Flustramine inspired synthesis and biological evaluation of pyrroloindoline triazole amides as novel inhibitors of bacterial biofilms

Article abstract of DOI:10.1039/c1ob05605k

Anti-biofilm agents have been developed based upon the flustramine family of alkaloids isolated from Flustra foliacea. A Garg interrupted Fischer indolization reaction was employed to access a core pyrroloindoline scaffold that was subsequently employed t

Full text of DOI:10.1039/c1ob05605k

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PAPER
Flustramine inspired synthesis and biological evaluation of pyrroloindoline
triazole amides as novel inhibitors of bacterial biofilms†
Cynthia Bunders,a John Cavanaghb and Christian Melander*a
Received 14th April 2011, Accepted 17th May 2011
DOI: 10.1039/c1ob05605k
Anti-biofilm agents have been developed based upon the flustramine family of alkaloids isolated from
Flustra foliacea. A Garg interrupted Fischer indolization reaction was employed to access a core
pyrroloindoline scaffold that was subsequently employed to create a pyrroloindoline triazole amide
library. Screening for the ability to modulate biofilm formation against strains of Gram-positive and
Gram-negative bacteria identified several compounds with low micromolar, non-toxic IC50 values.
Biofilms are populations of microorganisms that exist as an
organized community. They can be found in any environment in
which bacteria, moisture, nutrients, and a surface for attachment
are present. Biofilms are advantageous to bacteria because they
provide individual cells with shelter, food, and diversity, all
of which allow the bacteria to survive environmental selection
pressures.1 The National Institute of Health (NIH) estimates that
over 75% of microbial infections that occur in the human body
are linked to the formation of biofilms. Some common diseases
associated with the formation of biofilms are: lung infections
in cystic fibrosis (CF) patients, burn wounds, gastrointestinal
infections, urinary tract infections, and dental plaque.1,2 There
Fig. 1 A) Marine alkaloid natural product inspiration in the Melander
lab. B) Two of the most biologically active flustramine analogues. C)
is an immense need for innovative solutions to combat bacterial
Designed scaffold incorporating flustramine inspiration and allowing for
biofilms, as conventional methods such as antiseptics, antibiotics,
facile generation of analogues.
and other antimicrobials are becoming increasingly ineffective.3
One approach to the discovery of anti-biofilm agents is through
including the ability to eradicate bacteria native to the environment
the use of natural products as structural inspiration for analogue
they inhabit.6 It has also been reported that dihydroflustramine
design. Emboldened by our success with the 2-aminoimidazole
C inhibits production of reporter genes in an acyl homoserine
scaffold diverged from oroidin/ageliferin and the ethyl N-(2-
lactone (AHL)-based quorum sensing reporter assay (IC50 20 mg
phenyl) carbamate scaffold, we have begun to explore the potential
mL-1 or 62 mM), and is thus hypothesized to inhibit AHL-
of the indole derived flustramine family of natural products to
driven gene transcription.6 This suggests that this class of natural
serve as scaffolds for analogue design and to ultimately probe
products might have the ability to control quorum sensing. Given
bacterial biofilm modulation (Fig. 1).4 The flustramine family
that quorum sensing and biofilm formation have an undoubted
is a collection of monobrominated alkaloids that contain a
connection to one another, we envisioned that small molecules
pyrroloindoline or indolic core. These secondary metabolites were
based on the flustramine scaffolds might serve as potential
isolated from Flustra foliacea, a marine invertebrate, found in the
candidates to inhibit the formation of Gram-negative and Gram-
North Bryozoan Sea.5
positive biofilms.7
A major defining feature of the flustramine family is that it
The second determining factor that stimulated the selection of
possesses several members that have interesting biological activity,
the flustramine scaffold was that indole itself has been established
as an ubiquitous intercellular signal, and is produced by both
aDepartment of Chemistry, North Carolina State University, Raleigh, North
Gram-negative and Gram-positive bacteria.8 Indole has demon-
Carolina, 27695, USA. E-mail: Christian_Melander@ncsu.edu; Fax: +1
strated the ability to control numerous phenotypes in bacteria,
such as drug resistance, biofilm formation, virulence, and the
ability to alter phenotypes of other bacteria that do not produce
indole.9 Most recently, studies demonstrate that the concentration
of indole produced by Escherichia coli is dependent on pH and
temperature, and correspondingly determines the quantity of
919-515-5079; Tel: +1 919-513-2960
bDepartment of Molecular and Structural Biochemistry, North Car-
olina State University, Raleigh, North Carolina, 27695, USA.
E-mail: john_cavanagh@ncsu.edu
† Electronic supplementary information (ESI) available: 1H and 13CNMR,
ESI-MS, UV-Vis, IR, biofilm inhibition, and growth curves analyses. See
DOI: 10.1039/c1ob05605k
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biofilm formation.9,10 Therefore, it appears that indole signalling
is widespread and could be present among a majority of the
microbial community, and we thus posited that the flustramine
family, containing a deep-seated indole core, may lead us to
small molecules that have the ability to regulate bacterial biofilms
and ultimately deliver small molecule probes to dissect indole
signalling in vitro and in vivo.
Our approach to the key des-bromo pyrroloindoline 6 is
outlined in Scheme 1. The protected lactam 3 was synthesized with
a slight modification to Garg’s synthesis in that the toluenesulfonyl
(Ts) group was exchanged for an o-nitrobenzenesulfonyl (Ns)
protecting group. We found that in our system the Ts group
was difficult to remove at the end of the synthesis to generate
deprotected analogues for screening. The Ns group allowed for
the use of Fukuyama’s deprotection conditions which utilized
thiophenol, potassium carbonate, and CH3CN at room temper-
ature to provide the tricyclic aminal core in a facile way with
high yield (Scheme 2).16 The protected lactam 3 was alkylated
with propargyl bromide to give 4. The reduction of the protected
lactam to the hemiaminal 5 proceeded efficiently under standard
DIBAL-H conditions. The Fischer indolization reaction was then
carried out utilizing microwave irradiation to provide a more time
efficient reaction. The conditions employed in our synthesis were
5 min at 150 ◦C opposed to 1.5 h at 100 ◦C for conventional
heating, forming the tricyclic core 6 in 52% yield as originally
reported by Garg.15
To test the potential of the flustramine family to serve as a
template for the design of anti-biofilm compounds, we synthesized
flustramine C11 and evaluated its activity against three strains of
bacterial pathogens: Acinetobacter baumannii, E. coli, and methi-
cillin resistant Staphylococcus aureus (MRSA). A. baumannii, a
Gram-negative bacterium that forms robust films, was selected for
this study because of its rising prominence in hospital acquired
infections, especially those of military personnel in Iraq and
Afghanistan.12 This bacterium is difficult to eradicate and has
been found to have survived for weeks on surfaces, making it hard
to disinfect hospital and medical equipment. MRSA, a Gram-
positive bacterium that is resistant to numerous antibiotics was
investigated because it has emerged as a serious health problem
for patients with immunocompromised systems.13 E. coli, a Gram-
negative bacterium, is commonly found in the lower intestines of
warm-blooded organisms. Most E. coli strains are harmless, but
some can cause serious food poisoning (i.e. enterotoxigenic E. coli
(ETEC) aka travellers’ diarrhea). It has been reported that an
estimated 280 million episodes of ETEC diarrhea occur each year
in children under five in developing countries, with 20% of these
cases leading to death.14 To our delight we observed moderate
biofilm inhibition activity against A. baumannii (30% at 100 mM)
with flustramine C.
Based on this initial result we then began to analyze the
components of flustramine C to determine where modification
could be made to rapidly create a library of analogues for further
screening. In our experience with the 2-aminoimdazole scaffold,
we have noted that the most active biofilm inhibitors tend to be
amphipathic. Based on this observation we elected to keep the
aminal construct and modulate the identity of the reverse prenyl
group with an alkyne, which could be further elaborated through
click chemistry to introduce different hydrophobic components via
a triazole amide moiety (Fig. 1). Herein we describe the synthesis
and anti-biofilm activity of pyrroloindoline-triazole-amide (PTA)
analogues based on the flustramine family. These small molecules
were tested against the three above mentioned medically relevant
strains of bacteria (A. baumannii, E. coli, and MRSA) with the aim
of identifying molecules that inhibit bacterial biofilm formation
efficiently.
Scheme 1 Desired scaffold 1 based on flustramine family, and synthe-
sis of intermediate 6. Reagents and conditions: (a) i) BuLi, THF ii)
o-nitrobenzenesulfonylchloride, THF, -78 ◦C; (b) i) BuLi, hexamethyldis-
ilazane, THF ii) propargylbromide, THF, -78 ◦C; (c) DIBAL-H, CH2Cl2,
-78 ◦C; (d) AcOH : H2O (1 : 1), 150 ◦C mwave, 5 min.
To access the alkyne derived flustramine derivative as a base
scaffold for analogue design, we elected to employ Garg’s inter-
rupted Fischer indolization methodology.15 Our first attempt to
produce flustramine analogues centered on synthesizing bromo-
pyrroloindoline 1. This scaffold contains similar components
to those in flustramine A–E and it was envisioned that 3-
bromophenylhydrazine could be employed in the indolization
step to produce analogues with bromine at the six position. It
was quickly determined however, that we would have to produce
debromo analogues, as the interrupted Fischer indolization re-
action of 3-bromophenylhydrazine with key intermediate 5 was
unsuccessful despite numerous attempts and modifications to the
procedure.
Scheme 2 Click and deprotection conditions: (a) CuSO4·5H2O, sodium
ascorbate, azide, CH2Cl2, EtOH, H2O, rt (b) PhSH, K2CO3, CH3CN, rt.
With the core formed we then determined the optimal se-
quence of alkyne functionalization and protecting group removal
(Scheme 2). Initially, we performed the deprotection step first.
This method provided a 73% yield of the tricyclic aminal alkyne
intermediate 7. From this intermediate, three derivatives were
synthesized via the click reaction; however these analogues were
accessed in poor yield (~ 20%). Since the click reaction with
this alkyne was low yielding, we contemplated that it might
be beneficial to form the triazole (8) first then perform the
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Table 2 IC50 and percentage inhibition of compounds 23–26
deprotection to afford the desired products. This route improved
yields to 54–96% yield for the click reaction and 65–98% for the
deprotection step (Fig. 2). Although this route involved more
synthetic steps it provided the desired compounds in higher yields.
The synthesis proceeded with the use of an array of readily
available azide-amides (Fig. 2).17
IC50(mM)
Compound E. coli
IC50(mM)
A. baumannii
IC50(mM)
MRSA
IC50(mM)
S. aureus
23
24
25
26
20% @ 200
167.0 2.0
40% @ 200
206.4 5.0
13.5 1.5 < 5% @ 200
6.6 0.8
3.4 1.0
2.8 1.0a
33.3 1.8
12.5 1.3a
6.6 1.9.a
100.0 2.0a 90.0 5.0a
71.0 5.0a
20.0 2.0b
a Indicates inhibition of biofilm formation through a toxic mechanism
determined by growth curve analysis. b Indicates not complete toxicity,
bacterial growth delay is noted in the first 8 h, bacterial density is identical
at 24 h.
however this compound was determined to be toxic to planktonic
bacteria at this concentration.
When the library was screened against A. baumannii only
compounds 10 and 24 (IC50 = 192.5 mM and 206.4 mM respectively)
were able to efficiently inhibit A. baumannii bacterial biofilm
formation via a non-toxic mechanism. The lowest toxic IC50 value
determined was for compound 26 (IC50 = 20.0 mM). A number of
compounds were determined to affect the bacterial growth curve
(and are labeled as toxic).
Compounds 10 and 19 displayed moderate activity against
MRSA biofilm formation (IC50 78.0 mM, and 86.0 mM respec-
tively). The trend determined for compounds 9–12 against MRSA
revealed that increasing chain length augmented potency; however
the molecules also became toxic to planktonic bacterial growth.
Specifically, we noted that compounds 11 and 12 displayed low
micromolar IC50 values (25.0 mM and 5.4 mM respectively) but
were toxic to planktonic bacteria. Thus to generate non-toxic
PTA analogues against MRSA, the aliphatic appendage should
not exceed eight carbon units.
Compounds (23–25) that contained alkyl-derivatized-phenyl
rings also demonstrated exceptional non-toxic activity against
MRSA (Table 2), exhibiting IC50 values of 13.5 mM, 6.6 mM,
and 3.4 mM respectively. PTA analogue 26 displayed more potent
activity but was determined to affect early bacterial growth at
its IC50 concentration (2.8 mM); however we noted that after
24 h bacterial density is identical to the untreated control.
As we observed for the aliphatic analogues 9–12, the potency
of compounds 23–26 increased as a function of chain length,
however para-substituents longer than hexyl affected bacterial
growth.
Due to the exceptional activity of compounds 23–26 against
MRSA we envisioned that these compounds might have the
capability to affect the biofilm formation of other strains of
Staphylococcus aureus, a bacterium that is the leading cause
of bacterial infections worldwide. To our delight, three of
the four molecules efficiently inhibited biofilm formation by
a methicillin sensitive strain of S. aureus, with IC50 values
correlating to the chain length of the 4-alkylphenyl appendage
(Fig. 3). Compounds 24, 25, and 26 exhibited IC50 values of
32.0 mM, 12.5 mM, and 6.6 mM respectively; however, only
compound 24 inhibited S. aureus biofilm formation via a non-toxic
mechanism.
Fig. 2 18 member pyrroloindoline trizaole amide library with corre-
sponding click reaction yields. * = click yield with deprotected intermediate
7.
Biological screening
The evaluation of the PTA library commenced with screening each
small molecule for biofilm inhibition activity at 200 mM against
A. baumannii, E. coli, and MRSA. The activity of each molecule
was then assessed and dose response curves were obtained for
active analogues. Active molecules were also evaluated for their
toxicity to planktonic bacteria using growth curve and colony
count analysis. These experiments confirm whether the active
small molecule is simply killing planktonic bacteria, or modu-
lating biofilm formation through a non-microbicidal mechanism.
From our 18 compound library, two moieties emerged and were
identified as most active: analogues with aliphatic chains (Table 1)
and alkyl phenyl moieties (Table 2).
Against E. coli, compound 11 exhibited the lowest non-toxic
IC50 (35.7 mM). For this strain of Gram-negative bacteria the
PTA library illustrated that maximum activity is elicited with a
decyl aliphatic chain. Molecules 23–26 exhibited moderate activity
against E. coli with only compound 26 being active (IC50 71.0 mM);
Table 1 IC50 values and percentage inhibition of compounds 9–12 and
19
IC50(mM)
E. coli
IC50(mM)
A. baumannii
IC50(mM)
MRSA
Compound
9
30% @ 200
147.5 4.0
35.7 2.5
103.7 5.0
< 5% @ 200
25% @ 200
192.5 5.0
125.2 4.5a
40% @ 200
< 5% @ 200
< 5% @ 200
86.0 5.0
25.0 2.0a
5.4 1.0a
10
11
12
19
To further investigate structural components within the PTA
scaffold that drive biofilm inhibition, we exploited the interrupted
Fischer indolization reaction to access an alkyne furindoline
core 29 (Scheme 3).15 We utilized Garg’s methodology to access
78.0 3.0
a Indicates inhibition of biofilm formation through a toxic mechanism as
determined by growth curve analysis.
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Table 3 Biological evaluation of control triazole analogues 32 and 33 and
intermediates 6 and 7 at 200 mM. Flustramine C, structural inspiration,
was screened at 150 mM. Compound 25, our lead PTA analogue, is shown
for comparison purposes
E. coli biofilm
inhibition
A. baumannii
biofilm inhibition
MRSA biofilm
inhibition
Compound
6
<5%
<5%
<5%
30%
<5%
<5%
<5%
<5%
<5%
<5%
<5%
15%
< 5%
3.4 mM
7
Fig. 3 Correlation of 4-alkylphenyl PTA chain length and inhibition of
Gram-positive bacterial biofilm formation. Compound 23 (n = 2) for S.
aureus was not active at any tested concentrations but the graph is capped
at 50 mM, to show the relationship among the analogues.
32
33
Flustramine C
25
30%
20%
90.0 mMa
100.0 mMa
a Indicates inhibition of biofilm formation through a toxic mechanism
determined by growth curve analysis.
leading us to posit that both segments of the molecule are critical
for biological activity. Table 3 also includes flustramine C and our
lead PTA analogue 25 to reinforce that the tail or hydrophobic
region and substitution on the heterocycle are indeed critical to a
small molecule’s ability to inhibit bacterial biofilms.
Conclusions
Scheme 3 Synthesis of furindoline 31. IC50 values of compound 31.* =
Indicates inhibition of biofilm formation through a toxic mechanism as
determined by growth curve analysis. Reagents and conditions: (a) i)
BuLi, hexamethyldisilazane, THF ii) propargylbromide, THF, -78 ◦C;
(b) DIBAL-H, CH2Cl2, -78 ◦C; (c) AcOH : H2O (1 : 1), 60 ◦C, 4.5 h; (d)
CuSO4·5H2O, sodium ascorbate, azide 30, CH2Cl2, EtOH, H2O, rt.
In conclusion, we have accessed pyrroloindoline triazole amides
through an interrupted Fischer indolization reaction. Using this
approach, a set of 18 tricyclic indole derivatives with a wide variety
of functionality introduced through a click reaction was screened
against A. baumannii, E. coli, and MRSA. A structure activity
relationship was observed for compounds 23–26 based on the
chain length of the phenyl alkyl appendage and these compounds
exhibited low micromolar non-toxic IC50 values against two strains
of Gram-positive bacteria. We also note that some of the com-
pounds with long alkyl chains are also potent biofilm inhibitors;
however they do so via a toxic mechanism. This is potentially
due to compounds forming channels within the bacterial cell
wall. A furindoline analogue 31 was synthesized containing the
most active amide appendage, and was determined to be a less
active inhibitor of S. aureus and MRSA biofilm formation in
comparison to the pyrroloindoline analogue 25. This indicated
that the secondary nitrogen enhances activity presumably through
additional H-bond donor interactions with its biological target
or altered cell uptake properties. These PTA analogues may serve
as probes to potentially deconvolute bacterial signalling pathways
both in vitro and in vivo. Investigation of the most active PTA
analogues may probe the intriguing question of whether this class
of compounds can specifically block interactions that lead to
disease causing phenotypes and production of virulence factors,
in addition to biofilm formation. The PTA scaffold also contains
numerous synthetic handles that can be manipulated and further
optimized to generate more advanced analogues with enhanced
activity.
intermediate 27, which was then reduced to hemiacetal 28 and
cyclized to provide us with the furindoline core 29. Next, through
the click reaction, we reacted 29 with azide amide 30 to afford
furindoline 31, our targeted analogue of compound 25 (IC50
value of 3.4 mM against MRSA). To evaluate the effects of this
substitution we screened 31 for biofilm inhibition activity against
MRSA and. S. aureus. The furindoline core proved to be less active
as an inhibitor of both strains compared to its pyrroloindoline
analogue, but did exhibit IC50 values of 18.7 mM and 19.2 mM for
MRSA and S. aureus respectively. By replacing the nitrogen in our
heterocyclic core with oxygen there is a 5.5 fold decrease in MRSA
activity and a 1.6 fold decrease in S. aureus activity (Scheme 3).
Similar to analogue 25, compound 31 inhibited the Gram-positive
bacterial biofilms in a non-toxic manner for MRSA and through
a toxic mechanism for S. aureus.
To further decipher which functionality of the PTA library was
most critical for biological activity we synthesized two control
compounds (32 and 33). A one step click reaction between
propargyl amine and an azide amide produced the target control
compounds (Table 3). These compounds were examined for anti-
biofilm activity, along with intermediates 6 and 7 (which contain
only the tricyclic core). All control compounds were inactive
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J = 7.6 Hz), 2.40–2.37 (m, 1H), 2.25–2.20 (m, 1H), 1.65–1.56 (m,
2H), 1.29–1.23 (m, 6H), 0.87 (t, 3H, J = 7.6 Hz). 13C NMR (100
MHz, CDCl3) d 167.7, 148.9, 147.7, 147.6, 143.5, 133.9, 133.6,
132.2, 131.2, 131.0, 130.0 129.2, 128.9, 127.3, 124.5, 123.4, 123.1,
119.5, 109.5, 82.2, 58.4, 49.7, 47.5, 40.2, 36.6, 36.1, 33.8, 31.9, 31.4,
29.2, 22.8, 14.3; HRMS (ESI) calcd for C34H40N7O5S (M+H)+
658.2806, found 658.2807; IR vmax/cm-1 3398, 2921, 2859, 1644,
1539, 1166; lmax: 236 nm, 282 nm.
Experimental
Synthesis
All reagents used for chemical synthesis were purchased from
commercially available sources and used without further purifi-
cation. Chromatography was performed using 60-mesh standard
silica gel from Sorbtech. NMR solvents were obtained from
Cambridge Isotope Labs and were used as is. 1H NMR (300
MHz or 400 MHz) and 13C NMR (75 MHz or 100 MHz)
spectra were recorded at 25 ◦C on Varian Mercury spectrometers.
Chemical shifts (d) are given in ppm relative to the respective NMR
solvents; coupling constants (J) are in hertz (Hz). Abbreviations
used are s = singlet, bs = broad singlet, d = doublet, dd =
doublet of doublets, t = triplet, td = triplet of doublets, m =
multiplet, dab = ab doublet, ddab = ab doublet of doublet. Mass
spectra were obtained at the NCSU Department of Chemistry
Mass Spectrometry Facility. Infrared spectra were obtained on a
FT/IR-4100 spectrophotometer (nmax/cm-1). UV absorbance was
recorded on a Genesys 10 scanning UV/visible spectrophotometer
(lmax = nm).
General procedure for deprotection
25a (35 mg, 0.096 mmol) was placed in a vial and dissolved in 4 mL
of CH3CN. Then K2CO3 (53 mg, 0.386 mmol) was added to the
vial, then thiophenol (21.0 mg, 0.193 mmol) was added dropwise
and the reaction was stirred for 16 h at room temperature. The
reaction was then concentrated under reduced pressure to afford
crude aminal 25, which was purified by flash chromatography (1–
5% sat. NH3–MeOH–CH2Cl2) to afford 25 as an oil (66% yield).
1H NMR (300 MHz, CDCl3) d 7.62 (d, 2H, J = 8.4 Hz), 7.23 (d,
2H, J = 8.4 Hz), 7.00–6.92 (m, 2H), 6.72 (s, 1H), 6.64 (td, 1H,
J = 7.2 Hz, J = 1.2 Hz), 6.54 (t, 1H, J = 6.0 Hz), 6.32 (d, 1H, J =
7.5 Hz), 4.97 (s, 1H), 4.47–4.40 (m, 2H), 3.86–3.78 (m, 2H), 3.21
(dab, 2H, J = 14.4 Hz), 3.00–2.94 (m, 1H), 2.74–2.687 (m, 1H),
2.64 (t, 1H, J = 7.8 Hz), 2.14–2.08 (m, 1H), 2.00–1.90 (m, 1H),
1.61–1.55 (m, 2H), 1.30–1.25 (m, 6H), 0.87 (t, 3H, J = 7.2 Hz).
13C NMR (75 MHz, CDCl3) d 168.0, 151.1, 147.6, 145.2, 133.1,
131.3, 128.9, 128.5, 127.2, 123.7, 122.8, 118.8, 108.7, 82.2, 58.5,
49.7, 46.1, 41.8, 40.0, 36.1, 35.5, 31.9, 31.4, 29.1, 22.8, 14.3; HRMS
(ESI) calcd for C28H37N6O (M+H)+ 473.3023, found 473.3027; IR
General procedure for the preparation of intermediate 6
Hemiaminal 5 (250 mg, 0.805 mmol) was reacted with similar
synthetic methods developed by Garg15 with slight modification.
Microwave heating at 150 ◦C for five minutes was used opposed to
conventional heating. Purification by flash chromatography (10–
30% EtOAc : Hexanes) afforded 6 as an orange foam (160 mg,
1
52% yield). H NMR (300 MHz, CDCl3) d 8.04–8.01 (m, 1H),
v
max/cm-1 3384, 2923, 2853, 1644, 1609, 1546, 1455, 749; lmax: 238
7.73–7.64 (m, 3H), 7.17–7.10 (m, 2H), 6.79 (td, 1H, J = 7.5 Hz,
J = 0.9 Hz), 6.63 (dd, 1H, J = 8.4 Hz, J = 0.6 Hz), 5.50 (s, 1H),
5.00 (s, 1H (NH)), 3.68 (td, 1H, J = 7.8 Hz, J = 1.2 Hz), 3.27–3.18
(m, 1H), 2.59 (ddab, 2H, J = 7.8 Hz, J = 1.2 Hz), 2.48–2.40 (m,
1H), 2.92–2.30 (m, 1H), 1.92 (t, 1H, J = 2.7 Hz). 13C NMR (75
MHz, CDCl3) d 202.2, 148.7, 133.8, 133.2, 131.9, 130.7, 130.2,
129.4, 124.5, 123.3, 119.6, 109.7, 82.6, 80.3, 70.8, 57.4, 48.0, 35.7,
27.6; HRMS (ESI) calcd for C19H18N3O4S (M+)+ 384.1013, found
384.1010; IR vmax/cm-1 3405, 3287, 2916, 2364, 1616, 1539, 1462,
1357, 1168, 1056, 735; lmax: 226 nm, 280 nm.
nm, 296 nm.
Acknowledgements
The authors would like to thank the National Institute of Health
(GM055769 to JC and CM) for support of this work.
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Intermediate 6 (36 mg, 0.093 mmol) was dissolved in 1 : 1 ethanol–
water (2 mL total) and placed in a vial. Azide 30 (26 mg, 0.093
mmol) was then dissolved in dichloromethane (1 mL) and added to
the reaction vial. Next sodium ascorbate (11 mg, 0.056 mmol) and
copper sulfate (5 mg, 0.028 mmol) were added to the reaction vial.
The reaction mixture was then stirred for 16 h. The crude reaction
was quenched with water (5 mL) and diluted with EtOAc (5 ml).
The organic layer was washed with water (3 ¥ 5–10 mL), dried
over Na2SO4, and concentrated with reduced pressure to afford the
crude product, which was then purified by flash chromatography
(1–5% MeOH–CH2Cl2) afforded 25a as a yellow foam (57%
yield).1H NMR (400 MHz, CDCl3) d 8.00–7.96 (m, 1H), 7.70–
7.62 (m, 3H), 7.62–7.60 (m, 1H), 7.23 (s, 1H) 7.04 (td, 2H, J =
7.6 Hz, J = 1.2 Hz), 7.02 (s, 1H), 6.96 (d, 2H, J = 7.2 Hz), 6.76
(t, 1H, J = 6.0 Hz), 6.72 (td, 1H, J = 7.2 Hz, J = 1.2 Hz), 6.45 (d,
1H, J = 8.0 Hz) 5.48 (s, 1H), 4.82 (s, 1H), 4.56–4.48 (m, 2H), 3.88–
3.83 (m, 2H), 3.49–3.42 (m, 1H), 3.20–3.04 (m, 3H), 2.64 (t, 2H,
10 H. T. Han, J-H. Lee, M. H. Cho, T. K. Wood and L. Lee, Res.
Microbiol., 2011, 162, 108.
5480 | Org. Biomol. Chem., 2011, 9, 5476–5481
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The Royal Society of Chemistry 2011
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