoma cruzi trypomastigotes; f Not active up to 200 µM; BZ-
benznidazole, MT- miltefosine.
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3.2. Lethal Action studies in T cruzi
The ability of 12 to compromise the plasma membrane of T. cruzi was assessed
using the fluorescent probe SYTOX green method.[21] Compound 12 induced no
alteration in plasma membrane permeability within 7 hours of incubation (Figure 2).
Triton X-100 was used as the positive control.
Figure 2. Permeability of T. cruzi trypomastigotes, assessed with the vital dye SYTOX green.
Trypomastigotes were treated at the IC99 value of compound 12 and also with Triton X-100 (used as the
100 % permeabilization positive control). A control group (untreated) was also included.
Using flow cytometry analysis, the action of 12 on the mitochondrial function of
living T. cruzi parasites was also investigated, using two fluorescent probes, propidium
iodide and rhodamine 123.[21] At the IC50 and IC99 values, pyrone 12 induced
hyperpolarization of the mitochondrial membrane potential, increasing the fluorescent
levels by 25% and 26% (p<0.05), respectively, when compared to untreated parasites
(Figure 3A). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was used
as a positive control and caused an intense depolarization of the mitochondrial
membrane potential, as observed by the reduced fluorescence levels after 2 h
incubation (Figure 3B).
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Figure 3. Evaluation of the mitochondrial membrane potential of T. cruzi trypomastigotes incubated with
compound 12. (A) Flow cytometry graphs showing in y axis Rd123 fluorescence and x axis PI
fluorescence. Changes of Rd123 fluorescence determined by flow cytometry and reported as percentage
relative to untreated parasites. (B) Maximal and minimal fluorescence of Rd123 were achieved by non-
treatment and treatment with FCCP (10 µM), respectively. Trypomastigotes were treated at the IC50 and
IC99 values of compound 12 for 2 h. * p<0.05 related to positive control (FCCP).
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3.3. In vivo efficacy studies with T. cruzi
Five days post infection (5 d.p.i), BALB/c mice were treated with compound 12
at 30 mg/kg/day via intraperitoneal route for 10 consecutive days. Parasitemia was
determined in blood by light microscopy counting. Eight days post infection (8 d.p.i.), at
the first parasitemia onset, the compound showed no efficacy, demonstrating similar
number of parasites to the untreated group. After 14 d.p.i., at the second and last
parasitemia onset, compound 12 reduced the number of parasites in blood by 55%
(p<0.05) when compared to untreated animals (Figure 4A). Benznidazole was used as
a standard drug at 100 mg/kg via oral route and reduced parasite numbers by almost
100% at both parasitemia onsets. The survival curve demonstrated no death rate at
compound 12-treated and benznidazole-treated groups. Non-treated animals resulted
in a 20% death rate after 14 days (Figure 4B).
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Figure 4. In vivo efficacy of compound 12 in experimental acute-phase mice model of T. cruzi (Y
strain).The compound was administered intraperitoneally at 30 mg/kg/day and benznidazole (per oral
gavage) at 100 mg/kg/day at the same conditions. The parasitemia (blood) was determined by light
microscopy counting (A) and the survival rate was observed during 14 days (B). *p<0.05
3.4. Histopathological Examination
Healthy animals were intraperitoneally treated with compound 12 for 10
consecutive days at 30 mg/kg/day, and a histopathological examination of the organs
was performed. Figure 5A shows the liver of a representative animal from the
untreated group (control), demonstrating a healthy organ. The liver image of a treated
animal (Figure 5B) showed minimum hydropic degeneration (vacuolation) of
hepatocytes when compared to control. The other organs revealed normal architecture
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(data not presented), suggesting no detrimental changes and morphological
disturbances.
Figure 5. Histological sections of liver stained with hematoxylin and eosin demonstrating the vacuolization
of liver cells. Untreated animal (A); animal treated intraperitoneally with compound 12 at 30 mg/kg/day for
10 consecutive days (B). Vacuolization of liver cells (indicated by arrows).
4. DISCUSSION
2-Pyrone derivatives show promising antimicrobial and antiparasitic activities.
Previous studies have identified the 3,6-dialkyl-4-hydroxy-pyran-2-one marine microbial
metabolites pseudopyronines A and B as inhibitors of protozoan parasites Plasmodium
falciparum and Leishmania donovani.[14] In the search for new anti-parasitic hit
compounds, we now report the synthesis, antileishmanial and the antitrypanosomal
activities and a structure-activity relationship (SAR) study of twenty-seven alpha-
pyrones. The biological activities of these compounds were evaluated against
extracellular and intracellular amastigote forms of L. infantum and T. cruzi and for
cytotoxicity towards murine fibroblasts.
Among the twenty seven alpha-pyrones, over half (55%) exhibited in vitro
efficacy against T. cruzi parasites, with two compounds (11 and 12) demonstrating
superior antiparasitic potency and no toxicity to mammalian cells. Although compound
12 was effective against trypomastigotes, it failed to eliminate adequately the
intracellular amastigotes of T. cruzi. This fact might be ascribed to poor internalization
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into the macrophages (lack of receptors), intracellular degradation or even to the
resistance of the amastigote forms, which differ metabolically from trypomastigotes.
When compared to the standard drug benznidazole (BZ), pyrone 12 was approximately
6-fold more potent. The intracellular amastigotes are clinically relevant forms of T.
cruzi, but trypomastigote forms are also found in mammalians mainly in the acute
phase of the disease, resulting in recrudescence of the parasitemia, especially when
complete elimination of parasites is not achieved during treatment. Finding new drugs
to selectively affect trypomastigotes is considered a relevant therapeutic approach.[22]
Leishmania parasites were also susceptible to the alpha-pyrones. In the present
study, four compounds were effective against the intracellular amastigotes of
Leishmania; among them, hexanoyl pyrone 8 showed similar potency to the standard
drug miltefosine. Natural alpha-pyrones isolated from Raimondia cf. monoica showed
in vitro activities against Plasmodium falciparum and Leishmania panamensis, with IC50
values in a range between 0.4 to 10 µg/mL, but these compounds presented an
elevated mammalian cytotoxicity (CC50 values between 1 and 2 µg/mL).[23]
Considering the need for new hit compounds with wide-ranging antiparasitic activity,
the present study has identified two compounds (15 and 28) that inhibit the growth of
both L. infantum intracellular amastigotes and T. cruzi trypomastigotes. Although the
compounds activities were not considered very promising (e.g. IC50>10 µM), these
compounds could serve as future scaffolds for the synthesis of new derivatives.
Overall, limited cytotoxicity towards murine NCTC fibroblasts was observed for the
alpha-pyrones in this study - 22 compounds (81%) demonstrated cytotoxicity (CC50
values) higher than 100 µM and a total of 10 compounds completely lacked
mammalian toxicity up to 200 µM. This is very encouraging and suggests that a
reasonable therapeutic window already exists for the compounds, given the T. cruzi
activity is < 5 µM for the leading examples.
Pyrone 12 was selected for further analysis of two properties relevant to its
potential mechanism of antiparasitic action. Alteration of membrane permeability is
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recognized as one method that small molecules can exhibit antiparasitic activity. For
example, antimicrobial peptides[21] and marine guanidine alkaloids[24] demonstrate
the ability to increase the permeability of Trypanosoma and Leishmania parasite
membranes, respectively. In both cases, permeabilization leads to cell death. Also
relevant to the current study, was the report by Singh et al.[25] that pseudopyronine A
(5) alters bacterial membrane permeability. In the present study, pyrone 12 induced no
alteration in the permeability of the plasma membrane of T. cruzi trypomastigotes after
7 hours of incubation.
Differing from mammalian cells, the mitochondria of T. cruzi is a unique
organelle, and consequently, has been considered a promising target for new drugs.
For example, alpha-pyrones brevipolides A-F, isolated from the plant Hyptis brevipes,
were found to alter the mitochondrial transmembrane potential of mammalian cells.[26]
In our assays, we investigated the mitochondrial membrane potential of T. cruzi after
incubation with compound 12. By using flow cytometry analysis, we separated living
from dead cells using the fluorescent probe propidium iodide, and assessed
trypanosomal mitochondrial activity using the fluorescent probe rhodamine 123. When
compared to positive controls and untreated cells, hyperpolarization of the
mitochondrial membrane potential of living parasites in a dose-independent manner
after 1 h incubation was observed. Mitochondrial membrane potential (∆Ψm) is critical
for maintaining the physiological function of the respiratory chain to generate ATP. A
significant loss of ∆Ψm renders cells depleted of energy with subsequent death.[27]
The mitochondrial transmembrane potential (∆Ψm; negative inside and positive
outside) is the result of an electrochemical gradient maintained by two transport
systems. In mammalian cells, mitochondrial hyperpolarization (MHP) is an early event
of death, and appears to be mediated through inhibition of F0F1- ATPase or
dephosphorylation of cytochrome c oxidase.[28] In addition, MHP appears to be the
earliest change associated with several apoptosis pathways. Considering that
apoptosis-like or programmed cell death has been proposed in Trypanosoma
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cruzi,[29–31] future studies could investigate the apoptosis-like induction of compound
12 in T. cruzi. These results would direct the synthesis of more potent and selective
compounds.
Considering the need for new effective compounds against the trypomastigote
forms of T. cruzi, which causes recrudescence of the disease in benznidazole-treated
patients,[22] we investigated the in vivo efficacy of pyrone 12 in an acute-phase
experimental model of Chagas disease. Compound 12 was administered at a dose (30
mg/kg/day) for five consecutive days, and compared to the standard drug benznidazole
(100 mg/kg/day). At the first parasitemia onset [observed at the 8th day post-infection
(dpi)], 12 showed no efficacy (Figure 4). However by the second and last parasitemia
onset (14 dpi), the compound significantly reduced parasitemia by 55% (p<0.05). The
results suggest a delayed action of the pyrone, perhaps due to drug penetration issues.
Pharmacokinetic and pharmacodynamic (PK/PD) parameters, such as a short half-life
(T1/2), or a reduced maximal concentration in blood (Cmax), or even rapid metabolism,
could also be possible explanations for the moderate efficacy of 12 in T. cruzi-infected
animals. Future experimental PK studies will be performed in an effort to optimize
efficacy in animals.
Analysis of structure activity relationships (SAR) was next conducted. The main
features considered in the analyses were atom chain length / steric bulk and various
molecular properties such as lipophilicity, polar surface area and the presence or
absence of hydrogen bond donor / acceptor groups. An important caveat was that
since a phenotypic screening approach was being used certain assumptions were
necessarily made in this SAR analysis, for example, that all compounds were binding
to the same biological target or inhibiting the same pathway, and that permeability of
the various analogues was broadly similar. Compound properties were calculated using
DataWarrior.[32]
Compound activity seemed to be strongly affected by the length of the alkyl
chain (n atoms), with a linear arrangement of 12–14 atoms being favored. Longer chain
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examples appeared to be disfavored, being up to 50 fold less active, for example
compare 21 (n=16, IC50 = 26.6 µM) with 20 (n=14, IC50 = 4.9 µM); also 13 (n=16, IC50 =
66.3 µM) with 11 (n=12, IC50 = 1.3 µM) and 12 (n=14, IC50 = 2.6 µM). Shorter chain
examples also appeared less active, for example compare 11 (n=12, IC50 = 1.3 µM)
with 9 (n=8, IC50 = 25.5 µM). This suggests the presence of a hydrophobic binding site
having a defined depth, with the shorter chain analogues still being able to bind, but
having reduced potency due to decreased hydrophobic interactions.[33]
Steric bulk close to the pyranone core did not appear to be tolerated; for
example, compounds 14, 24 and 32 were all inactive, although interestingly as the
chain is extended, activity was recovered with examples 27 (IC50 = 81.4 µM), and
strikingly so with alkyl-cyclohexyl analogue 15 (IC50 = 4.4 µM). This suggests that the
binding site for the molecule can accommodate lipophilic bulk at certain points along
the alkyl chain. This information could prove important when designing a new set of
analogues for synthesis.
The introduction of hydrogen bond acceptor (HBA) and donor (HBD) groups
was also explored. For example, replacing a methylene unit with a keto group
appeared favorable, giving approximately a ten-fold improvement in activity, compare
19 (IC50 = 16.1 µM) with 11 (IC50 = 1.3 µM). Interestingly, amide derivatives seemed to
generally be much less favored. Although the direct analogues of compounds with
comparable chain lengths to 11 or 12 were not prepared, examples were seen where
the matched pairs of amides had significantly lower activity than the corresponding keto
analogues; compare 30 (inactive) with 13 (IC50 = 66.3 µM), and 28 (IC50 = 72.6 µM)
with 9 (IC50 = 25.7 µM).
Looking at predicted molecular characteristics, it seemed important that polar
surface area of the compounds be less than around 65Å2 in order to maintain activity.
This may reflect the permeability of the molecules or some characteristic of the binding
site – at this stage it is not possible to draw any more specific conclusions. Lipophilicity
does not seem to be directly relevant, with compounds having similar cLogP values
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ranging from very active (11, cLogP = 4.3, IC50 1.3 µM) to inactive (18, cLogP = 4.5,
and 30 cLogP = 4.9; both IA).
Whilst the compounds currently have encouraging potencies against T. cruzi
trypomastigotes, the most active analogues such as 12 are rather lipophilic and likely to
have modest solubilities and limited metabolic stability. This would be expected to
provide challenges when conducting extended in vivo dosing experiments. Having
some preliminary structure-activity data in hand for the series allows us to identify
areas where modification of structure with other motifs could be contemplated to
mitigate these potential liabilities and improve their overall drug-like properties.[34] For
example, synthesis of derivatives of 12 contain polyether-type chains would be
expected to maintain activity, yet have much improved physicochemical and metabolic
properties than the parent compound.
The alpha-pyrone analogues described in the present study represent new hit
compounds with in vitro efficacy against T. cruzi and L. infantum. Encouragingly,
pyrone 12 demonstrated moderate efficacy in an acute-phase experimental model of T.
cruzi. These preliminary results are being used to direct the synthesis of new and more
potent derivatives against Leishmania and Trypanosoma cruzi parasites.
5. Material and Methods
5.1. General experimental procedures
Infrared spectra were recorded using a Perkin-Elmer spectrum One Fourier-
transform IR spectrometer as a dry film. NMR spectra were recorded on a Bruker
1
Avance DRX-400 spectrometer operating at 400 MHz for H nuclei and 100 MHz for
13C nuclei. Proto-deutero solvent signals were used as internal references (DMSO-d6:
δH 2.50, δC 39.52; CDCl3: δH 7.26, δC 77.16). Standard Bruker pulse sequences were
utilized. HRMS data were acquired on either a VG-7070 or a Bruker micrOTOF Q II
mass spectrometer. Elemental analysis was performed at the Campbell Microanalytical
Laboratory, University of Otago, Dunedin, New Zealand. Column chromatography was
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performed using Davisil® silica gel. Thin layer chromatography was carried out on 0.2
mm thick plates of Kiesegel F254 (Merck).
5.2. General procedures for synthesis of compounds
5.2.1. General procedure A: synthesis of alkanoyl pyrones
4-Hydroxy-6-methyl-2-pyrone (7) (1 eq.) was dissolved in TFA (2 mL), the
appropriate acyl chloride (2.1 eq.) added, reaction vessel purged with nitrogen and
heated under reflux for 5 h. TFA was removed under reduced pressure to afford a
yellow-brown oil which was dissolved in Et2O or CH2Cl2, washed twice with brine and
dried over anhydrous MgSO4. Solvent removal under reduced pressure afforded the
crude product as a yellow-brown oil which was purified by silica gel column
chromatography eluting with CH2Cl2 to afford target compounds 8–15.
5.2.2. General procedure B: synthesis of alkyl pyrones
To a solution of acylated pyrone (8–15) (1 eq.) in THF (4 mL) was added HCl (2
N, 3.5 mL) followed by sodium cyanoborohydride (2.5 eq.). The mixture was stirred for
2–5 h and monitored by TLC. If reaction had not gone to completion after 2 h, an
additional 2 eq. of sodium cyanoborohydride was added and the reaction stirred for
another 3 h. Water (10 mL) was then added and the aqueous layer extracted with
CH2Cl2. The combined organic layers were washed twice with brine, dried over
anhydrous MgSO4 and the solvent removed under reduced pressure. Purification by
silica gel column chromatography (CH2Cl2/MeOH 95:5) afforded target compounds 16–
23.
5.2.3. General procedure C: synthesis of amido and thioamido pyrones
To a solution of 4-hydroxy-6-methyl-2-pyrone (7) (1 eq.) in toluene (5 mL) was
added isocyanate or isothiocyanate (5 eq.). Triethylamine (0.25 eq.) was then added
and the reaction mixture heated at 120 ºC for 6 h under nitrogen. The reaction was
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then cooled to room temperature followed by solvent removal under reduced pressure.
The solid was then dissolved in CH2Cl2 (10 mL) and purified by silica gel column
chromatography eluting with hexane followed by increments of CH2Cl2 to afford the
target compounds 24–34.
5.3. Synthesis of compounds
5.3.1. 3-Hexanoyl-4-hydroxy-6-methyl-2-pyrone (8)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.404
g, 3.2 mmol) with hexanoyl chloride (0.916 g, 6.8 mmol) followed by purification
o
afforded the title compound as white crystals (0.380 g, 53%). m.p. 64.0–65.0 C
o
(lit.66.0–66.5 C[16]); Rf (n-hexane/CH2Cl2, 1:1) 0.3; IR(ATR) νmax 3088, 2941, 1717,
1
1560, 1451 cm-1; H NMR (CDCl3, 400 MHz) δ 5.93 (1H, s, H-5), 3.07 (2H, t, J = 7.4
Hz, H2-9), 2.27 (3H, d, J = 1.0 Hz, H3-7), 1.70–1.62 (2H, m, H2-10), 1.38–1.34 (4H, m,
H2-11, H2-12), 0.91 (3H, t, J = 7.1 Hz, H3 -13); 13C NMR (CDCl3, 100 MHz) δ 208.2 (C-
8), 181.4 (C-4), 168.9 (C-6), 161.1 (C-2), 101.7 (C-5), 99.6 (C-3), 41.7 (C-9), 31.5 (C-
11), 23.8 (C-10), 22.6 (C-12), 20.8 (C-7), 14.1 (C-13); (+)-HRESIMS m/z 247.0937
[M+Na]+ (calcd for C12H16NaO4, 247.0941); Anal calcd for C12H16O4: C, 64.27; H, 7.19.
Found: C, 64.46; H, 7.19.
5.3.2. 4-Hydroxy-6-methyl-3-octanoyl-2-pyrone (9)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.202
g, 1.6 mmol) with octanoyl chloride (0.553 g, 3.4 mmol) followed by purification
afforded the title compound as pale yellow crystals (0.192 g, 48%). m.p. 67.5–68.5 °C
(lit. 67.5–68.0oC[16]); Rf (n-hexane/CH2Cl2, 1:1) 0.3; IR (ATR) νmax 3094, 2922, 1719,
1
1560, 1453 cm-1; H NMR (CDCl3, 400 MHz) δ 5.93 (1H, s, H-5), 3.07 (2H, t, J = 7.5
Hz, H2-9), 2.27 (3H, J = 1.0 Hz, H3-7), 1.69–1.62 (2H, m, H2-10), 1.39–1.29 (8H, m, H2-
11, H2-12, H2-13, H2-14), 0.88 (3H, t, J = 7.3Hz, H3 -15); 13C NMR (CDCl3, 100 MHz) δ
208.2 (C-8), 181.4 (C-4), 168.9 (C-6), 161.1 (C-2), 101.7 (C-5), 99.6 (C-3), 41.8 (C-9),
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31.8 (C-13), 29.3 (C-11, C-12), 24.1 (C-10), 22.8 (C-14), 20.8 (C-7), 14.2 (C-15); (+)-
HRESIMS m/z 275.1254 [M+Na]+ (calcd for C14H20NaO4, 275.1254); Anal calcd for
C14H20O4: C, 66.65; H, 7.99. Found: C, 66.86; H, 8.01.
5.3.3. 3-Decanoyl-4-hydroxy-6-methyl-2-pyrone (10)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.202
g, 1.6 mmol) with decanoyl chloride (0.648 g, 3.4 mmol) followed by purification
afforded the title compound as white crystals (0.266 g, 59%). m.p. 73.5–74.5 °C (lit.
1
74.0–74.5oC[16]); IR (KBr) 3094, 2915, 1713, 1555, 1452 cm-1; H NMR (400 MHz,
CDCl3) δ 5.93 (1H, s, H-5), 3.07 (2H, t, J = 7.3 Hz, H2-9), 2.27 (3H, s, H3-7), 1.69–1.61
(2H, m, H2-10), 1.39–1.27 (12H, m, H2-11, H2-12, H2-13, H2-14, H2-15, H2-16), 0.88
(3H, t, J = 6.5 Hz, H3-17); 13C NMR (100 MHz, CDCl3) δ 208.2 (C-8), 181.4 (C-2), 168.9
(C-4), 161.1 (C-6), 101.7 (C-5), 99.7 (C-3), 41.8 (C-9), 32.0 (C-15), 29.6 (C-12, C-13),
29.4 (C-11, C-14), 24.1 (C-10), 22.8 (C-16), 20.8 (C-7), 14.2 (C-17); (+)-HREIMS m/z
280.1672 [M+H]+ (calcd for C16H24O4, 280.1675); Anal calcd for C16H24O4: C, 68.54; H,
8.63. Found: C, 68.79; H, 8.55.
5.3.4. 3-Dodecanoyl-4-hydroxy-6-methyl-2H-pyran-2-one (11)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.337
g, 2.7 mmol) with dodecanoyl chloride (1.269 g, 5.8 mmol) followed by purification
afforded the title compound as white crystals (0.434 g, 52%). m.p. 80.0–81.0 °C (lit.
82.0–82.5oC[16]); Rf (n-hexane/CH2Cl2, 1:1) 0.3; IR (ATR) νmax 3094, 2921, 1722, 1555,
1
1449 cm-1; H NMR (CDCl3, 400 MHz) δ 5.93 (1H, s, H-5), 3.07 (2H, t, J = 7.3 Hz, H2-
9), 2.27 (3H, s, H3-7), 1.69–1.61 (2H, m, H2-10), 1.39–1.26 (16H, m, H2-11, H2-12, H2-
13, H2-14, H2-15, H2-16, H2-17, H2-18), 0.88 (3H, t, J = 7.0 Hz, H3 -17); 13C NMR
(CDCl3, 100 MHz) δ 208.2 (C-8), 181.4 (C-4), 168.9 (C-6), 161.1 (C-2), 101.7 (C-5),
99.6 (C-3), 41.8 (C-9), 32.1 (C-17), 29.8 (C-13, C-14), 29.6 (C-12, C-15), 29.5 (C-16),
29.4 (C-11), 24.1 (C-10), 22.8 (C-18), 20.8 (C-7), 14.3 (C-19); (+)-HRESIMS m/z
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331.1876 [M+Na]+ (calcd for C18H28NaO4, 331.1880); Anal calcd for C18H28O4: C, 70.10;
H, 9.15. Found: C, 70.20; H, 9.03.
5.3.5. 4-Hydroxy-6-methyl-3-tetradecanoyl-2-pyrone (12)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.337
g, 2.7 mmol) with tetradecanoyl chloride (1.431 g, 5.8 mmol) followed by purification
afforded the title compound as white needles (0.440 g, 49%). m.p. 84.5–85.0 °C; Rf (n-
1
hexane/CH2Cl2, 1:1) 0.3; IR (ATR) νmax 3093, 2915, 1713, 1562, 1471 cm-1; H NMR
(CDCl3, 400 MHz) δ 5.93 (1H, d, J = 1.0 Hz, H-5), 3.07 (2H, t, J = 7.2 Hz, H2-9), 2.26
(3H, s, H3-7), 1.69–1.61 (2H, m, H2-10), 1.39–1.26 (20H, m, H2-11, H2-12, H2-13, H2-14,
H2-15, H2-16, H2-17, H2-18, H2-19, H2-20), 0.88 (3H, t, J = 7.0 Hz, H3-21); 13C NMR
(CDCl3, 100 MHz) δ 208.2 (C-8), 181.4 (C-4), 168.9 (C-6), 161.1 (C-2), 101.7 (C-5),
99.6 (C-3), 41.8 (C-9), 32.1 (C-19), 29.8 (C-13, C-14, C-15, C-16), 29.6 (C-12, C-17),
29.5 (C-18), 29.4 (C-11), 24.1 (C-10), 22.8 (C-20), 20.8 (C-7), 14.3 (C-21); (+)-
HRESIMS m/z 359.2183 [M+Na]+ (calcd for C20H32NaO4, 359.2193); Anal calcd for
C20H32O4: C, 71.39; H, 9.59. Found: C, 71.45; H, 9.62.
5.3.6. 4-Hydroxy-6-methyl-3-palmitoyl-2-pyrone (13)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.337
g, 2.7 mmol) with palmitoyl chloride (1.594 g, 5.8 mmol) followed by purification
afforded the title compound as pale yellow crystals (0.422 g, 43%). m.p. 87.0–88.0 °C
(lit. 87.0–88.0ºC[17]); Rf (n-hexane/CH2Cl2, 1:1) 0.3; IR (ATR) νmax 3092, 2915, 1713,
1
1557, 1471 cm-1; H NMR (CDCl3, 400 MHz) δ 5.93 (1H, s, H-5), 3.07 (2H, t, J = 7.3
Hz, H2-9), 2.26 (3H, s, H3-7), 1.69–1.61 (2H, m, H2-10), 1.39–1.26 (24H, m, H2-11, H2-
12, H2-13, H2-14, H2-15, H2-16, H2-17, H2-18, H2-19, H2-20, H2-21, H2-22), 0.88 (3H,t, J
= 7.0 Hz, H3 -23); 13C NMR (CDCl3, 100 MHz) δ 208.2 (C-8), 181.4 (C-4), 168.9 (C-6),
161.1 (C-2), 101.7 (C-5), 99.7 (C-3), 41.8 (C-9), 32.1 (C-21), 29.8 (C-13, C-14, C-15,
C-16, C-17, C-18), 29.7 (C-19), 29.6 (C-12), 29.5 (C-20), 29.4 (C-11), 24.1 (C-10), 22.8
(C-22), 20.8 (C-7), 14.3 (C-23); (+)-HRESIMS m/z 387.2515 [M+Na]+ (calcd for
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C22H36NaO4, 359.2506); Anal calcd for C22H36O4: C, 72.49; H, 9.95. Found: C, 72.71; H,
9.89.
5.3.7. 3-(Cyclohexanecarbonyl)-4-hydroxy-6-methyl-2-pyrone (14)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.534
g, 4.2 mmol) with cyclohexanecarbonyl chloride (1.232 g, 8.4 mmol) followed by
purification afforded the title compound as white crystals (0.615 g, 62%). m.p. 71.0–
73.0 °C (lit. 72.0oC[18]); Rf (n-hexane/CH2Cl2, 1:1) 0.3; IR (ATR) νmax 3093, 2916,
1713, 1558, 1472 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.92 (1H, s, H-5), 3.70–3.65 (1H,
m, H-9), 2.26 (3H, d, J = 1.0 Hz, H3-7), 1.88–1.86 (2H, m, H2-10A, H2-14A), 1.83–1.80
(2H, m, H2-11A, H2-13A), 1.75–1.69 (1H, m, H2-12A), 1.47–1.33 (4H, m, H2-10B, H2-11B,
H2-13B, H2-14B), 1.27–1.20 (1H, m, H2 -12B); 13C NMR (CDCl3, 100 MHz) δ 211.3 (C-8),
182.2 (C-4), 168.8 (C-6), 160.8 (C-2), 101.8 (C-5), 98.9 (C-3), 47.3 (C-9), 28.8 (C-10,
C-14), 26.1 (C-12), 25.8 (C-11, C-13), 20.7 (C-7); (+)-HRESIMS m/z 259.0941 [M+Na]+
(calcd for C13H16NaO4, 259.0941); Anal calcd for C13H16O4: C, 66.09; H, 6.83. Found:
C, 66.06; H, 6.96.
5.3.8. 3-(3-Cyclohexylpropanoyl)-4-hydroxy-6-methyl-2-pyrone (15)
Using general procedure A, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.337
g, 2.7 mmol) with 3-cyclohexylpropanoyl chloride (1.013 g, 5.8 mmol) followed by
purification afforded the title compound as white crystals (0.375 g, 53%). m.p. 93–95
°C; Rf (n-hexane/CH2Cl2, 1:1) 0.3; IR (ATR) νmax 3097, 2921, 1722, 1555, 1450 cm-1; 1H
NMR (CDCl3, 400 MHz) δ 5.93 (1H, s, H-5), 3.07 (2H, t, J = 7.5 Hz, H2-9), 2.26 (3H, d,
J = 1.0 Hz, H3-7), 1.77–1.66 (6H, m, H2-12, H2-14, H2-15), 1.58–1.51 (2H, m, H2-10),
1.36–1.27 (1H, m, H-11), 1.25–1.13 (2H, m, H2-13), 0.98–0.88 (2H, m, H2-16); 13C
NMR (CDCl3, 100 MHz) δ 208.6 (C-8), 181.4 (C-4), 168.9 (C-6), 161.1 (C-2), 101.7 (C-
5), 99.6 (C-3), 39.5 (C-9), 37.5 (C-11), 33.3 (C-12, C-16), 31.5 (C-10), 26.7 (C-14),
26.4 (C-13, C-15), 20.8 (C-7); (+)-HRESIMS m/z 287.1250 [M+Na]+ (calcd for
24

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C15H20NaO4, 287.1254); Anal calcd for C15H20O4: C, 68.16; H, 7.63. Found: C, 68.29; H,
7.74.
5.3.9. 3-Hexyl-4-hydroxy-6-methyl-2-pyrone (16)
Using general procedure B, reaction of 8 (0.050 g, 0.22 mmol) with sodium
cyanoborohydride (0.035 g, 0.56 mmol) followed by purification afforded the title
compound as white crystals (0.035 g, 76%). m.p. 114.0–115.5 °C; Rf (CH2Cl2/MeOH,
1
97:3) 0.3; IR (ATR) νmax 2923, 2664, 1571, 1443 cm-1; H NMR (CDCl3, 400 MHz) δ
10.02 (1H, br s, OH), 6.21 (1H, s, H-5), 2.45 (2H, t, J = 7.6 Hz, H2-8), 2.21 (3H, d, J =
1.0 Hz, H3-7), 1.53–1.45 (2H, m, H2-9), 1.37–1.29 (6H, m, H2-10, H2-11, H2-12), 0.86
(3H, t, J = 6.5 Hz, H3-13); 13C NMR (CDCl3, 100 MHz) δ 168.4 (C-2), 167.2 (C-4), 159.9
(C-6), 103.5 (C-3), 101.8 (C-5), 31.9 (C-11), 29.4 (C-10), 28.2 (C-9), 23.2 (C-8), 22.8
(C-12), 19.8 (C-7), 14.2 (C-13); (+)-HRESIMS m/z 233.1154 [M+Na]+ (calcd for
C12H18NaO3, 233.1148); Anal calcd for C12H18O3: C, 68.54; H, 8.63. Found: C, 68.79; H,
8.68.
5.3.10. 4-Hydroxy-6-methyl-3-octyl-2-pyrone (17)
Using general procedure B, reaction of 9 (0.050 g, 0.20 mmol) with sodium
cyanoborohydride (0.031 g, 0.50 mmol) followed by purification afforded the title
compound as pale pink crystals (0.035 g, 75%). m.p. 103.0–104.5 °C (lit. 100–
102oC[19]); Rf (CH2Cl2/MeOH, 97:3) 0.3; IR (ATR) νmax 2921, 2662, 1571, 1445 cm-1; 1H
NMR (CDCl3, 400 MHz) δ 9.31 (1H, br s, OH), 6.12 (1H, d, J = 1.0 Hz, H-5), 2.43 (2H,
t, J = 7.5 Hz, H2-8), 2.21 (3H, d, J = 1.0 Hz, H3-7), 1.53–1.46 (2H, m, H2-9), 1.35–1.25
13
(10H, m, H2-10, H2-11, H2-12, H2-13, H2-14), 0.86 (3H, t, J = 6.7 Hz, H3-15); C NMR
(CDCl3, 100 MHz) δ 167.7 (C-2), 166.3 (C-4), 159.8 (C-6), 103.4 (C-3), 101.3 (C-5),
31.9 (C-13), 29.7 (C-12), 29.6 (C-11), 29.4 (C-10), 28.1 (C-9), 23.1 (C-8), 22.7 (C-14),
19.7 (C-7), 14.1 (C-15); (+)-HRESIMS m/z 261.1457 [M+Na]+ (calcd for C14H22NaO3,
261.1461); Anal calcd for C14H22O3: C, 70.56; H, 9.30. Found: C, 70.80; H, 9.38.
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5.3.11. 3-Decyl-4-hydroxy-6-methyl-2-pyrone (18)
Using general procedure B, reaction of 10 (0.050 g, 0.18 mmol) with sodium
cyanoborohydride (0.028 g, 0.45 mmol) followed by purification afforded the title
compound as white crystals (0.033 g, 70%). m.p. 101.5–102.5 °C (lit.103–104°C[20]);
1
Rf (CH2Cl2/MeOH, 97:3) 0.3; IR (ATR) νmax 2919, 2644, 1571, 1444 cm-1; H NMR
(CDCl3, 400 MHz) δ 9.69 (1H, br s, OH), 6.18 (1H, d, J = 1.0 Hz, H-5), 2.44 (2H, t, J =
7.4 Hz, H2-8), 2.21 (3H, s, H3-7), 1.53–1.46 (2H, m, H2-9), 1.36–1.24 (14H, m, H2-10,
H2-11, H2-12, H2-13, H2-14, H2-15, H2-16), 0.87 (3H, t, J = 6.8 Hz, H3-17); 13C NMR
(CDCl3, 100 MHz) δ 168.2 (C-2), 166.9 (C-4), 159.9 (C-6), 103.5 (C-3), 101.7 (C-5),
32.1 (C-15), 29.9 (C-10), 29.8 (C-11, C-12, C-13), 29.5 (C-14), 28.3 (C-9), 23.2 (C-8),
22.8 (C-16), 19.8 (C-7), 14.2 (C-17); (+)-HRESIMS m/z 289.1769 [M+Na]+ (calcd for
C16H26NaO3, 289.1774); Anal calcd for C16H26O3: C, 72.14; H, 9.84. Found: C, 72.28; H,
9.60.
5.3.12. 4-Hydroxy-6-methyl-3-undecyl-2-pyrone (19)
Using general procedure B, reaction of 11 (0.050 g, 0.17 mmol) with sodium
cyanoborohydride (0.026 g, 0.41 mmol) followed by purification afforded the title
compound as white crystals (0.034 g, 71%). m.p. 98.0–99.0 °C; Rf (CH2Cl2/MeOH,
1
97:3) 0.3; IR (ATR) νmax 2918, 2662, 1571, 1445 cm-1; H NMR (CDCl3, 400 MHz) δ
9.35 (1H, br s, OH), 6.15 (1H, d, J = 1.0 Hz, H-5), 2.44 (2H, t, J = 7.5 Hz, H2-8), 2.21
(3H, d, J = 1.0 Hz, H3-7), 1.53–1.43 (2H, m, H2-9), 1.35–1.24 (18H, m, H2-10, H2-11,
H2-12, H2-13, H2-14, H2-15, H2-16, H2-17, H2-18), 0.87 (3H, t, J = 6.8 Hz, H3-19); 13C
NMR (CDCl3, 100 MHz) δ 168.0 (C-2), 166.6 (C-4), 160.0 (C-6), 103.5 (C-3), 101.5 (C-
5), 32.1 (C-17), 29.9 (C-11, C-12, C-13, C-14), 29.8 (C-15, C-16), 29.5 (C-10), 28.2 (C-
9), 23.2 (C-8), 22.8 (C-18), 19.8 (C-7), 14.3 (C-19); (+)-HRESIMS m/z 317.208 [M+Na]+
(calcd for C18H30NaO3, 317.2087); Anal calcd for C18H30O3: C, 73.43; H, 10.27. Found:
C, 73.62; H, 10.01.
5.3.13. 4-Hydroxy-6-methyl-3-tetradecyl-2-pyrone (20)
26

ACCEPTED MANUSCRIPT
Using general procedure B, reaction of 12 (0.050 g, 0.15 mmol) with sodium
cyanoborohydride (0.024 g, 0.37 mmol) followed by purification afforded the title
compound as pale yellow crystals (0.030 g, 63%). m.p. 98.0–99.0 °C; Rf
(CH2Cl2/MeOH, 97:3) 0.3; IR (ATR) νmax 2918, 2662, 1571, 1445 cm-1; 1H NMR (CDCl3,
400 MHz) δ 9.33 (1H, br s, OH), 6.14 (1H, d, J = 1.0 Hz, H-5), 2.44 (2H, t, J = 7.5 Hz,
H2-8), 2.21 (3H, d, J = 1.0 Hz, H3-7), 1.53–1.46 (2H, m, H2-9), 1.35–1.24 (22H, m, H2-
10, H2-11, H2-12, H2-13, H2-14, H2-15, H2-16, H2-17, H2-18, H2-19, H2-20), 0.87 (3H, t, J
13
= 6.8 Hz, H3-21); C NMR (CDCl3, 100 MHz) δ 167.9 (C-2), 166.6 (C-4), 160.0 (C-6),
103.5 (C-3), 101.5 (C-5), 32.1 (C-19), 29.9 (C-10, C-11, C-12, C-13, C-14), 29.8 (C-15,
C-16, C-17), 29.5 (C-18), 28.3 (C-9), 23.2 (C-8), 22.8 (C-20), 19.8 (C-7), 14.3 (C-21);
(+)-HRESIMS m/z 317.208 [M+Na]+ (calcd for C18H30NaO3, 317.2087); Anal calcd for
C20H34O3.0.3H2O: C, 73.13; H, 10.64. Found: C, 72.78; H, 10.40.
5.3.14. 3-Hexadecyl-4-hydroxy-6-methyl-2-pyrone (21)
Using general procedure B, reaction of 13 (0.050 g, 0.14 mmol) with sodium
cyanoborohydride (0.022 g, 0.34 mmol) followed by purification afforded the title
compound as white solid (0.036 g, 75%). m.p. 107–109°C; Rf (CH2Cl2/MeOH, 97:3)
0.3; IR (ATR) νmax 2918, 2662, 1571, 1445 cm-1; 1H NMR (CDCl3, 400 MHz) δ 9.45 (1H,
br s, OH), 6.15 (1H, d, J = 1.0 Hz, H-5), 2.44 (2H, t, J = 7.5 Hz, H2-8), 2.21 (3H, s, H3-
7), 1.53–1.46 (2H, m, H2-9), 1.35–1.24 (26H, m, H2-10, H2-11, H2-12, H2-13, H2-14, H2-
15, H2-16, H2-17, H2-18, H2-19, H2-20, H2-21, H2-22), 0.88 (3H, t, J = 6.8 Hz, H3-23);
13C NMR (CDCl3, 100 MHz) δ 168.0 (C-2), 166.7 (C-4), 159.9 (C-6), 103.5 (C-3), 101.5
(C-5), 32.1 (C-21), 29.9 (C-10, C-11, C-12, C-13, C-14, C-15, C-16, C-17), 29.8 (C-18,
C-19), 29.5 (C-20), 28.3 (C-9), 23.2 (C-8), 22.8 (C-22), 19.8 (C-7), 14.3 (C-23); (+)-
HRESIMS m/z 317.208 [M+Na]+ (calcd for C18H30NaO3, 317.2087); Anal calcd for
C22H38O3.0.6H2O: C, 72.88; H, 10.94. Found: C, 72.82; H, 10.57.
5.3.15. 3-(Cyclohexylmethyl)-4-hydroxy-6-methyl-2-pyrone (22)
27

ACCEPTED MANUSCRIPT
Using general procedure B, reaction of 14 (0.050 g, 0.21 mmol) with sodium
cyanoborohydride (0.033 g, 0.53 mmol) followed by purification afforded the title
compound as white crystals (0.032 g, 68%). m.p. 196.5–198.0 °C; Rf (CH2Cl2/MeOH,
98:2) 0.3; IR (ATR) νmax 2920, 2652, 1561, 1448 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ
5.96 (1H, d, J = 1.0 Hz, H-5), 2.14 (2H, d, J = 6.8 Hz, H2-8), 2.13 (3H, s, H3-7), 1.65–
1.60 (2H, m, H2-11), 1.57–1.52 (4H, m, H2-12, H2-14), 1.51–1.41 (1H, m, H-9), 1.16–
1.09 (2H, m, H2-13), 0.94–0.85 (2H, m, H2 -10); 13C NMR (DMSO-d6, 100 MHz) δ 165.4
(C-2), 165.0 (C-4), 159.6 (C-6), 99.8 (C-3, C-5), 36.3 (C-9), 32.7 (C-10, C-14), 30.1 (C-
8), 26.1 (C-12), 25.8 (C-11, C-13), 19.2 (C-7); (+)-HRESIMS m/z 245.1143 [M+Na]+
(calcd for C13H18NaO3, 245.1148); Anal calcd for C13H18O3: C, 70.24; H, 8.16. Found:
C, 70.30; H, 8.29.
5.3.16. 3-(3-Cyclohexylpropyl)-4-hydroxy-6-methyl-2-pyrone (23)
Using general procedure B, reaction of 15 (0.050 g, 0.19 mmol) with sodium
cyanoborohydride (0.030 g, 0.47 mmol) followed by purification afforded the title
compound as pale yellow needles (0.034 g, 70%). m.p. 140.0–141.5 °C; Rf
1
(CH2Cl2/MeOH, 97:3) 0.2; IR (ATR) νmax 2921, 2659, 1574, 1445 cm-1; H NMR
(DMSO-d6 ,400 MHz) δ 5.96 (1H, J = 1.0 Hz, H-5), 2.21 (2H, t, J = 7.5 Hz, H2-8), 2.12
(3H, J = 1.0 Hz, H3-7), 1.65–1.55 (4H, m, H2-13, H2-16), 1.40–1.33 (2H, m, H2-9), 1.22–
13
1.07 (7H, m, H2-10, H-11, H2-14, H2-15), 0.86–0.76 (2H, m, H2-12); C NMR (DMSO-
d6, 100 MHz) δ 164.9 (C-2), 164.8 (C-4), 159.6 (C-6), 101.2 (C-3), 99.8 (C-5), 36.9 (C-
10), 36.7 (C-11), 32.9 (C-12, C-16), 26.2 (C-14), 25.9 (C-13, C-15), 24.8 (C-9), 22.9 (C-
8), 19.2 (C-7); (+)-HRESIMS m/z 273.1454 [M+Na]+ (calcd for C15H22NaO3, 273.1461);
Anal calcd for C15H22O3: C, 71.97; H, 8.86. Found: C, 71.83; H, 8.93.
5.3.17. 4-Hydroxy-6-methyl-2-oxo-N-phenyl-2-pyran-3-carboxamide (24)
Using general procedure C, reaction of 4-hydroxy-6-methyl-2-pyrone (7) (0.050
g, 0.4 mmol) with isocyanatobenzene (0.238 g, 2.0 mmol) and triethylamine (22 µL, 0.1
mmol) followed by purification by silica gel column chromatography, eluting with
28