The synthesis and biological evaluation of two analogues of the C-riboside showdomycin

Article abstract of DOI:10.1071/CH04273

Two novel analogues, 2 and 3, of the C-riboside showdomycin (1) have been prepared by exploiting the N-TIPS-substituted pyrrole 7 as a synthetic equivalent for the maleimide C3 anion. The tetraacetate precursor, 12, of target 2 as well as target 3 itself

Full text of DOI:10.1071/CH04273

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2005, 58, 86–93
www.publish.csiro.au/journals/ajc
The Synthesis and Biological Evaluation of Two Analogues
of the C-Riboside Showdomycin
Jens Renner,A Irma Kruszelnicki,B Beata Adamiak,C Anthony C. Willis,A Edward Hammond,D
Stephen Su,B Christopher Burns,B Edward Trybala,C Vito Ferro,D and Martin G. BanwellA,E
A Research School of Chemistry, Institute of Advanced Studies, Australian National University,
Canberra ACT 0200, Australia.
B Cytopia Pty Ltd, Baker Heart Research Institute, Commercial Road, Melbourne VIC 3004, Australia.
C Department of Clinical Virology, Göteborg University, 413 46 Göteborg, Sweden.
D Drug Design Group, Progen Industries Ltd, 2806 Ipswich Road, Darra QLD 4076, Australia.
E Corresponding author. Email: mgb@rsc.anu.edu.au
Two novel analogues, 2 and 3, of the C-riboside showdomycin (1) have been prepared by exploiting the N-TIPS-
substituted pyrrole 7 as a synthetic equivalent for the maleimide C3 anion. The tetraacetate precursor, 12, of target 2
as well as target 3 itself were subjected to single-crystal X-ray analyses. Analogues 2 and 3 as well as showdomycin
and its anomer (4) have each been evaluated in various assays for their cytotoxic, anti-bacterial, and anti-viral effects.
Manuscript received: 15 November 2004.
Final version: 13 January 2005.
Introduction
The C-riboside showdomycin (1; Scheme 1) is one of sev-
eral naturally occurring C-nucleosides that displays various
biological properties including anti-bacterial, anti-viral, and
anti-tumour activity.[1] Compound 1 was first isolated from
Streptomyces showdoensis by Nishimura in the early 1960s[2]
and has been the subject of many synthetic efforts since that
time.[3] Such studies have been prompted, at least in more
recent times, by an increasing recognition of the therapeutic
potential of C-nucleosides and investigations in the general
area are now focussed on variations in both the sugar and the
base.[4,5] The preparation of showdomycin analogues vary-
ing in the nature of the sugar residue has been hampered by
the paucity of flexible methods available for attachment of
maleimides to the anomeric centre of monosaccharides.[3o]
In 2002 we reported[6] that reaction of pyrroles with O-
glycosylimidates in the presence of certain Lewis acids leads
to the formation of C-glycosides. More recently, we have
exploited this sort of chemistry in the development of a
simple route to showdomycin[7] wherein the participating
N-triisopropylsilyl (TIPS)-substituted pyrrole[8] serves as a
synthetic equivalent for the maleimide C3 anion. Herein we
detail the extension of such chemistry to the preparation of
two showdomycin analogues, compounds 2 and 3, the first
involvingaglucosemoietyasthesugarresidueandthesecond
incorporating a cyclitol as a sugar surrogate. The outcomes
of the biological evaluation of compounds 2 and 3 are also
reported and compared with those obtained from simultane-
ous testing of synthetically derived showdomycin (1) and its
anomer (4).
Ј
Scheme 1.
Results and Discussion
Synthetic Studies
The synthesis of the ‘glucose’analogue, 2, of showdomycin is
shown in Scheme 2.Thus, the commercially available glucal 5
was converted into the corresponding and previously reported
α-epoxide 6 using protocols developed by Danishefsky and
Halcomb,[9] namely through reaction of the former com-
pound with dimethyldioxirane (DMDO) in CH2Cl2 at 0◦C.
Treatment of the highly electrophilic epoxide 6 with N-TIPS-
pyrrole (7) in the presence of indium trichloride (InCl3)[10]
thenaffordedtheC-glucoside8(37%)astheonlycharacteriz-
able product of reaction. Exhaustive debenzylation of product
© CSIRO 2005
10.1071/CH04273
0004-9425/05/020086

Two Analogues of Showdomycin
87
OBn
O
OBn
HO
HO
O
O
(a)
O
O
(a)
ϩ
TIPS
N
TIPS
N
ϩ
O
BnO
BnO
Br
7
Br
OBn
7
OBn
13
14
(b)
5
6
(b)
OAc
OH
N
TIPS
N
TIPS
OH
OBn
O
O
O
N
TIPS
(c)
N
TIPS
O
(c)
O
O
HO
OH
BnO
OH
Br
Br
OH
OBn
16
15
9
8
(d )
(d )
OAc
OAc
N
TIPS
NH
OAc
OAc
O
O
O
O
N
TIPS
NH
(e)
O
O
(e)
AcO
OAc
AcO
OAc
17
18
OAc
OAc
(f )
10
11
O
(f)
OAc
O
NH
O
O
(g)
OAc
3
NH
O
O
(g)
2
O
19
AcO
OAc
Scheme 3. Reagents and conditions: (a) see ref. [12]; (b) compound 7
(2.5 mol equiv. wrt 14), InCl3 (10 mol% wrt 14), CH2Cl2, 18◦C, 5 days;
(c) Ac2O (20 mol equiv.), Et3N (25 mol equiv.), CH2Cl2, 0–18◦C, 48 h;
(d) H2 (1 atm), 10% Pd on C, Et3N (4.9 mol equiv. wrt 16), EtOH,
18◦C, 16 h; (e) TBAF (1.0 mol equiv.), THF, 0◦C, 1 h; (f ) PCC (4.9 mol
equiv.), 4 Å molecular sieves, CH2Cl2, 18◦C, 16 h; (g) HCl (excess),
MeOH, 0–18◦C, 48 h.
OAc
12
Scheme 2. Reagents and conditions: (a) see ref. [9]; (b) compound 7
(2.6 mol equiv. wrt 6), InCl3 (10 mol% wrt 6), CH2Cl2, 0–18◦C, 19 h;
(c) H2 (1 atm), 10% Pd on C, EtOH, 18◦C, 22 h; (d) Ac2O (20 mol
equiv.), Et3N (25 mol equiv.), DMAP (12.5 mol%), CH2Cl2, 0–18◦C,
16 h; (e) TBAF (1.1 mol equiv.), THF, 0◦C, 1 h; ( f ) PCC (5 mol equiv.),
4 Å molecular sieves, CH2Cl2, 18◦C, 16 h; (g) HCl (excess), MeOH,
0–18◦C, 48 h.
this manner compound 2 was obtained in 44% yield and as a
clear, colourless oil. The spectroscopic data derived from this
compound were in full accord with the assigned structure. In
particular, the 300 MHz 1H NMR spectrum revealed the pres-
ence of an olefinic proton (doublet, J 0.9 Hz, at δ 6.68) thus
indicating that, under the deacetylation conditions used, no
intramolecular Michael addition of the C6ꢀ-hydroxyl to the
maleimide unit had occurred.
The synthetic route used to obtain the cyclitol-based show-
domycin analogue 3 is shown in Scheme 3 and started
with the cis-1,2-dihydrocatechol 13, a compound available
in bulk quantity and enantiomerically pure form through
enzymatic dihydroxylation of bromobenzene.[11] Thus, con-
version of this diol into the corresponding acetonide followed
by m-chloroperbenzoic acid (m-CPBA)-mediated epoxida-
tion afforded the previously reported[12] oxirane 14 (90%
from 13) in a fully regio- and stereo-controlled manner. Reac-
tion of the latter compound with the previously employed
pyrrole 7 in the presence of InCl3 gave the adduct 15 (54%)
in a completely selective fashion and by means of a pro-
cess involving nucleophilic attack at the allylic carbon.[13]
Acetylation of the hydroxyl group within this last compound
8wasachievedusingdihydrogeninthepresenceof10%Pdon
C to give the tetrol 9 (87%) which was immediately peracety-
lated using acetic anhydride to give the tetraacetate 10 in 73%
yield. Removal of the TIPS group within compound 10 was
achieved using tetra-n-butylammonium fluoride (TBAF) in
THF at 0◦C to deliver the 3-substituted pyrrole 11 in 96%
yield. Oxidation of this pyrrole to the corresponding and
crystalline 3-substituted maleimide 12 was accomplished in
67% yield using pyridinium chlorochromate (PCC) in the
presence of 4 Å molecular sieves. The NMR and mass spec-
troscopic data derived from compound 12 were in full accord
with the assigned structure but final confirmation of this was
established through a single-crystal X-ray analysis, details of
which will be published elsewhere. Efficient and complete
deacetylation of maleimide 12 so as to reveal the target C-
nucleoside 2 proved rather problematic, in part because of the
sensitive nature of the latter compound. Ultimately, the best
conditions we could identify for achieving this conversion
involved the use of methanolic HCl at 0–18◦C for 48 h and in

88
J. Renner et al.
O5
Table 1. Outcomes associated with the anti-bacterial testing of
compounds 1–4 using the disk diffusion methodA,B
Cell line
1C
2
3
4
N1
O3
Staphylococcus aureus
Bacillus subtilis
Escherichia coli
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
O4
Pseudomonas aeruginosa
O2
A See Experimental section for details.
B The symbol + indicates anti-bacterial effects, whereas the symbol −
indicates no anti-bacterial effects.
C At loadings of 100 µg disk−1, compound 1 formed inhibition zones of
24, 21, and 21 mm in diameter on plates growing S. aureus, B. subtilis,
and E. coli, respectively.
O1
Table 2. IC50 values [µM] determined for compounds 1–4 in
cytotoxicity testing against a range of cell linesA
Fig. 1. Molecular structure of compound 3 derived from X-ray
crystallographic data. Displacement ellipsoids show 50% probability
levels.
Cell lineB
1
2
3
4
CTLL2
TF-1
HepG2
HEK293
Mat-LyLu
Baf3-Tel-Jak2
Baf3-Tel-Jak3
K562
5.37
7.84
>20
3.27
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
was carried out under conventional conditions and gave the
acetate 16 in 63% yield. Subjection of compound 16 to hydro-
genation/hydrogenolysis through exposure to dihydrogen in
the presence of 10% Pd on C and Et3N resulted in complete
removal of the bromo-olefin unit and thus afforded cyclitol 17
in 81% yield.TheTIPS group within product 17 was removed
using TBAF and the resulting deprotected pyrrole 18 (80%)
then oxidized to the corresponding maleimide 19 (83%) using
PCC in the presence of 4 Å molecular sieves. Exhaustive
deprotection of the last compound was also achieved using
HCl in methanol to afford target 3 in 91% yield. Once again
the spectroscopic data obtained for this crystalline material
wereinfullaccordwiththeassignedstructurebutfinalconfir-
mation was provided through a single-crystal X-ray analysis
(see Fig. 1 and Experimental section).
5.18
4.76
4.22
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
approx. 15
>20
DU145
PC-3
>20
A DMSO was used as solvent in these assays.
B CTLL2, murine cytotoxic T-cells; TF-1, human erythroleukaemia
bone marrow cells; HepG2, human hepatoma cells; HEK293, human
embryonic kidney cells; Mat-LyLu, rat prostate adenocarcinoma cells;
Baf3-Tel-Jak2 and Baf3-Tel-Jak3, transformed cells; K562, human
chronicmyelogenousleukaemiacells;DU145andPC-3, humanprostate
carcinoma cells.
this trend involved the human kidney cell line HEK where
IC50 values for all compounds were in the range 3.27–
5.18 µM. Of course, the implied nephrotoxic properties of
these compounds are of concern in terms of their exploitation
as therapeutic agents.
Biological Studies
Anti-Bacterial Testing
Samples of showdomycin (1) and its anomer 4 along with
compounds 2 and 3 were tested for anti-bacterial activity
using a disk diffusion method. The target organisms were
Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
and Pseudomonas aeruginosa. Positive controls were set up
in a similar manner and involved varying concentrations of
the two known and potent antibiotics ampicillin and gentam-
icin. The outcomes of such tests, which are shown in Table 1,
reveal that while showdomycin is an anti-bacterial it is not
an especially effective one. The anomer, 4, of showdomycin
together with analogues 2 and 3 proved to be inactive as
anti-bacterials.
Anti-Viral Testing
Compounds 1–4 were also each tested for their ability to
inhibit the infectivity of herpes simplex virus type 1 (HSV-1)
inAfrican green monkey kidney (GMKAH1) and human epi-
dermal (HaCaT) cells. Thus, these compounds were added to
the cells together with the virus and incubated for 2–3 days.
Within the concentration range 0.08–50 µM the compounds
exerted no inhibitory effect both in GMK AH1 (Fig. 2) and
HaCaT (data not shown) cells. Thus, the number of viral
plaques that developed in the presence of compounds 1–4
was similar to that observed in control experiments. In addi-
tion, these compounds had no effect on the area of viral
plaques developed in GMK AH1 cells (data not shown). This
suggests they lack any capacity to interfere with cell-to-cell
transmission of the virus.
Cytotoxicity Testing
Each of compounds 1–4 was screened, at eight different
concentrations, against a panel of ten human and murine
cell lines as listed in Table 2. This revealed that while
showdomycin displayed some cytotoxicity against a good
proportion of these cell lines (IC50 range 3.27 to approx.
15 µM), compound 4 along with analogues 2 and 3 were
essentially inactive (IC50 > 20 µM). The only exception to
We also examined compounds 1–4 for their effect
on glycosylation of truncated HSV-1 glycoprotein gC in
insect cells. This gC construct contains four consensus

Two Analogues of Showdomycin
89
MW Control Compound 1 Compound 2 Compound 3 Compound 4
--------- -------------------- -------------------- -------------------- -------------------
120
100
80
60
40
20
0
C
E C1 E1 C5 E5 C1 E1 C5 E5 C1 E1 C5 E5 C1 E1 C5 E5
51
39
Fig. 3. Immunoblot analysis of truncated HSV-1 gC produced in insect
cells in the presence of compounds 1–4 (see Experimental section for
details). Cell-associated (C) or secreted extracellular (E) fractions of gC
produced in the presence of 10 µM (C1, E1) or 50 µM (C5, E5) were
electrophoresed under reducing conditions, blotted, and immunostained
with monoclonal anti-gC antibodies.
1
2
3
4
in the synthesis of analogues of the C-riboside natural prod-
uct showdomycin. Unfortunately, the analogues prepared as
described herein show very little useful biological activity,
perhaps because of their poor penetration of most cell plasma
membranes. As a consequence any further efforts in the area
ofC-nucleosidesynthesisforthepurposesofdevelopingdrug
entities should probably not involve maleimide derivatives
because of the poor therapeutic index such compounds are
likely to display.
0.1
1
10
100
Concentration (␮M)
Fig. 2. Effect of compounds 1–4 on HSV-1 infection of GMK AH1
cells (see Experimental section for details). The results are expressed as
a percentage of the number of viral plaques (p.f.u.) formed in the pres-
ence of compounds 1–4 relative to mock-treated controls. Two separate
experiments were carried out in duplicate for each compound.
Experimental
sequences for asparagine-linked glycans. Showdomycin has
been reported to interfere with the assembly of the core
region of asparagine-linked glycans, i.e. with the forma-
tion of GlcNAc-GlcNAc-P-P-dolichol.[14] Compounds 1–4
did not alter the electrophoretic mobility of secreted gC
bands as compared to control protein (Fig. 3). This sug-
gests they lack any capacity to affect glycosylation of viral
gC. It should be noted that in our experiments the high-
est concentration of showdomycin and analogues used was
50 µM (approx. 11 µg mL−1), i.e. the concentration that
was reported to inhibit formation of GlcNAc-GlcNAc-P-P-
dolichol by approximately 50%.[14] In addition these com-
pounds did not seem to inhibit the secretion of gC into
the culture supernatant (Fig. 3; see presence of extracellu-
lar gC fraction (E) in cells treated with each compound),
a feature frequently observed with known inhibitors of
N-glycosylation such as tunicamycin.
The above-mentioned test results are consistent with vari-
ous earlier observations which have revealed that the anomer
of showdomycin, namely compound 4, showed ‘no signifi-
cant activity when tested against several strains of bacteria,
fungi, yeasts, certain viruses, and a protozoa’.[4b] Similarly,
the carbocyclic analogue of showdomycin, namely that com-
pound in which the ring oxygen has been replaced by a
methylene unit, ‘failed to show any activity against 15 strains
of bacteria and four strains of fungi up to levels as high as
256 mcg/mL. Nine viral strains, including both DNA and
RNA types, were not significantly affected by [compound 4]
in tissue culture’. In a special plaque reduction assay against
a vaccinia virus challenge, slight activity was noted for
compound 4.[4a]
General Procedures
Unless otherwise specified, proton (1H) and carbon (13C) NMR spectra
were recorded on aVarian Gemini 300 orVarian Mercury 300 spectrom-
eter operating at 300 MHz for proton and 75 MHz for carbon nuclei.
Chemical shifts were recorded as δ values in parts per million (ppm).
Spectra were acquired in deuterated chloroform (CDCl3) at 20◦C unless
otherwise stated. For 1H NMR spectra recorded in CDCl3, the peak due
to residual CHCl3 (δH 7.26) was used as the internal reference while the
central peak (δC 77.0) of the CDCl3 triplet was used as the reference for
proton-decoupled 13C NMR spectra. 1H NMR spectroscopic data are
recorded as follows: chemical shift (δ) [relative integral, multiplicity,
coupling constant(s) J (Hz)] where multiplicity is defined as: s, singlet;
d, doublet; t, triplet; q, quartet or quintet; septet; m, multiplet, or com-
binations of the above. Infrared spectra (νmax) were recorded on either
a Perkin–Elmer 1800 Fourier-transform infrared spectrophotometer or
a Perkin–Elmer Spectrum One instrument. Samples were analyzed as
KBr discs (for solids) or as thin films on NaCl plates (for liquids/oils).
Low- and high-resolution mass spectra wererecorded on anAUTOSPEC
spectrometer or a Bruker Apex 4.7T FTICR-MS instrument. Opti-
cal rotations were determined on a Perkin–Elmer 241 polarimeter at
the sodium-D line (598 nm) using spectroscopic-grade CHCl3 (unless
otherwise specified) at 20◦C and at the concentrations (c; g/100 mL)
indicated. Measurements were carried out in a cell with a path length
of 1 dm. Melting points (mp) were recorded on a Reichert hot-stage
apparatus and are uncorrected. Analytical thin-layer chromatography
(TLC) was conducted on glass-backed 0.2 mm thick silica gel 60 F254
plates (Merck) and the chromatograms were visualized under a 254 nm
UV lamp and/or by treatment with an anisaldehyde–sulfuric acid–
ethanol (3/4.5/200 mL) dip or, occasionally, with a phosphomolybdic
acid–ceric sulfate–sulfuric acid–water (37.5 g/7.5 g/37.5 mL/720 mL)
dip, followed by heating. Flash chromatography was conducted accord-
ing to the method of Still and coworkers[15] using silica gel 60 (mesh
size 0.040–0.063 mm) as the stationary phase and the analytical reagent
(AR) grade solvents indicated. Many starting materials and reagents
were available from the Aldrich Chemical Co. or EGA–Chemie and
were used as supplied or, in the case of stable liquids, simply distilled.
Drying agents and other inorganic salts were purchased from AJAX
or BDH Chemicals. Reactions employing air- and/or moisture-sensitive
reagents and intermediates were carried out under an atmosphere of dry,
oxygen-free nitrogen in flame-dried apparatus. Tetrahydrofuran (THF)
and diethyl ether were dried using sodium metal and then distilled, as
required, from sodium benzophenone ketyl. Methanol (MeOH) was dis-
tilled from magnesium methoxide. CH2Cl2 was distilled from calcium
Conclusions
The synthetic studies detailed above serve to further rein-
force the utility of N-TIPS-substituted pyrrole as a synthetic
equivalent for the maleimide C3 anion and its applicability

90
J. Renner et al.
hydride. N,N-Dimethylformamide (DMF) was heated at reflux over cal-
cium hydride for 16 h then distilled and stored over 4 Å molecular sieves.
Organic solutions obtained from workup of reaction mixtures were dried
with anhydrous magnesium sulfate (MgSO4) then filtered and concen-
trated under reduced pressure on a rotary evaporator with the water bath
temperature generally not exceeding 40◦C.
clear, colourless oil, [α]D −4◦ (c 0.6 in CHCl3) (Found: M+•, 553.2704.
C27H43NO9Si requires M+•, 553.2707). νmax/cm−1 2948, 2869, 1754,
1626, 1561, 1464, 1432, 1227, 1091, 1038, 912, 884, 792. δH (300 MHz)
6.73 (1 H, t, J 1.5, Ar–H), 6.68 (1 H, t, J 2.3, Ar–H), 6.28 (1 H, dd, J 1.5
and 2.3, Ar–H), 5.28–5.14 (3 H, complex m), 4.43 (1 H, d, J 9.3), 4.26
(1 H, dd, J 4.5 and 12.3), 4.10 (1 H, m), 3.78 (1 H, m), 2.05 (3 H, s), 2.02
(3 H, s), 1.98 (3 H, s), 1.77 (3 H, s), 1.38 (3 H, septet, J 7.5), 1.04 (18 H,
dd, J 1.2 and 7.5). δC (75 MHz) 170.8, 170.4, 169.5, 169.0, 124.9, 123.2,
121.0, 109.4, 75.6, 75.1, 74.6, 72.1, 68.7, 62.4, 20.7, 20.6(1), 20.5(9),
20.4, 17.7, 11.5. m/z (EI, 70 eV) 553 (M+•, 7%), 408 (8), 374 (13), 360
(51), 318 (41), 173 (40), 149 (23), 86 (64), 55 (34), 43 (100).
Synthetic Studies
1,2-Anhydro-(3,4,6-tri-O-benzyl)-α-D-glucopyranose 6
Thetitlecompoundwaspreparedbyepoxidationofthecommercially
available glucal 5 with DMDO according to the method of Halcomb and
Danishefsky.[9] This highly sensitive product, which displayed spectro-
scopic characteristics in agreement with those reported previously,[9]
was used immediately in the next step of the reaction sequence.
1,5-Anhydro-(1-C-1H-pyrrol-3-yl)-D-glucitol
2,3,4,6-tetraacetate 11
A magnetically stirred solution of compound 10 (89 mg, 0.16 mmol)
in freshly distilled THF (5 mL) was cooled to 0◦C and TBAF (170 µL
of a 1 M solution in THF, 0.17 mmol) was added dropwise using a
syringe. The ensuing mixture was stirred at 0◦C until TLC analysis
indicated compete consumption of the starting material (approx. 1 h).
At this point the solvent was removed under reduced pressure and the
oily residue so obtained redissolved in CH2Cl2 (10 mL). The resulting
solution was washed with water (3 × 10 mL) and brine (1 × 10 mL) then
dried (MgSO4), filtered, and concentrated under reduced pressure. The
ensuing crude sample of pyrrole 11 (61 mg, 96%) was obtained as a
light-yellow oil, [α]D −9◦ (c 1.6 in CHCl3) (Found: M+•, 397.1371.
C18H23NO9 requires M+•, 397.1373). νmax/cm−1 3402, 2943, 2866,
1751, 1434, 1370, 1230, 1101, 1037, 913, 883, 799. δH (300 MHz) 8.58
(1 H, br s, NH), 6.78 (1 H, br s, Ar–H), 6.68 (1 H, d, J 1.8, Ar–H), 6.18
(1 H, m, Ar–H), 5.32–5.15 (3 H, complex m), 4.45 (1 H, d, J 8.7), 4.24
(1 H, dd, J 4.2 and 12.0), 4.10 (1 H, d, J 12.0), 3.78 (1 H, m), 2.04 (3
H, s), 2.02 (3 H, s), 1.99 (3 H, s), 1.84 (3 H, s). δC (75 MHz) 170.8,
1,5-Anhydro-[1-C-(1-triisopropylsilyl)-1H-pyrrol-3-yl]-
(3,4,6-tri-O-benzyl)-D-glucitol 8
A solution of epoxide 6 (104 mg, 0.24 mol) in CH2Cl2 (4 mL) was
cooled to 0◦C (ice/water bath) then treated with N-TIPS-pyrrole (7)[8]
(140 mg, 0.63 mmol) and InCl3 (6 mg, 0.03 mmol). Stirring was contin-
ued at 0◦C for 18 h then at 18◦C for 1 h. The solvent was then removed
underreducedpressureandtheresidualoilsubjectedtoflashchromatog-
raphy (silica, 1 : 4 v/v ethyl acetate–hexane elution). Collection of the
appropriate fractions (RF 0.4) afforded the title C-glycoside 8 (58 mg,
•
37%)asaclear, colourlessoil, [α]D + 21◦ (c1.1inCHCl3)(Found:M+
,
655.3690. C40H53NO5Si requires M+•, 655.3693). δH (300 MHz) 7.45–
7.19 (15 H, complex m, Ar–H), 6.85 (1 H, m, Ar–H), 6.78 (1 H, t, J 2.1,
Ar–H), 6.38 (1 H, m, Ar–H), 4.92 (3 H, m), 4.61 (3 H, m), 4.20 (1 H, m,
H1), 3.84–3.58 (6 H, complex m), 1.80 (1 H, s, OH), 1.44 (3 H, septet, J
7.2, SiC–H), 1.10 (18 H, d, J 7.2, CH3). δC (75 MHz) 138.9, 138.4,
138.3, 128.4, 128.3(4), 128.2(6), 128.0, 127.8(3), 127.7(6), 127.7,
127.5, 127.4, 125.3, 123.3, 122.5, 108.9, 86.2, 79.4, 78.0, 77.2, 75.8,
75.1, 75.0(6), 73.4, 69.2, 17.8(1), 17.7(9), 11.6. m/z (EI, 70 eV) 655
(M+•, 7%), 384 (2), 305 (3), 220 (15), 101 (88), 91 (100).
170.4, 169.5, 169.3, 118.9, 118.4, 116.9, 107.3, 75.6, 75.0, 74.6, 72.1,
•
68.7, 62.4, 20.7, 20.6(1), 20.5(7), 20.5(4). m/z (EI, 70 eV) 397 (M+
,
1%), 277 (10), 218 (12), 204 (73), 162 (67), 109 (13), 96 (44), 94 (16),
80 (16), 43 (100). This material was used, without further purification,
in the next step of the reaction sequence.
1,5-Anhydro-[1-C-(1-triisopropylsilyl)-1H-pyrrol-3-yl]-
D-glucitol 9
3-(2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl)-1H-pyrrole-
2,5-dione 12
A solution of compound 8 (120 mg, 0.18 mmol) in EtOH (5 mL)
containing 10% palladium on charcoal (30 mg) was stirred under H2
(1 atm) at 18◦C for 22 h. The ensuing mixture was filtered through a pad
ofCeliteandthefiltrateconcentratedunderreducedpressure.Theresult-
ing crude sample of tetrol 9 (61 mg, 87%) was obtained as a light-yellow
oil, [α]D + 7◦ (c 1.2 in MeOH) (Found: M+•, 385.2290. C19H35NO5Si
requires M+•, 385.2285). νmax/cm−1 3369, 2945, 2868, 1567, 1463,
1415, 1083, 1017, 883, 790. δH (300 MHz, CD3OD) 6.89 (1 H, m, Ar–
H), 6.77 (1 H, t, J 2.4,Ar–H), 6.35 (1 H, dd, J 1.5 and 2.4,Ar–H), 4.17 (1
H, m), 3.86 (1 H, d, J 12.0), 3.66 (1 H, m), 3.48–3.34 (4 H, complex m),
1.48 (3 H, septet, J 7.5), 1.11 (18 H, d, J 7.5, CH3). δC (75 MHz, CD3OD)
125.4, 125.3, 124.7, 110.6, 81.9, 79.9, 78.1, 75.9, 72.0, 63.2, 18.2, 12.8.
m/z (EI, 70 eV) 385 (M+•, 100%), 266 (47), 252 (69), 236 (28), 222 (23),
208 (27), 173 (19), 110 (31), 73 (34). This material was used, without
further purification, in the next step of the reaction sequence.
A magnetically stirred mixture of pyrrole 11 (61 mg, 0.15 mmol)
and molecular sieves (200 mg, 4 Å, powdered) in dry CH2Cl2 (8 mL)
was treated with PCC (162 mg, 0.75 mmol). After 16 h at 18◦C the reac-
tion mixture was filtered through a pad comprised of a 1 : 1 mixture
of type-60 silica gel and Celite which was then washed thoroughly
with ethyl acetate. The combined filtrates were concentrated under
reduced pressure to give a red oil that was subject to flash chromatog-
raphy (silica, 1 : 1 v/v ethyl acetate–hexane elution). Concentration of
the appropriate fractions (RF 0.4) then afforded the title maleimide 12
(43 mg, 67%) as a white crystalline solid, mp 183–185◦C, [α]D + 13◦
•
(c 0.9 in CHCl3) (Found: M+•, 427.1111. C18H21NO11 requires M+
,
427.1115). νmax/cm−1 3459, 2925, 2854, 1750, 1726, 1642, 1435, 1369,
1229, 1102, 1046. δH (300 MHz) 7.29 (1 H, br s, NH), 6.66 (1 H, s),
5.34 (1 H, t, J 9.5), 5.14 (1 H, t, J 9.5), 5.06 (1 H, t, J 9.5), 4.43 (1 H, d,
J 9.5), 4.26 (1 H, dd, J 4.7 and 12.5), 4.16 (1 H, d, J 12.5), 3.80 (1 H,
m), 2.09 (3 H, s), 2.05 (3 H, s), 2.02 (3 H, s), 1.99 (3 H, s). δC (75 MHz)
170.6, 170.1 (two signals superimposed), 169.5, 169.2, 169.1, 145.1,
130.7, 76.2, 73.3, 71.5, 71.1, 68.0, 61.8, 20.7, 20.6, 20.5 (two signals
superimposed). m/z (EI, 70 eV) 427 (M+•, 4%), 385 (94), 259 (61), 246
(32), 204 (79), 162 (33), 139 (100), 97 (47), 83 (37), 69 (49).
1,5-Anhydro-[1-C-(1-triisopropylsilyl)-1H-pyrrol-3-yl]-D-glucitol
2,3,4,6-tetraacetate 10
A magnetically stirred solution of tetrol 9 (61 mg, 0.16 mmol) in
dry CH2Cl2 (5 mL) was cooled to 0◦C then treated with Et3N (558 µL,
4 mmol),Ac2O (302 µL, 3.2 mmol), and DMAP (2 mg, 0.02 mmol).The
ensuing mixture was allowed to warm to 18◦C, stirred at this tempera-
ture for 16 h, then recooled to 0◦C and quenched with MeOH (1 mL).
The reaction mixture was then concentrated under reduced pressure
and the residue dissolved in CH2Cl2 (5 mL). The resulting solution
was washed with water (2 × 10 mL) and brine (1 × 10 mL) then dried
(MgSO4), filtered, and concentrated under reduced pressure to give a
yellow oil. Subjection of this material to column chromatography (silica,
1 : 3 v/v ethyl acetate–hexane elution) and collection of the appropriate
fractions (RF 0.35) afforded the title compound 10 (65 mg, 73%) as a
3-(α-D-Glucopyranosyl)-1H-pyrrole-2,5-dione 2
Compound 12 (30 mg, 0.07 mmol) was added to a solution of anhy-
drous HCl in MeOH [prepared by addition of acetyl chloride (142 µL) to
MeOH (10 mL) at 0◦C] at 0◦C.The resulting mixture was stirred at 18◦C
for 48 h and then the solvent removed under reduced pressure. The oily
residue thus obtained was subject to preparative TLC (1 mm thick silica
gel plate, 10 : 2 : 1 v/v/v ethyl acetate–MeOH–H2O elution). Extraction
of the lower chromophoric band gave a clear, colourless oil which was

Two Analogues of Showdomycin
91
subjected to flash chromatography (silica, 10 : 2 : 1 v/v/v ethyl acetate–
MeOH–H2O elution). Concentration of the appropriate fractions (RF
0.4) then afforded the title maleimide 2 (8 mg, 44%) as a clear, colour-
less oil, [α]D + 9◦ (c 0.8 in MeOH) (Found: M−, 259.0685. C10H13NO7
requires M−, 259.0692). νmax/cm−1 3325, 2914, 2846, 1717, 1588,
1408, 1354, 1087. δH (300 MHz, CD3OD/CD3CN) 6.68 (1 H, d, J 0.9),
4.18 (1 H, dd, J 0.9 and 9.2), 3.84 (1 H, dd, J 2.1 and 8.7), 3.68 (1 H,
m), 3.49–3.20 (4 H, complex m). δC (75 MHz, CD3OD/CD3CN) 174.9,
173.2, 148.5, 131.7, 82.5, 79.4, 75.3, 74.5, 71.5, 62.8. m/z (negative-ion
ESI) 259 (M−).
standunderH2 (1 atm)at18◦Cfor16 h, andthenfilteredthroughapadof
Celite.The solids thus retained were washed with EtOH (approx. 20 mL)
and the combined filtrates concentrated under reduced pressure to give
a grey solid. This material was dissolved in CH2Cl2 (10 mL) and the
resultingsolutionwashedwithwater(2 × 10 mL)andbrine(1 × 10 mL).
Subsequent drying of the separated organic phase (MgSO4) and evapo-
ration of the solvent afforded cyclohexane 17 (94 mg, 81%) as a clear,
colourless oil, [α]D −89◦ (c 1.1 in CHCl3) (Found: M+•, 435.2803.
C24H41NO4Si requires M+•, 435.2805). νmax/cm−1 2944, 2869, 1740,
1479, 1464, 1368, 1241, 1154, 1094, 1048, 884, 865, 779, 691, 661. δH
(300 MHz) 6.64 (1 H, t, J 2.7, Ar–H), 6.51 (1 H, t, J 1.5, Ar–H), 6.13
(1 H, dd, J 1.5 and 2.7, Ar–H), 5.11 (1 H, dd, J 8.1 and 11.4), 4.37 (1
H, m), 4.00 (1 H, dd, J 5.1 and 8.1), 2.51 (1 H, dt, J 3.3 and 11.6), 2.21
(1 H, broadened d, J 12.4), 1.83 (3 H, s, CH3), 1.82 (3 H, m), 1.60 (3
H, s, CH3), 1.39 (3 H, m, CHMe2), 1.38 (3 H, s, CH3), 1.07 (12 H,
s, 4 CH3), 1.05 (6 H, s, 2 CH3). δC (75 MHz) 169.7 (C=O), 125.4,
123.8, 120.8, 109.7, 108.9, 79.4 (C–O), 77.3 (C–O), 74.7 (C–O), 39.0,
27.9, 27.8 (CH2), 26.5, 26.4 (CH2), 21.0, 17.8(0), 17.7(8), 11.6. m/z
(EI, 70 eV) 435 (M+•, 14%), 375 (76), 289 (100), 247 (43), 173 (72),
105 (36).
(3aS,4R,5R,7aS)-7-Bromo-3a,4,5,7a-tetrahydro-5-
[1-(triisopropylsilyl)-1H-pyrrol-3-yl]-2,2-dimethyl-
benzo[d][1,3]dioxol-4-ol 15
N-TIPS-pyrrole (7)[8] (230 mg, 2.5 mol equiv.) was added in one
portion to a mixture of epoxide 14[12] (102 mg, 0.41 mmol) and InCl3
(9 mg, 0.04 mmol, 10 mol%) in CH2Cl2 (15 mL). The ensuing mixture
was stirred at 18◦C for 5 days then filtered through a pad of type-60
silica gel. The filtrate was concentrated under reduced pressure to give a
yellow oil which was subjected to flash chromatography (silica, 1 : 4 v/v
ethyl acetate–hexane elution). Collection of the appropriate fractions
(RF 0.2) afforded compound 15 (102 mg, 54%) as a clear, colourless oil
(Found: M+•, 471.1628. C22H36 81BrNO3Si requires M+•, 471.1627).
νmax/cm−1 3471, 2942, 2862, 1476, 1461, 1378, 1277, 1215, 1161,
1096, 1071, 1013, 879, 869, 789, 655. δH (300 MHz) 6.77 (1 H, t, J 2.1,
Ar–H), 6.66 (1 H, t, J 1.5, Ar–H), 6.26 (1 H, d, J 2.1, H6), 6.19 (1 H, dd,
J 1.5 and 2.1, Ar–H), 4.77 (1 H, dd, J 0.9 and 6.4, H7a), 4.21 (1 H, dd, J
6.7 and 9.1), 3.54 (1 H, t, J 9.4), 3.26 (1 H, dt, J 1.5 and 9.7), 2.14 (1 H, d,
J 1.5, OH), 1.57 (3 H, s, CH3), 1.45 (3 H, s, CH3), 1.43 (3 H, m, SiCH),
1.08 (18 H, d, J 7.3, CH3). δC (75 MHz) 136.9, 125.7, 123.2, 122.4,
117.5, 110.3 [C(CH3)2], 109.7, 79.2 (C–O), 77.8 (C–O), 73.9 (C–O),
43.0, 28.4 (CH3), 26.0 (CH3), 18.0 (CH3), 11.9 (SiCH). m/z (EI, 70 eV)
471 and 469 (M+•, 17 and 16%), 269 (87), 267 (67), 223 (59), 180
(100), 128 (52), 110 (48), 75 (39). This compound discolours rapidly
and was used immediately in the next step of the reaction sequence.
(3aR,4R,5R,7aR)-Hexahydro-2,2-dimethyl-5-(1H-pyrrol-
3-yl)benzo[d][1,3]dioxol-4-yl acetate 18
N-TIPS-protected pyrrole 17 (94 mg, 0.22 mmol) was dissolved in
freshly distilled THF (10 mL) and the resulting solution cooled to 0◦C
(ice bath). TBAF (216 µL of a 1.0 M solution in THF, 0.22 mmol) was
then added dropwise. After the addition was complete stirring was
continued at 0◦C for 1 h then the reaction mixture was diluted with
CH2Cl2 (10 mL) and washed with water (1 × 10 mL). The separated
aqueous phase was extracted with CH2Cl2 (3 × 10 mL) and the com-
bined organic phases were washed with brine (1 × 20 mL) before being
dried (MgSO4), filtered, and concentrated under reduced pressure. The
resulting clear, colourless oil was subject to flash chromatography (sil-
ica, 1 : 1 v/v ethyl acetate–hexane elution). Collection of the appropriate
fractions (RF 0.6) then afforded compound 18 (48 mg, 80%) as a clear,
colourless oil, [α]D −40◦ (c 1.3 in CHCl3) (Found: M+•, 279.1483.
C15H21NO4 requires M+•, 279.1471). νmax/cm−1 3392, 2984, 2936,
2874, 1732, 1454, 1434, 1378, 1244, 1220, 1153, 1099, 1043, 865, 793,
665. δH (300 MHz) 8.22 (1 H, br s, NH), 6.65 (1 H, q, J 2.4, Ar–H),
6.55 (1 H, q, J 1.8, Ar–H), 6.05 (1 H, q, J 1.8, Ar–H), 5.06 (1 H, dd, J
8.1 and 11.4), 4.38 (1 H, m), 4.02 (1 H, dd, J 4.8 and 8.1), 2.53 (1 H,
dt, J 2.7 and 11.4), 2.23 (1 H, br d, J 11.4), 1.88 (3 H, s, CH3), 1.85
(3 H, m), 1.58 (3 H, s, CH3), 1.38 (3 H, s, CH3). δC (75 MHz) 170.1
(C=O), 123.6, 117.4, 114.7, 109.0, 107.8, 79.2 (C–O), 77.7 (C–O), 74.6
(C–O), 38.9, 27.9, 27.2 (CH2), 26.5, 26.4 (CH2), 21.1. m/z (EI, 70 eV)
279 (M+•, 4%), 264 (4), 219 (41), 161 (26), 144 (54), 133 (100), 132
(37), 118 (16), 106 (21), 93 (24), 80 (23), 67 (16).
(3aS,4R,5R,7aS)-7-Bromo-3a,4,5,7a-tetrahydro-5-
[(1-(triisopropylsilyl)-1H-pyrrol-3-yl)]-2,2-dimethyl-
benzo[d][1,3]dioxol-4-yl acetate 16
Pyrrole 15 (60 mg, 0.13 mmol) was dissolved in dry CH2Cl2 (3 mL)
and Et3N (0.45 mL, 3.2 mmol) was added under a nitrogen atmosphere.
The ensuing mixture was cooled to 0◦C (ice/water bath) and Ac2O
(241 µL, 2.56 mmol) was added dropwise with a syringe.After the addi-
tion was complete, the reaction mixture was allowed to warm to 18◦C,
stirring was continued at this temperature for 48 h, then it was recooled
to 0◦C and MeOH (0.6 mL) added. The resulting mixture was washed
with water (2 × 5 mL) and brine (1 × 5 mL) then dried (MgSO4), fil-
tered, and concentrated under reduced pressure to afford a light-brown
oil.This material was subjected to flash chromatography (silica, 1 : 4 v/v
ethyl acetate–hexane elution). Collection of the appropriate fractions
(RF 0.4) afforded acetate 16 (41 mg, 63%) as a clear, colourless oil,
[α]D + 2◦ (c 1.2 in CHCl3). νmax/cm−1 2947, 2869, 2603, 2497, 1747
(C=O), 1640, 1563, 1480, 1464, 1373, 1227, 1167, 1078, 1017, 883,
870, 792, 692, 659, 580. δH (300 MHz) 6.66 (1 H, t, J 2.4, Ar–H), 6.56
(1 H, t, J 1.5, Ar–H), 6.30 (1 H, d, J 2.1, H6), 6.14 (1 H, dd, J 1.5 and
2.4, Ar–H), 5.16 (1 H, t, J 9.6), 4.73 (1 H, dd, J 1.5 and 6.0), 4.24 (1
H, dd, J 6.0 and 9.6), 3.43 (1 H, dt, J 1.5 and 9.6), 1.90 (3 H, s, OAc),
1.55 (3 H, s, CH3), 1.41 (3 H, s, CH3), 1.39 (3 H, m, SiCH), 1.05 (18 H,
d, J 7.5, CH3). δC (75 MHz) 169.4 (C=O), 136.9, 124.5, 122.6, 121.6,
116.6, 110.1, 110.0, 77.7 (C–O), 77.2 (C–O), 73.1 (C–O), 40.8 (C5),
27.8 (CH3), 26.3 (CH3), 20.9 (CH3), 17.7(4) (CH3), 17.7(1) (CH3), 11.6
(SiC). m/z (ESI) 534 and 536 [(M + Na)+, 95 and 100%].
(3aR,4R,5R,7aR)-Hexahydro-5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-
3-yl)-2,2-dimethylbenzo[d][1,3]dioxol-4-yl acetate 19
A magnetically stirred solution of pyrrole 18 (47 mg, 0.17 mmol)
in CH2Cl2 (12 mL) was treated with molecular sieves (150 mg, 4 Å,
powdered) then PCC (181 mg, 0.84 mmol, 4.9 mol equiv.). The ensuing
mixture was stirred at 18◦C for 16 h then diluted with CH2Cl2 (10 mL),
and filtered through a pad of type-60 silica gel. The solids thus retained
were washed with CH2Cl2 (10 mL) and ethyl acetate (10 mL) and the
combined filtrates concentrated under reduced pressure to give a brown
solid. This material was subjected to flash chromatography (silica,
1 : 1 v/v ethyl acetate–hexane elution) and collection of the appropri-
ate fractions (RF 0.35) afforded the title compound 19 (43 mg, 83%)
•
as a clear, colourless oil, [α]D −23◦ (c 0.35 in CHCl3) (Found: M+
,
309.1218. C15H19NO6 requires M+•, 309.1212). νmax/cm−1 3271,
2986, 2933, 1722, 1631, 1455, 1436, 1372, 1350, 1242, 1154, 1098,
1048, 1004, 936, 865, 788, 736, 675. δH (300 MHz) 8.04 (1 H, br s,
NH), 6.37 (1 H, s), 5.20 (1 H, dd, J 7.5 and 11.4), 4.39 (1 H, m), 4.03
(1 H, dd, J 4.8 and 7.5), 2.68 (1 H, t, J 9.5), 2.22 (1 H, d, J 7.5), 2.02
(3 H, s, CH3), 1.81 (3 H, m), 1.57 (3 H, s, CH3), 1.38 (3 H, s, CH3).
δC (75 MHz) 170.8 (C=O), 170.4 (C=O), 170.1 (C=O), 149.4, 127.5,
109.3, 78.2(C–O), 74.4(C–O), 73.9(C–O), 36.9, 27.7, 26.2, 25.6(CH2),
(3aR,4R,5R,7aR)-Hexahydro-5-[1-(triisopropylsilyl)-1H-pyrrol-
3-yl]-2,2-dimethylbenzo[d][1,3]dioxol-4-yl acetate 17
Compound 16 (136 mg, 0.27 mmol) was dissolved in EtOH (8 mL)
containing Et3N (185 µL, 1.32 mmol, 4.9 mol equiv.). The resulting
solution was treated with 10% palladium on charcoal (45 mg), allowed to

92
J. Renner et al.
25.2 (CH2), 20.8. m/z (EI, 70 eV) 309 (M+•, 1%), 294 (95), 234 (12),
192 (93), 174 (100), 156 (23), 59 (26).
lines. The target cells were murine IL-2 dependent cytotoxic T-cell line
CTLL-2, human GM-CSF dependent erythroleukaemic cell line TF-1,
human heptatocyte carcinoma cell line HepG2, human embryo kidney
cell line HEK293, rat prostate adenocarcinoma cell line MAT-LyLu,
human chronic myelogenous leukaemia cell line K562, human prostate
carcinoma cell line DU145, human prostate adenocarcinoma PC3 cell
line, and murine myeloid transformed Baf3-Tel-Jak2 and Baf3-Tel-Jak3
cell lines. Stock solutions of the test compounds were prepared at 20 mM
in neat DMSO. Dose response studies of the compounds were carried
out from 0.16 to 20 µM in 96-well T-C (tissue culture) plates. The cells
were incubated for 72 h at 37◦C in a 5% CO2 incubator. Inhibition of
cell proliferation by the above compounds was determined by measur-
ing conversion of Alamar-Blue fluorescent dye at excitation/emission
544/590 nm with a BMG FluoStar plate reader.
3-[(1R,2R,3R,4R)-2,3,4-Trihydroxycyclohexyl]-1H-pyrrole-
2,5-dione 3
Compound 19 (13 mg, 0.042 mmol) was dissolved in a methanolic
solution of HCl [prepared by adding acetyl chloride (25 µL) dropwise
to MeOH (1.5 mL) at 0◦C] at 0◦C. Stirring was continued at this temper-
ature for 20 min and then at 18◦C for 48 h. After this time TLC analysis
(50 : 2 : 1 v/v/v ethyl acetate–MeOH–H2O elution) showed complete
consumption of the starting material. Consequently, the reaction sol-
vent was removed under reduced pressure and the white solid thus
obtained subject to flash chromatography (silica, 50 : 2 : 1 v/v/v ethyl
acetate–MeOH–H2O elution). Collection of the appropriate fractions
(RF 0.2) then afforded compound 3 (9 mg, 91%) as a white crystalline
solid, mp 218◦C, [α]D +61◦ (c 0.3 in MeOH) (Found: M+•, 227.0800.
C10H13NO5 requires M+•, 227.0794). νmax/cm−1 3345, 2922, 1708,
1349, 1061. δH (300 MHz, D2O/CD3CN) 6.46 (1 H, s), 4.10 (1 H, br s),
3.85 (1 H, t, J 9.6), 3.47 (1 H, dd, J 2.7 and 9.6), 2.61 (1 H, t, J 9.6), 1.84
(1 H, d, J 10.5), 1.74–1.58 (3 H, complex m). δC (75 MHz, D2O/CD3CN)
174.8 (C=O), 174.7 (C=O), 152.0, 128.9, 76.3 (C–O), 72.4 (C–O), 70.5
(C–O), 41.1, 30.3, 25.3. m/z (EI, 70 eV) 227 (M+•, 8%), 209 (28), 191
(36), 167 (85), 140 (54), 124 (100), 111 (37), 81 (45), 70 (66), 68 (55).
Anti-Viral Testing
MonolayerculturesofAfricangreenmonkeykidney(GMKAH1)[19]
cells and human epidermal keratinocytes (HaCaT), cultivated in six-well
cluster plates, were used. The latter cell line was kindly provided by
Dr N. E. Fusenig (Heidelberg, Germany). The viral strain used was
herpes simplex virus type 1 (HSV-1) KOS321 strain.[20] For anti-viral
testing, 1 mL portions of serial fivefold dilutions of compounds 1–4 in
Eagle’s minimum essential medium (EMEM) and approximately 100–
200 plaque-forming units (p.f.u.) of the virus were simultaneously added
to confluent monolayers of GMK AH1 or HaCaT cells. Following the
virus adsorption period of 2 h at 37◦C, 1 mL portions of EMEM supple-
mented with the same concentrations of compounds 1–4 as well as with
4% fetal calf serum, 1% pooled human γ-globulin (Aventis Behring,
Marburg), and antibiotics were added. Following further incubation of
thecellsfor2–3daysat37◦C, theviralplaqueswerestainedandcounted.
The area of viral plaques was measured as described previously.[21]
The effect of compounds 1–4 on glycosylation of viral protein was
tested as follows. The compounds at 10 and 50 µM in SF900II insect
cell medium and the baculovirus construct expressing the truncated
form of HSV-1 glycoprotein C (gC) were simultaneously added to
insect sf9 cells. Following incubation of the cells for 2 days at 37◦C,
both culture medium and the infected cells were harvested and sub-
jected to immunoblot analysis by using the monoclonal anti-gC antibody
designated C2H12.
Crystallographic Studies
Crystal data for 3: C10H13NO5, M 227.22, T 200 K, orthorhombic,
space group P212121, Z 4, a 4.9048(1), b 9.3440(3), c 21.8641(5) Å,
V 1002.04(4) Å3, Dx 1.506 Mg m−3, 1378 unique data (2θmax 55.01◦),
1132 with I > 3.00σ(I), R 0.0261, Rw 0.0313, S 1.1305.
Images were measured on a Nonius Kappa CCD diffractometer
(MoKα, graphite monochrometer) and data extracted using the DENZO
package.[16] Structure solution was by direct methods (SIR97)[17] and
refinement was by full matrix least-squares on F using the CRYSTALS
program package.[18] Atomic coordinates, bond lengths and angles,
and displacement parameters have been deposited at the Cambridge
Crystallographic Data Centre (CCDC reference number 252069).
These data can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, by emailing data_request@ccdc.cam.ac.uk, or by con-
tacting the Cambridge Crystallographic Data Centre, 12, Union Road,
Cambridge CB2 1EZ, UK; fax +44 1223 336033.
Acknowledgments
Biological Studies
This work was generously supported by Progen Industries Ltd
and the Institute of Advanced Studies (Australian National
University). We thank the Alexander von Humboldt Foun-
dation for providing a Feodor Lynen Fellowship to J.R.
Dr Gregg Whited (Genencor International Inc., Palo Alto,
CA) is gratefully acknowledged for providing us with gen-
erous quantities of cis-1,2-dihydrocatechol 13. Dr Natasha
Hungerford is warmly thanked for preparing samples of
showdomycin (1) and its anomer (4). We are also grateful
to Dr Alison Edwards for carrying out a single-crystal X-ray
analysis of compound 12.
Anti-Bacterial Testing
Showdomycin (1) and the three analogues 2, 3, and 4 were tested for
anti-bacterial activity using a disk diffusion method. The target organ-
isms were Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
and Pseudomonas aeruginosa. The test compounds were impregnated
into the disks by pipetting 20 µL of one of three concentrations of the
test compound in MeOH onto the disk. The three concentrations gave
10, 33, and 100 µg of test compound per disk. After the MeOH was
allowed to evaporate, three disks of each concentration were placed on
a nutrient agar plate (Mueller–Hinton, 90 mm diameter) pre-swabbed
with one of the four bacteria. The cell suspensions used to swab the
plates were prepared by diluting overnight cultures to approximately
108 colony forming units mL−1 in 0.1% peptone. Controls were set up
in a similar design to the tested compounds with disks containing differ-
ent concentrations of two known antibiotics ampicillin and gentamicin.
The plates were then sealed, inverted, and incubated at 37◦C for 18 h.
After this time the plates were examined and the inhibition of bacterial
growth around the disks was observed. The results for compounds 1–4
are presented in Table 1. Not presented are the data for the controls
ampicillin and gentamicin, which were consistent with the antibacterial
profiles of these two compounds.
References
[1] (a) C. Lamberth, S. Blarer, Synlett 1994, 489, and references cited
therein. doi:10.1055/S-1994-22899
(b) S. Hanessian, R. Machaalani, Tetrahedron Lett. 2003,
44, 8321, and references cited therein. doi:10.1016/J.TETLET.
2003.09.103
[2] (a) H. Nishimura, M. Mayama, Y. Komatsu, H. Kato,
N. Shimaoka, Y. Tanaka, J. Antibiot. Ser. A 1964, 17, 148.
(b) Y. Nakagawa, H. Kano, Y. Tsukuda, H. Koyama, Tetrahedron
Lett. 1967, 4105. doi:10.1016/S0040-4039(01)89700-8
(c) K. R. Darnall, L. B. Townsend, R. K. Robins, Proc. NatlAcad.
Sci. USA 1967, 57, 548.
Cytoxicity Testing
Showdomycin (1) and the three analogues 2, 3, and 4 were tested
for their anti-proliferative activities against a panel of mammalian cell

Two Analogues of Showdomycin
93
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