Antifungal activity of 1,4-dialkoxynaphthalen-2-acyl imidazolium salts by inducing apoptosis of pathogenic candida spp.

pharmaceutics

Article

Antifungal Activity of 1,4-Dialkoxynaphthalen-2-Acyl

Imidazolium Salts by Inducing Apoptosis of Pathogenic

Candida spp.

Jisue Lee ¹^,^†, Jae-Goo Kim ²^,^†, Haena Lee , Tae Hoon Lee , Ki-Young Kim * and Hakwon Kim ¹^,*

1

2,

1

Department of Applied Chemistry, Global Center for Pharmaceutical Ingredient Materials,

Kyung Hee University, Seocheon, Giheung, Yongin, Gyeonggi-do 1732, Korea; cwltnc7552@naver.com (J.L.);

dlgosk97@naver.com (H.L.); thlee@khu.ac.kr (T.H.L.)

Graduate School of Biotechnology, Kyung Hee University, Seocheon, Giheung, Yongin,

2

Gyeonggi-do 1732, Korea; zxcv913@naver.com

*

Correspondence: kiyoung@khu.ac.kr (K.-Y.K.); hwkim@khu.ac.kr (H.K.); Tel.: +82-312012633 (K.-Y.K.);

82-312012459 (H.K.)

These authors contributed equally to this work.

+

†

Abstract: Even though Candida spp. are staying commonly on human skin, it is also an opportunistic

pathogenic fungus that can cause candidiasis. The emergence of resistant Candida strains and the

toxicity of antifungal agents have encouraged the development of new classes of potent antifungal

agents. Novel naphthalen-2-acyl imidazolium salts (NAIMSs), especially 1,4-dialkoxy-NAIMS from

1

,4-dihydroxynaphthalene, were prepared and evaluated for antifungal activity. Those derivatives

showed prominent anti-Candida activity with a minimum inhibitory concentration (MIC) of 3.125

to 6.26 g/mL in 24 h based on microdilution antifungal susceptibility test. Among the tested

compounds, NAIMS 7c showed strongest antifungal activity with 3.125 g/mL MIC value compared

with miconazole which showed 12.5 g/mL MIC value against Candida spp., and more importantly

>100 g/mL MIC value against C. auris. The production of reactive oxygen species (ROS) was

µ

Citation: Lee, J.; Kim, J.-G.; Lee, H.;

Lee, T.H.; Kim, K.-Y.; Kim, H.

Antifungal Activity of

µ

1

,4-Dialkoxynaphthalen-2-Acyl

µ

Imidazolium Salts by Inducing

Apoptosis of Pathogenic Candida spp..

Pharmaceutics 2021 , 13 , 312.

https://doi.org/10.3390/

increased and JC-1 staining showed the loss of mitochondrial membrane potential in C. albicans by

treatment with NAIMS 7c. The increased release of ultraviolet (UV) absorbing materials suggested

that NAIMS 7c could cause cell busting. The expression of apoptosis-related genes was induced in C.

albicans by NAIMS 7c treatment. Taken together, the synthetic NAIMSs are of high interest as novel

antifungal agents given further in vivo examination.

pharmaceutics13030312

Academic Editor: Hyo-Kyung Han

Keywords: 1,4-Dialkoxynaphthalen-2-acyl imidazolium salts; Candida sp.; antifungal agent; apoptosis

Received: 2 February 2021

Accepted: 24 February 2021

Published: 27 February 2021

1

. Introduction

Infections by invasive fungal pathogens result from immunosuppression, long-term

broad-spectrum antimicrobials, endocrinopathies, organ transplantation and use of in-

dwelling catheters [ ]. Candida spp. is a critical invasive fungal pathogen causing disease

in humans, normally responsible for 90% of mucosal infections and 60% of candidiasis

episodes [ ]. Although various compounds are currently used to control Candida infectious

diseases, including well-known azoles such as ﬂuconazole, miconazole and others, the

mortality of patients with Candida infection is above 15% [ ]. Antibiotic-resistant Candida

spp. have also arisen. The drugs currently used against fungal pathogens have limitations

because of their toxicity [ ]. For instance, Acetaminophen (APAP), amphotericin B deoxy-

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cholate (DAMB) and the triazoles may cause hepatic toxicity [8,9]. Therefore, there is an

urgent need for a new drug to treat Candida infection.

Imidazolium salts (IMS) have been reported to exhibit fungicidal activity [10]. Due

to its ionicity, IMS provides properties that are unusual and highly interesting for phar-

maceutical formulation including potential efﬁcacy against some bacteria and fungi [11].

4

.0/).

Pharmaceutics 2021, 13, 312. https://doi.org/10.3390/pharmaceutics13030312

https://www.mdpi.com/journal/pharmaceutics

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In addition, tuning the toxicity of IMSs as antitumor agents has attracted much atten-

tion [12]. Considering these and our previous results of antifungal compound study, we

decided to explore in further detail the potential activity of a new hybrid compound formed

by attaching an imidazole moiety to 1,4-dialkoxynaphthalen-2-acyl compound to enhance

antifungal activity.

Alagebrium, known as an advanced glycation end-products (AGEs) breaker that

reverse one of the main mechanisms of ageing, has a structure of phenacyl thiazolium

salt [13]. The phenacyl moiety is known to play an important role in biological activ-

ity [14,15]. Therefore, we were interested in the napththalenacyl moiety, similar to the

phenacyl moiety, as a pharmacophore of antifungal agents. The 1,4-dialkoxy naphthalenacyl

compound, derived from 1,4-naphthoquinone, became of particular interest (Figure 1).

Figure 1. Schematic design for synthesis of acylnaphthalene-imidazolium hybrid using pharmacophore-hybridization approach.

Induced endogenous fungal apoptotic responses could provide a basis for antifungal

therapies. Environmental stress (acetic acid and hydrogen peroxide) and an antifungal

agent (amphotericin B, hibicuslide C and coumarin) have been known to induce apop-

tosis in C. albicans [16–18]. Apoptosis is a kind of programmed cell death. Multicellular

organisms and even single-celled organisms, such as yeast, can exhibit many features of

apoptosis, including DNA fragmentation, reactive oxygen species (ROS) production and

the loss of mitochondrial membrane potential [19–21].

With the above considerations, a series of novel IMSs linked to a 2-acetyl-1,4-dialkoxyn

aphthalene moiety were efﬁciently synthesized through pharmacophore-hybridization

strategy (Figure 1) to ﬁnd promising drugs for dealing with Candida spp. infection. Through

microdilution antifungal susceptibility, NAIMS 7c showed the highest antifungal activity

among them by inducing Candida apoptosis and cell bursting.

2. Materials and Methods

2.1. General Remarks

The reactions were monitored by thin-layer chromatography (TLC) on Merck Silica

gel 60F254. Column chromatography was performed on Merck silica gel 200–300 mesh.

Melting points were determined on the melting point apparatus electrothermal A9100X1

1

13

and were uncorrected. We recorded H NMR (300 MHz) and C NMR (75 MHz) spectra on

a JEOL FT-NMR spectrometer (Tokyo, Japan), respectively. Spectra are referenced relative to

the chemical shift of tetramethylsilane (TMS). High-resolution mass spectra were obtained

with a JEOL JMS-700 mass spectrometer. All solvents and reagents were commercially

available from Acros Organics (Brookline, MA, USA), Aldrich (St. Louis, MO, USA) and

TCI (Tokyo, Japan) and were used as received. The chemicals 1-Methylimidazole (6a) from

Sigma-Aldrich (St. Louis, MO, USA) and 1-benzylimidazole (6b) from Aldrich are com-

mercially available. We readily obtained 1,4-Diacetoxynaphthalene (1a), 1,4-diacetoxy-5-

methoxynaphthalene (1b), 1,4-diisoamyloxynaphthalene (8), 4-acetoxy-2-acetyl-1-isoamylox

0

ynaphthalene (9 ) and 2-acetyl-1,4-diisoamyloxynaphthalene (9) from 1,4-naphthoquinone

Pharmaceutics 2021, 13, 312

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or 5-hydroxy-1,4-naphthoquinone [22]. We prepared 2-Bromoacetylnaphthalene and 2-

bromoacetyl-l-methoxynaphthalene from 2-acetylnaphthalene and 2-acetyl-1-methoxynapt

halene, respectively, according to the α-bromination procedure.

2.2. Synthetic Procedures and Analytical Data

2.2.1. Synthesis of 3-acetyl-4-hydroxynaphthalen-1-yl acetate (2a)

1,4-Diacetoxynaphthalene 1a (5 g, 20.4 mmol) was dissolved in boron triﬂuoride acetic

acid complex (20 mL, 144 mmol) and heated under reﬂux for 1 h. Then, it was cooled

to room temperature, quenched with water, extracted by ethyl acetate and washed with

water and brine. The combined organic layer was dried over anhydrous magnesium

sulfate, ﬁltered and concentrated in vacuo. The residue was puriﬁed by ﬂash column

1

chromatography to give the compound 2a as a pale-yellow solid. (4.9 g, 99%): H NMR

(

7

(

1

300 MHz, CDCl₃)

.65–7.60 (m, 1H), 7.55–7.50 (m, 1H), 7.35 (s, 1H), 2.59 (s, 3H), 2.42 (s, 3H); C NMR

75 MHz, CDCl₃) ppm 203.6, 169.6, 160.3, 137.6, 130.8, 130.4, 126.4, 125.8, 124.7, 120.9,

16.3, 111.8, 26.7, 20.7.

δ ppm 13.92 (s, 1H), 8.44 (d, 1H, J = 8.2 Hz), 7.71 (d, 1H, J = 8.0 Hz),

1

3

δ

2.2.2. Synthesis of 3-acetyl-4-hydroxy-5-methoxynaphthalen-1-yl acetate (2b)

Following the procedure described above for the preparation of 2a, compound 2b was

obtained from 5-methoxy-1,4-diacetoxynaphthalene (1b; 0.11 g, 0.4 mmol) as a pale-yellow

1

solid. (0.1 g, 92%): H NMR (300 MHz, CDCl )

δ ppm 14.33 (br s, 1H), 7.58 (dd, 1H,

3

J = 8.0 Hz, 8.2 Hz), 7.46 (s, 1H), 7.34 (d, 1H, J = 8.2 Hz), 6.95 (d, 1H, J = 8.0 Hz), 4.06 (s, 3H),

.68 (s, 3H), 2.45 (s, 3H).; ¹³C NMR (75 MHz, CDCl₃)

ppm 202.5, 169.7, 162.1, 159.7, 137.2,

33.3, 131.0, 117.7, 116.7, 113.4, 112.9, 107.0, 56.2, 27.7, 20.8.

2

δ

1

2

3

.2.3. Synthesis of 3-acetyl-4-alkyloxynaphthalen-1-yl acetate (3a–3d)

-Aetyl-4-methoxynaphthalen-1-yl acetate (3a)

Compound 2a (0.49 g, 2 mmol) was dissolved in dimethylformamide (DMF, 4 mL).

Then, iodomethane (0.19 mL, 3 mmol) and cesium carbonate (0.98 g, 3 mmol) were added

to the solution. The mixture was heated under reﬂux for 1.5 h and was cooled to room

temperature. It was quenched with water, extracted by ethyl acetate, and washed with

water and brine. The combined organic layer was dried over anhydrous magnesium

sulfate, ﬁltered and concentrated in vacuo. The residue was puriﬁed by ﬂash column

1

chromatography to afford the compound 3a as a yellow solid. (0.41 g, 80%): H NMR

(

1

300 MHz, CDCl₃)

s, 1H), 4.01 (s, 3H), 2.78 (s, 3H), 2.46 (s, 3H). C NMR (75 MHz, CDCl₃)

55.3, 142.5, 129.8, 128.7, 128.5, 126.8, 123.5, 121.4, 117.2, 63.6, 30.3, 20.4.

δ ppm 8.25–8.21 (m, 1H), 7.86–7.81 (m, 1H), 7.67–7.58 (m, 2H), 7.54

13

δ

ppm 198.0, 169.1,

3-Aetyl-4-(isoamyloxy)naphthalen-1-yl acetate (3b)

Following the procedure described above for the preparation of 3a, compound 3b was

obtained from 2a (0.49 g, 2 mmol) and 1-bromo-3-methyl butane (0.36 mL, 3 mmol) as a

1

yellow oil. (0.44 g, 71%): H NMR (300 MHz, CDCl )

δ

ppm 8.23–8.18 (m, 1H), 7.84–7.79

3

(

1

m, 1H), 7.66–7.56 (m, 2H), 7.49 (s, 1H), 4.07-4.01 (m, 2H), 2.76 (s, 3H), 2.45 (s, 3H), 1.89–1.82

m, 3H), 1.05 (d, 6H, J = 9.0 Hz). ¹³C NMR (75 MHz, CDCl₃)

ppm 199.2, 169.4, 154.6, 142.5,

29.9, 129.4, 128.7, 127.7, 127.0, 123.7, 121.6, 117.5, 76.2, 39.0, 30.5, 25.0, 22.6, 20.8.

δ

3-Acetyl-4-isopropoxynaphthalen-1-yl acetate (3c)

Following the procedure described above for the preparation of 3a, compound 3c was

obtained from 2a (0.49 g, 2 mmol) and 2-bromopropane (0.28 mL, 3 mmol) as a yellow

1

oil. (0.36 g, 63%): H NMR (300 MHz, CDCl ) δ ppm 8.23–8.18 (m, 1H), 7.83–7.80 (m, 1H),

3

7

.74–7.54 (m, 2H), 7.41 (s, 1H), 4.39–4.27 (m, 1H), 2.75 (s, 3H), 2.45 (s, 3H), 1.34 (d, 6H,

13

J = 6.0 Hz). C NMR (75 MHz, CDCl₃)

δ ppm 200.8, 169.3, 152.2, 142.3, 130.0, 129.5, 129.1,

128.4, 126.6, 124.1, 121.4, 117.2, 79.5, 60.2, 30.4, 22.2, 20.7.

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3-Acetyl-4-(isoamyloxy)-5-methoxynaphthalen-1-yl acetate (3d)

Following the procedure described above for the preparation of 3a, compound 3d was

1

obtained from 2b (0.1 g, 0.36 mmol) as a yellow solid. (0.98 g, 82%): H NMR (300 MHz,

CDCl₃)

δ

ppm 7.51 (dd, 1H, J = 8.2 Hz, 7.6 Hz), 7.45 (s, 1H), 7.41 (d, 1H, J = 8.4 Hz), 6.95

d, 1H, J = 7.6 Hz), 4.03 (s, 3H), 3.90 (t, 2H, J = 7.2 Hz), 2.76 (s, 3H), 2.44 (s, 3H), 1.87–1.75

m, 3H), 0.97 (d, 6H, J = 6.2 Hz). ¹³C NMR (75 MHz, CDCl₃)

ppm 200.5, 169.3, 157.2, 154.9,

42.4, 132.2, 129.7, 128.9, 120.8, 118.3, 114.0, 106.9, 56.1, 56.0, 38.9, 31.3, 29.6, 25.1, 22.7, 20.9.

(

1

δ

2

.2.4. General Synthesis of 2-acetyl-1,4-dialkoxynaphthalene (4)

-Acetyl-1,4-dimethoxynaphthalene (4a; CAS Registry Number 65131-13-7)

◦

3a (0.29 g, 1.12 mmol) was dissolved in methanol (6 mL) and was cooled to 0 C.

Then 1 wt % potassium hydroxide solution in methanol (5.4 mL) was added. After stir-

ring for 2 h, the mixture was neutralized by adding Amberlite IR-120(H) and stirred for

additional 15 m. Amberlite IR-120(H) was removed by ﬁltration and the ﬁltrate was con-

centrated in vacuo. The resulting residue was dissolved in DMF (2 mL), and iodomethane

(0.11 mL, 1.68 mmol) and cesium carbonate (0.5 g, 1.68 mmol) were added. The mixture

was heated under reﬂux. After monitoring the reaction complete by TLC, it was cooled to

room temperature, extracted with dichloromethane (DCM), washed with water and dried

over anhydrous magnesium sulfate. It was concentrated in vacuo and puriﬁed by ﬂash

column chromatography to give the product 4a as a yellow oil. (0.26 g, quantitative yield)-

1

H NMR (300 MHz, CDCl₃)

δ ppm 8.28–8.23 (m, 1H), 8.19–8.15 (m, 1H), 7.63–7.57 (m, 2H),

7

.08 (s, 1H), 4.01 (s, 3H), 3.96 (s, 3H), 2.81 (s, 3H). ¹³C NMR (75 MHz, CDCl3) δ ppm.

2-Acetyl-1,4-diisoamyloxynaphthalene (4b)

Following the procedure described above for the preparation of 4a, compound 4b

was obtained from 3b (1 g, 3.3 mmol) and 1-bromo-3-methylbutane (0.6 mL, 5 mmol) as a

1

yellow oil. (0.47 g, 42%): H NMR (300 MHz, CDCl )

δ

ppm 8.29–8.24 (m, 1H), 8.16–8.10

3

(

2

m, 1H), 7.60–7.55 (m, 2H), 7.01 (s, 1H), 4.17 (t, 2H, J = 6.3 Hz), 3.97 (t, 2H, J = 6.6 Hz), 2.78

s, 1H), 1.00 (d, 6H, J = 6.3 Hz), 0.98 (d, 6H, J = 5.4 Hz). ¹³C NMR (75 MHz, CDCl₃)

ppm

00.5, 151.0, 150.3, 128.9, 127.7, 127.4, 126.8, 122.9, 122.5, 102.6, 75.8, 66.6, 39.1, 37.8, 30.7,

δ

5.1, 24.9, 22.6, 22.5.

2-Acetyl-1,4-diisopropoxynaphthalene (4c)

Following the procedure described above for the preparation of 4a, compound 4c was

1

obtained from 3c (0.12 g, 0.42 mmol) as a yellow oil. (0.1 g, 83%): H NMR (300 MHz,

CDCl₃)

δ ppm 8.28–8.25 (m, 1H), 8.15–8.11 (m, 1H), 7.60–7.53 (m, 2H), 6.95 (s, 1H), 4.80–4.72

(

m, 1H), 4.31–4.25 (m, 1H), 2.76 (s, 3H), 1.45 (s, 3H), 1.43 (s, 3H), 1.31(d, 6H, J = 6.0 Hz).;

1

3

C NMR (75 MHz, CDCl₃)

δ ppm 202.5, 149.6, 147.7, 130.0, 129.5, 129.4, 127.2, 126.5, 123.5,

122.6, 104.4, 78.8, 70.5, 30.8, 22.3, 22.1.

2-Acetyl-1,4-diisoamyloxy-8-methoxynaphthalene (4d)

Following the procedure described above for the preparation of 4a, compound 4d

1

was obtained from 3d (0.09 g, 0.27 mmol) as a yellow solid. (0.09 g, 77%): H NMR

(300 MHz, CDCl₃)

δ ppm 7.90 (d, 1H, J = 8.2 Hz). 7.46 (dd, 1H, J = 8.2 Hz, 7.8 Hz), 7.00

(

2

(

1

s, 1H), 6.95 (d, 1H, J = 7.6 Hz), 4.15 (t, 2H, J = 6.4 Hz), 4.01 (s, 3H), 3.85 (t, 2H, J = 7.1 Hz),

1

3

.78 (s, 3H), 1.98–1.75 (m, 6H), 1.00 (d, 6H, J = 6.4 Hz), 0.96 (d, 6H, J = 6.2 Hz).; C NMR

ppm 202.1, 156.7, 150.7, 150.3, 131.1, 129.7, 127.6, 120.3, 114.9, 107.0,

03.7, 76.4, 66.7, 55.9, 38.9, 37.9, 31.5, 25.2, 25.1, 22.7, 22.6.

75 MHz, CDCl₃) δ

2

.2.5. General Synthesis of naphthalenacyl bromide (5a–5e)

-Bromoacetyl-1,4-dimethoxynaphthalne (5a)

4a

(0.2 g, 0.86 mmol) was dissolved in DCM (10 mL). Tetrabutylammonium tribromide

(

TBA-Br ; 0.36 g, 1.12 mmol) was added and the resulting solution was stirred under argon

3

atmosphere for 3 h. It was quenched with water, extracted with DCM and washed with

Pharmaceutics 2021, 13, 312

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water, 1M sodium bicarbonate solution and brine, successively. The combined organic

phase was dried over anhydrous sodium sulfate and concentrated in vacuo. It was puri-

ﬁed by ﬂash column chromatography to give the compound 5a as a yellow solid. (0.18

1

g, 67%) [23]: H NMR (300 MHz, CDCl )

δ ppm 8.30 (d, 1H, J = 8.3 Hz), 8.20 (d, 1H,

3

J = 7.9 Hz), 7.74 (t, 1H, J = 7.7 Hz), 7.65 (t, 1H, J = 7.3Hz), 6.82 (s, 1H), 4.82 (s, 2H), 3.99

13

(

s, 3H). C NMR (75 MHz, (CD ) SO) δ ppm 192.8, 152.0, 151.8, 129.3, 128.1, 127.2, 124.4,

3 2

123.2, 122.6, 102.1, 64.2, 55.7, 36.4, 36.4.

2-Bromoacetyl-1,4-diisoamyloxynaphthalene (5b)

Following the procedure described above for the preparation of 5a, compound 5b

1

was obtained from 4b (3.76 g, 11 mmol) as a yellow oil. (2.8 g, 62%): H NMR (300 MHz,

CDCl₃)

δ

ppm 8.31-8.27 (m, 1H), 8.12-8.09 (m, 1H), 7.63-7.57 (m, 2H), 6.99 (s, 1H), 4.79

s, 2H), 4.19-4.15 (m, 2H), 4.02-3.97 (m, 2H), 1.99–1.88 (m, 2H), 1.86–1.78 (m, 4H), 1.01

d, 6H, J = 6.6 Hz), 1.99 (d, 6H, J = 6.3 Hz). ¹³C NMR (75 MHz, CDCl₃)

ppm 193.9, 151.4,

50.4, 129.4, 128.5, 128.0, 127.2, 125.1, 123.0, 122.7, 102.7, 76.4, 66.8, 39.0, 37.8, 36.2, 25.2, 25.0,

(

1

δ

22.6, 22.6.

2-Bromoacetyl-1,4-diisopropoxynaphthalene (5c)

Following the procedure described above for the preparation of 5a, compound 5c was

1

obtained from 4c (0.17 g, 0.63 mmol) as a yellow oil. (0.13 g, 56%): H NMR (300 MHz,

CDCl₃)

δ ppm 8.30–8.27 (m, 1H), 8.10–8.08 (m, 1H), 7.60–7.55 (m, 2H), 6.92 (s, 1H), 4.79

1

3

(

1

2

s, 2H), 4.79–4.76 (m, 1H), 4.32–4.26 (m, 1H), 1.55–1.43 (m, 6H), 1.33–1.30 (m, 6H). C NMR

75 MHz, CDCl₃) ppm 195.7, 150.0, 147.7, 129.8, 129.4, 127.6, 126.9, 123.4, 122.9, 104.3,

00.5, 70.6, 36.0, 22.3, 22.0.

δ

-Bromoacetyl-1,4-diisoamyloxy-8-methoxynaphthalene (5d)

Following the procedure described above for the preparation of 5a, compound 5d

1

was obtained from 4d (0.67 g, 1.8 mmol) as a yellow solid. (0.53 g, 65%): H NMR (300

MHz, CDCl₃)

δ ppm 7.91 (d, 1H, J = 8.4 Hz), 7.50 (dd, 1H, J = 7.8 Hz, 8.4 Hz), 6.97 (d, 1H,

J = 7.8 Hz), 6.96 (s, 1H), 4.81 (s, 2H), 4.15 (t, 2H, J = 6.2 Hz), 4.02 (s, 3H), 3.86 (t, 2H,

1

3

J = 7.1 Hz), 1.98–1.72 (m, 6H), 1.00 (d, 6H, J = 6.5 Hz), 0.96 (d, 6H, J = 6.2 Hz). C NMR

CDCl , 75 MHz) ppm 195.1, 156.7, 151.1, 150.4, 131.5, 128.2, 127.0, 120.0, 115.1, 107.4,

03.9, 76.8, 66.9, 56.0, 38.9, 37.9, 37.1, 25.2, 25.1, 22.7, 22.6.

(

δ

3

1

2-Bromoacetyl-1-methoxynaphthalene (5e)

Following the procedure described above for the preparation of 5a, compound 5e was

obtained from 2-acetyl-1-methoxynaphthalene (0.5 g, 2.5 mmol) as a white solid. (0.49 g,

1

7

0%): H NMR (300 MHz, CDCl )

δ

ppm 8.21–8.18 (m, 1H), 7.88–7.85 (m, 1H), 7.74 (d, 1H,

3

1

3

J = 8.6 Hz), 7.65 (d, 1H, J = 8.4 Hz), 7.62–7.56 (m, 2H), 4.74 (s, 2H), 4.03 (s, 3H).; C NMR

75 MHz, CDCl₃) ppm 193.0, 157.7, 137.2, 128.7, 128.2, 127.5, 126.8, 125.6, 124.9, 124.6,

(

δ

123.3, 64.2, 36.3.

2

1

.2.6. General Synthesis of 1-substituted benzylimidazoles (6c and 6d)

-(4-Methoxybenzyl)-1H-imidazole (6c)

Imidazole (0.25 g, 3.68 mmol) and potassium carbonate (0.5 g, 3.68 mmol) were

dissolved in acetonitrile (15 mL). Then 4-methoxybenzyl chloride (0.5 mL, 3.68 mmol) was

added and the reaction mixture was stirred for 12 h at room temperature. After completion

of the reaction, it was concentrated under reduced pressure and puriﬁed by ﬂash column

chromatography to obtain compound 6c in the form of an ivory solid. (0.4 g, 60%) [24]:

1

H NMR (300 MHz, CDCl )

δ

ppm 7.52 (s, 1H), 7.12–7.07 (m, 3H), 6.89–6.86 (m, 3H), 5.04

ppm 159.4, 137.1, 129.6, 128.7, 128.0, 119.0,

3

(

s, 2H), 3.80 (s, 3H). ¹³C NMR (75 MHz, CDCl₃)

14.2, 55.2, 50.1.

δ

1

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1-(4-Nitrobenzyl)-1H-imidazole (6d)

Following the procedure described above for the preparation of 6c, compound 6d

was obtained from 4-nitrobenzyl bromide (0.5 g, 2.31 mmol) as a yellow solid. (0.46 g,

1

>

98%): H NMR (300 MHz, CDCl )

δ

ppm 8.22 (d, 2H, J = 8.6 Hz), 7.59 (s, 1H), 7.28 (d, 2H,

3

13

J = 6.5 Hz), 7.15 (s, 1H), 6.92 (s, 1H), 5.26 (s, 2H).; C NMR (75 MHz, CDCl₃)

43.4, 137.3, 129.9, 127.5, 123.8, 119.1, 49.5.

δ ppm 147.3,

1

2

.2.7. General Synthesis of NAIMSs (7a–7i, 10, 11)

-(2-(1,4-Dimethoxynaphthalen-2-yl)-2-oxoethyl)-1-methyl-1H-imidazol-3-ium bromide

3

(

NAIMS 7a)

Compound 5a (0.05g, 0.16mmol) and 1-methylimidazole (6a; 0.025 mL, 0.32 mmol)

were dissolved in acetonitrile (2 mL). The mixture was heated under reﬂux for 1 d. It was

concentrated under reduced pressure and recrystallized by acetonitrile and ether to give

1

the compound 7a as an ivory solid. (0.061 g, 98%) [25]: H NMR (300 MHz, (CD ) SO)

δ

3

2

ppm 9.10 (s, 1H), 8.26–8.22 (m, 2H), 7.78–7.76 (m, 4H), 7.19 (s, 1H), 5.98 (s, 2H), 4.09 (s, 3H),

3

.99 (s, 3H), 3.97 (s, 3H). ¹³C NMR (75 MHz, (CD ) SO)

δ

ppm 191.1, 152.8, 151.3, 137.8,

3

2

1

29.1, 128.9, 127.9, 124.1, 123.8, 123.2, 123.1, 122.2, 101.1, 64.1, 58.2, 55.8, 35.9. m.p. 227.6

◦

+

C decomposed. HRMS (FAB) m/z Calcd. for C H N O [M

−

Br] 311.1396, found

18

19

2

3

11.1395.

3

-(2-(1,4-Bis(isoamyloxy)naphthalen-2-yl)-2-oxoethyl)-1-methyl-1H-imiazol-3-ium

bromide (NAIMS 7b)

Following the procedure described above for the preparation of 7a, compound 7b

was obtained from 5b (0.12 g, 0.28 mmol) and imidazole 6a (0.045 g, 0.56 mmol) as an

1

ivory solid. (0.1 g, 71%): H NMR (300 MHz, (CD ) SO)

δ 9.12 (s, 1H), 8.24 (dd, 1H,

3

2

J = 3.1, 6.2 Hz), 8.17 (dd, 1H, J = 3.1, 5.8 Hz), 7.79–7.76 (m, 4H), 7.21 (s, 1H), 4.23–4.14

1

3

(

m, 4H), 3.97 (s, 3H), 1.97–1.85 (m, 4H), 1.81–1.74 (m, 2H), 0.99–0.97 (m, 12H). C NMR

(

1

(

75 MHz, (CD ) SO)

δ

191.1, 151.3, 150.6, 137.8, 129.0, 128.2, 127.8, 124.0, 123.6, 123.3, 123.1,

3

2

◦

22.3, 102.0, 75.5, 66.5, 57.9, 38.4, 37.2, 35.9, 24.8, 24.6, 22.6, 22.4. m.p. 68.9–70.8 C. HRMS

FAB) m/z Calcd. for C H N O [M − Br] 423.2648, found 423.2644.

+

26

35

2

3

1

-Benzyl-3-(2-(1,4-bis(isoamyloxy)naphthalen-2-yl)-2-oxoethyl)-1H-imiazol-3-ium

bromide (NAIMS 7c)

Following the procedure described above for the preparation of 7a, compound 7c was

obtained from 5b (0.05g, 0.12mmol) and 1-benzylimidazole (6b; 0.037 g, 0.24 mmol) to

1

give the compound 7c as an ivory solid. (0.07 g, >98%): H NMR (300 MHz, (CD ) SO)

δ

3

2

ppm 9.30 (s, 1H), 8.23 (dd, 1H, J = 2.9, 6.0 Hz), 8.17–8.15 (m, 1H), 7.92 (s, 1H), 7.80 (s, 1H),

7

2

δ

.77–7.74 (m, 2H), 7.51–7.43 (m, 5H), 7.21 (s, 1H), 5.98 (s, 2H), 5.58 (s, 2H), 4.22–4.16 (m, 4H),

.21–1.88 (m, 4H), 1.80–1.76 (m, 2H), 0.99–0.97 (m, 12H). ¹C NMR (75 MHz, (CD ) SO)

3

2

ppm 191.0, 151.3, 150.5, 137.5, 134.8, 129.0, 128.7, 128.2, 128.1, 127.8, 124.4, 123.5, 123.3,

◦

1

22.2, 122.0, 101.9, 75.5, 66.5, 58.1, 51.9, 38.4, 37.2, 24.7, 24.5, 22.5, 22.4. m.p. 87.7–88.8 C.

+

HRMS (FAB) m/z Calcd. for C H N O [M − Br] 499.2961, found 499.2960.

32

39

2

3

-(2-(1,4-Bis(isoamyloxy)naphthalen-2-yl)-2-oxoethyl)-1-(4-methoxybenzyl)-1H-imidazol-

-ium bromide (NAIMS 7d)

Following the procedure described above for the preparation of 7a, compound 7d

was obtained from 5b (0.06 g, 0.14 mmol) and 4-methoxybenzylimidazole (6c; 0.05 g,

1

0

8

.28 mmol) as an ivory solid. (0.045 g, 55%): H NMR (300 MHz, (CD ) SO)

δ

9.17 (s, 1H),

3

2

.23 (dd, 1H, J = 3.1, 6.2 Hz), 8.15 (dd, 1H, J = 3.3, 6.2 Hz), 7.86 (s, 1H), 7.77–7.74 (m, 3H), 7.44

(

d, 2H, J = 8.4 Hz), 7.18 (s, 1H), 7.02 (d, 2H, J = 8.6 Hz), 5.92 (s, 2H), 5.46 (s, 2H), 4.21–4.12

m, 4H), 1.90–1.87 (m, 4H), 1.80–1.74 (m, 2H), 0.98 (d, 6H, J = 2.9 Hz), 0.97 (d, 6H, J = 3.6

(

Hz). ¹³C NMR (75 MHz, (CD ) SO)

δ

191.0, 159.6, 151.4, 150.5, 137.2, 130.0, 129.0, 128.1,

3

2

127.8, 126.5, 124.4, 123.5, 123.3, 122.2, 121.8, 114.4, 101.9, 75.5, 66.5, 58.1, 55.2, 51.5, 38.4, 37.2,

Pharmaceutics 2021, 13, 312

7 of 20

◦

2

4.7, 24.5, 22.5, 22.4. m.p. 113.3–114.7 C. HRMS (FAB) m/z Calcd. for C H N O [M

−

33

41

2

4

+

Br] 529.3066, found 529.3068.

3

-(2-(1,4-Bis(isoamyloxy)naphthalen-2-yl)-2-oxoethyl)-1-(4-nitrobenzyl)-1H-imidazol-3-

ium bromide (NAIMS 7e)

Following the procedure described above for the preparation of 7a, compound 7e was

obtained from 5b (0.06 g, 0.14 mmol) and 1-(4-nitrobenzyl)imidazole (6d; 0.04 g, 0.21 mmol)

1

as an ivory solid. (0.06 g, 70%): H NMR (300 MHz, (CD ) SO)

δ

9.28 (s, 1H), 8.34 (d, 2H,

3

2

J = 6.7 Hz), 8.23–8.07 (m, 2H), 7.92 (s, 1H), 7.81 (s, 1H), 7.78–7.75 (m, 2H), 7.70 (d, 2H,

J = 7.1 Hz), 7.20 (s, 1H), 5.96 (s, 2H), 5.74 (s, 2H), 4.22–4.14 (m, 4H), 1.99–1.91 (m, 4H), 1.79–

1

(

.76

m, 2H), 0.99–0.96 (m, 12H).; ¹³C NMR (75 MHz, (CD ) SO)

δ 190.9, 151.4, 150.6, 147.6,

3

2

1

5

42.1, 137.9, 129.4, 129.0, 128.1, 127.8, 124.7, 124.1, 123.5, 123.3, 122.2, 122.1, 101.9, 75.6, 66.5,

◦

8.2, 51.0, 38.4, 37.2, 24.7, 24.5, 22.5, 22.4.; m.p. 160.2–160.6 C; HRMS (FAB) m/z Calcd. for

+

C H N O [M − Br] 544.2811, found 544.2812.

32

38

3

5

1

-Benzyl-3-(2-(1,4-diisopropoxynaphthalen-2-yl)-2-oxoethyl)-1H-imidazol-3-ium

bromide (NAIMS 7f)

Following the procedure described above for the preparation of 7a, compound 7f was

obtained from 5c (0.08 g, 0.22 mmol) and 1-benzylimidazole (6b; 0.06 g, 0.38 mmol) as an

1

ivory solid. (0.065 g, 59%): H NMR (300 MHz, CDCl )

δ

11.00 (s, 1H), 8.32–8.30 (m, 1H),

3

8

.10–8.07 (m, 1H), 7.63–7.60 (m, 2H), 7.47–7.42 (m, 5H), 7.12–7.10 (m, 2H), 7.04 (s, 1H), 6.08

1

3

(

1

4

s, 2H), 5.61 (s, 2H), 4.81–4.77 (m, 1H), 4.55–4.51 (m, 1H), 1.47–1.45 (m, 12H). C NMR

75 MHz, CDCl₃) ppm 192.2, 150.2, 149.8, 139.2, 132.5, 130.7, 129.6, 129.5, 129.1, 128.9,

28.4, 127.0, 125.2, 123.8, 123.1, 123.1, 120.8, 103.1, 79.7, 70.8, 58.6, 53.5, 22.4, 22.0. m.p.

δ

◦

+

91.7–192.5 C. HRMS (FAB) m/z Calcd. for C H N O [M

−

Br] 443.2335, found

28

31

3

43.2332.

3

-(2-(1,4-Bis(isoamyloxy)-8-methoxynaphthalen-2-yl)-2-oxoethyl)-1-methyl-1H-imidazol-

-ium bromide (NAIMS 7g)

Following the procedure described above for the preparation of 7a, compound 7g

was obtained from 5d (0.05 g, 0.11 mmol) and 1-methylimidazole (6a; 0.018 g, 0.22 mmol)

1

as an ivory solid. (0.051 g, 88%): H NMR (300 MHz, (CD ) SO)

δ

ppm 9.06 (s, 1H), 7.81

3

2

(

d, 1H, J = 8.4 Hz), 7.75 (s, 1H), 7.72 (s, 1H), 7.68–7.62 (m, 1H), 7.21 (d, 1H, J = 8.0 Hz), 7.17

s, 1H), 5.87 (s, 2H), 4.18–4.13 (m, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 3.95–3.92 (m, 2H), 1.89–1.87

1

3

(

1

4

m, 2H), 1.84–1.72 (m, 4H), 0.96 (d, 6H, J = 5.5 Hz), 0.95 (d, 6H, J = 5.6 Hz). C NMR

75 MHz, (CD ) SO) ppm 191.6, 156.9, 152.5, 150.0, 137.8, 131.3, 129.7, 124.3, 123.9, 123.0,

19.3, 114.0, 108.3, 102.7, 75.8, 66.4, 58.2, 56.0, 38.2, 37.2, 35.8, 24.8, 24.7, 22.7, 22.4. m.p.

δ

3

2

◦

+

12.9–114.7 C. HRMS (FAB) m/z Calcd. for C H N O [M

53.2752.

−

Br] 453.2753, found

27

37

2

4

1

3

-Benzyl-3-(2-(1,4-bis(isoamyloxy)-8-methoxynaphthalen-2-yl)-2-oxoethyl)-1H-imidazol-

-ium bromide (NAIMS 7h)

Following the procedure described above for the preparation of 7a, compound 7h was

obtained from 5d (0.1 g, 0.22 mmol) and imidazole 6b (0.05 g, 0.33 mmol) as an ivory solid.

1

(

0.1 g, 80%): H NMR (300 MHz, (CD ) SO)

δ

ppm 9.22 (s, 1H), 7.87 (s, 1H), 7.80 (d, 1H,

3

2

J = 8.4 Hz), 7.74 (s, 1H), 7.67–7.62 (m, 1H), 7.48–7.42 (m, 5H), 7.20 (d, 1H, J = 7.7 Hz), 7.17 (s,

H), 5.87 (s, 2H), 5.54 (s, 2H), 4.17–4.12 (m, 2H), 3.99 (s, 3H), 3.95–3.91 (m, 2H), 1.90–1.84

1

3

(

m, 4H), 1.75–1.73 (m, 2H), 0.96 (d, 6H, J = 6.6 Hz), 0.94 (d, 6H, J = 6.5 Hz). C NMR

75 MHz, (CD ) SO) ppm 191.5, 156.9, 152.4, 150.0, 137.5, 134.8, 131.3, 129.6, 129.0, 128.7,

28.2, 124.4, 124.3, 121.9, 119.3, 114.0, 108.2, 102.7, 75.9, 66.4, 58.5, 56.0, 51.9, 38.1, 37.2, 24.8,

(

δ

3

2

1

2

5

◦

+

4.7, 22.6, 22.3. m.p. 121.3–122.7 C. HRMS (FAB) m/z Calcd for C H N O [M

−

Br]

33

41

2

4

29.3066, found 529.3068.

Pharmaceutics 2021, 13, 312

8 of 20

3

-(2-(1,4-Bis(isoamyloxy)-8-methoxynaphthalen-2-yl)-2-oxoethyl)-1-(4-nitrobenzyl)-1H-

imidazol-3-ium bromide (NAIMS 7i)

Following the procedure described above for the preparation of 7a, compound 7i was

obtained from 5d (0.1 g, 0.22 mmol) and imidazole 6d (0.067 g, 0.33 mmol) as an ivory solid.

1

(

0.12 g, 88%): H NMR (300 MHz, (CD ) SO)

δ

ppm 9.27 (s, 1H), 8.34 (d, 2H, J = 8.0 Hz),

3

2

7

3

.90 (s, 1H), 7.81 (d, 1H, J = 8.4 Hz), 7.79 (s, 1H), 7.69 (d, 2H, J = 8.6 Hz), 7.68–7.62 (m, 1H),

.21 (d, 1H, J = 8.0 Hz), 7.17 (s, 1H), 5.90 (s, 2H), 5.73 (s, 2H), 4.17–4.13 (m, 2H), 3.99 (s, 3H),

.94 (t, 2H, J = 7.3 Hz), 1.87–1.83 (m, 4H), 1.77–1.73 (m, 2H), 0.96 (d, 6H, J = 6.3 Hz), 0.94

(

d, 6H, J = 6.2 Hz). ¹³C NMR (75 MHz, (CD ) SO)

δ ppm 191.4, 156.9, 152.5, 150.0, 147.6,

3 2

1

5

42.1, 137.9, 131.3, 129.7, 129.4, 124.6, 124.2, 124.0, 122.0, 119.3, 114.0, 108.2, 102.7, 75.9, 66.4,

◦

8.5, 56.0, 51.0, 38.2, 37.2, 24.8, 24.7, 22.6, 22.4. m.p. 158.3–159.8 C. HRMS (FAB) m/z Calcd.

+

for C H N O [M − Br] 574.2917, found 574.2916.

33

40

3

6

1-Benzyl-3-(2-(naphthalen-2-yl)-2-oxoethyl)-1H-imidazol-3-ium bromide (NAIMS 10)

Following the procedure described above for the preparation of 7a, compound 10 was

obtained from 2-bromoacetylnapththalene (0.1 g, 0.4 mmol) and imidazole 6b as a white

1

solid. (0.09 g, 56%): H NMR (300 MHz, (CD ) SO)

δ ppm 9.24 (s, 1H), 8.79 (s, 1H), 8.19

3

2

(

7

d, 1H, J = 8.0 Hz), 8.13 (d, 1H, J = 8.2 Hz), 8.06 (d, 1H, J = 8.2 Hz), 7.91 (s, 1H), 7.76 (s, 1H),

.75–7.69 (m, 2H), 7.47–7.45 (m, 5H), 6.16 (s, 2H), 5.56 (s, 2H). ¹³C NMR (75 MHz, (CD ) SO)

3 2

δ

ppm 191.1, 137.5, 135.4, 134.8, 131.9, 130.9, 130.4, 129.6, 129.3, 129.0, 128.8, 128.7, 128.2,

◦

1

27.8, 127.4, 124.4, 123.1, 122.2, 55.5, 52.0. m.p. 170.8 C decomposed. HRMS (FAB) m/z

+

Calcd. for C H N O [M − Br] 327.1497, found 327.1497.

22

19

2

1

-Benzyl-3-(2-(1-methoxynaphthalen-2-yl)-2-oxoethyl)-1H-imidazol-3-ium bromide

(

NAIMS 11)

Following the procedure described above for the preparation of 7a, compound 11 was

obtained from 2-bromoacetyl-l-methoxynaphthalene 5e (0.1 g, 0.36 mmol) and imidazole

1

6b

as an ivory solid. (0.12 g, 79%): H NMR (300 MHz, (CD ) SO)

δ

ppm 9.39 (s, 1H),

3

2

8

.29 (d, 1H, J = 7.5 Hz), 8.07 (d, 1H, J = 8.4 Hz), 7.95 (s, 1H), 7.91–7.88 (m, 2H), 7.84

1

3

(s, 1H), 7.78–7.70 (m, 2H), 7.49–7.40 (m, 5H), 6.04 (s, 2H), 5.61 (s, 2H), 4.16 (s, 3H). C NMR

(

1

75 MHz, (CD ) SO)

δ

ppm 191.1, 158.8, 137.5, 137.2, 134.8, 129.4, 129.0, 128.7, 128.3, 128.2,

3

2

◦

27.2, 127.1, 124.6, 124.5, 124.4, 123.6, 123.4, 121.9, 64.2, 58.4, 51.9. m.p. 159.5–159.8 C.

+

HRMS (FAB) m/z Calcd. for C H N O [M − Br] 357.1603, found 357.1604.

23

21

2

2.3. Fungal Strains and Culture

Candida tropicalis var. tropicalis (KCTC17762), Candida glabrata (KCTC7219), Candida

tropicalis (KCTC7212), Candida albicans (KCTC7270), Candida albicans (KCTC7965) and Can-

dida auris (KCTC17810) were purchased from KCTC (Korean Collection for Type Cultures,

Candida parapsilosis var. parapsilosis (KACC45480) and Candida albicans (KACC30071) were

purchased from KACC (Korean Agricultural Culture Collection, Suwon, Korea). All strains

◦

were kept in 20% glycerol at

1

(

−

70 C. They were cultured in YPD containing yeast extract

0 g/L (BD Difco, Franklin Lakes, NJ, USA), peptone 20 g/L (BD Difco) and 2% D-glucose

◦

w/v) (Daejung, Gyeonggi-do, Suwon, Korea) at 30 C for 24–48 h [26].

2.4. Antifungal Susceptibility Microdilution Assay

Microdilution assay was performed based on the description of CLSI document M27-

A [26–28]. Compounds were prepared in DMSO (Dimethyl sulfoxide; Junsei, Tokyo,

Japan) at a concentration of 10 mg/mL as a stock solution. A colony of each strain was

◦

inoculated in 3 mL of YPD broth at 30 C overnight, and the medium was changed to new

4

fresh medium. The strains were adjusted at 3.0

concentrations ranging from 1.5625 to 100

×

10 CFU/mL with the ﬁnal compound

µ

g/mL. Growth control without treatment and

sterilized medium control were included in each experiment and miconazole was used as

the positive control [26,29].

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2.5. Cell Viability Assay

Cell viability was estimated according to previously reported method with slightly

modiﬁcation [30]. A normal cell line, HaCaT (ATCC, VA, USA), was added to a 96-

4

well plate at 1.0

×

10 cells per well and incubated for 24 h. Various concentrations of

NAIMS 7c (3.125–100 µg/mL) were added and further incubated for 24 h. MTT (3-(4,5-

dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO, USA) in

◦

PBS was added into each well, followed by incubation for 3 h at 37 C. The medium was

then removed, and cells were suspended in 100 µL DMSO for 10 m. Viable cells were

calculated from optical density (OD₅₄₀) values measured using a microplate reader (BioTek

Instruments, Winooski, VT, USA).

2.6. Fungal Cell Growth Test

Inoculation and culture conditions in this study were same as those in the above-

mentioned antifungal susceptibility microdilution assay. The compound concentrations

then ranged from 1.56 to 25

µ

g/mL. We cultured 96-well plates (SPL, Gyeonggi-do, Korea)

◦

at 30 C and OD₆were measured using a microplate reader (BioTek Instruments) at

00

indicated time [31].

2.7. ROS Detection

ROS detection cell-based assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used

according to manufacturer’s instructions. Cells were incubated with/without antimycin

positive control) or indicated concentrations of NAIMS 7c for 2 h. Cells were rinsed

(

with ice cold cell-based assay buffer and then incubated with ROS straining buffer. Dihy-

droethidium (DHE) ﬂuorescence was measured using an excitation wavelength 480 nm

and an emission wavelength 580 nm according to manufacturer’s instructions by VICTOR2

(

Perkin Elmer, Waltham, MA, USA). Antimycin A was used as positive control [32].

2

.8. Measurement of Mitochondrial Membrane Potential (4Ψm)

6

C. albicans was added to the wells of a 24-well plate at a density of 5.0

×

10 CFU/mL.

NAIMS 7c (1.56, 3.125 and 6.25

stained with 5

µg/mL) was added and further incubated for 2 h. Cells were

◦

µM of JC-1 (Biotium, CA, USA) for 30 m at 30 C. After washing, photo-

graphic images were acquired under an inverted ﬂuorescence microscope (EVOS FL Cell

Imaging System, Thermo Fisher, Waltham, MA, USA) using the microscope program [17].

2.9. Detection for Release of UV Absorbing Materials at 260 and 280 nm

Overnight cultured C. albicans was washed with PBS to stop further proliferation.

◦

Cells were treated with indicated concentration of NAIMS 7c for 1 h at 30 C. Lysate

released was measured with the Ultrospec 3000 at 260 and 280 nm. The values were

adjusted by subtracting the optical density measured for the corresponding negative

control, which was obtained by same compound concentrations in PBS without cells at the

same wavelength [33,34].

2.10. Quantitative Reverse Transcriptase PCR (qRT-PCR) analysis

qRT-PCR was performed following the method with slight modiﬁcation [35].

Total RNA was extracted with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA,

USA). Real-time PCR was performed in 96-well PCR plates (Bio-rad, Hercules, CA, USA)

using 2

×

RT Pre-MIX kit (Biofact, Daejeon-si, Korea) and CFX Connect Real-Time PCR

Detection System (Bio-rad). Primer sequences used in this study are listed in Table 1.

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Table 1. Primer list used for qRT-PCR.

Gene

Oligomer

References

F: TAGGTTTGGAAGCTGCTGG

R: CCTGGGAACATGGTAGTAC

F: CAACACAACACAGTAGCACC

R: GTTGTGGATAAAGGTGGTTCG

F: TACAACCAACACATCAACCAG

R: ATTAGTTGGACCGGAAGATG

F: TATAATAGACCTTCTGGAC

R: TTGGTGGACGAGAATAATG

ACT1

YPK1

[36]

This study

[37]

HAC1

MCA1

[38]

2.11. Statistical Analysis

All data are expressed as the mean

were determined using the Student’s t-test or one-way ANOVA, and p < 0.05 was

±

S.D. Signiﬁcant differences among the groups

considered signiﬁcant.

3. Results

3.1. Chemistry

First, we tried to synthesize 2-acetyl-1,4-dialkoxynaphthalnes

to target NAIMS by direct alkylation of 1,4-dihydroxynaphthalene followed by Friedel–

Crafts acylation. However, Friedel–Crafts acylation was found to be inefﬁcient due to its

low yield. Therefore, another synthetic route to produce was suggested as shown in

Scheme 1. The compounds were synthesized in high yield from 1,4-diacetoxynaphthalene

) by Fries rearrangement. Then, alkylation of compound with the corresponding

alkyl halide afforded compound . The removal of the acetyl group with KOH followed

by O-alkylation produced key intermediate in two steps with good yield. Various

bromination reactions of compound were tested to ﬁnd a condition optimized for the

synthesis of bromoacyl intermediate . Finally, we found that compound was obtained in

4, a key intermediate

7

4

2

(

1

3

4

α-

4

5

the best yield when reacted with TBA-Br . N-Arylimidazoles such as 6a and 6d, which are

3

not commercially available, were synthesized from imidazole and 4-substituted benzyl halide

in acetonitrile. The coupling reaction of the 2-bromoacyl intermediates

5 with corresponding

imidazoles 6 gave nine new 1,4-dialkoynaphthalen-2-acyl imidazolium salts 7a–i.

Scheme 1. Preparation of 1,4-dialkoxy-NAIMSs 7a–7i.

Reagents and conditions. (i) BF 2CH COOH (7 eq), reﬂux, 1h; (ii) Cs CO (1.5 eq),

3

2

3

◦

R -X (1.5 eq), DMF (0.5M), 70 C, 2.5 h; (iii) KOH (0.67 eq), MeOH (0.5 M), 0 C, 1h,

then Amberlite IR-120(H), 0 C, 15 m; (iv) Cs CO (1.5 eq), R -X (1.5 eq), DMF (0.5M),

1

◦

2

3

1

Pharmaceutics 2021, 13, 312

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◦

0 C, 1.5 h; (v) TBA-Br (1.2–1.3 eq), CH Cl (0.05 M), rt, 3 h; (vi) imidazole (1–2 eq),

3 2 2

7

CH CN, reﬂux, 1d.

3

To investigate a simple relationship between structure and activity of NAIMSs,

0

we prepared compounds

8,

9

,

9

, NAIMS 10 and NAIMS 11, as shown in Figure 2. Com-

0

pounds 8, 9 and

9

have no imidazolium moiety, and NAIMS 10 and NAIMS 11 have

no dialkoxy substituents in a naphthalene ring. The same coupling reaction of readily

available 2-bromoacetylnaphthalene or 2-bromoacetyl-1-methoxynaphthalene (5e) with

imidazole 6b gave, respectively, NAIMS 10 and NAIMS 11.

Figure 2. Structure of naphthalene compounds without an imidazolium moiety or a dialkoxy group in the naphthalene ring

made for comparison with 1,4-dialkoxy-NAIMS 7.

3.2. Antifungal Activity of NAIMS 7c Against Candida spp.

The growth inhibitory activity of 7a–i, 10 and 11 against Candida spp. was evaluated

using broth microdilution assays. The MIC values of miconazole were used as a positive

control and conﬁrmed over 12.5 g/mL to Candida spp. after 24 h incubation. Among the

µ

tested compounds, NAIMS 7b, NAIMS 7c, NAIMS 7d and NAIMS 7e showed stronger

antifungal activity against C. albicans than miconazole. NAIMS 7c showed strongest an-

tifungal activity compared with other derivatives; MIC values ranged from 3.125 to 6.25

µg/mL against all Candida spp. used in this study. In particular, NAIMS 7c showed

3

.125 g/mL MIC value in 24 h assay against C. auris (KCTC17810) which possessed native

µ

resistance to miconazole (Table 2). To check the stability of antifungal activity of synthetic

compounds, MIC values were detected every day for 72 h. NAIMS 7c had 3.125 and

6

.25

zole, by contrast, showed sustainable anti-Candida activity of 25

Table 3). NAIMS 7c showed stable antifungal activity compared with other derivatives.

µ

g/mL MIC values for C. albicans (KACC30071) at 48 and 72 h, respectively. Micona-

µg/mL MIC value for 72 h

(

The yield of NAIMS 7c was fortunately higher than NAIMS 7b and NAIMS 7d, and this

suggested that NAIMS 7c is more cost-beneﬁcial than other derivatives (Scheme 1). In vitro

cell viability of NAIMS 7c against HaCaT was performed via MTT assays. NAIMS 7c had

IC₅₀of 21.39 µg/mL against HaCaT. Based on results, cell growth test was also evaluated

for the serial diluted concentration of NAIMS 7c in C. albicans (KCTC7965, KCTC7270 and

KACC30071) for 48 h (Figure 3). Regardless of the time, NAIMS 7c at 6.25 µg/mL strongly

inhibited cell growth compared with miconazole.

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Table 2. Minimum inhibitory concentration (MIC) values (µg/mL) of fungal strains after 24 h treatment.

C. tropicalis

var. tropicalis

C. parapsilosis var.

C. albicans

C. glabrata

C. tropicalis

C. auris

parapsilosis

Compound

KCTC7965

KCTC17762

KACC45480

KCTC7219

KCTC7212

KCTC17810

Miconazole

NAIMS 7c

NAIMS 7g

NAIMS 7h

8

12.5

3.125

12.5

50

12.5

6.25

25

12.5

3.125

6.25

12.5

6.25

25

12.5

3.125

6.25

50

>100

25

>100

3.125

6.25

25

>100

6.25

12.5

>100

3.125

100

>100

6.25

12.5

>100

6.25

100

>100

6.25

3.125

6.25

>100

3.125

12.5

>100

6.25

12.5

>100

6.25

N/A

>100

3.125

6.25

12.5

>100

6.25

0

9

NAIMS 10

NAIMS 11

NAIMS 7e

NAIMS 7b

NAIMS 7i

NAIMS 7a

NAIMS 7d

NAIMS 7f

25

6.25

3.125

6.25

>100

3.125

25

100

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C. albicans (KACC30071)

1

.4

.2

1

NAIMS 7c

Miconazole

DMSO

0.8

0.6

0.4

0.2

0

*

***

*

**

0

2

4

6

8

10

24

48

Time [h]

C. albicans (KCTC7270)

1

.4

.2

1

NAIMS 7c

Miconazole

DMSO

0

.8

.6

.4

.2

0

*

**

*

0

2

4

6

8

10

24

48

Time [h]

Figure 3. The growth of C. albicans was inhibited by NAIMS 7c. Microdilution assay was performed

at 3.125 g/mL for 48 h. Data represent mean SD from three independent experiments. ** p < 0.05

and *** p < 0.01.

µ

±

3.3. Inducing ROS Level and the Loss of Mitochondria Membrane Potential by NAIMS 7c.

To identify antifungal activity of NAIMS 7c against C. albicans, ROS production

in C. albicans was detected after NAIMS 7c treatment. NAIMS 7c induced higher ROS

Pharmaceutics 2021, 13, 312

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production than did antimycin. The treatment of 0.78

DHE ﬂuorescence with the maximum at 1.56

mechanism of NAIMS 7c, C. albicans was stained with JC-1 dye to detect the mitochondria

membrane potential. NAIMS 7c decreased a red ﬂuorescence at 1.56 g/mL in C. albicans.

µg/mL of NAIMS 7c increased the

µg/mL (Figure 4). For further study about the

µ

This suggested that NAIMS 7c causes damage to mitochondria and alters the cellular state

of C. albicans (Figure 5). Based on these results, NAIMS 7c induces loss of mitochondria

membrane potential and increases the release of ROS in C. albicans.

2

9700

9500

9300

9100

8900

8700

8500

0

Antimycin

0.78

1.56

3.12

NAIMS 7c [μg/ml]

Figure 4. NAIMS 7c induced the production of reactive oxygen species (ROS) in C. albicans. The amount of ROS of C.

albicans (KCTC7965) was detected after 2 h. Antimycin was used for the positive control. Data represent mean

three independent experiments.

±

SD from

Figure 5. NAIMS 7c changed the mitochondrial membrane potential in C. albicans. We treated

.56 g/mL of NAIMS 7c for 2 h. Cells were detected by ﬂuorescence microscope. Red ﬂuorescence

indicates dye aggregated in the mitochondria, and green indicates dye scattered in the cytoplasm.

1

µ

Pharmaceutics 2021, 13, 312

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3.4. The Cell Lysis and the Apoptosis of C. albicans by NAIMS 7c

To determine the induction of the loss of mitochondria membrane potential by NAIMS

c treatment, changes in UV absorbing materials were detected by spectrophotometer with

7

NAIMS 7c in the C. albicans culture medium supernatant. The use of 12.5

treatment signiﬁcantly increased the absorbance at 260 and 280 nm after 1 and 2 h

Figure 6). These results show that treatment of NAIMS 7c at over the MIC value can lead

µg/mL of NAIMS

7c

(

to lysis of Candida spp. and might induce the apoptosis of Candida spp. Three different

Candida-apoptosis related genes were used to detect the effect of NAIMS 7c in C. albicans.

YPK1 is a serine/threonine protein kinase that affects diverse cellular activities, including

sphingolipid homeostasis. HAC1 is a transcription factor and plays a major role in stress-

related transcriptional response. MCA1 is a cysteine protease involved in apoptosis in

response to stresses. The expression of these three genes was dramatically increased by

NAIMS 7c treatment (Figure 7).

Figure 6. NAIMS 7c induced the cell lysis of C. albicans. The release-detecting assay was performed

after 1 or 2 h. Data represent mean ± SD from three independent experiments. ** p < 0.05.

Pharmaceutics 2021, 13, 312

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Figure 7. NAIMS 7c induced the expression of apoptosis- related genes of C. albicans. mRNA of C.

albicans (KCTC7965) was obtained after 2 h treatment. Miconazole and NAIMS 7c were treated at

6

.25 and 1.56

µg/mL, respectively. Data represent mean

±

SD from three independent experiments.

*

** p <0.01.

4. Discussion

Candida spp. are the most common organism recovered from the skin and blood of

hospitalized patients. Although the need to treat them is increasing, the range of antifungal

agents available is limited because of their toxicity. In addition, resistant strains and new

species that show innate resistance to antifungal agents have been reported [39,40]. Therefore,

it is necessary to introduce new antifungal agents to limit the spread of pathogenic fungi.

For the synthesis of 1,4-dialkoy-NAIMS, we developed a highly efﬁcient synthesis

method including the synthesis of 1,4-dialkoynaphthalen-2-acyl intermediate

4, the α-

bromination reaction of compound , and the S 2 reaction of compound and imidazole 6,

4

5

N

such as a quarternization of imidazole. We found that compound 4 was produced in better

yield by a novel synthetic method including Fries-rearrangement of compound 1 followed

by alkylation than the direct alkylation of 1,4-dihydroxynaphthalene and subsequent

Friedel–Crafts acylation. In addition, it was conﬁrmed that the α-bromination reaction of

compound 4 was best under the reaction conditions of TBA-Br .

3

0

Among the 14 synthesized compounds (7a

–

i,

8

,

9,

9

,

10

,

11), NAIMS 7b

,

7c, and 7d

showed superior activity compared to other synthetic NAIMSs or miconazole. In particular,

NAIMS 7c is best when considering its antifungal activity, stability and synthesis efﬁciency.

As shown in Figure 8, the energy calculation (Gaussian B3LYP, 6-311 [d, p]) predicts NAIMS

7c presenting a slightly curved structure similar to phospholipids. Based on these results,

we might propose a ﬁrst-pass reasoning on the relationship between structure and activity

as follows. For antifungal activity, the imidazolium ring must be essential. The naphthalene

ring requires a 1,4-dialkoxy group, in which the optimal alkyl group is an isoamyl group.

Furthermore, it seems better to have no methoxy groups in the naphthalene A ring and

no electron withdrawing groups such as NO in the benzene ring in order for NAIMS

2

to be activated.

Pharmaceutics 2021, 13, 312

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Figure 8. Graphical representation of the structure of NAIMS 7c by DFT calculation (Gaussian B3LYP,

6

331[d,p]). Grey (carbon), white (hydrogen), red (oxygen) and blue (nitrogen) bonds.

Miconazole was used as an antifungal agent which has a fungicidal activity against

planktonic Candida spp., and the cytotoxic concentration of NAIMS 7c can be signiﬁcantly

lower than miconazole [41 42]. NAIMS 7c had IC₅₀of 21.39 and 7.56 g/mL against

HaCaT and C. albicans (KACC30071), respectively. Miconazole had IC₅₀of 13.10 and

7.25 g/mL against HaCaT and C. albicans, respectively. Accordingly, the selective index

IC₅₀HaCaT/IC₅₀C. albicans) of NAIMS 7c was higher than miconazole. This result shows

,

µ

1

µ

(

that NAIMS 7c can be more effective to treat Candida spp. and safe. Additional in vivo

research should be provided to determine the property of any potential candidate for

therapeutic applications in the future [43]. Thus, NAIMS 7c could serve as a promising

lead compound for further research. The 1,4-dialkoxy naphthalene-2-acyl compound

9

without the imidazolium moiety and the naphthalene-2-acyl imidazolium 10 without the

dialkoxy substituent on the naphthalene ring showed no antifungal activity. However,

its hybrid NAIMS 7c found an excellent antifungal agent. Therefore, as mentioned in the

literature, the pharmacophore-hybridization approach is thought to be a useful tool for

new drug design and development [44].

NAIMS 7c led to induced ROS production, the loss of mitochondria membrane po-

tential, the release of UV absorbing materials and up-regulated apoptotic gene expression.

ROS and mitochondria play an essential part in apoptosis. These results suggest that

NAIMS 7c induced apoptosis in C. albicans.

In summary, a series of novel imidazolium salts containing 2-acetylnaphthalene moiety

were designed, prepared and evaluated for antifungal activity. The results presented in

this study conclusively demonstrate that NAIMS 7c has a long-term enhanced antifungal

activity against Candida spp., including especially C. albicans and C. auris, which possess

native resistance to miconazole, as compared with other compounds including miconazole.

Further studies of structure–activity relationships and a wide range of biological activities

are ongoing and will be reported in the future.

Author Contributions: Conceptualization, K.-Y.K. and H.K.; methodology, K.-Y.K., H.K., and J.-G.K.;

validation, K.-Y.K. and H.K.; formal analysis, K.-Y.K. and H.K.; investigation, J.L., H.L. and J.-G.K.;

resources, J.L., K.-Y.K., H.K. and J.-G.K.; data curation, T.H.L., K.-Y.K. and H.K.; writing—original

draft preparation, J.L. H.L. and J.-G.K.; writing—review and editing, T.H.L., K.-Y.K., H.K., J.L. and

J.-G.K.; supervision, K.-Y.K. and H.K.; project administration, T.H.L., K.-Y.K. and H.K. All authors

have read and agreed to the published version of the manuscript.

Funding: This work was supported by the GRRC program of Gyeonggi province [GRRC-kyunghee20

2

0(B04)].

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available on request from the

corresponding author.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Pharmaceutics 2021, 13, 312

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Article Doi

Antifungal activity of 1,4-dialkoxynaphthalen-2-acyl imidazolium salts by inducing apoptosis of pathogenic candida spp.

DOI: 10.3390/pharmaceutics13030312

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