Antifungal agents: Design, synthesis, antifungal activity and molecular docking of phloroglucinol derivatives

Article abstract of DOI:10.3390/molecules23123116

Pseudoaspidinol is a phloroglucinol derivative with Antifungal activity and is a major active component of Dryopteris fragrans. In our previous work, we studied the total synthesis of pseudoaspidinol belonging to a phloroglucinol derivative and investigated its antifungal activity as well as its intermediates. However, the results showed these compounds have low antifungal activity. In this study, in order to increase antifungal activities of phloroglucinol derivatives, we introduced antifungal pharmacophore allylamine into the methylphloroglucinol. Meanwhile, we remained C1–C4 acyl group in C-6 position of methylphloroglucinol using pseudoaspidinol as the lead compound to obtain novel phloroglucinol derivatives, synthesized 17 compounds, and evaluated antifungal activities on Trichophyton rubrum and Trichophyton mentagrophytes in vitro. Molecular docking verified their ability to combine the protein binding site. The results indicated that most of the compounds had strong antifungal activity, in which compound 17 were found to be the most active on Trichophyton rubrum with Minimum Inhibitory Concentration (MIC) of 3.05 μg/mL and of Trichophyton mentagrophytes with MIC of 5.13 μg/mL. Docking results showed that compounds had a nice combination with the protein binding site. These researches could lay the foundation for developing antifungal agents of clinical value.

Full text of DOI:10.3390/molecules23123116

molecules
Article
Antifungal Agents: Design, Synthesis, Antifungal
Activity and Molecular Docking of
Phloroglucinol Derivatives
Xingxing Teng 1, Yuanyuan Wang 1, Jinhua Gu 1, Peiqi Shi 1, Zhibin Shen 2 and Lianbao Ye 1,3,
*
1
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China;
tengxingxing563@126.com (X.T.); 15768985885@163.com (Y.W.); huaqianphar@126.com (J.G.);
spq15989276019@163.com (P.S.)
School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China;
szb8113@126.com
Key Laboratory of New Drug Discovery and Evaluation of Ordinary Universities of Guangdong Province,
Guangzhou Key Laboratory of Construction and Application of New Drug Screening Model Systems,
Guangzhou key Laboratory of Construction and Application of New Drug Screening Model Systems and
Guangdong Province Engineering Technology Center for Molecular Probes & Biomedical Imaging,
Guangdong Pharmaceutical University, Guangzhou 510006, China
2
3
*
Correspondence: yelb7909@163.com; Tel.: +86-20-39352139
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 5 November 2018; Accepted: 24 November 2018; Published: 28 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Pseudoaspidinol is a phloroglucinol derivative with Antifungal activity and is a major
active component of Dryopteris fragrans. In our previous work, we studied the total synthesis of
pseudoaspidinol belonging to a phloroglucinol derivative and investigated its antifungal activity as
well as its intermediates. However, the results showed these compounds have low antifungal activity.
In this study, in order to increase antifungal activities of phloroglucinol derivatives, we introduced
antifungal pharmacophore allylamine into the methylphloroglucinol. Meanwhile, we remained
C1–C4 acyl group in C-6 position of methylphloroglucinol using pseudoaspidinol as the lead
compound to obtain novel phloroglucinol derivatives, synthesized 17 compounds, and evaluated
antifungal activities on Trichophyton rubrum and Trichophyton mentagrophytes in vitro. Molecular
docking verified their ability to combine the protein binding site. The results indicated that most of
the compounds had strong antifungal activity, in which compound 17 were found to be the most
active on Trichophyton rubrum with Minimum Inhibitory Concentration (MIC) of 3.05
µg/mL and
of Trichophyton mentagrophytes with MIC of 5.13 g/mL. Docking results showed that compounds
µ
had a nice combination with the protein binding site. These researches could lay the foundation for
developing antifungal agents of clinical value.
Keywords: phloroglucinol derivatives; antifungal activity; molecular docking; allylamine;
squalene epoxidase
1. Introduction
Fungi are widely distributed in nature and frequently present as pathogens in the animal and
plant kingdoms [1]. In recent decades, despite progress in antifungal therapy, infectious diseases
caused by a variety of clinically significant species of fungi remain a major global health concern,
due to the development of antifungal drug resistance [2,3]. However, resistance to several antifungal
agents among a variety of clinically significant species of fungi is becoming an increasingly major
global problem and is reaching an alarming level on a global scale [4]. Hence, searching for efficient,
nontoxic or low toxic chemotherapeutic agents with potent broad-spectrum antifungal activity is
Molecules 2018, 23, 3116; doi:10.3390/molecules23123116
www.mdpi.com/journal/molecules

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an important goal in new drug research [
5
,6]. Currently, the main targets of the antifungal agents
are the cytochrome P450 sterol 14a-demethylase (CYP51), squalene epoxidase (SE) and
β-1,3-glucan
synthase, in which azole and triazole drugs are CYP51 inhibitors widely used as fungal antibiotics and
antimycobacterial activity, spinosins are
squalene epoxidase (SE) [7–10].
β-1,3-glucan synthase inhibitors and allylamine agents act on
Dryopteris fragrans is mainly used as a folk medicine at present. It has been found that the main
active compounds from Dryopteris fragrans have a significant effect on a variety of skin diseases
caused by fungi [11,12]. Our group isolated a variety of phloroglucinols from the plant and activity
experiment showed that the phloroglucinols had strong antifungal activity such as the minimal
inhibition concentration (MIC) values activity of aspidin BB against some clinical S. aureus was ranged
from 15.63 mg/mL to 62.5 mg/mL [13–16]. In addition, in our current research, phlorglucinols,
especially 2-methyl-6-acyl phloroglucinol derivatives. Pseudoaspidinol belongs to phlorglucinols and
shows good antifungal activity and its key intermediate methyl phlorglucinol can be used to generate
diverse compounds. In our previous study, a preliminary structure-activity study found that the
hydroxyl group of methyl phloroglucinol and the butyryl group at the C-4 position are the active
groups of the compound, and C-4 or C-6 is the active site. Introduction of different groups at this
position may alter its properties and biological activity. In order to increase antifungal activity of
phloroglucinol derivatives, in this study, we introduced antifungal pharmacophore allylamine into the
methyl phloroglucinol and remained C1–C4 acyl group in C-6 position using pseudoaspidinol as lead
compound to obtain novel phloroglucinol derivatives 4–17 (Figure 1).
Figure 1. Design of compounds.
We found in the literature that the main antifungal target of allylamine antifungal drugs is
squalene oxidase, especially the representative drug naftifine [17]. It is particularly noteworthy that
naftifine is the first marketed drug for allylamine antifungal drugs and is not replaceable in antifungal
action. Unfortunately, the widespread use of allylamine has led to the development of severe drug
resistance, which has significantly reduced their efficacy [18]. These compounds were synthesized
and evaluated for their antifungal activity against Trichophyton rubrum and Trichophyton mentagrophytes
in vitro. Molecular docking simulations were conducted to confirm the mechanism of action between
the compound and squalene epoxidase (SE).
2. Results and Discussions
2.1. Chemistry
The synthetic route of compounds was demonstrated in Scheme 1. Methyl phloroglucinol (
3) was
synthesized using 1,3,5-hydroxybenzene ( ) as raw material through Vilsmeier–Haack reaction and
1

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reduction reaction. The formyl chloride, acetyl chloride, propionyl chloride and butyryl chloride are
introduced into the C-4 position of methyl phloroglucinol under the catalysis of aluminum trichloride
and nitrobenzene to obtain the compound
The 4-chloro-2-butenamine hydrochloride is introduced into the benzene ring of the compound
obtain the compound . Compound reacted with the above four acid chloride to make compound
12 by Friedel–Crafts reaction using AlCl3 as catalyst under mild reaction conditions. On the one
4
–7. In addition, we are at normal temperature conditions.
3
to
8
8
9
–
hand, compound 12 is achieved in DMF by adding potassium carbonate and methyl iodide to give
compounds 13 and 14 under reflux. On the other hand, the methylnaphthalene group of compounds
15–16 were obtained by reacting a compound 12, 1-Chloromethyl naphthalene in the presence of K2CO3
and potassium iodide in ethanol. Finally, 1-chloromethylnaphthalene is introduced into a compound
12 to give the final product compound 17.
Scheme 1. Synthesis of phloroglucinol derivatives. (a) POCl3, DMF, EtoAc. (b) NaBH3CN, THF, PH 4.
(c) AlCl3, CS2, reflux. (d) AlCl3, CH2Cl2, rt. (e) CH3I, K2CO3, DMF, reflux. (f) PEG-400, K2CO3, KI,
EtOH, reflux.
In our previous studies, the reduction of aldehyde groups on the benzene ring to methyl
groups mostly uses NaBH3CN as a catalyst and tetrahydrofuran as a solvent, but this reduction
reaction is sensitive to pH, and the reaction at room temperature requires at least 10 h [1]. In the
subsequent synthesis optimization, we used zinc powder as a catalyst, diethyl ether and ethyl acetate
as a common solvent. This improvement makes the whole reaction not need to adjust pH, and the
normal temperature reaction can be completely finished within one hour—even the yield increased
from 60% to 90%. It is well known that Friedel–Crafts reaction is part of the most important reactions.
In the past, when butyryl group was introduced into methyl phloroglucinol, if only AlCl3 and a single
solvent carbon disulfide were unreactive, the addition of nitrobenzene had a momentous influence
on the reaction. In this study, there are hydroxyl groups on both sides of the C-4 position of methyl
phloroglucinol. This electron donating group will offset the passivation of the acyl ring to the benzene
ring. The second acyl group is submitted at the C-6 position in the reaction, so the reaction yield
is low. It is worth mentioning that after the introduction of an acyl group on the benzene ring
of the compound
Friedel-Crafting. Subsequently, we introduce 4-chloro-2-butenamine hydrochloride into the C-6
position of compound to obtain compound , then carry out acylation reaction to obtain compound
12. Surprisingly, the synthesis of compound 12 did not require the addition of nitrobenzene,
and the solvent was changed from carbon disulfide to dichloromethane. This reaction stilled be
4–7, the acyl group as the electron withdrawing group was completely inert to
2
8
9
–
9–

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and the yield was partially improved. This change is more in line with the contemporary concept
of green chemistry. Compound 12 has a lone pair of electrons on the nitrogen atom, so it is easy
to methylate, then the ratio of methyl iodide and DMF is changed to obtain compounds 13 and 14
After exploring various methods, we add PEG-400 and potassium iodide, enhancing the activity of
1-chloromethylnaphthalene and introducing a methylnaphthalene into the nitrogen atom of compound
12 under mild normal temperature conditions to obtain a secondary amine compound 15 and a tertiary
amine compound 16. In the final synthesis of compound 17, methylnaphthalene is more likely to be
included onto compound 13 than when a methyl group is introduced on compound 15. Since the
introduction of a methyl group by a secondary amine usually requires refluxed reaction, this causes
instability of the naphthalene and excessive impurities.
.
2.2. Evaluation of Antifungal Activity
The antifungal results of all synthetic compounds, pseudoaspidinol and Naftifine were shown
in Table 1. The reading of the minimum inhibitory concentration results complied with by observing
the growth effect of the two fungal strains on the 96-well plate (seen in Figure 2). Pseudoaspidinol
exhibited strong activity against T. mentagrophyte and T. rubrum, with IC50 values of 21.06
and 19.89 g/mL, respectively. Compound exhibited poor activity against T. mentagrophytes and
T. rubrum. Subsequently, the activity of the compound showed that only the compound and the
compound had an antifungal effect. The antifungal activity of compound was 62.5 g/mL, while the
IC50 of compound against T. rubrum was 39.89 g/mL. Activity of against T. mentagrophytes was
similar. Disappointingly, Compound was lower active against both fungi. The activity of Compound
12 was not optimized compared to the activity of Compound , but the activity of Compound 12
was decreased by nearly 1/3 compared with Compound , and the activity against both fungi was
39.24 g/mL and 39.17 g/mL, respectively. Surprisingly, the activity of Compound was practically
four times lower than that of Compound . Compounds 15 and 17 showed the best activity against
µg/mL
µ
3
4–7
6
7
6
µ
7
µ
8
7
6
µ
µ
9
4
Trichophyton rubrum and T. mentagrophyte, and the antifungal effect was much higher than that of the
lead compound pseudomicinol.
Table 1. Antifungal activities of compounds on Trichophyton rubrum (Tr) and Trichophyton mentagrophytes (Tm).
Compound
Trichophyton rubrum (MIC)
Trichophyton mentagrophyte (MIC)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
41.38 µg/mL
19.57 µg/mL
160 µg/mL
160 µg/mL
160 µg/mL
62.5 µg/mL
39.89 µg/mL
160 µg/mL
78.14 µg/mL
160 µg/mL
36.42 µg/mL
37.23 µg/mL
39.24 µg/mL
160 µg/mL
5.12 µg/mL
160 µg/mL
3.05 µg/mL
21.06 µg/mL
1.036 µg/mL
33.63 µg/mL
39.82 µg/mL
160 µg/mL
160 µg/mL
160 µg/mL
62.5 µg/mL
39.72 µg/mL
160 µg/mL
79.53 µg/mL
160 µg/mL
37.46 µg/mL
39.54 µg/mL
39.17 µg/mL
160 µg/mL
8.13 µg/mL
160 µg/mL
5.13 µg/mL
19.89 µg/mL
1.072 µg/mL
Pseudoaspidinol
Naftifine

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Figure 2.
(A) Complete antifungal effect. (B) Growth effect of fungal strains: Trichophyton rubrun.
(C) Growth effect of fungal strains: Trichophyton mentagrophytes.
The results showed that the butyryl group at the C-4 position of methyl phloroglucinol may be
a pharmacophore of pseudomicinol. Subsequently, it was found that most of the antifungal activity
results of the compound 9–12 after the para-introduction of the acyl group after the introduction
of the allylamine group at the C-6 position of the methyl phloroglucinol skeleton was optimized
(Figure 3A,B). This clearly indicates that the allylamine group may not be suitable as a single antifungal
group, but it has some antifungal optimization effects. In addition, the naphthylmethyl group of N
in allylamine can improve the antifungal activity. Obviously, structurally modified compounds 15
and 17 exhibited excellent activity against Trichophyton rubrum and T. mentagrophyte, compared to
pseudomicinol (Figure 3C,D). From the optimal compound 17, it can be assumed that the antifungal
key pharmacophore of the allylamine antifungal agent such as Naftifine is methylnaphthalene. Further,
research work on structure-activity relationship is currently under investigation and will be reported
in due course.
Figure 3. Activities of all compounds against Trichophyton rubrun and Trichophyton mentagrophytes.
(
A
) The antifungal results of all compounds against Trichophyton rubrun. (
results of all compounds against Trichophyton mentagrophytes. ( ) The strong antifungal results of
) The strong antifungal results of compounds against
B) The antifungal
C
compounds against Trichophyton rubrun. (
Trichophyton mentagrophytes.
D

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2.3. Docking Studies
Currently, the main targets of the antifungal agents are the cytochrome P450 sterol
14a-demethylase (CYP51), squalene epoxidase (SE) and -1,3-glucansynthase. The mechanism of
molecular docking was targeted at the positive drug, Naftifine. Docking experiments select SE (PDB
ID: 2AIB) to perform molecular docking since allylamine antifungal agents mainly acts on SE [ 10].
The docking score of compounds is shown in Table 2. The results show compounds 13 had higher
scores than pseudoaspidinol in accord with the results of antifungal activities and compound 17
demonstrate better score and antifungal activities, and the 3D interactions of compounds 13 with
β
7–
4–
8
–
9–
2AIB are shown in Figure 4. As can be seen from the diagram, compounds formed the hydrogen
bonding interactions of allylamine NH with TYR 47 and phenolic hydroxyl with MET 35 and TYR 33,
hydrophobic interaction of allyl with LEU 41, MET 59 and ALA 88, and Pi-Pi interaction of naphthalene
ring with LYS 94. Compound 17 shows the best antifungal activity owing to Pi-Pi interaction of
a naphthalene ring and hydrophobic interactions of N–CH3 with ILE 60 (seen in Figure 5). In order
to understand the binding mode in a dynamic environment, molecular dynamics simulation was
executed for the compound 17 with the crystal structure of 2AIB. Compound 17 exhibited a stable
binding conformation throughout the MD run as confirmed through the ligand Root Mean Squared
Deviation (RMSD), and the ligand tends to stable during 15–35 ps (RMSD < 0.4 Å) (Figure 6).
Figure 4. Binding modes of compounds 13–17 with 2AIB. The structural framework of compounds
was marked with amaranth color. Protein skeleton of 2AIB was marked with green color. The atoms
involved in the path are identified as colored stick. (Oxygen (orange), nitrogen (blue), hydrogen in
hydroxyl (white)).

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Figure 5. (A) Optimal binding modes of compound 17 in squalene epoxidase (PDB ID 2AIB); (B) 2D
interaction plot of 2AIB and compound 17. The structural framework of compound 17 was marked
with green color. Protein skeleton of 2AIB was marked with red color. The atoms involved in the path
are identified as colored stick. (Oxygen (red), nitrogen (blue), hydrogen (white)).
Figure 6. Plots of RMSD for all of the backbone atoms (Å) vs. simulation time (ps) for 2AIB in complex
with compound 17.
Table 2. Docking results of synthesized compounds.
Compounds
(−) Binding Energy ∆G (kcal/mol) 2AIB
8
9
10
11
12
13
14
15
6.23
5.65
6.02
6.18
5.98
6.00
6.44
9.93
11.27
11.80
5.75
9.32
16
17
Pseudoaspidinol
Naftifine

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3. Materials and Methods
3.1. Synthesis of Compounds
The solvents and reagents used throughout this study were purchased from commercial vendors
and were dried and purified by conventional methods prior to use. Nuclear magnetic resonance (NMR)
spectra was recorded on a Bruker AC-400P spectrometer (Bruker, Karlsruhe, Germany) with TMS as
the internal standard and was analyzed by MestReNova (Mestrelab Research, Santiago de Compostela,
Coruña, Spain) [19]. Infrared spectra were recorded using potassium bromide disks on a Hitachi
270-50 spectrophotometer (Hitachi Limited, Tokyo, Japan) and ESI mass spectra were made on an
API-3000 LC-MS spectrometer (Applied Biosystems, Shanghai, China). Thin-Layer Chromatography
(TLC) analysis was performed on silica gel 60 F254 silica plates (Merck KGaA, Darmstadt, Germany).
1H-NMR, 13C-NMR and MS of compounds 2–17 were found in Supplementary Materials.
Synthesis of
3
.
To the solution of 1-formyl-2,4,6-trihydroxy benzene (2) (1.54 g, 10 mmol) in
tetrahydrofuran (20 mL) we added methyl orange (1–2 drops) followed by the addition of sodium
cyanoborohydride (1.90 g, 30 mmol). The pH of the reaction mixture was maintained at 4.0, throughout
the reaction, by addition of 1 N HCl. The reaction mixture was stirred at room temperature for 12 h
prior to extraction with ethyl acetate (30 mL
solution (30 mL 3) and finally dried over Na2SO4. Solvent was evaporated to get the crude product,
which was purified by column chromatography with hexane/EtOAc (50:50 v/v).
×
4). The ethyl acetate layer was washed with brine
×
Synthesis of . 2,4,6-trihydroxytoluene (2 g, 14.28 mmol) was placed in a 250 mL three-necked flask,
7
carbon disulfide (10 mL) and anhydrous aluminum trichloride powder (5.7 g, 42.84 mmol) were added,
and nitrobenzene (6 mL) was added with stirring, and heated to reflux. After 0.5 h, then butyryl
chloride (1.68 g, 15.7 mmol) was slowly added dropwise, and the mixture was refluxed for 1.5 h, cooled
to room temperature, and poured into a mixture of ice water (10 mL of concentrated hydrochloric acid,
70 g of ice water) with stirring, and distilled for 30 min. The mixture was filtered while hot, and the
yellow needle-like crystals were precipitated in the filtrate. After sufficient cooling, the mixture was
filtered, and the remaining oil was repeatedly subjected to hot extraction several times. The combined
crystals were dried to obtain 2-methyl-6-butyrylphloroglucol.
Synthesis of 8. At room temperature, 2-methylbenzene-1,3,5-triol (421.04 mg, 3 mmol) and anhydrous
aluminum trichloride powder (799.98 mg, 6 mmol) were slowly added to a solution of methylene
chloride (20 mL), stirred evenly and refluxed for 1 h. Simultaneously, 4-chloro-2-butenamine
hydrochloride (468.41 mg, 3.31 mmol) was dissolved in a 10% (by mass) aqueous sodium
hydroxide solution(10 mL), stirred for 10 min to neutralize the hydrochloride, extracted three
times(15 mL) with methylene chloride and dried over anhydrous sodium sulfate. The neutralized
solution of 4-chloro-2-butenamine in methylene chloride was slowly added to a flask containing
◦
2-methylbenzene-1,3,5-triol at 0 C. At room temperature, the reaction was stirred until complete.
The solvent was recovered, dissolved in deionized water, extracted with ethyl acetate five times
(20 mL), dried over anhydrous sodium sulfate and the ethyl acetate removed under reduced pressure
to obtain the crude product as a yellow solid. This crude product was isolated by silica gel column
chromatography (hexane/EtOAc 1:1 v/v) to obtain compound 8.
Synthesis of 11. A mixture of 8 (627.62 mg, 3 mmol) and aluminum chloride (1.201 g, 9 mmol) in 20 mL
of methylene chloride was added to propionyl chloride (305 mg, 3.3 mmol). The reaction mixture
was stirred at 42 ◦C and monitored by TLC. Once the reaction was complete (5 h), the mixture was
poured onto ice water (70.00 g) and hydrochloric acid (2.00 g) was added. The solvent was recovered,
dissolved in deionized water, extracted with ethyl acetate five times (20 mL), dried over anhydrous
sodium sulfate and the ethyl acetate removed under reduced pressure to obtain the crude product.
The residue was subsequently purified by silica gel column chromatography (hexane/EtOAc 3:2 v/v)
to give a pure product.

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Synthesis of 13. The intermediate of compound 12 (558.30 mg, 2 mmol) was then dissolved in dry
DMF (20 mL), followed by the addition of K2CO3 (2.66 g, 12 mmol) and Me2SO4 (2.51 g, 12 mmol).
The reaction mixture was refluxed for 4 h, after which the mixture was filtered to obtain compound 13
.
Synthesis of 15. PEG-400 (5 d) and potassium carbonate were added to a mixture of compound 12 (1.40 g,
5 mmol) in EtOH (15 mL). The solution of 1-Chloromethyl naphthalene (1.77 g, 10 mmol) and potassium
iodide (1.60 g, 10 mmol) in EtOH (5 mL) was then added dropwise. The reaction was stirred at room
temperature for 36 h and neutralized with a saturated solution of NaHCO3. Following extraction with
dichloromethane, the organic layer was washed with brine, dried over anhydrous sodium sulfate,
concentrated, and purified by column chromatography (methyl alcohol/dichloromethane, 1:20 v/v,
Rf = 0.57) to obtain the intermediary of compound 15.
2,4,6-Trihydroxybenzaldehyde (
DMSO): (ppm) 11.46 (s, 2H), 10.66 (s, 1H), 9.93 (s, 1H), 5.79 (s, 2H), 2.50 (dt, J = 3.5, 1.7 Hz,
1H). 13C-NMR (101 MHz, DMSO):
(ppm) 192.4 (s), 167.5 (s), 165.7 (s), 159.3 (s), 102.9 (s), 94.9 (s),
2
). Yield: 82%. Yellow power. m.p.: 195.4–198.3 ◦C. 1H-NMR (400 MHz,
δ
δ
87.9 (s). EI-MS: 153.36 [M
O, (41.75) 41.52.
−
H]−. Anal. calcd for C7H6O4 (154.03): C, (54.34) 54.55; H, (3.91) 3.92;
2-Methylbenzene-1,3,5-triol (
DMSO):
166.4 (s), 153.5 (d, J = 7.8 Hz), 103.1 (s), 94.0 (s), 86.3 (s), 8.2 (s). EI-MS: 139.52 [M
3
). Yield: 78%. Yellow power. m.p.: 213.4–216.5 ◦C. 1H-NMR (400 MHz,
δ
(ppm) 8.79 (s, 2H), 8.66 (s, 1H), 5.77 (s, 2H), 2.50 (s, 3H). 13C-NMR (101 MHz, DMSO):
δ(ppm)
−
−
H] . Anal. calcd for
C7H8O3 (140.05): C, 60.00 (59.97); H, 5.75 (5.73); O, 34.25 (34.30).
◦
1
2,4,6-Trihydroxy-3-methylbenzaldehyde (
(400 MHz, DMSO):
(ppm) 8.79 (s, 2H), 8.66 (s, 1H), 5.77 (s, 2H), 2.50 (s, 3H). 13C-NMR (101 MHz,
DMSO): (ppm) 192.3 (s), 166.0 (s), 165.2 (s), 164.9 (s), 102.5 (s), 101.9 (s), 94.6 (s), 8.4 (s). EI-MS: 169.52
[M + H]+. Anal. calcd for C8H8O4 (168.04): C, 57.14 (57.08); H, 4.80 (4.83); O, 38.06 (38.09).
4). Yield: 58%. White solid. m.p.: 223.7–224.5 C. H-NMR
δ
δ
◦
1-(2,4,6-Trihydroxy-3-methylphenyl)ethan-1-one (
5
). Yield: 76%. Yellow power. m.p.: 206.9–208.1 C.
1H-NMR (400 MHz, DMSO):
13C-NMR (101 MHz, DMSO):
δ(ppm) 10.50 (s, 1H), 10.25 (s, 1H), 6.00 (s, 1H), 2.53 (s, 3H), 1.82 (s, 3H).
δ
(ppm) 205.0 (s), 164.4 (d, J = 2.9 Hz), 163.1 (s), 104.0 (s), 102.8 (s), 94.3 (s),
28.6 (s), 7.8 (s). EI-MS: 183.90 [M + H]+. Anal. calcd for C9H10O4 (182.06): C, 59.34 (59.23); H, 5.53 (5.59);
O, 35.13 (35.18).
1-(2,4,6-Trihydroxy-3-methylphenyl)propan-1-one (
10.20 (s, 1H), 6.00 (s, 1H), 2.99 (q, J = 7.2 Hz, 2H), 1.82 (s, 3H), 1.04 (t, J = 7.2 Hz, 3H). 13C-NMR
6 δ(ppm) 10.48 (s, 1H),
). 1H-NMR (400 MHz, DMSO):
(101 MHz, DMSO): δ(ppm) 206.1 (s), 163.7 (s), 162.7 (s), 160.2 (s), 103.8 (s), 101.8 (s), 94.4 (s), 36.8 (s),
9.3 (s), 7.7 (s). EI-MS: 197.93 [M + H]+. Anal. calcd for C10H12O4 (196.07): C, 61.22 (61.31); H, 6.17 (5.20);
O, 32.62 (33.58).
◦
1-(2,4,6-Trihydroxy-3-methylphenyl)butan-1-one (
1H-NMR (400 MHz, DMSO):
2.97 (t, J = 7.3 Hz, 2H), 1.84 (s, 3H), 1.60 (dd, J = 14.7, 7.3 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13C-NMR
7
). Yield: 68%. Yellow power. m.p.: 162.0–164.2 C.
δ(ppm) 14.01 (s, 1H), 10.52 (s, 1H), 10.26 (s, 1H), 6.01 (s, 1H),
(101 MHz, DMSO): δ(ppm) 205.1 (s), 163.4 (s), 162.3 (s), 159.7 (s), 103.5 (s), 101.4 (s), 94.0 (s), 45.1 (s),
17.8 (s), 13.9 (s), 7.3 (s). EI-MS: 211.53 [M + H]+. Anal. calcd for C11H14O4 (210.09): C, 62.85 (62.79);
H, 6.71 (6.83); O, 30.44 (30.38).
1
(E)-2-(4-Aminobut-2-en-1-yl)-4- methylbenzene-1,3,5-triol (
8
). Yield: 95%. H-NMR (400 MHz, CDCl3):
δ
(ppm) 7.01 (s, 1H), 6.48 (s, 1H), 6.33–6.28 (m, 1H), 4.12 (dd, J = 14.3, 7.1 Hz, 1H), 2.24 (d, J = 34.0 Hz,
2H), 2.09 (d, J = 38.0 Hz, 2H), 1.57 (s, 3H). 13C-NMR (101 MHz, DMSO):
153.2 (s), 129.6 (s), 123.0 (s), 107.2 (s), 101.7 (s), 94.5 (s), 46.6 (s), 25.4 (s), 8.4 (s). EI-MS: 209.56 [M
Anal. calcd for C11H15NO3 (209.11): C, 63.14 (63.12); H, 7.23 (7.24); N, 6.69 (6.71); O, 22.94 (22.93).
δ
(ppm) 156.5 (s), 155.6 (s),
−
−
H] .

Molecules 2018, 23, 3116
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1
(E)-3-(4-Aminobut-2-en-1-yl)-2,4,6-trihydroxy-5-methylbenzaldehyde (9). Yield: 53%. H-NMR (400 MHz,
DMSO):
9.9, 1.6 Hz, 1H), 4.78 (s, 1H), 3.39–3.24 (m, 4H), 2.16 (s, 3H), 1.46 (s, 2H). 13C-NMR (101 MHz, DMSO):
(ppm) 193.2 (s), 161.9 (s),156.8 (s), 155.9 (s), 129.6 (s), 123.0 (s), 106.4 (s), 101.2 (s), 94.5 (s), 46.6 (s),
24.8 (s), 8.4 (s). EI-MS: 236.21 [M
H]−. Anal. calcd for C12H15NO4 (237.10): C, 60.75 (60.73);
H, 6.37 (6.38); N, 5.90 (5.89); O, 26.97 (27.00).
δ(ppm) 9.15 (s, 1H), 8.53 (s, 1H), 8.12 (s, 1H), 6.22 (dt, J = 24.2, 10.0, 1.5 Hz, 1H), 5.71 (dt, J = 24.1,
δ
−
1
(E)-1-(3-(4-Aminobut-2-en-1-yl)-2,4,6-trihydroxy-5-methylphenyl)ethan-1-one (10). Yield: 58%. H-NMR
(400 MHz, DMSO): (ppm) 9.36 (s, 1H), 7.02 (s, 1H), 6.55 (s, 1H),5.65 (dt, J = 10Hz, 1H), 5.42 (dt, J = 9 Hz
1H), 3.35 (d, J = 3Hz, 2H), 3.23 (d, J = 3 Hz, 2H), 2.32 (d, J = 4Hz, 6H), 1.26 (s, 2H). 13C-NMR (101 MHz,
δ
,
DMSO): δ(ppm) 205.7 (s), 162.1 (s), 161.6 (s), 161.2 (s), 129.6 (s), 123.0 (s), 107.0 (s), 106.6 (s), 104.5 (s),
46.6 (s), 28.6 (s), 24.8 (s), 8.1 (s). EI-MS: 251.73 [M + H]+. Anal. calcd for C13H17NO4 (251.12):
C, 62.14 (61.13); H, 6.82 (6.83); N, 5.57 (5.56); O, 25.47 (27.48).
(E)-1-(3-(4-Aminobut-2-en-1-yl)-2,4,6-trihydroxy-5-methylphenyl)propan-1-one (11). Yield: 76%. Yellow
power. m.p.: 155.6–157.4 ◦C. 1H-NMR (400 MHz, DMSO):
δ(ppm) 8.65 (s, H), 8.53 (s, 1H),
5.83 (dt, J = 20.9, 12.3 Hz,1H), 5.57 (dt, J = 20.9, 9.1 Hz,1H), 4.25 (s, 1H), 3.64 (d, 2H), 3.62 (d, 2H),
2.64 (q, J = 8.4 Hz, 2H), 2.50 (s, 3H), 1.31 (t, J =8.5 Hz, 3H), 1.79 (s, 3H). 13C-NMR (101 MHz, DMSO):
δ
(ppm) 208.4 (s), 162.8 (s), 161.4 (d, J = 11.0 Hz), 129.6 (s), 123.0 (s), 106.6 (s), 106.1 (s), 102.8 (s),
46.6 (s), 35.5 (s), 24.8 (s), 8.4 (s), 8.1 (s). EI-MS: 265.55 [M + H]+. Anal. calcd for C14H19NO4 (265.13):
C, 63.38 (63.39); H, 7.22 (7.23); N, 5.28 (5.27); O, 24.12 (24.11).
(E)-1-(3-(4-Aminobut-2-en-1-yl)-2,4,6-trihydroxy-5-methylphenyl)butan-1-one (12). Yield: 82%. White power.
m.p.: 98.5–102.3 ◦C. 1H-NMR (400 MHz, DMSO):
δ(ppm) 9.86 (s, 1H), 6.14 (dt, J = 24.3, 12.4 Hz, 1H),
5.71 (dt, J = 21.4, 11.4 Hz, 1H), 4.84 (s, 1H), 4.04 (s, 1H), 3.31 (m, J = 12.6, 4.1 Hz, 4H), 2.96 (t, J = 9.2 Hz,
2H), 2.16 (s, 3H), 1.60 (m, 2H), 1.43 (d, J = 65.2 Hz, 2H), 0.94 (t, J = 13.1 Hz, 3H). 13C-NMR (101 MHz,
DMSO): δ(ppm) 205.5 (s), 163.8 (s), 162.7 (s), 160.1 (s), 129.6 (s), 123.0 (s), 104.0 (s), 101.8 (s), 94.4 (s),
45.5 (s), 41.8 (s), 24.8 (s), 18.3 (s), 14.3 (s), 7.7 (s). EI-MS: 279.57 [M + H]+. Anal. calcd for C15H21NO4
(279.15): C, 64.50 (64.49); H, 7.58 (7.59); N, 5.01 (4.99); O, 22.91 (22.93).
(E)-1-(2,4,6-Trihydroxy-3-methyl-5-(4-(methylamino)but-2-en-1-yl)phenyl)butan-1-one (13). Yield: 49%.
1H-NMR (400 MHz, DMSO):
δ
(ppm) 9.55 (s, 1H), 6.02 (dt, J = 11.2, 9.8 Hz, 1H), 5.71 (dt, J = 11.2,
9.84 Hz, 1H), 5.41 (s, 1H), 4.08 (s, 1H), 3.97 (s, 3H), 3.41–3.31 (m, 4H), 2.96 (t, J = 8.5 Hz, 2H), 2.16 (s, 3H),
1.67–1.47 (m, 2H), 1.34 (s, 1H), 0.94 (t, J = 10.48 Hz, 3H). 13C-NMR (101 MHz, DMSO):
(ppm) 207.8 (s),
δ
163.1 (s), 161.6 (d, J = 1.6 Hz), 127.3 (s), 124.6 (s), 106.7 (s), 106.5 (s), 103.4 (s), 55.4 (s), 41.8 (s),
36.2 (s), 24.8 (s), 19.6 (s), 13.5 (s), 8.1 (s). EI-MS: 293.81 [M + H]+. Anal. calcd for C16H23NO4 (293.16):
C, 65.51 (65.50); H, 7.90 (7.91); N, 4.77 (4.76); O, 21.81 (21.83).
(E)-1-(3-[4-(Dimethylamino)but-2-en-1-yl]-2,4,6-trihydroxy-5-methylphenyl)butan-1-one (14). Yield: 51%.
1H-NMR (400 MHz, DMSO):
δ
(ppm) 7.75 (s, H), 6.04 (dt, J = 21.2, 11.3 Hz, 1H), 5.67 (dt, J = 18.2, 12.0,
9.04 Hz, 1H), 5.55 (s, 1H), 4.80 (s, 1H), 3.33 (dd, J = 9.8, 1.4 Hz, 2H), 3.08–2.90 (m, 4H), 2.75 (s, 6H),
2.16 (s, 3H), 1.68–1.45 (m, 2H), 0.94 (t, J = 10.5 Hz, 3H). 13C-NMR (101 MHz, DMSO):
(ppm) 208.4 (s),
δ
161.9 (d, J = 1.6 Hz), 159.3 (s), 157.7 (s), 131.7 (s), 126.8 (s), 111.8 (s), 106.5 (s), 106.5 (s), 49.1 (s), 46.3 (s),
45.4 (s), 25.4 (s), 17.1 (s), 13.8 (s), 7.4 (s). EI-MS: 307.57 [M + H]+. Anal. calcd for C17H25NO4 (307.18):
C, 66.43 (66.42); H, 8.20 (8.21); N, 4.56 (4.58); O, 20.82 (20.79).
(E)-1-(2,4,6-Trihydroxy-3-methyl-5-(4-((naphthalen-2-ylmethyl)amino)but-2-en-1-yl)phenyl)butan-1-one (15).
Yield: 32%. 1H-NMR (400 MHz, DMSO):
δ(ppm) 9.46 (s, 1H), 8.05–7.91 (m, 2H), 7.69 (dt, J = 11.8,
2.3 Hz, 1H), 7.56 (dt, J = 12.0, 2.4 Hz, 1H), 7.29 (dd, J = 18, 14.48, 7.28 Hz, 2H), 6.98 (dd, J = 12.0, 2.5 Hz,
1H), 6.22 (dt, J = 22.8, 9.9, 1.4 Hz, 1H), 5.78 (dt, J = 24.2, 10.0, 1.6 Hz, 1H), 5.01 (s, 1H), 4.20 (s, 2H),
3.66 (s, 1H), 3.49 (dd, J = 9.8, 1.6 Hz, 1H), 3.40 (dd, J = 9.84, 1.6 Hz, 1H), 3.33 (dd, J = 9.84, 1.4 Hz, 2H),
2.96 (t, J = 12.8 Hz, 2H), 2.16 (s, 4H), 1.69–1.45 (m, 2H), 0.94 (t, J = 10.6 Hz, 3H). 13C-NMR (101 MHz,
DMSO):
δ(ppm) 161.8 (s), 159.1 (s), 157.7 (s), 133.9 (s), 133.7 (s), 131.1 (s), 129.9 (s), 129.5 (s), 129.1 (s),
128.4 (s), 127.1 (d, J = 7.6 Hz), 125.9 (s), 124.3 (s), 111.0 (s), 106.8 (s), 106.1 (d, J = 34.1 Hz), 51.2 (s),

Molecules 2018, 23, 3116
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49.1 (s), 44.9 (s), 25.4 (s), 17.5 (s), 13.7 (s), 7.8 (s). EI-MS: 419.53 [M
−
H]−. Anal. calcd for C26H29NO4
(419.21): C, 74.44 (74.42); H, 6.97 (6.98); N, 3.34 (3.35); O, 15.25 (15.25).
(E)-1-(3-(4-(bis(Naphthalen-2-ylmethyl)amino)but-2-en-1-yl)-2,4,6-trihydroxy-5-methylphenyl)butan-1-one
16). Yield: 40%. 1H-NMR (400 MHz, DMSO):
(ppm) 8.25–7.81 (m, 4H), 7.68 (dt, J = 12.0,
(
δ
2.5 Hz, 2H), 7.55 (dt, J = 11.9, 2.4 Hz, 2H), 7.28 (dt, J = 17.9, 14.3, 7.2 Hz, 4H), 7.18 (s, 1H),
6.97 (dd, J = 12.0, 2.3 Hz, 2H), 6.92 (s, 1H), 6.25 (dt, J = 24.2, 9.9, 1.6 Hz, 1H), 5.72 (dt, J = 22.9, 9.8,
1.4 Hz, 1H), 4.68 (s, 1H), 4.09 (s, 4H), 3.32 (dd, J = 9.9, 1.5 Hz, 2H), 3.11–2.88 (m, 4H), 2.15 (s, 3H),
1.71–1.46 (m, 2H), 0.94 (t, J = 10.5 Hz, 3H). 13C-NMR (101 MHz, DMSO):
δ(ppm) 208.8 (s), 161.6 (s),
159.2 (s), 157.5 (s), 138.0 (s), 137.2 (s), 133.4 (s), 132.1 (s), 131.9 (s), 130.1 (d, J = 26.4 Hz), 130.2–130.7
(m), 128.1 (d, J = 37.0 Hz), 128.2 (d, J = 13.1 Hz), 127.3 (d, J = 8.6 Hz), 126.4 (d, J = 29.7 Hz),
126.2 (d, J = 8.3 Hz), 126.1 (s), 125.7 (s), 124.4 (d, J = 22.4 Hz), 124.2 (s), 111.5 (s),106.5 (s), 106.3 (s),
60.2 (s), 53.4 (s), 49.5 (s), 46.5 (s), 25.7 (s), 17.6 (s), 13.8 (s), 7.5 (s).EI-MS: 559.53 [M
for C37H37NO4 (559.27): C, 79.40 (79.39); H, 6.66 (6.68); N, 2.50 (2.49); O, 11.43 (11.44).
−
H]−. Anal. calcd
(E)-1-(2,4,6-Trihydroxy-3-methyl-5-(4-(methyl(naphthalen-2-ylmethyl)aminbut-2-en-1-yl)phenyl)butan-1-one
1
(
17). Yield: 75%. H-NMR (400 MHz, DMSO):
δ
(ppm) 8.47 (s, 1H), 8.05–7.89 (m, 2H), 7.69 (dt, J = 11.8
,
2.3 Hz, 1H), 7.56 (dt, J = 11.9, 2.4 Hz, 1H), 7.28 (dd, J = 17.9, 14.3, 7.2 Hz, 2H), 6.98 (dd, J = 11.2, 2.4 Hz,
1H), 5.92 (dt, J = 21.8, 9.4, 1.3 Hz, 1H), 5.71 (dt, J = 24.2, 9.8, 1.3 Hz, 1H), 4.82 (s, 1H), 4.09 (s, 2H),
3.51 (s, 1H), 3.32 (dd, J = 9.7, 1.4Hz, 2H), 3.13–2.83 (m, 4H), 2.26 (s, 3H), 2.16 (s, 3H), 1.70–1.44 (m, 2H),
0.94 (t, J = 10.6 Hz, 3H). 13C-NMR (101 MHz, DMSO):
δ
(ppm) 207.0 (s), 162.5 (s), 162.0 (s), 136.5 (s),
136.3 (s), 133.4 (s), 132.5 (s), 131.7–131.0 (m), 127.6–129.2 (m), 128.5 (s), 126.9 (s), 106.7 (s), 106.1 (s),
103.4 (s), 60.3 (s), 58.7 (s), 47.8 (s), 47.5 (s), 24.9 (s), 19.7 (s), 13.6 (s), 8.1 (s). EI-MS: 433.30 [M
H]−.
−
Anal. calcd for C27H31NO4 (433.23): C, 74.80 (74.79); H, 7.21 (7.19); N, 3.23 (3.22); O, 14.76 (14.80).
3.2. Evaluation of Antifungal Activity
The antifungal activity of the compounds was evaluated against two standard human pathogenic
fungal strains, Trichophyton rubrum (CMCC (F) T1d) and Trichophyton mentagrophytes (CMCC (F) T5c),
purchased from the Chinese Academy of Medical Sciences (Hospital for Pathology and Dermatolog,
Science and Peking Union Medical College). A solution of Naftifine (1.0 mg/mL) was used as a positive
control, while pseudo mallow phenol (1.0 mg/mL) was used as a comparison. The minimum inhibitory
concentration (MIC) of the compound was determined by a micro-broth dilution method according
to the method defined by the National Clinical Laboratory Standards Committee (NCCLS National
Clinical Laboratory Standards Committee 2002) [20]. For the assay, the MIC was determined in
accordance with Clinical and Laboratory Standards Institute (CLSI) method M38-A2, in which the test
compound monomer stock solution (32 mg/mL) and the positive drug naftifine were diluted 100-fold
with RPMI-1640 liquid medium, and the concentration of the solvent DMSO after dilution (v/v)
less than 1% to reduce the interference of the solvent on the experimental results. Then, the diluted
compound monomer stock solution was subjected to lateral dilution in RPMI-1640 liquid medium.
A blank control and growth control were placed in sterile 96-well plates. Finally, the 96 well plate
mixed with the bacterial liquid and the drug solution was incubated at 35 ◦C for 4 days at a constant
temperature, and the results were visually observed. The results were read by two people. The lowest
concentration of the drug-free wells at the lowest drug concentration was the minimum inhibitory
concentration (MIC). Each strain was tested three times.
3.3. Docking Studies
Docking studies of all derivatives were implemented in the program AutoDock 4.2.6 [21].
The X-ray crystallographic structure of SE (PDB ID: 2AIB) was taken from PDB (www.rcsb.org/pdb)
and was invoked as a protein structure [22,23]. Beginning of docking, all the water and ligands
were removed and the random hydrogen atoms were added. All the compounds were constructed
using ChemDraw Ultra 14.0. Most parameters were default values except the calculation for the

Molecules 2018, 23, 3116
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Lamarckian genetic algorithm (LGA). The grid box for docking size was set to 55
×
55
×
55 points in x,
y and z directions. The docking experiment was implemented by AutoDock program. There were
100 multiples to independent docking runs, then each experiment was repeated 20 times, to obtain
the best performance of the docking programs. Molecular dynamics of the complexes resulting from
docking of compounds 13–17 were performed using Discovery Studio 3.5 Module Standard Dynamic
Cascade (Sun Yat-sen University, Guangzhou, China). The CHARMm force field and the Moony–Rone
charge were added to the composite, and then the H2O molecules and the NaCl molecules were added
to make the NaCl concentration of the complex system 0.145, which was reached the osmotic pressure
of the human body. After an initial default relaxation protocol, an MD production run was made with
a time step of 2.0 ps [24].
4. Conclusions
In conclusion, our strategy to introduce antifungal pharmacophore allylamine into the methyl
phloroglucinol and remain acyl group in the C-6 position using pseudoaspidinol as the lead compound
to obtain novel phloroglucinol derivatives 4–17 was successful in leading to compounds, which
showed inhibition of T. mentagrophytes and T. rubrum. Furthermore, the first basic efficacy analysis of
Naftifine groups in the study. These compounds could be useful as lead compounds in developing
novel antifungal agents.
Supplementary Materials: The following are available online, 1H-NMR, 13C-NMR and MS of compounds 2–17.
Author Contributions: L.Y. contributed to the conception and design of the experiments, analysis of the data and
revised the paper, X.T. synthesized title compounds, and selected the data. Z.S. analyzed the data. Y.W. and P.S.
performed the biological studies. J.G. performed the docking studies.
Funding: The work was supported by Special Innovation Project of Guangdong Education Department (Natural
Science) (2017KTSCX107), Guangdong Natural Science Foundation (2015A03031356), Science and Technology
Planning Project of Guangdong Province (2017ZC0199), Key Laboratory of New Drug Discovery and Evaluation
of Ordinary Universities of Guangdong Province (2017KSYS002), Guangzhou Key Laboratory of Construction
andAapplication of New Drug Screening Model Systems (201805010006), Guangdong Province Precise Medicine
and Big Data Engineering Technology Research Center for Traditional Chinese Medicine, Guangdong Province
Engineering Technology Center for Molecular Probes & Biomedical Imaging.
Acknowledgments: The authors are grateful to Tan and Sun Yet-Sen University for molecular docking experiment.
Conflicts of Interest: The authors declare no competing interests.
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Sample Availability: Samples of the compounds 2–17 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).