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

Article abstract of DOI:10.3390/antibiotics9070381

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 na?ve 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.

Full text of DOI:10.3390/antibiotics9070381

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
3
4
1
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
2
7
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
2
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
6
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
2 of 31
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
3 of 31
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
1
1
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
3
5
1
1
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
4 of 31
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
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
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
1
3
3
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
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
3
MHz, CDCl
3
1
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
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),

Antibiotics 2020, 9, 381
5 of 31
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
3
3
1
1
OCH
2
3
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
1
1
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
2
4
3
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
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.

Antibiotics 2020, 9, 381
6 of 31
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
1
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
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
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
1
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.

Antibiotics 2020, 9, 381
7 of 31
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
3
6) δ
1
1
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
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
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
3
13
Hz, H‐5). C‐RMN (DMSO‐d
6

Antibiotics 2020, 9, 381
8 of 31
(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
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
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
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
3
3
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
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
4
7
1
1
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
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
10 of 31
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
11 of 31
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
2
2
3
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
2

Antibiotics 2020, 9, 381
14 of 31
, 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
100
50
>100
6.25
12.5
>100
25
>100
25
50
>100
25
12.5
12.5
1.5
>100
100
>100
25
>100
0.06
>100
0.25
100
2
>100
25
100
100
50
25
100
25
6.25
>100
50
100
12.5
25
50
50
100
12.5
12.5
100
50
>100
25
25
25
50
1
2
3
4
5
6
7
8
9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
100
>100
>100
>100
100
50
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
>100
100
>100
50
>100
>100
>100
25
12.5
6.25
100
>100
>100
100
>100
12.5
100
100
6.25–12.5
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

Antibiotics 2020, 9, 381
15 of 31
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
1
3
4
6
7
8
9
3
5
6
90.15 ± 14.3 150.80 ± 23.7 3648 ± 5425
1
1
1
1
1
1
2
2
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).

Antibiotics 2020, 9, 381
16 of 31
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.

Antibiotics 2020, 9, 381
17 of 31
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
25
50
25
1.5
26
12.5
50
50
50
>100
>100
>100
A. gandavensis
B. cereus
B. subtilis
3.12
12.5
6.25
12.5
100
100
50
6.25
6.25
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
12.5
>100
>100
>100
>100
6.25
>100
>100
>100
>100
50
6.25
12.5
6.25
6.25
25
25
>100
>100
>100
>100
6.25
12.5
6.25
>100
>100
>100
>100
3.125
6.25
6.25
12.5
25
25
S. pyogenes
A. baumannii
C. farmer
C. rodentium
E. coli
H. pylori
K. pneumonia
P. aeruginosa
>100
>100
>100
>100
6.25
>100
>100
>100
>100
>100
>100
3.12
>100
>100
>100
>100
3.125
>100
>100
>100
>100
>100
>100
3.12
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
50
6.25–12.5 6.25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>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.

Antibiotics 2020, 9, 381
18 of 31
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.

Antibiotics 2020, 9, 381
19 of 31
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,

Antibiotics 2020, 9, 381
20 of 31
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)

Antibiotics 2020, 9, 381
21 of 31
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

Antibiotics 2020, 9, 381
22 of 31
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.

Antibiotics 2020, 9, 381
23 of 31
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.

Antibiotics 2020, 9, 381
24 of 31
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.

Antibiotics 2020, 9, 381
25 of 31
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.

Antibiotics 2020, 9, 381
26 of 31
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.

Antibiotics 2020, 9, 381
27 of 31
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

Antibiotics 2020, 9, 381
28 of 31
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
29 of 31
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.
References
1.
2.
3.
4.
Brown, E.D.; Wright, G.D. Antibacterial drug discovery in the resistance era. Nature 2016, 529, 336–343,
doi:10.1038/nature17042.
Arendrup, M.C.; Patterson, T.F. Multidrug‐Resistant Candida: Epidemiology, Molecular Mechanisms, and
Treatment. J. Infect. Dis. 2017, 216, S445–S451, doi:10.1093/infdis/jix131.
Derosa, G.; Maffioli, P. Alkaloids in the nature: Pharmacological applications in clinical practice of
berberine and mate tea. Curr. Top. Med. Chem. 2014, 14, 200–206, doi:10.2174/1568026613666131213155252.
Xiao, D.; Liu, Z.; Zhang, S.; Zhou, M.; He, F.; Zou, M.; Peng, J.; Xie, X.; Liu, Y.; Peng, D. Berberine Derivatives
with Different Pharmacological Activities via Structural Modifications. Mini Rev. Med. Chem. 2018, 18, 1424–
1441, doi:10.2174/1389557517666170321103139.
5.
6.
7.
Iwasa, K.; Kim, H.‐S.; Wataya, Y.; Lee, D.‐U. Antimalarial activity and structure‐activity relationships of
protoberberine alkaloids. Eur. J. Med. Chem. 1998, 33, 65–69, doi:10.1016/S0223‐5234(99)80077‐4.
Vennerstrom, J.L.; Lovelace, J.K.; Waits, V.B.; Hanson, W.L.; Klayman, D.L. Berberine derivatives as
antileishmanial drugs. Antimicrob. Agents Chemother. 1990, 34, 918–921, doi:10.1128/aac.34.5.918.
Wang, Y.‐X.; Yang, L.; Wang, H.‐Q.; Zhao, X.‐Q.; Liu, T.; Li, Y.‐H.; Zeng, Q.‐X.; Li, Y.‐H.; Song, D.‐Q.
Synthesis and Evolution of Berberine Derivatives as a New Class of Antiviral Agents against Enterovirus
71
through
the
MEK/ERK
Pathway
and
Autophagy.
Molecules
2018,
23,
2084,
doi:10.3390/molecules23082084.
8
9
1
.
Bodiwala, H.S.; Sabde, S.; Mitra, D.; Bhutani, K.K.; Singh, I.P. Synthesis of 9‐substituted derivatives of
berberine as anti‐HIV agents. Eur. J. Med. Chem. 2011, 46, 1045–1049, doi:10.1016/j.ejmech.2011.01.016.
Yu, P.; Li, D.; Ni, J.; Zhao, L.; Ding, G.; Wang, Z.; Xiao, W. Predictive QSAR modeling study on berberine
derivatives with hypolipidemic activity. Chem. Biol. Drug Des. 2018, 91, 867–873, doi:10.1111/cbdd.13150.
.
0. Xiao, D.; He, F.; Peng, D.; Zou, M.; Peng, J.; Liu, P.; Liu, Y.; Liu, Z. Synthesis and Anticancer Activity of 9‐
O‐Pyrazole Alkyl Substituted Berberine Derivatives. Anticancer Agents Med. Chem. 2018, 18, 1639–1648,
doi:10.2174/1871520618666180717121208.
11. Vieira, S.; Castelli, S.; Falconi, M.; Takarada, J.; Fiorillo, G.; Buzzetti, F.; Lombardi, P.; Desideri, A. Role of
13‐(di)phenylalkyl berberine derivatives in the modulation of the activity of human topoisomerase IB. Int.
J. Biol. Macromol. 2015, 77, 68–75, doi:10.1016/j.ijbiomac.2015.02.051.

Antibiotics 2020, 9, 381
30 of 31
1
1
1
1
1
2. Wang, J.; Yang, T.; Chen, H.; Xu, Y.‐N.; Yu, L.‐F.; Liu, T.; Tang, J.; Yi, Z.; Yang, C.‐G.; Xue, W.; et al. The
synthesis and antistaphylococcal activity of 9, 13‐disubstituted berberine derivatives. Eur. J. Med. Chem.
017, 127, 424–433, doi:10.1016/j.ejmech.2017.01.012.
3. Fan, T.‐Y.; Wang, Y.‐X.; Tang, S.; Hu, X.‐X.; Zen, Q.‐X.; Pang, J.; Yang, Y.‐S.; You, X.‐F.; Song, D.‐Q. Synthesis
and antibacterial evaluation of 13‐substituted cycloberberine derivatives as a novel class of anti‐MRSA
agents. Eur. J. Med. Chem. 2018, 157, 877–886, doi:10.1016/j.ejmech.2018.08.050.
4. Park, K.D.; Lee, J.H.; Kim, S.H.; Kang, T.H.; Moon, J.S.; Kim, S.U. Synthesis of 13‐(substituted benzyl)
berberine and berberrubine derivatives as antifungal agents. Bioorg. Med. Chem. Lett. 2006, 16, 3913–3916,
doi:10.1016/j.bmcl.2006.05.033.
5. Olleik, H.; Yahiaoui, S.; Roulier, B.; Courvoisier‐Dezord, E.; Perrier, J.; Pérès, B.; Hijazi, A.; Baydoun, E.;
Raymond, J.; Boumendjel, A.; et al. Aurone derivatives as promising antibacterial agents against resistant
Gram‐positive pathogens. Eur. J. Med. Chem. 2019, 165, 133–141, doi:10.1016/j.ejmech.2019.01.022.
6. Olleik, H.; Nicoletti, C.; Lafond, M.; Courvoisier‐Dezord, E.; Xue, P.; Hijazi, A.; Baydoun, E.; Perrier, J.;
Maresca, M. Comparative Structure–Activity Analysis of the Antimicrobial Activity, Cytotoxicity, and
Mechanism of Action of the Fungal Cyclohexadepsipeptides Enniatins and Beauvericin. Toxins 2019, 11,
2
514, doi:10.3390/toxins11090514.
1
1
1
2
2
2
2
7. Chiumento, S.; Roblin, C.; Kieffer‐Jaquinod, S.; Tachon, S.; Leprètre, C.; Basset, C.; Aditiyarini, D.; Olleik,
H.; Nicoletti, C.; Bornet, O.; et al. Ruminococcin C, a promising antibiotic produced by a human gut
symbiont. Sci. Adv. 2019, 5, eaaw9969, doi:10.1126/sciadv.aaw9969.
8. Benkhaled, B.T.; Hadiouch, S.; Olleik, H.; Perrier, J.; Ysacco, C.; Guillaneuf, Y.; Gigmes, D.; Maresca, M.;
Lefay, C. Elaboration of antimicrobial polymeric materials by dispersion of well‐defined amphiphilic
methacrylic SG1‐based copolymers. Polym. Chem. 2018, 9, 3127–3141, doi:10.1039/C8PY00523K.
9. Oyama, L.B.; Girdwood, S.E.; Cookson, A.R.; Fernandez‐Fuentes, N.; Privé, F.; Vallin, H.E.; Wilkinson, T.J.;
Golyshin, P.N.; Golyshina, O.V.; Mikut, R.; et al. The rumen microbiome: An underexplored resource for
novel antimicrobial discovery. NPJ Biofilms Microbiomes 2017, 3, 33, doi:10.1038/s41522‐017‐0042‐1.
0. Borie, C.; Mondal, S.; Arif, T.; Briand, M.; Lingua, H.; Dumur, F.; Gigmes, D.; Stocker, P.; Barbarat, B.;
Robert, V.; et al. Enediynes bearing polyfluoroaryl sulfoxide as new antiproliferative agents with dual
targeting of microtubules and DNA. Eur. J. Med. Chem. 2018, 148, 306–313, doi:10.1016/j.ejmech.2018.02.030.
1. Razafimanjato, H.; Benzaria, A.; Taïeb, N.; Guo, X.‐J.; Vidal, N.; Di Scala, C.; Varini, K.; Maresca, M. The
ribotoxin deoxynivalenol affects the viability and functions of glial cells. Glia 2011, 59, 1672–1683,
doi:10.1002/glia.21214.
2. Olleik, H.; Baydoun, E.; Perrier, J.; Hijazi, A.; Raymond, J.; Manzoni, M.; Dupuis, L.; Pauleau, G.; Goudard,
Y.; Villéon, B. de L.; et al. Temporin‐SHa and Its Analogs as Potential Candidates for the Treatment of
Helicobacter pylori. Biomolecules 2019, 9, 598, doi:10.3390/biom9100598.
3. Di Pasquale, E.; Salmi‐Smail, C.; Brunel, J.‐M.; Sanchez, P.; Fantini, J.; Maresca, M. Biophysical studies of
the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic
membranes: Importance of the distribution coefficient in membrane selectivity. Chem. Phys. Lipids 2010,
163, 131–140, doi:10.1016/j.chemphyslip.2009.10.006.
2
4. Tan, C.M.; Therien, A.G.; Lu, J.; Lee, S.H.; Caron, A.; Gill, C.J.; Lebeau‐Jacob, C.; Benton‐Perdomo, L.;
Monteiro, J.M.; Pereira, P.M.; et al. Restoring methicillin‐resistant Staphylococcus aureus susceptibility to
β‐lactam antibiotics. Sci. Transl. Med. 2012, 4, 126ra35, doi:10.1126/scitranslmed.3003592.
25. Korb, O.; Stützle, T.; Exner, T.E. Empirical scoring functions for advanced protein‐ligand docking with
PLANTS. J. Chem. Inf. Model. 2009, 49, 84–96, doi:10.1021/ci800298z.
2
6. Giordano, A.; del Gaudio, F.; Johansson, C.; Riccio, R.; Oppermann, U.; Di Micco, S. Virtual Fragment
Screening Identification of a Quinoline‐5,8‐dicarboxylic Acid Derivative as a Selective JMJD3 Inhibitor.
Chemmedchem 2018, 13, 1160–1164, doi:10.1002/cmdc.201800198.
2
7. Lee, J.; Cheng, X.; Swails, J.M.; Yeom, M.S.; Eastman, P.K.; Lemkul, J.A.; Wei, S.; Buckner, J.; Jeong, J.C.; Qi,
Y.; et al. CHARMM‐GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and
CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput.
2016, 12, 405–413, doi:10.1021/acs.jctc.5b00935.
28. Jo, S.; Kim, T.; Iyer, V.G.; Im, W. CHARMM‐GUI: A web‐based graphical user interface for CHARMM. J.
Comput. Chem. 2008, 29, 1859–1865, doi:10.1002/jcc.20945.
29. Chu, M.H.; Xiao, R.; Yin, Y.; Wang, X.; Chu, Z.; Zhang, M.; Ding, R.; Wang, Y. Berberine: A Medicinal
Compound for the Treatment of Bacterial Infections. Clin. Microbiol. 2014, doi:10.4172/2327‐5073.1000150.