Total synthesis confirms laetirobin as a formal diels-alder adduct

Article abstract of DOI:10.1002/asia.200900306

Laetirobin, isolated from a parasitic fungus host-plant relationship, was synthesized in six practical steps with an overall yield of 12% from commercially available 2,4-dihydroxyacetophenone. Because the product is a pseudosymmetric tetramer of benzo[b]-

Full text of DOI:10.1002/asia.200900306

FULL PAPERS
DOI: 10.1002/asia.200900306
Total Synthesis Confirms Laetirobin as a Formal Diels–Alder Adduct
Oliver Simon,[a] Bastien Reux,[a] James J. La Clair,[b] and Martin J. Lear*[a]
Dedicated to the 80th Anniversary of Chemistry in Singapore (1929–2009)
Abstract: Laetirobin, isolated from a
parasitic fungus host–plant relationship,
was synthesized in six practical steps
with an overall yield of 12% from
commercially available 2,4-dihydroxya-
cetophenone. Because the product is a
pseudosymmetric tetramer of benzo[b]-
furans, each step of the synthesis was
designed to involve tandem operations.
Highlights include: 1) the double Sono-
gashira reaction of a bis
practical copper(I)-mediated formation
of a bis(benzo[b]furan), and 3) the bio-
mimetic [4+2] dimerization and unex-
pected cationic [5+2] annulation of
(alkyne), 2) the
gem-diaryl alkene precursors. Prelimi-
nary structure–activity relationship
data between the isomeric [4+2] and
[5+2] tetramers revealed only the natu-
ral product to possess promising anti-
cancer potential. Specifically, laetirobin
is capable of blocking tumor cell divi-
sion (mitosis) and invoking pro-
grammed cell death (apoptosis).
AHCTUNGTRENNUNG
Keywords: antitumor agents · bio-
mimetic synthesis · cycloaddition ·
dimerization · total synthesis
Introduction
Soon after elucidating the structure of (Æ)-1,[1] we recog-
nized that laetirobin could arise naturally through homodi-
Using convenient immunoaffinity-fluorescent (IAF) labeling
and cell phenotype screening techniques, we recently isolat-
ed and characterized (Æ)-laetirobin (1) as a novel cytostatic
agent from the fruiting bodies of the fungus Laetiporus sul-
phureus.[1] As an unprecedented fusion of four 5-aceto-6-hy-
droxybenzo[b]furan units, laetirobin (Æ)-1 potently inhibit-
ed the cell division of HCT116, Neuro-2a, and HeLa tumor
cell lines with IC50 values of 0.1–0.5 nm. Interestingly, the
natural product (Æ)-1 was only produced in fungi found
growing parasitically on the black locust tree, Robinia pseu-
doacacia; sources of fungi found growing in saprobic envi-
ronments, or sources of the living plant host alone, did not
produce (Æ)-1.
meric Diels–Alder cyclization of the bisACTHNGUTERN(UNG benzo[b]furanyl)
alkene 2 (Scheme 1). In attempts to identify 2 in natural
specimens, we reexamined our L. sulphureus extracts. Al-
though conclusive NMR data was not obtained, we found
0.9 mg of an unstable compound from the EtOAc extract of
the XRI2009 specimen with an exact mass of m/z 376.0938,
which could be correlated to a calculated value of m/z
376.0947 for the putative natural precursor 2 (C22H16O6).[1]
Our primary natural sources of laetirobin allowed us to
complete structure elucidation and initial cellular activity
studies. However, we needed a practical supply of (Æ)-1 for
mode-of-action and preclinical studies. Natural harvesting
[a] O. Simon, Dr. B. Reux, Prof. M. J. Lear
Department of Chemistry, Faculty of Science and
Medicinal Chemistry Program of the Life Sciences Institute
National University of Singapore
Singapore 117543 (Singapore)
Fax : (+65)6516-3998
E-mail: chmlmj@nus.edu.sg
[b] Dr. J. J. La Clair
Xenobe Research Institute
3371 Adams Avenue
San Diego, California 92116 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900306.
Scheme 1. Biosynthetic hypothesis to form laetirobin [(Æ)-1] via the im-
plicated alkene 2.
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Chem. Asian J. 2010, 5, 342 – 351

was considered to be unlikely to achieve sufficient quanti-
ties. Similarly, culturing of the host–parasite relationship was
considered impractical. We thus decided to develop a total
synthesis capable of providing access to gram-scale quanti-
ties of (Æ)-1. Herein, we describe a biomimetic approach
through a formal Diels–Alder dimerization of the proposed
natural precursor 2. Subsequent in vitro evaluation of the
formal [4+2] adduct (Æ)-1 in tumor cells, not only con-
firmed the unusual mitotic activity,[1] but also revealed cyto-
toxic activity associated with programmed cell death (apop-
tosis).
Results and Discussion
Given these preliminary observations and our interest in ex-
ploring the mode of action of laetirobin, we elected to
pursue a biomimetic synthesis[2] of (Æ)-1 through dimeriza-
tion[3] of alkene 2.[4] Although several retrosynthetic routes
to 2 were evaluated, we recog-
Scheme 2. Symmetry-driven strategy to form the bis
alkene (2) via the bis(alkyne) tertiary carbinols 3 and 4.
ACHTUNGTREN(NUGN benzo[b]furanyl)
AHCTUNGTRENNUNG
nized that the alkene 2 could
be prepared from the tertiary
Table 1. Representative bisACTHNUTRGNE(UNG alkyne) carbinols to improve purification and stability.
carbinols 3 and 4 in an appro-
priately functionalized form (as
given by R1 in Scheme 2). Bis-
ACHTUNGTRENNUNG(benzo[b]furan) formation and
subsequent elimination of water
or an alcohol (R1OH) from 3
would then facilitate the syn-
thesis of 2 and hence (Æ)-1.
The underlying tactic here was
to maximize the efficiency of
the assembly of (Æ)-1 through
a sequence of dual functionali-
zation steps. In effect, this pro-
cess would reduce the custom-
Product
R1 [a]
Protection conditions[b]
Yield[c]
6a
6b
4b
TMS
TBS
Bn
TMSCl, im, DMF, room temperature, 30 min
79%
11%
67%
TBSOTf, 2,6-lutidine, CH2Cl2, room temperature, 2 h
BnBr, NaH, TBAI, DMF, room temperature, 8 h[d]
[a] TBS=tert-butyldimethylsilyl; Bn=benzyl. [b] Ethynylmagnesium bromide was first treated with 5 in THF
at reflux for 1 h, after which the indicated protection step was performed. [c] Yields of isolated products after
silica gel chromatography. [d] Reaction proceeds with concomitant cleavage of the TMS group.
ary number of synthetic operations by half.
We began our studies by making an appropriately func-
tionalized bisACHTUNGTRENNUNG(alkyne) 4. Unfortunately, the known carbinol
The synthesis of 11 began by identifying a means to di-
rectly monoiodinate commercially available 2,4-dihydroxya-
cetophenone (7) without resorting to protecting group ma-
nipulations (Scheme 3). After several conditions were
screened, iodine monochloride (ICl) gave the known mono-
iodide 8[9] in 50% yield under the procedure of Blagg and
co-workers.[10] In glacial acetic acid, the desired 5-iodo com-
pound 8 was formed as a 1:1 mixture along with the 3-iodo
side product 12. Alternative solvents led to a decrease in the
desired selectivity; for example, diethyl ether and acetoni-
trile gave 1:2 and 2:3 ratios, respectively, of 8 to 12, as well
as minor amounts of diiodide 13. We therefore settled on
using ICl in acetic acid and purified 8 on an 8–10 g scale by
silica gel chromatography.
4a (R1 =H),[5] prepared by addition of excess ethynyl mag-
nesium bromide to methyl acetate, was difficult to isolate
and co-evaporated with solvents typically used during chro-
matographic and distillation techniques. Although a subli-
mation method could be used,[5] we searched for a derivative
of 4 that was easier to handle. A solution arose through the
reaction of commercial 4-trimethylsilyl-3-butynyl-2-one (5)
with ethynylmagnesium bromide directly followed by pro-
tection of the carbinol (Table 1). The use of the silylated
ynone 5 was found to be important to prevent suspected
side reactions resulting from Michael-type additions and
alkyne deprotonations, which allowed the bis-silylated com-
pounds 6a[6] and 6b[7] to be prepared cleanly. Eventually, we
discovered conditions to form multigram amounts of the
crystallizable derivative 4b (R1 =Bn) directly from 5.[8] No-
tably, the benzylated derivative 4b conferred appropriate
stability and reactivity to prepare the bis
ACHTUNGTREN(NUGN benzo[b]furanyl)
carbinol 11 at gram-scale quantities (Scheme 3).
Chem. Asian J. 2010, 5, 342 – 351
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343

M. J. Lear et al.
FULL PAPERS
oughly degassed solvents and stoichiometric amounts of
freshly prepared 4b and 9, palladium coupling proceeded re-
producibly at multigram scales (Table 2, entry 5). We have
since adapted this process to provide over 8 g of compound
10.
Having a supply of compound 10 on hand, we attempted
to form 11 under concomitant tosyl deprotective/benzo[b]-
furan conditions (Scheme 3). Treatment of 10 with potassi-
um carbonate in methanol, however, yielded only the depro-
tected material
3 in an uncontrolled fashion (0–75%
yield).[9] This reaction also proved to be effective only on
scales of less than 50 mg. Further attempts at a deprotective
cyclization to form directly the bisACTHNUTRGNEUNG(benzo[b]furan) 11 were
unsuccessful. After further screening, we determined that
the stepwise conversion of intermediate 10 to 11 via 3 was
more reliable. Thus, the phenol 3 was first generated from
the tosylate 10 by using freshly activated magnesium in
methanol[12] and then cyclized to 11. The key bis
ACTHUNRTGNE(GNU phenolic)
closure to 11, however, proved to be difficult to achieve at
gram scales. Palladium-based cyclization methods often af-
forded low yields.[11,13] Subsequent studies indicated that the
Scheme 3. Gram-scale synthesis of the bisACTHNUTRGNEU(GN benzo[b]furan) 11. a) ICl,
AcOH, room temperature, 2 h, 50%; b) TsCl, acetone, reflux, 45 min,
94%; c) 4b [0.5 equiv], PdCl2, PPh3, CuI, Et3N, DMF, 808C, 4 h, 81%;
d) K2CO3, wet CH3OH, reflux, 1 h; e) Mg, CH3OH, room temperature,
20 h, 61%; f) CuI [25 mol%], Et3N, DMF, 608C, 2 h, 75 %.
addition of 25 mol% of copper(I) iodide to the bis
provided an effective entry to the desired bis-
(benzo[b]furan) 11 through a modified Stephens–Castro re-
action.[14]
We soon found the seemingly simple generation of the
ACHTUNGTRENNUNG(alkyne)
3
AHCTUNGTRENNUNG
We then protected the phenolic moieties of 8 as the bis-
A
proposed alkene 2, or the direct dimerization of carbinol 11
to (Æ)-1,[3a] to be a nontrivial task (Scheme 4). We planned
to generate 2 by treating 11 to Brønsted or Lewis acid con-
ditions that would eliminate benzyl alcohol. Under varying
solvents, mild Brønsted acids, including acetic and citric
acid, gave recovered starting material. Stronger acids, in-
cluding p-toluenesulfonic acid (pTsOH) and trifluoroacetic
acid (TFA), returned complicated mixtures. Eventually, the
selection of hydrochloric acid in the presence of protic
media proved to be successful in generating (Æ)-1. Synthetic
2, however, was neither isolated nor observed (by LC–MS)
in all attempts. Optimally, the prolonged exposure of 11 to
1% hydrochloric acid in wet methanolic dichloromethane
provided the desired [4+2] adduct laetirobin (Æ)-1 in a
clean, direct, and reproducible manner (Scheme 4). Synthet-
ic (Æ)-1 was found to be identical in all respects to natural
(Æ)-1 by NMR spectroscopic, X-ray crystallographic, and
HRMS analyses.[1]
bisACHTUNGTRENNUNG(acetate) versions of 9 were found to be incompatible
with palladium coupling methods. Tosyl protection not only
provided stable and reactive aryl materials for the palladi-
um-coupling step, but was also orthogonal to the benzyl
ether protection in 4b. The key one-pot double-Sonogashira
reaction between the bisACTHNUTRGENNU(G alkyne) 4b and the bisACHTUGNTREN(NUGN tosylate) 9
was studied under the PdII/CuI conditions of Larock.[11] Rep-
resentative experiments are provided in Table 2. On a small
scale (less than 100 mg of 4b), microwave irradiation gave
reliable results with stoichiometric amounts of 9 (Table 2,
entries 1 and 2). As scales increased to above 100 mg, micro-
wave heating resulted in lower yields, requiring the reaction
to be conducted in multiple batches in series (Table 2,
entry 3). Although thermal heating of 4b returned yields
below 40% on small scales (Table 2, entry 4), by using thor-
Table 2. One-pot Sonogashira coupling of 4b and 9 to afford 10.[11]
When we employed Lewis acids such as boron tribromide
or boron trichloride to generate 2 from 11, product (Æ)-15,
initially suspected to be the [4+2] regioisomer of laetirobin
(1), was isolated (Scheme 4). Subsequent X-ray and NMR
studies showed (Æ)-15 to be a [5+2] adduct. The structural
isomer (Æ)-15 presumably arose by stepwise, cationic cycli-
zation of a molecule of 2 with the stabilized carbocation 14
(Scheme 4). This Lewis acid catalyzed intermolecular [5+2]
cyclization differs from the known methods of forming
seven-membered carbocycles.[15,16] To validate this cationic
chemistry and to screen conditions to isolate alkene precur-
Entry
Scale[a]
Equiv. of 9[b]
Conditions[c]
Yield of 10[d]
1
2
3
4
5
25 mg
25 mg
250 mg[f]
25 mg
1.72 g
2
mw 1008C, 25 min
mw 1008C, 25 min
mw 1008C, 25 min
thermal 608C, 6 h[g]
thermal 808C, 4 h[h]
68%
40–60%
63%
39%
81%
3, 4, or 6[e]
2
2
2
[a] Quantity of 4b used. [b] Relative to one equivalent of 4b. [c] Each re-
action was conducted in DMF/Et3N (1:3) with PdCl2 [10 mol%], PPh3
[0.2 equiv], and CuI [0.1 equiv]. mw=microwave. [d] Yields of isolated
product after silica gel chromatography. [e] Similar yields were obtained.
[f] Ten microwave vials of 4b (10ꢁ25 mg) were used in sequence and
then combined on workup. [g] Similar yields were obtained when con-
ducted at 808C or 1008C. [h] DMF/Et3N was thoroughly degassed for at
least 1 h prior to the reaction.
sors, we prepared the simplified bis
ACHTUNGERTN(NUNG benzo[b]furanyl) carbi-
nol 16 (Scheme 5).[17] Indeed, treatment with Burgess re-
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Chem. Asian J. 2010, 5, 342 – 351

Laetirobin as a Formal Diels–Alder Adduct
Scheme 5. Suggested [4+2] and [5+2] formal dimerization modes of the
simplified bis(benzo[b]furanyl) carbinol 16. a) Et3NSO2NCO2Me, THF,
AHCTUNGTRENNUNG
room temperature, 20 h, 89%; b) BCl3, CH2Cl2, À788C, 5 min, 32%. X-
ray crystal structures are shown below each adduct: (Æ)-18 crystallized
as a 1:1 mixture of benzo[b]furanyl conformers.
As the adducts are inherently fluorescent at comparable
wavelengths, the cellular uptake of each structural isomer
(Æ)-1 and (Æ)-15 was quantified by fluorescence densitome-
try (Figure 1). Isomers (Æ)-1 and (Æ)-15 reached an intra-
cellular saturation of 10Æ1 mm and 18Æ3 mm, respectively,
within 15 min, by the addition of 500 mL of 1 nm solution to
a 35 mm diameter dish containing 106 cellscmÀ2 (see the Ex-
perimental Section). These results suggested that both (Æ)-1
and (Æ)-15 rapidly concentrated up to 5000 fold within live
HCT116 tumor cells.
During the 3 h treatment of live HCT116 cells (Figure 1),
laetirobin (Æ-1) was observed to travel from the nucleolus
(nu; (Æ)-1 appearing red) to the vesicles (ly; (Æ)-1 appear-
ing green). The change in color fluorescence from red to
green was attributed to a combination of biomolecular bind-
ing events and changes in the environment within each sub-
cellular region. Subsequent treatment of cells with a panel
of organelle stains indicated that the targeted vesicles (ly)
were lysosomes (see the Supporting Information for co-
Scheme 4. Total synthesis of laetirobin (1) and discovery of iso-laetirobin
(15) by the dimerization of bisACTHNUGRTENUNG(benzo[b]furanyl) carbinol 11. a) 1% (v/v)
1m HCl, CH2Cl2/MeOH (95:5), room temperature, 30 h, 64%; b) BCl3,
CH2Cl2, À788C, 5 min, 24%. X-ray crystal structures are shown below
each adduct as a 1:1 solvent complex: (Æ)-1·CHCl3 and (Æ)-15·CH3OH.
agent led to the [4+2] adduct (Æ)-18 efficiently, presumably
via the alkene intermediate 17.[18] Treatment with boron tri-
chloride alternatively led to the [5+2] adduct (Æ)-20, pre-
sumably by stepwise addition of 17 onto the cationic inter-
mediate 19 followed by proton loss. The structures of (Æ)-
18 and (Æ)-20 were both confirmed by X-ray crystallogra-
phy, but again isolation of an alkene precursor (17 in this
case) remained elusive.
Intrigued by the structure–activity differences that the
pseudosymmetrical tetrameric benzofuran compounds may
possess, we examined the relative behavior of the [4+2] and
[5+2] adducts in human colorectal carcinoma HCT116 cells.
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M. J. Lear et al.
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Figure 2. Laetirobin (1) selectively induces apoptosis. a) An image depict-
ing an HCT116 cell that underwent apoptosis after treatment with 10 nm
1 for 24 h. The cell was stained for membranes with BODIPY-FL ceram-
ide (green) and oligonucleotides with Syto-60 (red). Condensed chroma-
tin (cc) and blebs (bl) are noted. Bar denotes 10 mm. b) A caspase-3
assay[19] indentified increased proteolytic, hence, apoptotic activity after
24 h exposure to (Æ)-1 and not (Æ)-15, relative to untreated cells (nega-
tive control) and cells inhibited with Ac-DEVD-CHO (positive control).
c,d) Compacting of chromatin was evaluated in HCT116 cells using a
FACS assay. Hoechst 33342 and propidium iodide were used as stains to
distinguish normal (N), apoptotic (A), and dead (D) cell populations.[20]
Figure 1. Three-color confocal microscopic images depicting the uptake
of laetirobin [(Æ)-1] and iso-laetirobin [(Æ)-15] in HCT116 tumor cells.
Both compounds fluoresce at multiple wavelengths as given by: red (R:
633 nm laser with emission filtered at 692Æ40 nm); green (G: 543 nm
laser and emission filtered at 593Æ40 nm); and blue (B: 405 nm laser
with emission filtered at 447Æ60 nm) fluorescence. Each image was col-
lected under identical optical parameters (intensity of excitation, mode
of filtration, mode of image capture, magnification and time of expo-
sure). Cellular regions are noted by: (er) endoplasmic reticulum, (ly)
vesicles/lysosomes, and (nu) nucleolus. Scale bar denotes 10 mm.
Conclusions
Through tandem functionalization steps, we achieved a scal-
able and reproducible total synthesis of laetirobin (1) in six
steps from 2,4-dihydroxyacetophenone (7) with an overall
yield of 12% (Schemes 3 and 4). The success of the
[4+2] dimerization of 11 is noteworthy, and we believe that
it proceeds through the naturally implicated, but unverified,
alkene 2. Among plausible biomimetic Diels–Alder reac-
tions,[2,3] there are few accounts[4a–d] on achieving normal
[4+2] or inverse [2+4] reactivity with substituted benzo[b]-
furanyl alkenes akin to 2 (cf. Scheme 1).[4e–h] Stepwise, asyn-
chronous Diels–Alder mechanisms (e.g., via diradical or
zwitterionic dimers of 2) might also be operating, and the
cationic [5+2] dimerization of diaryl carbinols (cf. 11 and
16) was an unexpected finding.[15,16]
Clearly, definitive annulation mechanisms to form the re-
sultant adduct (Æ)-1 via alkenes (like 2) or carbinols (like
11) remains to be proven in a natural or synthetic context;
for example, our empirical data suggest that a protic envi-
ronment is required, and we have not observed nor isolated
the regioisomeric [4+2] adduct of (Æ)-1. Thus, rationales fa-
voring stepwise over concerted mechanisms, and vice-versa,
may be argued equally given the current evidence. In addi-
staining images). This result suggested that a change in pH
commonly observed in the lysosomes was a possible factor
for this color change.
In contrast, iso-laetirobin [(Æ)-15] was first observed
throughout the cytoplasm and the nucleolus (nu), and trav-
eled to the endoplasmic reticulum (er) over the 3 h period
(Figure 1). This event was again accompanied by an increase
in green fluorescence. Although the fluorescence changes of
(Æ)-1 and (Æ)-15 cannot be ascribed to a specific molecular
event at this point, these studies indicated a distinct differ-
ence in the intracellular trafficking of the two related tetra-
meric isomers.
The differences in bioactivity were particularly evident
within cells treated with (Æ)-1 after 3 h, whereby laetirobin
displayed a clear cytotoxic response (GI50 value of 0.16Æ
0.03 mm) and apoptotic activity after 24 h (Figure 2a). This
phenotype was not evident in cells treated with the isomer
(Æ)-15 (GI50 value of 90.4Æ6.3 mm) over the same time
period. The entry of cells treated with (Æ)-1 into an apop-
totic pathway was confirmed by measuring increases in cas-
pase-3 activity[19] (Figure 2b) and chromatin condensation[20]
(Figure 2c,d).
tion, biosynthetic rationales to forming bisACTHNUGRTENUNG(benzo[b]furan)s
resembling 2 or 11 are also arguable within fungus–plant
parasitism; for example, it is difficult to discern the producer
organism(s), and intervening bacteria may be participating
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Chem. Asian J. 2010, 5, 342 – 351

Laetirobin as a Formal Diels–Alder Adduct
in the production of (Æ)-1.[21] The unique biosynthetic as-
sembly of laetirobin [(Æ)-1] remains an intriguing question
to be answered.
column chromatography (5:1 hexane/EtOAc) to afford compound 9 in
94% yield (5.3 g, 8.66 mmol) as white crystals. IR (KBr): n˜ =2960, 2927,
1695, 1596, 1585, 1466, 1355 cmÀ1 1H NMR (500 MHz, CDCl3): d=8.01
;
(s, 1H), 7.75 (dd, J=1.4, 6.8 Hz, 2H), 7.71 (dd, J=1.4, 7.0 Hz, 2H), 7.36
(d, J=8.2 Hz, 2H), 7.34 (dd, J=8.2 Hz, 2H), 7.09 (s, 1H), 2.50 (s, 3H),
2.45 (s, 3H), 2.44 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=195.7,
152.6, 148.0, 146.9, 146.7, 141.2, 133.1, 132.3, 131.5, 130.6, 130.3, 129.1,
128.8, 117.9, 88.4, 30.7, 22.1 ppm; EIMS: m/z (%): 586 (10) [M+], 417
(32), 154 (89), 138 (15), 91 (100); HRMS (EI) m/z 585.9623 [calcd M+
585.9617 for C22H19IO7S2].
In closing, it is appropriate to note that laetirobin [(Æ)-1]
is a poorly water-soluble compound that readily racemizes
under physiological conditions and, therefore, is in need of
therapeutic development. Although preliminary structure–
activity relationship (SAR) data between (Æ)-1 and (Æ)-15
are given herein, the detailed pharmacophoric profiling of
laetirobin scaffolds and the biomolecular targeting of natu-
ral product (Æ)-1 are continuing. Currently, laetirobin [(Æ)-
1] stands as the first in its structural class. It is a new, potent
antimitotic agent (IC50 =0.1–0.5 nm) with moderate cytotox-
icity (GI50 =0.16Æ0.03 mm) that displays the ability to
invoke programmed cell death (apoptosis), all desirable at-
tributes in the development of a potential anticancer agent.
{(3-methylpenta-1,4-diyn-3-yloxy)methyl}benzene (4b): A solution of 4-
trimethylsilyl-3-butynl-2-one (13.02 g, 92.8 mmol) in anhydrous THF
(80 mL) was added to a 0.5m solution of ethynylmagnesium bromide in
THF (557 mL, 278.4 mmol) at 08C. After the addition, the reaction mix-
ture was slowly warmed up to room temperature and then heated at
reflux for 1 h. The resulting solution was cooled to room temperature, di-
luted with Et2O (500 mL), and treated slowly with saturated NH4Cl
(300 mL). The organic layer was collected and the aqueous layer was ex-
tracted twice with Et2O (500 mL). The combined organic layers were
washed sequentially with saturated NH4Cl (300 mL), water (300 mL),
and brine (300 mL). The organic layer was dried over Na2SO4 and the
solvent was removed on a rotary evaporator at a temperature below
238C (Note: the product is highly volatile). The resulting brownish oil
was dissolved in anhydrous DMF (240 mL) and cooled to 08C. NaH
(5.6 g. 139 mmol) was added to this solution in portions. After 15 min,
nBu4NI (3.4 g, 9.3 mmol) and benzyl bromide (17.5 g, 102.0 mmol) were
added sequentially. After 18 h, the solvent was removed on a rotary
evaporator at 408C. The crude product was dissolved in water and ex-
tracted three times with Et2O (700 mL). The combined organic layers
were washed with 1m HCl (500 mL), water (500 mL) and brine (500 mL).
The organic layer was dried over Na2SO4 and concentrated by rotary
evaporation. The crude product was purified by flash chromatography
(98:2 hexane/EtOAc) to afford intermediate 4b as a viscous colorless oil,
which crystallized upon storage at À208C for 24 h affording white crystals
of compound 4b in 67% yield (11.5 g, 62.2 mmol). IR (KBr): n˜ =3281,
Experimental Section
Chemical synthesis: All reactions were performed under argon atmos-
phere and stirred magnetically in oven-dried glassware fitted with a
rubber septa. Commercial anhydrous solvents were used throughout and
transferred under an argon atmosphere. Additionally, CH2Cl2 was dried
by distilling over CaH2, and THF was dried by distilling over sodium ben-
zophenone ketyl. Inorganic salts and acids were used in aqueous solution
and are reported in % w/v. Unless otherwise stated, all reagents were ob-
tained from Aldrich, Alfa Aesar, or TCI America and used without fur-
ther purification. Flash chromatography and dry column vacuum chroma-
tography (DCVC) were performed using EMD (40–63 mm particle size)
or Silicycle silica gel (25–40 mm particle size), respectively. Thin layer
chromatography analyses were performed using pre-coated Merck Silica
Gel 60 F254 glass slides and visualized with ultraviolet light or a ceric am-
monium molybdate (CAM) and KMnO4 stain. Rf values were obtained
by elution in the stated solvent ratios (v/v). All solvent mixtures are re-
ported as (v/v) unless noted otherwise. Mass spectral analyses were re-
corded on Finnigan LCQDECA or ThermoFinnigan MAT900XL. High-
resolution mass spectrometric (HRMS) analyses were referenced against
perfluorokerosene. Copies of relevant spectra as well as additional confo-
cal images pictures are provided in the Supporting Information.
3246, 3007, 2861, 2116, 1378 cmÀ1 1H NMR (500 MHz, CDCl3): d=7.39–
;
7.25 (m, 5H), 4.76 (s, 2H), 2.58 (s, 2H), 1.85 ppm (s, 3H); 13C NMR
(125 MHz, CDCl3): d=138.0, 128.6, 128.3, 127.9, 82.7, 72.8, 68.6, 65.8,
30.9 ppm; EIMS: m/z (%): 153 (65), 141 (20), 91 (100), 77 (63); HRMS
(EI) m/z 183.0808 [calcd MÀH 184.0888 for C13H12O].
A small colorless block (0.8ꢁ0.64ꢁ0.16 mm3) of synthetic intermediate
4b was used for X-ray crystallographic data collection at 223(2) K using
MoKa radiation. 7372 reflections were collected and 2458 were unique
(Rint =0.0271). No symmetry higher than monoclinic was observed and
the centrosymmetric alternative P21/c was chosen on the basis of the re-
sults of refinement. Direct methods were used to solve the structure and
all non-hydrogen atoms were refined anisotropically. All H atoms were
placed in idealized locations. For C13H12O: monoclinic, P21/c, a=
12.5415(10), b=6.7365(5), c=13.0122(11) ꢂ, b=102.770(2)8, V=
1072.15(15) ꢂ3, Z=4, 1=1.141 MgmÀ3, R1=0.0468, wR2=0.1108 [I>
2s(I)]. CCDC 752640 contains the supplementary crystallographic data
for structure 4. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre at www.ccdc.cam.ac.uk/data_
request/cif.
2,4-dihyroxy-5-iodoacetophenone (8): Iodine monochloride (10.65 g,
65.6 mmol) in acetic acid (30 mL) was added over 30 min to a solution of
2,4-dihydroxyacetophenone (7) (9.5 g, 62.5 mmol) in glacial acetic acid
(77 mL). After 4 h, the reaction was quenched by addition of a saturated
solution of sodium thiosulfate. The aqueous layer was extracted four
times with ethyl acetate and the combined organic layers were washed
four times with water (40 mL), dried over Na2SO4, and concentrated by
rotary evaporation. The residue was purified by silica gel column chro-
matography (hexane/ethyl acetate, 8:2) to afford iodide 8 (8.688 g, 50%).
IR (KBr): n˜ =3291, 2917, 2735, 1636, 1615, 1270 cmÀ1
;
1H NMR
(300 MHz, CDCl3): d=12.47 (s, 1H), 8.02 (s, 1H), 6.59 (s, 1H), 2.56 ppm
(s, 3H); 13C NMR (300 MHz, CDCl3): d=201.68, 165.20, 160.86, 140.75,
116.80, 103.54, 73.42, 26.23 ppm; EIMS: m/z (%): 278 (80) [M+],263
(100), 136 (25), 108 (12), 41 (37); HRMS (EI) m/z 277.9440 [calcd M+
277.9440 for C8H7IO3].
1,1’-(5,5’-(3-(benzyloxy)-3-methylpenta-1,4-diyne-1,5-diyl)bis(2,4-tosyl-
5,1-phenylene))diethanone (10): A solution of aryl iodide 9 (10.98 g,
18.71 mmol), bis(acetylene) 4b (1.724 g, 9.36 mmol), PPh3 (589 mg,
2.24 mmol), palladium dichloride (199.0 mg, 1.12 mmol), and cuprous
iodide (213.0 mg, 1.12 mmol) in a mixture of degassed DMF/triethyla-
mine (23 mL/70 mL) was heated to 808C for 4 h. 1m HCl (200 mL) was
added, and the mixture was extracted three times with 100 mL ethyl ace-
tate. The combined organic layers were washed successively with 1m HCl
(70 mL), water (70 mL), and brine (70 mL). The organic layer was dried
over Na2SO4 and evaporated under vacuum. The residue was purified by
silica gel chromatography (hexane/ethyl acetate, 65:35, to hexane/ethyl
acetate, 50:50) to afford compound 10 (8.30 g, 81%) as a yellowish wax.
2,4-bisACHTUNGTRENNUNG(tosyloxy)-5-iodoacetophenone (9): A mixture of 2,4-dihydroxy-5-
iodoacetophenone 8 (2.7 g, 9.71 mmol), p-toluenesulfonyl chloride (5.7 g,
29.90 mmol), and anhydrous K2CO3 (11.5 g, 83.21 mmol) in anhydrous
acetone (150 mL) was heated at reflux for 45 min. The reaction mixture
was cooled, filtered through celite, and washed with acetone, and the fil-
trate was concentrated by rotary evaporation. The residue was dissolved
in water and extracted three times with CH2Cl2 (200 mL). The combined
organic layers were washed with 1m HCl (70 mL), water (70 mL), and
brine (70 mL). The organic layer was dried over Na2SO4 and concentrat-
ed by rotary evaporation. The resulting product was purified by flash
IR (NaCl): n˜ =3064, 2925, 1700, 1593, 1480, 1384 cmÀ1
;
1H NMR
(500 MHz, CDCl3): d=7.75 (s, 2H), 7.71 (d, J=8.3 Hz, 4H), 7.69 (d, J=
Chem. Asian J. 2010, 5, 342 – 351
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
347

M. J. Lear et al.
FULL PAPERS
8.4 Hz, 4H), 7.34 (d, J=8.3 Hz, 4H), 7.23 (d, J=8.3 Hz, 4H), 7.11 (s,
4H), 4.73 (s, 2H), 2.49 (s, 6H), 2.46 (s, 6H), 2.34 (s, 6H), 1.83 ppm (s,
3H); 13C NMR (75 MHz, CDCl3): d=195.8, 152.1, 147.4, 146.8, 137.9,
135.3, 134.8, 132.0, 131.5, 130.4, 130.2, 128.7, 128.5, 128.2, 127.9, 117.7,
116.2, 94.2, 77.9, 68.9, 66.8, 30.6, 30.5, 21.9, 21.8 ppm; ESIMS: m/z (%):
1123 (100) [M+Na+], 861 (17), 707 (31); HRMS (ESI) m/z 1123.1775
[calcd M+Na+ 1123.1768 for C57H48O15S4Na].
Synthetic laetirobin [(Æ)-1] was evaluated against natural material in var-
ious deuterated solvents and solvent mixtures because deprotonated
forms and conformers exist during NMR measurements. For consistent
results, (Æ)-1 was allowed to equilibrate in [D5]pyridine for 12–18 h at
room temperature before NMR data were accumulated. Spectra of the
synthetic and natural products are given in the Supporting Information.
For synthetic (Æ)-1: 1H NMR (500 MHz, [D5]pyridine): d=13.03 (s, OH,
1H), 13.00 (s, OH, 2H), 12.99 (s, OH, 1H), 12.96 (s, OH, 1H), 8.23 (s,
1H), 8.23 (s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 7.59 (brm, 1H), 7.33 (s, 1H),
7.32 (brs, 1H), 7.31 (brs, 1H), 7.27 (s, 1H), 6.97 (brs, 1H), 6.88 (brs,
1H), 6.82 (brm, 1H), 5.72 (s, 1H), 4.98 (s, 1H), 4.72 (t, J=5.7 Hz, 1H),
3.09–3.01 (m, 2H), 2.95–2.84 (m, 2H), 2.70 (s, 3H), 2.67 (s, 3H), 2.65 (s,
3H), 2.48 ppm (s, 3H); 13C NMR (125 MHz, [D5]pyridine): d=205.17,
205.14, 205.10, 204.77, 161.98, 161.95, 161.75, 161.73, 160.21, 160.00,
159.39,158.96, 158.20, 154.61, 144.10, 129.03, 127.50, 127.41, 125.51,
125.07, 121.91, 121.56, 120.32, 118.37, 118.33, 118.01, 116.60, 106.84,
106.64, 105.30, 100.71, 100.30, 100.27, 100.17, 64.60, 44.16, 35.26, 31.91,
27.39, 27.38, 26.10 ppm.
1,1’-[5,5’-{3-(benzyloxy)-3-methylpenta-1,4-diyne-1,5-diyl}bis(2,4-hydroxy-
5,1-phenylene)]diethanone (3): For reliability, magnesium turnings were
activated by stirring over 1m HCl for 5 min, filtered, washed with acetone
and diethyl ether, and dried under high vacuum for 2 h. A solution of 10
(8.00 g, 7.26 mmol) and freshly activated magnesium turnings (7.06 g,
290.42 mmol) in methanol (150 mL) was then stirred for 20 h. 10% Citric
acid (300 mL) was added, and the mixture was extracted three times with
ethyl acetate (200 mL). The combined organic layers were washed twice
with water (200 mL) and brine (200 mL). The organic layer was dried
over Na2SO4 and evaporated under vacuum. The resulting crude product
was purified by flash column chromatography (3:2 hexane/EtOAc) to
afford compound 3 in 61% yield (2.14 g, 4.41 mmol) as a clear oil. IR
For natural (Æ)-1: 1H NMR (500 MHz, [D5]pyridine): d=13.03 (s, OH,
1H), 13.00 (s, OH, 2H), 12.96 (s, OH, 1H), 8.23 (s, 1H), 8.22 (s, 1H),
8.18 (s, 1H), 8.10 (s 1H), 7.58 (brs, 1H), 7.33 (s, 1H), 7.31 (brs, 1H),
7.27 (brs, 1H), 6.97 (brs, 1H), 6.87 (brs, 1H), 6.82 (brm, 1H), 5.71 (s,
1H), 4.98 (brs, 1H), 4.72 (t, J=5.7 Hz, 1H), 3.35 (m, 2H), 3.06–3.01 (m,
2H), 2.94–2.86 (m, 2H), 2.69 (s, 3H), 2.66 (s, 3H), 2.64 (s, 3H), 2.47 ppm
(s, 3H); 13C NMR (125 MHz, [D5]pyridine): d=205.17, 205.14, 205.10,
204.79, 162.01, 161.98, 161.78, 161.76, 160.23, 160.02, 159.42, 158.98,
158.22, 154.62, 144.13, 129.05, 127.52, 127.43, 125.51, 125.08, 121.93,
121.57, 120.33, 118.39, 118.35, 118.03, 116.62, 106.86, 106.65, 105.32,
100.74, 100.32, 100.29, 100.19, 64.62, 44.18, 35.28, 31.92, 27.40, 27.38,
26.11 ppm.
(NaCl): n˜ =2922, 1640, 1490, 1411, 1368, 1293 cmÀ1 1H NMR (500 MHz,
;
CDCl3): d=12.72 (s, OH, 2H), 7.78 (s, 2H), 7.40 (d, J=7.5 Hz, 2H), 7.35
(t, J=7.4 Hz, 2H), 7.30 (d, J=7.4 Hz, 1H), 6.49 (s, 2H), 4.89 (s, 2H),
2.57 (s, 6H), 2.05 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=202.6,
166.0, 163.0, 136.0, 128.7, 128.2, 128.0, 127.1, 114.8, 104.1, 103.6, 101.1,
94.1, 78.4, 68.9, 67.3, 31.5, 26.5 ppm; ESIMS: m/z (%): 967 (74)
[2MÀHÀ],637 (12), 505 (28), 483 (100) [MÀHÀ], 375 (14) [M-OBnÀ];
HRMS (ESI) m/z : 483.1455 [calcd MÀHÀ 483.1449 for C29H23O7].
1,1’-[2,2’-{1-(benzyloxy)ethane-1,1-diyl}bis(6-hydroxybenzofuran-5,2-
diyl)]diethanone (11): A solution of dialkyne 3 (2.0 g, 4.13 mmol), cupr-
ous iodide (200.0 mg, 1.05 mmol), and triethylamine (4.2 g, 41.5 mmol) in
anhydrous dimethylformamide (30 mL) was heated at 608C for 2 h. The
reaction mixture was poured into 1m HCl (100 mL) and extracted three
times with Et2O (30 mL). The combined organic layers were washed with
1m HCl (50 mL), saturated NaHCO3 (50 mL), water (50 mL), and brine
(50 mL). The organic layer was dried over Na2SO4, and the solvent was
removed on a rotary evaporator. The resulting crude product was puri-
fied by flash column chromatography (3:1 hexane/EtOAc) to afford 11 in
75% yield (1.51 g, 3.11 mmol) as a light yellow oil. IR (NaCl): n˜ =3435,
A small orange block (0.16ꢁ0.10ꢁ0.08 mm3) of synthetic (Æ)-1 was used
for X-ray crystallographic data collection at 223(2) K using MoKa radia-
tion. 14236 reflections were collected and 9076 were unique (Rint
=
0.0342). No symmetry higher than triclinic was observed and the centro-
ꢀ
symmetric alternative, P1 was chosen on the basis of the results of the re-
finement. Direct methods were used to solve the structure and all non-
hydrogen atoms were refined anisotropically. All H atoms were placed in
ꢀ
2927, 1645, 1448, 1371 cmÀ1 1H NMR (500 MHz, CDCl3): d=12.44 (s,
;
idealized locations. For C44H32O12
:
triclinic, P1, a=10.9989(12), b=
13.8100(14), c=14.2340(15) ꢂ, a=112.139(2), b=90.723(3), g=
94.576(3)8, V=1994.1(4) ꢂ3, Z=2, 1=1.452 Mg/m3, R1=0.0709, wR2=
0.1967 [I>2s(I)]. CCDC 752639 contains the supplementary crystallo-
graphic data for synthetic structure (Æ)-1. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre at
www.ccdc.cam.ac.uk/data_request/cif.
2H), 7.96 (s, 2H), 7.32 (m, 5H), 6.99 (s, 2H), 6.79 (s, 1H), 6.78 (s, 1H),
4.50 (s, 2H), 2.67 (s, 6H), 2.09 ppm (s, 3H); 13C NMR (75 MHz, CDCl3):
d=204.2, 161.3, 159.8, 157.9, 128.8, 128.6, 127.9, 127.7, 124.3, 120.9, 117.4,
105.4, 100.2, 75.1, 66.9, 65.6, 27.1, 23.8 ppm; ESIMS: m/z (%): 483 (28)
[MÀHÀ], 375 (100) [MÀOBnÀ]; HRMS (ESI) m/z: 483.1416 [calcd
MÀHÀ 483.1438 for C29H23O7].
Iso-laetirobin [(Æ)-15]: A 1.0m solution of BCl3 in hexane (154 mL,
0.154 mmol) was added to 11 (50 mg, 0.103 mmol) in anhydrous CH2Cl2
(5 mL) at À788C. The reaction mixture was stirred at this temperature
for 15 min. The reaction was terminated by the addition of water (5 mL)
and the resulting mixture was slowly warmed to 238C over 1.5 h. The re-
action mixture was extracted three times with CH2Cl2 (10 mL). The com-
bined organic layers were washed with 5% NaHCO3 (10 mL), water
(10 mL), and brine (10 mL). The organic layer was dried over Na2SO4,
and the solvent was removed on a rotary evaporator. The resulting crude
product was purified by preparative HPLC on a Waters SunFire C18
OBD 19ꢁ50 mm, 5 mm with a gradient 10 to 100% CH3CN in 30 min to
Synthetic laetirobin [(Æ)-1]: Bis
ACHTUNGTNER(NUNG benzo[b]furan) 11 (797.0 mg,
1.645 mmol) was dissolved in mixture of 5% MeOH in CH2Cl2
a
(50 mL). Subsequently, 1.0m HCl (0.5 mL) was added and the mixture
was stirred for 30 h at 238C under ambient atmosphere. The reaction
mixture was poured into saturated NaHCO3 solution (30 mL) and ex-
tracted three times with CH2Cl2 (20 mL). The combined organic layers
were washed with water (20 mL) and brine (20 mL). The organic layer
was dried over Na2SO4 and the solvent was removed on a rotary evapora-
tor. The resulting crude product was purified by preparative HPLC on a
Waters SunFire C18 OBD 19ꢁ50 mm, 5 mm with a gradient 10 to 100%
CH3CN in 30 min to afford synthetic (Æ)-1 in 64% yield (399 mg,
0.530 mmol) as a light yellow solid. IR (KBr): n˜ =3516, 2925, 1644, 1465,
afford (Æ)-15 in 24% yield, as
a
yellow-brownish oil (9.5 mg,
1H NMR
0.013 mmol). IR: n˜ =3434, 1638, 1452, 1355,1241 cmÀ1
;
1369, 1254 cmÀ1 1H NMR (500 MHz, CDCl3): d=12.45 (s, OH, 2H),
;
(500 MHz, CDCl3): d=12.85 (s, 1H), 12.57 (s, 1H), 12.55 (s, 1H), 12.39
(s, 1H), 7.93 (s, 1H), 7.89 (s, 1H), 7.85 (s, 1H), 7.27 (s, 1H), 7.01 (s, 1H),
6.77 (s, 1H), 6.58 (s, 1H), 6.30 (s, 1H), 6.28 (s, 1H), 6.19 (s, 1H), 4.80 (s,
1H), 4.18 (d, J=18.0 Hz, 1H), 3.68 (d, J=18.0 Hz, 1H), 2.73 (s, 3H),
2.68 (s, 3H), 2.62 (s, 3H), 2.01 (d, J=2.0 Hz, 3H) 1.64 ppm (s, 3H);
13C NMR (125 MHz, CDCl3): d=204.19, 204.13, 204.04, 202.66, 165.92,
163.83, 161.45, 158.59, 155.64, 153.47, 150.83, 127.83, 124.30, 120.16,
116.86, 115.07, 110.14, 105.28, 105.20, 100.32, 99.97, 99.80, 98.82, 47.53,
27.20, 27.15, 27.05, 25.27, 11.69 ppm; ESIMS: m/z (%): 751 (100)
[MÀHÀ]; HRMS (EI) m/z: 752.1892 [calcd M+ 752.1894 for C44H32O12].
12.44 (s, OH, 1H), 12.37 (s, OH, 1H), 7.92 (s, 1H), 7.91 (s, 1H), 7.90 (s,
1H), 7.68 (s, 1H), 7.00 (s, 1H), 6.96 (s, 1H), 6.96 (s, 2H), 6.57 (s, 1H),
6.48 (s, 1H), 6.47 (s, 1H), 6.47 (s, 1H), 4.47 (t, J=5.5 Hz, 1H), 2.76 (m,
1H), 2.69 (m, 1H), 2.66 (s, 3H), 2.65 (s, 6H), 2.36 (m, 1H), 2.35 (s, 3H),
2.32 ppm (m, 1H); 13C NMR (125 MHz, CDCl3): d=204.2, 204.2, 204.1,
204.0, 161.5, 161.5, 161.4, 161.3, 159.8, 159.7, 159.7, 159.6, 158.7, 158.2,
157.3, 153.7, 144.7, 123.9, 123.9, 123.6, 123.3, 121.2, 120.8, 120.7, 119.5,
117.4, 117.2, 117.2, 115.9, 106.0, 105.6, 104.4, 100.4, 100.1, 100.1, 100.0,
43.5, 34.7, 31.3, 27.1, 27.1, 27.1, 26.9, 25.5 ppm. EIMS: m/z (%): 751 (33)
[MÀHÀ]; HRMS (EI) m/z: 752.1882 [calcd M+ 752.1894 for C44H32O12].
348
www.chemasianj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2010, 5, 342 – 351

Laetirobin as a Formal Diels–Alder Adduct
A small block (0.40ꢁ0.20ꢁ0.18 mm3) of synthetic intermediate (Æ)-15
was used for X-ray crystallographic data collection at 223(2) K using
MoKa radiation. 12405 reflections were collected and 8291 were unique
(Rint =0.0276). No symmetry higher than triclinic was observed and the
from The Cambridge Crystallographic Data Centre at www.ccdc.cam.a-
c.uk/data_request/cif.
A
A 1.0m solution of BCl3 in hexane (320 mL,
0.320 mmol) was added to 12 (60 mg, 0.216 mmol) in anhydrous CH2Cl2
(5 mL) at À788C. The deep violet reaction mixture was stirred at this
temperature for 15 min. The reaction was terminated by the addition of
water (10 mL) and slowly warmed to 238C over 1.5 h. The reaction mix-
ture was extracted three times with CH2Cl2 (10 mL). The combined or-
ganic layers were washed with 5% NaHCO3 (10 mL), water (10 mL) and
brine (10 mL). The organic layer was dried over Na2SO4, and the solvent
was removed on a rotary evaporator. Silica gel chromatography gave
18.0 mg of a yellow powder (32%, 0.035 mmol) of the [5+2] cycloadduct
(Æ)-20. For unambiguous structure determination, 13.0 mg of this materi-
al was further purified by preparative HPLC on a Waters SunFire C18
OBD 19ꢁ50 mm, 5 mm column with a gradient 10 to 100% CH3CN in
30 min to afford 4.7 mg of (Æ)-20. IR (NaCl): n˜ =2897, 1638, 1442, 1420,
ꢀ
centrosymmetric alternative P1 was chosen on the basis of the results of
the refinement. Direct methods were used to solve the structure and all
non-hydrogen atoms were refined anisotropically. All H atoms were
ꢀ
placed in idealized locations. For C44H32O12: triclinic, P1, a=11.9232(17),
b=12.0133(16), c=13.613(2) ꢂ, a=97.200(3), b=109.150(3), g=
90.874(3)8, V=1824.1(4) ꢂ3, Z=2, 1=1.400 Mg/m3, R1=0.0759, wR2=
0.2006 [I>2s(I)]. CCDC 752641 contains the supplementary crystallo-
graphic data for synthetic structure (Æ)-15. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre at
www.ccdc.cam.ac.uk/data_request/cif.
1,1-diACHTUNGTRENNUNG(benzo[b]furan-2-yl)ethanol (16): A solution of benzo[b]furan
(100 mg, 0.85 mmol) in freshly distilled THF (3 mL) was added to a solu-
tion of n-butyllithium (0.6 mL of a 1.6m solution in hexane, 0.94 mmol)
in THF (3 mL) at À788C. The solution was stirred at À788C for 1 h,
transferred via cannula to a solution of 2-acetylbenzo[b]furan (136.0 mg,
0.85 mmol) in THF (6 mL), and cooled to À788C. After 2 h at À788C,
the reaction mixture was poured into water (30 mL). The aqueous layer
was extracted three times with diethyl ether (15 mL). The combined or-
ganic layers were washed with water (10 mL) and brine (10 mL). The or-
ganic layer was dried over Na2SO4, and the solvent was removed on a
rotary evaporator. The resulting crude product was purified by prepara-
tive HPLC on a Waters SunFire C18 OBD 19ꢁ50 mm, 5 mm with a gradi-
ent 10 to 100% CH3CN in 30 min to afford 16 in 41% yield (97 mg,
1253 cmÀ1 1H NMR (300 MHz, CDCl3): d=.64 (m, 1H), 7.53 (m, 1H),
;
7.46 (m, 2H), 7.38 (m, 2H), 7.29 (s, 2H), 7.17 (m, 5H), 7.01 (d, J=
8.07 Hz, 1H), 6.66 (t, J=7.56 Hz, 1H), 6.27 (s, 1H), 6.15 (s, 1H), 5.98 (d,
J=7.74 Hz, 1H), 5.01 (s, 1H), 4.25 (d, J=18 Hz, 1H), 3.76 (dd, J=
1.6 Hz, 18 Hz, 1H) 2.06 ppm (d, J=2 Hz, 3H); 13C NMR (125 MHz,
CDCl3): d=158.97, 157.98, 155.88, 154.77, 154.56, 153.86, 153.55, 150.66,
130.96, 128.29, 127.16, 125.54, 122.82, 122.68, 122.03, 121.57, 121.11,
118.32, 111.66, 111.39, 110.90, 110.14, 109.55, 106.74, 104.95, 103.72, 52.64,
47.66, 34.66, 29.93, 11.77, 11.76 ppm. ESIMS: m/z (%): 591 (57), 575
(100), 570 (24), 535 (31), 519 (71) [MÀH+], 419 (27), 247 (20); HRMS
(ESI) m/z: 519.1593 [calcd MÀH+ 519.1591 for C36H23O4].
A small colorless block (0.32ꢁ0.18ꢁ0.08 mm3) of the [5+2] cycloadduct
(Æ)-20 was used for X-ray crystallographic data collection at 223(2) K
using MoKa radiation. 16383 reflections were collected and 5751 were
unique (Rint =0.0241). No symmetry higher than triclinic was observed
0.35 mmol) as
a white solid. IR (KBr): n˜ =3387, 3063, 1450, 1373,
1250 cmÀ1 1H NMR (300 MHz, CDCl3): d=7.54 (d, J=7.23 Hz, 2H),
;
7.44 (d, J=7.89 Hz, 2H), 7.24 (m, 4H), 6.70 (s, 2H) 3.04 (bs, 1H),
2.09 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=158.9, 154.8, 127.9,
124.4, 122.9, 121.2, 111.4, 103.1, 70.1, 25.9 ppm; ESIMS: m/z (%): 277
(100) [MÀ], 159 (28), 113 (22); HRMS (ESI) m/z: 277.0883 [calcd MÀHÀ
277.0870 for C18H13O3].
ꢀ
and the centrosymmetric alternative P1 was chosen on the basis of the
results of the refinement. Direct methods were used to solve the struc-
ture and all non-hydrogen atoms were refined anisotropically. All H
ꢀ
atoms were placed in idealized locations. For C36H24O4: triclinic, P1, a=
1,1,4-tri
G
N
9.6753(4), b=11.4318(5), c=12.7153(5) ꢂ, a=72.7480(10), b=
79.5670(10), g=69.6640(10)8, V=1254.61(9) ꢂ3, Z=2, 1=1.378 Mg/m3,
R1=0.0454, wR2=0.1130 [I>2s(I)]. CCDC 752643 contains the supple-
mentary crystallographic data for synthetic structure (Æ)-20. These data
can be obtained free of charge from The Cambridge Crystallographic
Data Centre at www.ccdc.cam.ac.uk/data_request/cif.
18]: To a solution of compound 16 (30.0 mg, 0.108 mmol) in freshly dis-
tilled THF (4 mL) was added Burgess reagent (77.0 mg, 0.324 mmol).The
reaction mixture was stirred for 20 h. The reaction was poured into water
(20 mL) and extracted three times with diethyl ether (10 mL). The com-
bined organic layers were washed with water (10 mL) and brine (10 mL).
The organic layer was dried over Na2SO4 and the solvent was removed
on a rotary evaporator. The resulting crude product was purified by prep-
arative HPLC on a Waters SunFire C18 OBD 19ꢁ50 mm, 5 mm with a
gradient 10 to 100% CH3CN in 30 min to afford (Æ)-18 in 89% yield
(25 mg, 0.048 mmol) as a white solid. IR (NaCl): n˜ =3064, 2892, 1691,
Cell cultures: HCT116 cells (ATCC CCL-247) were propagated in
McCoyꢃs SA media (GIBCO-BRL), supplemented with 10% inactivated
FCS (FCS=fetal calf serum), and penicillin/streptomycin (GIBCO-
BRL). All cells were incubated at 378C in a 5% CO2 atmosphere. For
routine passage, cells were split 1:6 when they reached confluence, gener-
ally every 1–3 days. Cells were plated in poly-d-lysine-coated glass-bot-
tomed dishes (MatTek Corporation) for microscopic analyses.
1594, 1457, 1241 cmÀ1 1H NMR (300 MHz, CDCl3): d=7.52 (m, 3H),
;
7.39 (m, 2H), 7.31 (m, 3H), 7.19 (m, 4H), 7.11 (m, 1H), 7.05 (s, 1H),
6.87 (t, J=7.40 Hz, 1H), 6.70 (d, J=7.38 Hz, 1H), 6.36 (s, 1H), 5.97 (s,
1H), 5.26 (s, 1H) 2.87 ppm (m, 4H); 13C NMR (125 MHz, CDCl3): d=
159.42, 158.10, 155.06, 155.03, 154.19, 153.80, 153.25, 151.98, 129.50,
129.03, 128.39, 128.12, 124.42, 123.22, 122.83, 121.34, 121.29, 120.66,
111.49, 111.23, 111.01, 110.55, 106.10, 104.92, 103.99, 99.99, 49.34, 45.07,
33.33, 22.61 ppm; ESIMS: m/z (%): 559 (15) [M+K+], 543 (60) [M+Na+],
521 (100) [M+H+], 419 (11), 261 (13) [M/2+H+]; HRMS (ESI) m/z:
543.1569 [calcd M+Na+ 543.1567 for C36H24O4Na].
Cytostatic and cytotoxicity assays: HCT116 cells were grown to
106 cellscmÀ2 in 35 mm glass-bottomed dishes (MatTek Corporation).
Each compound was dissolved at 5 mgmLÀ1 and then diluted to generate
10ꢁ stocks in the required media (DMEM or McCoyꢃs SA media). An
aliquot (50 mL) of each 10ꢁ stock was added to cells covered with media
(450 mL), and the cells were incubated for 12 h at 378C in 5% CO2 at-
mosphere. A live/dead cell staining with a mixture of calcein AM and
ethidium homodimer-1 or C12 resazurin (Invitrogen) and SYTOX Green
(Invitrogen) was used as an end point. Screening for each compound or
extract was typically conducted at 10-fold intervals using stock solutions
from 0.1 ngmLÀ1 to 100 mgmLÀ1. Data are reported as IC50 values based
on the concentration of compound required to reduce the growth to
50% of that observed in the control (DMSO). Once a window of activity
was found, a second round of screening was conducted by reducing the
concentration window to span single digit concentrations within the iden-
tified range. In addition, the cytotoxicity of each compound, expressed as
the concentration at which viability was reduced to 50% (GI50 value),
was screened using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.
A small colorless block (0.48ꢁ0.12ꢁ0.08 mm3) of the [4+2] cycloadduct
(Æ)-18 was used for X-ray crystallographic data collection at 223(2) K
using MoKa radiation. 18023 reflections were collected and 5865 were
unique (Rint =0.0421). No symmetry higher than monoclinic was ob-
served, and the centrosymmetric alternative P21/c was chosen on the
basis of the results of the refinement. Direct methods were used to solve
the structure and all non-hydrogen atoms were refined anisotropically.
All H atoms were placed in idealized locations. For C36H24O4: monoclin-
ic, P21/c, a=10.0478(6), b=28.418(2), c=9.3323(6) ꢂ, b=105.343(2)8,
V=2569.7(3) ꢂ3, Z=4, 1=1.346 Mg/m3, R1=0.0626, wR2=0.1441 [I>
2s(I)]. CCDC 752642 contains the supplementary crystallographic data
for synthetic structure (Æ)-18. These data can be obtained free of charge
Chem. Asian J. 2010, 5, 342 – 351
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
349

M. J. Lear et al.
FULL PAPERS
Cell uptake studies: Uptake studies were conducted by treating cells at a
density of 106 cellscmÀ2 in a 35 mm glass-bottomed dish (MatTek Corpo-
ration). Each compound was added as a 10ꢁ stock in media such that
the net DMSO content remained less than 0.5%. Compounds were
added at a defined stage of the cell cycle and were periodically imaged
throughout this process. Time-lapse imaging was collected by incubating
the cells at 378C under a 5% CO2 atmosphere while on a conventional
fluorescent microscope (Nikon TE 2000). Images were collected at
15 min intervals with a 500 ms exposure to ensure minimal phototoxicity
to the cells or photobleaching of the compounds. Multiple images were
taken per plate and each imaging study was conducted in triplicate and
replicated on multiple passes of cells.
incubating the same cells in parallel without (Æ)-1. The adherent cells
were washed with phosphate buffered saline (PBS), harvested with
0.25% trypsin (GIBCO-BRL), and pelleted by centrifugation at 500ꢁg
for 5 min. The cell pellets were resuspended at approximately 5ꢁ106 cells
per 1.0 mL of 10 mm tris(hydroxymethyl)aminomethane (TRIS) at
pH 7.5, 2m NaCl, 1 mm ethylenediaminetetraacetic acid (EDTA), 0.01%
triton X-100, frozen at À788C, thawed, disrupted by repetitive passage
through at 30 gauge needle, and centrifuged at 5000 rpm for 5 min. Ali-
quots (50 mL) of the resulting supernatant, used as is or inhibited by the
addition of 1 mL of 1 mm Ac-DEVD-CHO (Ac-DEVD-CHO=acetyl-
Asp-Glu-Val-Asp-1-aldehyde; a caspase inhibitor from Promega, Cat.
No. G5961) in DMSO, were added to individual wells of a 96-welled
plate containing 50 mL of 25 mm Z-DEVD-R110 (Z-DVD-R110=bis-(N-
CBZ-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide)rhodamine 110; a
substrate of EnzChek Caspase-3 Assay kit #2 from Invitrogen, Cat. No.
E13184) and 10 mm 1,4-dithiothreitol (DTT) in 20 mm 1,4-piperazine bi-
s(ethanesulfonic acid) buffer (PIPES) at pH 7.4, 4 mm EDTA, and 0.2%
Cell densitometry: Densitometry measurements were collected on a con-
ventional fluorescence microscope (Nikon TE2000) and processed using
Image J software (NIH). Through the addition of 500 mL of 1 nm solution
to a 35 mm diameter dish containing 106 cellscmÀ2, the intracellular con-
centrations of 10Æ1 mm for (Æ)-1 and 18Æ3 mm for (Æ)-15 were obtained
by averaging data over 100–150 cells. The concentrations were standar-
dized against fluorescent microspheres (Invitrogen). The values are un-
corrected against changes in the absorption and fluorescence of each
compound. The two isomeric products displayed comparable emission
(fluorescence) spectra, as measured in 1 nm DMSO. The absorption band
of iso-laetirobin [(Æ)-15] was broad and had a maximum at 472 nm with
an extinction coefficient of 36200Æ500 LmolÀ1 cmÀ1. Iso-laetirobin [(Æ)-
15] fluoresced with a maximum at 531 nm after excitation at 400 nm and
gave a quantum yield of 0.32 (referenced to 0.70 for rhodamine B). The
absorption band of laetirobin [(Æ)-1] was also broad with a maximum at
478 nm with an extinction coefficient of 39100Æ400 cmÀ1 LmolÀ1 in
DMSO. The fluorescence of (Æ)-1 when excited at 400 nm in DMSO had
two peaks. The first appeared with a maximum of fluorescence at 434 nm
with a quantum yield of 0.12 (referenced to 0.70 for rhodamine B). The
second had a maximum at 547 nm with a quantum yield of 0.45 (refer-
enced to 0.70 for rhodamine B). The absorption and emission behavior
of (Æ)-1 and (Æ)-15 displayed solvatochromism, as well as sensitivity to
pH value and salts. The observed colorimetric cellular and solvent re-
sponses are being investigated, and their possible origins will be reported
elsewhere.
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid
(CHAPS). The plate was transferred to a Bioassay 7000 plate reader
(Perkin–Elmer) and evaluated by measuring fluorescence by excitation
at 495 nm and emission at 520 nm. Readings were made at 238C at 5 min
intervals over 45 min. Each experiment was reproduced in triplicate.
Data from these studies are provided in Figure 2b.
Chromatin condensation assays:[20] HCT116 cells, grown to 106 cellscmÀ2
in a 75 mm2 tissue culture dish (BD Falcon), were treated with 25 mL of
1 nm (Æ)-1 in McCoyꢃs SA media (GIBCO-BRL), supplemented with
10% inactivated FCS, and penicillin/streptomycin (GIBCO-BRL) for
12 h at 378C under a 5% CO2 atmosphere. A negative control was pre-
pared by incubating the same cells without (Æ)-1. Cells were washed
with PBS, harvested with 0.25% trypsin (GIBCO-BRL), and pelleted by
centrifugation at 500ꢁg for 5 min. The cells were resuspended at
~106 cellsmLÀ1 in cold PBS. A 1 mL aliquot was stained by treatment
with 1 mL of 5.0 mgmLÀ1 Hoechst 33342 in water and 1 mL of 1 mgmLÀ1
propidium iodide in water followed by incubation on ice for 30 min. The
cells were then analyzed by flow cytometry on FACStar flow cytometer
(Becton Dickinson) using excitation at 355 nm and 488 nm and measur-
ing the fluorescence emission at 450Æ40 nm and 585Æ42 nm, respective-
ly. Over 5000 events were collected for each analysis. Each experiment
was reproduced in triplicate using three independent experiments. Data
from these studies are provided in Figure 2c.
Fluorescence microscopy: Confocal fluorescence images were collected
on a self-built inverted microscope (Xenobe Research Institute) or a
Leica (Wetzlar, Germany) DMI6000 inverted confocal microscope with a
Yokogawa (Tokyo, Japan) spinning-disk confocal head, Orca ER High
Resolution B&W Cooled CCD camera (6.45 mm/pixel at 1X) (Hamamat-
su, Sewickley, PA), Plan Apochromat 40ꢁ/1.25 na and 63ꢁ/1.4 na objec-
tive, and a Melles Griot (Carlsbad, CA) Argon/Krypton 100 mW air-
cooled laser for 488, 568, and 647 nm excitations. Under standard fluores-
cence techniques, the concentrations of compounds (Æ)-1 and (Æ)-15 in
live cells were determined by comparison with 100 mm polystyrene micro-
spheres (Polysciences Inc.) that were impregnated with (Æ)-1 at 10.0Æ
0.5 nm, 100.0Æ0.2 nm, 1.00Æ0.1 mm, 5.00Æ0.1 mm, 10.0Æ0.1 mm, 25.0Æ
0.1 mm, and 50.0Æ0.1 mm, as verified by spectrophotometric analyses.
Cells were cultured in 35 mm glass-bottomed dishes (MatTek Corpora-
tion) and imaged live after treatment with (Æ)-1 or (Æ)-15 and washing
with fresh media. Data from these studies are provided in Figure 1, and
an enhanced-resolution image is given in Figure S1 of the Supporting In-
formation. The subcellular localization of compounds (Æ)-1 and (Æ)-15
was confirmed by co-staining with established organelle compounds by
treating cells exposed to (Æ)-1 or (Æ)-15 to either Syto-60 (nucleus), Ly-
soTracker Red DND-99 (lysosomes), BODIPY TR glibenclamide (endo-
plasmic reticulum), or MitoTracker Red 580 (mitochondria) for 20 min,
washing the cells three times with media, and imaging the cells in two
colors (green from compound (Æ)-1 or (Æ)-15 and red from the organ-
elle stain). Selected co-staining images are provided in Figure S2 of the
Supporting Information.
Acknowledgements
We thank the Ministry of Education of Singapore for funding (AcRF
Tier-2 Grant T206B1112) and the National University of Singapore for
PhD and postdoctoral research scholarships to O.S. and B.R., respective-
ly.
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350
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: July 23, 2009
Published online: December 23, 2009
Chem. Asian J. 2010, 5, 342 – 351
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