The furan route to tropolones: Probing the antiproliferative effects of β-thujaplicin analogs

Article abstract of DOI:10.1039/c2ob26553b

A direct route to analogs of the naturally occurring tropolone β-thujaplicin has been developed in just four steps from furan. Using this method, a series of derivatives were synthesized and evaluated. Several of these compounds demonstrated very high levels of potency against bacterial and fungal pathogens with good selectivity over mammalian cells.

Full text of DOI:10.1039/c2ob26553b

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COMMUNICATION
The furan route to tropolones: probing the antiproliferative effects of
β-thujaplicin analogs†
E. Zachary Oblak,a Erin S. D. Bolstad,b Sophia N. Ononye,a Nigel D. Priestley,b M. Kyle Haddena and
Dennis L. Wright*a
Received 6th August 2012, Accepted 18th September 2012
DOI: 10.1039/c2ob26553b
have been no attempts to probe key structure–activity relation-
ships through the synthesis and evaluation of β-thujaplicin
analogs.
A direct route to analogs of the naturally occurring tropolone
β-thujaplicin has been developed in just four steps from
furan. Using this method, a series of derivatives were syn-
thesized and evaluated. Several of these compounds demon-
strated very high levels of potency against bacterial and
fungal pathogens with good selectivity over mammalian cells.
The mechanism(s) by which these small natural products are
able to elicit such a wide range of effects in both prokaryotic and
eukaryotic systems is unclear but may relate to the ability of the
tropolone unit to chelate metal ions. Accordingly, the antiproli-
ferative activity is typically abrogated by etherification or
acylation of the tropolone hydroxyl functionality.2 Recent inves-
tigations have uncovered several specific pathways affected by
thujaplicin including p27/Skp2 in a melanoma line,7 androgen
receptor signaling in prostrate cells8 and Cyr1/Ras1 in Candida
ablicans.9 In addition, potential metalloenzyme targets such as
carboxypeptidase A10 have been identified, again highlighting
the chelating potential of the tropolone in targeting a bound
metal ion such as zinc. From the perspective of a drug develop-
ment effort, thujaplicin is much more reminiscent of a lead
structure, characterized by low molecular weight, minimal func-
tionalization and multiple target effects.11
Introduction
Natural products containing a tropolone nucleus have been
known since the early 1950s when a novel non-benzenoid aro-
matic structure was independently proposed by Dewar and
Nozoe for the plant derived metabolite β-thujaplicin (hinoki-
tiol).1 This nucleus also appears in more complex terpenes such
as colchicine, which is used clinically for the treatment of gout
(Fig. 1). Recently, there has been renewed interest in the mono-
terpenoid tropolones because of the wide range of biological
activities associated with the parent natural products.2 β-Thuja-
plicin and its positional isomers have been shown to possess sig-
nificant antibacterial,3 antifungal,4 and anticancer5 activities and
may serve as exciting leads for new therapeutics. Despite wide-
spread interest in the synthesis of these natural products,6 there
Results and discussion
Chemistry
We have been interested in developing an efficient and flexible
route to substituted tropolones that would allow us to probe key
structure–activity relationships between these lead-like com-
pounds and the variety of biological functions associated with
them. In doing so, we aim to achieve a route by which these
leads can be developed into efficient, potent and selective
agents. Herein, we describe a new route to substituted tropolones
and its application to the synthesis of β-thujaplicin analogs.
Over the past several years, we have investigated the formal
[4 + 3] cycloadduct produced from the reaction of furan and
tetrabromocyclopropene (TBCP) (Scheme 1).12 Hydrolysis of
the primary adduct gives in high yield the dibromoenone 1, an
intermediate that can be used for a variety of applications.
Fig. 1 Tropolone natural products.
aDepartment of Pharmaceutical Sciences, University of Connecticut,
Storrs, CT 06269, USA. E-mail: dennis.wright@uconn.edu
bPromiliad Biopharma, 950 West Fork Petty Creek Rd., Alberton,
MT 59820-9437, USA
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26553b
This highly functionalized intermediate is both complimentary
to and functionally distinct from the oxyallyl cation adduct of
furan that had previously been used by Cha in an elegant syn-
thesis of β-thujaplicin.6a Because of the renewed interest in sub-
stituted tropolones, we were intrigued by the possibility of using
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work-up with aqueous hydroxide led to direct formation of the
tropolone and complete suppression of the bromotropone
product. Utilizing this optimized method, β-thujaplicin was pre-
pared in just four steps from furan in 56% overall yield.
With an effective synthesis of β-thujaplicin in hand, we turned
our attention to exploiting this synthesis to access a small array
of thujaplicin analogs. In addition to probing the scope of the
tropolone synthesis, we would also be in position to develop
some preliminary structure–activity relationships for these small
but highly active natural products (Table 1).
The introduction of the β-substituent after cycloaddition
renders the generation of analogs particularly straightforward.
In prior work with dibromoenone 1, we had shown that the
β-bromide could be regioselectively substituted either though
nucleophilic addition/elimination or palladium catalyzed cross-
coupling reactions. Accordingly, 1 was converted into nine
different enones 2b–j containing various alkyl and aryl groups at
C3. Exposure of these compounds to samarium diiodide fol-
lowed by a basic work-up as previously described gave the
tropolones 6b–j in very good yields. This route to β-thujaplicin
analogs only requires two parallel operations from a common
intermediate.
Scheme 1 Synthesis of an oxabicyclic building block.
Biological activities
Scheme 2 Direct synthesis of tropolones.
With a small array of β-thujaplicin analogs in-hand we were in a
position to evaluate the role of β-substitution on the activity of
the compounds (Table 2). A panel of cell-based assays was
selected to evaluate the efficacy of the compounds in two impor-
tant bacterial pathogens (methicillin-resistant Staphylococcus
aureus (MRSA)13 and Enterococcus faecalis14), a fungal patho-
gen (Candida glabrata15) that is resistant to most common anti-
fungal agents and a human breast cancer line (MCF-7). The
results of the assays are shown in Table 2 and demonstrate that
key structure–activity relationships exist across this class of
natural product analogs. Firstly, the assays confirm that the
β-substituted tropolones function as very effective antimicrobial
agents and show very high levels of activity against both bac-
terial and fungal pathogens. Several of the derivatives show MIC
values <0.1 μg mL−1 against MRSA and C. glabrata, notor-
iously difficult organisms to target. For example, compounds
6a–6f are ten-fold more potent against MRSA than vancomycin
and compounds 6a–6d are 3–5 times more potent than vanco-
mycin against sensitive lines of E. faecalis. While several of the
compounds show moderate activity against the cancer cell line,
there is still very strong pathogenic selectivity with at least a
1000-fold difference relative to the antibacterial (MRSA)
potency. Secondly, the lack of inhibition of a non-proliferating
human dermal fibroblast cell line emphasizes that specific targets
are involved rather than simple sequestration of metal ions. In
general, structure–activity relationships show that increasing
steric bulk at the β-position correlates strongly with activity and
is sensitive to substitution (compare 6h and 6i). Finally, it is
interesting that the SAR follows closely regardless of the organ-
ism, possibly indicating conserved or structurally similar targets
in both prokaryotic and eukaryotic cells.
1 as a versatile precursor to β-thujaplicin analogs. Key to utiliz-
ing bridged adducts derived from 1 would be the development
of a highly regioselective, reductive ether cleavage that would
preserve the vicinal oxygen functionality at C1 and C7. This
route would be strategically distinct from the elimination-based
cleavages used with oxyallyl cation adducts which necessitates
the prior incorporation of additional oxygen functionality. We
initially set out to examine the inherent regioselectivity in the
reductive cleavage of the ether bridge from a C1-ketyl and evalu-
ate the potential of such a reaction en route to the tropolone
nucleus.
A potential precursor to β-thujaplicin was prepared by conju-
gate addition/elimination of an isopropyl zinc cuprate to the
dibromoenone 1. Exposure of the enone to samarium diiodide
resulted in a rapid reaction and complete consumption of starting
material. Interestingly, neutral or mildly acidic work-up of the
reaction directly yielded a mixture of two aromatic products, the
bromotropone 5 and β-thujaplicin 6a (Scheme 2).
It is assumed that both products initially arise from a highly
regioselective opening of the C4–O8 bond to generate enolate 4.
The exclusive opening of this bond is likely driven by formation
of this extended enolate in contrast to the cross-conjugated
enolate that would arise from opening of the C7–O8 bond.
Kinetic protonation of the enolate should initially lead to the
diene 4 that is poised for easy aromatization either through dehy-
dration to give 5 or dehydrohalogenation to give the tropolone
6a. It was presumed that dehydration to produce the tropone
would be more facile in acidic media while under more basic
conditions the desired dehydrobromination to the tropolone
would be preferred. Gratifyingly, switching to a strongly basic
Through the use of the readily available dibromoenone 1, we
have been able to develop a highly flexible route to β-thujaplicin
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Table 1 Synthesis of tropolones
Enone 2
R
Methoda
Yieldb [%]
Tropolone 6
Yieldb [%]
2a
2b
2c
2d
2e
2f
Isopropyl
Cyclopropyl
sec-Butyl
tert-Butyl
Cyclopentyl
Phenyl
A
B
C
C
A
D
88
70
77
82
92
80
6a
6b
6c
6d
6e
6f
75
61
71
77
68
72
2g
D
78
6g
54
2h
D
73
6h
62
2i
2j
D
D
69
72
6i
6j
65
67
a A: RZnI, CuCN, TMSCl, BF3·Et2O. B: RMgBr, CuI (1.1 equiv.). C: RLi, CuCN (1.1 equiv.), TMSCl. D: RB(OH)2, Pd(PPh3)2Cl2, Cs2CO3. b Yield
of isolated products. THF = tetrahydrofuran. DMPU = N,N′-dimethylpropylene urea. TMS = trimethylsilyl.
analogs based on a one-pot reductive ether cleavage/aromatiza-
tion strategy. The new route was adapted for the formation of
several new tropolone derivatives that show compelling struc-
ture–activity relationships. High levels of potency and selectivity
suggest that these compounds could serve as excellent leads in
developing novel antimicrobial agents.
permanganate (KMnO4) and heat as developing agents. Flash
chromatography was performed using Baker silica gel (60 Å par-
ticle size). NMR spectra were recorded on Bruker-500 and cali-
brated using residual undeuterated solvent as an internal
reference (CHCl3 at δ 7.26 ppm 1H NMR, d 77.0 ppm 13C
NMR). The following abbreviations were used to explain the
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, hept = heptet, b = broad. High-resolution mass
spectra (HRMS) were provided by the Notre Dame Mass Spectro-
metry Laboratory by electrospray ionization of flight reflectron
experiments. IR Spectra were recorded on Shimadzu FTIR 8400
spectrometer. Melting points (m.p.) are uncorrected and were
recorded on a Mel-Temp 3.0 digital melting point apparatus.
Experimental
Synthesis
General. All reactions were carried out under an inert argon
atmosphere with dry solvents under anhydrous conditions unless
otherwise noted. Commercial grade reagents and solvents were
used without further purification except as indicated below.
Hexanes, tetrahydrofuran (THF), diethyl ether (Et2O), and
dichloromethane (CH2Cl2) were used directly from a Baker
cycletainer system. Reagents were purchased at the highest com-
mercial quality and used without further purification, unless
otherwise noted. Yields refer to chromatographically and
spectroscopically (1H NMR) homogenous materials, unless
otherwise stated. Dibromoenone 1 and α-bromoenones 2f, 2g,
and 2h were prepared according to literature procedures. Reac-
tions were monitored by thin layer chromatography (TLC)
carried out on Whatman Partisil K6F silica gel 60 Å precoated
plates using UV light as the visualizing agent and an acidic
mixture of anisaldehyde or basic aqueous potassium
General procedure for zinc cuprate additions
To a suspension of activated zinc dust (4.2 g, 64.3 mmol) in
50 mL THF was added chlorotrimethylsilane (0.61 mL,
4.6 mmol) at room temperature. After stirring for 10 min, the
iodoalkane (30.8 mmol) was added dropwise at room tempera-
ture and stirred for 1 h. A separate round bottomflask, charged
with freshly purified CuCN (2.89 g, 30.8 mmol) and lithium
chloride (2.79 g, 64.3 mmol), was flame dried and allowed to
cool to room temperature before the addition of THF (45 mL).
The resulting light green solution was stirred for 30 min at
room temperature, cooled to
0 °C, and the organozinc
reagent was added dropwise via cannula. After 30 min, the
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Table 2 Biological evaluation of thujaplicin analogs
Tropolone 6
Fibroblasts
NAb
MCF-7a
42.3
E. faecalis
MRSA
0.04
C. glabrata
1.3
0.7
NDc
58.0
1.3
0.03
2.6
NAb
NAb
46.9
33.5
1.4
0.7
0.09
0.09
0.7
0.4
NAb
NAb
47.7
53.8
24
25
0.09
0.1
0.8
0.8
NDc
NAb
248
31
65
NAb
NAb
NDc
39.1
NAb
98.0
28
14
64
18
3.6
258
36
NAb
4.6
Vancomycin
NDc
NDc
4
1
NDc
a Inhibition of human strains reported with IC50 values in μM, activity against pathogenic species is shown as MIC values (μg mL−1). b NA = Not
active at 100 μM. c Not determined.
solution was cooled to −78 °C and BF3·Et2O (8.07 mL,
10.3 mmol) was added followed by the dropwise addition of
dibromoenone 1 (3.00 g, 10.3 mmol) in 5 mL THF. The reaction
was stirred at this temperature for 45 min before the solution was
poured over sat. aq. NH4Cl (150 mL) and 25% aq. NH3
(30 mL). The resulting mixture was stirred for 1 h at room temp-
erature before it was extracted with EtOAc (3 × 50 mL). The
combined organics were washed with sat. aq. NH4Cl (25 mL),
H2O (25 mL), and brine (25 mL), dried over Na2SO4, filtered
and concentrated in vacuo. Flash chromatography of the crude
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(1S,5R)-4-sec-Butyl-3-bromo-8-oxa-bicyclo[3.2.1]octa-3,6-dien-
2-one (2c). Dibromoenone 1 (500 mg, 1.79 mmol) was subject
to the higher-order cuprate conditions with sec-butyllithium to
afford 2c as a colorless solid (354 mg, 77%, 1 : 1 mixture of dia-
stereomers): Rf = 0.49 (15% hexanes in EtOAc); mp =
68–71 °C; IR (KBr): ν: 2964, 2931, 1697, 1587, 1577, 1460,
residue (SiO2, EtOAc in hexanes) provided the desired
α-bromoenones.
(1S,5R)-3-Bromo-4-isopropyl-8-oxa-bicyclo[3.2.1]octa-3,6-dien-
2-one (2a). Dibromoenone 1 (3.0 g, 10.3 mmol) was subject to
the zinc-cuprate conditions with 2-iodopropane to afford 2a as a
pale yellow solid (2.3 g, 88%): Rf = 0.44 (15% hexanes in
EtOAc); mp = 74–76 °C; IR (NaCl): ν: 2968, 2872, 1698, 1588,
1465, 1325, 1242, 1179, 1071, 1020, 926 cm−1; 1H NMR
(500 MHz, CDCl3) δ 6.76 (ddd, J = 5.8, 2.0, 0.7 Hz, 1H), 6.49
(dd, J = 5.7, 2.2 Hz, 1H), 5.30 (d, J = 2.0 Hz, 1H), 5.06 (dd, J =
2.3, 0.6 Hz, 1H), 3.15 (hept, J = 6.9 Hz, 1H), 1.22 (d, J =
6.9 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 186.12, 168.47, 140.48, 130.39, 113.51, 86.80,
79.79, 33.12, 19.76, 18.35; HRMS (ESI) calcd for C10H12BrO2
[M + H]+: 243.0021; found: 243.0016.
1
1070, 927 cm−1; H NMR (500 MHz, CDCl3) δ 6.76 (d, J =
5.7 Hz, 1H), 6.56–6.45 (m, 1H), 5.25 (dd, J = 10.0, 1.9 Hz, 1H),
5.08 (s, 1H), 3.06–2.93 (m, 1H), 1.61 (dd, J = 7.4, 1.5 Hz, 1H),
1.42 (ddt, J = 21.5, 13.7, 7.3 Hz, 1H), 1.22 (d, J = 6.9 Hz,
1.5H), 1.04 (d, J = 6.9 Hz, 1.5H), 0.99 (t, J = 7.5 Hz, 1.5H),
0.87 (t, J = 7.4 Hz, 1.5H). 13C NMR (126 MHz, CDCl3) δ
186.07, 185.93, 168.03, 167.74, 140.67, 140.50, 130.32, 130.30,
114.94, 114.77, 86.88, 86.84, 80.04, 79.76, 40.69, 40.00, 28.05,
26.52, 17.62, 16.58, 12.05, 11.78; HRMS (ESI) calcd for
C11H14BrO2 [M + H]+: 257.0177; found: 257.0172.
(1S,5R)-3-Bromo-4-cyclopentyl-8-oxa-bicyclo[3.2.1]octa-3,6-dien-
2-one (2e). Dibromoenone 1 (500 mg, 1.79 mmol) was subject
to the zinc–cuprate conditions with iodocyclopentane to afford
2e as a white solid (474 mg, 92%): Rf = 0.47 (15% hexanes in
EtOAc); mp = 70–73 °C; IR (KBr): ν: 2973, 2874, 1697, 1577,
(1S,5R)-4-tert-Butyl-3-bromo-8-oxa-bicyclo[3.2.1]octa-3,6-dien-
2-one (2d). Dibromoenone 1 (500 mg, 1.79 mmol) was subject
to the higher-order cuprate conditions with tert-butyllithium to
afford 2d as a pale yellow solid (377 mg, 82%): Rf = 0.51 (15%
hexanes in EtOAc); mp = 68–71 °C; IR (KBr): ν: 2966, 2879,
1066, 927 cm−1 1H NMR (500 MHz, CDCl3) δ 6.78–6.73
;
1701, 1552, 1211, 1083, 935 cm−1 1H NMR (500 MHz,
;
(m, 1H), 6.48 (dd, J = 5.7, 2.2 Hz, 1H), 5.20 (d, J = 1.8 Hz,
1H), 5.05 (d, J = 2.1 Hz, 1H), 3.20–3.10 (m, 1H), 2.09–1.99 (m,
1H), 1.83–1.64 (m, 5H), 1.55 (ddd, J = 18.7, 12.5, 8.7 Hz, 1H),
1.35 (ddd, J = 12.3, 10.5, 5.4 Hz, 1H). 13C NMR (126 MHz,
CDCl3) δ 185.82, 167.22, 140.26, 130.46, 114.39, 86.76, 80.49,
44.31, 30.88, 29.23, 25.95, 25.35; HRMS (ESI) calcd for
C12H13BrO2 [M + H]+: 269.0177; found: 269.0172.
CDCl3) δ 6.84–6.79 (m, 1H), 6.48 (dd, J = 5.7, 2.2 Hz, 1H),
5.49 (d, J = 1.9 Hz, 1H), 5.05 (d, J = 2.2 Hz, 1H), 1.38 (s, 9H);
13C NMR (126 MHz, CDCl3) δ 185.93, 169.29, 140.47, 130.08,
114.47, 86.15, 82.54, 77.29, 77.04, 76.79, 36.69, 28.32; HRMS
(ESI) calcd for C11H14BrO2 [M + H]+: 257.0177; found:
257.0172.
General procedure for Suzuki coupling. Dibromoenone 1
(500 mg, 1.79 mmol), aryl boronic acid (2.86 mmol), and
cesium carbonate (700 mg, 2.15 mmol) were added to 10 : 1
THF–H2O (10 mL) and the mixture was thoroughly degassed by
bubbling argon through the solution (10 min). Bis(triphenylpho-
sphine) palladium(II) dichloride (146 mg, 10 mol%) was added
and the mixture was again degassed with argon. The homo-
genous solution was heated at 70 °C for 5 h before being cooled
to room temperature. Water was added and the mixture was
extracted with EtOAc (3 × 15 mL). The combined organic layers
were washed with brine (25 mL), dried over Na2SO4, filtered
and concentrated in vacuo. Flash chromatography of the crude
residue (SiO2, EtOAc in hexanes) provided the desired
α-bromoenones.
Representative procedure for higher-order cuprate additions.
Freshly purified and flame-dried CuCN (88 mg, 0.982 mmol)
was suspended in dry THF (4.0 mL) and treated with the appro-
priate organolithium or organomagnesium reagent (1.96 mmol)
at −78 °C. The resulting solution was stirred at −78 °C for 1 h
before the addition of chlorotrimethylsilane (0.45 mL,
3.6 mmol) and dropwise addition of dibromoenone 1 (250 mg,
0.893 mmol) in THF (2 mL). The reaction mixture was stirred
for 45 min before being quenched with sat. aq. NH4Cl (5 mL).
The mixture was extracted with EtOAc (3 × 20 mL) and the
combined organics were washed with brine (20 mL), dried over
Na2SO4, filtered and concentrated in vacuo. Flash chromato-
graphy of the crude residue (SiO2, EtOAc in hexanes) provided
the desired α-bromoenones.
(1S,5R)-3-Bromo-4-cyclopropyl-8-oxa-bicyclo[3.2.1]octa-3,6-dien-
2-one (2b). Dibromoenone 1 (250 mg, 0.893 mmol) was subject
to the higher-order cuprate conditions with cyclopropylmagne-
sium bromide to afford 2b as a white solid (151 mg, 70%): Rf =
0.24 (10% hexanes in EtOAc); mp = 115–117 °C IR (KBr):
ν: 1689, 1570, 1257, 1008, 930, 845 cm−1; 1H NMR (500 MHz,
CDCl3) δ 6.69 (dd, J = 5.7, 1.9 Hz, 1H), 6.53 (dd, J = 5.7,
2.2 Hz, 1H), 5.06 (d, J = 2.2 Hz, 1H), 4.62 (d, J = 1.9 Hz, 1H),
2.13 (tt, J = 8.4, 5.1 Hz, 1H), 1.24 (tdd, J = 8.7, 6.7, 5.0 Hz,
1H), 1.10 (tdd, J = 8.5, 6.8, 5.2 Hz, 1H), 1.00 (ddt, J = 9.8, 6.7,
5.0 Hz, 1H), 0.89–0.81 (m, 1H); 13C NMR (126 MHz, CDCl3)
δ 185.14, 165.60, 139.53, 131.25, 114.19, 86.91, 78.82, 15.74,
8.28, 6.81; HRMS (ESI) calcd for C10H10BrO2 [M + H]+:
240.9864; found: 240.9859.
(1S,5R)-3-Bromo-4-(2,5-dimethoxyphenyl)-8-oxa-bicyclo[3.2.1]-
octa-3,6-dien-2-one (2i). Dibromoenone 1 (500 mg, 1.79 mmol)
was subject to the coupling conditions with 2,5-dimethoxy-
phenylboronic acid to afford 2i as a white solid (416 mg, 69%):
Rf = 0.31 (15% hexanes in EtOAc); mp = 129–132 °C; IR
(KBr): ν: 2974, 1699, 1494, 1463, 1286, 1228, 1211,
1
1047 cm−1; H NMR (500 MHz, CDCl3) δ 6.98–6.93 (m, 2H),
6.90 (d, J = 9.0 Hz, 1H), 6.86 (d, J = 2.9 Hz, 1H), 6.55 (dd, J =
5.7, 2.2 Hz, 1H), 5.41 (d, J = 1.8 Hz, 1H), 5.17 (dd, J = 5.4, 4.8
Hz, 1H), 3.84 (s, 3H), 3.78 (d, J = 3.4 Hz, 3H); 13C NMR
(126 MHz, CDCl3) δ 186.18, 161.14, 153.38, 150.22, 141.27,
129.80, 124.65, 116.57, 115.86, 115.11, 112.46, 86.80, 84.34,
56.12, 55.90; HRMS (ESI) calcd for C15H13BrNaO4 [M + Na]+:
358.9895; found: 358.9889.
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(1S,5R)-3-Bromo-4-(2,3,4-trimethoxyphenyl)-8-oxa-bicyclo[3.2.1]-
octa-3,6-dien-2-one (2j). Dibromoenone 1 (500 mg, 1.79 mmol)
was subject to the coupling conditions with 2,3,4-trimethoxy-
phenylboronic acid to afford 2j as a pale yellow solid (473 mg,
72%): Rf = 0.28 (15% hexanes in EtOAc); mp = 105–108; IR
(KBr): ν: 2840, 1699, 1595, 1490, 1463, 1411, 1299, 1209,
give pale yellow crystals). A mixed melting point of the subli-
mate and authentic β-thujaplicin melted at 48–51 °C: IR (KBr):
ν: 3209, 2962, 2929, 1610, 1541, 1473, 1270, 1220, 1105,
1
948 cm−1; H NMR (500 MHz, CDCl3) δ 7.34–7.29 (m, 2H),
7.22 (d, J = 10.7 Hz, 1H), 6.95 (d, J = 10.1 Hz, 1H), 2.89 (hept,
J = 6.9 Hz, 1H), 1.28 (s, 3H), 1.27 (s, 3H) 13C NMR (126 MHz,
CDCl3) δ 171.27, 170.90, 159.97, 137.22, 127.62, 123.23,
122.24, 38.91, 23.36.; HRMS (ESI) calcd for C10H13O2 [M + H]+:
165.0916; found: 165.0906.
1
1076, 1012 cm−1; H NMR (500 MHz, CDCl3) δ 6.98 (d, J =
8.7 Hz, 1H), 6.95 (dd, J = 5.6, 1.6 Hz, 1H), 6.71 (d, J = 8.7 Hz,
1H), 6.53 (dd, J = 5.7, 2.2 Hz, 1H), 5.37 (d, J = 1.8 Hz, 1H),
5.14 (dd, J = 2.2, 0.5 Hz, 1H), 3.89–3.87 (m, 9H); 13C NMR
(126 MHz, CDCl3) δ 186.17, 161.17, 155.22, 150.99, 142.11,
141.10, 129.88, 124.39, 121.85, 115.45, 107.16, 86.64, 84.52,
61.49, 60.99, 55.97; HRMS (ESI) calcd for C16H15BrNaO5
[M + Na]+: 389.0001; found: 388.9995.
(2E,4Z,6Z)-6-Cyclopropyl-2-hydroxycyclohepta-2,4,6-trienone
(6b). The α-bromoenone 2b (215 mg, 0.891 mmol) was subject
to the general procedure and purification A to afford 6b as a
yellow oil (88 mg, 61%): IR (KBr): ν: 3197, 2947, 2929, 1605,
1530, 1482, 1269, 967 cm−1 1H NMR (500 MHz, CDCl3)
;
δ 7.26 (d, J = 10.3 Hz, 1H), 7.20 (d, J = 10.7 Hz, 1H),
7.13–7.08 (m, 1H), 6.83 (d, J = 10.2 Hz, 1H), 1.96 (ddd, J =
13.4, 8.4, 5.1 Hz, 1H), 1.17–1.12 (m, 2H), 0.90–0.86 (m, 2H);
13C NMR (126 MHz, CDCl3) δ 170.75, 170.71, 156.33, 136.69,
126.90, 121.83, 121.54, 20.10, 10.96.; HRMS (ESI) calcd for
C10H11O2 [M + H]+: 163.0759; found: 163.0754.
General procedure for SmI2-mediated ring opening
(purification A)
The α-bromoenone (250 mg, 1.03 mmol) was dissolved in THF
(20.0 mL, 0.05 M) and freshly distilled DMPU (2.00 mL) was
added. Argon was bubbled through the solution for 20 min, after
which time SmI2 (30.9 mL, 3.09 mmol, 3.0 equiv., 0.1 M in
THF) was rapidly added at room temperature. After 30 s, the
reaction was quenched with 10% NaOH (20 mL) and stirred for
6 h at room temperature. THF was removed by rotary evapo-
ration and the resulting aqueous layer was cooled to 0 °C,
acidified to pH = 2 with 2 N HCl and extracted with Et2O
(3 × 25 mL). The combined organics were washed with sat.
aq. Rochelle’s salt (3 × 20 mL) and H2O (3 × 20 mL). [Note:
It is necessary to wash excessively with Rochelle’s salt to break
up samarium chelates and wash with water to remove all traces
of DMPU.] The alkyl tropolones were extracted from the organ-
ics by washing the ether layer with 10% NaOH (3 × 20 mL)
then acidifying the alkaline extract to pH = 2 with 2 N HCl. The
acidic aqueous layer was washed once with ether followed by
extraction with CH2Cl2 (3 × 25 mL). The combined organics
were washed with brine, dried over Na2SO4, filtered and concen-
trated in vacuo to give essentially pure tropolones.
(2E,4Z,6Z)-6-sec-Butyl-2-hydroxycyclohepta-2,4,6-trienone (6c).
The α-bromoenone 2c (202 mg, 0.786 mmol) was subject to the
general procedure and purification A to afford 6c as a yellow oil
(99 mg, 71%): IR (KBr): ν: 3196, 2962, 2930, 1610, 1548,
;
1471, 1265, 943 cm−1 1H NMR (500 MHz, CDCl3) δ
7.35–7.25 (m, 2H), 7.21 (d, J = 10.7 Hz, 1H), 6.90 (d, J =
9.9 Hz, 1H), 2.58 (dt, J = 14.0, 6.9 Hz, 1H), 1.60 (dd, J = 14.7,
7.3 Hz, 2H), 1.23 (d, J = 6.9 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H);
13C NMR (126 MHz, CDCl3) δ 171.04, 170.93, 158.92, 137.15,
128.21, 123.65, 122.38, 46.35, 30.45, 21.35, 12.04; HRMS
(ESI) calcd for C11H15O2 [M + H]+: 179.1072; found: 179.1069.
(2E,4Z,6E)-6-tert-Butyl-2-hydroxycyclohepta-2,4,6-trienone (6d).
The α-bromoenone 2d (200 mg, 0.778 mmol) was subject to the
general procedure and purification A to afford 6d as a yellow oil
(107 mg, 77%): IR (KBr): ν: 3210, 2995, 1608, 1498, 1475,
1
1265 cm−1; H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 1.2 Hz,
1H), 7.32 (d, J = 10.6 Hz, 1H), 7.22 (d, J = 10.8 Hz, 1H), 7.16
(d, J = 10.1 Hz, 1H), 1.34 (s, 9H); 13C NMR (126 MHz, CDCl3)
δ 171.45, 170.34, 161.85, 136.97, 126.35, 123.28, 122.18,
38.22, 30.94; HRMS (ESI) calcd for C11H15O2 [M + H]+:
179.1072; found: 179.1062.
General procedure for SmI2-mediated ring opening
(purification B)
The general procedure was followed as above without extracting
the tropolones from the ether layer (attempts at the acid/base
purification led to significantly lower yields due to partitioning
of the tropolones between the aqueous and organic layers). After
extraction with ether (3 × 25 mL), the combined organics
were washed with sat. aq. Rochelle’s salt (3 × 20 mL), H2O
(3 × 20 mL), brine (25 mL), dried over Na2SO4, filtered and con-
centrated in vacuo. Flash chromatography (SiO2) of the crude
material using 10% EtOAc in hexanes as the eluent provided the
substituted tropolones. [Note: Rf values for the tropolones are
not reported because the compounds streak on TLC plates and
accurate values could not be obtained.]
(2E,4Z,6Z)-6-Cyclopentyl-2-hydroxycyclohepta-2,4,6-trienone
(6e). The α-bromoenone 2e (210 mg, 0.780 mmol) was subject
to the general procedure and purification B to afford 6e as a
thick yellow oil (100 mg, 68%); IR (KBr): ν: 3192, 2984, 1608,
1
1542, 1471, 1263, 733 cm−1; H NMR (500 MHz, CDCl3) δ
7.35 (d, J = 1.5 Hz, 1H), 7.31–7.26 (m, 1H), 7.24–7.18 (m, 1H),
7.01–6.95 (m, 1H), 3.04–2.94 (m, 1H), 2.14–2.06 (m, 2H),
1.88–1.81 (m, 2H), 1.75–1.69 (m, 2H), 1.65–1.59 (m, 2H); 13C
NMR (126 MHz, CDCl3) δ 171.07, 170.79, 158.27, 137.00,
128.24, 123.63, 122.38, 50.54, 34.75, 25.87; HRMS (ESI) calcd
for C12H15O2 [M + H]+: 191.1072; found: 191.1067.
β-Thujaplicin (6a). The α-bromoenone 2a (200 mg,
0.823 mmol) was subject to the general procedure and purifi-
cation A to afford β-thujaplicin as a pale yellow oil (101 mg,
75%): (a sample of 6a was sublimed (80 °C, ∼0.1 mmHg) to
(2E,4Z,6E)-2-Hydroxy-6-phenylcyclohepta-2,4,6-trienone (6f).
The α-bromoenone 2f (200 mg, 0.722 mmol) was subject to the
general procedure and purification B to afford 6f as a white solid
(103 mg, 72%): mp = 87–91 °C IR (KBr): ν: 3186, 1606, 1498,
8602 | Org. Biomol. Chem., 2012, 10, 8597–8604
This journal is © The Royal Society of Chemistry 2012

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1
1465, 1436, 1245, 920 cm−1; H NMR (500 MHz, CDCl3) δ
Anti-proliferative effects of tropolones against MCF-7 and
hDF. MCF-7 cells were maintained in a 1 : 1 mixture of Dulbecco’s
modified Eagle’s medium : Ham’s F-12 (Gibco) supplemented
with non-essential amino acids, L-glutamine (2 mM), strepto-
mycin (500 μg mL−1), penicillin (100 units mL−1) and 10% fetal
bovine serum. Human adult fibroblasts (hDF) were cultured in
DMEM (Life Technologies) supplemented with 10% FBS. Cells
were grown to confluence in a humidified atmosphere (37 °C,
5% CO2). MCF-7 cells were seeded at a density of 3000/well
whereas hDF cells were seeded at 5000/well in clear 96-well
plates and allowed to attach (37 °C, 5% CO2). Compounds, at
varying concentrations in DMSO, were added (1% DMSO final
concentration) and cells returned to the incubator (37 °C, 5%
CO2) for 72 h. At 72 h, the number of viable cells was deter-
mined using an MTS/PMS cell proliferation kit (Promega) per
the manufacturer’s instructions. Cells incubated in 1% DMSO
were used as 100% proliferation and values were adjusted
accordingly. Data was subjected to graphical and statistical
analysis using SigmaPlot or GraphPad software package and
GI50 values obtained for each compound are the result of at least
two separate experiments performed in triplicate.
7.61 (s, 1H), 7.56–7.52 (m, 2H), 7.52–7.37 (m, 4H), 7.32 (d, J =
10.9 Hz, 1H), 7.23 (d, J = 10.0 Hz, 1H); 13C NMR (126 MHz,
CDCl3) δ 172.23, 169.79, 151.61, 142.68, 137.36, 129.13,
128.92, 128.66, 127.82, 123.79, 123.43; HRMS (ESI) calcd for
C13H11O2 [M + H]+: 199.0759; found: 199.0753.
(2E,4Z,6E)-2-Hydroxy-6-(naphthalen-1-yl)cyclohepta-2,4,6-
trienone (6g). The α-bromoenone 2g (200 mg, 0.611 mmol) was
subject to the general procedure and purification B to afford 6g
as pale yellow solid (82 mg, 54%): mp = 129–134 °C IR (KBr):
ν: 3096, 1697, 1681, 1290, 1265, 802 cm−1
;
1H NMR
(500 MHz, CDCl3) δ 8.06 (dd, J = 7.9, 1.3 Hz, 1H), 7.88 (d, J =
8.2 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.61 (td, J = 7.5, 1.4 Hz,
1H), 7.53–7.45 (m, 2H), 7.45–7.44 (m, 1H), 7.43 (d, J = 2.1 Hz,
1H), 7.38–7.32 (m, 2H), 7.28 (dd, J = 7.0, 1.1 Hz, 1H); 13C
NMR (126 MHz, CDCl3) δ 171.33, 141.97, 139.19, 133.27,
132.22, 132.16, 131.97, 130.86, 130.04, 128.16, 127.65, 127.56,
125.97, 125.58, 125.48, 125.07; HRMS (ESI) calcd for
C17H13O2 [M + H]+: 249.0916; found: 249.0902.
(2E,4Z,6E)-2-Hydroxy-6-(4-methoxyphenyl)cyclohepta-2,4,6-
trienone (6h). The α-bromoenone 2h (200 mg, 0.651 mmol)
was subject to the general procedure and purification B to afford
6h as an pale yellow solid (92 mg, 62%): mp = 116–118 °C: IR
Antifungal assay. C. glabrata was stored as a suspension in
50% glycerol at −78 °C. For susceptibility testing, a streak of
stock culture was made on SDA agar and grown at 30 °C for
48 h. One pure colony of the test organism was recovered from
the plate, suspended in appropriate media and grown in a 5 mL
shake flask culture. A sample of the shake flask culture was
diluted to 1 × 105 cells mL−1 in media and added to 96-well test
plates (100 μL per well) containing test compounds dispensed in
DMSO (1 μL). Amphotericin and ketoconazole were used as
controls. After an incubation period determined from the strain
specific doubling time, Alamar Blue (10 μL) was added, allowed
to incubate; each well was scored for dye reduction (Davey
et al., 1998). The MIC value was taken as the lowest concen-
tration of test compound that inhibits growth such that less than
1% reduction of the blue resazurin (λmax 570 nm) component of
the Alamar Blue to the pink resorufin (λmax 600 nm) was
observed.
1
(KBr): ν: 3192, 1601, 1514, 1456, 1292, 1180 cm−1; H NMR
(500 MHz, CDCl3) δ 7.59 (d, J = 1.6 Hz, 1H), 7.52–7.47 (m,
2H), 7.41 (dd, J = 14.5, 6.7 Hz, 1H), 7.26 (dd, J = 9.2, 7.4 Hz,
1H), 7.24–7.19 (m, 1H), 7.01–6.93 (m, 2H), 3.85 (s, 3H);
13C NMR (126 MHz, CDCl3) δ 171.93, 169.76, 160.44, 151.24,
137.25, 134.82, 129.16, 128.21, 123.11, 123.09, 114.35, 55.36;
HRMS (ESI) calcd for C14H13O3 [M + H]+: 229.0865; found:
229.0860.
(2E,4Z,6E)-2-Hydroxy-6-(2,5-dimethoxyphenyl)cyclohepta-
2,4,6-trienone (6i). The α-bromoenone 2i (130 mg,
0.386 mmol) was subject to the general procedure and purifi-
cation B to afford 6i as a thick yellow oil (65 mg, 65%): IR
1
(KBr): ν: 3206, 1659, 1503, 1229, 1160, 980 cm−1; H NMR
(500 MHz, CDCl3) δ 7.52 (s, 1H), 7.40 (dd, J = 12.8, 8.0 Hz,
1H), 7.35–7.30 (m, 1H), 7.15 (d, J = 9.9 Hz, 1H), 6.93 (t, J =
3.3 Hz, 2H), 6.84 (t, J = 1.6 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H);
13C NMR (126 MHz, CDCl3) δ 172.17, 169.53, 153.68, 150.12,
149.25, 136.72, 132.61, 130.65, 125.47, 124.09, 116.15, 114.85,
112.68, 56.23, 55.85; HRMS (ESI) calcd for C15H15O4
[M + H]+: 259.0970; found: 259.0965.
Antibacterial assay. Minimum inhibitory concentrations
(MIC) against E. faecalis and MRSA were determined using a
broth microdilution approach based upon CLSI (formerly
NCCLS) standards and the use of the colorimetric reporter
Alamar Blue. The MIC value is the lowest concentration of test
compound that inhibits growth such that less than 1% reduction
of the blue resazurin (λmax 570 nm) component of the Alamar
Blue to the pink resorufin (λmax 600 nm) is observed.
(2E,4Z,6E)-2-Hydroxy-6-(2,3,4-trimethoxyphenyl)cyclohepta-
2,4,6-trienone (6j). The α-bromoenone 2i (130 mg,
0.386 mmol) was subject to the general procedure and purifi-
cation B to afford 6i as a thick yellow oil (65 mg, 65%): IR
Acknowledgements
1
(KBr): ν: 3206, 1659, 1503, 1229, 1160, 980 cm−1; H NMR
We would like to thank the National Science Foundation for
support of this work. We would also like to acknowledge
Dr Philip Pelphrey and Dr David Bolstad for preliminary syn-
thetic experiments.
(500 MHz, CDCl3) δ 7.52 (s, 1H), 7.40 (dd, J = 12.8, 8.0 Hz,
1H), 7.35–7.30 (m, 1H), 7.15 (d, J = 9.9 Hz, 1H), 6.93 (t, J =
3.3 Hz, 2H), 6.84 (t, J = 1.6 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H);
13C NMR (126 MHz, CDCl3) δ 172.17, 169.53, 153.68, 150.12,
149.25, 136.72, 132.61, 130.65, 125.47, 124.09, 116.15,
114.85, 112.68, 56.23, 55.85; HRMS (ESI) calcd for C15H15O4
[M + H]+: 259.0970; found: 259.0965.
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