Antibacterial activity of substituted 5-methylbenzo[c]phenanthridinium derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.09.097

Antibiotic resistance has prompted efforts to discover antibiotics with novel mechanisms of action. FtsZ is an essential protein for bacterial cell division, and has been viewed as an attractive target for the development of new antibiotics. Sanguinarine is a benzophenanthridine alkaloid that prevents cytokinesis in bacteria by inhibiting FtsZ self-assembly. In this study, a series of 5-methylbenzo[c]phenanthridinium derivatives were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. The data indicate that the presence of a 1- or 12-phenyl substituent on 2,3,8,9-tetramethoxy-5-methylbenzo[c]phenanthridinium chloride significantly enhances antibacterial activity relative to the parent compound or sanguinarine.

Full text of DOI:10.1016/j.bmcl.2012.09.097

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7080–7083
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antibacterial activity of substituted
5-methylbenzo[c]phenanthridinium derivatives
Ajit Parhi a, Cody Kelley b, Malvika Kaul c, Daniel S. Pilch c, Edmond J. LaVoie b,
⇑
a TAXIS Pharmaceuticals Inc., North Brunswick, NJ 08902, USA
b Department of Medicinal Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
c Department of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2012
Revised 20 September 2012
Accepted 25 September 2012
Available online 2 October 2012
Antibiotic resistance has prompted efforts to discover antibiotics with novel mechanisms of action. FtsZ is
an essential protein for bacterial cell division, and has been viewed as an attractive target for the devel-
opment of new antibiotics. Sanguinarine is a benzophenanthridine alkaloid that prevents cytokinesis in
bacteria by inhibiting FtsZ self-assembly. In this study, a series of 5-methylbenzo[c]phenanthridinium
derivatives were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and
Enterococcus faecalis. The data indicate that the presence of a 1- or 12-phenyl substituent on 2,3,8,9-tet-
ramethoxy-5-methylbenzo[c]phenanthridinium chloride significantly enhances antibacterial activity rel-
ative to the parent compound or sanguinarine.
Keywords:
Benzo[c]phenanthridine
FtsZ-targeting
Staphylococcus aureus
Enterococcus faecalis
Ó 2012 Elsevier Ltd. All rights reserved.
Multidrug-resistant (MDR) bacterial pathogens like methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
enterococci (VRE) represent an increasing nosocomial health con-
cern for both patients and healthcare professionals.1,2 The growing
problem of resistance to current clinical antibiotics has created a
critical need for novel therapeutic antibiotics with unique modes
of action. FtsZ is a key protein involved in bacterial cell division
(cytokinesis) and is highly conserved among bacterial patho-
gens.3–5 The essential role that FtsZ plays in bacterial cell division
makes this protein a promising therapeutic target. Interest in the
development of small molecules that target FtsZ is reflected by
several recent reviews on this topic.6–9
Sanguinarine 1 (Fig. 1) is a plant alkaloid that has been identi-
fied as a small molecule that alters bacterial FtsZ Z-ring formation
and has antibacterial activity.10 Recent studies have also demon-
strated that the structurally-related alkaloid, berberine also affects
FtsZ self-polymerization.11,12 In the present study, the effect of var-
ied substituents at the 1- and 12-positions of several 5-meth-
ylbenzo[c]phenanthridinium derivatives on antibacterial activity
versus S. aureus and E. faecalis was evaluated.
provide the 5-nitro derivative. Hydrolysis of the mesylates fol-
lowed by treatment with methyl iodide provided 1-nitro-5,6-
dimethoxynaphthalene, which was reduced to 5,6-dimethoxy-1-
naphthylamine. Treatment of this naphthylamine with the acid
chloride of either 6-bromo-2,3-methylenedioxybenzoic acid or 6-
bromo-2,3-dimethoxybenzoic acid provided the benzamide deriv-
atives, which were converted to their tertiary amides by treatment
with NaH and then methyl iodide. These tertiary benzamide inter-
mediates were converted to their respective 5-methylbenzo[c]phe-
nanthridin-6-ones under Heck cyclization conditions. Subsequent
treatment of these 5-methylbenzo[c]phenanthridin-6-ones with
LAH, followed with acidification with HCl provided 2 and 6.
The method used for the preparation of several 1-substituted 5-
methylbenzo[c]phenanthridinium chlorides is outlined in Scheme
2. 1-Nitro-5,6-dimethoxynaphthalene was converted to 1-nitro-
4-bromo-5,6-dimethoxynaphthalene, which was treated with
various boronic acids under Suzuki coupling conditions to pro-
vide 1-susbtituted-2,3-dimethoxy-5-nitronaphthalene derivatives.
Reduction of the nitro group and acylation using either 2,3-dime-
thoxy- or 2,3-methylenedioxy-6-bromobenzoyl chloride provided
the benzamide derivatives. Formation of the tertiary N-methylben-
zamides, followed by cyclization to their 5-methylbenzo[c]phe-
nanthridin-6-ones, and reduction with LAH with subsequent
treatment with HCl provided the desired 1-substituted-5-meth-
ylbenzo[c]phenanthridinium chlorides.
The 5-methylbenzo[c]phenanthridinium derivatives synthe-
sized and evaluated for antibacterial activity are listed in Table 1.
The methods used for preparation of 2 and 6 are summarized in
Scheme 1. 2,3-Dihydroxynaphthalene was converted to its dimes-
ylate and then treated with nitric acid in acetic anhydride to
The synthesis of 12-susbtituted 5-methylbenzo[c]phenanthrid-
inium chlorides was accomplished as illustrated in Scheme 3. We
prepared 1-amino-5,6-dimethoxynaphthalene from 1-nitro-
⇑
Corresponding author. Tel.: +1 848 445 2674; fax: +1 732 445 6312.
E-mail address: elavoie@pharmacy.rutgers.edu (E.J. LaVoie).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.097

A. Parhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7080–7083
7081
5-methylbenzo[c]phenanthridin-6-ones. Reduction of these inter-
mediates with LAH followed by treatment with HCl provided 5,
8, and 10.
O
O
O
O
N
Sanguinarine, 1, exhibited significant activity against both the
methicillin-sensitive and methicillin-resistant strains of S. aureus
(MSSA and MRSA, respectively) used in this study. It also had mod-
est activity against both the vancomycin-sensitive and vancomy-
cin-resistant strains of E. faecalis (VSE and VRE, respectively).
Compound 2, which resembles berberine with regard to the rela-
tive location of its methylenedioxy and methoxyl substituents,
was significantly less active against the MSSA and MRSA strains,
and did not exhibit appreciable activity against the VSE and VRE
strains. The addition to 2 of a phenyl substituent at the 1-position
or a biphenyl substituent at either the 1- or 12-position (3–5) dra-
matically increased antibacterial activity against all the S. aureus
and E. faecalis strains examined. The tetramethoxy analog of these
alkaloids 6 was also prepared and evaluated for antibacterial activ-
ity. These analogs were viewed as attractive, as they would be less
likely to cause adverse effects in mammalian cells by possible
N
H3CO
O
CH3
OCH3
Berberine
O
1
Figure 1. Structures of sanguinarine 1 and berberine.
5,6-dihydroxynaphthalene by initially forming the 5,6-dimethoxy
derivative, followed by reduction of the nitro substituent. Treat-
ment of this 1-naphthylamine with iodine in pyridine initially at
0 °C and allowing it to warm to room temperature provided
4-iodo-5,6-dimethoxy-1-naphthylamine. Suzuki coupling with
various organoboronates was performed with this iodo derivative.
Conversion of the resulting 1-naphthylamine to its secondary bro-
mobenzamide and then to its N-methyl tertiary benzamide, fol-
lowed by Heck cyclization provided the desired 12-substituted
Table 1
1- and 12-Substituted 5-methylbenzo[c]phenanthridinium compounds synthesized and evaluated
X12 X1
R2
R3
N
R8
CH3
R7
Cl
.
Compd
R2
R3
R7
R8
X1
X12
1
2
–O–CH2–O–
–OCH3
–O–CH2–O–
–O–CH2–O–
H
H
H
H
–OCH3
–OCH3
3
4
–OCH3
–OCH3
–O–CH2–O–
–O–CH2–O–
H
H
–OCH3
5
6
–OCH3
–OCH3
–OCH3
–OCH3
–O–CH2–O–
–OCH3
H
H
–OCH3
–OCH3
H
H
7
8
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
H
H
9
H
10
11
12
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
H
H
13
14
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
H
H
OCH3
OCH3
OCH3
N(CH3)2
15
–OCH3
–OCH3
–OCH3
–OCH3
H
16
17
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
H
H
O
N
18
–OCH3
–OCH3
–OCH3
–OCH3
H

7082
A. Parhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7080–7083
OMe
OMe
OH
OH
OMe
OMe
Br
OH
OH
d,e
f
a,b,c
N
R8
Me
NO2
NH2
g,h
R7
O
OMe
OMe
OMe
OMe
i
N
N
R8
Me
R8
Me
R7
O
R7
2, 6
Scheme 1. Preparation of 2,3-dimethoxy-7,8-methylenedioxy-5-methylbenzo[c]phenanthridinium chloride 2 and 2,3,7,8-tetramethoxy-5-methylbenzo[c]phenanthridinium
chloride 6. Reagents and conditions: (a) MsCl, Et3N, CHCl3, 0 °C; (b) Ac2O, HNO3, 37–39 °C; (c) NaOH, H2O, 100 °C; (d) MeI, K2CO3, DMF, 50 °C; (e) hydrazine monohydrate, Pd/
C (10%), EtOH, 85 °C; (f) (i) 5–bromobenzo[d][1,3]dioxole-4-carboxylic acid towards the preparation of 2 or 6-bromo-2,3-dimethoxybenzoic acid towards the preparation of 6,
oxalyl chloride, CH2Cl2, 45 °C; (ii) 6,7-dimethoxyaniline, Et3N, CH2Cl2, rt; (g) MeI, NaH, DMF, rt; (h) Pd(OAc)2, p(o-tolyl)3, Ag2CO3, DMF, 155 °C; (i) LAH, THF; then HCl (10%).
R1
Br
R1
OH
OH
OMe
OMe
OMe
OMe
OMe
OMe
a,b
d
c
NO2
NH2
NO2
NO2
3b,4b,7b,9b,
3a,4a,7a,9a,
11b-13b,15b-18b
11a-13a,15a-18a
R1
R1
R1
OMe
OMe
OMe
OMe
OMe
g
i
e,f
Br
OMe
N
N
R8
Me
R8
Me
N
R8
Me
Cl
R7
R7
O
R7
3c,4c,7c,9c,11c-13c,15c-18c
O
3,4,7,9,11-18
3d,4d,7d,9d,11d-13d,15d-18d
h
14d
Scheme 2. Methods used for the preparation of 1-substituted-5-methylbenzo[c]phenanthridinium chlorides. Reagents and conditions: (a) Br2, CH2Cl2, 0 °C; (b) MeI, K2CO3,
DMF, 50 °C; (c) R1–B(OH)2, Pd(PPh3)4, Na2CO3 toluene/H2O, 90 °C; for 11a,13a PdOAc, PCy3, K2PO4 toluene/H2O, 90 °C; for 12a vinyltributyltin, Pd(PPh3)4, THF, reflux (d)
hydrazine monohydrate, Pd/C (10%), EtOH, 85 °C; for 12b SnCl2, EtOH; for 13b H2 (g) Pd/C (10%); (e) (i) 6-bromo-2,3-dimethoxybenzoic acid, oxalyl chloride, CH2Cl2, 45 °C; for
3,4 6-bromo-2,3-methylenedioxybenzoic; oxalyl chloride, CH2Cl2, 45 °C (ii) the naphthylamine in CH2Cl2 was added to the appropriate acid chloride, Et3N, rt; (f) MeI, NaH,
DMF, rt; (g) Pd(OAc)2, p(o-tolyl)3, Ag2CO3, DMF, 155 °C; (h) H2 (g), Pd/C (10%), EtOH (i) LAH, THF, 0 °C; then HCl (10%).
R12
I
OMe
OMe
OMe
OMe
OMe
OMe
OH
OH
d
c
e,f
a,
NH2
NH2
NH2
OMe
OMe
NO2
R12
R12
R12
OMe
OMe
OMe
OMe
h
g
Br
N
N
R8
Me
N
R8
Me
R8
Me
Cl
R7
R7
O
R7
O
5,8,10
Scheme 3. Methods used for the preparation of 12-substituted 5-methylbenzo[c]phenanthridinium chlorides. Reagents and conditions: (a) MeI, K2CO3, DMF, 50 °C; (b)
hydrazine monohydrate, Pd/C (10%), EtOH, 85 °C; (c) I2, pyridine, dioxane, 0 °C to rt; (d) R1–B(OH)2, Pd(PPh3)4, Na2CO3, toluene/H2O, 90 °C; (e) (i) 6-bromo-2,3-
methylenedioxybenzoic acid towards the preparation of 5 or 2,3-dimethoxybenzoic acid towards the preparation of 8 and 11, oxalyl chloride, CH2Cl2, 45 °C; (ii) Requisite 6,7-
dimethoxyaniline, Et3N, CH2Cl2, rt; (f) MeI, NaH, DMF, rt; (g) Pd(OAc)2, p(o-tolyl)3, Ag2CO3, DMF, 155 °C; (h) (i) LAH, THF, 0 °C; (ii) HCl (10%).
intercalation into DNA. While 6 is similar to berberine, having only
weak antibacterial activity (MIC >64 g/ml), the antibacterial
activity could be substantially increased by the addition of a phe-
1- or 12-position. The 1-biphenyl derivative 9 was particularly
active against these four bacterial strains. Several additional
1-substituted derivatives of 6 were evaluated for antibacterial
activity. The cyclohexen-1-yl (13), 3,4,5-trimethoxyphenyl (15),
l
nyl substituent (7,8) or a biphenyl substituent (9,10) at either its

A. Parhi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7080–7083
7083
Table 2
benzo[c]phenanthridines had greater activities versus VRE than
any of the listed clinical antibiotics (Table 2).
Antibacterial activities of various 1- and 12-substituted benzo[c]phenanthridinium
derivatives against S. aureus and E. faecalis
These data indicate that several 1- and 12-susbtituted 5-meth-
ylbenzo[c]phenanthridinium have significant antimicrobial activ-
ity against multidrug-resistant (MDR) strains of S. aureus and E.
faecalis. Studies are in progress to further investigate the struc-
ture–activity of 5-methylbenzo[c]phenanthridinium derivatives
and related compounds with regard to antimicrobial and FtsZ-tar-
geting activity.
aMIC (
lg/mL)
S. aureus
8325-4
(MSSA)
S. aureus
ATCC 33591
(MRSA)
E. faecalis
ATCC 19433
(VSE)
E. faecalis
ATCC 51575
(VRE)
1
2
3
2
32
1
2
32
2
8
64
8
16
>64
8
4
5
2
2
2
2
8
4
8
8
Acknowledgements
6
7
32
1
32
1
>64
4
>64
4
This study was supported by research agreements between
TAXIS Pharmaceuticals, Inc. and both Rutgers, The State University
of New Jersey (E.J.L.) and the University of Medicine and Dentistry
of New Jersey (D.S.P). S. aureus 8325-4 was the generous gift of Dr.
Glenn W. Kaatz (John D. Dingell VA Medical Center, Detroit, MI).
The Brucker Avance III 400 MHz NMR spectrometer used in this
study was purchased with funds from NCRR Grant No.
1S10RR23698-1A1. Mass spectrometry was provided by the
Washington University Mass Spectrometry Resource with support
from the NIH National Center for Research Resources Grant No.
P41RR0954.
8
9
10
11
12
13
14
15
1
2
8
4
16
8
8
16
8
32
8
16
32
8
16
8
16
8
8
32
8
0.5
0.5
1
1
2
1
4
1
1
8
0.06
1
0.13
0.06
0.03
0.5
0.5
2
2
4
2
4
1
1
32
>64
2
>64
64
>64
32
8
16
17
18
16
32
>64
>64
>64
>64
>64
Oxacillin
Vancomycin
Erythromycin
Tetracycline
Clindamycin
1
1
0.5
2
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