Structure-activity relationships of 2-aryl-1H-indole inhibitors of the NorA efflux pump in Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2008.06.093

The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or Rh-mediated methodologies, or selective electrophilic substitution. All three methods were based on elaborations from simple indole precursors. SAR studies on

Full text of DOI:10.1016/j.bmcl.2008.06.093

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4294–4297
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of 2-aryl-1H-indole inhibitors of the NorA
efflux pump in Staphylococcus aureus
b
b
b
Joseph I. Ambrus a, Michael J. Kelso a, John B. Bremner a, , Anthony R. Ball , Gabriele Casadei , Kim Lewis
*
a School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia
b Department of Biology and Antimicrobial Discovery Center, Northeastern University, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or
Rh-mediated methodologies, or selective electrophilic substitution. All three methods were based on
elaborations from simple indole precursors. SAR studies on these indoles and 2-phenyl-1H-indole in
Staphylococcus aureus as NorA efflux pump inhibitors indicated 5-nitro-2-(3-methoxycarbonyl)phenyl-
1H-indole was a slightly more potent inhibitor than the lead INF55. A promising new antibacterial lead
compound against S. aureus (2-phenyl-1H-indol-5-yl)-methanol, was also found.
Received 23 April 2008
Revised 25 June 2008
Accepted 26 June 2008
Available online 2 July 2008
Keywords:
2-Arylindoles
Ó 2008 Elsevier Ltd. All rights reserved.
NorA efflux pump inhibitors
Antibacterial
Efflux pumps compromise the efficacy of a wide range of antibi-
otics by actively extruding them from bacterial cells.1,2 The pumps
can be expressed in many different forms in both Gram-positive3
and Gram-negative bacteria,4 and for some species a variety of
pumps may be present with different or overlapping substrates.
For the important community and nosocomially acquired human
pathogen Staphylococcus aureus a number of pumps have been
identified, including NorA, which has been shown to play a role
in the development of clinical multidrug resistance (MDR) by this
organism.5 One promising strategy for combating MDR in S. aureus
is to treat infections with a combination of a NorA efflux pump
inhibitor and a conventional antibiotic, with the pump inhibitor
serving to restore the antibiotic’s potency by reducing its efflux
from bacterial cells.6
4
7
6' 5'
2' 3'
3
R
5
6
4'
a-e
N
H
N
H
1
2-6
R
Method
a
Compound
NO2 (INF55)
SO3H
2
3
4
5
6
b
c
SO2NH2
SO2OCH3
d
e
SO2On-Pr
Reported classes of NorA inhibitors include flavones and flavon-
olignans,7 pyrroloquinoxalines,8 4-arylpyridine-3,5-dicarboxylate
esters,9 N-aryl ureas,10, and indoles.11
17
Scheme 1. Reagents and conditions: (a) NaNO3, H2SO4 (concd), ꢀ20 °C;
(b) i—ClSO3H, 0 °C to rt, 30 min; ii—NaOH, H2O, rt and then 1 M HCl; (c)
(i) followed by (ii) NH3/THF, rt; (d) (i) followed by (ii) MeOH, pyridine, rt;
(e) (i) followed by (ii) n-PrOH, pyridine rt.
From the indole class, 5-nitro-2-phenylindole (2, INF55)
(Scheme 1) represents a promising lead structure capable of pro-
ducing a 4-fold increase in S. aureus susceptibility to ciprofloxacin
when co-administered with the antibiotic at a concentration of
recently reported as NorA inhibitors12 suggesting that the indole-
NH is not essential for activity. In 2006, we reported preliminary
structure–activity data showing that various substituents around
the 2-aryl ring of INF55 improved NorA inhibitory potency.13 For
example, 50-benzyloxy-20-hydroxymethyl-5-nitroindole was found
to potentiate the activity of the mild antibacterial agent berberine
(a known substrate of NorA) more than 15-fold against the NorA
overexpressing S. aureus strain K2361 at a concentration of
1.5 l
g/mL.11
Structure–activity relationships for INF55 are only just starting
to emerge. In one theoretical COMFA (3D-QSAR) analysis, it was
suggested that replacement of the indole 5-nitro group with other
electron-withdrawing substituents should be favorable for activ-
ity.10 A series of 2-arylbenzo[b]thiophenes related to INF55 were
0.8
lg/mL (cf. INF55 potentiates berberine activity to the same
* Corresponding author. Tel.: +61 2 42214255; fax +61 2 42214287.
degree at the higher concentration of 3.0
lg/mL).
E-mail address: john_bremner@uow.edu.au (J.B. Bremner).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.093

J. I. Ambrus et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4294–4297
4295
R2
The current work discloses our latest systematic SAR explora-
R2
R1
R1
tion around the 2-aryl ring of INF55 and details the first experi-
mental description of the effects of substituting the indole
5-nitro group. The goal of this study was to obtain a deeper under-
standing of the SAR of INF55 and analogs with a view to increasing
potency against NorA in S. aureus. Furthermore, we sought to
broaden our knowledge of substituent tolerance around the
INF55 nucleus in order to advance our long-term goals of develop-
ing potent dual-action antimicrobials,14 including both non-cleav-
able hybrid molecules15 and cleavable ‘‘mutual” prodrugs16 which
combine antibacterial agents and NorA-inhibiting moieties into
single molecules.
INF55 2 was synthesized in one step (Scheme 1) from commer-
cially available 2-phenylindole 1 using the known regioselective
nitration procedure.17 Compounds 3–6 were each synthesized in
two steps starting from 1 and proceeding via a 5-chlorosulfonyl-
2-phenylindole intermediate 1a (Scheme 2). The intermediate
was cleanly accessed using a previously unreported, highly regio-
selective 5-chlorosulfonation of 2-phenylindole. Briefly, 1 was stir-
red with neat chlorosulfonic acid at 0 °C and allowed to warm to rt
with stirring over 30 min. The reaction mixture was then poured
slowly onto crushed ice and the product filtered, washed with
water, and dried under vacuum (80%). As 1a degraded when left
at rt over a period of several days, the freshly prepared crude
5-chlorosulfonyl-2-phenylindole (1a) was then reacted with
appropriate nucleophiles to furnish the 5-sulfonyl-2-phenylindole
derivatives 3–6 (Scheme 1) in good yields (3 = 72%, 4 = 70%,
5 = 57%, 6 = 76%).
i) a
R3
+
I
R3
N
N
H
ii) b-e
H
7-9
10-13
14-25
Compound
R1
Compound
R2
R3
H
CN
CO2Me
H
H
H
7
8
9
10
11
12
13
H
H
CO2Me
H
CO2Me
R1
R2
R3
Compound/Method
CN
H
H
H
H
H
H
14
CO2Me
H
H
15
b
c
CO2H
CH2OH
H
16
H
17
CO2Me
18
d
e
H
CH2OH
H
19
NO2
NO2
H
CO2Me
H
H
20
d
We propose that the regioselective 5-chlorosulfonation of 2-
phenylindole proceeds via the electrophilic aromatic substitution
mechanism shown in Scheme 2. Under strongly acidic conditions
(e.g., neat ClSO3H) indoles are known to protonate at the indole
C3 position which serves to protect this normally reactive site from
electrophiles. C3 protonation of 2-phenylindole would yield a 2-
phenylindolinium cation that ꢀcould potentially react directly with
a weak nucleophile like ClSO3 to form an indoline-2-chlorosulfo-
nate adduct.
CH2OH
21
H
H
H
H
CO2Me
CH2OH
CO2Me
CH2OH
22
d
e
H
23
NO2
NO2
24
d
25
Scheme 3. Reagents and conditions: (a) [Rh(coe)2Cl2]2, [p-CF3-C6H4]3P, Cs(OPiv)2,
1,4-dioxane, 120 °C;19 (b) 1 M LiOH, THF, 75 oC; (c) LiAlH4, THF, rt; (d) LiBH4, THF, rt;
(e) NaNO3, H2SO4 (concd), ꢀ20 oC.17
A related indoline-2-sulfonate adduct has been reported as a
participant in the regioselective formation of 5-substituted in-
doles.18 What is perhaps more likely is that the indoline-2-chloro-
sulfonate is formed in a concerted process that proceeds via the
cyclic 6-membered transition state shown in Scheme 2. The result-
ing indoline-2-chlorosulfonate, which contains an ortho-substi-
pointing (14–43%) but nevertheless provided sufficient quantities
of pure materials for use in our study. 2D NOESY spectra of com-
pounds 18 and 22 confirmed that indole C2 arylation had occurred
in preference to arylation at C3 (i.e., NOEs were observed between
the indole H3 and H4 signals for both 18 and 22).
+
tuted aniline ring, should direct a ClSO2 electrophile to the
indole C5 position (i.e., para to the aniline nitrogen), with indole
1a returned after quenching with water.
The methyl ester 15 was smoothly hydrolyzed to the carboxylic
acid 16 under standard conditions using LiOH/THF, and 15 was also
reduced without incident to the hydroxymethyl derivative 17
using LiAlH4 (Scheme 3). Esters 18 and 22 were reduced in essen-
tially quantitative yield to their respective hydroxylmethyl deriva-
tives 19 and 23 using LiBH4. The same esters 18 and 22 could be
regioselectively nitrated at ꢀ20 °C in NaNO3/H2SO4 mixtures17 to
provide the 5-nitro-2-arylindole derivatives 20 and 24 in 80%
and 81%, yields respectively. Compounds 22 and 24 were compre-
hensively characterized by 2D NMR spectroscopy, with NOESY
spectra confirming that mononitration had occurred selectively
at the indole C5 position for both compounds (i.e., observed NOEs
included NH/H7, H6/H7, and H3/H4). Esters 20 and 24 were subse-
quently reduced to their respective hydroxymethyl derivatives 23
and 25 in high yields (90% and 79%, respectively) using LiBH4
(Scheme 3).
Compounds 14, 15, 18, and 22 were prepared by direct C2 ary-
lation of commercially available indoles 7–9 with the respective
aryl iodides 10–13 using the recently reported Rh-catalyzed cou-
pling procedure (Scheme 3).19 Yields for the couplings were disap-
O
Ph
H
H
S
O
ClSO H
+
3
Cl
N
H
O
NH
1
H
H
+
ClO2S
"
O
(i) "ClSO2
(ii) H2O
O
S
Cl
N
H
N
H
O
Ph
Indoline-2-chlorosulfonate
(Directs Chlorosulfonation
to 5-position)
1a
A Pd-mediated intramolecular cyclization approach was used to
access the 20-substituted-2-arylindoles 31-35. All attempts to ac-
cess these analogs via Rh-catalyzed 2-arylation of indoles with
ortho-substituted aryl iodides failed. N-benzoylindoles 26 and 27
Scheme 2. Proposed mechanism for the regioselective 5-chlorosulfonation of
2-phenylindole in neat chlorosulfonic acid.

4296
J. I. Ambrus et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4294–4297
Table 2
R4
R1
Bacterial inhibition and potentiation results for 2-aryl analogs of INF55 (2) with
berberine in S. aureus
R1
R1
a
i) b
N
N
H
MIC (lM) of inhibitor
N
ii) c-e
R2
R1
COPh
O
4'
26-27
28-29
30-35
NH
2' 3'
Compound
R1
Compound/Method
Compound
R1
R1
R4
Compound R1
R2
D
norA K1758
WT 8325-4
NorA++ K2378
H
H
H
CO2H
CO2H
CO2Me
CH2OH
26
27
28
29
30
31*
32
33
34
35*
plus berberine plus berberine plus berberine
c
20-C02Me
20-CH2OH NP
30-C02Me
40
30-CH2OH NP
40-C02Me
NP
40-CH2OH NP
0.63
NP
NO2 20-CH2OH 24
NP
20
45
5
45
20
22
1.3
34
47
20
45
10
45
40
45
1.3
NP
47
NO2
NO2
NO2
H
32
33
18
19
22
23
225
34
3513
20
21
24
25
H
H
H
H
H
H
NO2
d
c
H
NO2
NO2
CO2Me
CH2OH
H
e
NO2 20-C02Me
NO2 30-C02Me
NO2 30-CH2OH
NO2 40-C02Me
NO2 40-CH2OH
0.51
1.0
1.0
Scheme 4. Reagents and conditions: (a) Pd(OAc)2, AcOH, 110 °C;20 (b) tBuOK,
tBuOH/H2O, 82 °C;20 (c) AcCl, MeOH, reflux; (d) LiAlH4, THF, rt; (e) BH3-THF, rt.13
9
NP
5
5
17
2
9
NP
2
*
Compounds synthesized previously.13
NP denotes no antibacterial potentiation. The inhibitor concentrations are the
minimum for potentiation of berberine MIC by factors of 3.3 (K1758), 3.3 (8325-4),
and 13.3 (K2378).
(Scheme 4) were converted to their respective 6-oxo-6H-isoindo-
lo[2,1-a]indoles 28 and 29 using the palladium (II) acetate pro-
moted oxidative intramolecular cyclization procedure reported
by Itahara.20 Ring-opening of compounds 28 and 29 by lactam
hydrolysis (t-BuOK, t-BuOH, H2O)20 yielded the 20-carboxylic acids
3020 and 31,13 respectively. Compound 30 was converted to the
methyl ester 32 under standard conditions (AcCl/MeOH) and to
the hydroxymethyl derivative 33 using LiAlH4. The acid 2-(20-car-
boxyphenyl)-5-nitroindole 31 was converted to its methyl ester
34 using AcCl/MeOH and to its hydroxymethyl derivative 3513
using BH3.THF.
The potentiation activities of compounds 1–6 and 14–17 (Table
1) were determined against the wild type S. aureus strain (8325-4),
a mutant strain deleted in NorA (K1758) and a NorA overexpress-
ing strain (K2378) with the antibacterial agent berberine using a
checkerboard assay which involved co-administration of varying
concentrations of the inhibitor and berberine.21
ecules. The inhibitor ‘MIC’ is defined as the lowest concentration of
inhibitor required to potentiate berberine’s MIC by at least a factor
of two. The MIC values for berberine (as the chloride salt) alone
against S. aureus were 269
lM (8325-4) and 67 lM (K1758)), and
2152 M (K2378). The activities of compounds 18–25 and 32–35
l
(Table 2) were determined against the same three strains of S. aur-
eus but using our previously published methodology13 (even
though not as complete as the full checkerboard assay), in order
to enable a better comparison with the prior results on compounds
with other 2-aryl substituents. Briefly, for this methodology, cells
were grown in the presence of a sub-inhibitory concentration of
berberine [81 lM (8325-4), 20 lM (K1758), and 161 lM (K2378)]
and varying concentrations of the test compounds. Bacterial
growth was monitored by measuring the absorption at 600 nm
and MIC values were determined. MICs represent the minimum
concentrations of the test compounds (in combination with the
fixed concentration of berberine) that completely inhibited cell
growth during an 18 h incubation at 37 °C. Differences in the val-
ues for INF55 (2) in Tables 1 and 2 reflect the difference in the
potentiation assay used, although the different result with the
NorA knockout mutant K1758 (Table 1) was not resolved.
This assay provides access to combinatorial interactions (syner-
gistic, antagonistic, indifferent, or additive) between the two mol-
Table 1
Bacterial inhibition and potentiation results for C5-substituted analogs of INF55 (2)
with berberine in S. aureus
MIC (lM) of inhibitor
R
The inherent antibacterial activity of each compound in the
absence of berberine was >1000
in this assay), except for the sulfonate esters 5 and 6 (MIC values
of 174 and 79.3 M, respectively, against all three S. aureus strains),
and 17. The alcohol 17 showed an MIC of 14.0 M against the NorA
M against both the wild type and NorA
lM (the testing limit employed
N
H
l
l
Compound
R
D
norA K1758
WT 8325-4
plus berberine
NorA++ K2378
plus berberine
knockout strain, and 28.0
l
plus berberine
overexpressing strains.22 Interestingly, in further testing, 17 was
shown to be inactive as an antibacterial against a panel of other
Gram-negative and Gram-positive pathogens.23
1
2
3
4
5
6
14
15
16
17
H
NO2
SO3H
SO2NH2
SO2OCH3
SO2On-Pr
CN
CO2Me
CO2H
CH2OH
NP
NP
NP
NP
IA
65 (4)
3.3 (2)
NP
NP
IA
IA
3.6 (2)
NP
NP
IA
259.0 (4)
6.5 (2)
NP
NP
IA
IA
7.1 (2)
NP
NP
IA
Compounds 1–6 and 14–17 (Table 1) varied only with respect to
substituents at the indole-C5 position allowing for direct conclu-
sions to be drawn regarding the importance of the 5-NO2 group
of INF55. Three of the compounds tested (5, 6, and 17) showed
intrinsic antibacterial activity, and thus the potentiation of the
MIC value could not be accurately determined for these analogs.
Structure–activity conclusions regarding the importance of the
C5 position of INF55 for inhibitory activity against the NorA pump
include:
IA
NP
NP
NP
IA
NP denotes no antibacterial potentiation; IA denotes intrinsic antibacterial activity.
Potentiation values are shown in brackets and are the ratios of berberine MICs with
and without inhibitor present.

J. I. Ambrus et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4294–4297
4297
(1) Removing the C5 substituent (1) is deleterious to activity.
References and notes
(2) Carbonyl based electron-withdrawing groups at C5 (15 and
16) abolish all activity.
(3) Inhibitors with sulfonic acids/esters/amides at C5 (com-
pounds 3–6) show no potentiation. Compounds 5 and 6 dis-
play mild intrinsic antibacterial activity.
1. Marquez, B. Biochimie 2005, 87, 1137.
2. Lomovskaya, O.; Zgurskaya, H. I.; Totrov, M.; Watkins, W. J. Nat. Rev. Drug
Discov. 2007, 6, 56.
3. Kaatz, G. W. In Frontiers in Antimicrobial Resistance; White, D. G., Alekshun, M.
N., McDermott, P. F., Eds.; ASM Press: Washington, DC, 2005; p 275.
4. Poole, K. Clin. Microbiol. Infect. 2004, 10, 12.
5. Kaatz, G. W.; Seo, S. M.; Ruble, C. A. Antimicrob. Agents Chemother. 1993, 37,
1086.
(4) Substitution with a nitrile group leads to retention of potency
with 14 showing similar potentiation activity to INF55.
6. Bremner, J. B. Pure Appl. Chem. 2007, 79, 2143.
7. Guz, N. R.; Stermitz, F. R.; Johnson, J. B.; Beeson, T. D.; Willen, S.; Hsiang, J.-F.;
Lewis, K. J. Med. Chem. 2001, 44, 261.
8. Vidaillac, C.; Guillon, J.; Moreau, S.; Arpin, C.; Lagardere, A.; Larrouture, S.;
Dallemagne, P.; Caignard, D.-H.; Quentin, C.; Jarry, C. J. Enzyme Inhib. Med.
Chem. 2007, 22, 620.
9. Marquez, B.; Neuville, L.; Moreau, N. J.; Genet, J.-P.; dos Santos, A. F.; Cano de
Andrade, M. C.; Goulart Sant’Ana, A. E. Phytochemistry 2005, 66, 804.
10. Markham, P. N.; Mulhearn, D. C.; Neyfakh, A. A.; Crich, D.; Jaber, M.-R.; Johnson,
M. E.; Influx, Inc., USA. Application: WO, 2000, 32196, 119 pp.
11. Markham, P. N.; Westhaus, E.; Klyachko, K.; Johnson, M. E.; Neyfakh, A. A.
Antimicrob. Agents Chemother. 1999, 43, 2404.
12. Fournier dit Chabert, J.; Marquez, B.; Neville, L.; Joucla, L.; Broussous, S.;
Bouhours, P.; David, E.; Pellet-Rostaing, S.; Marquet, B.; Moreau, N.; Lemaire,
M. Bioorg. Med. Chem. 2007, 15, 4482.
13. Samosorn, S.; Bremner, J. B.; Ball, A.; Lewis, K. Bioorg. Med. Chem. 2006,
14, 857.
14. Bremner, J. B.; Ambrus, J. I.; Samosorn, S. Curr. Med. Chem. 2007, 14, 1459.
15. Ball, A. R.; Casadei, G.; Samosorn, S.; Bremner, J. B.; Ausubel, F. M.; Moy, T. I.;
Lewis, K. ACS Chem. Biol. 2006, 1, 594.
16. Singh, G.; Dev Sharma, P. Indian J. Pharm. Sci. 1994, 56, 69.
17. Noland, W. E.; Rush, K. R.; Smith, L. R. J. Org. Chem. 1966, 31, 65.
18. Russell, H. F.; Harris, B. J.; Hood, D. B.; Thompson, E. G.; Watkins, A. D.;
Williams, R. D. Org. Prep. Proced. Int. 1985, 17, 391.
19. Wang, X.; Lane, B. S.; Sames, D. J. Am. Chem. Soc. 2005, 127, 4996.
20. Itahara, T. Bull. Chem. Soc. Jpn. 1981, 54, 305.
Compounds 20, 21, 24, 25, 34, and 35 (Table 2)24 systematically
explore the effects of methyl ester and hydroxymethyl substitu-
ents at the 20, 30, and 40 positions of the 2-aryl ring of INF55. Com-
pounds 18, 19, 22, 23, 32, and 33 explore these same effects in
compounds lacking the 5-NO2 of INF55. One of the goals of this
study was to identify analogs of INF55 that retained or improved
NorA inhibitory potency while containing functional groups useful
for covalent attachment to antimicrobial agents. We were particu-
larly interested in analogs bearing hydroxymethyl groups since
these could be incorporated into dual-action NorA inhibitor-anti-
bacterial ‘mutual’ prodrugs bearing labile ester linkages. Success
of this strategy of course requires that the alcohol-bearing INF55
analog released from such prodrugs is a potent NorA inhibitor.
The methyl ester analogs were intermediates in the synthesis of
the hydroxymethyl analogs and were tested here to add depth to
the SAR. The analogous series lacking the 5-NO2 group was ex-
plored in an attempt to identify inhibitors which might avoid the
known toxicity problems of nitroaromatic compounds.
Table 2 shows that all compounds lacking 5-NO2 groups (i.e., 18,
19, 22, 23, 32, and 33) were significantly less potent than INF55.
Another trend to emerge was that, apart from 24 with respect to
25, each of the methyl ester analogs was more potent than its cor-
responding hydroxymethyl derivative. The 30-CO2Me derivative 20
proved to be the most active compound identified in this study,
displaying slightly higher potency than INF55 against all three S.
aureus strains. Another important finding was that analog 25 bear-
ing a 40-CH2OH group was almost equipotent with INF55 against
the wild type and NorA overexpressing strain. This suggests that
25 is the best compound to progress into our studies of mutual
prodrugs employing labile ester linkages. It should be mentioned
that there is a possibility that non-covalent complex formation be-
tween berberine and the indole-based inhibitors reported here
may play a role in the potentiation observed.26
In conclusion, the SAR data for indole-based NorA efflux pump
inhibitors have been deepened by this study and three new potent
inhibitors, 14, 20, and 25, have been uncovered. Inhibitor 25 repre-
sents a promising candidate for incorporation into dual action mu-
tual prodrugs targeting the NorA pump. In addition, a new indole
derivative 17 was identified with specific antibacterial activity
against S. aureus. The mode of action of this compound and the rea-
son for its selective activity against this organism remains to be
investigated.
21. A checkerboard assay was conducted to specify the degree of potentiation of
berberine by the test compounds and to determine the specificity of these
compounds for the NorA efflux pump. Serial 2-fold dilutions of berberine
and a test compound were mixed in each well of a 96-well microtiter plate
so that each row (and column) contained a fixed amount of one agent and
increasing amounts of the second agent. The resulting plate presents
pattern in which every well contains unique combination of
concentrations between the two molecules. The concentrations of
berberine ranged from 100 to 12.5 g/mL (S. aureus 8325-4) and 200 to
800 g/mL (S. aureus K2378), while inhibitor concentrations ranged from
1.56 to 100 g/mL. Each plate also contained a row and column in which a
a
a
l
l
l
serial dilution of each agent was present alone. Cells were added to each
well at
a
final concentration of 5 ꢁ 105 CFU/mL, and plates incubated at
37 °C for 20 h. Growth was assayed by absorption at 600 nm with
a
microtiter plate reader (Spectramax PLUS384, Molecular Devices). An OD
less than 0.05 was considered to reveal no growth.
22. For the antimicrobial susceptibility assay, cells (105 CFU/mL) were inoculated
into broth and dispensed at 0.2 mL/well in 96-well microtiter plates. MICs
were determined by serial 2-fold dilution of the test compound. The MIC was
defined as the concentration of an antimicrobial that completely inhibited cell
growth during an 18–20 h incubation at 37 °C. Growth was assayed with a
microtiter plate reader (Spectramax PLUS384; Molecular Devices) by
monitoring absorption at 600 nm.
23. Compound 17 was tested against the following panel of Gram-negative
pathogens with MICs given in brackets in
lg/mL; the highest concentration
tested was 200 g/mL: Escherichia coli O157:H7, BW25113, and UT189 (all
l
>200); Salmonella typhimurium CS132 (>200); Pseudomonas aeruginosa PA01
and PA14 (both > 200). This compound was also tested against a number of
Gram-positive pathogens as follows with MICs in brackets in
highest concentration tested was 200 g/mL for all species apart from the
Clostridium species, where the highest concentration tested was 100 g/mL:
lg/mL; the
l
l
Enterococcus faecalis MMH594, V583, and OG1RF (all >200); Clostridium
difficile VPI10463, CD196, and CD2001 (all>100), and Clostridium perfringens
(>100).
Acknowledgments
24. Biological testing of compounds 30 and 31 was not undertaken as the latter
compound with an ortho-carboxylic acid group had been tested previously13
and showed poor potentiation of berberine, while the former compound was
only made as a precursor for the corresponding methyl ester 32 in order to
obtain a complete set of methyl esters for SAR purposes.
We thank the University of Wollongong, Australia and North-
eastern University, Boston, for supporting this work. The award
of an APA scholarship (Joseph Ambrus) and NHMRC C.J. Martin Fel-
lowship (Michael Kelso) are also gratefully acknowledged. Kim Le-
wis, Gabriele Casadei, and Anthony Ball were supported by Grant
R21 AI59483-01 from the NIH. We thank Glenn Kaatz for kindly
providing S. aureus strains.
25. The MIC of this compound with berberine was also determined, for comparison
purposes, against S. aureus K2361 and found to be 1.56
l
g/mL, broadly
commensurate with the previously obtained value (3
pump overexpressing strain.
l
g/mL)13 in this NorA
26. Zloh, M.; Gibbons, S. Theor. Chem. Acc. 2007, 117, 231.