Synthesis and evaluation of the antibacterial activities of 13‐substituted berberine derivatives

Article

Synthesis and Evaluation of the Antibacterial

Activities of 13‐Substituted Berberine Derivatives

Hamza Olleik ¹^,², Taher Yacoub , Laurent Hoffer , Senankpon Martial Gnansounou ⁴^,⁵,

3

4

1

4

Kehna Benhaiem‐Henry , Cendrine Nicoletti , Malika Mekhalfi , Valérie Pique ,

Josette Perrier , Akram Hijazi , Elias Baydoun , Josette Raymond , Philippe Piccerelle ,

Marc Maresca * and Maxime Robin *

1

6

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4

1

,

4,

1

Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, 13397 Marseille, France;

hamza.olleik@live.com (H.O.); cendrine.nicoletti@univ‐amu.fr (C.N.); mekhalfimalika@yahoo.fr (M.M.);

josette.perrier@univ‐amu.fr (J.P.)

Department of Biology, American University of Beirut, Beirut 1107‐2020, Lebanon; eliasbay@aub.edu.lb

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3

Aix Marseille Univ, CNRS, INSERM, Institut Paoli‐Calmettes, CRCM, 13397 Marseille, France;

taher.yacoub7@gmail.com (T.H.); laurent.hoffer@inserm.fr (L.H.)

Aix Marseille Univ, Avignon Université, CNRS, IRD, IMBE, 13397 Marseille, France ;

4

gnansounoumartial@yahoo.fr (S.M.G.); h.kehna@gmail.com (K. B.‐H.); valerie.pique@univ‐amu.fr (V.P.);

philippe.piccerelle@univ‐amu.fr (P.P.)

Laboratoire d’études et de Recherches en Chimie Appliquée (LERCA), Université d’Abomey‐Calavi

5

(

UAC), Cotonou 01 BP 2009, Bénin

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Doctoral School of Science and Technology, Research Platform for Environmental Science (PRASE),

Lebanese University, Beirut 5, Lebanon; Akram.Hijazi@ul.edu.lb

Hôpital Cochin, Service de Bactériologie, Université Paris 5, 75014 Paris, France; josette.raymond@aphp.fr

Correspondence: m.maresca@univ‐amu.fr (M.M.); maxime.robin@univ‐amu.fr (M.R.)

7

*

Received: 3 March 2020; Accepted: 1 July 2020; Published: 6 July 2020

Abstract: The biological activities of berberine, a natural plant molecule, are known to be affected

by structural modifications, mostly at position 9 and/or 13. A series of new 13‐substituted berberine

derivatives were synthesized and evaluated in term of antimicrobial activity using various

microorganisms associated to human diseases. Contrarily to the original molecule berberine, several

derivatives were found strongly active in microbial sensitivity tests against Mycobacterium, Candida

albicans and Gram‐positive bacteria, including naïve or resistant Bacillus cereus, Staphylococcus aureus

and Streptococcus pyogenes with minimal inhibitory concentration (MIC) of 3.12 to 6.25 μM. Among

the various Gram‐negative strains tested, berberine’s derivatives were only found active on

Helicobacter pylori and Vibrio alginolyticus (MIC values of 1.5–3.12 μM). Cytotoxicity assays

performed on human cells showed that the antimicrobial berberine derivatives caused low toxicity

resulting in good therapeutic index values. In addition, a mechanistic approach demonstrated that,

contrarily to already known berberine derivatives causing either membrane permeabilization, DNA

fragmentation or interacting with FtsZ protein, active derivatives described in this study act through

inhibition of the synthesis of peptidoglycan or RNA. Overall, this study shows that these new

berberine derivatives can be considered as potent and safe anti‐bacterial agents active on human

pathogenic microorganisms, including ones resistant to conventional antibiotics.

Keywords: berberine derivatives; anti‐bacterial agents; microbial sensitivity tests; structure‐activity

relationship; resistant strain; FtsZ protein

Antibiotics 2020, 9, 381; doi:10.3390/antibiotics9070381

www.mdpi.com/journal/antibiotics

Antibiotics 2020, 9, 381

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1

. Introduction

According to the World Health Organization (WHO) “we are heading towards a post‐antibiotic

era in which common infections and minor injuries can once again kill” [1]. Indeed, antibiotic

resistance is rising dangerously worldwide, threatening our ability to cure common infectious

diseases that become harder, and sometimes impossible, to treat. Antibiotic resistance is associated

to higher medical costs, prolonged hospital stays, and increased mortality. In 2017, WHO published

its first ever list of antibiotic‐resistant “priority pathogens”—a catalogue of 12 families of bacteria that

pose the greatest threat to human health. This list is divided into three categories, i.e., critical, high

and medium priority groups, according to the urgency of need for new antibiotics. The critical

priority group includes Acinetobacter, Pseudomonas and Klebsiella strains that are resistant to a large

number of antibiotics, including carbapenems. The high priority group includes Enterococcus,

Staphylococcus, Helicobacter and Salmonella strains that are becoming resistant to various antibiotics.

Finally, the medium priority group includes resistant strains of Streptococcus and Shigella. Although

it was not mentioned in the WHO list of priority pathogens due to the fact that tuberculosis is targeted

by other dedicated programs, the rise in the number of resistant and multiresistant Mycobacterium

tuberculosis is also alarming as it infects approximately 25% of the world population and causes

millions deaths per year. In addition to bacterial infections, infections caused by fungi such as Candida

albicans remain an important cause of death in hospitalized and immunocompromised or critical ill

patients [1]. Similar to bacteria, an increase in the prevalence of Candida albicans strains resistant to

antifungal drugs is observed worldwide [2]. In this context, there is an urgent need to find safe natural

or synthetic molecules active against pathogenic microorganisms, including resistant ones. Berberine

is a natural molecule present in some medicinal plants, mainly of the Berberidaceae family (Berberis

aquifolium, B. aristata, B. asiatica, B. chitria, B. lyceum, B. vulgaris) but also in other species such as

Argemone mexicana, Coptis japonica, Hydrastis canadensis, Papaver dubium, Thalictrum flavum [3].

Berberine and its derivatives [4] are known for having various biological activities such as

antimalarial [5], antileishmanial [6], antiviral [7,8], hypolipidemic [9], anticancer [10,11] and

antimicrobial effects [12–14]. Most of the active derivatives of berberine were obtained by substitution

of various group at the position 9 and/or 13 resulting in higher activities compared to the parent

molecule. For example, regarding antimicrobial activity, it has been shown that substitution at the

1

3th position by phenyl (A), ether link (B) or cycloberberine (C) (Figure 1) resulted in a higher activity

against Candida species and S. aureus [14], including the methicillin‐resistant strain MRSA [13].

Figure 1. Chemical structure of reported antimicrobially active derivatives of berberine obtained by

modification by phenyl (A), ether link (B) or cycloberberine (C) at position 13.

Based on that, in the present study, a series of new derivatives were synthetized by substitution

at position 13 and tested in term of their antimicrobial (antibacterial and antifungal) activity,

innocuity on human cells and mechanism of action. The results showed that substitutions with

polyphenol rings and chalcone moiety leaded to the more active and safer compounds. In addition,

mechanistic studies revealed that antimicrobial activity of derivatives relies on their ability to inhibit

the synthesis of cell wall and RNA.

Antibiotics 2020, 9, 381

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2

. Materials and Methods

2

.1. Chemistry

1

13

H‐ and C‐NMR spectra were recorded on an Avance III nanobay‐300 MHz instrument (Bruker,

1

13

Bremen, Germany, 300 MHz for H, 75 MHz for C). Chemical shifts are reported in ppm relative to

the solvent in which the spectrum was recorded [ H: δ (d

C: δ (d

analysis facilities of Spectropole (https://fr‐chimie.univ‐amu.fr/spectropole) with a Thermo Finnigan

San Jose, CA, USA) EA 1112 apparatus; all compounds had purity higher than 95%. Microwave‐

1

6

‐DMSO) = 2.50 ppm, δ (CDCl ) = 7.27 ppm;

3

1

3

6

‐DMSO) = 39.52 ppm, δ (CDCl

3) = 77.16 ppm]. Combustion analyses were performed at the

(

assisted reactions were performed in a CEM Discover microwave reactor with a focused field (CEM

Corporation, Matthews, NC, USA). Silica gel F‐254 plates (0.25 mm; Merck, Darmstadt, Germany)

were used for thin‐layer chromatography (TLC), and silica gel 60 (200–400 mesh; Merck) was used

for flash chromatography. Unless otherwise stated, reagents were obtained from commercial sources

and were used without further purification. Dihydroberberine (compound 1), 13‐hydroxyberberine

(compound 2), 8‐acetonyl dihydroberberine (compound 3) (yield: 85,40 and 85%) were synthesized

by a procedure described in the literature [14].

2

.1.1. General Procedure for the Synthesis of Compounds 4–20

Berberine chloride (3.71 g, 10 mmol) was dissolved in 5N NaOH (20 mL) under stirring at room

temperature. Acetone (5 mL) was added dropwise at that temperature and stirred for 1 h,

precipitation occurred during that time and the reaction mixture was filtered and washed with 80%

MeOH to give the desired acetonylberberberine (compound 3) (3.34 g, 85%). Compound 3 (1 g, 2.5

mmol) was used without further purification, dissolved in acetonitrile, NaI (0.5 g, 3.3 mmol) was

added with various methylbromide (3 mmol) at 80 °C for 4 h. The reaction mixture was concentrated

under vaccuo and chromatographed on silica gel (CH

2Cl

2

/CH OH, 90/10 v/v) to give compound 4–

3

2

0.

1

3‐(Acetic acid ethylester) Berberine (compound 4)

1

Yellow solid; yield: 26%; mp: 216 °C [12]; H‐NMR (CDCl

3) δ ppm: 10.56 (1H, s, H‐8), 7.86 (1H,

d, J = 9.2 Hz, H‐12), 7.73 (1H, d, J = 9.2 Hz, H‐11), 7.24 (1H, s, H‐1), 6.90 (1H, s, H‐4), 6.10 (2H, s, ‐

OCH O‐), 5.20 (2H, brs, H‐6), 4.37 (3H, s, OCH ‐9), 4.35 (2H, q, J = 7.1 Hz, H‐3′), 4.28 (2H, s, H‐1′), 4.07

3H, s, OCH ‐10), 3.26 (2H, t, J = 5.1 Hz, H‐5), 1.37 (3H, t, J = 7.1 Hz, H‐4′). C‐RMN (CDCl

2

3

13

(

3

3) δ ppm:

1

4

70.6 (C‐2′), 150.7 (C‐10), 150.2 (C‐3), 147.5 (C‐2), 147.2 (C‐8), 146.5 (C‐9), 137.6 (C‐13a), 134.11 (C‐4a),

33.42 (C‐12a), 125.93 (C‐12), 125.68 (C‐13), 121.79 (C‐8a), 119.79 (C‐13b), 119.34 (C‐11), 109.12 (C‐4),

08.66 (C‐1), 102.13 (‐OCH2O‐), 63.12 (OCH

3‐C9), 62.29 (C‐3′), 57.41 (C‐6), 56.97 (OCH3‐C10), 37.15 (C‐

′), 28.50 (C‐5), 14.2 (C‐4′). Anal. calcd. for C24

H

24BrNO6: C, 57.38; H, 4.82; N, 2.79. Found: C, 57.44; H,

.87; N, 2.63.

1

3‐(Acetic Acid) Berberine (compound 5)

1

Yield 53%; H‐RMN (CDCl

3

) δ ppm: 10.56 (1H, s, H‐8), 7.86 (1H, d, J = 9.2 Hz, H‐12), 7.73 (1H, d,

O‐), 5.20 (2H, brs, H‐6), 4.37

‐9), 4.35 (2H, q, J = 7.1 Hz, H‐3′), 4.28 (2H, s, H‐1′), 4.07 (3H, s, OCH ‐10), 3.26 (2H, t, J =

.1 Hz, H‐5), 1.37 (3H, t, J = 7.1 Hz, H‐4′). ¹³C‐RMN (CDCl

) δ ppm: 168.89 (COOH’), 150.66 (C‐10),

J = 9.2 Hz, H‐11), 7.24 (1H, s, H‐1), 6.90 (1H, s, H‐4), 6.10 (2H, s, ‐OCH

3H, s, OCH

2

(

3

5

1

3

49.21 (C‐3), 147.46 (C‐2), 147.21 (C‐8), 145.31 (C‐9), 137.60 (C‐13a), 134.11 (C‐4a), 133.11 (C‐12a),

30.24 (C‐13), 125.93 (C‐12), 121.79 (C‐8a), 119.79 (C‐13b), 119.34 (C‐11), 109.12 (C‐4), 108.66 (C‐1),

02.01 (‐OCH

2

O‐), 63.12 (OCH

3

‐C9), 57.41 (C‐6), 56.97 (OCH ‐C10), 40.03 (C‐1′), 28.50 (C‐5). Anal.

3

calcd. for C22

H

20BrNO6: C, 55.71; H, 4.25; N, 2.95. Found: C, 55.73; H, 4.32; N, 2.89.

1

9

3‐(4‐Fluorobenzyl)berberine (compound 6)

1

Yellow solid; yield: 55%; mp: 238 °C; H‐RMN (CDCl

3

) δ ppm: 10.45 (1H, s, H‐8), 7.73 (1H, d, J =

.3 Hz, H‐11), 7.60 (1H, d, J = 9.3 Hz, H‐12), 7.08 (2H, m, H‐4′), 7.08 (2H, m, H‐3′), 6.92 (1H, s, H‐1),

Antibiotics 2020, 9, 381

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6

4

1

.89 (1H, s, H‐4), 6.02 (2H, s, ‐OCH

.04 (3H, s, OCH ‐10), 3.29 (2H, t, J = 5.7 Hz, H‐5). C‐RMN (CDCl

C, C‐5′), 150.52 (C‐10), 149.93 (C‐3), 147.25 (C‐8), 147.10 (C‐2), 146.3 (C‐9), 137.37 (C‐13a), 133.81 (d,

2

O‐), 5.20 (2H, brs, H‐6), 4.65 (2H, s, H‐1′), 4.39 (3H, s, OCH

3

‐9),

13

3

3) δ ppm: 161.72 (d, JCF = 246.5 Hz,

JCF = 3.3 Hz, 1C, C‐2′), 133.81 (C‐4a), 133.30 (C‐12a), 129.61 (C‐13), 129.40 (d, JCF =7 .8 Hz, 1C, C‐3′),

1

25.63 (C‐12), 121.91 (C‐8a), 120.74 (C‐11), 119.86 (C‐13b), 116.35 (d, JCF = 21.5 Hz, 1C, C‐4′), 108.64 (C‐

), 108.64 (C‐4), 101.96 (‐OCH O‐), 63.07 (OCH ‐C9), 57.50 (C‐6), 56.86 (OCH ‐C10), 35.68 (C‐1′), 28.53

: C, 61.84; H, 4.42; N, 2.67. Found: C, 61.75; H, 4.46; N, 2.61.

2

3

(C‐5). Anal. calcd. for C27

H

23BrFNO

4

1

3‐(4‐Cyanobenzyl)berberine (compound 7)

1

Dark green solid; yield: 60%; H‐RMN (300 MHz, CDCl

3) δ ppm: 10.49 (1H, s, H‐8), 7.73 (1H, d,

J = 9.2 Hz, H‐11), 7.67 (1H, d, J = 8.4 Hz, H‐4′), 7.51 (1H, d, J = 9.2 Hz, H‐12), 7.31 (1H, d, J = 8.4 Hz, H‐

′), 6.90 (1H, s, H‐4), 6.77 (1H, s, H‐1), 6.02 (2H, s, ‐OCH O‐), 5.20 (2H, brs, H‐6), 4.65 (2H, s, H‐1′), 4.35

3H, s, OCH ‐9), 4.04 (3H, s, OCH ) δ ppm: 150.64

‐10), 3.30 (2H, t, J = 5.7 Hz, H‐5). ¹³C‐RMN (CDCl

C‐10), 150.20 (C‐3), 147.29 (C‐2), 147.20 (C‐8), 146.38 (C‐9), 143.77 (C‐2′), 137.74 (C‐13a), 133.76 (C‐4a),

3

2

(

1

3

33.21 (C‐12a), 133.21 (C‐4′), 128.88 (C‐3′), 128.46 (C‐13), 125.87 (C‐12), 121.96 (C‐8a), 120.47 (C‐11),

19.71 (C‐13b), 118.29 (C‐CN), 111.43 (C‐5′), 108.76 * (C‐4), 108.44 * (C‐1),102.12 (‐OCH O‐), 63.13

‐C‐9), 57.86 (C‐6), 56.89 (OCH ‐C‐10), 36.81 (C‐1′), 28.48 (C‐5). Anal. calcd. for C28 : C,

2

(OCH

3

H

23BrN

2O

4

6

3.29; H, 4.36; N, 5.27. Found: C, 63.02; H, 4.41; N, 5.22.

1

3‐(4‐Iodomethylbenzyl)berberine (compound 8)

1

Yellow solid; yield: 28%; H‐RMN (300 MHz, CDCl

3) δ ppm: 10.41 (1H, s, H‐8), 7.72 (1H, d, J =

9

6

.3 Hz, H‐11), 7.59 (1H, d, J = 9.3 Hz, H‐12), 7.38 (2H, d, J = 8.3 Hz, H‐4′), 7.06 (2H, d, J = 8.3 Hz, H‐3′),

.91 (1H, s, H‐1), 6.88 (1H, s, H‐4), 6.00 (2H, s, ‐OCH O), 5.20 (2H, brs, H‐6), 4.64 (2H, s, H‐1′), 4.45

‐9), 4.03 (3H, s, OCH

‐10), 3.30 (2H, t, J = 5.8 Hz, H‐5). ¹³C‐RMN (300

) δ ppm: 150.46 (C‐10), 150.03 (C‐3), 147.19 (C‐2), 146.49 (C‐8), 146.30 (C‐9), 138.34 (C‐5′),

2

(2H, s, H‐6′), 4.41 (3H, s, OCH

3

MHz, CDCl

3

1

6

37.85 (C‐2′), 137.58 (C‐13a), 133.65 * (C‐4a), 133.55 * (C‐12a), 129.83 (C‐4′), 129.81 (C‐13), 128.38 (C‐3′),

25.76 (C‐12), 121.80(C‐8a), 111.82 (C‐11), 119.92 (C‐13b), 108.79 (C‐1), 108.57 (C‐4), 63.21 (‐OCH O‐),

3.21 (OCH ‐C9), 58.13 (C‐6), 56.83 (OCH ‐C10), 36.36 (C‐1′), 28.37 (C‐5), 4.73 (C‐6′). * may be reverse.

: C, 52.03; H, 3.90; N, 2.17. Found: C, 51.93; H, 3.98; N, 2.09.

2

3

Anal. calcd. for C28

H

25BrINO

4

1

3‐(4‐Ethenylbenzyl)berberine (compound 9)

1

Yellow solid; yield: 18%; H‐RMN (DMSO‐d

H‐11), 7.81 (1H, d, J = 9.4 Hz, H‐12), 7.17 (1H, s, H‐4), 7.03 (1H,s, H‐1), 6.88 (1H, d, J = 7.9, H‐7’), 6.83

1H, brs, H‐3′), 6.56 (1H, brd, J = 7.9, H‐6′), 6.10 (2H, s, ‐OCH2O‐ [2,3]),), 6.02 (2H, s, ‐OCH2O‐ [4′,5′]),

.88 (2H, brs, H‐6), 4.64 (2H, s, H‐1′), 4.12 (3H, s, OCH3‐9), 4.03 (3H, s, OCH3‐10), 3.16 (3H, t, J = 5.1

6

) δ ppm: 10.02 (1H, s, H‐8), 8.10 (1H, d, J = 9.4 Hz,

(

4

13

Hz, H‐5). C‐RMN (DMSO‐d

6) δ ppm: 150.17 (C‐10), 149.17 (C‐3), 147.90 (C‐4′), 146.40 (C‐2), 146.02

(

C‐5′), 145.40 (C8), 144.18 (C‐9), 137.14 (C‐13a),), 134.01 (C‐4a), 132.83 (C‐2′), 132.76 (C‐12a), 130.11 (C‐

1

6

5

3), 126.12 (C‐11), 121.67 (C‐12), 121.29 (C‐8a), 121.05 (C‐7′), 120.02 (C‐13b), 108.65 * (C‐3′), 108.58 * (C‐

′), 108.47 (C‐4), 108.15 (C‐1), 102.08 (‐OCH2O‐ [2,3]), 101.17 (‐OCH2O‐ [4′,5′]), 62.21 (OCH3‐C‐9),

6.98 (C‐6), 56.93 (OCH3‐C‐10), 35.14 (C‐1′), 27.24(C‐5). * may be reverse. Anal. calcd. for

C29

H

26BrNO : C, 65.42; H, 4.92; N, 2.63. Found: C, 65.36; H, 5.01; N, 2.58.

4

1

3‐(4‐Sulfamoylbenzyl)berberine (compound 10)

1

Green solid; yield: 73%; H‐RMN (DMSO‐d

6) δ ppm: 10.05 (1H, s, H‐8), 8.08 (1H, d, J = 9.3 Hz, H‐

1

1), 7.80 (2H, d, J = 8.0Hz, H‐4’), 7.73 (1H, d, J = 9.3 Hz, H‐12), 7.38 (2H, d, J = 8.0Hz, H3’), 7.17 (1H, s,

H‐4), 6.89 (1H, s, H‐1), 6.08 (2H, s, ‐OCH2O‐), 4.89 (2H, s, H‐6), 4.84 (2H, brs, H‐1′), 4.13 (3H, s, OCH3‐

9

1

), 4.02 (3H, s, OCH3‐10), 3.18 (3H, t, J = 5.1 Hz, H‐5). ¹³C‐RMN (DMSO‐d

49.25 (C‐3), 146.40 (C‐2), 145.61 (C8), 144.36 (C‐9), 143.20 * (C‐2′), 142.59 * (C‐5′), 137.34 (C‐13a), 134.10

6) δ ppm: 150.22 (C‐10),

(

1

C4a), 132.57 (C12a), 128.57 (C‐3′), 126.29 (C‐11), 126.29 (C‐4′), 121.47 (C‐12), 121.30 (C‐8a), 119.89 (C‐

3b), 108.51 (C‐4), 108.00 (C‐1), 102.07 (‐OCH2O‐), 62.06 (OCH3‐C‐9), 56.95 (C‐6), 56.95 (OCH3‐C‐10),

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3

4

5.41 (C‐1′), 27.23 (C‐5). * (C13) et (C‐4″) not observed. Anal. calcd. for C27

.30; N, 4.78. Found: C, 55.33; H, 4.38; N, 4.88.

H

25BrN

2O6S: C, 55.39; H,

1

3‐(4‐Aminomethylbenzyl)berberine (compound 11)

1

Reddish solid; yield: 10%; H‐RMN (CDCl

3

) δ ppm: 10.05 (1H, s, H‐8), 8.17 (2H,brs, NH ), 8.06

2

(

1H, d, J = 9.4 Hz, H‐11), 7.72 (1H, d, J = 9.4 Hz, H‐12), 7.45 (2H, d, J = 8.4 Hz, H‐4′), 7.24 (2H, d, J = 8.4

Hz, H‐3′), 6.93 (1H, s, H‐1), 7.18 (1H, s, H‐4), 6.08 (2H, s, ‐OCH O), 4.88 (2H, brs, H‐6), 4.76 (2H, brs,

H‐1′), 4.14 (3H, s, OCH ‐9), 4.03 (2H, sl, H‐6′), 4.03 (3H, s, OCH

‐10), 3.17(2H, t, J = 5.8 Hz, H‐5). ¹³C‐

RMN (CDCl ) δ ppm: 150.16 (C‐10), 149.19 (C‐3), 147.26 (C‐2), 146.97 (C‐8), 146.30 (C‐9), 145.12 (C‐2′),

37.69 (C‐13a), 134.08 (C‐5′), 133.69 (C‐4a), 133.37 (C‐12a), 129.53 (C‐4′), 128.93 (C‐13), 128.26 (C‐3′),

25.74 (C‐12), 121.90 (C‐8a), 120.53 (C‐11), 119.78 (C‐13b), 108.69 * (C‐4), 108.56 * (C‐1), 102.06 (‐

O‐), 62.05 (OCH ‐C‐9), 57.02 (C‐6), 56.99 (OCH ‐C‐10), 41.85 (C‐6′), 40.35 (C‐1′), 27.21 (C‐5).

Anal. calcd. for C28 : C, 62.81; H, 5.08; N, 5.23. Found: C, 62.78; H, 5.12; N, 5.24.

2

3

1

OCH

2

3

H

27BrN

2O4

1

3‐(4‐Formylbenzyl)berberine (compound 12)

1

Red solid; yield: 75%; H‐RMN (CDCl

2H, d, J = 8.3 Hz, H‐4′), 7.71 (1H, d, J = 9.3 Hz, H‐11), 7.54 (1H, d, J = 9.3 Hz, H‐12), 7.36 (2H, d, J = 8.3

Hz, H‐3′), 6.90 (1H, s, H‐4), 6.83 (1H, s, H‐1), 6.01 (2H, s, ‐OCH O‐), 5.20 (2H, brs, H‐6), 4.79 (2H, s, H‐

3

) δ ppm: 10.02 (1H, s, H‐CHO), 10.46 (1H, s, H‐8), 7.90

(

2

13

1

′), 4.41 (3H, s, OCH

3

‐9), 4.03 (3H, s, OCH

3

‐10), 3.32 (2H, t, J = 5.5 Hz, H‐5). C‐RMN (CDCl ) δ ppm:

3

91.44 (C‐CHO), 150.58 (C‐10), 150.14 (C‐3), 147.26 (C‐2), 146.97 (C‐8), 146.30 (C‐9), 145.20 (C‐2′),

37.69 (C‐13a), 135.45 (C‐5′), 133.69 (C‐4a), 133.37 (C‐12a), 130.78 (C‐4′), 128.93 (C‐13), 128.71 (C‐3′),

25.74 (C‐12), 121.90 (C‐8a), 120.53 (C‐11), 119.78 (C‐13b), 108.69 * (C‐4), 108.56 * (C‐1), 102.06 (‐

OCH

2O‐), 63.16 (OCH

3

‐C‐9), 57.98 (C‐6), 56.86 (OCH ‐C‐10), 36.91 (C‐1′), 28.40 (C‐5). Anal. calcd. for

3

C28H24BrNO

5: C, 62.93; H, 4.53; N, 2.62. Found: C, 62.84; H, 4.67; N, 2.60.

1

3‐[(2H‐1,3‐Benzodioxol‐5‐yl)methyl]berberine (compound 13)

1

Yellow solid; yield: 40%; H‐RMN (DMSO‐d

6) δ ppm: 10.02 (1H, s, H‐8), 8.10 (1H, d, J = 9.4 Hz,

H‐11), 7.81 (1H, d, J = 9.4 Hz, H‐12), 7.17 (1H, s, H‐4), 7.03 (1H,s, H‐1), 6.88 (1H, d, J = 7.9, H‐7’), 6.83

1H, brs, H‐3′), 6.56 (1H, brd, J = 7.9, H‐6′), 6.10 (2H, s, ‐OCH O‐ [2,3]), 6.02 (2H, s, ‐OCH O‐ [4′,5′]),

.88 (2H, brs, H‐6), 4.64 (2H, s, H‐1′), 4.12 (3H, s, OCH ‐9), 4.03 (3H, s, OCH ‐10), 3.16 (3H, t, J = 5.1

) δ ppm: 150.17 (C‐10), 149.17 (C‐3), 147.90 (C‐4′), 146.40 (C‐2), 146.02

C‐5′), 145.40 (C8), 144.18 (C‐9), 137.14 (C‐13a), 134.01 (C‐4a), 132.83 (C‐2′), 132.76 (C‐12a), 130.11 (C‐

(

2

4

3

13

Hz, H‐5). C‐RMN (DMSO‐d

6

(

1

6

3), 126.12 (C‐11), 121.67 (C‐12), 121.29 (C‐8a), 121.05 (C‐7′), 120.02 (C‐13b), 108.65 * (C‐3′), 108.58 * (C‐

′), 108.47 (C‐4), 108.15 (C‐1), 102.08 (‐OCH O‐ [2,3]), 101.17 (‐OCH O‐ [4′,5′]), 62.21 (OCH ‐C‐9), 56.98

‐C‐10), 35.14 (C‐1′), 27.24(C‐5). * may be reverse. Anal. calcd. for C29 : C,

2

3

(

C‐6), 56.93 (OCH

3

H

26BrNO

4

6

1.10; H, 4.40; N, 2.54. Found: C, 61.02; H, 4.48; N, 2.51.

13[(2,3,4‐Trimethoxyphenyl)methyl]berberine (compound 14)

1

Brown solid; yield: 51%; H‐RMN (DMSO‐d

6) δ ppm: 10.02 (1H, s, H‐8), 8.12 (1H, d, J =9.3 Hz,

H‐11), 7.77 (1H, d, J = 9.3 Hz, H‐12), 7.16 (1H, s, H‐4), 6.92 (1H, s, H‐1), 6.65 (1H, d, J = 8.6 Hz, H‐7’),

.34 (1H, d, J = 8.6 Hz, H‐6′), 6.08 (2H, s, ‐OCH2O‐),4.88 (2H, brs, H‐6), 4.51 (2H, brs, H‐1′), 4.13 (3H,

s, OCH3‐9), 4.04 (3H, s, OCH3‐10), 3.91 (3H, s, OCH3‐5′), 3.76 * (3H, s, OCH3‐3′), 3.75 * (3H, s, OCH3‐

6

1

3

4

1

′), 3.16 (3H, t, J = 5.7 Hz, H‐5). C‐RMN (DMSO‐d

6

) δ ppm: 152.64 (C‐3′), 150.61 (C‐5′), 150.12 (C‐10),

49.13 (C‐3), 146.38 (C‐2), 145.21 (C8), 144.14 (C‐9), 142.04 (C‐4′), 137.03 (C‐13a), 133.93 (C‐4a), 132.94

(

1

3

C‐12a), 130.11 (C‐13), 126.22 (C‐11), 124.42 (C‐2′), 122.72 (C‐7′), 121.46 (C‐12), 121.11 (C‐8a), 119.98 (C‐

3b), 108.40 (C‐1), 108.04 (C‐4), 107.84 (C‐6′), 102.02 (‐OCH2O‐),62.04 (OCH3‐C‐9), 60.43 * (OCH3‐C‐

′), 60.30 * (OCH3‐C‐4′), 56.91 (C‐6), 56.91 (OCH3‐C‐10), 55.76 (OCH3‐C‐5′), 30.51 (C‐1′), 27.19(C‐5). *

may be reverse. Anal. calcd. for C30

H

30BrNO : C, 60.41; H, 5.07; N, 2.35. Found: C, 60.36; H, 5.10; N,

7

2

.35.

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13[(3,4,5‐Trimethoxyphenyl)methyl]berberine (compound 15)

1

Brown solid; yield: 50%; H‐RMN (DMSO‐d

6) δ ppm: 10.03 (1H, s, H‐8), 8.13 (1H, d, J = 9.3 Hz,

H‐11), 7.88 (1H, d, J = 9.3 Hz, H‐12), 7.17 * (1H, brs, H‐4), 7.15 * (1H,brs, H‐1), 6.47 (2H, s, H‐3′, H‐7’),

6

1

.11 (2H, s, ‐OCH2O‐),4.89 (2H, brs, H‐6), 4.69 (2H, s, H‐1′), 4.14 (3H, s, OCH3‐9), 4.04 (3H, s, OCH3‐

13

0), 3.65 (9H, brs, OCH3‐4′, OCH3‐5′, OCH3‐6′), 3.17 (2H, brt, J = 4.8 Hz, H‐5). C‐RMN (DMSO‐d )

6

δ ppm: 153.22 (C‐4′), 153.22 (C‐6′), 150.05 (C‐10), 149.12 (C‐3), 146.38 (C‐2), 145.35 (C8), 144.08 (C‐9),

1

37.14 (C‐13a), 136.27 (C‐5′), 134.68 (C‐2′), 133.91 (C‐4a), 132.86 (C‐12a), 130.07 (C‐13), 126.05 (C‐11),

21.54 (C‐12), 121.22 (C‐8a), 120.08 (C‐13b), 108.35 * (C‐1), 108.33 * (C‐4), 105.69 (C‐3′), 105.69 (C‐7′),

01.99 (‐OCH2O‐),61.97 (OCH3‐C‐9), 59.96 (OCH3‐C‐5′), 56.93 (C‐6), 56.87 (OCH3‐C‐10), 56.10

(OCH3‐C‐4′), 56.10 (OCH3‐C‐6′), 35.55 (C‐1′), 27.23(C‐5).). * may be reverse. Anal. calcd. for

C30H30BrNO

7: C, 60.41; H, 5.07; N, 2.35. Found: C, 60.21; H, 5.02; N, 2.33.

4

‐[2‐(Berberin‐13‐yl) acetyl]‐2,6‐dimethoxyphenyl Benzoate (compound 16)

1

Yellow solid; yield: 16%; H‐RMN (CDCl

3) δ ppm: 10.03 (1H, s, H‐8), 8.19(1H, d, J = 9.5 Hz, H‐

1

1), 8.15 (2H, m, H‐3″), 7.82 (1H, d, J = 9.5 Hz, H‐12), 7.80 (1H, m, H‐5″), 7.65 (2H, m, H‐4 ″), 7.64 (2H,

s, H‐4’), 7.19 (1H, s, H‐4), 6.89 (1H, s, H‐1), 6.11 (2H, s, ‐OCH2O‐), 5.37 (2H, brs, H‐6), 4.90 (2H, br s,

H‐1′), 4.14 (3H, s, OCH3‐9), 4.07 (3H, s, OCH3‐10), 3.90 (3H, s, OCH3‐5’), 3.15 (3H, t, J = 5.6 Hz, H‐5).

13

C‐RMN (CDCl

3) δ ppm: 196.91 (C‐2’), 163.24 (C‐1″), 152.24 (C‐5′), 153.31 (C‐10), 149.44 (C‐3), 146.79

(C‐2), 145.69 (C‐8), 144.20 (C‐9), 134.35 (C‐4a), 133.99 (C‐5″), 133.59 (C‐12a), 132.86 * (C‐6′), 132.84 * (C‐

3

1

′), 129.90 (C‐3″), 129.14 (C‐4″), 128.02 (C‐13), 127.66 (C‐2″), 126.28 (C‐11), 121.52 (C‐12), 120.90 (C‐8a),

19.93 (C‐13b), 108.52 (C‐4), 107.91 (C‐1), 105.74 (C‐4’), 102.13 (‐OCH2O‐), 62.07 (OCH3‐C‐9), 57.02

(OCH3‐C‐10), 56.78 (C‐6), 56.50 (OCH3‐C‐5’), 27.24 (C‐5). * may be reverse. Anal. calcd. for

C37H32BrNO

9: C, 62.19; H, 4.51; N, 1.96. Found: C, 62.20; H, 4.48; N, 2.00.

4

‐[2‐(Berberin‐13‐yl)acetyl]‐3‐(benzoyloxy)phenyl Benzoate (compound 17)

1

Yellow solid; yield: 55%; H‐RMN (DMSO‐d

6) δ ppm: 9.97 (1H, s, H‐8), 8.52 (1H, d, J = 9.4 Hz, H‐

4

7

’), 8.03 (4H, m, H‐3″), 8.20 (4H, m, H‐3″), 8.13(1H, d, J = 9.4 Hz, H‐11), 7.89 (1H, d, J = 9.4 Hz, H‐12),

.80 (2H, m, H‐5″), 7.66 (4H, m, H‐4″), 7.62 (1H, brs, H‐7′), 7.61 (1H, dd, J = 9.4 Hz, J = 2.0 Hz, H‐5′),

.15 (1H, s, H‐4), 6.86 (1H, s, H‐1), 6.16 (2H, s, ‐OCH

2

O‐), 5.25 (2H, s, H‐1′), 4.82 (2H, brs, H‐6), 4.10

13

(

3H, s, OCH

3

‐9), 4.06 (3H, s, OCH ‐10), 3.08 (3H, t, J = 4.9 Hz, H‐5). C‐RMN (DMSO‐d ) δ ppm: 196.23

3

6

C‐2’), 164.18, 163.98 (C‐1″), 154.74 (C‐6′), 150.27 (C‐10), 149.44 (C‐3), 146.91 (C‐2), 145.56 (C‐8), 144.13

C‐9), 137.06 (C‐13a), 134.43 (C‐4a), 134.10, 134.07 (C‐5″), 132.77 (C‐12a), 132.19 (C‐4′), 129.98,129.76

C‐4″), 129.05, 128.85 (C‐3″), 128.42, 128.34 (C‐2″), 127.08 (C‐13), 126.97 (C‐3′), 126.09 (C‐11), 121.52 (C‐

1

2), 120.87 (C‐8a), 120.40 (C‐5′), 119.78 (C13b), 118.62 (C‐7′), 108.50 (C‐1), 107.89 (C‐4), 102.20 (‐

OCH O‐), 62.02 (OCH ‐C‐9), 57.04(OCH ‐C‐10), 56.67 (C‐6), 43.88 (C‐1′), 27.24 (C‐5). Anal. calcd. for

: C, 65.12; H, 4.16; N, 1.81. Found: C, 65.10; H, 4.21; N, 1.80.

2

3

C

42H

32BrNO

9

8

‐{4‐[(Berberin‐13‐yl)methyl]phenoxy}‐2H‐chromen‐2‐one (compound 18)

1

Yellow solid; yield: 12%; H‐RMN (DMSO‐d

6) δ ppm: 10.06 (1H, s, H‐8), 8.09 (1H, d, J = 9.5 Hz,

H‐12), 8.00 (1H, d, J = 9.5Hz, H3″), 7.78(1H, d, J = 9.5 Hz, H‐11), 7.65 (1H, d, J = 8.8Hz, H‐4″), 7.47 (2H,

d, J = 8.3Hz, H3’), 7.21 (2H, d, J = 8.3Hz, H‐4’), 7.17 (1H, s, H‐4), 7.08 (1H, d, J = 2.5, H‐7″), 7.02 (1H,

dd, J = 2.5Hz, J = 8.8Hz, H‐5″), 6.95 (1H, s, H‐1), 6.30 (1H, d, J = 9.5, H‐2″), 6.07 (2H, s, ‐OCH2O‐), 5.20

(

1H, s, H‐6’), 4.88 (2H, brs, H‐6), 4.76 (2H, s, H‐1′), 4.12 (3H, s, OCH3‐9), 4.02 (3H, s, OCH3‐10), 3.16

13

3H, t, J = 5.5 Hz, H‐5). C‐RMN (DMSO‐d

6) δ ppm: 161.44 (C‐6″), 160.26 (C‐1″), 155.32 (C‐8″), 152.22

C‐10), 149.21 (C‐3), 146.39 (C‐2), 145.50 (C‐8), 144.31 (C‐3″),144.23 (C‐9), 139.12 (C‐2’), 137.17 (C‐13a),

1

5

37.76 (C‐5′), 134.06 (C‐4a), 132.69 (C‐12a), 129.89 (C‐13), 129.89 (C‐4″), 129.54 (C‐3’), 128.84 (C‐4’),

26.17 (C‐11), 121.67 (C‐12), 121.26 (C‐8a), 119.98 (C‐13b), 112.96 (C‐5″), 112.63 (C‐2″), 112.53 (C‐9″),

08.51 (C‐4), 108.08 (C‐1), 102.06 (‐OCH2O‐),101.56 (C‐7″), 69.62 (C‐6’), 62.07 (OCH3‐C‐9), 56.95 (C‐6),

6.90 (OCH3‐C‐10), 35.28 (C‐1’), 27,26 (C‐5). Anal. calcd. for C37

H

30BrNO : C, 65.30; H, 4.44; N, 2.06.

7

Found: C, 65.25; H, 4.48; N, 2.02.

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9

‐{[4‐(1,3‐Benzothiazol‐2‐yl)phenyl]methyl}berberine (compound 19)

1

Red solid; yield: 20%; H‐RMN (DMSO‐d

6) δ ppm: 10.06 (1H, s, H‐8), 8.13 (1H, brd, J = 7.8, H‐7″),

8

.11 (1H, d, J = 9.4 Hz, H‐11), 8.09 (2H, d, J = 8.1 Hz, H‐4’), 8.04 (1H, brd, J = 7.8 Hz, H‐4″), 7.81 (1H, d,

J = 9.4 Hz, H‐12), 7.55 (1H, brt, J = 7.8 Hz, H‐5″), 7.47(1H, brt, J = 7.8Hz, H‐6″), 7.39 (2H, d, J = 8.1Hz,

H3’), 7.18 (1H, s, H‐4), 6.99 (1H, s, H‐1), 6.08 (2H, s, ‐OCH2O‐), 4.90 (2H, brs, H‐6), 4.86 (2H, s, H‐1′),

1

3

4

1

.14 (3H, s, OCH3‐9), 4.03 (3H, s, OCH3‐10), 3.19 (3H, t, J = 5.3 Hz, H‐5). C‐RMN (DMSO‐d

6

) δ ppm:

66.70 (C‐2″), 153.50 (C‐3a″), 150.19 (C‐10), 149.23 (C‐3), 146.40 (C‐2), 145.54 (C8), 144.29 (C‐9), 143.73

(

C‐2′), 137.27 (C‐13a), 134.40 (C‐7a″), 134.10 (C4a), 132.63 (C12a), 131.43 (C‐5′), 129.45 (C‐13), 129.01

C‐3′), 127.81 (C‐4′),126.62(C‐5″), 126.25 (C‐11), 125.51 (C‐6″), 122.77 (C‐4″), 122.29 (C‐7″), 121.54 (C‐

1

5

4

2), 121.22 (C‐8a), 119.88 (C‐13b), 108.46 (C‐1), 108.11 (C‐4), 102.02 (‐OCH2O‐),62.01 (OCH3‐C‐9),

6.89 (C‐6), 56.89 (OCH3‐C‐10), 35.49 (C‐1′), 27.22 (C‐5). Anal. calcd. for C34

H

27BrN

2

O S: C, 63.85; H,

4

.26; N, 4.38. Found: C, 63.75; H, 4.12; N, 4.40.

9

‐{[4‐(1,3,4‐Oxadiazol‐2‐yl)phenyl]methyl}berberine (compound 20)

1

Yellow solid; yield: 57%; H‐RMN (DMSO‐d

6) δ ppm: 10.06 (1H, s, H‐8), 9.33 (1H, s, H‐2″), 8.09

(

1H, d, J = 9.5 Hz, H‐11), 8.02(2H, d, J = 8.3 Hz, H4′), 7.79 (1H, d, J =9.5 Hz, H‐12), 7.42(2H, J = 8.3 Hz,

H3′), 7.18 (1H, s, H‐4), 6.94 (1H,s, H‐1), 6.08 (2H, s, ‐OCH O‐ [2,3]), 4.90 (2H, brs, H‐6), 4.87 (2H, brs,

H‐1′), 4.14 (3H, s, OCH ‐9), 4.03 (3H, s, OCH ‐10), 3.18 (2H, t, J = 5.2 Hz, H‐5). C‐RMN (DMSO‐d

ppm: 163.35 (C‐1″), 154.47 (C‐2″), 150.22 (C‐10), 149.3 (C‐3), 146.5 (C‐2), 145.61 (C8), 144.35 (C‐9),

2

13

3

6) δ

1

43.52 (C‐2′), 137.92 (C‐5′), 137.34 (C‐13a), 134.11 (C‐4a), 132.63 (C‐12a), 129.34 (C‐13), 129.11 (C‐3′),

27.40 (C‐4′), 126.28 (C‐11), 121.79 (C‐12), 121.51 (C‐8a), 119.89 (C‐13b), 108.50 (C‐4), 108.11 (C‐1),

02.07 (‐OCH

2O‐ [2,3]), 62.55 (OCH

3

‐C‐9), 57.44 (C‐6), 57.44 (OCH ‐C‐10), 36.08 (C‐1′), 27.27(C‐5).

3

Anal. calcd. for C29

H

24BrN

3O5: C, 60.64; H, 4.21; N, 7.32. Found: C, 60.51; H, 4.25; N, 7.29.

N‐({4‐[(Berberin‐13‐yl)methyl]phenyl}methylidene)hydroxylamine (compound 21)

Compound 12 (0.4 g, 0.74 mmol), hydroxylamine hydrochloride (0.16 g, 2.2 mmol, 3 eq) in

EtOH/H

2O (10/2 mL) was added in sodium acetate (0.24 g, 3 mmol, 4 eq). The mixture was stirred at

6

0 °C for 1 h, poured onto water (50 mL). The solution was extracted with ether (3  20 mL) and the

organic phase was removed under vacuum and reprecipitate in CHCl

3

. The yellow precipitate was

1

collected washed with CHCl

3

to give pure compound 21. Yellow solid; yield: 10%; H‐RMN (DMSO‐

d6

) δ ppm: 11.21 (1H, s, OH), 10.02 (1H, s, H‐8), 8.09 (1H, d, J = 9.4 Hz, H‐11), 7.78 (1H, d, J = 9.4 Hz,

H‐12), 8.12 (1H, s, H‐6′), 7.57 (2H, d, J = 8.1Hz, H‐4′),7.21 (2H, d, J = 8.1 Hz, H‐3′), 7.16 (1H, s, H‐1),

6

4

.96 (1H, s, H‐4), 6.08 (2H, s, ‐OCH

2

O‐), 4.88 (2H, brs, H‐6), 4.76 (2H, s, H‐1′), 4.13 (3H, s, OCH

3

‐9),

13

.02 (3H, s, OCH ‐10), 3.17 (2H, t, J = 5.7 Hz, H‐5). C‐RMN (DMSO‐d

3

6) δ ppm: 150.19 (C‐10), 149.22

(

1

C‐3), 147.65 (C‐6′), 146.40 (C‐2), 145.49 (C‐8), 144.30(C‐9), 140.32 (C‐2′), 137.22 (C‐13a), 134.04 (C‐5′),

33.12 (C‐4a), 131.67 (C‐12a), 129.68 (C‐13), 128.41 (C‐4′), 127.04 (C‐3′), 126.21 (C‐12), 121.59 (C‐11),

21.27 (C‐8a), 119.97 (C‐13b), 108.49 (C‐4), 108.15 (C‐1), 102.05 (‐OCH O‐), 62.05 (OCH ‐C‐9), 57.00

‐C‐10) 56.93 (C‐6), 35.43 (C‐1′), 27.25 (C‐5). Anal. calcd. for C27 : C, 60.34; H, 4.69; N,

.21. Found: C, 60.29; H, 4.71; N, 5.23.

2

3

(OCH

3

H

25BrN2O

5

(2E)‐3‐{4‐[(Berberin‐13‐yl)methyl]phenyl}prop‐2‐enoic Acid (compound 22)

Compound 12 (1 g, 1.8 mmol), malonic acid (0.4 g, 4.1 mmol, 2.2 eq) in MeOH/ethyl acetate (5/2

mL) was added in acetic acid (15 mL). Then was added piperidine (0.5 mL, 4 mmol, 2.7 eq) and the

reaction mixture was submitted to microwave irradiation in a CEM microwave apparatus (Power

time, Acetic acid, Tmax: 110 °C, 30 min). The solution was poured onto ice/water (200 mL) and the

precipitate was collected by sucion and washed with excess water to give 22 as a reddish powder.

1

Red solid; yield: 50%; H‐RMN (DMSO‐d

6) δ ppm: 10.04 (1H, s, H‐8), 8.09 (1H, d, J = 9.4 Hz, H‐11),

7

=

.76 (1H, d, J = 9.4 Hz, H‐12), 7.68 (2H, d, J = 8.2 Hz, H‐4′), 7.58 (1H, d, J = 16.0 Hz, H‐6′), 7.22 (2H, d, J

8.2 Hz, H‐3′), 7.17 (1H, s, H‐1), 6.93 (1H, s, H‐4), 6.53 (1H, d, J = 16.0 Hz, H‐7′), 6.08 (2H, s, ‐OCH O‐

‐9), 4.02 (3H, s, OCH ‐10), 3.16 (2H, t, J = 5.7

) δ ppm: 167.78 (C‐8′), 150.18 (C‐10), 149.19 (C‐3), 146.38 (C‐2), 145.49

2

)

, 4.87 (2H, brs, H‐6), 4.77 (2H, s, H‐1′), 4.12 (3H, s, OCH

3

13

Hz, H‐5). C‐RMN (DMSO‐d

6

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(C‐8), 144.26 (C‐9), 142.39 (C‐6′), 141.13 (C‐2′), 137.20 (C‐13a), 134.03 (C‐5′), 133.12 (C‐4a), 132.65 (C‐

1

7

2a), 129.68 (C‐13), 128.71 (C‐4′), 128.55 (C‐3′), 126.18 (C‐12), 121.58 (C‐11), 121.24 (C‐8a), 120.40 (C‐

′), 119.95 (C‐13b),), 108.47 (C‐4), 108.12 (C‐1), 102.04 (‐OCH O‐), 62.04 (OCH ‐C‐9), 56.95 (C‐6), 56.90

‐C‐10), 27.24(C‐5). Anal. calcd. for C30 : C, 62.51; H, 4.55; N, 2.43. Found: C, 62.41; H,

.58; N, 2.47.

2

3

(OCH

3

H

26BrNO

6

4

(

2E)‐3‐{4‐[(Berberin‐13‐yl)methyl]phenyl}‐N‐[2‐(4‐sulfamoylphenyl)ethyl]prop‐2‐enamide

compound 23)

Compound 22 (0.2 g, 0.36 mmol), 1‐(3‐dimethyl‐aminopropyl)‐3‐ethylcarbodiimide

hydrochloride: EDCI (0.06 g, 0,4 mmol, 1,1 eq) and 1‐hydroxybenzotriazole: HOBT (0.05 g, 0.4 mmol,

1

.1 eq) in DMF (10 mL) were stirred for 2.5 h at r.t., then was added 4‐(2‐aminoethyl) benzene‐

sulfonamide (0.1 g, 0.45 mmol, 1.3 eq) under stirring for 12 h. The reaction mixture was poured onto

ice/water (100 mL), the precipitate was filtered off and washed with excess water to give 23 as a dark

1

green powder. Green solid; yield: 16%; H‐RMN (DMSO‐d

6) δ ppm: 10.04 (1H, s, H‐8), 8.21 (1H, t, J =

5

7

NH

.7 Hz, NH), 8.09 (1H, d, J = 9.3 Hz, H‐11), 7.77 (1H, d, J = 9.3 Hz, H‐12), 7.73 (2H, d, J = 8.2 Hz, H5″),

.55 (2H, d, J = 8.0 Hz, H‐4′), 7.41 (2H, d, J = 8.2 Hz, H‐4″), 7.40 (1H, d, J = 15.8 Hz, H‐6′), 7.30 (2H, s,

), 7.21 (2H, d, J = 8.0 Hz, H‐3′), 7.17 (1H, s, H‐4), 6.94 (1H, s, H‐1), 6.57 (1H, d, J = 15.8 Hz, H‐7′),

2

6

1

.08 (2H, s, ‐OCH

2

O‐), 4.87 (2H, brs, H‐6), 4.76 (2H, s, H‐1′), 4.12 (3H, s, OCH

3

‐9), 4.02 (3H, s, OCH

3

‐

)

13

0), 3.44 (2H, m, H‐1″), 3.16 (2H, t, J = 5.7 Hz, H‐5), 2.85 (2H,t, J = 7.0 Hz, H‐2″). C‐RMN (DMSO‐d

6

δ ppm: 164.9 (C‐8′), 150.2 (C‐10), 149.2 (C‐3), 146.4 (C‐2), 145.5 (C‐8), 144.2 (C‐9), 143.6 (C‐6′’), 142.0

(C‐2′), 140.54 (C‐6′), 138.0 (C‐3″), 137.2 (C‐13a), 134.0 (C‐4a), 133.5 (C5′), 132.7 (C‐12a), 129.7 (C‐13),

1

3

6

29.1 (C‐4″), 128.6 (C‐3′), 128.2 (C‐4′), 126.2 (C‐11), 125.6 (C5″), 122.2 (C‐7′), 121.6 (C‐12), 121.2 (C‐8a),

19.9 (C‐13b), 108.5 (C‐4), 108.1 (C‐1), 102.3 (‐OCH O‐), 62.0 (OCH ‐C‐9), 56.9 (C‐6), 56.9 (OCH ‐C‐10),

S: C, 60.16; H, 4.78; N, 5.54. Found: C,

2

3

5.4 (C‐1′), 34.7 (C‐2″), 27.2 (C‐5). Anal. calcd. for C38

H36BrN

3O7

0.21; H, 4.81; N, 5.44.

N‐{3‐[(2E)‐3‐{4‐[(Berberin‐13‐yl)methyl]phenyl}prop‐2‐enoyl]‐4‐hydroxyphenyl}acetamide

compound 24)

(

Compound 12 (0.44 g, 0.8 mmol, 0.7 eq) and N‐(3‐acetyl‐4‐hydroxyphenyl)acetamide (0.2 g, 1.1

mmol, 1.2 eq) were solubilized in methanol (20 mL). To this solution was added LiOH (0.15 g, 6 mmol,

eq.) and the solution was submitted to microwave irradiation using a CEM apparatus (mode open

8

vessel, new method, mode standard, hold time: 2 min., power: 300 W, run time: 20 min.). At the end

of the irradiation the solvent was removed under vacuum to give a red mixture. Addition of 1N HCl

(

50 mL) under ice give a yellow precipitate that was filtered with ether to give a yellow solid of 24.

1

Yellow solid; yield: 40%; H‐RMN (DMSO‐d

6) δ ppm: 11,62 (1H, s, OH‐2″), 10.05 (1H, s, H‐8), 9.92

(1H, s, NH), 8.18 (1H, d, J = 2.5 Hz, H‐6″), 8.10 (1H, d, J = 9.4 Hz, H‐11), 7.80 (2H, d, J = 7.2 Hz, H‐3′),

7

.79 (1H, d, J = 9.4 Hz, H‐12), 7.77 (1H, d, J = 15.6 Hz, H‐6′), 7.73 (1H, d, J = 15.6 Hz, H‐7′), 7.65 (1H,

dd, J = 8.8 Hz, J = 2.5 Hz, H‐4″), 7.28 (2H, d, J = 7.2 Hz, H‐4′), 7.18 (1H, s, H‐4), 6.96 (1H, s, H‐1), 6.96

1H, d, J = 8.8 Hz, H‐3″), 6.09 (2H, s, ‐OCH O‐), 4.88 (2H, brs, H‐6), 4.81 (2H, s, H‐1′), 4.13 (3H, s, OCH

), 4.03 (3H, s, OCH ‐10), 3.17 (2H, t, J = 5.4 Hz, H‐5), 2.02 (3H,s, CH

) δ ppm: 193.2 (C‐8′),168.2 (CO‐NHCOCH ), 156.9 (C‐2″), 150.6 (C‐10), 149.5 (C‐3), 146.2 (C‐2), 145.2

(

9

2

3

‐

13

3

‐NHCOCH ). C‐RMN (DMSO‐

3

d6

3

(

C‐8), 144.5 (C‐9), 143.6 (C‐6′), 142.5 (C‐2′), 142.4 (C‐5′), 137.5 (C‐13a), 134.3 (C‐4a), 132.9 (C‐12a), 129.9

C‐13), 129.1 (C‐3′), 128.4 (C‐4′), 127.6 (C‐4″), 125.9 (C‐11), 122.7 (C‐7′), 121.6 (C‐8a),121.6 (C‐1″), 121.4

C‐5″), 121.3 (C‐12), 120.6 (C‐6″), 120.2 (C‐13b), 117.4 (C‐3″), 108.2 (C‐4), 107.9 (C‐1), 101.8 (‐OCH

1.7 (OCH ‐C‐9), 56.6 (C‐6), 56.6 (OCH ‐C‐10), 35.2 (C‐1′), 27.0 (C‐5), 23.4 (C‐CH NHCOCH ). Anal.

calcd. for C38 : C, 64.32; H, 4.69; N, 3.95. Found: C, 64.30; H, 4.671; N, 4.00.

2O‐),

6

3

H

33BrN

2O7

(

2E)‐3‐{4‐[(Berberin‐13‐yl)methyl]phenyl}‐1‐(2‐hydroxy‐4‐methoxyphenyl)prop‐2‐en‐1‐one

compound 25).

Using the same protocol as for 24 with 2‐hydroxy‐4‐methoxyacetophenone gives 25. Green solid;

1

yield: 78%; H‐RMN (DMSO‐d

6) δ ppm: 10.08 (1H, s, H‐8), 8.27 (1H, d, J = 9.1 Hz, H‐6″), 8.10 (1H, d, J

Antibiotics 2020, 9, 381

9 of 31

=

9.4 Hz, H‐11), 8.01 (2H, d, J = 15.5 Hz, H‐a),7.92 (2H, d, J = 8.3Hz, H‐4’), 7.80 (2H, d, J = 15.5 Hz, H‐

b), 7.78 (1H, d, J = 9.4 Hz, H‐12), 7.28 (2H, d, J = 8.3Hz, H3’), 7.18 (1H, s, H‐4), 6.96 (1H, s, H‐1), 6.56

1H, dd, J = 2.5Hz, J = 9.1Hz, H‐5″), 6.53 (1H, d, J =2.5, H‐3″), 6.08 (2H, s, ‐OCH2O‐), 4.90 (2H, brs, H‐

′), 4.81 (2H, brs, H‐6), 4.13 (3H, s, OCH3‐9), 4.02 (3H, s, OCH3‐10), 3.85 (3H, s, OCH3‐4″),3.17 (3H, t,

(

1

13

J =5.7 Hz, H‐5). C‐RMN (DMSO‐d

49.28 (C‐3), 146.46 (C‐2), 145.59 (C‐8), 144.30 (C‐9), 143.52 (C‐b, 142.19 (C‐2’), 137.25 (C‐13a), 134.10

C‐4a), 133.59 (C‐5′), 132.78 (C‐6″), 132,66 (C‐12a), 129.83 (C‐4’), 129.63 (C‐13), 128.67 (C‐3’), 126.23 (C‐

6) δ ppm: 191.82 (C‐b’), 166.04 (C‐2″), 165.68 (C‐4″), 150.24 (C‐10),

1

(

1

5

1), 121.64 (C‐12), 121,29 (C‐8a), 121.26 (C‐a), 119,97 (C‐13b), 113.91 (C‐1″), 108.54 (C‐4), 108.14 (C‐1),

07.49 (C‐5″), 102.11 (‐OCH2O‐), 100.95 (C‐3″),62.10 (OCH3‐C‐9), 56,98 (C‐6), 56,93 (OCH3‐C‐10),

5.81 (OCH3‐4″), 35.59 (C‐1’), 27,29 (C‐5). Anal. calcd. for C37

H

32BrNO : C, 65.11; H, 4.73; N, 2.05.

7

Found: C, 65.02; H, 4.76; N, 2.01.

2

‐{4‐[(Berberin‐13‐yl)methyl]phenyl}‐7‐methoxy‐4H‐chromen‐4‐one (compound 26)

Compound 25 (0.17 g, 0.24 mmol) was solubilized in a solution of DMSO (20 mL) and Iodine (17

mg, 10% weight). The solution was submitted to microwave irradiation (CEM, Power time, Tmax:

1

40 °C, 30 min). The solution was poured on ice/1N HCl (100 mL) to give a yellow precipitate of 26

1

which was washed with excess water. Yellow solid; yield: 18%; H‐RMN (DMSO‐d

6) δ ppm: 10.06

(1H, s, H‐8), 8.10 (1H, d, J = 9.3 Hz, H‐11), 8.06 (2H, d, J = 8.3Hz, H‐4’), 7.95 (1H, d, J = 8.5Hz, H‐5″),

7

4

7

1

.79 (1H, d, J = 9.3 Hz, H‐12), 7.38 (2H, d, J = 8.3Hz, H3’), 7.29 (1H, d, J = 2.2Hz, H8″), 7.18 (1H, s, H‐

), 7.08 (1H, dd, J = 8.5Hz, J = 2.2Hz, H‐6″), 6.97 (1H, s, H3″), 6.95 (1H, s, H‐1), 6.08 (2H, s, ‐OCH2O‐),

.90 (2H, brs, H‐6), 4.85 (2H, s, H‐1′), 4.12 (3H, s, OCH3‐9), 4.02 (3H, s, OCH3‐10), 3.92 (3H, s, OCH3‐

″), 3.18 (3H, t, J = 5.1 Hz, H‐5). ¹³C‐RMN (DMSO‐d

) δ ppm: 163.8 (C‐7″), 161.7 (C‐2″), 157.5 (C‐9″),

6

50.3 (C‐10), 149.3 (C‐3), 146.5 (C‐2), 145.6 (C8), 144.2 (C‐9), 143.5 (C‐2′), 137.4 (C‐13a), 134.2 (C4a),

32.6 (C12a), 129.5 (C‐5′), 128.8 (C‐3′), 127.1 (C‐4′), 126.4 (C‐5″), 126.1 (C‐11), 121.6 (C‐12), 121.3 (C‐8a),

19.9 (C‐13b), 117.1 (C‐10″), 114.7 (C‐6″), 108.6 (C‐4), 107.9 (C‐1), 107.6 (C‐3″), 102.1 (‐OCH2O‐), 101.1

(C‐8″), 62.2 (OCH3‐C‐9), 57.1 (C‐6), 56.9 (OCH3‐C‐10), 56.2 (OCH3‐C‐7″), 35.7 (C‐1′), 27.5 (C‐5). * (C13)

et (C‐4″) not observed. Anal. calcd. for C37 : C, 65.30; H, 4.44; N, 2.06. Found: C, 65.20; H,

H

30BrNO

7

4

2

.49; N, 1.99.

.2. Biology

.2.1. Microorganism Strains Used and Growth Conditions

Reference strains used in the study were obtained either from the American Type Culture

Collection (ATCC, Molsheim Cedex France), the German Leibniz Institute (DSMZ, Braunschweig,

Germany) or the French Pasteur Institute (CIP, Paris, France). Environmental and pathogenic Gram‐

negative bacterial strains used were: Acinetobacter baumannii (CIP 110431), Citrobacter farmeri (ATCC

5

4

1633), Citrobacter rodentium (ATCC 51116), Escherichia coli (ATCC 8739), Helicobacter pylori (ATCC

3504), Klebsiella pneumoniae (DSMZ 26371), Pseudomonas aeruginosa (ATCC 9027), fluoroquinolone‐

and carbapenem‐resistant Pseudomonas aeruginosa (CIP 107398), Salmonella enterica (CIP 80.39), Shigella

flexneri (ATCC 12022), Vibrio alginolyticus (DSM 2171) and Vibrio diabolicus (from Dr Aurélie

Tasiemski, Institut Pasteur Lille). Environmental and pathogenic Gram‐positive bacterial strains used

were: Arthrobacter gandavensis (DSM 2447), Bacillus subtilis (ATCC 6633), nisin‐resistant B. subtilis

(

DSMZ 347), Clostridium perfringens (ATCC 13124), Clostridium difficile (DSM 1296), vancomycin‐

resistant Enterococcus faecalis (DSMZ 13591), Lactococcus lactis (DSM 20481), Staphylococcus aureus

ATCC 6538P), methicillin‐resistant S. aureus strain MRSA USA300 (ATCC BAA‐1717 USA 300 CA‐

(

MRSA) and Streptococcus pyogenes (DSM 20565). Mycobacterium smegmatis (mc2155, ATCC 700084)

was used as model of Mycobacterium species. Bacteria were cultured as previously described [15–18]

Briefly, most of the bacterial strains were routinely grown on Luria Bertani (LB) agar plates and LB

broth at 37 °C in aerobic condition except H. pylori that was grown in BHI in micro‐aerobic condition

using microaerobic BD GasPak generator (Sigma‐Aldrich, Lyon, France). M. smegmatis was cultured

in Middlebrook 7H10 agar plate and Middlebrook 7H10 broth at 37 °C in aerobic condition. E. faecalis,

C. difficile, C. perfringens and S. pyogenes were cultured in Brain Heart Infusion (BHI) agar plates and

Antibiotics 2020, 9, 381

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BHI broth at 37 °C in anaerobic chamber (Coy Laboratory Products, Grass Lake, MI, USA). Fungal

strain Candida albicans (DSM 10697) was grown on potato dextrose (PD) agar plates.

2

.2.2. Antimicrobial Activity Assay

Antimicrobial activity of berberine and its derivatives was evaluated by determination of their

minimal inhibitory concentration (MIC) using two‐fold serial dilutions in bacterial liquid media

following the National Committee of Clinical Laboratory Standards (NCCLS, 1997) as previously

described [15–18]. Briefly, for most of the bacteria, single colonies of the different bacterial strains

cultured on specific agar plates were used to inoculate 3 mL of Mueller‐Hinton (MH), except C.

difficile, C. perfringens, H. pylori, E. faecalis, and S. pyogenes that were cultured in 3 mL of BHI and or

M. smegmatis that was cultured in 3 mL of Middlebrook 7H10 broth. Tubes were then incubated

overnight (for approximately 16 h) at 37 °C under stirring (200 rpm), except for M. smegmatis that was

left to grow for 48–72 h. Optical density (OD) of the bacterial suspensions were then read at 600 nm,

adjusted to 1 with medium before bacteria were diluted 1/100 in 3 mL of fresh medium and incubated

at 37 °C, 200 rpm until bacteria reached log phase growth (OD600nm around 0.6). In the case of C.

difficile, C. perfringens, E. faecalis, H. pylori, S. pyogenes and M. smegmatis, MIC were performed directly

using over‐night growing suspension. In all cases, bacteria were diluted in appropriate medium to

5

reach bacterial density around 10 cells/mL. 100 μL per well of bacterial suspension were then added

into sterile polypropylene 96 well microplates (Greiner BioOne, Dominique Dutscher, Brumath,

France). Bacteria were exposed to increasing concentrations of berberine or berberine’s derivatives

obtained by serial dilution (from 0 to 100 μM, 1:2 dilution). Stock solutions of berberine and

berberine’s derivatives at 10 mM were prepared in DMSO. Volume of DMSO corresponding to the

highest dose of berberine’s derivatives tested (1% DMSO final concentration) was used as negative

control and was found inactive. Plates were incubated at 37 °C for 18–24 h for all bacteria except M.

smegmatis that where incubated for 72 h. All bacterial strains were tested in aerobic conditions except

C. difficile, C. perfringens, E. faecalis and S. pyogenes that were incubated in an anaerobic chamber (Coy

Laboratory Products, Grass Lake, MI, USA). MIC on H. pylori was performed in a microaerobic

atmosphere generated using anaerobic BD GasPak system (Sigma‐Aldrich, Lyon, France). For C.

albicans, liquid suspension was prepared by resuspending colonies collected from LB plates in sterile

3

NaCl 0.85% solution. C. albicans were then diluted at 1–2  10 cells/mL in RMPI media buffered with

MOPS at final concentration of 0.165 mol/L (pH 7.0) before being added to the 96‐wells plates in the

presence of increasing concentrations of test compounds. Plates were then incubated at 35 °C during

2

(

4 h before reading. At the end of the incubation, OD600nm was measured using microplate reader

Biotek, Synergy Mx, Colmar, France). The MIC was defined as the lowest concentration of drug that

inhibited visible growth of the organism. Experiments were conducted in independent triplicate (n =

). Antimicrobial activity of berberine and berberine’s derivatives was also evaluated by

3

determination of the minimal bactericidal concentration (MBC), i.e., the minimum concentration of

the test compound killing 99.9% of the bacteria as previously described [15]. MBCs were measured

by streaking 10 μL of wells with no observed growth of previously prepared MIC plates on agar

plates. After incubation in the proper condition, numbers of colonies were counted. Concentrations

where ≤ 1 colony grew were considered to be the MBC.

2

.2.3. Cytotoxic Assay on Human Cells

The toxicity of berberine and berberine’s derivatives on human cells was evaluated using

resazurin assay as previously described [15–17,19–21]. Human cells used were BEAS (ATCC CRL‐

9

609), Caco2 (ATCC HTB‐37) and HepG2 (ATCC HB‐8065) corresponding respectively to human

airway epithelial cells, intestinal epithelial cells and liver cells. Cells were cultured in Dulbecco’s

modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L‐

glutamine and 1% antibiotics (Thermo Fisher Scientific, Illkirch‐Graffenstaden, France). Cells were

2

routinely grown on 25 cm flasks and maintained in a 5% CO

2

incubator at 37 °C. For toxicity assay,

cells were detached using trypsin–EDTA solution (Thermo Fisher Scientific), counted using Mallasez

counting chamber and seeded into 96‐well cell culture plates (Greiner bio‐One, Dominique Dutscher,

Antibiotics 2020, 9, 381

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Brumath, France) at approximately 10,000 cells per well. The cells were left to grow for 48–72 h at 37

C in a 5% CO incubator until they reached confluence. Media from wells was then aspirated and

°

2

cells were treated with 100 μL of culture media containing increasing concentrations of berberine or

berberine’s derivatives obtained by serial dilution (from 0 to 50 μM, 1:2 dilution), DMSO (0.5% DMSO

final concentration) was used as negative control. After 48 h incubation at 37 °C in a 5% CO

2

incubator, cell viability was evaluated using resazurin based in vitro toxicity assay kit (Sigma‐

Aldrich) following manufacturer’s instructions. Briefly, cell wells were emptied, and cells were

++

treated with 100 μL of resazurin diluted 1:10 in sterile PBS containing calcium and magnesium (PBS ,

pH 7.4). After 4 h incubation at 37 °C, fluorescence intensity (excitation wavelength of 530

nm/emission wavelength of 590 nm) was measured using Synergy Mx microplate reader (Biotek).

The fluorescence values were normalized by the controls (DMSO treated cells) and expressed as

percent viability. The IC50 values of berberine and berberine’s derivatives on cell viability (i.e., the

concentration of derivative causing a reduction of 50% of the cell viability) were calculated using

®

GraphPad Prism 7 software (San Diego, CA, USA). t‐Test and two ways ANOVA analyses were

used to address the significant differences between mean values with significance set at p < 0.05.

2

.2.4. Bacterial Membrane Permeabilization Assay.

Membrane permeabilization by berberine derivatives was evaluated using the cell‐impermeable

DNA/RNA probe propidium iodide as previously explained [15,16,22,23]. Logarithmic growing

bacterial suspensions were prepared from overnight bacterial suspension by 1 in 10 dilution. After 3

h incubation at 37 °C, 200 rpm, bacterial suspensions were centrifuged for 5 min at 3000 rpm. Cell

9

pellets were then resuspended in sterile PBS at about 10 cells/mL. Propidium iodide (Sigma Aldrich)

was then added to the suspension at a final concentration of 60 μM. 100 μL of this suspension were

then transferred into 96‐well black plates (Dominique Dutscher) and exposed to increasing

concentrations of berberine’s derivative (from 0 to 100 μM, 1:2 dilution). Volume of DMSO

corresponding to 100 μM of derivative was used as negative control and was found inactive. All steps

of the assay were performed in aerobic condition except for H. pylori which was done in microaerobic

condition generated with GasPak unit (Sigma Aldrich). Bacteria were incubated at 37 °C and the

kinetics of fluorescence variations (excitation at 530 nm and emission at 590 nm) were then recorded

over time using a Synergy Mx microplate reader (Biotek). Cetyl trimethylammonium bromide

(CTAB) at 300 μM was used as positive control giving 100% permeabilization. Results were expressed

in % of permeabilization. All experiments were done in triplicate.

2

.2.5. DNA Fragmentation Assay

Overnight grown bacteria were pelleted at 6000 rpm for 10 min. Bacterial pellets were

resuspended in 577 μL of TE buffer (Tris 50 mM, EDTA 50 mM). 30 μL of SDS (at 10%) and 3 μL of

proteinase‐K (at 20 mg/mL) were then added to lyse the cells. After 1 h incubation at 37 °C, 180 μL of

NaCl 5 M were added and tubes were vortexed for 30 s. Chloroform/isoamyl alcohol (24/1, v/v) was

then added. Tubes were vortexed for 30 s and centrifuged at 6000 rpm for 5 min. The supernatants

containing extract DNA were then transferred to new tubes containing 1/1 volume of

phenol/chloroform/isoamyl alcohol (25/24/1, v/v/v). After vortexing for 5 min and centrifuging at 6000

rpm for 5 min, the supernatants were transferred to new tubes containing 0.6 mL of isopropanol.

Tubes were mixed gently until a white DNA precipitate was obtained. Then, the tubes were

centrifuged at 10,000 rpm for 10 min. The supernatants were discarded, and the pellets were

resuspended in 100 μL of 70% ethanol. The tubes were centrifuged for 5 min at 10,000 rpm and let to

dry. Finally, the DNA pellets were resuspended in 50 μL of TE buffer. DNA concentrations were

determined at 260 nm and DNA solutions obtained from different bacteria were all adjusted to a

concentration of 170 ng/μL. Then 20 μL of the DNA solution were transferred to tubes and were

treated with berberine’s derivatives at a final concentration corresponding to 4‐times their MIC. After

6

h of incubation at 37 °C, DNA were run onto a 1% agarose gels before being stained using sybr safe

and being observed using an Uvitec Cambridge gel viewer (Cambridge, England, United Kingdom).

Incubation of DNA for 6 h with sulfuric acid at 10% was used as positive control of fragmentation.

Antibiotics 2020, 9, 381

12 of 31

2

.2.6. Docking Studies

The crystal structure of FtsZ protein from S. aureus in complex with PC190723 inhibitor was

selected for the molecular docking studies [24]. The corresponding PDB file 4DXD was prepared

using MOE version 2016 (http://chemcomp.com). The binding site was defined as all residues with at

least one atom within 14 Å radius from the PC190723 inhibitor. The studied compounds were also

prepared using MOE in order to both generate 3D conformers and compute required partial charges.

PLANTS was used as the docking engine with its “chemplp” scoring function to generate and

evaluate the poses [25]. Its docking algorithm is based on a class of stochastic optimization algorithms

called ant colony optimization (ACO). This kind of algorithm, which mimics the behavior of ants

finding the shortest path between food and their nest, can be used to efficiently sample the

conformational space for docking purpose. Docking simulations of reference ligand PC190723 into

its FtsZ structure (PDB code: 4DXD) were beforehand performed to validate the docking protocol.

This control study is used to determine the best docking parameters for the target [26]. The PC190723

ligand was successfully redocked into its binding site with a RMSD value of less than 1 Å between

crystallized conformation and predicted pose (Supplementary Material Figure S1). All generated

poses were subjected to visual analysis using Pymol (http://pymol.org) and the binding mode of

interest was selected accordingly. The latter should highlight shared binding mode for the berberine

core (5 fused‐rings substructure) for all considered compounds (15, 16, 24, 25) and be reasonable in

terms of docking score and explicit interactions with the binding site.

2

.2.7. Molecular Dynamics Simulations

The selected binding modes from molecular docking were subsequently subjected to molecular

dynamics simulations as final refinement stage. All MD simulations were performed in explicit

solvent with periodic conditions with the AMBER package version 18. The initial protein coordinates

were taken from the ligand‐free crystal structure of FtsZ protein from S. aureus (PDB code: 4DXD).

MD simulations were performed for each ligand of interest: compounds 15, 16, 24, 25 and the native

ligand from the 4DXD structure considered as positive control. In contrast to native ligand, the initial

coordinates for compounds 15, 16, 24, 25 were taken from previous docking simulations. The

CHARMM‐GUI web services (http://www.charmm‐gui.org) were used to read the PDB structures

and generate inputs and scripts to run the MD simulations [27,28]. For each ligand, the topology and

parameter files were generated starting from an external SDF file with the CHARMM General Force

Field method. The system was solvated with a pre‐equilibrated solvation box of TIP3P water

molecules around the protein. Chloride and potassium ions were then added to neutralize the system.

Finally, periodic boundary conditions were automatically applied based on the system shape and

size. For each considered system, unfavorable contacts were first removed by a short energy

minimization protocol (max 5000 minimization cycles), followed by equilibration and production

simulations. Equilibration stage consisted of 1 ns simulation (time step 1 fs) with enabled constraints

on protein and ligands. Production stage consisted of 50 ns simulation (time step 2 fs) without any

constraints. Snapshots of the coordinates were saved every 5000 steps (10 ps) leading to 5000

instantaneous conformations for each trajectory. Trajectories were analyzed using the VMD tool

using both built‐in and in house Tcl scripts. The main considered criterion was the RMSD of both

protein backbone and ligands over the trajectory, after initial superimposition of the CA atoms of the

protein in the same referential.

2

.2.8. Evaluation of the Effect of Berberine’s Derivatives on the Synthesis of Macromolecules by

Bacteria Using Fluorescent Microscopy

Microscopic observation was used to evaluate the effect of berberine’s derivatives on the

synthesis of macromolecules by bacteria as previously described [15,16]. Briefly, overnight cultures

of bacteria were diluted 1:100 in culture media and were grown at 37 °C with shaking at 200 rpm

until they reached an OD600nm of about 0.2. Conventional antibiotics known to inhibit

macromolecule’s synthesis (i.e., amoxicillin, gemifloxacin, rifampicin and tetracycline) or berberine’s

Antibiotics 2020, 9, 381

13 of 31

derivatives were then added at a concentration equivalent to 4‐times their MIC and bacteria were

incubated at 37 °C for 2 h. At the end of the incubation, bacteria were stained for 15 min on ice for

membrane and DNA using 12 μg/mL FM4‐64FX (Thermo Fisher Scientific) and 2 μg/mL DAPI

(Sigma‐Aldrich), respectively. After staining, bacteria were pelleted by centrifugation (centrifugation

for 30 s at 7500 rpm) and resuspended in sterile PBS. Bacteria were pelleted a second time and

resuspended in 500 μL of sterile PBS. Bacteria were then pelleted a last time and fixed with PBS

containing paraformaldehyde (PFA) at 4% (v/v) for 15 min on ice in the dark. After centrifugation for

1

min at 7500 rpm, the samples were resuspended in 50 μL of mounting solution (Vector Laboratories,

Peterborough, UK). Eight μL were transferred onto microscope glass slides and bacteria were

observed and images collected using an IX71 Fluoview confocal microscope (Olympus, Rungis,

France) with excitation at 543 nm and 405 nm for FM4‐64FX and DAPI, respectively.

3

. Results

3

.1. Synthesis

The synthetic route of this new series of 13‐substituted berberines is outlined in Scheme 1,

starting from commercially available berberine chloride (BBR) converted to its 8‐acetonylberberine

compound 3) with NaOH 5M and acetone in 83% yield [14]. Compound 3 was then treated with

various bromides and NaI in CH CN yielding 13‐substituted berberine derivatives 4–24.

(

3

Scheme 1: General pathways for the synthesis of berberine derivatives 1–20. Reagents and conditions:

a) Pyridine/NaBH , rt, 2 h; (b) mCPBA, CH Cl , −20 °C, 1 h then Na SO , 0 °C, 1 h; (c) 5N NaOH,

acetone, rt, 1 h; (d) various bromides, NaI, CH CN, 80 °C, 4 h.

(

4

2

3

Scheme 2. General pathways for the synthesis of berberine derivatives 21–26. Reagents and conditions:

a) NH OH.HCl, AcONa, EtOH/H O 60 °C, 1 h. (b) Malonic acid, AcOH, piperidine cat., MWI, 110

(

2

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, MWI

°C, 30 min.; (c) EDCI, HOBt, DMF, rt, 12 h; (d) LiOH, MeOH, MWI 80 °C, 20 min.; (e) DMSO/I

2

140 °C, 30 min.

Using 4‐formyl berberine (compound 12) as starting material, we also obtained berberine

bearing a sulfonamide moiety in two steps (compounds 22 and 23). Construction of new dimeric

chalcone‐berberine scaffold (compounds 24 and 25) was done by aldol condensation using

compound 12 as starting material. Intramolecular cyclization of compound 25 of its chalcone moiety

into flavone lead to a new scaffold flavone‐berberine (compound 26) (Scheme 2). NMR spectra of the

synthetized derivatives are shown in the Supplementary Materials in Figure S1.

3

.2. Antibacterial Activity

Antimicrobial activity of the twenty‐six different berberine’s derivatives produced in this study

was compared to the activity of the original berberine molecule through a first screening assay

performed on selected relevant microorganisms representative of Gram‐negative (E. coli and P.

aeruginosa), Gram‐positive (B. subtilis and S. aureus), Mycobacterium (M. smegmatis) and fungi (Candida

albicans) (Table 1).

Table 1. Evaluation of the antimicrobial activity of the berberine derivatives. Antibacterial and

antifungal activities of the berberine’s derivatives were tested using selected representing strains of

Gram‐positive, Gram‐negative, Mycobacterium and fungi as explained in the Materials and Methods

Section. Values in the table correspond to Minimal Inhibitory Concentration (MIC) expressed in μM.

Berberine was used as reference molecule.

Compound

S. aureus B. subtilis E. coli P. aeruginosa M. smegmatis C. albicans

Amphotericin B

Berberine

Gemifloxacin

‐

>100

0.06

100

12.5–25

100

>100

100

12.5–25

100

6.25

3.125

>100

25

100

6.25–12.5

25

‐

1.1

100

‐

>100

0.02

>100

50

>100

100

50

>100

6.25

12.5

>100

25

>100

25

50

>100

25

12.5

1.5

>100

100

>100

25

>100

0.06

>100

0.25

100

2

>100

25

100

50

25

100

25

6.25

>100

50

100

12.5

25

50

100

12.5

100

50

>100

25

50

1

2

3

4

5

6

7

8

9

>100

100

>100

100

>100

100

50

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

>100

100

>100

50

>100

25

12.5

6.25

100

>100

100

>100

12.5

100

6.25–12.5

3.125–6.25

1.5–3.12

100

50

>100

6.25

50–100

3.125

12.5

100

6.25

50

As previously published [29], results showed that the natural molecule berberine possesses low

to no antimicrobial activity. Oppositely, among the 26 synthetized berberine’s derivatives, many

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possess higher antimicrobial activity compared to berberine (i.e compounds 2, 5, 6, 8, 9, 11, 13–19, 21

and 23–26). This first antimicrobial activity screening revealed that tested berberine’s derivatives

were particularly active against Gram‐positive strains (B. subtilis and S. aureus) with MIC values as

low as 1.5 μM for compound 19 for example. M. smegmatis, a model of Mycobacterium, was also found

sensitive to berberine’s derivatives, the lowest MIC value being of 6.25 μM for compound 9. Some

derivatives were also found active against C. albicans, the lowest value of MIC being 6.25 μM for

compound 19. Importantly, none of the tested derivatives were found active on the two Gram‐

negative strains tested (E. coli and P. aeruginosa). We selected the best derivatives, based on their high

antimicrobial activity (lower MIC value) and/or larger spectrum of activity (i.e., active against

different microorganisms tested in Table 1) to further study them. The selected derivatives were

compounds 2, 6, 8, 9, 11, 13, 14, 16–19, 23, 25 and 26.

3

.3. Toxicity Evaluation

The next step was to evaluate the toxicity of these selected derivatives using human cells and to

compare it to the original berberine molecule (Table 2).

Table 2. Evaluation of the toxicity of the most active berberine derivatives. Toxicity of the most active

berberine’s derivatives identified in Table 1 was tested using human cells as explained in the Materials

and Methods section. Values in the table correspond to the Inhibitory Concentration 50 (IC50), i.e., the

concentration of derivatives causing a decrease in cell viability of 50%. IC50 are expressed in μM

(

means +/−SD). Berberine was used as reference molecule.

Compound Caco2 BEAS

Berberine 21.56 ± 2.7

HEPG2

40.45 ± 5.4 526.1 ± 151.9

64.61 ± 4.5 392.2 ± 114.5

56.25 ± 5.0 376.4 ± 161.4

23.38 ± 4.5 1627 ± 855.1

2

6

8

9

34.18 ± 3.8

36.91 ± 4.3

69.90 ± 8.1

24.77 ± 3.6

12.79 ± 1.6

61.45 ± 10.0

1

3

4

6

7

8

9

3

5

6

90.15 ± 14.3 150.80 ± 23.7 3648 ± 5425

1

2

31.43 ± 3.8

86.14 ± 9.2

64.80 ± 4.9

53.40 ± 8.4

18.26 ± 1.3

11.95 ± 1.3

77.52 ± 7.2

10.54 ± 0.7

16.90 ± 1.5

73.62 ± 8.7

86.22 ± 7.6

42.41 ± 9.8

9.59 ± 1.2

4.92 ± 0.6

197.9 ± 35.0

2217 ± 2876

450.9 ± 171

2138 ± 2243

60.75 ± 11.6

31.17 ± 7.1

79.16 ± 7.7 1288 ± 711.1

8.87 ± 1.5 123.1 ± 9.7

253 ± 128.4 519 ± 237.2 2259 ± 1797

Results showed that toxicity of berberine’s derivatives is cell‐type dependent. Thus, in human

intestinal epithelial cells (Caco‐2 cells), compounds 18, 19 and 25 were the only ones found more toxic

than berberine (i.e., giving lower IC50 values) all other compounds being less toxic than the original

molecule. For human airway epithelial cells (BEAS cells), in addition to compounds 18, 19 and 25,

compounds 8, 9 and 13 were also found more toxic than berberine whereas other derivatives were

found less toxic. Finally, in human liver epithelial cells (HepG2 cells), in addition to compounds 9,

1

3, 18, 19 and 25, compounds 2, 6 and 16 were also found more toxic than berberine. Based on good

antimicrobial activity and low toxicity, compounds 8, 16, 17, 23, 25 and 26 were selected and further

studied.

The therapeutic indexes (TI) of selected derivatives were calculated by dividing their IC50 value

on a specific human cell type by their MIC values found on S. aureus that gave the lower MIC values

(Figure 2 and Table 3).

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Figure 2. Graphical representation of the therapeutic index of the more active and less toxic berberine

derivatives. Best berberine derivatives were selected based on their high antimicrobial activity and

low toxicity. Dose‐dependent effects of the selected derivatives in term of human cell toxicity and

antibacterial activity against S. aureus were plotted using the Graph Pad Prism software. Results are

expressed as means +/−SD with n = 3.

Table 3. Determination of the therapeutic index of the more active and less toxic berberine derivatives.

Best berberine derivatives were selected based on their high antimicrobial activity and low toxicity.

The therapeutic index of each derivative was calculated by dividing its IC50 value on a specific human

cell type by its MIC value on S. aureus.

Compound

8

16

Caco2 11.18 10.36 8.54

BEAS 3.74 13.79 6.78

17

23

25

26

12.40 3.37 20.24

12.66 2.83 41.52

HEPG2 260.30 72.14 342.00 206.00 39.39 180.70

TI were found dependent on both the specific compound and human cells considered. Thus, for

human intestinal epithelial cells (Caco‐2 cells), TI ranged from 3.37 to 20.24 with the following order

of safety: compound 26 > 23 > 8 > 16 > 17 > 25. For human airway epithelial cells (BEAS cells), TI

ranged from 2.83 to 41.52 with the following order of safety: compound 26 > 16 > 23 > 17 > 8 > 25.

Finally, for human liver epithelial cells (HepG2 cells), TI ranged from 39.39 to 342.00 with the

following order of safety: compound 17 > 8 > 23 > 26 > 16 > 25. Overall, these data showed that

although antimicrobial berberine’s derivatives gave good TI values, suggesting that they could

potentially be used to treat systemic infections without taking the risk of toxicity for the patients.

Compounds 8, 16, 17, 23, 25 and 26 were further tested using a large panel of Gram‐negative

and Gram‐positive environmental and pathogenic bacteria, including resistant strains, listed in Table

4

. Regarding Gram‐positive bacteria, results showed that selected compounds possess good

antimicrobial activity (i.e., MIC of 3.12–6.25 μM) against important human pathogens such as B.

cereus, S. aureus or S. pyogenes. Other Gram‐positive pathogenic bacteria, such as C. difficile, C.

perfringens or E. faecalis were found less sensitive giving MIC values superior or equal to 12.5 μM.

Importantly, Gram‐positive strains resistant to conventional antibiotics, i.e., nisin‐resistant B. subtilis,

methicillin‐resistant S. aureus and vancomycin‐resistant E. faecalis, were found sensitive to berberine’s

derivatives. Interestingly, results confirmed that derivatives were inactive on most of the tested

Gram‐negative bacteria, except H. pylori and V. alginolyticus that were found sensitive with MIC

values as low as 1.5–3.12 μM. Determination of the Minimal Bactericidal Concentration (MBC) of the

selected compounds showed that their MBC values were closed or identical to their MIC values

(

Table S1), indicating that they are bactericidal rather than bacteriostatic.

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Table 4. Evaluation of the spectrum of antimicrobial activity of the most active berberine derivatives.

Antibacterial and antifungal activities of the most active berberine derivatives identified in Table 1

were further tested using a large panel of microbial strains as explained in the Materials and Methods

section. Values in the table correspond to Minimal Inhibitory Concentration (MIC) expressed in μM.

Compound

8

16

100

12.5

25

12.5

100

25

17

12.5

25

12.5

12.5–25

100

23

50

12.5

25

50

25

1.5

26

12.5

50

>100

A. gandavensis

B. cereus

B. subtilis

3.12

12.5

6.25

12.5

100

50

6.25

12.5

25

B. subtilis (Nisin‐resistant)

C. difficile

C. perfringens

E. faecalis (Vancomycin‐resistant)

L. lactis

Gram‐positive

50

25

50

100

50

12.5

3.12–6.25 6.25–12.5 3.12–6.25 6.25 1.5–3.12 6.25–12.5

S. aureus

S.aureus (Methicillin‐resistant)

6.25

12.5

>100

6.25

>100

50

6.25

12.5

6.25

25

>100

6.25

12.5

6.25

>100

3.125

6.25

12.5

25

S. pyogenes

A. baumannii

C. farmer

C. rodentium

E. coli

H. pylori

K. pneumonia

P. aeruginosa

>100

6.25

>100

3.12

>100

3.125

>100

3.12

>100

50

>100

50

6.25–12.5 6.25

>100

Gram‐negative

P. aeruginosa (Quinolone‐resistant)

S. enterica

S. flexneri

V. diabolicus

V. alginolyticus

>100

6.25

1.5–3.12 6.25

3

.4. Mechanistic Analysis

Compounds 16 and 25 showing the higher activity on a large number of bacteria, they were

further investigated in term of mechanism of action using different approaches, their structurally‐

related inactive derivatives (i.e., compounds 15 and 24) being used as controls (Figure 3).

Figure 3. Comparison of the structure of closely related compounds found active or inactive.

Structure of compounds 15 (inactive) and 16 (active) and compounds 24 (inactive) and 25 (active) are

shown for comparison.

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3

.4.1. Membrane Permeabilisation Assay

Previous studies have shown that some berberine’s derivatives have the ability to cause

membrane permeabilization [15,16,22]. For this reason, the effect of the compounds 15, 16, 24 and 25

on the bacterial membrane integrity was investigated (Figure 4). As expected, inactive derivatives

(

i.e., compounds 15 and 24) caused low to no membrane permeabilization (data not shown). Active

derivatives (i.e., compounds 16 and 25) caused membrane permeabilization, with the following order

of efficiency: V. alginolyticus = S. aureus > H. pylori > B. subtilis with a maximal permeabilization of 80–

1

00; 50–60 and 10–15%, respectively after 120 min exposure to 100 μM of derivative.

In all cases, compound 16 was more active than compound 25. Importantly, although membrane

damages were observed at high doses, at 4‐times their MIC compounds 16 and 25 caused low to no

membrane permeabilization, except for V. alginolyticus where 20–40% permeabilization was

observed, demonstrating that the antibacterial effect of these compounds rely on other mechanism(s).

Figure 4. Concentration and time‐dependent effect of berberine derivatives on the bacterial

membrane integrity. The dose‐ and time‐dependent effects of compounds 16 and 25 on bacterial

membrane integrity were studied using B. subtilis (A–C), H. pylori (D–F), S. aureus (G–I) or V.

alginolyticus ((J–L). Graphs (A,D,G,J) show the dose‐dependent effect of 60 min incubation at 37 °C

with increasing concentrations of derivatives. Graphs (B,E,H,K) show the time‐dependent effect at 37

°

C of derivative at 4‐times their MIC. Graphs (C,F,I,L) show the time‐dependent effect at 37 °C of

derivative at 100 μM. In all cases, results were expressed as percentage of membrane permeabilization

using CTAB at 300 μM as positive control giving 100% permeabilization. Results corresponding to

means ± SD (n = 3) were fitted using the Graph Pad Prism software.

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3

.4.2. DNA Fragmentation Assay

The ability of compounds 16 and 25 to cause DNA fragmentation was then evaluated, known

antibacterial berberine’s derivatives obtained by modification at position 13 with alkyl and diphenyl

alkyl possessing such activity [11]. Results showed that incubation of bacterial DNA extracted from

B. subtilis or V. alginolyticus for 6 h at 37 °C with compounds 16 and 25 at 4‐times their MIC did not

caused DNA fragmentation (Figure S2). Similarly, compounds 16 and 25 failed at causing the

fragmentation of DNA extracted from S. aureus and H. pylori (data not shown) ruling out the

implication of such mechanism in their antibacterial effect.

3

.4.3. Molecular Docking

FtsZ protein from S. aureus (FtsZ, PDB code: 4DXD) has been previously shown, using in vitro

biological assays, to be the target of antibacterial berberine’s derivatives [30]. Since FtsZ protein is

involved in septum formation and bacterial cell division, the interaction of berberines with this

bacterial protein was proposed to explain their antibacterial activity [30]. For this reason, molecular

docking studies were conducted on the FtsZ protein from S. aureus. The berberine core (5 fused‐rings

substructure) and compounds 15, 16, 24, 25 were investigated. Based on docking experiments (Figure

5

), the core berberine substructure was predicted to be deeply buried in a hydrophobic pocket from

the binding site, with an overlap with the known PC190723 inhibitor from reference 4DXD structure.

A

B

Figure 5. (A) Binding mode of compound 25 depicted as a 2D diagram. The figure was produced

using the “Ligand interaction” feature of MOE. (B) The proposed binding mode of compound 25 in

the S. aureus FtsZ protein. Compound 25 and surrounding protein sidechains are displayed in stick

representation. The backbone from the protein is shown as cartoon. The picture was generated using

Pymol (http://pymol.org) software.

More precisely, the ABC rings (from the dioxole end) of the berberine core were superimposed

on the chlorothiazolopyridine moiety of PC190723. Interestingly, the variable sidechain was also

directed toward the exit of the binding site, allowing large modifications as highlighted by the

diversity of the sidechains from reported active compounds in this study. While compound 25 was

successfully predicted as the best compound (score criterion, favorable contacts with the site) among

the five considered structures, the models were not able to explain the observed activity differences

between close analog pairs (15/16 and 24/25). The potent compound 25 is expected to create additional

favorable contacts with the binding site, especially a hydrogen bond between its hydroxyl group and

the polar sidechain from E305 (Figure 5). At this stage, docking analysis suggested that at least

compound 25 is able to interact with FtsZ, a protein involved in bacterial division. It should be noted

that this predicted binding mode for the berberine core was previously proposed for the

cycloberberine analog series [13]. These predicted binding modes from docking were then subjected

to molecular dynamics simulations as final refinement (Figure 6). The monitoring of RMSD criterion

over the trajectory clearly highlighted that the docking binding modes were not reliable, as the

complexes were not stable starting from the beginning of the production trajectories. As a control,

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the same molecular dynamics simulations were performed on the original 4DXD complex and small

RMSD values were obtained in this case in contrast to the berberine series.

Figure 6. RMSD diagrams from the molecular dynamics simulations for each considered compound

(

15, 16, 24 and 25). The RMSD values over the trajectory are displayed in orange for the protein and

in blue for the ligand. The RMSD values of the reference ligand from 4DXD structure are also

displayed for information (data from an independent simulation).

3

.4.4. Macromolecule Synthesis Inhibition Assay

Since the antibacterial activity of compounds 16 and 25 does not rely on membrane

permeabilization, DNA fragmentation or FtsZ interaction (i.e., the reported mechanisms of action of

already known berberine derivatives) their effect on bacterial synthesis of macromolecules was finally

evaluated using fluorescent microscopy as previously described (Tables 5 and 6. Figures 7–12) [16,17].

Table 5. Description of the morphological changes caused by conventional antibiotics or berberine’s

derivative 16 and 25 in B. subtilis. Morphological changes of B. subtilis caused by conventional

antibiotics and compounds 16 or 25 illustrated in Figures 7–9 are summarized in this table.

Known/Possible

Condition

Observed Morphology

Target



No cell elongation

1 condensed DNA in each cell

2–5 cells connected

Control

‐



Cell enlargement (1.5 times compared to control)

2 condensed DNA in each cell

2–4 cells connected

Amoxicillin

Cell wall biosynthesis



Two morphologies:

‐

Cell elongation (3–4 times compared to control)

Spore like (circular cells)

Gemifloxacin

Rifampicin

DNA synthesis



Decondensed DNA

Single cells

RNA biosynthesis



Cell elongation (2.5 times compared to control)

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DNA condensed in one thick filament crossing the cells some with dot



like shape



2–3 cells connected

Cell membrane thickening



Cell elongation (1.5–3 times compared to control)

DNA condensed in one thin filament across the cells

Tetracycline

Protein biosynthesis



1–3 cells connected



Cell enlargement (1.5–6 times compared to control)

2 morphologies of DNA:

1

2

6

5

Cell wall biosynthesis

RNA biosynthesis

‐

2 condensed DNA in each cell

Decondensed DNA spanning the cell



2–4 cells connected



Cell elongation (1–2.5 times compared to control)

DNA condensed and cross the cells with dot like shape

2–8 cells connected



Cell membrane thickening

Table 6. Description of the morphological changes caused by conventional antibiotics or berberine’s

derivative 16 and 25 in V. alginolyticus. Morphological changes of V. alginolyticus caused by conventional

antibiotics and compounds 16 or 25 illustrated in Figures 10–12 are summarized in this table.

Condition

Known/Possible Target

Observed Morphology

No cell elongation



Control

‐

1–2 condensed DNA in each cell

2–7 cells connected

Cell elongation (1–3 times compared to control)

Thick condensed and decondensed DNA spanning the cell

1–2 cells connected

Amoxicillin

Gemifloxacin

Rifampicin

Cell wall biosynthesis

DNA synthesis

Cell membrane thickening

Cell elongation (3–9 times compared to control)

Thick condensed DNA spanning the cell

1–2 cells connected

Cell elongation (2.5 times compared to control)

Condensed DNA spanning the cell

1–2 cells connected

RNA biosynthesis

Protein biosynthesis

Cell wall biosynthesis

Cell membrane pigmentation

Cell elongation (1.5–3 times compared to control)

DNA condensed in one thin filament spiring the cells

1–3 cells connected

Cell elongation (2–4 times compared to control)

Decondensed DNA spanning cell

2–4 cells connected

Tetracycline

1

6

5

Cell membrane thickening

Cell elongation (2–7 times compared to control)

Condensed DNA spanning the cell

1–2 cells connected

2

RNA biosynthesis

Cell membrane pigmentation

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Figure 7. Morphological bacterial changes caused by conventional antibiotics in B. subtilis.

Morphological changes of B. subtilis caused by conventional antibiotics were studied as explained in

the Materials and Methods section. B. subtilis was incubated for 2 h at 37 °C in aerobic conditions with

4

 MIC of conventional antibiotics with known bacterial target. Control (A–C) was bacteria with no

antibiotics added. Antibiotics used were: amoxicillin ((D–F), inhibiting cell wall synthesis),

gemifloxacin ((G–I), inhibiting DNA synthesis), fifampicin ((J–L), inhibiting RNA synthesis) and

tetracycline ((M–O), inhibiting protein synthesis). Images shown are representative of overall

observed effects and correspond to bacterial membrane stained with FM4‐64FX (red signal)

(

A,D,G,J,M), bacterial DNA stained with DAPI (B,E,H,K,N) and merged membrane and DNA signals

C,F,I,L,M,O). The white scale bar on the bottom right corner corresponds to 4 μm.

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Figure 8. Morphological bacterial changes caused by compound 16 in B. subtilis. Morphological

changes of B. subtilis caused by compound 16 were studied as explained in the Materials and Methods

section. B. subtilis was incubated for 2 h at 37 °C in aerobic conditions with 4 X MIC of 16 (D–F).

Control (A–C) was bacteria with no antibiotics added. At the end of the incubation, bacteria were

fixed and membrane (A,D,G) and DNA (B,E,H) were stained using FM4‐64FX (red signal) and DAPI

(

blue signal), respectively. Membrane and DNA signals were merged in the figure in (C,F,I). The effect

of amoxicillin, an inhibitor cell wall synthesis, is shown for comparison in (G–I). Bacteria were

observed using confocal microscope and images representative of observed effect were taken. The

white scale bar on the bottom right corner corresponds to 4 μm.

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Figure 9. Morphological bacterial changes caused by compound 25 in B. subtilis. Morphological

changes of B. subtilis caused by compound 25 were studied as explained in the Materials and Methods

section. B. subtilis was incubated for 2 h at 37 °C in aerobic conditions with 4 X MIC of 25 (D–F).

Control (A–C) was bacteria with no antibiotics added. At the end of the incubation, bacteria were

fixed and membrane (A,D,G) and DNA (B,E,H) were stained using FM4‐64FX (red signal) and DAPI

(

blue signal), respectively. Membrane and DNA signals were merged in the figure in (C,F,I). The effect

of rifampicin, inhibiting RNA synthesis, is shown for comparison in (G–I). Bacteria were observed

using confocal microscope and images representative of observed effect were taken. The white scale

bar on the bottom right corner corresponds to 4 μm.

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Figure 10. Morphological bacterial changes caused by conventional antibiotics in V. alginolyticus.

Morphological changes of V. alginolyticus caused by conventional antibiotics were studied as

explained in the Materials and Methods section. V. alginolyticus was incubated for 2 h at 37 °C in

aerobic conditions with 4  MIC of conventional antibiotics with known bacterial target. Control (A–

C) was bacteria with no antibiotics added. Antibiotic used were: amoxicillin (D–F), inhibiting cell wall

synthesis), gemifloxacin (G–I), inhibiting DNA synthesis), rifampicin (J–L), inhibiting RNA synthesis)

and tetracycline (M–O), inhibiting protein synthesis). Images shown are representative of overall

observed effects and correspond to bacterial membrane stained with FM4‐64FX (red signal)

(

A,D,G,J,M), bacterial DNA stained with DAPI (B,E,H,K,N) and merged membrane and DNA signals

C,F,I,L,M,O). The white scale bar on the bottom right corner corresponds to 4 μm.

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Figure 11. Morphological bacterial changes caused by compound 16 in V. alginolyticus. Morphological

changes of V. alginolyticus caused by compound 16 were studied as explained in the Materials and

Methods section. V. alginolyticus was incubated for 2 h at 37 °C in aerobic conditions with 4 X MIC of

1

6 (D–F). Control (A–C) was bacteria with no antibiotics added. At the end of the incubation, bacteria

were fixed and membrane (A,D,G) and DNA (B,E,H) were stained using FM4‐64FX (red signal) and

DAPI (blue signal), respectively. Membrane and DNA signals were merged in the figure in (C,F,I).

The effect of amoxicillin, an inhibitor cell wall synthesis, is shown for comparison in (G–I). Bacteria

were observed using confocal microscope and images representative of observed effect were taken.

The white scale bar on the bottom right corner corresponds to 4 μm.

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Figure 12. Morphological bacterial changes caused by compound 25 in V. alginolyticus. Morphological

changes of V. alginolyticus caused by compound 25 were studied as explained in the Materials and

Methods section. V. alginolyticus was incubated for 2 h at 37 °C in aerobic conditions with 4 X MIC of

2

5 (D–F). Control (A–C) was bacteria with no antibiotics added. At the end of the incubation, bacteria

were fixed and membrane (A,D,G) and DNA (B,E,H) were stained using FM4‐64FX (red signal) and

DAPI (blue signal), respectively. Membrane and DNA signals were merged in the figure in (C,F,I).

The effect of rifampicin, inhibiting RNA synthesis, is shown for comparison in (G–I). Bacteria were

observed using confocal microscope and images representative of observed effect were taken. The

white scale bar on the bottom right corner corresponds to 4 μm.

This technique based on the specific fluorescent labelling of bacterial followed by microscopic

observation represents an alternative to radioactivity and was successfully used by others and us to

evaluate the effect of various antibacterial agents on macromolecule synthesis [16,17]. The assay relies

on the comparison of the morphological changes caused by test compounds and conventional

antibiotics with known mechanism of action used as controls. The assay was performed on B. subtilis

(

Figures 7–9) and V. alginolyticus (Figures 10–12) as models of Gram‐positive and Gram‐negative

sensitive strains. Bacteria were exposed to compounds 16, 25 or conventional antibiotics at 4‐times

their MIC values for 2 h before labelling of their membrane using FM 4–64 (red fluorescence) or their

DNA using DAPI (blue fluorescence) and microscopic observation. Results showed that compound

1

6 caused morphological changes in B. subtilis (Table 5) and V. alginolyticus (Table 6) similar to the

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ones caused by amoxicillin known to inhibit cell wall synthesis, whereas compound 25 caused

bacterial morphological changes similar to the ones observed with rifampicin known to inhibit the

synthesis of RNA.

4

. Discussion

A series of 13‐substituted berberine derivatives were synthesized and evaluated in term of

antimicrobial activity, toxicity, and mechanism of action. Evaluation of the antimicrobial activity and

toxicity of the derivatives showed that biological activities were affected by the substituent present

at position 13. Analysis of the structure‐activity relationship showed that with the simple para

substitution in compounds 6–12, only compound 8 possessing an iodomethyl group and compound

9

with an allyl group had interesting bactericidal activities. Unfortunately, compounds 8 and 9 were

also found more toxic than berberine. Regarding compounds 13–17 which are multi‐substituted on

the phenyl ring, only compound 15 (3,4,5 trimethoxy phenyl) was found inactive. Replacement of the

4

‐methoxy group of compound 15 by a 4‐benzoate group (as in compound 16) or with benzoate at

position 2 and 4 (as in compound 17) increased the antimicrobial activity. Increasing the size of

substituent on the phenyl ring seems to enhance the antimicrobial activity without increasing the

toxicity of the compound. The presence of a coumarin (as in compound 18) or benzothiazole (as in

compound 19) dramatically increased the antimicrobial activity but also the toxicity of the molecules.

Introducing a sulfonamide (in compound 23) gave activity comparable to that of compound 22 and

reduced toxicity compare to berberine. The presence of a 4‐acetamido chalcone at position 4 (as in

compound 24) leaded to an inactive molecule, whereas the presence of a 5‐methoxy chalcone at the

same positon (as in compound 25) led to an active form. Finally, compound 26 corresponding to the

first in class flavones‐berberine was found less active than compound 25. The antimicrobial activity

of the berberine’s derivatives described in the present study was meanly directed against Gram‐

positive bacteria, including the ones part of the high priority group of the WHO’s list such as

Staphylococcus, Enterococcus and Streptococcus, but also against Mycobacterium and C. albicans. Gram‐

negative bacteria were not sensitive to the berberine derivatives except V. alginolyticus and H. pylori

that were found sensitive to compounds 8, 16, 17, 23‐26. The antibacterial action of these compounds

on H. pylori is very interesting since this bacteria is classified as priority pathogen by the WHO due

to its prevalence (i.e., 50% of the adults worldwide and even 80–90% of adults in developing

countries, making it the second cause of bacterial infection after caries) and its severe complications

(i.e., gastric ulcers in 10% of infected patients and gastric cancer in 1% of the patients that is estimated

to kill 700,000 persons per year) [31]. Mechanistic approaches were performed for compounds 16 (3,5‐

dimethoxy‐4‐benzoate phenyl derivative) and 25 (5‐methoxychalcone derivative) demonstrated that,

contrary to previously described 13‐substituted berberine derivatives, these derivatives do not cause

membrane permeabilization or DNA fragmentation. Docking studies were used to predict the

binding mode of several compounds from this series including the potent compound 25. The global

binding mode, with the berberine moiety deeply buried within the binding site of FtsZ, was

previously described for an analog cycloberberine series [13]. However, refinement of these predicted

binding modes using molecular dynamics simulations suggested that they were not reliable. Given

this and the fact that the activity cliffs could not be explained by the models (compounds 15 vs. 16;

compounds 24 vs. 25), the latter were not validated for this new series of compounds. Taken together

mechanistic studies showed that the new active berberine’s derivatives described in this study are

not acting through mechanisms of action reported for already known berberine’s derivatives (i.e.,

membrane permeabilization, DNA fragmentation or FtsZ interaction). Morphological microscopic

approach demonstrated that compounds 16 and 25 have an original mechanism of action for

berberine’s derivatives relying on their ability to inhibit the synthesis of the cell wall and of RNA,

respectively.

5

. Conclusions

In conclusion, the good activity of the 13‐substituted berberine derivatives developed in the

present study against pathogenic bacteria, including resistant strains, associated to their low toxicity

Antibiotics 2020, 9, 381

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against human cells, suggest that these molecules should be further evaluated and considered to treat

bacterial infection, particularly in the context of world‐wild increase in antibiotic resistance of

bacteria.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: NMR

spectra of compounds 5–26; Figure S2: DNA fragmentation assay; Table S1: Evaluation of the bactericidal

activity of the most active berberine derivatives.

Author Contributions: Conceptualization, H.O., M.M. and M.R.; methodology, H.O., M.M., V.P., L.H., P.P.,

M.M. and M.R.; software, L.H. and P.P.; validation, M.M. and M.R.; formal analysis, H.O., C.N., V.P., T.Y., L.H.,

P.P., M.M. and M.R..; investigation, H.O., S.M.G., K.B.H., C.N., M.M., V.P., T.Y., L.H., P.P., M.M. and M.R.;

resources, M.M. and M.R.; data curation, L.H., M.M. and M.R.; writing—original draft preparation, H.O., L.H.,

M.M. and M.R.; writing—review and editing, H.O., S.M.G., K.B.H., C.N., M.M., V.P., J.P., A.H., E.B., J.R., T.Y.,

L.H. P.P., M.M. and M.R.; supervision, J.P., A.H., E.B., M.M. and M.R.; project administration, M.M. and M.R.;

funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript.

Funding: This project has received financial support from the CNRS through the MITI interdisciplinary

programs (Défi Biomimétisme 2019, HelicAMP) attributed to M. Maresca.

Acknowledgments: We would like to thank Aurélie Tasiemski (Institut Pasteur de Lille, U1019‐UMR 8204‐

CIIL—Center for Infection and Immunity of Lille, F‐59000 Lille, France) for providing us the V. diabolicus strain

used in this study. We would also like to thank Gilles Iacazio from the AVB platform (iSm2, Marseille) were

antifungal activities were tested. Finally, we also thank the Datacenter IT and Scientific Computing (DISC) and

Molecular Modeling (Int3D) platforms of the CRCM.

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

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Article Doi

Synthesis and evaluation of the antibacterial activities of 13‐substituted berberine derivatives

DOI: 10.3390/antibiotics9070381

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