Cytosporone E: Racemic synthesis and preliminary antibacterial testing

Article abstract of DOI:10.1016/j.bmc.2004.10.018

The antibiotic cytosporone E (isolated from the broth of the endophytic fungi CR 200 (Cytospora sp.) and CR 146 (Diaporthe sp.)) was synthesized as a racemic mixture. The key step in the synthesis is the Meyers ortho-alkylation of a chiral aromatic oxazoline. Preliminary antibiotic activity shows antibiosis against Gram-positive bacteria but not Gram-negative bacteria as previously reported.

Full text of DOI:10.1016/j.bmc.2004.10.018

Bioorganic & Medicinal Chemistry 13 (2005) 1409–1413
Cytosporone E: racemic synthesis and preliminary
antibacterial testing
Jeffrey D. Hall,a Nathan W. Duncan-Gould,a Nasar A. Siddiqi,a Jennifer N. Kelly,b
L. Alexis Hoeferlin,a Susan J. Morrisonb and Justin K. Wyatta,*
aDepartment of Chemistry and Biochemistry, College of Charleston, 66 George Street, Charleston, SC 29424, USA
bDepartment of Biology, College of Charleston, 66 George Street, Charleston, SC 29424, USA
Received 10 September 2004; accepted 6 October 2004
Available online 28 October 2004
Abstract—The antibiotic cytosporone E (isolated from the broth of the endophytic fungi CR 200 (Cytospora sp.) and CR 146 (Dia-
porthe sp.)) was synthesized as a racemic mixture. The key step in the synthesis is the Meyers ortho-alkylation of a chiral aromatic
oxazoline. Preliminary antibiotic activity shows antibiosis against Gram-positive bacteria but not Gram-negative bacteria as previ-
ously reported.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Equipotent Antibiotic Activity
O
HO
HO
O
There is a continuing search for new and more effective
antibiotics as bacteria become resistant to the current
antibiotic arsenal. In 2000 Clardy and co-workers1 iso-
lated the secondary metabolites cytosporone A–E as
racemates from the broth of two endophytic fungi; CR
200 (Cytospora sp.) and CR146 (Diaporthe sp.). Two
of these, cytosporone D (1) and E (2) (Fig. 1), were
shown to have equipotent antibacterial activity against
representative strains of Staphylococcus aureus, Entero-
coccus faecalis, Escherichia coli, and the fungus Candida
albicans. These activities are significant because as bacte-
ria develop resistance to available antibiotics, new anti-
bacterial compounds must be discovered. For example,
Staphylococcus aureus (bacteria that can cause pneumo-
nia, sepsis, osteomyelitis, meningitis, arthritis, and toxic
shock syndrome) shows widespread resistance to amox-
icillin and decreased susceptibility to vancomycin is of
great concern.2 The intestinal bacterium Enterococcus
faecalis, a frequent cause of nosocomial infections,
including urinary tract, and wound infections, is increas-
ingly resistant to vancomycin, which has been consid-
ered the last line of defense.3,4
HO
HO
O
O
(CH2)6CH3
OH (CH2)6CH3
OH
Cytosporone D (1)
Cytosporone E (2)
No Antibiotic Activity
HO
O
O
OH (CH2)6CH3
Cytosporone C (3)
Figure 1. Selected cytosporones.
This report focuses on the synthesis of cytosporone E,
which has a phthalide carbon backbone, a structure
common in many naturally occurring biologically active
substances.5 Cytosporone D is equipotent to E; however
it is interesting to note that it has been proposed that the
trihydroxy functionality is likely the source of antibio-
sis.1 This was proposed by comparing cytosporone D
to cytosporone C (3), which only differ by the central
phenolic hydroxy moiety.
Keywords: Phthalide; Antibiotic; Meyers ortho-Alkylation; Chiral
oxazoline; Endophytic fungus.
*
Corresponding author. Tel.: +1 843 953 6587; fax: +1 843 953 1404;
e-mail: wyattj@cofc.edu
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.018

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J. D. Hall et al. / Bioorg. Med. Chem. 13 (2005) 1409–1413
2. Results and discussion
H3CO
H3CO
CO2H
EDC, HOBt
+
Our synthesis of the cytosporone E focuses on using the
powerful Meyers ortho-alkylation of chiral aromatic
oxazolines as the key step to complete the phthalide
backbone. Ohzeki and Moriꢀs6 racemic synthesis of this
compound relied on the Snieckusꢀs ortho-lithiation of an
achiral aromatic amide. These two methods are compli-
mentary; however, the chiral oxazoline methodology is
foreseen to directly yield the chiral benzylic center in a
future chiral synthesis of the antibiotic.7
CH2Cl2, Et3N
(91%)
H2N
CO2CH3
OCH3
•HCl
8
9
O
H3CO
LiBH4
N
H
CO2CH3
THF, rt
(94%)
H3CO
OCH3
10
Formation of the aromatic oxazoline was initially ap-
proached via imidate ester 6 as described by Meyers
et al.8 (Scheme 1). Amide formation was accomplished
by converting 3,4,5-trimethoxybenzoyl chloride (4) to
amide 5 using ammonium acetate9 in 40% yield. The
amide was then reacted with Meerweinꢀs reagent
(Et3OÆBF4) to give an intermediate imidate ester 6,
which then reacted with L-valinol to yield chiral oxazo-
line 7 in 56% yield from the amide. The overall yield of
the oxazoline was 29% from 4; therefore, we approached
the synthesis of the oxazoline from a different angle.
O
MsCl, Et3N
H3CO
H3CO
OH
7
N
H
CH2Cl2, rt
(97%)
OCH3
11
Scheme 2. Revised synthesis of the chiral aromatic oxazoline.
The revised synthesis10 of oxazoline 7 (Scheme 2) started
with 3,4,5-trimethoxybenzoic acid (8) and was coupled
with L-valine methyl ester hydrochloride (9) using
EDC, HOBt, triethylamine to afford ester 10 in 91%
yield. Reduction of the methyl ester using lithium boro-
hydride afforded alcohol 11 in 94% yield. Cyclization
was accomplished using methane sulfonyl chloride and
triethylamine to give the desired oxazoline 7 in 97%
yield. The three steps gave an improved overall 83%
yield from 8.
N
a. sec -BuLi, THF
6h, -78 ˚C
H3CO
H3CO
7
O
b. CH3(CH2)6CHO
-78 ˚C to rt
OH
H3CO (CH2)6CH3
12
OH
N
H3CO
H3CO
With oxazoline 7 in hand we proceeded to complete the
Meyers ortho-alkylation (Scheme 3). This was accom-
plished via ortho-lithiation with sec-BuLi at ꢀ78°C for
NH4Cl (sat.)
(56%)
O
H
(CH2)6CH3
OCH3
13
H3CO
H3CO
COCl
NH4OAc
acetone
H3CO
H3CO
CONH2
O
O
H3CO
H3CO
3N HCl
( 40%)
reflux 30 min
(99%)
OCH3
OCH3
(CH2)6CH3
OCH3
14
4
5
NH2•BF4
OEt
O
Et3O•BF4
CH2Cl2
H3CO
H3CO
HO
HO
BBr3, CH2Cl2
O
-30 ˚C to rt
(90%)
(CH2)6CH3
OCH3
OH
6
2
Scheme 3. Formation of cytosporone E.
N
H3CO
H3CO
O
L
-Valinol
6h followed by addition of n-octanal. A mild acidic
work-up (satd NH4Cl) rearranges8 the intermediate
alcohol 12 to afford a 1:1 diastereomeric mixture of
imino lactone 13 in 56% yield. We are currently investi-
gating methods to improve the alkylation and the
diastereoselectivity of this reaction.7 Hydrolysis of this
(2 steps 56%)
OCH3
7
Scheme 1. Initial synthesis of the chiral aromatic oxazoline.

J. D. Hall et al. / Bioorg. Med. Chem. 13 (2005) 1409–1413
Table 1. Cytosporone activity against representative strains of Gram-
negative bacteria
1411
4. Experimental
Gram-negative bacteriaa
Diameter of the zone of
inhibition (mm 1mm)
The NMR spectra were recorded on a Varian Mercury
300 VXR NMR (1H at 300MHz and 13C at 75MHz).
A Mattson Genesis II FT-IR using an ATR sampling
method was used to obtain infrared spectra. Melting
point in an open ended capillary was uncorrected and
recorded using a Mel-Temp II apparatus. Optical rota-
tion was measured using an Autopol IV Automatic
Polarimeter. Elemental analyses were performed by
Quantitative Technologies Inc. of Whitehouse, NJ,
USA. THF and Et2O were distilled from lithium alum-
inum hydride or sodium/benzophenone ketyl; CH2Cl2
was purified from molecular sieves, Et3N was distilled
from CaH2, acetone was distilled from K2CO3, and all
other reagents were purchased and used as obtained.
Column chromatography separations were obtained on
Silica gel (230–400mesh). All reactions were done with
flame-dried glassware and under N2 gas unless otherwise
noted. EDC is 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride, and HOBt is 1-hydroxybenzo-
triazole hydrate.
lg Cytosporone E/disk
40
60
80
E. coli
None
None
None
None
None
None
None
None
None
None
None
None
P. aeruginosa
M. smegmatis
C. freundii
a Purchased from Difco Laboratories, Detroit, MI. The ATCCÒ
strains are as follows: Esherichia coli 25922; Pseudomonas aeruginosa
27853; Mycobacterium smegmatis wild type; Citrobacter freundii
8090.
Table 2. Cytosporone activity against representative strains of Gram-
positive bacteria
Gram-negative bacteriaa
Diameter of the zone of
inhibition (mm 1mm)
lg Cytosporone E/disk
40
60
80
4.1. 3,4,5-Trimethoxybenzamide 5
E. faecalis
S. aureus
10mm
13mm
13mm
12mm
11mm
13mm
14mm
12mm
10mm
13mm
14mm
11mm
To a solution of 3,4,5-trimethoxybenzoyl chloride
(10.4g, 44.mmol) in acetone (150mL) was added in
one portion ammonium acetate (10.5mL, 136mmol).
After stirring for 48h at rt the mixture was filtered
and the filtrate was concentrated in under reduced pres-
sure. The brown residue was recrystallized with hot
methanol (150mL) to yield a white solid (3.79g, 40%
yield). This material matched the known compound.
Mp 174–176°C, lit.12 177°C. 1H NMR (CDCl3): d
7.04 (s, 2H), 3.91 (s, 6H), 3.89 (s, 3H), 1.83 (s, 2H);
13C NMR (CDCl3): d 169.2, 153.2, 141.4, 128.7, 104.9,
61.2, 56.6.
S. epidermidis
B. subtilis
a Purchased from Difco Laboratories, Detroit, MI. The ATCCÒ
strains are as follows: Enterococcus faecalis 19433; Staphylococcus
aureus 25923; Staphylococcus epidermidis 12228; Bacillus subtilis wild
type.
intermediate in refluxing 3N HCl affords the 4,5,6-tri-
methoxyphthalide (14) in 99% yield. Removal of the meth-
oxy groups was conducted using boron tribromide11 at
ꢀ30°C to room temperature to afford cytosporone E
(2) in 90% yield (1H NMR and 13C NMR coincide with
the natural product1). The overall six-step yield of cyto-
sporone E from 3,4,5-trimethoxybenzoic acid is 41%.
4.2. (4S)-4-Isopropyl-2-(3,4,5-trimethoxyphenyl)-2-oxaz-
oline 7 via imidate ester 6
It was noted1 that cytosporone E was effective against
Gram-positive and Gram-negative strains of bacteria.
Our initial screenings of representative strains of
Gram-positive and Gram-negative bacteria contradict
the initial results. Table 1 illustrates that cytosporone
E is not effective against Gram-negative bacteria at 40,
60, or 80lg of antibiotic per disc. However, Table 2
shows that the antimicrobial is effective against Gram-
positive bacteria at 40, 60, and 80lg of antibiotic per
disc.
4.2.1. Formation of Meerweinꢀs salt (Et3OÆBF4).13 To a
stirred solution of BF3ÆOEt2 (14.3mL, 113mmol) in
diethyl ether (29.0mL) in a three neck RB flask
equipped with a refluxcondenser was added epic-
hlorohydrin (6.68mL, 85.2mmol) dropwise over 1min.
Upon addition the reaction was refluxed for 1h and then
cooled to rt and allowed to stand overnight to produce
colorless crystals. The ether was cannulated off and fresh
anhydrous ether (15mL) was added to rinse the crystals
and then this was cannulated off. This step was repeated
two more times. The crystals were dried under high vac-
uum to afford 12.3g (76% yield) of triethyl oxonium flu-
oroborate. (This yield varied between 75% and 90%.)
These were used directly in the formation of the imidate
ester above.
3. Conclusion
Racemic cytosporone E was synthesized in 41% yield
starting from 3,4,5-trimethoxybenzoic acid using the
Meyers ortho-alkylation of a chiral aromatic oxazoline
as the key step to form the phthalide carbon framework.
It has been noted that cytosporone E has antibiotic
activity against both Gram-positive and Gram-negative
bacteria. However, our samples were found to only be
active against Gram-positive bacteria.
4.2.2. Oxazoline 7 formation. To a stirred solution of tri-
ethyl oxonium fluoroborate (12.3g, 63.2mmol) in 1,2-
dichloroethane (50mL) was added a solution of amide
6 (17.6g, 63.2mmol) in 1,2-dichloroethane (250mL).
After stirring the combined solutions for 24h a solution
of L-valinol (7.17g, 69.5mmol) in 1,2-dichloroethane

1412
J. D. Hall et al. / Bioorg. Med. Chem. 13 (2005) 1409–1413
(40mL) was added. After refluxing for 24h the reaction
was quenched by pouring it into 500mL of 5% NaH-
CO3. The layers were separated and the aqueous phase
was extracted three times with CH2Cl2. The combined
organic layers were washed with brine, dried over
Na2SO4, and then concentrated under reduced pressure.
The crude oil was purified by flash chromatography (5–
25% EtOAc/hexanes) to afford oxazoline 7 as a light yel-
low oil in 72% yield (10.0g, 45.5mmol). 1H NMR
(CDCl3): d 7.21 (s, 2H), 4.40 (m, 1H), 4.12 (m, 2H),
3.91 (s, 6H), 3.88 (s, 3H), 1.89 (m, 1H), 1.03 (d,
J = 6.83Hz, 3H), 0.93 (d, J = 6.83Hz, 3H). 13C NMR
(CDCl3): d 163.0, 152.9, 140.5, 123.1, 105.3, 72.6,
70.0, 60.9, 56.2, 32.7, 19.0, 17.9. IR (oil) 1653,
H2O. The separated aqueous layer was extracted three
times with EtOAc. The combined organic layers were
washed with 1M NaOH, brine, dried over Na2SO4,
and then concentrated under reduced pressure. The
crude oil was purified using flash chromatography (35–
70% EtOAc/hexanes) afforded alcohol 11 in 94% yield
1
(0.533g). Mp 163–168°C. H NMR (CDCl3): d 7.00 (s,
2H), 6.28 (br d, J = 8.2Hz, 1H), 3.91 (s, 6H), 3.88 (s,
3H), 3.82 (m, 2H), 2.59 (br s, 1H), 2.02 (m, 1H), 1.04
(d, J = 5.7Hz, 3H), 1.02 (d, J = 5.8Hz, 3H). 13C NMR
(CDCl3): d 168.1, 153.1, 141.0, 130.0, 104.4, 63.7, 60.9,
57.6, 56.3, 29.2, 19.6, 19.1. IR (solid) 3291, 1630,
20
D
20
365
20
436
1128cmꢀ1
.
½aꢁ ꢀ0.266, ½aꢁ ꢀ0.884, ½aꢁ ꢀ0.516,
20
633
½aꢁ ꢀ0.228 (c 1.0, CH2Cl2). Anal. Calcd for
20
20
1587, 1505, 1128cmꢀ1
.
½aꢁ ꢀ0.404, ½aꢁ ꢀ1.79,
C15H23NO5: C, 60.59; H, 7.80; N, 4.71. Found: C,
60.72; H, 8.12; N, 4.63.
D
365
20
436
20
633
½aꢁ ꢀ0.938, ½aꢁ ꢀ0.334 (c 1.0, CH2Cl2). Anal. Calcd
for C15H21NO4: C, 64.50; H, 7.58; N, 5.01. Found: C,
64.01; H, 7.70; N, 4.94.
4.5. (4S)-4-Isopropyl-2-(3,4,5-trimethoxyphenyl)-2-oxaz-
oline 7 via amide alcohol 11
4.3. Methyl (2S)-2-(3,4,5-trimethoxybenamido)-3-methyl-
butanoate 10
To a solution of alcohol 11 (0.158g, 0.531mmol) in
CH2Cl2 (2.70mL) at 0°C was added Et3N (2.34mL,
1.68mmol) then MsCl (0.0822mL, 1.06mmol) dropwise
then warmed to rt. After 16h the solvent was removed
under reduced pressure. The oily residue was redissolved
in a 4:1 EtOAc/H2O solution. The aqueous layer was ex-
tracted three times with EtOAc and then the combined
organic layers were washed with brine and dried over
Na2SO4. After concentrating the organic layer under re-
duced pressure, the oily residue was purified via flash
chromatography (30–37% EtOAc/hexanes) to afford
oxazoline 7 as an oil in 97% yield (0.143g). Spectral data
matched that of the oxazoline via the imidate ester. See
Section 4.2.
To a 50mL round bottom flask was added 3,4,5-tri-
methoxybenzoic acid (8) (0.447g, 2.11mmol) and 9mL
of CH2Cl2. This suspension was stirred at 0°C, and then
EDC (0.809g, 4.22mmol) and HOBt (0.570g,
4.22mmol) were added. In a second 50mL round bot-
tom flask was added L-valine methyl ester hydrochloride
(9) (0.500g, 3.17mmol), 9mL of CH2Cl2 and Et3N
(0.588mL, 4.22mmol). (The solution goes from having
a suspension to clear and then back to having a white
suspension.) After 10min the methyl ester solution was
cannulated into the 3,4,5-trimethoxybenzoic acid solu-
tion. Upon mixing the ice bath was removed and the
reaction was stirred for 24h. Then the CH2Cl2 was evap-
orated under reduced pressure and the residue was dis-
solved in a 4:1 mixture of EtOAc/H2O mixture. The
organic layer was then washed two times with 1M
HCl, one time each with satd NaHCO3, H2O, and brine.
The organic layer was dried over Na2SO4, filtered, and
then concentrated under reduced pressure. The residue
was then purified using flash chromatography (15%
EtOAc/hexanes) to yield 10 as an oil in 91% (0.625g,
4.6. (S,Z)-2-(1-Heptyl-5,6,7-trimethoxyisobenzofuran-
3(1H)-ylideneamino)-3-methylbutan-1-ol 13
To
a stirred solution of oxazoline 7 (0.105mg,
0.376mmol) in THF (2.51mL) at ꢀ78°C was added
sec-BuLi (0.418mL of a 1.08M solution in hexanes,
0.451mmol).
After
6h,
n-octanal
(0.070mL,
0.451mmol) was added dropwise at ꢀ78°C. After
45min at ꢀ78°C the reaction was allowed to warm to
rt overnight. The reaction was quenched with the addi-
tion of NH4Cl and stirred for 15min at rt and then di-
luted with Et2O. The aqueous layer was extracted one
time with Et2O then the combined organic layers were
washed with brine and dried over Na2SO4 and concen-
trated under reduced pressure. The crude oil was puri-
fied by flash chromatography (30% EtOAc/hexanes) to
afford 13 as a 1:1 mixture of diastereomers as an oil in
56% yield (0.086g). Characterization of the top diaster-
eomer will be reported for simplicity. (Based on TLC
80% EtOAc/hexanes the top diastereomer has an
Rf = 0.38 and the bottom diastereomer has an
1
2.03mmol). Mp 132–135°C. H NMR (CDCl3): d 7.03
(s, 2H), 6.58 (br d, J = 8.2Hz, 1H), 4.76 (dd, J = 8.6
and 4.9Hz, 1H), 3.92 (s, 6H), 3.89 (s, 3H), 3.79 (s,
3H), 2.28 (m, 1H), 1.02 (d, J = 7.3Hz, 3H), 0.99 (d,
J = 7.0Hz, 3H). 13C NMR (CDCl3): d 172.8, 166.9,
153.2, 141.1, 129.5, 104.4, 60.9, 57.5, 56.3, 52.2, 31.6,
19.0, 18.0. IR (solid) 3288, 1747, 1630, 1124cmꢀ1
.
20
D
20
20
20
633
½aꢁ +0.218, ½aꢁ365 +0.616, ½aꢁ436 +0.466, ½aꢁ +0.180 (c
1.0, CH2Cl2). Anal. Calcd for C16H23NO6: C, 59.06;
H, 7.13; N, 4.31. Found: C, 59.10; H, 7.27; N, 4.18.
4.4. N-((S)-1-Hydroxy-3-methylbutan-2-yl)-3,4,5-tri-
methoxybenzamide 11
1
Rf = 0.23.) H NMR (CDCl3): d 7.16 (s, 1H), 5.45 (dd,
To a stirred solution of ester 10 (0.626g, 1.92mmol) in
THF (28.6mL) at 0°C was added LiBH4 (1.92mL of a
2M solution in THF, 3.85mmol) dropwise and then
warmed to rt. After 20h the reaction was quenched with
2M HCl and then concentrated under reduced pressure.
The residue was dissolved in a 4:1 mixture of EtOAc/
J = 7.4 and 3.1Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.89
(s, 3H), 3.78–3.70 (m, 3H), 2.49 (br s, 1H), 2.06 (m,
1H), 1.87 (m, 1H), 1.60 (m, 1H), 1.22 (m, 10H), 0.97
(d, J = 6.6Hz, 3H), 0.91 (d, J = 6.8Hz, 3H), 0.88 (t,
J = 7.0Hz, 3H). 13C NMR (CDCl3): d 168.8, 155.1,
147.4, 144.5, 131.7, 125.9, 101.1, 82.3, 64.6, 63.7, 61.0,

J. D. Hall et al. / Bioorg. Med. Chem. 13 (2005) 1409–1413
1413
60.6, 56.4, 34.0, 31.8, 30.3, 29.3, 29.1, 24.7, 22.6, 19.7,
19.4, 14.1. IR (oil) 3210, 1688, 1614, 1478, 1346,
1108cmꢀ1. Anal. Calcd for C23H37NO5: C, 67.78; H,
9.15; N, 3.44. Found: C, 67.64; H, 9.39; N, 3.22.
of agar plates that had been heavily inoculated with
the test organisms; then the plates were incubated over-
night. The diameter of the clear zone that resulted (i.e.,
the zone of inhibition) was measured in millimeters. The
zone of inhibition results from Cytosporone E diffusing
into the medium surrounding the disc. As a standard,
one disc was treated only with the solvent system and
carried through the process described above. This disc
showed no activity as expected if all the ethanol had
evaporated. The antibiotic activity of each sample was
determined in triplicate.
4.7. 3-Heptyl-4,5,6-trimethoxyphthalide 14
To a stirred solution of 13 (0.079g, 0.194mmol) in THF
(0.200mL) was added 3M HCl (2.8mL) and then ref-
luxed at 110°C for 30min. The reaction was cooled
and diluted with CH2Cl2 and the two layers were sepa-
rated. The aqueous layer was extracted three times with
CH2Cl2. The organic layers were combined and dried
over Na2SO4 and then concentrated under reduced pres-
sure. Flash column chromatography (30% EtOAc/hexa-
nes) afforded 14 as a light yellow oil in 99% yield
Acknowledgements
We would like to thank the Department of Chemistry
and Biochemistry at the College of Charleston, the
ACS-PRF# 39541-GB1 grant, and the BRIN the
NIH# RR-P20 RR 016461 grant from the BRIN pro-
gram of the National Center for Research Resources
for funding this project.
1
(0.062g). H NMR (CDCl3): d 7.12 (s, 1H), 5.46 (dd,
J = 2.9 and 7.8Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.92
(s, 3H), 2.25–2.14 (m, 1H), 1.76–1.63 (m, 1H), 1.46–
1.22 (m, 10H), 0.87 (t, J = 6.9Hz, 3H). 13C NMR
(CDCl3): d 170.6, 155.5, 147.6, 146.8, 135.4, 121.4,
102.5, 80.2, 61.1, 60.8, 56.4, 33.4, 31.7, 29.2, 29.1, 24.7,
22.6, 14.0. IR (oil) 1766, 1618, 1108cmꢀ1. Anal. Calcd
for C18H26O5: C, 67.06; H, 8.13. Found: C, 66.99; H,
8.28.
References and notes
1. Brady, S. F.; Wagenaar, M. M.; Singh, M. P.; Janso, J. E.;
Clardy, J. Org. Lett. 2000, 2, 4043.
4.8. Cytosporone E 3
2. (a) Levy, S. B. The Challenge of Antibiotic Resistance;
Scientific American, 1998, March; (b) Srinivasan, A.;
Dick, J. D.; Perl, T. M. Clin. Microbiol. Rev. 2002, 15, 430.
3. Huycke, M. M.; Sahm, D. F.; Gilmore, M. S. Emerging
Infect. Dis. 1998, 4, 239, http://www.cdc.gov/ncidod/eid/
vol4no2/huycke.htm.
4. Cetinkaya, Y.; Falk, P.; Mayhall, C. G. Clin. Microbiol.
Rev. 2000, 13, 686.
5. (a) Barton, D. H. R.; deVries, J. X. J. Chem. Soc. 1963,
1916; (b) Shamma, M. The Isoquinoline Alkaloids; Aca-
demic: New York, 1972.
To a solution of 14 (0.218g, 0.676mmol) in CH2Cl2
(2.25mL) at ꢀ30°C was added BBr3 (2.70mL of a
1.0M solution in CH2Cl2, 2.70mmol). After 1h the reac-
tion is warmed to rt for 2h after which it was poured
into ice-water and the aqueous layer was extracted with
CH2Cl2 twice. The combined organic layers were
washed with brine and dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified
by flash chromatography (5% MeOH/CHCl3) to afford
Cytosporone E in 90% yield (0.170g). 1H and 13C
NMR spectra were identical with those of the natural
product.
6. Ohzeki, T.; Mori, K. Biosci. Biotechnol. Biochem. 2003, 67,
2584.
7. The current chiral oxazoline yielded a 1:1 mixture of
diastereomers in the alkylation step (see Scheme 3), and we
are currently investigating different chiral oxazolines to
improve this step and finish the chiral synthesis of
cytosporone E.
8. Meyers, A. I.; Hanagan, M. A.; Trefonas, L. M.; Baker,
R. J. Tetrahedron 1983, 39, 1991.
9. Finan, P. A.; Fothergill, G. A. J. Chem. Soc. 1962,
2824.
10. Gilbertson, S. R.; Fu, Z. J. Org. Chem. 2001, 66, 7240.
11. Ogawa, Y.; Hosaka, K.; Chin, M.; Zhengxiong, C.;
Mitsuhashi, H. Heterocycles 1989, 29, 865.
12. TCI Organic Chemicals Catalog. 2004–2005.
13. Meerwein, H. Org. Synth. 1973, 5, 1080, Coll.
4.9. Antibiotic testing
The antibiotic testing against representative Gram-posi-
tive and Gram-negative bacteria was accomplished
using the Kirby–Bauer method. Cytosporone E, in a
solution of 1:1 ethanol–sterile water, was applied to ster-
ile standardized filter paper discs to correspond to the
varying desired amounts of 40, 60, and 80lg of antibi-
otic per disc. The discs were placed in an incubator at
37°C for 40min to allow the ethanol to evaporate.
The discs were then aseptically applied to the surface