Synthesis of functionalised 2-aryl-5-nitro-1H-indoles and their activity as bacterial NorA efflux pump inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.09.019

In order to develop structure-activity relationships and to provide access to antibacterial agents for dual action studies, a variety of aryl group-substituted 2-aryl-5-nitro-1H-indoles were synthesized and the activity of the compounds assessed as inhibitors of the NorA multidrug resistance pump in the bacterium Staphylococcus aureus. The NorA protein from the major facilitator superfamily of efflux pumps confers resistance to a variety of structurally dissimilar antimicrobials such as norfloxacin, ethidium bromide, berberine and acriflavin. The compound [4-benzyloxy-2-(5-nitro-1H-2-yl)-phenyl]-methanol was the most potent pump inhibitor.

Full text of DOI:10.1016/j.bmc.2005.09.019

Bioorganic & Medicinal Chemistry 14 (2006) 857–865
Synthesis of functionalised 2-aryl-5-nitro-1H-indoles and
their activity as bacterial NorA efflux pump inhibitors
Siritron Samosorn,a John B. Bremner,a,* Anthony Ballb and Kim Lewisb
aInstitute for Biomolecular Science and Department of Chemistry, University of Wollongong, NSW 2522, Australia
bDepartment of Biology, Northeastern University, Boston, MA 02155, USA
Received 27 August 2005; accepted 2 September 2005
Available online 3 October 2005
Abstract—In order to develop structure–activity relationships and to provide access to antibacterial agents for dual action studies, a
variety of aryl group-substituted 2-aryl-5-nitro-1H-indoles were synthesized and the activity of the compounds assessed as inhibitors
of the NorA multidrug resistance pump in the bacterium Staphylococcus aureus. The NorA protein from the major facilitator super-
family of efflux pumps confers resistance to a variety of structurally dissimilar antimicrobials such as norfloxacin, ethidium bromide,
berberine and acriflavin. The compound [4-benzyloxy-2-(5-nitro-1H-2-yl)-phenyl]-methanol was the most potent pump inhibitor.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the synthesis of 5-nitro-2-phenyl-1H-indole, including
a palladium-assisted reaction of 4-nitro-2-bromoaniline
and a-piperidinostyrene,7 the Fischer indole synthesis
of acetophenone with phenylhydrazine and subsequent
nitration,8 and reaction of 4-nitroaniline and dimethyl-
(2-oxo-2-phenyl-ethyl)-sulfonium bromide followed by
cyclisation.9 However, there are limitations with respect
to functional group tolerances in these approaches.
Another approach to such 2-aryl indoles involves a pre-
formed indole nucleus and subsequent introduction of a
functionalised 2-aryl group via N-acylation, palladium-
induced oxidative cyclisation, and ring opening of the
2-nitro-6H-isoindolo[2,1-a]indol-6-one intermediate.
In the vast heterocyclic structural space, the indole
nucleus occupies a position of major importance. Many
indole derivatives, including fused derivatives, form the
basis of a range of pharmaceuticals1,2 and a high level of
activity continues in the search for new indole-based
medicinal agents.3,4 In this context and as part of a wid-
er programme on the development of dual action anti-
bacterial agents, we have investigated the synthesis of
5-nitro-2-aryl-1H-indoles. The known compound 5-ni-
tro-2-phenyl-1H-indole (INF55) is an inhibitor of the
NorA efflux pump in the human pathogenic bacterium
Staphylococcus aureus.5 While some related 2-aryl deriv-
atives (for example, an o-anisole group) have been pre-
dicted by CoMFA (3D-QSAR) analysis to be NorA
pump inhibitors,6 there is a need to access a greater
range of 2-aryl indole derivatives with a variety of func-
tionalisation in the aryl substituent group, particularly
with respect to groups in the ortho position to the point
of indole attachment in the aryl ring. Such derivatives
would be of value in establishing structure–activity rela-
tionships in the pump inhibitors, and also in providing
handles for linking other antibacterial agents for dual
action studies. Several routes have been described for
The NorA protein of S. aureus is a drug/proton antipor-
ter belonging to the major facilitator family of trans-
porters and extrudes chemically unrelated substances
such as norfloxacin, ciprofloxacin, ethidium bromide,
berberine and acriflavin.10 The effects of functionalised
INF55 can be reported by a decrease of the minimum
inhibitory concentration (MIC) of berberine.11 Berber-
ine alkaloids are amphipathic cations, a preferred type
of MDR transporter substrate.10 Berberine is a plant
secondary metabolite and a phytoalexin produced in re-
sponse to stressors like microbial invasion.12,13 Interest-
ingly, Berberis plants that make berberine also produce
an MDR inhibitor, 50-methoxyhydnocarpin (50-
MHC).14 The synergistic couple of berberine and 50-
MHC produces an effective antimicrobial action.
Designing inhibitors to block resistance conferred by
Keywords: Indoles; 5-Nitro-2-phenyl-1H-indole derivatives; NorA
efflux pump inhibitors; Antibacterial agent potentiators.
*
Corresponding author. Tel.: +61 2 4221 4255; fax: +61 2 4221
4287; e-mail: john_bremner@uow.edu.au
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.019

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S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
COOH
multidrug transporters could be an attractive strategy
for developing a new anti-infective.
4
3
O2N
O2N
a
2
+
6
N
N
Application of this methodology, with subsequent func-
tional group manipulations, to the synthesis of new aryl-
group functionalised 5-nitro-2-aryl-1H-indoles, together
with their assessment as NorA pump inhibitors, are now
described in this article.
H
7
R
2
O
3
R
1
a R = H
b R = OCH3
c R = OCH2Ph
b
1
HOOC
11
10b
O2N
1'
10
c
O2N
R
2'
10a
3
N
2. Results and discussion
6
N
4
8
H
7
R
O
7
4
2.1. Synthesis of 2-aryl-5-nitro-1H-indoles
Scheme 1. Reagents: (a) DCC, DMAP, DCM; (b) Pd(OAc)2, AcOH;
(c) t-BuOK, t-BuOH, H2O.
Commercially available 5-nitro-1H-indole (1) was used
as the starting point for the syntheses followed by direct
N-acylation using the carboxylic acids 2, and then palla-
dium cyclisation and hydrolytic ring opening of 4 to the
carboxylic acid derivatives 7 (Scheme 1).
corresponding alcohols 8 without affecting the nitro sub-
stituent. Subsequently, conversion of the alcohols to the
azides 9 and then the amines 10 was also established; no
indolic NH protection was required in these sequences
(Scheme 2). Selective reduction of the benzoic acid
derivatives 7a–7c to the corresponding alcohols 8a–8c
was achieved with borane in tetrahydrofuran.19,20 The
reaction was remarkably facile and proceeded in high
yield. The 1H NMR analysis of 8a–8c confirmed the for-
mation of the benzyl alcohol derivatives with a distinc-
tive signal ascribed to the methylene protons in the
range of d 4.63–4.77, and the loss of the signal assigned
to the carbonyl carbon in the starting materials 7a–7c
was observed in the 13C NMR spectra.
The direct N-acylation of 1 with carboxylic acids 2a–2c,
which were prepared according to the reported proce-
dure,15 afforded 3a–3c in high yields. The intramolecular
ring-closure of the N-acylated indoles 3a–3c to the isoin-
dolo-indolones 4a–4c (Scheme 1) was accomplished via
a palladium(II) acetate-promoted oxidative intramolec-
ular cyclisation, following the method for the intramo-
lecular ring-closure of 1-aroylindoles reported
previously by Itahara.16 The compounds 4a–4c were ob-
tained in moderate yields. The structure of 4a was con-
firmed by NMR spectroscopic analysis and mass
1
spectrometry. The H NMR spectrum revealed a dis-
tinctive signal attributed to H-11 at d 6.79. In 4b, this
signal appeared at d 6.77.
Treatment of alcohols 8a–8b with sodium azide and tri-
phenylphosphine21 afforded the azides 9a–9b in moder-
ate yields. Reduction of the azides 9a–9b to the amines
10a–10b was achieved using NaBH4. The initial reduc-
tion attempt involved the reduction of 9a with NaBH4
in THF with dropwise addition of MeOH.22 Two prod-
ucts were obtained, one of which was the amine 10a, and
the other was the new cyclised product11 (Scheme 3).
In the case of the cyclisation of 3c, 4c was obtained in
20% yield together with the unexpected products 5 and
6 in 22% and 35% yields, respectively.
O2N
O2N
OH
1
The H NMR spectrum of 11 revealed the absence of
N
N
a signal which could be ascribed to the indolic NH pro-
ton and the appearance of a downfield singlet integrat-
ing for two protons at d 5.12, which was attributed to
6
5
O
O
OH
Debenzylation can be accomplished under acidic condi-
tions at reflux.17 Therefore, the cleavage of the benzyl-
oxy substituent in 3c and 4c could both occur together
with the cyclisation of 3c. Compound 5 can be converted
to 4c in good yield by benzylation of the phenoxide an-
ion with benzyl bromide. The structure of 4c was con-
firmed by the presence of a singlet signal attributed to
HOOC
HOH2C
1'
O2N
O2N
a
2'
N
H
7
N
H
R
R
8
a R = H
b R = OCH3
c R = OCH2Ph
1
H-11 at d 6.79 in the H NMR spectrum.
b
Ring-opening by amide hydrolysis18 of compounds 4a–
4c then afforded the acids 7a–7c in high yield. The 13C
NMR spectra of these acids revealed a signal ascribed
to the carbonyl carbon of the carboxylic acids in the
range of d 167.9–171.7.
H2NH2C
N3H2C
1'
2'
O2N
O2N
2'
1'
c
N
H
N
H
9
R
R
10
Further selective functional group manipulations in the
indole derivatives were based on the carboxylic acids 7,
starting with carboxylic acid group reduction to the
Scheme 2. Reagents: (a) BH3-THF; (b) NaN3, PPh3, CCl4-DMF; (c)
NaBH4, HS(CH2)3SH.

S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
859
N3H2C
S. aureus strains was 15 lg/mL for K1758, 125 lg/mL
for 8325-4 and >500 lg/mL for K2361 (Table 2). All
inhibitors, with the exception of 7a, increased S. aureus
susceptibility to berberine (Table 1). Compound 8c had
the highest activity against the strain overexpressing the
NorA pump. At a concentration of 0.8 lg/ml, it poten-
tiated berberine more than 15-fold.
1
11
O2N
O2N
10
7
NaBH4
9
+
10a
3
N
N
4
8
H
9a
6
11
Scheme 3. Reduction of azide 9a with NaBH4.
Compound 9b was almost as active as 8c, and both
showed higher activity as compared to the known inhib-
itors INF271 and INF55. Compounds 10a and 10b were
highly active against the strain deleted in NorA and the
wild type, but relatively ineffective against K2361 over-
expressing NorA. This suggests that these two inhibitors
are more effective in blocking some additional pumps in
S. aureus, rather than NorA.
the methylene protons (H-6). The 1H NMR spectrum of
10a showed an upfield signal integrating for two protons
at d 3.99 as a singlet, which was assigned to the methy-
lene protons adjacent to the amine group, and a signal
ascribed to the indolic NH was apparent at d 13.63.
When reduction of the azides 9a–9b was undertaken
using NaBH4 and 1,3-propanedithiol23 in i-PrOH and
triethylamine at low temperature, to avoid cyclisation,
the amines 10a–10b were obtained in high yields.
The nature of the functional group in the 2-aryl substi-
tuent is clearly significant in determining relative poten-
cy. With a neutral group at the 10 position in the aryl
ring and an ether substituent in the 40 position, an in-
crease in potency was observed. In the case of the pri-
mary amines 10a and 10b, the presence of the 4-
methoxy group in the latter decreased potency as an
inhibitor. Protonation of the primary amino group at
physiological pH may be a factor in determining poten-
cy between the strains in the case of 10a.
Since the palladium-induced oxidative cyclisation of 3c
gave 4c in low yield and two by-products 5 and 6, the
synthesis of benzyloxy analogues of the azide and amine
derivatives from 4c was omitted.
The various 2-aryl-5-nitro-1H-indoles prepared were
then evaluated as inhibitors of the NorA MDR efflux
pump in S. aureus.
H
N
O
O2N
2.2. Assessment of NorA MDR pump inhibitory activity
HN
H3CO
N
H
The NorA inhibitory activity of synthetic indole deriva-
tives 7a–b, 8a–c, 9a–b and 10a–c was assayed in the wild
type S. aureus 8325-4, in a mutant deleted in NorA
(K1758), and in a strain overexpressing the pump from
a recombinant plasmid (K2361). The inhibitors did not
have a direct activity (MICs P 50 lg/mL), except for
9a (MIC 3 lg/mL) and 10b (25 lg/mL) against S. aureus
K1758. The MIC of berberine alone against the
INF55
INF271
Next, we tested the ability of the most potent inhibi-
tor, 8c to potentiate a variety of chemically unrelated
antimicrobial compounds, which are substrates of
NorA. Compound 8c was comparable in its potency
to the known inhibitor INF55 (Table 2). Potentiation
of a number of substrates was highly significant, for
example, the MIC of berberine changed from
>500 lg/mL in
a strain overexpressing NorA to
Table 1. Potentiation of berberine by MDR inhibitors in Staphylo-
coccus aureus
2 lg/mL in the presence of 8c; and the potency of
the fluoroquinolone antibiotic norfloxacin increased
16-fold.
Compound
MIC (lg/mL) of inhibitor
DnorA
WT
NorA++
K1758 plus
berberine
(3 lg/mL)
8325-4 plus
berberine
(30 lg/mL)
K2361 plus
berberine
Potentiation of berberine accumulation by 8c into cells
of S. aureus was measured by following fluorescence of
berberine bound to DNA (Fig. 1). The rate of berberine
accumulation increased sharply in the presence of 8c in
all strains tested. The initial accumulation rate was high-
er with 8c as compared to INF55.
(30 lg/mL)
7a
8a
>50
6.25
0.8
12.5
3
>50
12.5
6
>50
12.5
3
9a
7b
12.5
12.5
1.5
0.4
0.4
0.4
0.8
0.2
>50
6.25
1.5
0.8
12.5
12.5
3
8b
9b
0.4
0.4
<0.2
0.8
3
8c
3. Conclusions
10a
10b
INF271
INF55
The synthesis of a range of new 2-aryl-5-nitroindole
derivatives was achieved using a palladium-mediated
cyclisation to establish the crucial indole C2-aryl C
bond formation. These compounds were then shown
to be effective NorA MDR pump inhibitors, apart
from an acid derivative. Overall, compounds 8a–8c,
0.8
3
Inhibitors were added to S. aureus cells growing in MH broth in the
presence of sub-inhibitory amounts of berberine and inhibition of
growth was measured by recording the optical density after 18 h of
incubation.

860
S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
Table 2. Potentiation of antimicrobials by an indole-based MDR inhibitor 8c.
MIC (lg/mL) MIC (lg/mL)plus INF55 (5 lg/mL) Fold change MIC (lg/mL)plus 8c (5 lg/mL) Fold change
Wild-type (8325-4)
Berberine
125
1
1
125
10
1
125
10
40
4
Ciprofloxacin
Ethidium bromide
Norfloxacin
0.1
0.03
0.25
0.1
0.1
0.25
4
130
4
1
NorA++ (K2361)
Berberine
ꢀ 500
4
31
2
250
8
2
250
8
Ciprofloxacin
Ethidium bromide
Norfloxacin
0.5
0.5
2
0.5
0.1
1
62
8
310
16
16
DnorA (K1758)
Berberine
15
0.5
0.1
0.03
0.5
30
0.5
30
2
Ciprofloxacin
Ethidium bromide
Norfloxacin
0.24
2.4
0.12
0.03
0.24
0.24
0.24
8
0.5
8
No change
Conditions as described in legend to Table 1.
800
700
600
500
400
300
200
100
0
distilled from sodium in the presence of benzophenone.
Other solvents used were of AR grade, and were used as
received, except for dichloromethane (DCM), which was
of LR grade and was distilled before use. The term
petroleum spirit (PS) refers to petroleum spirit within
the boiling range of 40–60 ꢁC. Flash chromatography
was performed under medium pressure using silica gel
60 (230–400 mesh, Merck); this silica gel was also used
with vacuum liquid chromatography (VLC). Preparative
TLC was done on Merck Silica gel 60 F254 with a thick-
ness of 0.2 mm on aluminium sheet. Reactions were
monitored by thin-layer chromatography (TLC) on
Merck Silica gel 60 F254 with a thickness of 0.2 mm on
aluminium sheet, and the compounds were detected by
examination under ultraviolet light and by exposure to
iodine vapour. Organic solvents were dried over anhy-
drous sodium sulfate and the solvent was removed un-
0
5
10
15
20
der reduced pressure with a Buchi rotary evaporator.
¨
Solvent ratios are vol/vol. All compounds were judged
Time (min)
1
to be greater than 95 % purity by H NMR and TLC
Figure 1. Accumulation of berberine in S.aureus. Accumulation of
berberine in S. aureus cells. The uptake of berberine with no addition
(s h D) or with the addition of INF55 (d j m); 8c (Æ Ç .); in DnorA
(s d Æ), WT (h j Ç) and NorA++ (D m.) was measured by increase
in fluorescence, and is expressed as relative fluorescence units (RFUs).
Berberine was present at a concentration of 30 lg/mL and INF55 and
8c were added at 10 lg/mL.
analysis. High resolution (EI) MS (for M+) was run
using a VG Autospec spectrometer operating at 70 eV
and a source temperature of 250 ꢁC with PFK reference.
1
The H and 13C NMR were determined at 299.92 and
75.42 MHz with a Varian Unity-300 spectrometer, and
at 499.91 and 125.71 MHz with Varian Inova-500 spec-
trometer. Unless otherwise stated, the spectra were ob-
tained from solutions in CDCl3 and referenced to
TMS (proton) and the chloroform mid-line (77) (car-
bon). Chemical shifts of the outer peaks are given for
specified multiplet patterns in the 1H NMR spectra.
The assignments were made by standard gradient corre-
lation spectroscopy (gCOSY), gradient heteronuclear
single quantum correlation (gHSQC) and gradient het-
eronuclear multiple bond correlation (gHMBC) spec-
troscopy. The same superscript assignments may be
reversed for the signals designated in the same com-
pound. The infrared spectrum was measured (KBr disc)
with an AVATAR 370 FT-IR spectrometer. The melt-
ing point (mp) determinations were recorded with a
Reichert melting point apparatus and are reported
uncorrected.
9a–9b and 10a–10b potentiated the action of the anti-
bacterial agent berberine by blocking the NorA MDR
pump in S. aureus. The alcohol 8c was the most effec-
tive inhibitor in the 2-aryl-5-nitroindole series against
all strains of S. aureus. Compound 8c also potentiated
the action of other antibiotics (e.g., ciprofloxacin) in
S. aureus.
4. Experimental
4.1. General chemistry methods
All starting materials were used without further purifica-
tion. Tetrahydrofuran (THF) and diethyl ether were

S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
861
4.2. Synthesis
(CO). HMRS (EI); m/z calcd for C22H16N2O4 [M]+:
372.1110; found: 372.1105.
4.2.1. Preparation of 4-benzyloxybenzoic acid 2c.24,25 To
a
suspension of p-hydroxybenzoic acid (3.0 g,
4.2.3. Palladium cyclisation of compounds 3a–3c: typical
procedure. A solution of 3a, 3b, or 3c (0.75 mmol) and
palladium (II) acetate (0.75 mmol) in glacial acetic acid
(20 mL) was heated at 110 ꢁC under a nitrogen atmo-
sphere for 11–20 h (3a, 11 h; 3b, 16 h; 3c, 20 h). The
reaction was monitored by TLC until there was no
change in the concentration of cyclised product. The
reaction was allowed to cool to rt. The black suspension
was filtered through Celite and washed with acetone and
then the filtrate was evaporated. The residue was added
to ice water (200 mL), the precipitate filtered, dried and
chromatographed on silica gel by VLC (40–60% DCM
in PS) to give the desired products (4a–4c).
22.8 mmol) and dry K2CO3 (7.0 g, 50.0 mmol) in dry
DMF (60 mL) was added dropwise benzyl bromide
(5.4 mL, 45.8 mmol) under a nitrogen atmosphere. The
reaction mixture was heated (with a reflux condensor at-
tached) at 80 ꢁC for 6 h. The mixture was evaporated,
added to ice water (500 mL) and acidified with 5 M
HCl to pH1. The suspension was filtered, washed several
times with H2O, then dried and chromatographed on sil-
ica gel by VLC (60% DCM in PS) to give benzyl 4-(ben-
zyloxy) benzoate (5.62 g, 81%) as an off-white solid, mp
93–95 ꢁC. To the suspension of the dibenzylated product
(5.0 g, 15.7 mmol) in MeOH (50 mL) was added 30%
aqueous solution of KOH (10 mL) and heated at reflux
for 6 h. The solvent was evaporated, added to ice water
(300 mL) and acidified to pH1. The white precipitate
was filtered, washed thoroughly with water and then
20% DCM in PS (100 mL) to remove the dibenzylated
starting material, and dried to give the acid 2c (3.25 g,
91%) as white needles, mp 185–187 ꢁC (lit.25 187–
190 ꢁC).
4.2.3.1. 2-Nitro-isoindolo[2,1-a]indol-6-one (4a). Yield:
100.1 mg (50%) as pale yellow needles, mp 268.8 ꢁC
1
(decomposed). H NMR (CDCl3, 300 MHz): d 6.79 (d,
J = 0.6 Hz, 1H, H-11), 7.43–7.49 (ddd, J = 8.4, 5.9,
2.6 Hz, 1H, H-9) 7.62-7.65 (m, 2H, H-10, H-8), 7.84
(dt, J = 7.5, 0.9 Hz, 1H, H-7), 7.98 (d, J = 8.7 Hz, 1H,
H-4), 8.23 (dd, J = 8.7, 2.1 Hz, 1H, H-3), 8.41 (d,
J = 2.1 Hz, 1H, H-1). 13C NMR (125 MHz, DMSO-
d6): d 104.2 (C11), 112.6 (C4), 118.8 (C1), 121.8 (C3),
122.8 (C7), 125.6 (C10), 130.2 (C9)a, 132.5 (C10a),
133.8 (C11a), 134.6 (C6a), 135.1 (C8)a, 136.1 (C4a),
141.1 (C10b), 143.9 (C2), 162.0 (C6). HRMS (EI); m/z
calcd for C15H8N2O3 [M]+: 264.0535; found: 264.0533.
4.2.2. N-Acylation of 5-nitro-1H-indole (1): typical
procedure. To a solution of indole 1 (0.2 mmol),
DMAP (0.2 mmol) and carboxylic acid 2a, 2b, or 2c
(0.4 mmol) in DCM (2 mL) at 0 ꢁC under a nitrogen
atmosphere were added a stirred solution of DCC
(0.4 mmol) in DCM (1 mL). The solution was warmed
to rt and stirred for 4–6 h, and was monitored by
TLC until no starting material remained. The resulting
suspension was concentrated in vacuo. The residue
was treated with MeOH (20 mL), the mixture filtered
and the precipitate was then washed with MeOH
and dried to give the desired product (3a–3b). The fil-
trate was concentrated and the residue was recrystal-
lised from MeOH. In the case of 3c, a different
workup procedure was used. After evaporation of
the solvent, the residue was chromatographed on silica
gel (40% DCM in PS) to obtain 3c.
4.2.3.2. 9-Methoxy-2-nitro-isoindolo[2,1-a]indol-6-one
(4b). Yield: 163.7 mg (74%) as pale yellow needles, mp
1
238–240 ꢁC. H NMR (300 MHz, CDCl3 + CD3OD): d
3.95 (s, 3H, OCH3), 6.77 (s, 1H, H-11), 6.91 (dd,
J = 8.7, 2.1 Hz, 1H, H-8), 7.11 (d, J = 2.7 Hz, 1H, H-
10), 7.74 (d, J = 8.4 Hz, 1H, H-7), 7.95 (d, J = 8.7 Hz,
1H, H-4), 8.20 (dd, J = 8.7, 2.1 Hz, 1H, H-3), 8.40 (d,
J = 2.1 Hz, 1H, H-1). 13C NMR (75 MHz, CDCl3
+
CD3OD): d 55.8 (OCH3), 103.0 (C11), 107.7 (C10),
112.6 (C4), 115.1 (C8), 118.3 (C1), 121.7 (C3), 125.0
(C10a), 127.5 (C7), 134.1 (C11a), 136.3 (C4a), 136.5
(C6a), 140.8 (C10b), 144.0 (C2), 162.3 (CO), 165.1
(C9). HRMS (EI); m/z calcd for C16H10N2O4 [M]+:
294.0641; found: 294.0648.
4.2.2.1. 1-Benzoyl-5-nitro-1H-indole (3a). Yield:
48.4 mg (91%); mp 158–159 ꢁC (Lit.15 158–159 ꢁC).
4.2.2.2. 1-(4-Methoxybenzoyl)-5-nitro-1H-indole (3b).
Yield: 56.1 mg (95%); mp 199–200 ꢁC (lit.15 199–
200 ꢁC).
4.2.3.3. 9-Benzyloxy-2-nitro-isoindolo[2,1-a]indol-6-
one (4c). Yield: 51.5 mg (20%) as pale yellow needles,
mp > 250 ꢁC. IR (KBr): mmax 1736, 1615, 1597, 1516,
1342, 1245, 1130, 1077 cmꢁ1 1H NMR (300 MHz,
.
4.2.2.3.
1-(4-Benzyloxybenzoyl)-5-nitro-1H-indole
CDCl3 + CD3OD): d 5.21 (s, 2H, OCH2), 6.79 (s, 1H,
H-11), 7.01 (dd, J = 8.7, 2.1 Hz, 1H, H-8), 7.22 (d,
J = 2.1 Hz, 1H, H-10), 7.43-7.50 (m, 5H, ArH), 7.76
(d, J = 8.7 Hz, 1H, H-7), 7.97 (d, J = 8.7 Hz, 1H, H-6),
8.22 (dd, J = 8.7, 2.4 Hz, 1H, H-3), 8.42 (d, J = 2.1 Hz,
1H, H-1) 13C NMR (CDCl3 + CD3OD or DMSO-d6)
spectrum showed only methine and methylene carbon
signals without quaternary carbon signals due to precip-
itation whilst the experiment was underway. 13C NMR
(125 MHz, DMSO-d6): d 70.9 (CH2), 105.4, 109.9,
113.1, 117.5, 119.7, 126.6, 128.2, 128.7, 129.4 (>1C).
HRMS (EI); m/z calcd for C22H14N2O4 [M]+:
370.0954; found: 370.0944.
(3c). Yield: 64.7 mg (87%) as off-white needles, mp
177–179 ꢁC. 1H NMR (300 MHz, CDCl3): d 5.19 (s,
2H, OCH2), 6.77 (dd, J = 3.8, 0.8 Hz, 1H, H-3), 7.12
(d, J = 9.0 Hz, 2H, H-30), 7.36–7.48 (m, 5H, ArH),
7.55 (d, J = 3.6 Hz, 1H, H-2), 7.77 (d, J = 8.7 Hz, 2H,
H-20), 8.25 (dd, J = 9.3, 2.1 Hz, 1H, H-6), 8.41 (d,
J = 9.0 Hz, H-7), 8.54 (d, J = 2.1 Hz, 1H, H-4). 13C
NMR (75 MHz, CDCl3): d 70.3 (OCH2), 108.3 (C3),
115.0 (2C, C30), 116.2 (C7), 117.2 (C4), 119.6 (C6),
125.4 (C10), 127.5 (2C, C30), 128.4 (2C, C400), 128.8
(C200), 130.4 (C3a), 130.5 (C2), 132.1 (2C, C20), 135.8
(C100), 139.1 (C7a), 144.2 (C5), 162.5 (C40), 167.9

862
S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
4.2.3.4. 9-Hydroxy-2-nitro-isoindolo[2,1-a]indol-6-one
(C40), 128.9 (C60), 129.9 (C30), 138.5 (C20), 139.3
(C7a), 140.5 (C5), 142.2 (C10), 171.7 (CO). HRMS
(EI); m/z calcd for C15H10N2O4 [M]+: 282.0641; found:
282.0643.
(5). Yield: 47.5 mg (22%) as pale yellow needles, mp 199-
201 ꢁC. 1H NMR (300 MHz, DMSO-d6): d 6.82 (dd,
J = 8.6, 2.3 Hz, 1H, H-8), 7.07 (s, 1H, H-11), 7.16 (d,
J = 2.1 Hz, 1H, H-10), 7.60 (d, J = 8.7 Hz, 1H, H-7),
7.81 (d, J = 9.0 Hz, 1H, H-4), 8.13 (dd, J = 8.7, 2.4 Hz,
1H, H-3), 8.45 (d, J = 1.8 Hz, 1H, H-1). 13C NMR
(75 MHz, DMSO-d6): d 103.6 (C11), 109.6 (C10),
112.1 (C4), 116.7 (C8), 118.6 (C1), 121.5 (C3), 122.7
(C10), 127.6 (C7), 134.1 (C11a), 135.9 (C4a), 136.2
(C6a)a, 140.6 (C10b)a, 143.4 (C2), 161.6 (CO), 163.9
(C9). HRMS (EI); m/z calcd for C15H8N2O4 [M]+:
280.0484; found: 280.0480.
4.2.5.2. 4-Methoxy-2-(5-nitro-1H-indol-2-yl)benzoic
acid (7b). Yield: 1.113 g (94%) as a pale yellow solid,
mp 206–208 ꢁC. 1H NMR (300 MHz, DMSO-d6): d
3.86 (s, 3H, OCH3), 6.78 (dd, J = 2.1, 0.6 Hz, 1H, H-
3), 7.08 (dd, J = 7.8, 2.7 Hz, 1H, H-50), 7.11 (s, 1H, H-
30), 7.50 (d, J = 8.7 Hz, 1H, H-7), 7.89 (d, J = 7.8 Hz,
1H, H-60), 8.00 (dd, J = 9.0, 2.4 Hz, 1H, H-6), 8.55 (d,
J = 2.4 Hz, 1H, H-4), 12.07 (s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6): d 55.7 (OCH3), 103.3 (C3),
111.5 (C7), 114.0 (C50), 116.4 (C30), 116.7 (C6), 117.1
(C4), 123.8 (C2), 127.4 (C3a), 132.3 (C60), 134.1
(C10)a, 139.6 (C7a), 140.7 (C5), 141.5 (C20)a, 161.3
(C40), 167.9 (CO). HRMS (EI); m/z calcd for
C16H12N2O5 [M]+: 312.0746; found: 312.0749.
4.2.3.5. 1-(4-Hydroxybenzoyl)-5-nitro-1H-indole (6).
Yield: 73.7 mg (35%) as a pale yellow solid, mp 201–
203 ꢁC. 1H NMR (300 MHz, DMSO-d6): d 6.92 (d,
J = 3.3 Hz, 1H, H-3), 6.91–6.93 (m, 2H, ArH), 7.64–
7.67 (m, 2H, ArH), 7.73 (d, J = 3.3 Hz, 1H, H-2), 8.17
(dd, J = 9.3, 2.4 Hz, 1H, H-6), 8.27 (d, J = 9.0 Hz, 1H,
H-7), 8.60 (d, J = 1.8 Hz, 1H, H-4), 10.53 (br s, 1H,
OH). 13C NMR (75 MHz, DMSO-d6): d 108.1 (C3),
115.6 (2 · ArCH), 115.8 (C7), 117.3 (C10), 119.3 (C6),
123.0 (C4), 130.3 (C2), 131.7 (C3a), 132.5 (2 · ArCH),
138.6 (C7a), 143.4 (C5), 162.0 (C40), 167.6 (CO). HRMS
(EI); m/z calcd for C15H10N2O4 [M]+: 282.0641; found:
282.0638.
4.2.5.3. 4-Benzyloxy-2-(5-nitro-1H-indol-2-yl)benzoic
acid (7c). Yield: 167.0 mg (96%) as a pale yellow solid,
m.p. 223–235 ꢁC. 1H NMR (300 MHz, DMSO-d6): d
5.24 (s, 2H, OCH2), 6.78 (s, 1H, H-3), 7.16 (dd,
J = 8.6, 2.6 Hz, 1H, H-50), 7.24 (d, J = 2.7 Hz, 1H, H-
30), 7.30–7.50 (m, 5H, ArH), 7.52 (d, J = 9.0 Hz, 1H,
H-7), 7.85 (d, J = 8.7, 1H, H-60), 8.00 (dd, J = 8.9,
2.3 Hz, 1H, H-6), 8.55 (d, J = 2.4 Hz, H-4), 12.20 (s,
1H, NH), 12.57 (s, 1H, COOH). 13C NMR (75 MHz,
DMSO-d6): d 69.6 (OCH2), 103.2 (C3), 111.5 (C7),
114.6 (C50), 116.6 (C6), 117.0 (C30), 117.1 (C4), 124.1
(C2), 127.3 (C3a), 127.7 (2 · ArCH), 128.0 (ArCH),
128.4 (2 · ArCH), 132.1 (C60), 133.8 (C20), 136.4
(C100), 139.6 (C7a), 140.6 (C5), 141.1 (C10), 160.2
(C50), 167.9 (CO). HRMS (EI); m/z calcd for
C22H16N2O5 [M]+: 388.1059; found: 388.1069.
4.2.4. Conversion of 5 to 4c. To a suspension of 5
(200.0 mg, 0.71 mmol) and caesium carbonate
(230.0 mg, 0.70 mmol) in dry DMF (20 mL) was added
dropwise with stirring benzyl bromide (0.1 mL,
0.84 mmol). The reaction was heated at 80 ꢁC under a
nitrogen atmosphere for 7 h. The reaction was allowed
to cool to rt and then filtered. The precipitate was
washed thoroughly with water and dried to give a pale
1
yellow solid 4c (196.1 mg, 74%). H NMR and HRMS
(EI) data for 4c were the same as those noted for this
compound above.
4.2.6. Selective reduction of acids 7a–7c: typical proce-
dure. To a solution of 7a or 7b (3.54mmol; 0.52 mmol
for 7c) in dry THF (90 mL for 7a or 7b; 15 mL for 7c)
was slowly added 1 M BH3-THF complex solution
(7.1 mL, 7.1 mmol for 7a or 7b; 1.0 mL, 1.0 mmol for
7c) at 0 ꢁC under a nitrogen atmosphere. After vigorous
stirring for 2 h at room temperature, the excess hydride
was carefully destroyed by slowly adding a solution of
50% THF in H2O (20 mL for 7a or 7b; 2 mL for 7c) until
no gas bubbling was observed in the reaction mixture.
The aqueous layer was saturated with anhydrous
K2CO3. The THF layer was separated and the aqueous
layer was extracted with diethyl ether (3 · 20mL). The
combined THF and ether extract was dried, then evap-
orated, and the residue was chromatographed on silica
gel by VLC (2% MeOH in DCM) to give the desired
alcohols (8a–8c).
4.2.5. Ring opening of compounds 4a–4c: typical proce-
dure. A solution of t-BuOH (192 mL for 4a or 4b;
22.5 mL for 4c) and H2O (19.2 mL 4a or 4b; 2.3 mL
for 4c) containing t-BuOK (37.9 mmol 4a or 4b;
4.45 mmol for 4c) was added to 4a or 4b (3.79 mmol;
0.45 mmol for 4c) and heated at 82 ꢁC for 12 h. The mix-
ture was evaporated and added to ice water (400 mL 4a
or 4b; 50 mL for 4c). The solution was acidified to pH 1
with 5 M HCl and then saturated with solid NaCl. The
solution was stirred vigorously for 2 h and then extract-
ed with diethyl ether (3 · 400mL 4a or 4b; 3 · 50 mL for
4c). The combined ether extract was dried and evaporat-
ed to give the desired acids (7a–7c).
4.2.5.1. 2-(5-Nitro-1H-indol-2-yl)benzoic acid (7a).
Yield: 1.016 g (95%) as a pale yellow solid, mp 250–
252 ꢁC. 1H NMR (300 MHz, DMSO-d6): d 6.98 (s,
1H, H-3), 7.33–7.45 (m, 2H, H-40, H-50), 7.52 (d,
J = 9.0 Hz, 1H, H-7), 7.67 (br.d, J = 7.2 Hz, 1H, H-30),
7.75 (br d, J = 7.5 Hz, 1H, H-60), 7.94 (dd, J = 9.03,
2.4 Hz, 1H, H-6), 8.52 (d, J = 2.4, H-4). 13C NMR (75
MHz, DMSO-d6): d 102.2 (C3), 117.8 (C7), 116.3 (C6),
116.8 (C4), 127.5 (C3a), 127.8 (C50), 128.5 (C2), 128.7
4.2.6.1. [2-(5-Nitro-1H-indol-2-yl)-phenyl]-methanol
(8a). Yield: 940.5 mg (99%) as bright yellow needles,
mp 132-134 ꢁC. 1H NMR (300 MHz, CDCl3): d 4.77
(s, 2H, CH2O), 6.91 (d, J = 1.2 Hz, 1H, H-3), 7.36–
7.47 (m, 4H, H-7, ArH), 7.79 (br d, J = 7.8 Hz, 1H,
ArH), 8.12 (dd, J = 8.9, 2.4 Hz, 1H, H-6), 8.61 (d,
J = 2.4 Hz, 1H, H-4), 10.92 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3): d 65.1 (CH2), 103.5 (C3), 111.2

S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
863
(C7), 117.5 (C6), 117.6 (C4), 127.8 (C3a), 128.8 (ArCH),
129.4 (ArCH), 130.3 (ArCH), 131.1 (ArCH), 132.8 (C2),
135.8 (C20), 139.6 (C7a), 141.4 (C10), 141.8 (C5). HRMS
(EI); m/z calcd for C15H12N2O3 [M]+: 268.0848; found:
268.0838.
4.2.7.2. 2-(2-Azidomethyl-5-methoxy-phenyl)-5-nitro-
1H-indole (9b). Yield: 209.88 mg (61%) as bright yellow
needles, mp 138–140 ꢁC. H NMR (300 MHz, CDCl3):
1
d 3.79 (s, 3H, OCH3), 4.26 (s, 2H, CH2), 6.78 (dd,
J = 2.1, 0.9 Hz, 1H, H-3), 6.89 (dd, J = 8.7, 2.7 Hz,
1H, H-40), 7.12 (d, J = 2.7 Hz, 1H, H-60), 7.29 (d,
J = 8.7 Hz, 1H, H-30), 7.37 (d, J = 8.4 Hz, 1H, H-7),
8.03 (dd J = 9.0, 2.1 Hz, 1H, H-6), 8.52 (d,
J = 2.1 Hz, 1H, H-4), 9.77 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3): d 53.6 (CH2), 55.5 (OCH3), 104.6
(C3), 111.2 (C7), 114.2 (C50), 116.1 (C60), 117.7 (C4),
117.9 (C6), 124.6 (C2), 127.7 (C3a), 132.6 (C30),
133.5 (C10)a, 139.5 (C7a), 140.0 (C20)a, 142.3 (C5),
160.1 (C50). HRMS (EI); m/z calcd for C16H13N5O3
[M]+: 323.1018; found: 323.1024.
4.2.6.2. [4-Methoxy-2-(5-nitro-1H-indol-2-yl)-phenyl]-
methanol (8b). Yield: 997.8 mg (95%) as bright yellow
1
needles, mp 206–208 ꢁC. H NMR (300 MHz, CDCl3):
d 3.89 (s, 3H, OCH3), 4.70 (s, 2H, CH2OH), 6.91 (d,
J = 1.8 Hz, 1H, H-3), 6.92 (dd, J = 8.4, 2.7 Hz, 1H, H-
50), 7.30 (d, J = 2.7 Hz, 1H, H-30), 7.34 (d, J = 8.7 Hz,
1H, H-60), 7.46 (d, J = 8.7 Hz, 1H, H-7), 8.10 (dd,
J = 8.7, 2.1 Hz, 1H, H-6), 8.62 (d, J = 2.1 Hz, 1H, H-
4), 11.02 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): d
55.5 (OCH3), 64.6 (CH2O), 103.5 (C3), 111.3 (C7),
113.9 (C50), 115.7 (C30), 117.6 (C6), 117.7 (C4), 127.7
(C3a), 128.3 (C2), 132.6 (C60), 134.2 (C10)a, 139.6
(C20)a, 141.4 (C7a), 141.8 (C5), 160.1 (C40). HRMS
(EI); m/z calcd for C16H14N2O4 [M]+: 298.0954; found:
298.0941.
4.2.8. Reduction of azides 9a–9b: typical procedure. To
a solution of 9a or 9b (0.34 mmol), triethylamine
(TEA) (0.09 mL, 0.68 mmol) and 2 drops of 1,3-prop-
anedithiol (ca 0.1 mL, 1.0 mmol) in 35% MeOH in i-
PrOH (12 mL) was added sodium borohydride
(128.6 mg, 3.4 mmol) at 0 ꢁC. After 2 h, more sodium
borohydride (64.3 mg, 1.7mmol) was added and the
mixture was stirred for a further 10 min. The reaction
mixture was then evaporated, the residue was added to
H2O (50 mL) and the mixture was extracted with 40%
diethyl ether in PS (2 · 40 mL). The aqueous layer was
basified to pH 11 with a saturated NaOH solution and
extracted with DCM (3 · 50 mL). The combined DCM
extract was dried, concentrated and then chromato-
graphed on silica gel by VLC (4% MeOH in DCM).
The amine products (10a–10b) were washed with
40% diethyl ether in PS to remove traces of 1,3-
propanedithiol.
4.2.6.3. [4-Benzyloxy-2-(5-nitro-1H-indol-2-yl)-phen-
yl]-methanol (8c). Yield: 163.5 mg (85%) as bright yellow
1
needles, mp 70–72 ꢁC. H NMR (300 MHz, CDCl3): d
4.63 (s, 2H, CH2OH), 5.08 (s, 2H, OCH2), 6.80 (dd,
J = 2.0, 0.8 Hz, 1H, H-3), 6.90 (dd, J = 8.4, 2.7 Hz,
1H, H-50), 7.23–7.42 (m, 8H, H-7, H-30, H-60, ArH),
8.03 (dd, J = 9.0, 2.1 Hz, 1H, H-6), 8.54 (d, J = 2.4 Hz,
1H, H-4), 10.92 (s, 1H, NH). 13C NMR (75 MHz,
CDCl3): d 64.6 (CH2OH), 70.2 (OCH2), 103.5 (C3),
111.3 (C7), 114.7 (C50), 116.7 (C60), 117.6 (C6), 117.7
(C4), 112.4 (2 · ArCH), 127.7 (C2), 128.2 (ArCH),
128.5 (C3a), 128.7 (2 · ArCH), 132.6 (C30), 134.2
(C100), 136.4 (C20), 139.6 (C7a), 141.3 (C5), 141.9
(C10), 159.3 (C40). HRMS (EI); m/z calcd for
C22H18N2O4 [M]+: 374.1267; found: 374.1256.
4.2.8.1. 2-(5-Nitro-1H-indol-2-yl)-benzylamine (10a).
Yield: 81.6 mg (90%) as a brown yellow solid, mp 127–
129 ꢁC, 1H NMR (300 MHz, CDCl3): d 3.99 (s, 2H,
CH2), 6.87 (br s, 1H, H-3), 7.26–7.46 (m, 4H, H-7,
ArH), 7.78 (d, J = 7.5 Hz, 1H, ArH), 8.08 (dd, J = 8.7,
2.4 Hz, 1H, H-6), 8.61 (d, J = 2.1 Hz, 1H, H-4), 13.63
(s, 1H, NH). 13C NMR (75 MHz, CDCl3): d 46.0
(CH2), 102.5 (C3), 111.2 (C7), 117.1 (C6), 117.6 (C4),
127.9 (C3a), 128.5 (ArCH), 128.7 (ArCH), 130.3
(ArCH), 131.4 (ArCH), 133.1 (C2), 136.4 (C20), 139.6
(C7a), 141.4 (C5), 142.7 (C10). HRMS (EI); m/z calcd
for C15H12N5O2 [M]+: 267.1008; found: 267.0095.
4.2.7. Synthesis of azides 9a–9b: typical procedure. A
mixture of 8a or 8b (1.06 mmol), sodium azide
(1.16 mmol) and triphenyl phosphine (2.08 mmol) in a
solution of 25% CCl4 in DMF (10 mL) was heated at
90 ꢁC under a nitrogen atmosphere for 5 h. The reaction
mixture was then cooled to room temperature, quenched
by adding H2O (10 mL) and stirred for 10 min. The mix-
ture was diluted with diethyl ether (40 mL) and washed
thoroughly with H2O. The organic layer was dried, con-
centrated, and then chromatographed on silica gel by
VLC (30% DCM in PS) to give the azides (9a–9b).
4.2.8.2. 4-Methoxy-2-(5-nitro-1H-indol-2-yl)-benzyl-
amine (10b). Yield: 91.0 mg (91%) as a yellow solid,
1
4.2.7.1. 2-(2-Azidomethyl-phenyl)-5-nitro-1H-indole
(9a). Yield: 231.0 mg (74%) as bright yellow needles,
mp 170–178 ꢁC. H NMR (300 MHz, CDCl3): d 3.86
(s, 3H, OCH3), 3.91 (s, 2H, CH2), 6.84 (br s, 1H, H-
3), 6.86 (dd, J = 7.9, 2.6 Hz, 1H, H-50), 7.23 (d,
J = 8.1 Hz, 1H, H-60), 7.28 (s, 1H, H-60), 7.40 (d,
J = 9.3 Hz, 1 H, H-7), 8.06 (dd, J = 9.3, 2.4 Hz, 1H,
H-6), 8.59 (d, J = 2.1 Hz, 1H, H-4), 13.66 (s, 1H,
NH). 13C NMR (75 MHz, DMSO-d6): d 42.5 (CH2),
55.3 (OCH3), 103.4 (C3), 111.9 (C7), 114.1 (C50),
114.6 (C30), 116.7 (C6), 117.0 (C4), 127.6 (C3a),
129.0 (C2), 131.8 (C60), 132.9 (C10)a, 139.6 (C20)a,
140.7 (C5), 141.4 (C7a), 158.6 (C40). HRMS (EI); m/
1
mp 146–148 ꢁC. H NMR (300 MHz, CDCl3): d 4.40
(s, 2H, CH2N3), 6,86 (dd, J = 2.1 Hz, 0.6 Hz, 1H, H-
3), 7.44–7.54 (m, 4H, H-7, ArH), 7.69 (dd, J = 6.2,
1.7 Hz, 1H, ArH), 8.13 (dd, J = 9, 2.1 Hz, 1H, H-6),
8.61 (d, J = 2.1 Hz, 1H, H-4), 9.53 (s, 1H, NH). 13C
NMR (75 MHz, CDCl3,): d 54.0 (CH2N3), 104.7 (C3),
111.1 (C7), 117.8 (C6), 117.9 (C4), 127.8 (C3a), 129.2
(ArCH), 129.5 (ArCH), 130.7 (ArCH), 131.2 (ArCH),
132.1 (C2), 132.3 (C10), 139.4 (C7a), 140.0 (C20), 142.1
(C5). HRMS (EI); m/z calcd for C15H12N5O2 [M]+:
294.0991; found: 294.0987.
z
calcd for C16H15N3O3 [M]+: 297.1113; found:
297.1101.

864
S. Samosorn et al. / Bioorg. Med. Chem. 14 (2006) 857–865
4.2.9. Reduction of azide 9a with NaBH4 in THF/MeOH.
To a mixture of 9a (260.0 mg, 0.89 mmol) and sodium
borohydride (25.0 mg, 0.66 mmol) in dry THF (10 mL)
heated at reflux was slowly added MeOH (1.0 mL) and
the mixture was stirred for 2 h. Further sodium borohy-
dride (25.0 mg, 0.66 mmol) was then added and stirring
was continued for 3 days. The mixture was cooled to
room temperature and then 1 M HCl (3 mL) was added
until the gas bubbling ceased. The mixture was basified
to pH11 with a saturated NaOH solution and then
extracted with DCM (3 · 20 mL). The combined DCM
extract was dried, concentrated and then chromato-
graphed on silica gel by VLC (PS, PS and DCM, then
MeOH and TEA) to give starting material 9a
(70.2 mg, 0.24 mmol; eluent: 30% DCM in PS), 2-ni-
tro-6H-isoindolo[2,1-a]indole (11) (30.1 mg, 14%; elu-
ent: 40% DCM in PS) as a yellow solid and the
benzylamine 10a (70.6 mg, 30%; eluent: 30:1:2 DCM/
MeOH/TEA) as a brown solid.
defined as the concentration of an antimicrobial or anti-
microbial plus inhibitor that completely inhibited cell
growth during an 18 h incubation at 37 ꢁC. Growth
was assayed with a microtitre plate reader (Spectramax
PLUS384; Molecular Devices) by monitoring absorp-
tion at 600 nm.
4.3.3. Berberine uptake assay28. Cells were cultured with
aeration at 37 ꢁC to an optical density at 600 nm
(OD600) of 1.5, pelleted by centrifugation for 2 min at
9000 RPMs and washed with fresh MH broth. The
above procedure was repeated twice. The cells were then
resuspended to an OD600 of 0.4 in MH broth. Assays
were performed in 96-well flat-bottomed white plates
(NUNC) in a final volume of 200 lL. Berberine was
added at 30 lg/mL and inhibitor when present was
added at 10 lg/mL. Fluorescence was measured with a
Spectramax GeminiXS spectrofluorometer (Molecular
Devices) at a 355-nm excitation wavelength and a 517-
nm emission wavelength.
1
Compound 11: mp 234–236 ꢁC. H NMR (300 MHz,
CDCl3): d 5.12 (s, 2H, H-6), 6.75 (s, 1H, H-11), 7.33
(d, J = 9.0 Hz, 1H, H-4), 7.38–7.51 (m, 3H, ArH), 7.74
(d, J = 7.2 Hz, 1H, ArH), 8.09 (dd, J = 9.0, 2.1 Hz,
1H, H-3), 8.58 (d, J = 2.1 Hz, 1H, H-1). 13C NMR
(75 MHz, CDCl3): d 48.8 (C6), 93.6 (C11), 108.8 (C4),
117.3 (C3), 118.7 (C1), 121.6 (ArCH) 123.7 (ArCH),
128.3 (ArCH), 128.6 (ArCH), 132.0 (C10b), 136.6
(C11a), 141.3 (C6a, C10a), 141.6 (C2), 147.1 (C4a).
HRMS (EI); m/z calcd for C15H10N2O2 [M]+:
250.0742; found: 250.0732.
Acknowledgments
We wish to thank the University of Wollongong, Aus-
tralia, and Srinakharinwirot University, Thailand, for
supporting this work. The award of UPA and IPRS
scholarships (Siritron Samosorn) is also gratefully
acknowledged. Kim Lewis and Anthony Ball were sup-
ported by Grant R21 AI059483-01 from the NIH. We
thank Glenn Kaatz for kindly providing S. aureus
K1758 and K2361. We also thank Penelope Markham
for providing INF271.
4.3. Bacterial strains and growth conditions
The following bacterial strains were used in this study:
S. aureus 8325-426 (wild-type), K175827 (DnorA) and
K2361 (K1758 with pK364:norA). Cells were grown
in Mueller–Hinton broth (MH) with aeration at
37 ꢁC. Growth of K1758 was supplemented with eryth-
romycin (25 lg/mL) and K2361 with chloramphenicol
(25 lg/mL).
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