Synthesis and biological evaluation of 7-deoxy analogues of the human rhinovirus 3c protease inhibitor thysanone

Article abstract of DOI:10.1002/ejoc.201301515

The synthesis of (±)-7-deoxythysanone and three analogues has been developed. The key oxa-Pictet-Spengler reaction enabled the synthesis of the naphthopyran precursor, which could be readily converted to 7-deoxythysanone and three related analogues. The biological activity of these compounds was also evaluated against HRV 3C protease, the results of which suggest that inhibition of the enzyme requires the presence of the 7-oxa functionality. Several 7-deoxy analogues of the 3C protease inhibitor thysanone have been synthesised by using an oxa-Pictet-Spengler reaction as the key step. The biological activity of these compounds was also evaluated against human rhinovirus (HRV) 3C protease, the results of which suggest that inhibition of the enzyme requires the presence of the 7-oxa functionality. Copyright

Full text of DOI:10.1002/ejoc.201301515

FULL PAPER
DOI: 10.1002/ejoc.201301515
Synthesis and Biological Evaluation of 7-Deoxy Analogues of the Human
Rhinovirus 3C Protease Inhibitor Thysanone
Katrin Schünemann,[a,b] Daniel P. Furkert, Stephen Connelly, John D. Fraser,
[a]
[c]
[b]
[a]
[a]
Jonathan Sperry, and Margaret A. Brimble*
Keywords: Viruses / Oxa-Pictet–Spengler reaction / Quinones / Biological activity / Inhibitors
The synthesis of (Ϯ)-7-deoxythysanone and three analogues
has been developed. The key oxa-Pictet–Spengler reaction
enabled the synthesis of the naphthopyran precursor, which
could be readily converted to 7-deoxythysanone and three
related analogues. The biological activity of these com-
pounds was also evaluated against HRV 3C protease, the re-
sults of which suggest that inhibition of the enzyme requires
the presence of the 7-oxa functionality.
Introduction
which inhibits HRV 3C protease with an IC50 value of
[
7]
13 μg/mL.
The human rhinovirus (HRV) is the major cause for the
common cold in the western world and yet, only symptom-
atic treatment is available to medicate rhinovirus infec-
[
1]
tions. It is estimated that the total economic impact of
cold-related work loss exceeded $40 billion in 2003 in the
United States alone.[ Therefore, the discovery of an effec-
tive therapeutic agent to cure HRV infections continues to
2]
[
3]
be of major interest to the pharmaceutical industry.
Rhinoviruses are non-enveloped, single-stranded posi-
tive-sense RNA viruses with an icosahedral capsid. Upon
replication of the viral RNA genome by virally encoded
RNA-dependent RNA polymerase (RDRP) through a
Figure 1. (–)-Thysanone (1) and (Ϯ)-7-deoxythysanone (2).
[
4]
Currently, two total syntheses and one formal synthesis
[
8]
of the natural product 1 have been published. We have an
ongoing interest in the synthesis of 1 and its analogues.
Over the past decade, we have published the total synthesis
of 1 as well as the syntheses of a series of analogues.[
In this current study, we report the synthesis of 7-deoxy-
thysanone (2) and analogues to identify the role of the 7-
oxa function in the mechanism of inhibition of HRV 3C
protease by 1.
double-stranded RNA intermediate, translation of the
8e,8f,9]
RNA is undertaken by host cell ribosomes.[5] However,
translation is not initiated by a (5Ј)G-cap as usual, but
rather is initiated by one single internal ribosome entry site
(IRES) and, thus, one single polyprotein precursor is pro-
duced. This polyprotein precursor is self-processed by the
encoded viral proteases 2A and 3C to generate functional
proteins and enzymes.[ This renders HRV 3C protease an
interesting target in the quest to discover an effective thera-
peutic agent for the treatment of rhinovirus infections.
During a screening of fermentation broths of the fungus
Thysanophora penicilloides for lead structures for HRV 3C
protease inhibition, chemists at Merck isolated the pyran-
onaphthoquinone antibiotic (–)-thysanone (1, Figure 1),
6]
Results and Discussion
We planned to synthesize 2 by using a key oxa-Pictet–
[10]
Spengler reaction of naphthalene 3 with dimethoxymeth-
ane (4) to provide the complete carbon framework of the
target compound 2 (Scheme 1). This approach comple-
[
[
[
a] School of Chemical Sciences, The University of Auckland,
3 Symonds St., Auckland, New Zealand
2
E-mail: m.brimble@auckland.ac.nz
b] Department of Molecular Medicine and Pathology,
The University of Auckland,
85 Park Road, Auckland, New Zealand
c] Department of Molecular Biology, The Scripps Research
Institute, BCC 265,
1
0550 Nr. Torrey Pines Rd., La Jolla, CA 92037, USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301515.
Scheme 1. Retrosynthetic analysis of (Ϯ)-7-deoxythysanone (2).
122
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 122–128

Synthesis and Biological Evaluation of Analogues of Thysanone
ments a related oxa-Pictet–Spengler strategy employed dur- ing results suggested that further electronic activation of the
ing a formal synthesis of thysanone.[
8c,8d]
C-3 position of the homobenzylic alcohol is required to fa-
With this strategy in mind, we decided to start with an cilitate electrophilic substitution at C-3 by the intermediate
investigation of the key oxa-Pictet–Spengler reaction by oxonium ion.
using the easily accessible homobenzylic alcohols 9, 10 and
13 with dimethoxymethane (Scheme 2). The homobenzylic
alcohols 9 and 10 were synthesized from the common inter-
mediate naphthol 6, which is itself readily available from 1-
[
11]
naphthol (5) in two steps by allylation
followed by
Claisen rearrangement at 180 °C.[ Naphthol 6 was pro-
12]
tected as either tert-butyldimethylsilyl (TBS) ether 7 by
using sodium hydride and TBSCl in 99% yield[ or as
13]
methyl ether 8 by using dimethyl sulfate and potassium car-
bonate in 97% yield.[ The Wacker oxidation of both 7
14]
and 8 followed by reduction gave homobenzylic alcohols 9
and 10. Homobenzylic alcohol 13 was synthesised in three Scheme 3. Failed oxa-Pictet–Spengler reactions.
steps from commercially available naphthoquinone (11),
Despite the failure of these model studies, we next pur-
sued the synthesis of 2 from homobenzylic alcohol 3 start-
which was converted to allylnaphthalene 12 by slight modi-
fication to the conditions described by Ray et al.[ Allylin-
dium iodide, prepared in situ from allyl bromide, indium
15]
[
17]
ing from 5-methoxy-1-naphthol (20; Scheme 4). O-Allyl-
ation of 20 by using potassium carbonate, allyl bromide and
and sodium iodide, was treated with 11 to afford the corre-
[
18]
tetrabutylammonium iodide (TBAI) followed by Claisen
sponding hydroquinone.[
9b,16]
A one-pot methylation with
rearrangement at 180 °C afforded naphthol 21 in 89% yield
dimethyl sulfate and potassium hydroxide afforded naphth-
alene 12 in 80% over two steps. The Wacker oxidation of
[
19]
over two steps (Scheme 4).
To introduce the C-4 oxa
functionality, naphthol 21 was oxidized to naphthoquinone
12 followed by reduction afforded homobenzylic alcohol 13
2
2 in 55% yield by using freshly prepared salcomine and
in 76% yield over two steps.
[
20]
oxygen.
The reductive methylation of 22 then afforded
homobenzylic alcohol precursor 23 in 67% yield. Wacker
oxidation followed by sodium borohydride reduction then
gave homobenzylic alcohol 3 in 86% yield over two steps.[
21]
Scheme 2. Reagents and conditions: (i) allyl bromide, TBAI,
K
9
2
CO
6%; (iii) NaH, TBSCl, THF, 99%; (iv) Me
reflux, 97%; (v) PdCl , CuCl, O , R = TBS: 71%, R = Me: 76%;
vi) NaBH , MeOH, 9: quantitative, 10: quantitative; (vii) a: In,
allyl bromide, NaI, THF; b: Me SO , KOH, THF/H O, 80 %;
viii) PdCl , CuCl, O , 76%; (ix) NaBH , MeOH, quantitative.
3
, acetone, reflux, quantitative; (ii) neat, 180 °C, 20 min,
2
SO , K CO , acetone,
4
2
3
2
2
Scheme 4. Reagents and conditions: (i) allyl bromide, TBAI,
(
4
K
2
CO
mine, O
b: KOH, Me
vi) NaBH , MeOH, 90%.
3
, acetone, reflux, 99%; (ii) neat, 180 °C, 3 h, 90%; (iii) salco-
, MeCN, 55%; (iv) a: Na , TBAI, THF, H O, 1 h;
SO , 67%, (v) PdCl , CuCl, O , DMF/H O, 96 %;
2
4
2
2
2
S
2
O
4
2
(
2
2
4
2
4
2
2
2
(
4
With homobenzylic alcohols 9, 10 and 13 in hand, we
next investigated the oxa-Pictet–Spengler reaction with dif-
Gratifyingly, the oxa-Pictet–Spengler reaction of homo-
ferent solvents, acids and reaction temperatures. Disap- benzylic alcohol 3 with dimethoxymethane (DMM) and
pointingly, although we attempted a plethora of reaction boron trifluoride diethyl etherate proceeded smoothly and
conditions to effect the oxa-Pictet–Spengler reaction, none delivered naphthopyran 24 in 89% yield (Scheme 5). The
of the desired product was observed (Scheme 3). The de- oxidative demethylation of 24 by using silver(II) oxide af-
composition of starting materials 9, 10 and 13 was observed forded the first 7-deoxythysanone analogue pyrano-
with various Brønsted and Lewis acids (boron trifluoride naphthoquinone (Ϯ)-25 in an excellent 97% yield. The
diethyl etherate, TBS trifluoromethanesulfonate and para- stereoselective oxidation at C-1 according to the established
[
8a,8b,8e,8f]
toluenesulfonic acid). Furthermore, only the methoxy procedure
afforded a second analogue, pyrano-
[
8a]
methyl ether (MOM) protected homobenzylic alcohols were naphthoquinone (Ϯ)-26, in 56% yield. The methyl ether
isolated when Amberlyst-15® was used. These disappoint- of (Ϯ)-25 was also cleaved by using boron trichloride to
Eur. J. Org. Chem. 2014, 122–128
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
123

M. A. Brimble et al.
FULL PAPER
afford a third analogue, pyranonaphthoquinone (Ϯ)-27, in namely, (Ϯ)-27 and (Ϯ)-2, exhibit greater cytotoxicity than
excellent yield. The stereoselective oxidation of (Ϯ)-27 gave the analogues (Ϯ)-25 and (Ϯ)-26 for which the C-9 hydroxy
(Ϯ)-7-deoxythysanone (2) in 84% yield.
group is protected as a methyl ether.
Conclusions
We have achieved an efficient synthesis of (Ϯ)-7-deoxy-
thysanone (2) and three analogues (Ϯ)-25, (Ϯ)-26 and (Ϯ)-
27 by the oxa-Pictet–Spengler reaction of homobenzylic
alcohol 3 with dimethoxymethane 4 to afford pyrano-
naphthalene 24. Subsequent screening of (Ϯ)-7-deoxythys-
anone (2) and analogues (Ϯ)-25, (Ϯ)-26 and (Ϯ)-27 estab-
lished that all 7-deoxyanalogues of thysanone (1) do not
inhibit HRV 3C protease; this suggests that the C-7 oxa
function is an important structural feature, which possibly
anchors 1 to HRV 3C protease by hydrogen bonding. The
structure activity results reported herein are significant for
the future design of HRV 3C protease inhibitors based on
the thysanone (1) scaffold.
Experimental Section
General Details: Unless otherwise stated, all nonaqueous reactions
and distillations were performed under a nitrogen or argon atmo-
sphere in oven- or flame-dried glassware. Tetrahydrofuran (THF)
was freshly distilled from sodium/benzophenone. Dichloromethane
Scheme 5. Reagents and conditions: i) DMM, BF
9%; (ii) AgO, HNO , THF/H O, 97%; (iii) a: Br
b: H O/THF, 56% over two steps; (iv) BCl , CH Cl
O/THF, 84% over two
3
·OEt
2
2
, 0 °C Ǟ room
2
, CH
2
Cl
2
,
8
3
2
, (BzO)
2
, CCl
4
;
2
3
2
temp., 98%; (v) a: Br
2 2 4 2
, (BzO) , CCl ; b: H
2 2
(CH Cl ) and toluene were freshly distilled from calcium hydride.
steps.
Tetramethylethylenediamine (TMEDA) was distilled from calcium
hydride and stored under a nitrogen atmosphere over magnesium
sulfate. Reactions performed at low temperature were either cooled
in an acetone–dry-ice bath for temperatures below 0 °C or a water-
ice bath for 0 °C. Flash chromatography was performed with
All four 7-deoxyanalogues of thysanone (1), namely, (Ϯ)-
5, (Ϯ)-26, (Ϯ)-27 and (Ϯ)-7-deoxythysanone (2) were sub-
2
jected to HRV 3C protease screening as well as to cyto-
toxicity assays (Table 1). For HRV 3C protease screening,
0.063–0.1 mm silica gel (Davisil R LC60A 40–63 Micron) with the
our previously reported assay that uses a solid-phase-based indicated solvent. Preparatory TLC was performed on 500 μm Un-
fluorescent substrate was used.[ The cytotoxicity (LD ) iplateTM (Analtech) silica gel (20ϫ20 cm) thin layer chromatog-
22]
50
was detected by using a cell-proliferation assay. The human raphy plates. Infrared spectra were recorded with a Perkin–Elmer
lung adenocarcinoma epithelia cell line NCI-H441 was
grown in the presence of the synthesised inhibitors (Ϯ)-25,
Ϯ)-26, (Ϯ)-27 and (Ϯ)-2 and treated with H-thymidine for
detection of cell proliferation. Unfortunately, all analogues
lacked activity against HRV 3C protease, which suggests
that the inhibition of HRV 3-C protease by thysanone (1) is
very reliant on the 7-oxa functionality. Interestingly, both
R Spectrum 1000 Fourier transform infrared spectrometer. Values
–
1
are expressed in wavenumbers (cm ) and were recorded in the
–
1
3
range 4000 to 450 cm . NMR spectra were recorded at 21 °C in
(
3 6 6
CDCl or C D
with either a Bruker® Avance 300 spectrometer
operating at 300 MHz for 1H nuclei and 75 MHz for 13C nuclei
or a Bruker® DRX400 or Bruker® 400 spectrometer operating at
1
13
4
00 MHz for H nuclei and 100 MHz for C nuclei. All chemical
shifts are reported in parts per million (ppm) relative to tetrameth-
analogues that contain a free hydroxy function at C-9, ylsilane (TMS, δ = 0 ppm) and were measured relative to the sol-
vent in which the sample was analysed (CDCl
3
/TMS δ = 0.00 ppm
1
13
for H NMR and CDCl
3
δ = 77.16 ppm for C NMR; [D
6
]acetone:
Table 1. Determination of the IC50 and LD50 values of 7-deoxy-
thysanone analogues.
1
13
δ = 2.05 ppm for H NMR and δ = 29.84Ϯ0.01 ppm for
NMR). Coupling constants (J) are reported in Hertz (Hz).
C
1
H
NMR spectroscopic data is reported as chemical shift in ppm, fol-
lowed by multiplicity (s” singlet, “d” doublet, “dd” doublet of dou-
blets, “ddd”, doublet of doublets of doublets, “dt” doublet of trip-
lets, “t” triplet, “q” quartet, “quint.” quintuplet, “sept.” septet,
“
m” multiplet, “br” broad), coupling constant where applicable
1
3
and relative integral. C NMR spectroscopic data is reported as
chemical shift in ppm. High-resolution mass spectra were recorded
with a Bruker micrOTOF-QII mass spectrometer.
Compound
R1
R2
IC50 [μm] LD50 [μm]
(
(
(
(
Ϯ)-7-deoxythysanone (2)
H
OH
H
OH
H
n.d.
n.d
n.d.
n.d.
13.2Ϯ0
107.3Ϯ2
155.3Ϯ0
8.1Ϯ2
Ϯ)-25
Ϯ)-26
Ϯ)-27
Me
Me
H
3
-{1Ј-[(tert-Butyldimethylsilyl)oxy]naphthalen-2Ј-yl}propan-2-ol (9):
Copper(I) chloride (80 mg, 0.80 mmol) and palladium(II) chloride
23 mg, 0.13 mmol) were dissolved in DMF/water (6:1, 12 mL).
(
124
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Synthesis and Biological Evaluation of Analogues of Thysanone
Oxygen was bubbled through the mixture for 2 h, followed by the
addition of [(2Ј-allylnaphthalen-1Ј-yl)oxy](tert-butyl)dimethylsilane
Na]+ 237.0886; found 237.0890. MS (EI): m/z (%) = 214 (60), 171
(100), 156 (50) 141 (50), 128 (50), 115 (30). To a mixture of 1-(1-
methoxynaphthalen-2-yl)propan-2-one (0.85 g, 4.0 mmol) in meth-
anol (40 mL) was added sodium borohydride (185 mg, 4.9 mmol).
The mixture was stirred for 1 h and then quenched with water
(40 mL). The aqueous layer was extracted with ethyl acetate (3ϫ
(7, 200 mg, 0.67 mmol) in DMF (10 mL). Oxygen was bubbled
through the mixture for further 2 h, and the reaction mixture was
stirred overnight under an atmosphere of oxygen. Aqueous hydro-
chloric acid (1 m, 30 mL) was added, and the aqueous layer was
extracted with ethyl acetate (3ϫ 50 mL). The combined organic 40 mL), and the combined organic extracts were washed with brine
extracts were washed with water (3ϫ 50 mL) and brine (50 mL)
and then dried with magnesium sulfate. The solvent was removed
(40 mL) and then dried with magnesium sulfate. The solvent was
removed in vacuo, and the residue was purified by flash chromatog-
in vacuo. The residue was purified by flash chromatography (SiO
hexanes/EtOAc 9:1) to afford 3-{1Ј-[(tert-butyldimethylsilyl)oxy]-
2
,
raphy (SiO
(0.87 g, 4.0 mmol, quant.) as a colourless oil. R
4:1): 0.12. IR (film): ν˜ max = 3213 (OH), 3053 (Ar), 2967 (Ar), 2931
2
, hexanes/EtOAc 9:1) to afford the title compound
f
(hexanes/EtOAc
naphthalen-2Ј-yl}propan-2-one (149 mg, 0.47 mmol, 71%) as a yel-
1
low oil. R
CDCl
.5 Hz, 1 H), 7.27–7.21 (m, 2 H), 7.03 (d, J = 8.5 Hz, 1 H), 3.66
f
(hexanes/EtOAc 9:1): 0.29. H NMR (400 MHz, (Ar), 2841 (Ar), 1598, 1572, 1507, 1446, 1368, 1341, 1258, 1245,
–
1
1
3
): δ = 7.92–7.85 (m, 1 H), 7.62–7.55 (m, 1 H), 7.29 (d, J =
1195, 1114, 1085, 989, 949, 931, 808, 750, 707 cm . H NMR
(400 MHz, CDCl ): δ = 8.08 (d, J = 8.6 Hz, 1 H), 7.82 (d, J =
s, 2 H), 1.89 (s, 3 H), 0.94 (s, 9 H), –0.03 (s, 6 H) ppm. C NMR 7.7 Hz, 1 H), 7.58 (d, J = 8.4 Hz, 1 H), 7.53–7.42 (m, 2 H), 7.32
100 MHz, CDCl ): δ = 207.0, 149.1, 134.5, 128.5, 128.3, 127.8, (d, J = 8.4 Hz, 1 H), 4.21–4.08 (m, 1 H), 3.94 (s, 3 H), 2.97–2.95
8
(
(
3
1
3
3
(m, 2 H), 2.26 (br s, 1 H), 1.27 (d, J = 6.1 Hz, 3 H) ppm. 1 C NMR
(100 MHz, CDCl ): δ = 154.1, 134.3, 129.0, 128.1, 128.1, 127.1,
126.2, 125.8, 124.4, 122.2, 68.8, 62.0, 40.1, 23.4 ppm. HRMS (ESI):
3
1
26.0, 125.2, 123.3, 122.2, 120.1, 46.2, 29.2, 26.2, 18.8, –3.1 ppm.
+
HRMS (ESI): calcd. for C19
H
26NaO
26KO
27
O
2
Si [M + H] 315.1775; found
3
+
315.1779; calcd. for C19
337.1606; calcd. for C19
353.1351. MS (EI): m/z (%) = 314 (10), 257 (100), 239 (15), 215 m/z (%) = 216 (65), 172 (100), 157 (80), 141 (65), 128 (65), 115 (20).
H
2
Si [M + Na] 337.1594; found
+
+
H
2
Si [M + K] 353.1334; found
16 2
calcd. for C14H O [M + H] 239.1043; found 239.1046. MS (EI):
(
15), 199 (10), 185 (10), 75 (35). To a mixture of 3-{1Ј-[(tert-butyldi-
1
-(1Ј,4Ј-Dimethoxynaphthalen-2Ј-yl)propan-2-ol (13): Copper(I)
chloride (525 mg, 5.3 mmol) and palladium(II) chloride (148 mg,
.8 mmol) were dissolved in DMF/water (6:1, 70 mL). Oxygen was
bubbled through the mixture for 2 h, followed by the addition of
2 (1.0 g, 4.4 mmol) in DMF (40 mL). Oxygen was bubbled
methylsilyl)oxy]naphthalen-2Ј-yl}propan-2-one (0.50 g, 1.6 mmol)
in methanol (10 mL) was added sodium borohydride (72 mg,
0
1
.9 mmol). The mixture was stirred for 1 h and then quenched with
water (10 mL). The aqueous layer was extracted with ethyl acetate
3ϫ 10 mL), and the combined organic extracts were washed with
1
(
through the mixture for a further 2 h, and the reaction was stirred
overnight under an atmosphere of oxygen. Aqueous hydrochloric
acid (1 m, 100 mL) was added, and the aqueous layer was extracted
with ethyl acetate (3ϫ 100 mL). The combined organic extracts
were washed with water (3ϫ 100 mL) and brine (100 mL) and then
dried with magnesium sulfate. The solvent was removed in vacuo,
brine (10 mL) and then dried with magnesium sulfate. The solvent
was removed in vacuo, and the residue was purified by flash
chromatography (SiO
pound (0.51 g, 1.6 mmol, quant.) as a colourless oil. R
EtOAc 9:1): 0.09. IR (film): ν˜ max = 3412 (OH), 2966 (Ar), 2929
2
, hexanes/EtOAc 9:1) to afford the title com-
f
(hexanes/
(
Ar), 2839 (Ar), 1598, 1572, 1446, 1368, 1258, 1245, 1085, 989, 807,
–1
1
and the residue was purified by flash chromatography (SiO , hex-
7
7
4
1
48 cm . H NMR (400 MHz, CDCl
3
): δ = 8.10–8.04 (m, 1 H),
2
anes/EtOAc 9:1) to afford 1-(1Ј,4Ј-dimethoxynaphthalen-2Ј-yl)-
.80–7.34 (m, 1 H), 7.49–7.40 (m, 3 H), 7.30 (d, J = 8.5 Hz, 1 H),
.15–4.05 (m, 1 H), 3.00–2.86 (m, 2 H), 1.22 (d, J = 6.0 Hz, 3 H),
.14–1.10 (m, 9 H), 0.21–0.18 (m, 6 H) ppm. 13C NMR (100 MHz,
propan-2-one (0.82 g, 3.4 mmol, 76%) as a yellow oil. R
f
(hexanes/
EtOAc 4:1): 0.16. IR (film): ν˜ max = 2929, 2832, 1672, 1589, 1584,
–
1
1
1
(
461, 1438, 1398, 1263, 1125, 1069, 1002 cm . H NMR
400 MHz, CDCl ): δ = 8.25–8.21 (m, 1 H), 8.05–8.00 (m, 1 H),
7.56–7.51 (m, 1 H), 7.49–7.44 (m, 1 H), 6.54 (s, 1 H), 3.95 (s, 3 H),
3
CDCl ): δ = 149.2, 134.2, 129.2, 128.3, 127.7, 125.6, 125.0, 123.5,
1
23.3, 122.0, 68.7, 40.8, 26.3, 26.2, 23.1, –2.9 ppm. HRMS (ESI):
3
+
calcd. for C19
calcd. for C19
H28NaO
2
Si [M + Na] 339.1751; found 339.1750;
1
3
+
3.87 (s, 2 H), 3.85 (s, 3 H), 2.20 (s, 3 H) ppm. C NMR (100 MHz,
CDCl ): δ = 206.9, 152.2, 147.6, 128.6, 126.9, 126.3, 125.5, 122.7,
H28KO
2
Si [M + K] 355.1490; found 355.1493.
3
1
-(1Ј-Methoxynaphthalen-2Ј-yl)propan-2-ol (10): Copper(I) chloride
122.6, 121.9, 105.6, 67.1, 55.8, 45.8, 29.4 ppm. HRMS (ESI): calcd.
(0.87 g, 8.8 mmol) and palladium(II) chloride (0.25 g, 1.4 mmol)
for C15
H
17
O
3
[M + H]+ 245.1172; found 245.1170; calcd. for
+
were dissolved in DMF/water (6:1, 130 mL). Oxygen was bubbled
through the mixture for 2 h, followed by the addition of 2-allyl-1-
methoxynaphthalene 8 (1.45 g, 7.3 mmol) in DMF (40 mL). Oxy-
gen was bubbled through the mixture for a further 2 h, and the
reaction mixture was stirred overnight under an atmosphere of oxy-
gen. Aqueous hydrochloric acid (1 m, 100 mL) was added, and the
aqueous layer was extracted with ethyl acetate (3ϫ 100 mL). The
combined organic extracts were washed with water (3ϫ 100 mL)
and brine (100 mL) and dried with magnesium sulfate. The solvent
was removed in vacuo, and the residue was purified by flash
C
C
15
H
H
16NaO
16KO
3
[M + Na] 267.0992; found 267.0998; calcd. for
[M + K]+ 283.0731; found 283.0745. MS (EI) m/z (%)
15
3
= 244 (100), 229 (20), 201 (25), 169 (25), 141 (20), 128 (19), 115
(18), 55 (19). To a mixture of 1-(1Ј,4Ј-dimethoxynaphthalen-2Ј-yl)-
propan-2-one (0.80 g, 3.3 mmol) in methanol (50 mL) was added
sodium borohydride (149 mg, 3.9 mmol). The mixture was stirred
for 1 h and then quenched with water (50 mL). The aqueous layer
was extracted with ethyl acetate (3ϫ 50 mL), and the combined
organic extracts were washed with brine (50 mL) and then dried
with magnesium sulfate. The solvent was removed in vacuo, and
chromatography (SiO
oxynaphthalen-2-yl)propan-2-one (1.19 g, 5.6 mmol, 76%) as a yel-
2
, hexanes/EtOAc 9:1) to afford 1-(1-meth-
the residue was purified by flash chromatography (SiO
EtOAc 9:1) to afford the title compound (0.81 g, 3.3 mmol, quant.)
(hexanes/EtOAc 4:1): 0.38. H NMR (400 MHz, as a colourless oil. R (hexanes/EtOAc 4:1): 0.02. IR (film): ν˜ max
): δ = 8.11–8.07 (m, 1 H), 7.85–7.82 (m, 1 H), 7.61 (d, J = 3403, 2963, 2932, 2838, 1627, 1595, 1508, 1460, 1417, 1394, 1345,
.4 Hz, 1 H), 7.54–7.45 (m, 2 H), 7.26 (d, J = 8.4 Hz, 1 H), 3.90
2
, hexanes/
1
low oil. R
CDCl
f
f
=
3
–
1
8
1263, 1225, 1161, 1119, 1093, 1030, 1000, 978, 829, 767, 712 cm .
13
1
(
2
1
s, 5 H), 2.20 (s, 3 H) ppm. C NMR (100 MHz, CDCl
3
): δ =
06.7, 154.2, 134.7, 128.5, 128.3, 128.1, 126.3, 126.2, 124.5, 123.4,
22.2, 62.1, 45.5, 29.5 ppm. HRMS (ESI): calcd. for C14 [M
[M +
3
H NMR (400 MHz, CDCl ): δ = 8.24–8.19 (m, 1 H), 8.03–7.99
(m, 1 H), 7.57–7.40 (m, 2 H), 6.62 (s, 1 H), 4.23–4.10 (m, 1 H),
H
15
O
2
3.97 (s, 3 H), 3.89 (s, 3 H), 2.94 (d, J = 6.2 Hz, 2 H), 2.29 (br s, 1
+
13
+
H] 215.1067; found 215.1061; calcd. for C14
H14NaO
2
H), 1.28 (d, J = 6.2 Hz, 3 H) ppm. C NMR (100 MHz, CDCl
3
):
Eur. J. Org. Chem. 2014, 122–128
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
125

M. A. Brimble et al.
FULL PAPER
δ = 162.5, 152.1, 128.7, 126.8, 126.6, 125.9, 125.2, 122.5, 121.9,
then brine (20 mL). The organic layer was dried with magnesium
sulfate. The solvent was removed in vacuo, and the residue was
1
06.3, 68.9, 62.0, 55.8, 40.6, 23.4 ppm. HRMS (ESI): calcd. for
+
C
C
=
15
H
H
18NaO
18KO
3
[M + Na] 269.1148; found 269.1146; calcd. for
[M + K] 285.0888; found 285.0892. MS (EI): m/z (%) ford the title compound (103 mg, 0.38 mmol, 96%), which was di-
2
purified by flash chromatography (SiO , hexanes/EtOAc 7:3) to af-
+
15
3
246 (60), 201 (25), 187 (100), 170 (10), 150 (10) 128 (15), 115 rectly transferred to the next step. To a mixture of 1-(1Ј,4Ј,5Ј-trime-
thoxynaphthalen-2Ј-yl)propan-2-one (0.50 g, 1.8 mmol) in meth-
(13).
anol (20 mL) was added sodium borohydride (100 mg, 2.6 mmol).
The mixture was stirred for 1 h and then quenched with water
2
-Allyl-5-methoxynaphthalene-1,4-dione (22): To a mixture of 2-
allyl-5-methoxynaphthalen-1-ol (21, 250 mg, 1.17 mmol) in aceto-
nitrile (5 mL) was added salcomine (50 mg, 0.15 mmol). Oxygen
was bubbled through the mixture for 4 h. The solvent was removed
in vacuo, and the residue was purified by flash chromatography
(
2
(
20 mL). The aqueous layer was extracted with ethyl acetate (3ϫ
0 mL), and the combined organic extracts were washed with brine
20 mL) and then dried with magnesium sulfate. The solvent was
removed in vacuo, and the residue was purified by flash chromatog-
raphy (SiO , hexanes/EtOAc 9:1) to afford the title compound
0.45 g, 1.63 mmol, 90%) as a colourless oil. R (hexanes/EtOAc
): δ = 7.63 (dd, J = 8.0,
.8 Hz, 1 H), 7.40 (t, J = 8.0 Hz, 1 H), 6.84 (dd, J = 8.0, 0.8 Hz, 1
(
0
(
SiO
.64 mmol, 55%) as a yellow oil. R
film): ν˜ max = 3073, 3010, 2980, 2848, 1663, 1649, 1629, 1584, 1478,
2
, hexanes/EtOAc 9:1) to afford the title compound (145 mg,
2
f
(hexanes/EtOAc 7:3): 0.52. IR
(
7
0
f
1
:3): 0.21. H NMR (400 MHz, CDCl
3
1
8
449, 1438, 1402, 1370, 1280, 1256, 1171, 1063, 1051, 977, 921,
82, 775 cm . H NMR (300 MHz, CDCl ): δ = 7.79–7.74 (m, 1
3
–1 1
H), 6.66 (s, 1 H), 4.19–4.11 (m, 1 H), 3.95 (s, 3 H), 3.92 (s, 3 H),
H), 7.70–7.63 (m, 1 H), 7.32–7.27 (m, 1 H), 6.70 (t, J = 1.5 Hz, 1
3
6
1
5
.84 (s, 3 H), 2.92–2.88 (m, 2 H), 2.47 (br s, 1 H), 1.27 (d, J =
.3 Hz, 3 H) ppm. C NMR (100 MHz, CDCl ): δ = 157.6, 153.7,
3
47.8, 131.5, 127.4, 126.9, 117.8, 114.8, 109.0, 106.4, 68.8, 61.7,
H), 5.97–5.81 (m, 1 H), 5.25–5.15 (m, 2 H), 4.00 (s, 3 H), 3.32–3.26
13
(
m, 2 H) ppm. 13C NMR (100 MHz, CDCl
3
): δ = 185.3, 184.7,
1
5
2
2
2
1
6
59.6, 147.3, 137.6, 134.9, 134.6, 133.2, 120.1, 119.6, 118.9, 117.9,
6.6, 33.2 ppm. HRMS (ESI): calcd. for C14
29.0859; found 229.0848; calcd. for C14
51.0679; found 251.0678; calcd. for C14
67.0418; found 267.0425. MS (EI): m/z (%) = 228 (100), 213 (85),
99 (20), 181 (50), 157 (30), 141 (50), 121 (55), 104 (30), 76 (60),
3 (20).
7.1, 56.6, 40.5, 23.4 ppm. The spectroscopic data was in agreement
+
H
12NaO
12KO
13
O
3
[M + H]
[21]
with that reported in the literature.
H
H
3
[M + Na]+
[M + K]+
5,9,10-Trimethoxy-3-methyl-3,4-dihydro-1H-benzo[g]isochromene
3
(
24): To a mixture of 3 (180 mg, 0.64 mmol) and dimethoxymeth-
ane (170 μL, 1.96 mmol) in dry Et O (5 mL) was added BF ·OEt
2
3
2
at –78 °C under an atmosphere of nitrogen. The reaction mixture
was warmed to room temperature over 18 h and then quenched
with saturated aqueous ammonium chloride solution (10 mL). The
aqueous layer was extracted with ethyl acetate (3ϫ 20 mL), and
the combined organic extracts were washed with brine (20 mL) and
then dried with magnesium sulfate. The solvent was removed in
2
5
-Allyl-1,4,5-trimethoxynaphthalene (23): To a mixture of 2-allyl-
-methoxynaphthalene-1,4-dione (22, 450 mg, 2.0 mmol) in THF
(20 mL) were added TBAI (cat.) and sodium dithionate (2.0 g,
1
1.8 mmol) in water (10 mL). The mixture was stirred for 30 min.
Potassium hydroxide (2.5 g, 45.3 mmol) in water (10 mL) and di-
methyl sulfate (4.3 mL, 45.3 mmol) were then added, and the mix-
ture was stirred for 2 h. Aqueous ammonia (10 mL) was added,
and the mixture was stirred for a further 30 min. The phases were
separated, and the aqueous layer was extracted with ethyl acetate
vacuo, and the residue was purified by flash chromatography (SiO
hexanes/EtOAc 9:1) to afford the title compound (167 mg,
.58 mmol, 89%) as a colourless oil. R (hexanes/EtOAc 7:3): 0.31.
IR (film): ν˜ max = 2930, 2837, 1654, 1619, 1597, 1573, 1502, 1460,
2
,
0
f
1
1
446, 1362, 1337, 1281, 1262, 1222, 1179, 1127, 1115, 1094, 1065,
(3ϫ 30 mL). The combined organic extracts were washed with
–1 1
044, 1005, 991, 970, 838, 811, 761 cm . H NMR (400 MHz,
): δ = 7.66 (dd, J = 8.0, 0.7 Hz, 1 H), 7.36 (t, J = 8.0 Hz, 1
aqueous HCl (1 m, 50 mL), water (50 mL) and then brine (50 mL).
The solvent was removed in vacuo, and the residue was purified by
CDCl
3
H), 6.83 (d, J = 8.0 Hz, 1 H), 5.25 (d, J = 15.8 Hz, 1 H), 4.87 (d,
J = 15.8 Hz, 1 H), 4.00 (s, 3 H), 3.86 (s, 3 H), 3.79 (s, 3 H), 3.79–
flash chromatography (SiO
tle compound (346 mg, 1.34 mmol, 67%) as a yellow oil. R
anes/EtOAc 7:3): 0.72. IR (film): ν˜ max = 3076, 2933, 2838, 1724,
2
, hexanes/EtOAc 19:1) to afford the ti-
f
(hex-
3
1
.73 (m, 1 H), 3.06 (dd, J = 16.6, 2.9 Hz, 1 H), 2.65 (dd, J =
6.6, 10.9 Hz, 1 H), 1.43 (d, J = 6.1 Hz, 3 H, Me) ppm. C NMR
13
1
1
618, 1599, 1583, 1508, 1461, 1448, 1379, 1346, 1262, 1237, 1127,
069, 1006, 913, 847, 810, 754 cm . H NMR (400 MHz, CDCl ):
3
(100 MHz, CDCl
3
): δ = 156.2, 149.2, 147.9, 130.0, 125.8, 125.8,
–1 1
1
24.9, 119.2, 114.7, 105.6, 70.6, 65.2, 61.8, 61.0, 56.2, 30.9, 21.9
δ = 7.69–7.65 (m, 1 H), 7.40 (t, J = 8.0 Hz, 1 H), 6.86–6.83 (m, 1
H), 6.66 (s, 1 H), 6.09–5.97 (m, 1 H), 5.16–5.07 (m, 2 H), 3.97 (s,
+
21 4
ppm. HRMS (ESI): calcd. for C17H O [M + H] 289.1434; found
+
2
3
3
89.1434; calcd. for C17
11.1262; calcd. for: C17
27.1014. MS (EI): m/z (%) = 288 (100), 273 (10), 257 (14), 244
H20NaO
4
[M + Na] 311.1254; found
3
H), 3.93 (s, 3 H), 3.84 (s, 3 H), 3.57 (dt, J = 6.5, 1.5 Hz, 2 H)
[M + K]+ 327.0993; found
H20KO
4
13
ppm. C NMR (100 MHz, CDCl
3
): δ = 157.5, 153.5, 147.2, 137.2,
1
31.7, 128.5, 126.7, 116.2, 114.9, 108.5, 106.3, 62.0, 57.1, 56.6, 34.2
(
15), 229 (82), 214 (12), 199 (10), 128 (10), 115 (12).
+
ppm. HRMS (ESI): calcd. for C16
H
19
O
3
[M + H] 259.1329; found
+
1,7-Dideoxythysanone-9-methylether [(؎)-25]: To a mixture of 24
(30 mg, 0.10 mmol) in THF (3 mL) were added freshly prepared
AgO (124 mg, 1.14 mmol) and aqueous nitric acid (6 n, 0.35 mL,
2
2
2
59.1330; calcd. for C16
81.1147. MS (EI): m/z (%) = 258 (100) [M] , 243 (60), 228 (20),
H
18NaO
3
[M + Na] 281.1148; found
+
12 (40).
2
.10 mmol). The reaction mixture was stirred for 5 min and then
®
1-(1Ј,4Ј,5Ј-Trimethoxynaphthalen-2Ј-yl)propan-2-ol (3): A mixture
filtered through Celite . The filtrate was diluted with H
and the aqueous layer was extracted with CH Cl (3ϫ 10 mL). The
combined organic extracts were washed with brine and dried with
MgSO . The solvent was removed in vacuo, and the residue was
purified by flash chromatography (SiO , hexanes/EtOAc 7:3Ǟ1:1)
to afford the title compound (25 mg, 97 μmol, 97%) as a yellow
oil. R (hexanes/EtOAc 7:3): 0.24. IR (film): ν˜ max = 2922, 2851,
1654, 1644, 1584, 1574, 1474, 1445, 1332, 1291, 1276, 1261, 1209,
2
O (10 mL),
of freshly prepared copper(I) chloride (46 mg, 0.47 mmol) and pal-
ladium(II) chloride (13 mg, 74 μmol) in DMF/H O (6:1.7 mL) was
2
2
2
stirred for 1 h as oxygen was bubbled through the mixture. Naphth-
alene (23, 100 mg, 0.39 mmol) was added in DMF (3 mL). Oxygen
was bubbled through the mixture for 4 h, and the reaction mixture
was then stirred overnight under an atmosphere of oxygen. The
mixture was hydrolysed with 1 m aqueous HCl (20 mL), and the
aqueous layer was extracted with EtOAc (3ϫ 20 mL). The com-
bined organic extracts were washed with water (3ϫ 20 mL) and
4
2
f
–
1 1
1200, 1187, 1137, 1124, 1091, 1034, 954, 917, 780 cm . H NMR
(400 MHz, CDCl ): δ = 7.76 (dd, J = 7.8, 1.0 Hz, 1 H), 7.66 (t, J
3
126
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 122–128

Synthesis and Biological Evaluation of Analogues of Thysanone
=
7.8 Hz, 1 H), 7.28 (dd, J = 7.8, 1.0 Hz, 1 H), 4.84 (dd, J = 18.7, by preparative TLC (SiO
.2 Hz, 1 H), 4.50 (ddd, J = 18.7, 4.0, 3.2 Hz, 1 H), 4.00 (s, 3 H), compound (9 mg, 38 μmol, 84%) as a yellow oil. R
.71–3.62 (m, 1 H), 2.74–2.67 (m, 1 H), 2.32–2.22 (m, 1 H), 1.37 EtOAc 1:1): 0.55. IR (film): ν˜ max = 3350, 2976, 2923, 2853, 1650,
): δ = 184.1,
83.2, 159.8, 144.3, 139.8, 135.0, 134.4, 119.7, 119.4, 117.9, 69.7,
3.8, 56.6, 29.3, 21.4 ppm. HRMS (ESI): calcd. for C15 [M
[M +
Na] 281.0784; found 281.0787. MS (EI): m/z (%) = 258 (100), 243
30), 225 (30), 187 (20), 128 (35).
2
, hexanes/EtOAc 1:1) to afford the title
2
3
(
f
(hexanes/
d, J = 6.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl
1623, 1596, 1579, 1447, 1405, 1393, 1360, 1311, 1276, 1231, 1207,
1179, 1167, 1142, 1120, 1081, 1071, 1050, 1021, 1008, 984, 875,
857, 836, 722 cm . H NMR (400 MHz, CDCl ): δ = 11.94 (s, 1
3
H), 7.66–7.59 (m, 2 H), 7.27 (dd, J = 7.8, 2.0 Hz, 1 H), 6.07 (d, J
= 3.8 Hz, 1 H), 4.41–4.31 (m, 1 H), 3.27 (d, J = 2.8 Hz, 1 H), 2.79
(dd, J = 19.6, 3.4 Hz, 1 H), 2.31–2.23 (m, 1 H), 1.41 (d, J = 6.3 Hz,
3
1
6
+
–
1 1
H
15
O
4
+
H] 259.0965; found 259.0967; calcd. for C15
H
14NaO
4
+
(
1
3
3
1
H) ppm. C NMR (100 MHz, CDCl
45.1, 140.4, 136.6, 131.9, 119.5, 114.8, 86.7, 62.7, 29.6, 21.1 ppm.
[M + Na]+ 283.0577; found
12NaO
3
): δ = 188.5, 183.7, 161.8,
7
(
3
-Deoxythysanone-9-methylether (؎)-26: To a mixture of (Ϯ)-25
10 mg, 39 μmol) in CCl (8 mL) were added bromine (1 m in CCl
(cat.). The reaction mixture was irradi-
4
4
,
HRMS (ESI): calcd. for C14
83.0584.
H
5
2 2
9 μL, 39 μmol) and Bz O
2
ated with a desk lamp and heated to reflux for 20 min. The solvent
was removed in vacuo, and THF (2 mL) and H O (1 mL) were
added. The reaction mixture was stirred for 1 h and then diluted
with H
CH Cl
with MgSO
was purified by preparative TLC (SiO
ford the title compound (6 mg, 22 μmol, 56%) as a yellow oil. R
HRV 3C Protease Assay:[22] A typical protease assay was performed
at room temperature for 1 h in a 50 μL reaction containing rHRV
3C protease (50 μg/mL), substrate (52 μm) and tris(2-carboxyethyl)-
phosphine (TCEP, 5 μm) in phosphate buffered saline (PBS) pH 6.8
under continuous rotation. The reaction mixture was centrifuged,
and a portion of the supernatant (10 μL) was added to PBS pH
6.8 (200 μL) in a 96-well opti plate. The fluorescence intensity (FI)
2
2
O (10 mL), and the aqueous layer was extracted with
(3ϫ 10 mL). The combined organic extracts were dried
2
2
4
. The solvent was removed in vacuo, and the residue
2
, hexanes/EtOAc 1:1) to af-
f
(hexanes/EtOAc 1:1): 0.21. IR (film): ν˜ max = 3468, 2974, 2844, was measured with a Perkin–Elmer EnSpire multimode plate
1
1
7
656, 1645, 1585, 1472, 1448, 1435, 1413, 1331, 1289, 1263, 1194,
122, 1084, 1069, 1054, 1029, 1012, 955, 922, 856, 849, 795, 749,
reader with an excitation wavelength of 495 nm and an emission
wavelength of 516 nm and 100 excitation flashes. A blank was mea-
–1 1
35, 674, 554 cm . H NMR (400 MHz, CDCl
3
): δ = 7.76 (dd, J sured that contained enzyme only in PBS at pH 6.8, and an assay
=
1
8.0, 1.0 Hz, 1 H), 7.68 (t, J = 8.0 Hz, 1 H), 7.31 (dd, J = 8.0,
.0 Hz, 1 H), 6.05 (s, 1 H), 4.38–4.28 (m, 1 H), 4.01 (s, 3 H), 3.40
br s, 1 H), 2.72 (dd, J = 19.2, 3.3 Hz, 1 H), 2.25 (ddd, J = 19.2,
with (–)-thysanone (1) was run as a control (detected IC50 for 1:
53Ϯ0 μm).
(
1
(
1
1.2, 0.9 Hz, 1 H), 1.40 (d, J = 6.3 Hz, 3 H) ppm. 1 C NMR
100 MHz, CDCl ): δ = 184.7, 183.2, 160.0, 142.1, 141.2, 135.2,
34.2, 128.6, 119.4, 118.3, 87.2, 63.0, 56.6, 29.1, 21.1 ppm. HRMS
3
Cytotoxicity Assay: Materials: Complete RPMI 1640: Roswell Park
Memorial Institute media 1640 pH 7.4 (Invitrogen, USA) sup-
plemented with sodium hydrogen carbonate (1.5 mg/mL), penicillin
(Gibco, Invitrogen, USA; 50 U/mL), streptomycin (Gibco, Invi-
trogen, USA; 50 μg/mL), l-glutamine (Gibco, Invitrogen, USA;
2 mm), sodium pyruvate (Gibco, Invitrogen, USA; 110 μg/mL) and
10% foetal calf serum (FCS). Foetal calf serum: heat-inactivated
3
+
(ESI): calcd. for C15
H14NaO
5
[M + Na] 297.0733; found 297.0745.
1
4
0
,7-Dideoxythysanone (؎)-27: To a mixture of (Ϯ)-25 (12 mg,
6 μmol) in CH Cl (0.2 mL) was added BCl (1 m in CH Cl ,
2
.14 μL, 0.14 mmol) at –78 °C. The cooling bath was removed, and for 30 min at 56 °C and filtered (Invitrogen, USA). Cells: NCI-
2
2
3
2
the reaction mixture was stirred for 30 min. Aqueous NaHCO
5 mL) was slowly added, and the aqueous layer was extracted with
CH Cl (3ϫ 5 mL). The combined organic extracts were dried with
MgSO . The solvent was removed in vacuo, and the residue was
purified by preparative TLC (SiO , hexanes/EtOAc 1:1) to afford
the title compound (11 mg, 45 μmol, 98%) as a yellow oil. R (hex-
anes/EtOAc 1:1): 0.65. IR (film): ν˜ max = 2975, 7919, 2850, 1663,
647, 1623, 1580, 1466, 1386, 1366, 1346, 1278, 1249, 1088, 743
3
H441 (human lung adenocarcinoma epithelial cell line). Thymidine
solution: [methyl- H] thymidine, 12.5 μCi/mL, GE Healthcare
3
(
2
2
TRK 120. Assays: The NCI-H441 human lung adenocarcinoma
epithelial cell line was used for cytotoxicity assays. The cell line was
maintained in complete RPMI 1640 supplemented with sodium hy-
drogen carbonate (1.5 mg/mL), penicillin (50 U/mL), streptomycin
(50 μg/mL), l-glutamine (2 mm), sodium pyruvate (110 μg/mL) and
10% heat-inactivated FCS (cRPMI; all from Gibco, Invitrogen,
USA). Cells were harvested with 0.5% v/v trypsin/EDTA, washed
4
2
f
1
–1 1
cm . H NMR (400 MHz, CDCl
(
(
3
): δ = 11.90 (s, 1 H), 7.66–7.57
m, 2 H), 7.24 (dd, J = 7.6, 1.8 Hz, 1 H), 4.90–4.82 (m, 1 H), 4.52
ddd, J = 18.8, 4.1, 3.2 Hz, 1 H), 3.75–3.62 (m, 1 H), 2.81–2.70 (m, dilutions of inhibitor (80 μg/mL Ǟ 0.63 μg/mL) in cRPMI. For
2
in cRPMI and incubated at 37 °C, 5% CO with serial twofold
1
3
1
H), 2.37–2.34 (m, 1 H), 1.39 (d, J = 6.2 Hz, 3 H) ppm. C NMR
measurement of cytotoxicity, 40000 cells/well were added to flat-
(
1
100 MHz, CDCl
3
): δ = 188.7, 183.1, 161.6, 143.4, 142.6, 136.5, bottom 96-well plates for 36 h, and the cytotoxicity was quantified
3
32.1, 124.5, 119.4, 114.8, 69.8, 63.0, 29.8, 21.3 ppm. HRMS (ESI): by adding 0.25 μCi/well methyl-[ H] thymidine (GE Healthcare,
+
calcd. for C14
for C14 12NaO
[M – H] 243.0663; found 243.0677. MS (EI): m/z (%) =
H
13
O
4
[M + H] 245.0808; found 245.0806; calcd.
TRK 120) to the cells for an additional 6 h. The plates were har-
vested onto glass filter mats by using a Tomtec Harvester 96 and
dried, and Betaplate scint was added prior to the measurement of
radioactivity with a Microbeta TriLux counter (all materials from
Perkin–Elmer).
+
H
4
[M + Na] 267.0628; found 267.0641; calcd. for
–
14 11 4
C H O
2
9
44 (100), 229 (10), 214 (20), 200 (95), 172 (55), 144 (20), 115 (30),
2 (27), 63 (13).
(؎)-trans-7-Deoxythysanone (2): To a mixture of (Ϯ)-27 (11 mg,
Supporting Information (see footnote on the first page of this arti-
4
5
5 μmol) in CCl
0 μmol) and Bz
4
(9 mL) were added bromine (1 m in CCl
4
, 50 μL,
cle): Details of synthesis and spectroscopic data for intermediates,
H and C NMR spectra.
1
13
2
O
2
(cat.). The reaction mixture was irradiated
with a desk lamp and heated to reflux for 20 min. The solvent was
removed in vacuo, and THF (3 mL) and H O (1.5 mL) were added.
The reaction mixture was stirred for 1 h and then diluted with H
10 mL), and the aqueous layer was extracted with CH Cl (3ϫ
0 mL). The combined organic extracts were dried with MgSO
The solvent was removed in vacuo, and the residue was purified
2
Acknowledgments
2
O
(
1
2
2
4
.
The authors are grateful to the Maurice Wilkins Centre for Mo-
lecular Biodiscovery for financial support.
Eur. J. Org. Chem. 2014, 122–128
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
127

M. A. Brimble et al.
FULL PAPER
hedron 2002, 58, 183–189; d) D. Barker, M. A. Brimble, P. Do,
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