Synthesis and antifungal activity of polycyclic pyridone derivatives with anti-hyphal and biofilm formation activity against Candida albicans

Article abstract of DOI:10.1016/j.bmcl.2021.127845

Thirty-five pyridone derivatives were synthesized, with derivatization conducted on polycyclic pyridone scaffolds, including cis- or trans-oxydecalin and other cyclic structures, by domino-Knoevenagel-electrocyclic reactions. The anti-fungal activities of the synthesized compounds were tested against Candida albicans. Ten compounds inhibited hyphal formation without inhibiting growth. Pyridones with anti-hyphal formation activity (4c, 6d, 12a and 12c) were tested for their ability to inhibit biofilm formation. Compound 6d showed both anti-hyphal and biofilm inhibition activity.

Full text of DOI:10.1016/j.bmcl.2021.127845

Bioorg. Med. Chem. Lett. 37 (2021) 127845
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal activity of polycyclic pyridone derivatives with
anti-hyphal and biofilm formation activity against Candida albicans
,
Hitoshi Kamauchia *, Yu Kimuraa, Mikoto Ushiwataria, Mitsuaki Suzukib, Taishi Sekia,
Koichi Takaoa, Yoshiaki Sugitaa
a Department of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1-1 Keyaki-dai, Sakado, Saitama 350-0295, Japan
b Department of Chemistry, Faculty of Science, Josai University, 1-1 Keyaki-dai, Sakado, Saitama 350-0295, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thirty-five pyridone derivatives were synthesized, with derivatization conducted on polycyclic pyridone scaf-
folds, including cis- or trans-oxydecalin and other cyclic structures, by domino-Knoevenagel-electrocyclic re-
actions. The anti-fungal activities of the synthesized compounds were tested against Candida albicans. Ten
compounds inhibited hyphal formation without inhibiting growth. Pyridones with anti-hyphal formation activity
(4c, 6d, 12a and 12c) were tested for their ability to inhibit biofilm formation. Compound 6d showed both anti-
hyphal and biofilm inhibition activity.
Pyridone
Hyphal formation
Biofilm formation
Candida albicans
XTT reduction assay
Candida albicans is a dimorphic fungus that transforms from a yeast
to a hyphal form. The capacity of C. albicans to switch from yeast to
hypha is associated with antifungal drug resistance, virulence, invasion
and colonization, and the development of spatially organized architec-
tures of highly structured mature biofilms.1 Biofilm formation by fungi
undergoing the yeast-to-hypha transition is thus a target of antifungal
drugs. Several small-molecule inhibitors of hyphal formation have been
discovered but the number of lead compounds are poor compared to
typically used antifungal agents (e.g., azole and polyene type antibi-
otics)2 and thus anti-hyphal and anti-biofilm formation pharmacophores
are required.
reactions initiated by reagent- and substrate-controlled site-selective
activation of different pairs of functional groups strategically placed
around a linear template.
Starting from this interesting pharmacological profile, we have
designed pyridone derivatives with cyclic scaffolds following the
approach of combining in a single molecule two different pharmaco-
phores. C-5 substituted pyridone with pyran structure is selected as the
common scaffold with trichodin A. Targets of derivatization were A)
substituent at C-5, B) cis or trans oxydecalin unit, C) presence of prenyl
unit at C-21 and D) presence of chlorine at C-6 (Fig. 2).
These pyridone derivatives with cyclic scaffolds were synthesized by
intermolecular electrocyclic reactions and their antifungal activities
towards hyphal formation and biofilm formation were tested using
C. albicans.
Natural pyridone derivatives isolated from marine-derived fungi are
attractive lead compounds for anti-hyphal formation agents. For
example, didymellamide A was isolated from Stagonosporopsis cucurbi-
tacearum and showed antifungal activity against azole-resistant
C. albicans.3 Trichodin A was isolated from Trichoderma sp. and
showed antifungal activity against C. albicans.4 These compounds both
contain the pyridone moiety and other cyclic structures (Fig. 1).
The skeletal diversity of a small molecule library is important for
discovering bioactive leads. Diversity-oriented synthesis (DOS) and
scaffold diversity synthesis (SDS) have been used to increase structural
diversity.5 A derivatized lead compound with a skeletal scaffold could
fill the three-dimensional (3D) surfaces of chemical space and interact
with biological macromolecules in a selective manner. Skeletal diver-
sification has previously been achieved by intramolecular cyclization
Polycyclic pyridone derivatives were synthesized from 4-hydroxy-2-
pyridone via domino-Knoevenagel-electrocyclic reactions.6 C-5-
substituted 4-hydroxy-2-pyridones (2a-2d) were prepared from aceto-
nitrile derivatives (1a-1d) and malonyl chloride according to the liter-
ature.7 Knoevenagel condensation of pyridone and aldehyde gave
intermediates. An additional electrocyclic reaction between the heter-
odiene at C-4 and C-3 to C-13 and the dienophile at C-18 to C-19 in the
intermediate afforded the polycyclic pyridone skeleton. This electro-
cyclic reaction yielded an oxydecalin ring with an altered ring juncture
corresponding to the aldehyde and base.8 Functional group diversity (C-
3, prenyl group, chlorine) was introduced by using the corresponding
* Corresponding author.
E-mail address: kamauchi@josai.ac.jp (H. Kamauchi).
https://doi.org/10.1016/j.bmcl.2021.127845
Received 29 November 2020; Received in revised form 22 January 2021; Accepted 29 January 2021
Available online 9 February 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

H. Kamauchi et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127845
Fig. 1. Structures of polycyclic pyridones isolated from natural resources.
Fig. 2. Molecular design of pyridone derivatives.
Fig. 3. NOESY correlations (red arrows) of 3b and 5b.
acetonitrile, aldehyde, and by dechlorination.
(ꢀ )-Citronellal was used for this reaction to yield tricyclic pyridone
derivatives with trans-annulation of pyran derivatives (3a-3d) through a
hetero Diels–Alder reaction (Scheme 1). The stereochemistry 3b was
determined from the coupling constants of 1H NMR and NOESY corre-
lations. The large coupling constants between H-13 and H-18 (11.5 Hz)
indicated that the ring juncture was trans. The NOESY correlations be-
tween H-13 and H-15, H-14
α and H-18 determined their relative
configuration (Fig. 3). The other tricyclic pyridone derivatives had the
same relative configuration because they were generated using the same
synthetic process.
The cis-annulations of oxydecalin derivatives (4a-4d, 5a-5d) were
generated using 2-prenyloxy or 2-geranyloxybenzaldehyde (Scheme 2).
The hetero Diels-Alder reaction did not proceed in the presence of
triethylamine as base and an intermediate was obtained, whereas the
use of ethylene diammonium diacetate (EDDA) as a base under reflux
generated the oxydecalin skeleton. The coupling constant between H-13
and H-18 of 5b (6.0 Hz) indicated that the stereochemistry of the ring
juncture was cis. The NOESY correlations between H-17α and H-20, H-
18 and H-21 provided the relative configuration of 5b (Fig. 3). The other
tetracyclic pyridone derivatives (4a-4d and 5a-5d) had the same rela-
tive configuration because they were generated using the same synthetic
process.
Scheme 1. Synthesis of tricyclic pyridone derivatives.
Use of the aldehyde with an E-alkene at C-2′ and C-3′ (citral and 3-
methyl-2-butenal) generated bicyclic pyridone derivatives (6a-6d and
2

H. Kamauchi et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127845
Scheme 2. Synthesis of the tetra- and bicyclic pyridone derivatives.
7a-7d) by oxa-electrocyclization (Scheme 2).
Manikandan reported that electrocyclization using EDDA at reflux or
microwave condition provided 2- and 4-pyridone derivatives.8 13C NMR
spectroscopic data allowed determination of whether these compounds
were 2- or 4-pyridone. The δC value for C-5 of the 3-phenyl-2-pyridone
derivative was around 110 ppm, whereas that of the 3-phenyl-4-pyri-
done derivative was around 120 ppm.9 Since δC in C-5 of all synthe-
sized polycyclic pyridones was around 110 ppm, these compounds were
likely 2-pyridone derivatives. 6a was crystallized from n-hexane-EtOAc
to give colorless needles. X-ray structure of 6a suggested synthesized
bicyclic pyridones were 2-pyridones (deposited at the Cambridge Crys-
tallographic Data Centre, reference number CCDC 2056814). 2-Pyri-
done has been regarded as the prototype for the lactam ꢀ lactim
tautomerization. 6a was existed as lactim form in the crystal structure
(Fig. 4).
Polycyclic 6-hydropyridone derivatives (9a-9c, 10a-10c, 11a-11c,
12a-12c and 13a-13c) were also synthesized as the same pathway from
6-hydropyridone intermediates (8a-8c) prepared by dechlorination by
using Pd/C (Schemes 1 and 2).7 Dechlorination of thienylpyridone (2d)
didn’t progress due to the presence of sulfur atom. Thirty-five pyridones
derivatized by substitution at C-5 (phenyl, 4-methylphenyl, 4-methox-
yphenyl, thienyl), by the presence of chlorine at C-6, and by alteration of
the scaffold of the pericyclic pyridone skeleton, were prepared (Table 1).
The anti-hyphal formation activities of these compounds were tested
using C. albicans (SC-5314). Hyphal formation was induced by growth in
spider medium (nutrient broth, mannitol and K2PO4). Farnesol, a
quorum-sensing molecule in C. albicans, inhibits hyphal formation and
was used as a positive control.10 Colonies were visually observed at the
Fig. 4. Lactam ꢀ lactim tautomerization and X-ray structure of 6a.
3

H. Kamauchi et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127845
Table 1
Synthesized pyridone derivatives.
bottom of wells of a 96 well microplate in the presence of all samples,
showing that these compounds did not inhibit the growth of C. albicans.
The results are shown in Fig. 5. Four compounds (4c, 6d, 12a and
sulfophenyl)–2H-tetrazolium-5-carboxanilide) reduction assay.11 Min-
ocycline was used as the positive control. Compound 6d reduced biofilm
formation between 6.25 and 25 μg/mL (Fig. 7) whereas 12a and 12c
12c) at 25
μ
g/mL inhibited hyphal growth by over 50%. Six compounds
showed mild inhibitory activity (respective inhibitory activities of 18.7
and 17.9% at 25 g/mL) and 4c did not inhibit biofilm formation.
(3d, 4d, 5c, 6a, 6c and 7c) showed minor activity (30–50% inhibition)
μ
whereas the other derivatives showed no inhibitory activity. The four
In conclusion, polycyclic pyridones were synthesized with the
structural features of trichodin A. Four derivatives (4c, 6d, 12a and 12c)
inhibited hyphal formation by C. albicans, and 6d also inhibited biofilm
formation. These results suggest that 6d may inhibit biofilm formation
by modulating the hyphal formation and reducing cellular adhesion.
Our findings illustrate the potential of pyridones as antifungal agents.
active derivatives showed dose dependency in the range 3.12 to 25
mL (Fig. 6).
μg/
Structure activity-relationships based on these results suggest that
five of the seven bicyclic pyridones without prenyl sidechains (6a-6d
and 12a-12c) showed inhibitory activity. This hit rate was superior to
that obtained using other polycyclic pyridone derivatives. Moreover, 6-
hydro pyridone exhibited enhanced inhibitory activity compared with
6a and 6c and with 12a and 12c.
Declaration of Competing Interest
The four active compounds (4c, 6d, 12a and 12c) were investigated
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
◦
in biofilm formation tests. Biofilms were grown at 37 C using RPMI
1640 medium and evaluated by the XTT (2,3-bis-(2-methoxy-4-nitro-5-
4

H. Kamauchi et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127845
Fig. 7. Inhibition of biofilm formation by C. albicans using 6d. *, **, ***: P <
0.05, 0.01, 0.001 vs. control. Minocycline was tested in 10
positive control.
μg/mL and used as
Acknowledgment
This work was supported by JSPS KAKENHI Grant Number
JP20K16029.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127845.
Fig. 5. Hyphal growth of C. albicans (SC5314) in spider medium. (A) Measured
hyphal length in the presence of the active pyridones and farnesol at 25
μ
g/mL.
g/mL on C. albicans
SC5314. Farnesol (far) was used as a positive control. ***: P < 0.001 vs. control.
Scale bars represent 50 m.
(B) The inhibitory effect of the active pyridones at 25
μ
μ
Fig. 6. Inhibition of C. albicans hyphal formation using 4c, 6d, 12a and 12c. ***: P < 0.001 vs. control.
5

H. Kamauchi et al.
Bioorganic & Medicinal Chemistry Letters 37 (2021) 127845
in probe and drug discovery. Angew Chemie - Int Ed. 2016;55(27):7586–7605.
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