Synthesis and pharmacological evaluation of childinin E and several derivatives as anti-hyphal formation inhibitors against Candida albicans

Article abstract of DOI:10.1016/j.tetlet.2020.152588

The natural highly substituted benzophenone childinin E (1) was previously isolated from the fungus Daldinia childiae. Here we describe the total synthesis of childinin E and several derivatives using a linear seven-step sequence. The antifungal propertie

Full text of DOI:10.1016/j.tetlet.2020.152588

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Synthesis and pharmacological evaluation of childinin E and several deriva‐
tives as anti-hyphal formation inhibitors against Candida albicans
Hitoshi Kamauchi, Momoka Hirata, Koichi Takao, Yoshiaki Sugita
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S0040-4039(20)31087-X
https://doi.org/10.1016/j.tetlet.2020.152588
TETL 152588
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Tetrahedron Letters
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19 October 2020
Please cite this article as: Kamauchi, H., Hirata, M., Takao, K., Sugita, Y., Synthesis and pharmacological
evaluation of childinin E and several derivatives as anti-hyphal formation inhibitors against Candida albicans,
Tetrahedron Letters (2020), doi: https://doi.org/10.1016/j.tetlet.2020.152588
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Synthesis and pharmacological evaluation of childinin E and several derivatives as
anti-hyphal formation inhibitors against Candida albicans
Hitoshi Kamauchi, Momoka Hirata, Koichi Takao, Yoshiaki Sugita
Laboratory of Bioorganic Chemistry, Department of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, 1-1
Keyaki-dai, Sakado, Saitama 350-0295, Japan
———
ARTICLE INFO
ABSTRACT
 Corresponding author. Tel./fax: +81-49-271-7256; e-mail: kamauchi@josai.ac.jp
Article history:
Received
Received in revised form
Accepted
The natural highly substituted benzophenone childinin E (1) was previously isolated from the
fungus Daldinia childiae. Here we describe the total synthesis of childinin E and several
derivatives using a linear seven-step sequence. The antifungal properties of the synthesized
compounds against Candida albicans were evaluated by an anti-hyphal formation test. Childnin
E and two derivatives exhibited anti-hyphal formation activity.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
hyphal formation
Candida albicans
benzophenone
Daldinia spp.
The fungus Daldinia is a rich source of polyketides with unique structures. The highly functionalized benzophenones daldinals A
and B and childinins D and E (1-4) have been isolated from D. concentrica and D. childiae (Fig. 1) 1, 2 but their bioactivities are poorly
investigated. For example, only the nitric oxide production inhibitory activities of daldinals have been evaluated.3 Natural highly
functionalized benzophenones show various bioactivities. Pestalone, isolated from a marine derived fungus, shows in vitro cytotoxicity
OH
2'
O
OH
O
CHO
6
3'
1
MeO
OMe
MeO
OMe
7
5
4
1'
6'
4'
2
3
5'
OH
OH
7'
1
2
childinin E
daldinal A
OMe
O
CHO
OMe
COOH
Fig. 1 Structures of benzophenones isolated frOom Daldinia spp.
MeO
OMe
MeO
OMe
OH
daldinal B
OMe
childinin D
3
4
4
against tumor cell lines and antibiotic activity against drug-resistant Staphylococcus aureus and Enterococcus faecium, and
5
xanthochymol isolated from Garcinia xanthochymus fruit shows antifungal activity against Candida albicans. Natural highly
functionalized benzophenones thus exhibit promising antibiotic properties.
The hyphal-yeast transition of C. albicans is an important antifungal target and represents the first step in the development of
spatially highly structured and dynamic organized mature biofilms. Biofilms are virulence factors that aid in the adhesion to epithelial
and endothelial cell types that resist to current antifungal therapeutics. 6 Farnesol, an autoregulatory molecule produced by C. albicans,
7
inhibits the induction of hyphal growth by inhibiting adenylate cyclase (Cyr1). However, few compounds have been identified with
anti-hyphal formation activity compared to typically used antifungal agents (e.g., azole and polyene type antibiotics).
Here we describe the synthesis of natural benzophenones isolated from Daldinia spp. and several derivatives, and their in vitro
antifungal activities against C. albicans determined using an anti-hyphal formation assay.
To synthesize the target compounds, we first elaborated the synthesis of 3-methoxy benzophenone derivatives using a multi-step
reaction to probe the general feasibility of the synthetic approach. The strategy behind our synthesis is summarized in Scheme 1. The
benzophenone scaffold is intended to be assembled using two building blocks. Building block A is a 2,3,5-substituted benzaldehyde and
building block B is a 2,3,5-substituted bromobenzene. The two blocks are linked by reaction with a lithiated arene of building block B
and subsequent oxidation. The introduction of formyl group at C-6 was placed at a faster stage in the synthesis.

2
Tetrahedron
OH
2'
O
7
R3
6
MeO
OMe
3'
1
5
4
1'
6'
4'
2
3
5'
OMe
7'
R3
OP
R2
OMe
MeO
R1
OMe
A
C
R
1 = CHO
1 = Br
B
D
R
2 = Br, R3 = CHO
R
R2 = CHO, R3 = H
P = protective group
Scheme 1. Retrosynthetic analysis of benzophenone derivatives.
Starting from commercial o-vanillin (5), benzaldehyde (7) was prepared by selective methylation at C-5 via intermediate 6, and the
hydroxy group was protected with MOM-Cl, yielding 8 as building block A.8 Compound 11 as building block B was prepared by
Vilsmeier formylation of 1-bromo-3,5-dimethoxybenzene (9) followed by and acetal protection (Scheme 2).9
Bromine–lithium exchange of 11 employing n-BuLi, followed by reaction with 8, did not provide the expected benzhydrol 12. The
same reaction using 9 in place of 11 also resulted in no reaction.
We therefore changed our strategy and exchanged the substituted groups on bromobenzene and benzaldehyde (Scheme 1). We
OH
OMOM
CHO
OH
OH
MeO
CHO
MeO
MeO
CHO
MeO
CHO
a
e
b-d
N(Me)2
7
5
6
8
O
O
CHO
O
O
MOMO
MeO
OH
Br
OMe
Br
OMe
g
f
h
Br
OMe
OMe
OMe
OMe
OMe
11
OMe
9
10
12
Scheme 2. Synthesis of benzophenone derivatives: a) HCHO, NH(Me)2, reflux, 4 h; b) Ac2O, reflux, overnight; c) HCl, rt, 1.5 h; d) SnCl2,
reflux, 0.5 h, 53% over four steps; e) MOMCl, DIPEA, rt, 3 h, 85%; f) DMF, POCl3, 0°C to 100°C, overnight, 61%; g) ethylene glycol,
pTsOH, reflux, 3 h, 71%. h) n-BuLi, 8, −78°C to rt.
prepared a 2,3,5-substituted bromobenzene as building block C and a 3,5-substituted benzaldehyde as building block D. The
benzophenone scaffold was synthesized as described above. The target compound was non-substituted at C-6 due to the complicated
process required for formylation at C-6.
CHO
MeO
OMe
OR
OMOM
Br
OH
OH
OH
16
MeO
OMe
MeO
MeO
Br
MeO
Br
a, b
c
d
OMe
CHO
13
14
15
17
20
R = MOM
R = H
h
OMOM
O
OH
O
OH
O
MeO
OMe
MeO
OMe
MeO
OMe
e
f
g
OMe
OMe
OH
1
childinin E
18
19
Scheme 3. Synthesis of childinin E: a) hydrazine monohydrate, diethylene glycol, 110°C, 15 min; b) KOH, 150°C, 2 h, 99% over two
steps; c) MOMCl, DIPEA, rt, 3 h, 77%; d) n-BuLi, 16, −78°C to rt, 15 min, 41%; e) MnO2, rt, overnight, 41%; f) BCl3, 0°C to rt,
overnight, 74%; g) BBr3, −78°C to reflux, 1 h, 25%; h) HCl, rt, 1 h, 67%.

Building block 14 was prepared by a two-step reaction using the procedure of Nazih and co-workers from 5-bromovanillin (13)
(Scheme 3).10 The phenolic hydroxy group at C-2 was protected by MOM. Bromine–lithium exchange of 15 employing n-BuLi,
followed by reaction with 3,5-dimethoxybenzaldehyde (16) at −78°C to rt, gave benzhydrol 17 in 41% yield. Oxidation of benzylic
position of 17 with MnO2 provided benzophenone derivative 18. The MOM protecting group was cleaved using a Lewis acid in good
yield to give 3-O-methyl-childinin E (19).
Finally, the methoxy groups in 19 were removed using BBr3. There are three methoxy groups in 19: one each at C-3, C-5 and C-3’.
1
Demethylation proceeded selectively at C-3 or C-5 and yielded childinin E (1) as a white solid. The H, 13C and MS spectroscopic
properties of synthetic 1 matched those reported for the natural product.
In contrast, the formylation of 18 or 19 by classical Vilsmeier formylation, or dichloro-methylation using TiCl4 or SnCl4/CH2Cl2
were unsuccessful. To investigate the structure-activity relationship of these benzophenone derivatives, we focused on synthesizing
several derivatives by introducing a number of hydroxy groups using the above synthetic route. The MOM group in benzhydrol 17 was
removed using 10% aqueous HCl in dioxane to yield 2’-hydroxy benzhydrol 20 (Scheme 3). The 2’-methoxy derivatives were
synthesized from 2,3-dimethoxy-5-methylbromobenzene 21 as the building block (Scheme 4), prepared by methylating 14 with
iodomethane.10 2’-O-Methyl-benzhydrol 22 and 2’-O-methyl-benzophenone 23 were obtained by bromine–lithium exchange of 21
followed by reaction with 16 and oxidation of benzylic position of 22 with MnO2.

4
Tetrahedron
OH
OMe
MeO
Br
MeO
Br
a
b
14
21
OMe
OMe
O
OH
MeO
OMe
MeO
OMe
c
OMe
OMe
22
23
Scheme 4. Synthesis of 2’-methoxy benzophenone derivatives:
a) MeI, K2CO3, 80°C, overnight, quant.; b) n-BuLi, 16, −78°C to
rt, 15 min, 20%; c) MnO2, rt, overnight, 55%.
The anti-hyphal formation activities of the synthesized benzophenone and its derivatives (1, 19, 20, 22 and 23) were tested against C.
albicans (SC-5314) using spider medium and N-acetylglucosamine (GlcNac) medium. Hyphal formation of C. albicans is triggered by
several external signals and can be controlled by the choice of medium. GlcNac medium activates the MAPK signaling pathway,
suppressing the Cph1 gene,11 whereas the cAMP-PKA signaling pathway is activating by spider medium, suppressing the Efg1
transcription factor gene.12 The induced hyphal cells were picked and measured their length from photo. Evaluation was performed by
using the average of hyphal length for each compound.
The results of the anti-hyphal formation test are shown in Fig. 2 (all photo was represented in supplementary data). In spider
medium, 1, 19 and 20 showed anti-hyphal formation activity at 25 g/mL, and 1 and 19 also showed inhibitory activity in GlcNac
medium, but this activity was weaker than in spider medium. Compound 20 show inhibitory activity only in spider medium.
Compounds 22 and 23 showed no inhibitory activity.
We investigated the dose-dependencies of 1 and 19 and found that both compounds showed inhibitory activities of over 25% at 6.25
g/mL in spider medium whereas activity was observed at 12.5 g/mL in GlcNac medium (Fig. 3). Toxicity was checked using
minimum inhibitory concentration (MIC) values by performing susceptibility testing according to the CLSI M27-A2 microdilution
method using spider and GlcNac media. The MIC values of 1 and 19 were 100 g/mL, whereas 20 showed no toxicity under both
Fig. 2 Benzophenone derivative inhibition of hyphal formation
by C. albicans. Hyphal growth of C. albicans in spider and
GlcNac media in the presence of DMSO (A and E), 1 (B and F),
19 (C and G) and 20 (D and H) after 24 h incubation at 37°C.
Measured hyphal length of C. albicans in spider (I) and GlcNac
(J) media in the presence of the synthesized compounds.
Compounds were tested at 25 g/mL, except for farnesol (far)
in GlcNac medium (50 g/mL), used as a positive control. *,
***: P< 0.05, 0.001 vs. control. Scale bars represent 50 μm.
conditions.

These results indicated that the phenolic hydroxy group is essential for anti-hyphal formation inhibitory activity. All compounds
active in spider medium had a hydroxy group at C-2’ whereas compounds active in GlcNac medium also required a carbonyl group at
C-7, perhaps because a different signaling pathway is activated in spider and GlcNac media.
Thus, we completed the total synthesis of childinin E and some structural analogues and evaluated their antifungal properties by
inhibiting hyphal formation of C. albicans. The important substituted groups in benzophenone with anti-hyphal formation activity
(hydroxy group at C-2’ and carbonyl group at C-7) were discovered. The further extension of this study could develop new class of
antifungal compounds.
Declaration of Competing Interest
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.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Number JP20K16029.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
References and notes
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5. Jackson DN, Yang L, Wu SB, Kennelly EJ, Lipke PN. Antimicrob. Agents Chemother. 2015; 59: 6032–6038.
6. Toenjes KA, Munsee SM, Ibrahim AS, Jeffrey R, Edwards JE, Johnson DI. Antimicrob. Agents Chemother. 2005; 49: 963–972.
7. Lindsay AK, Deveau A, Piispanen AE, Hogan DA. Eukaryot Cell. 2012; 11: 1219–1225.
8. Sinhababu AK, Borchardt RT. Synth. Commun. 1983; 13: 677–683.
9. Wang Z, Wang Y, Wang B, Li W, Huang L, Li X. J. Med. Chem. 2015; 58: 8616–8637.
10. Nazih A, Benezra C, Lepoittevin JP. Chem. Res. Toxicol. 1993; 6: 215–222.
11. Naseem S, Gunasekera A, Araya E, Konopka JB. J. Biol. Chem. 2011; 286: 28671–28680.
12. Braun BR, Johnson AD. Genetics. 2000; 155: 57–67.
Fig. 3 Inhibition of hyphal formation by C. albicans using 1
#,
(A) and 19 (B). ***: P< 0.001 vs. control in spider medium,
###: P< 0.05, 0.001 vs. control in GlcNac medium.

6
Tetrahedron
Highlights
・The first total synthesis of childinin E was achieved from the commercial benzaldehyde.
・Five benzophenone derivatives were obtained by using the synthetic route of childinin E.
・ The antifungal properties of the synthesized compounds against Candida albicans were evaluated by an anti-
hyphal formation test.
・Childnin E and two derivatives exhibited anti-hyphal formation activity.
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Synthesis and pharmacological evaluation of
childinin E and several derivatives as anti-
hyphal formation inhibitors against Candida
albicans
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Hitoshi Kamauchi*, Momoka Hirata, Koichi Takao, Yoshiaki Sugita
Laboratory of Bioorganic Chemistry, Department of Pharmaceutical Sciences, Faculty of Pharmacy and
Pharmaceutical Sciences, Josai University, 1-1 Keyaki-dai, Sakado, Saitama 350-0295, Japan