Synthesis of berberineefflux pump inhibitor hybrid antibacterials

Article abstract of DOI:10.1080/00397910903457415

This article describes the compact synthesis of two isomeric dual-action hybrid antimicrobials where the 13-position of the antibacterial berberine has been linked via 3′-and 4′-methylene bridges to INF55 (5-nitro-2-phenylindole), an inhibitor of the bacterial NorA multidrug-resistance pump. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910903457415

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Synthesis of Berberine—Efflux Pump
Inhibitor Hybrid Antibacterials
John B. Bremner a & Michael J. Kelso a
a School of Chemistry, University of Wollongong , Wollongong,
Australia
Published online: 05 Nov 2010.
To cite this article: John B. Bremner & Michael J. Kelso (2010) Synthesis of Berberine—Efflux Pump
Inhibitor Hybrid Antibacterials, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 40:23, 3561-3568, DOI: 10.1080/00397910903457415
To link to this article: http://dx.doi.org/10.1080/00397910903457415
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Synthetic Communications1, 40: 3561–3568, 2010
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910903457415
SYNTHESIS OF BERBERINE—EFFLUX PUMP
INHIBITOR HYBRID ANTIBACTERIALS
John B. Bremner and Michael J. Kelso
School of Chemistry, University of Wollongong, Wollongong, Australia
This article describes the compact synthesis of two isomeric dual-action hybrid
antimicrobials where the 13-position of the antibacterial berberine has been linked via 30-
and 40-methylene bridges to INF55 (5-nitro-2-phenylindole), an inhibitor of the bacterial
NorA multidrug-resistance pump.
Keywords: Antibacterial; 2-arylindole; berberine; dual-action; hybrid; INF55
INTRODUCTION
A recent promising strategy for combating bacterial drug resistance arising
from multidrug-resistance (MDR) efflux pumps is to covalently link a pump inhibi-
tor to an antibacterial agent that is normally a pump substrate.[1,2] Dual-action
hybrid antibacterials of this type potentially carry the advantage of synchronous
and equimolar delivery of an antibacterial and an efflux pump inhibitor to sites of
infection[3] and may slow the onset of resistance since they challenge bacteria to
acquire resistance phenotypes at two independent targets.[4]
In the first demonstration of this concept we synthesized a prototype hybrid
antibacterial SS14 1,[1] which contained the antibacterial alkaloid berberine substi-
tuted at its 13-position with a non-cleavable 20-CH2-linkage to 5-nitro-2-phenyindole
(INF55), a known inhibitor of the NorA MDR pump. In recent work, we have com-
pared the antimicrobial activity and conformations of 1 with its 30- and 40-substituted
regioisomers 2 and 3 (Figure 1) in order to probe the effects of varying the relative
orientation of the INF55 and berberine components in hybrids.[5] This article
describes the synthesis of the two new dual-action hybrid antibacterials 2 and 3.
RESULTS AND DISCUSSION
Our previous synthesis of 1[1] involved pre-assembling 20-bromomethyl-
5-nitro-2-phenylindole and subsequently reacting this with 8-allyldihydroberberine[6]
as the final step. It was envisaged that similar reactions of 8-allyldihydroberberine
with the analogous 30- and 40-bromomethyl-5-nitro-2-phenylindoles (8 and 9,
Received October 11, 2009.
Address correspondence to Michael J. Kelso, School of Chemistry, University of Wollongong,
Wollongong, NSW 2522, Australia. E-mail: mkelso@uow.edu.au
3561

3562
J. B. BREMNER AND M. J. KELSO
Figure 1. Structures of dual action antibacterial hybrids 1–3 and the NorA MDR pump inhibitor INF55.
Scheme 1) would provide access to 2 and 3, respectively. Our route to 20-
bromomethyl-5-nitro-2-phenylindole[7] was not suitable for accessing 8 and 9 since
it only provided 2-arylindoles bearing ortho substituents in the phenyl ring for later
conversion to bromomethyl groups. An alternative strategy was thus implemented
for the preparation of the key bromide precursors 8 and 9.
Numerous methods are known for preparing 2-arylindoles[8,9] but the recently
reported Rh(III)-catalyzed direct C2-arylation of unprotected NH-indoles with func-
tionalized iodobenzenes[10] appeared as the most direct route towards 2-arylindoles
with appropriate functionality in place for elaboration to 8 and 9. Accordingly,
methyl esters of 3-iodo- and 4-iodobenzoic acid were reacted with indole
(Scheme 1) using the optimized catalytic conditions reported by Sames and collea-
gues[10] for related reactions. These reactions consistently provided only modest
Scheme 1. Reagents and conditions: (a) [Rh(coe)2Cl]2, CsOPiv, (p-CF3-C6H4)3P, 1,4-dioxane, reflux 3
days, 4 14–18%, 5 22–28%; (b) NaNO3, H2SO4 (conc.), ꢀ20 ꢁC, slow addition, 6 81%, 7 96%; (c) LiBH4,
THF, RT, 24 h, quant., (d) PPh3, CBr4, THF-Et2O, 1 h, 8 79%, 9 94% (over 2 steps), (e) (i) CH3CN, 60 ꢁC,
24 h, (ii) 100–110 ꢁC, 2–3 days (sealed vial), Brꢀ=Clꢀ exchange, 40–45%.

SYNTHESIS OF BERBERINES
3563
yields (14–28%) of the 2-arylated indole esters 4 and 5. Unreacted indole was the
only other species isolated from the reactions. Optimization attempts met with lim-
ited success although it was observed that carrying the reactions out in 1,4-dioxane
at reflux under N2 for 3 days afforded similar or better yields compared to the
reported method of stirring in a sealed tube at 120 ꢁC. This finding made the reac-
tions more practical on a multigram scale and allowed production of usable quanti-
ties of 4 and 5 despite the low yields. Evidence that arylation had occurred at the
indole C2-positions was unambiguously confirmed from g-DQFCOSY and ROESY
NMR experiments.
Esters 4 and 5 underwent nitration at low temperature at their respective indole
5-positions to afford the 5-nitro-2-phenylindole methyl esters 6 and 7 in excellent
yields (81% and 96%, respectively).[11] Regioselective nitration at the indole 5-posi-
tions was confirmed from g-DQFCOSY and ROESY NMR experiments. The
nitration procedure was adapted from the method reported by Noland et al. for
the nitration of 2-phenylindole.[12] The modified procedure involved dissolving a
stoichiometric quantity of NaNO3 in conc. H2SO4 and adding this solution via syr-
inge pump over 2 h to a dilute solution of the indoles 4 and 5 in conc. H2SO4 at
ꢀ20 ꢁC. Reactions were quenched immediately after the addition was complete by
pouring onto a large quantity of crushed ice. Filtration followed by trituration with
4:1 MeOH:H2O afforded pure 6 and 7. It was found that nitration had to be carried
out after Rh(III)-catalyzed C2-arylation as 5-nitroindole did not undergo coupling
with 30- and 40-methyl iodobenzoates. This may have been due to the increased acid-
ity of the indolic NH group containing a strongly electron-withdrawing nitro group
at C5 resulting in indolide anion formation in the presence of cesium pivalate.
Selective reduction of the methyl esters 6 and 7 with LiBH4 in THF to their
corresponding benzylic alcohols[11] was quantitative as observed by TLC (SiO2;
1:1 Petroleum Spirit:EtOAc). The crude alcohols were taken on directly to the key
benzylic bromide precursors 8 and 9 by reaction with CBr4 and PPh3 under standard
conditions. Bromides 8 and 9 were obtained in yields of 79% and 94%, respectively,
(over two steps) and were used immediately in the final coupling step to minimize
decomposition. Reactions of 8 and 9 with 8-allyldihydroberberine[6] proceeded as
expected and produced 40–45% yields of the target hybrids 2 and 3 after preparative
RP-HPLC. It has been proposed that these reactions proceed via a three-step cascade
comprising an enamine alkylation, [3,3]-sigmatropic rearrangement and final retro-
ene reaction.[6] Careful monitoring of the reactions of 8 and 9 with 8-allyldihydrober-
berine by analytical RP-HPLC and mass spectrometry confirmed that the enamine
alkylation step with 8 and 9 was clean and quantitative after stirring overnight at
60 ꢁC, and that increased heating to at least 100 ꢁC was required to effect the sub-
sequent electrocyclic reactions. Reactions were stopped when the RP-HPLC peak
corresponding to the alkylated enamine had disappeared from the HPLC trace (typi-
cally after 3 days). Preparative RP-HPLC of 2 and 3 was carried out in the presence
of HCl to initially provide mixed Clꢀ=Brꢀ salts which were subsequently converted
to pure Clꢀ salts by stirring with a quaternary ammonium chloride anion exchange
resin in CH3OH at room temperature.
In summary, a compact 5-step synthesis of two new berberine-containing anti-
bacterial hybrids 2 and 3 has been developed. A feature of the synthesis was acces-
sing the required indolic benzylic bromide precursors 8 and 9 using an initial

3564
J. B. BREMNER AND M. J. KELSO
Rh(III)-catalyzed direct C2-arylation of indole without NH group protection or C2
functionalization and subsequent selective functional group manipulations.
EXPERIMENTAL
A summary of the preparation of compounds 4–7 was provided as part of our
earlier communication.[11] Full experimental details are now provided here.
General
Indole (Sigma Aldrich) was recrystallized from hot petroleum spirit immedi-
ately prior to use. [Rh(coe)2Cl]2 and (p-CF3-C6H4)3P were purchased from Strem
Chemicals Inc. (MA, USA) and used without further purification. Cesium pivalate
(CsOPiv) was freshly prepared and dried according to the method of Campo and
Larock.[13] Methyl 3-iodobenzoate (CAS Registry Number 618-91-7) and methyl
4-iodobenzoate (CAS Registry Number 619-44-3) were prepared in 95þ% yields
from the corresponding iodobenzoic acids (Sigma Aldrich) by refluxing in CH3OH
containing a catalytic quantity of conc. H2SO4 for 2 days. Anhydrous 1,4-dioxane
was purchased from Sigma Aldrich. Anhydrous tetrahydrofuran (THF) and diethyl
ether were freshly distilled from sodium benzophenone ketyl. Acetonitrile was
freshly distilled from CaH2. All other solvents were of analytical reagent (AR) grade
and used without further purification. The term petroleum spirit refers to petroleum
spirit within the boiling range 40–60 ꢁC. Column chromatography was performed
using silica gel 60 (230–400 mesh, Merck). Reaction monitoring by thin layer chro-
matography (TLC) was carried out using Merck Silica Gel 60 F254 (0.2 mm) plates.
Compounds were visualized by examination under UV light and by staining with
cerium ammonium molybdate. Reaction monitoring by RP-HPLC was carried out
using a Waters 600 chromatography system fitted with a Waters 486 UV-Vis detec-
tor. The separations were performed using gradient elutions (30% solvent B to 100%
solvent B over 30 mins) with solvents A (100% H2O, 0.1% HCl) and B (10% H2O,
90% CH3CN, 0.1% HCl) on a Phenomenex C18 4.6 ꢂ 150 mm (5 mm) column run
at 1.0 mL ꢃ minꢀ1. Preparative RP-HPLC was carried out using a Waters LC-150
chromatography system fitted with a Waters 2489 dual wavelength detector. The
separations were performed using gradient elutions (30% solvent B to 100% solvent
B over 30 mins) on a Waters RadPak C18 150 ꢂ 40 mm column at a flow rate of
40 mL ꢃ minꢀ1 with detection at 254 nm. High resolution EIMS (for Mþ) were
recorded using a VG Autospec spectrometer operating at 70 eV and a source tem-
perature of 250 ꢁC with PFK reference. High resolution ESI-TOF-MS (for Mþ) were
recorded using a factory modified Waters QToF UltimaTM Mass Spectrometer
1
(Wyntheshawe, UK). H and 13C NMR spectra were recorded on either a Varian
Unity-300 (299.92 and 75.42 MHz, respectively) or a Varian-Inova-500 (499.91
and 125.71 MHz, respectively) spectrometer. NMR spectra recorded in CD3COCD3
were referenced to the acetone 1H methyl signal at 2.09 ppm and 13C methyl signal at
30.60 ppm. Spectra recorded in CDCl3 were referenced to the CHCl3 1H signal at
7.26 ppm and 13C signal at 77.0 ppm. Spectra recorded in DMSO-d6 were referenced
to the DMSO 1H methyl signal at 2.49 ppm and 13C methyl signal at 39.5 ppm. Spec-
1
tra recorded in DMF-d7 were referenced to the DMF H formyl signal at 8.01 ppm

SYNTHESIS OF BERBERINES
3565
and 13C carbonyl signal at 167.7 ppm. Melting points were determined using a
Reichert melting point apparatus and are uncorrected. The representative synthetic
procedures described below can be used interchangeably for accessing the 30- and
40-isomers of 2 and 3 and their respective intermediates.
Methyl 4-(1H-Indol-2-yl)benzoate (5)[11]
Indole (8.0 g, 68.3 mmol, 1.0 eq), methyl 4-iodobenzoate (21.56 g, 81.95 mmol,
1.2 eq), [Rh(coe)2Cl]2 (990 mg, 1.38 mmol, 0.02 eq), (p-CF3-C6H5)3P (1.83 g,
4.1 mmol, 0.06 eq) and cesium pivalate (22.37 g, 95.6 mmol, 1.4 eq; freshly prepared
and dried according to the method of Campo and Larock[13]) were weighed into sep-
arate vials and dried under high vacuum in a desiccator for 24 h. The powders were
added successively to a dry 3-neck 250 mL round bottom flask fitted with a ther-
mometer and a condenser and placed under an atmosphere of dry N2. Anhydrous
1,4-dioxane (60 mL; pre-purged with N2 for 1 h immediately prior to use) was added
to the flask and the reaction brought to reflux. The reaction was monitored by TLC
(SiO2; petroleum spirit:EtOAc 5:1) and stopped after refluxing for 3 days when no
further progress was observed. The dark mixture was filtered through a plug of silica
and the filter cake rinsed thoroughly with EtOAc. After evaporating the solvent, the
crude residue was triturated with 50:1 petroleum spirit:Et2O (3 ꢂ 250 mL) and recrys-
tallized from EtOAc=petroleum spirit to yield 4.74 g of 5 (28%) as small grey crystals:
1
mp 204–206 ꢁC; H NMR (CD3COCD3, 500 MHz) d 10.82 (br.s, 1H), 8.07 (d, 2H,
J ¼ 8.5 Hz), 7.98 (d, 2H, J ¼ 8.5 Hz); 7.61 (d, 1H, J ¼ 8 Hz), 7.45 (d, 1H, J ¼ 7.5 Hz),
7.16 (t, 1H, J ¼ 7 Hz), 7.07–7.04 (m, 2H), 3.90 (s, 3H); 13C NMR (CD3COCD3,
125 MHz) d 166.9, 138.8, 137.8, 137.4, 130.8, 130.0, 129.5, 125.6, 123.5, 121.5,
120.8, 112.2, 102.0, 52.5; HRMS [found: (EIþ) Mþ m=z 251.0942; C16H13NO2
requires 251.0946].
Methyl 3-(1H-Indol-2-yl)benzoate (4)[11]
1
Method of preparation as for 5: Yield ¼ 14 ꢀ18%, mp 148–150 ꢁC; H NMR
(CDCl3, 300 MHz) d 8.59 (br.s, 1H), 8.34 (s, 1H), 7.98 (d, 1H, J ¼ 8.1 Hz); 7.87 (d,
1H, J ¼ 8.1 Hz), 7.66 (d, 1H, J ¼ 8.1 Hz), 7.50 (t, 1H, J ¼ 7.5 Hz), 7.42 (d, 1H,
J ¼ 8.4 Hz), 7.23 (t, 1H, J ¼ 7.2 Hz), 7.15 (t, 1H, J ¼ 6.9 Hz), 6.91 (s, 1H), 3.91 (s,
3H); 13C NMR (CDCl3, 75 MHz) d 166.9, 137.0, 136.6, 132.7, 130.8, 129.5, 129.1,
128.8, 128.5, 125.8, 122.7, 120.8, 120.4, 111.0, 100.7, 52.3; HRMS [found: (EIþ)
Mþ m=z 251.0946; C16H13NO2 requires 251.0946].
Methyl 4-(5-Nitro-1H-indol-2-yl)benzoate (7)[11]
Indole 5 (2.5 g, 9.95 mmol) was dissolved in conc. H2SO4 (170 mL) in a 500 mL
round bottom flask and cooled to ꢀ20 ꢁC. A solution of NaNO3 (896 mg, 10.55 mmol,
1.06 eq) in conc. H2SO4 (60 mL) was then added dropwise to the cooled solution
(syringe pump) over 2 h. TLC analysis (SiO2; petroleum spirit:EtOAc 1:1) immedi-
ately after the addition was finished confirmed complete consumption of 5. The reac-
tion was poured rapidly onto a large amount of crushed ice in a 2 L beaker (Careful!
large amount of heat is given off due to exothermic dissolution of H2SO4) and stirred

3566
J. B. BREMNER AND M. J. KELSO
until all the ice had melted. The crude product was filtered and recrystallized from
MeOH=H2O to yield 2.82 g of 7 (96%) as a bright yellow solid: mp 280–286 ꢁC;
1H NMR (CD3COCD3, 500 MHz) d 11.58 (br.s, 1H), 8.61 (s, 1H), 8.12–8.04 (m,
5H), 7.64 (d, 1H, J ¼ 10 Hz), 7.36 (s, 1H), 3.93 (s, 3H); 13C NMR (CD3COCD3,
125 MHz) d 166.7, 142.9, 141.6, 141.0, 136.52, 131.0, 130.6, 129.2, 126.3, 118.5,
118.3, 112.6, 103.72, 52.4; HRMS [found: (EIþ) Mþ m=z 296.0792; C16H12N2O4
requires 296.0797].
Methyl 3-(5-Nitro-1H-indol-2-yl)benzoate (6)[11]
Method of preparation as for 7: Yield ¼ 81%, mp 216–220 ꢁC; 1H NMR
(CDCl3, 300 MHz) d 8.59 (br.s, 1H), 8.34 (s, 1H), 7.96 (d, 1H, J ¼ 7.8 Hz), 7.87 (d,
1H, J ¼ 8.7 Hz), 7.66 (d, 1H, J ¼ 8.1 Hz), 7.50 (t, 1H, J ¼ 7.8 Hz), 7.42 (d, 1H,
J ¼ 8.4 Hz), 7.23 (t, 1H, J ¼ 7.2 Hz), 7.15 (t, 1H, J ¼ 8.1 Hz), 6.91 (s, 1H), 3.96 (s,
3H); 13C NMR (CDCl3, 75 MHz) d 166.9, 137.0, 136.6, 132.7, 130.9, 129.5, 129.1,
128.8, 128.5, 125.8, 122.7, 120.8, 120.4, 111.0, 100.7, 52.3; HRMS [found: (EIþ)
Mþ m=z 296.0792; C16H12N2O4 requires 296.0797].
2-(4-Bromomethyl-phenyl)-5-nitro-1H-indole (9)
A solution of the methyl ester 7 (82 mg, 0.28 mmol) in dry THF (5.0 mL) was
treated with LiBH4 (31 mg, 1.4 mmol, 5.0 eq). The reaction was monitored by TLC
(SiO2; petroleum spirit:EtOAc 1:1) and upon completion (typically 24 h) was slowly
quenched by the dropwise addition of saturated aqueous NH4Cl. After bubbling had
ceased, the mixture was diluted with H2O (50 mL) and extracted with EtOAc
(4 ꢂ 50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated
1
to yield the benzylic alcohol as a bright yellow powder. mp 215–218 ꢁC; H NMR
(CD3COCD3, 500 MHz) d 11.39 (br.s, 1H), 8.56 (s, 1H), 8.04 (d, 1H, J ¼ 8.5 Hz),
7.89 (d, 2H, J ¼ 8.5 Hz), 7.59 (d, 1H, J ¼ 8.5 Hz), 7.51 (d, 2H, J ¼ 8.5 Hz), 7.17 (s,
1H), 4.72 (s, 2H), 4.35 (s, 1H); 13C NMR (CD3COCD3, 125 MHz) d 144.0, 142.7,
141.3, 130.8, 129.5, 128.0, 126.2, 117.8, 117.7, 112.2, 110.6, 101.5, 64.3. The crude
alcohol (27 mg, 0.1 mmol, 1.0 eq) and CBr4 (100 mg, 0.3 mmol, 3.0 eq) were added
to a dry flask under Ar and 5.0 mL of anhydrous Et2O=THF (1:1) was added. After
stirring for 10 min, PPh3 (79 mg, 0.3 mmol, 3.0 eq) was added to the stirring mixture
in one portion. The reaction was stirred at rt for 1 h (monitored by TLC on SiO2;
petroleum spirit:EtOAc 1:2) before concentrating in vacuo. The crude residue was
purified by column chromatography (SiO2; petroleum spirit=EtOAc 4:1!3:1) and
the product triturated with petroleum spirit (to remove traces of CBr4 and PPh3)
to yield the benzylic bromide 9 (31 mg; 94%) as a yellow powder that was used
1
immediately to prepare 3. H NMR (CD3COCD3, 500 MHz) d 11.40 (br.s, 1H),
8.57 (s, 1H), 8.05 (d, 1H, J ¼ 8.5 Hz), 7.91 (d, 2H, J ¼ 7.5 Hz), 7.61–7.57 (m, 3H),
7.22 (s, 1H), 4.71 (s, 2H).
2-(3-Bromomethyl-phenyl)-5-nitro-1H-indole (8)
Method of preparation as for 9: Yield ¼ 79%, 1H NMR (CD3COCD3,
500 MHz) d 11.45 (br.s, 1H), 8.58 (d, 1H, J ¼ 2 Hz), 8.06 (dd, 1H, J ¼ 9.25, 2 Hz),

SYNTHESIS OF BERBERINES
3567
8.02 (s, 1H), 7.86–7.88 (m, 1H), 7.61 (d, 1H, J ¼ 9.25 Hz), 7.51–7.53 (m, 2H), 7.25
(s, 1H), 4.75 (s, 2H).
9,10-Dimethoxy-13-[3-(5-nitro-1H-indol-2-yl)benzyl]-5,6-
dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium Chloride (2)
8-Allyldihydroberberine[6] (39 mg, 0.10 mmol, 1.1. eq) and
8
(31 mg,
0.094 mmol, 1.0 eq) were added to a dry 5 mL screw-cap vial under Ar. Anhydrous
CH3CN (freshly distilled from CaH2) (2.5 mL) was then added and the vial capped
and stirred at 60 ꢁC. After 24 h, an aliquot was removed from the reaction and
RP-HPLC analysis showed clean enamine alkylation. The vial was then heated to
100 ꢁC and the reaction monitored by RP-HPLC for 2 to 3 days until all of the alky-
lated enamine intermediate (tR ¼ 28.0 mins) had been consumed. The crude reaction
was purified by preparative RP-HPLC to yield the mixed Clꢀ=Brꢀ salt of 2
(tR ¼ 22.5 mins) after lyophilisation. The mixed salt was redissolved in MeOH and
stirred at rt for 2 h with excess Amberlite IRA-904 quaternary ammonium Clꢀ anion
exchange resin. The resin was filtered and solvent removed in vacuo to yield 37 mg of
1
2 (45%) as a yellow amorphous solid: mp 220 ꢁC (decomp.); H NMR (DMSO-d6,
500 MHz) d 12.79 (s, 1H), 10.08 (s, 1H), 8.48 (s, 1H), 8.06 (d, 1H, J ¼ 7.3 Hz),
7.94 (d, 1H, J ¼ 7 Hz), 7.88–7.79 (m, 3H), 7.53 (d, 1H, J ¼ 7 Hz), 7.47 (m, 1H),
7.21–7.08 (m, 4H), 6.07 (s, 2H), 4.93 (s, 2H), 4.82 (s, 2H), 4.12 (s, 3H), 4.00 (s,
3H), 3.20 (s, 2H) ; 13C NMR (DMSO-d6, 125 MHz) d 150.1, 149.2, 146.3, 145.8,
144.2, 141.0, 140.3, 140.2, 137.5, 134.0, 132.7, 131.8, 129.9, 129.7, 128.0, 127.7,
127.6, 126.1, 125.0, 124.0, 121.8, 121.7, 120.2, 117.0, 116.9, 111.9, 108.7, 108.3,
101.9, 101.0, 61.8, 57.4, 57.3, 35.8, 27.4; ESI-TOF-MS m=z 586.1998 (C35H28N3O6
requires 586.1978).
9,10-Dimethoxy-13-[4-(5-nitro-1H-indol-2-yl)benzyl]-5,6-
dihydrobenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium Chloride (3)
Method of preparation as for 2: Yield ¼ 40%, mp 230 ꢁC (decomp.); 1H NMR
(DMF-d7, 500 MHz) d 13.23 (br.s, 1H), 10.28 (s, 1H), 8.54 (s, 1H), 8.16 (d, 2H,
J ¼ 7.5 Hz), 7.96–8.00 (m, 2H), 7.70 (d, 2H, J ¼ 9 Hz), 7.38 (d, 2H, J ¼ 7.5 Hz),
7.25 (s, 1H), 7.20 (s, 1H), 7.13 (s, 1H), 6.15 (s, 2H), 5.13 (s, 2H), 4.92 (s, 2H), 4.19
(s, 3H), 4.11 (s, 3H), 3.34 (s, 2H); 13C NMR (DMF-d7, 125 MHz) d 156.3, 155.2,
152.6, 150.7, 147.5, 147.1, 146.7, 145.3, 143.6, 140.0, 138.9, 136.2, 134.7, 134.4,
134.1, 133.7, 132.2, 131.8, 127.6, 127.3, 126.3, 122.3, 122.2, 117.3, 114, 113.9,
107.9, 106.4, 67.3, 62.9, 62.2, 41.0, 33.1; ESI-TOF-MS m=z 586.1998 (C35H28N3O6
requires 586.1978).
ACKNOWLEDGMENTS
We thank the University of Wollongong, Australia, for supporting this work.
Award of an NHMRC CJ Martin Fellowship ID252922 to M. Kelso is gratefully
acknowledged. This work was supported by NIH grant R01 AI076372.

3568
J. B. BREMNER AND M. J. KELSO
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