Probing for the pharmacophore of the cytotoxic neoclerodane salvileucalin B

Article abstract of DOI:10.1071/CH12358

The novel [4.3.1]propelladiene 2, which embodies the key structural elements of the pentacyclic core of the cytotoxic neoclerodane salvileucalin B (1), has been prepared using a rhodium-catalysed intramolecular Bchner reaction as the key step. Compound 2 and the readily obtained derivatives 12-17 all proved to be essentially inactive when tested against a panel of four human cancer cell lines. Furthermore, not one of these compounds was a P-gp inhibitor.

Full text of DOI:10.1071/CH12358

CSIRO PUBLISHING
Aust. J. Chem. 2012, 65, 1679–1686
Full Paper
http://dx.doi.org/10.1071/CH12358
Probing for the Pharmacophore of the Cytotoxic
Neoclerodane Salvileucalin B
A
A
,
C
A
Nora Heinrich, Martin G. Banwell,
Anthony C. Willis,
A
B
B
Ian A. Cade, Robert J. Capon, and Xiao-Cong Huang
A
Research School of Chemistry, Institute of Advanced Studies, The Australian
National University, Canberra, ACT 0200, Australia.
B
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, St Lucia, Qld 4072, Australia.
C
Corresponding author. Email: mgb@rsc.anu.edu.au
The novel [4.3.1]propelladiene 2, which embodies the key structural elements of the pentacyclic core of the cytotoxic
neoclerodane salvileucalin B (1), has been prepared using a rhodium-catalysed intramolecular B u¨ chner reaction as the key
step. Compound 2 and the readily obtained derivatives 12–17 all proved to be essentially inactive when tested against a
panel of four human cancer cell lines. Furthermore, not one of these compounds was a P-gp inhibitor.
Manuscript received: 31 July 2012.
Manuscript accepted: 31 August 2012.
Published online: 31 October 2012.
Introduction
In 2008 Takeya et al. reported the isolation of salvileucalin B
1, Fig. 1), a neoclerodane, from the aerial parts of Salvia
leucantha (Mexican bush sage) of the subgenus Calosphace
Labiatae). Its structure, including absolute configuration, was
2
and that this, as well as related systems, could display similar
[1]
activities to the natural product. Herein, therefore, we report the
syntheses of compound 2 and various of its reduced forms
together with the outcomes of the preliminary biological screen-
ing of this suite of salvileucalin B analogues.
(
(
established through a combination of spectroscopic techniques
including X-ray analysis and vibrational circular dichroism. The
structurally novel propellane core associated with natural
product 1 is believed to be formed by an intramolecular
Diels–Alder reaction of the triene moiety associated with the
co-occurring spirocyclic neoclerodane salvileucalin A, a pro-
Results and Discussion
Synthetic Studies
The synthetic sequence used to prepare compound 2 is shown in
Scheme 1. The key step employed in preparing the associated
propellane framework was an intramolecular B u¨ chner reac-
[
2]
posal that is supported by very recent synthetic studies.
[
5]
Compound 1 was shown to exert cytotoxic activity against the
A549 and HT-29 human adenocarcinoma cell lines with half
maximal inhibitory concentration (IC ) values of 15.7 and
tion, a process used to great effect by Reisman et al. in their
[
recently completed total synthesis of the natural product.
6]
5
0
Thus, commercially available indane-2-one (3) was subjected to
a Horner–Wadsworth–Emmons (HWE) reaction with the anion
derived from trimethyl phosphonoacetate and the resulting
[
1]
5
.6 mM, respectively. Arguably, these effects can be attributed
to the presence of the a-alkylidene-g-butyrolactone-type moiety
[
3]
[7]
embedded within the polycyclic framework.
Accordingly, we speculated that the basic pharmacophore
associated with salvileucalin B is embodied within substructure
a,b-unsaturated ester 4 (90 %) then hydrogenated to give the
[4]
[7]
corresponding saturated system 5 (99 %).
compound 5 with the acetonitrile anion
b-ketonitrile 6 (98 %) that engaged in a diazotransfer reaction
upon treatment with imidazolesulfonyl azide and pyridine.
Exposure of the resulting a-diazoketone 7 (83 %) to dirhodium
tetraacetate in refluxing dichloromethane (DCM) afforded the
B u¨ chner product 8 (86 %), the structure of which was estab-
lished by a single-crystal X-ray analysis (see Experimental
section and Supplementary Material for details).
Treatment of
afforded the
[
8]
[
9]
O
O
O
O
y
O
O
While ketone 8 was readily converted into the corresponding
enol triflate 9 (93 %) by treating the former compound with
LiHMDS in the presence of N-phenyltriflimide (PhNTf ), the
O
1
2
2
Fig. 1. Structures of salvileucalin B (1) and key substructure 2.
next stage of the reaction sequence proved rather problematic. In
y
When the B u¨ chner reaction was effected using copper-based catalysts only small quantities of compound 8 were obtained.
Journal compilation Ó CSIRO 2012
www.publish.csiro.au/journals/ajc

1
680
N. Heinrich et al.
Trimethyl
phosphonoacetate
NaH
O
H
2
CO Me
R
2
Pd on C
O
THF, 0 to 18°C
EtOAc, 18°C
99 %
90 %
LiHMDS, MeCN
THF, Ϫ78°C
98 %
3
4
5 R ϭ OMe
R ϭ CH CN
6
2
Imidazolesulfonyl azide
pyridine, MeCN, 40°C
8
3 %
OTf
O
O
N2
PhNTf2
LiHMDS
NC
NC
Rh (OAc)
4
2
CN
THF/DCM, Ϫ78°C
DCM, 40°C
86 %
93 %
9
8
7
DIBAl-H
DCM, Ϫ40°C
O
OTf
OH OTf
O
CO, Pd0
OHC
DIBAl-H
DCM, Ϫ40°C
6 % (from 9)
Et N, LiCl
3
THF, 18°C
89 %
1
10
11
2
Scheme 1. Synthesis of the core, 2, of salvileucalin B (1).
O1
particular, attempts to effect reduction of the nitrile 9, via the
corresponding aldehyde 10, to the primary alcohol 11 were
complicated by the tendency of the intermediate aldehyde to
engage in an apparently reversible rearrangement reaction
O2
C3
z
C4
involving the norcaradiene unit. As a consequence, only a
1
6 % yield of compound 11 could be realised when using
C2
C1
DIBAl-H as the reducing agent. Nevertheless, sufficient mate-
rial could be accumulated by such means to allow the comple-
tion of the synthesis of target 2. Thus, reaction of alcohol 11 with
C5
C9
C10
0
C12
carbon monoxide in the presence of Pd , triethylamine, and
C11
lithium chloride at 188C afforded the lactone 2 in 89 % yield. All
the derived spectroscopic data were in accord with the assigned
structure but final confirmation of this followed from a single-
crystal X-ray analysis. The associated ORTEP diagram is shown
in Fig. 2 and further details are provided in the Experimental
section.
C13
C6
C8
C7
C14
The generation of a series of partially reduced forms of
compound 2 was readily achieved by exposing it to hydrogen in
the presence of various catalysts (see Scheme 2 and Table 1),
and by such means the polyhydro-forms 12–17 of compound 2
were produced. The structures of these were secured by con-
ducting the usual range of spectroscopic analyses, and that of 17
Fig. 2. ORTEP derived from the single-crystal X-ray analysis of com-
pound 2. Anisotropic displacement ellipsoids display 30 % probability
levels. Hydrogen atoms are drawn as circles with small radii.
(wherein the cyclopropane ring associated with precursor 2 had
been hydrogenolysed) was confirmed by single-crystal X-ray
analysis (see Experimental section for details).
[
10]
Biological Evaluation of Compounds 2 and 12–17
Interestingly, a related pair of partially reduced compounds
and a cyclopropane ring-cleavage product were observed when
the ester 18 derived from triflate 9 was exposed to hydrogen in
the presence of Raney-cobalt (Scheme 3). In particular, a
chromatographically separable mixture of compounds 19
Compounds 2 and 12–17 were tested against the cell lines
SW620, SW620 Ad300, KB-3-1, and KB-V1 which are,
respectively, a human colon cancer cell line, a daughter cell line
overexpressing P-gp (prepared by culturing SW620 in the
presence of adriamycin), a human cervical cancer cell line, and a
daughter cell line overexpressing P-gp (prepared by culturing
KB-3-1 in the presence of vinblastine). P-gp is an ATP binding
cassette (ABC) transporter protein that drives multi-drug
(37 %), 20 (25 %), and 21 (5 %) was obtained and the structure
of the last of these was secured by single-crystal X-ray analysis
[
10]
(see Experimental section for details).
z
The details of this process will be the subject of a separate report.

The Pharmacophore of Salvileucalin B
1681
O
O
when tested in the SW620 cell line. Clearly, even though
many of these synthetic materials embody an a-alkylidene-g-
butyrolactone residue they are not particularly cytotoxic.
The origins of this situation are under investigation.
In a separate set of evaluations to test whether or not
compounds 2 and 12–17 posses any capacity to inhibit P-gp-
based multidrug resistance mechanisms displayed by numerous
human cancer cell lines, they were subjected to a flow
cytometry-based calcein AM assay. Once again, however, no
significant activity was observed with all the compounds dis-
playing calcein FAR (fluorescence arbitrary ratio) values in the
range 0.54 to 0.71 as compared with the value of 43.5 measured
for the positive control verapamil at the same concentration.
2
See Table 1
O
O
O
O
O
O
ϩ
ϩ
Conclusions
12
13
14
The intramolecular B u¨ chner reaction has provided a highly
effective means for assembling the [4.3.1]propellane substruc-
ture 2 associated with the cytotoxic neoclerodane natural
product salvileucalin B (1). Furthermore, reduction of the
former compound under a variety of conditions has provided a
suite of partially or fully saturated derivatives, namely
compounds 12–17, which have been used to probe for the
pharmacophore of natural product 1.
O
O
O
O
O
O
ϩ
ϩ
H
17
15
16
Scheme 2. Compounds produced by reduction of compound 2 under the
The lack of cytotoxic effects displayed by compounds 2 and
2–17 suggest that the second lactone ring and the furan moiety
conditions described in Table 1.
1
incorporated within the natural product 1 are contributing in a
significant way to its biological properties. Accordingly, we are
now studying means by which the meso compound 2 can be
elaborated, using desymmetrising acylation or alkylation
protocols, so as to incorporate one or both of these residues
and, thereby, deliver enantiomerically enriched compounds
displaying useful cytotoxicities. Results will be reported in
due course.
Table 1. Products from reaction of compound 2 with hydrogen in the
presence of various catalysts
A
Entry Reaction conditions
Product (Yield [%])
1
2
3
H
H
H
2
2
2
, Raney-Co, MeOH/NH
, Rh on Al , EtOAc, 188C
, Pd on C, EtOAc, 188C
3
, 188C 12 (5)
13 (40) 14 (50)
2
O
3
15 (25) 16 (50) 17 (8)
16 (11) 17 (54)
–
A
Experimental
See Experimental section for details.
General Experimental Protocols
1
Unless otherwise specified, proton ( H) and carbon ( C) NMR
OTf
NC
CO Me
2
13
NC
CO, MeOH
spectra were recorded at 188C in base-filtered CDCl on a
3
0
Pd
Varian spectrometer operating at 400 MHz for proton and
1
1
00 MHz for carbon nuclei. H NMR data are recorded as
9
18
follows: chemical shift (d) (multiplicity, coupling constant(s)
J (Hz), relative integral) where multiplicity is defined as: s ¼
singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, or
combinations of the above. The signal due to residual CHCl3
H , Raney-Co
2
MeOH/NH3
3
7 %
25 %
5 %
CO Me
appearing at dH 7.26 and the central resonance of the CDCl3
‘
1
triplet’ appearing at d 77.0 were used to reference the H and
CO Me
CO Me
C
2
2
2
1
3
NC
NC
NC
C NMR spectra, respectively. Infrared spectra (nmax) were
recorded on a Perkin–Elmer 1800 Series FTIR Spectrometer.
Samples were analysed as thin films on KBr plates. A VG Fisons
AutoSpec mass spectrometer was used to obtain low- and high-
resolution electron impact (EI) mass spectra. Low- and high-
resolution electrospray ionisation (ESI) mass spectra were
obtained on a VG Quattro II triple-quadrupole MS instrument
operating in positive ionisation mode. Melting points were
measured on an Optimelt automated melting point system and
are uncorrected. Analytical thin-layer chromatography (TLC)
was performed on aluminium-backed 0.2 mm thick silica gel 60
ϩ
ϩ
19
20
21
Scheme 3. Compounds produced by reduction of compound 18 with
hydrogen in the presence of Raney-cobalt.
resistance through enhanced drug efflux. Unfortunately, an
authentic sample of salvileucalin B was not available to us and
nor were the cell lines used by Takeya in testing this natural
F254 plates as supplied by Merck. Eluted plates were visualised
[1]
product. In the event, each of compounds 2 and 12–17 had
IC50 values of .100 mM in the relevant assays (see the Exper-
imental section and Supplementary Material for details) except
in the case of compound 15 which displayed an IC50 of 83 mM
using a 254 nm UV lamp and/or by treatment with a suitable dip
followed by heating. These dips included phosphomolybdic
acid/ceric sulfate/sulfuric acid (conc.)/water (37.5 g : 7.5 g :
37.5 g : 720 mL) or potassium permanganate/potassium

1
682
N. Heinrich et al.
carbonate/5 % aqueous sodium hydroxide/water (3 g : 20 g :
mL : 300 mL). Flash chromatographic separations were car-
This material was used without purification in the next step of
the reaction sequence.
5
[
11]
ried out following protocols defined by Still et al. with silica
gel 60 (40–63 mm) as the stationary phase and using the AR- or
HPLC-grade solvents indicated. Starting materials and reagents
were generally available from the Sigma–Aldrich, Merck, TCI,
Strem, or Lancaster chemical companies and were used as
supplied. Drying agents and other inorganic salts were
purchased from the AJAX, BDH, or Unilab chemical
companies. Tetrahydrofuran (THF), methanol, and DCM were
dried using a Glass Contour solvent purification system that was
Compound 6
LiHMDS (2.1 mL of 1 M solution in THF, 2.10 mmol) was
added to THF (6 mL) and the resulting solution stirred magneti-
cally while being cooled to ꢂ788C under a nitrogen atmosphere.
Dry acetonitrile (82 mL, 1.58 mmol) was then added dropwise to
this solution and after 1 h a solution of the ester 5 (200 mg,
1.05 mmol) in THF (4 mL) was added slowly. The ensuing
mixture was allowed to warm to 188C and stirred at this
temperature for 16 h before being treated with diethyl ether
[
12]
based upon a technology originally described by Grubbs et al.
Where necessary, reactions were performed under an argon
atmosphere.
(
(
(
(
10 mL), HCl (5 mL of a 1 M aqueous solution), and brine
10 mL). The separated aqueous phase was extracted with ether
3 ꢀ 10 mL) and the combined organic phases washed with brine
2 ꢀ 20 mL) before being dried (MgSO ), filtered, and
Specific Experimental Procedures
4
concentrated under reduced pressure. Subjection of the ensuing
light-yellow oil to flash chromatography (silica, 1 : 1 v/v
pentane/diethyl ether elution) afforded, after concentration of
Compound 4
[7]
Following the procedure of Ksander, a magnetically stirred
a suspension of NaH (1.31 g of a 60 % dispersion in mineral oil,
the appropriate fraction (R 0.1 in 15 : 85 v/v ethyl acetate/
F
5
4.5 mmol) in THF (45 mL) was cooled to 08C and trimethyl
hexane), the b-keto nitrile 6 (205 mg, 98 %) as a white, crystal-
line solid, mp 107–1088C (Found: M , 199.1000. C H NO
phosphonoacetate (8.8 mL, 9.92 g, 54.5 mmol) then added drop-
wise. The resulting thick white suspension was stirred at 188C
for 0.5 h and then cooled to 08C before being treated, dropwise,
with a solution of 2-indanone (3) (3.00 g, 22.7 mmol, Aldrich) in
THF (45 mL). The ensuing brown reaction mixture was stirred at
þꢁ
1
3 13
þꢁ
requires: M , 199.0997). dH (CDCl , 300 MHz) 7.22–7.12
3
(
2
(
complex m, 4H), 3.46 (s, 2H), 3.19 (dd, J 20.4 and 9.6, 2H),
.94 (m, 1H), 2.82 (d, J 9.6, 2H), 2.59 (dd, J 20.4 and 8.8, 2H). dC
CDCl , 75 MHz) 196.8 (C), 142.1 (C), 126.5 (CH), 124.5 (CH),
3
1
88C for 1 h before being poured into brine (100 mL) and
1
KBr/cm 3026, 2920, 2258, 1729, 1398, 1109, 1088, 1053. m/z
13.7 (C), 47.8 (CH ), 38.8 (CH ), 34.5 (CH ), 32.2 (CH). n
2
2
2
max
extracted with diethyl ether (2 ꢀ 40 mL). The combined organic
ꢂ1
phases were washed with brine (1 ꢀ 100 mL) and then dried
þꢁ
(
EI, 70 eV) 199 (M , 2 %), 129 (5), 116 (100), 91 (6).
(MgSO ), filtered, and concentrated under reduced pressure.
4
Subjection of the brown oil thus obtained to flash chromato-
graphy (silica, pentane - 7 : 3 v/v pentane/diethyl ether
gradient elution) afforded, after concentration of the appropriate
Compound 7
A magnetically stirred solution of b-keto nitrile 6 (380 mg,
.91 mmol) in acetonitrile (20 mL) was treated with imidazole
1
fractions (R 0.4 in 85 : 15 v/v hexane/ethyl acetate), the title
F
[
compound 4 (4.18 g, 98 %) as a white crystalline solid, mp
7]
sulfonyl azide (396 mg, 2.29 mmol) and pyridine (0.77 mL,
9.54 mmol) and the ensuing pale-yellow reaction mixture was
heated at 408C for 20 h and then concentrated under reduced
pressure (CAUTION – diazo compounds are potentially explo-
sive) to give an orange oil. Subjection of this material to flash
column chromatography (silica, 3 : 2 v/v pentane/diethyl ether)
þꢁ
þꢁ
8
1
7–898C (Found: M , 188.0835. C H O requires: M
,
88.0837). dH (CDCl , 300 MHz) 7.41 (d, J 8.0, 1H), 7.32
1
2 12 2
3
(d, J 4.0, 1H), 7.24 (t, J 8.0, 1H), 7.15 (m, 1H), 6.70 (s, 1H),
3
1
.72 (s, 3H), 3.54 (s, 2H), 3.45 (s, 2H). d (CDCl , 75 MHz)
C 3
71.4 (C), 144.7 (C), 143.4 (C), 140.9 (C), 130.1 (CH), 126.3
gave, after concentration of the appropriate fractions (R 0.7 in
F
(
CH), 124.4 (CH), 123.5 (CH), 120.6 (CH), 52.0 (CH ), 41.3
3
ꢂ1
65 : 35 v/v hexane/ethyl acetate), compound 7 (415 mg,
1.84 mmol, 96 %) as fine-yellow needles, mp 908C (with
decomposition) (Found: M , 225.0908. C H N O requires:
(CH ), 36.8 (CH ). n
KBr/cm 3056, 3020, 2923, 1740,
þꢁ
2
2
max
1
1
461, 1435, 1169, 753, 717. m/z (EI, 70 eV) 188 (M , 32 %),
49 (12), 129 (100), 128 (75), 84 (35).
þꢁ
1
3 11 3
þꢁ
M
, 225.0902). d (CDCl , 300 MHz) 7.22–7.12 (complex m,
H 3
4
(d, J 8.0, 2H), 2.66 (dd, J 15.6 and 8.0, 2H). d (CDCl , 75 MHz)
H), 3.17 (dd, J 15.6 and 8.0, 2H), 2.97 (septet, J 8.0, 1H), 2.82
Compound 5
C
3
1
89.5 (C), 142.1 (C), 126.5 (CH), 124.5 (CH), 108.4 (C), 44.9
CH ), 38.8 (CH ), 35.5 (CH) (signal due to carbon bearing the
A magnetically stirred solution of the a,b-unsaturated ester 4
4.18 g, 22.23 mmol) in ethyl acetate (100 mL) was treated with
(
diazo group not observed). nmax KBr/cm 2934, 2227, 2136,
1
(
2
2
ꢂ1
1
0 % palladium on carbon (1.18 g) and the flask containing the
þꢁ
673, 1374, 1228, 1187, 1004. m/z (EI, 70 eV) 225 (M , 1 %),
97 (15), 129 (15), 116 (100), 115 (55), 91 (22).
resulting mixture was degassed and then filled with hydrogen
three times. The reaction mixture was then left stirring under an
atmosphere of hydrogen for 5 h before being filtered through
Celite. The filtrate was concentrated under reduced pressure to
1
Compound 8
[7]
give the title ester 5 (4.22 g, 99 %) as a crystalline solid, mp
A magnetically stirred dispersion of Rh (OAc) (19 mg,
2
4
þꢁ
þꢁ
3
1
3
1
2–358C (Found: M , 190.0991. C H O requires: M
,
90.0994). d (CDCl , 300 MHz) 7.22–7.10 (complex m, 4H),
0.04 mmol, 5 mol %) in DCM (70 mL) was heated at reflux
and then treated with a solution of compound 7 (200 mg,
0.89 mmol) in DCM (20 mL) at a rate of 0.2 mmol h (using
a syringe pump). After addition was complete, the reaction
mixture was cooled and then concentrated under reduced
pressure to give a white to purple coloured solid. Recrystallisa-
tion (THF) of this material gave compound 8 (430 mg, 86 %) as a
1
2 14 2
H
3
ꢂ1
.70 (s, 3H), 3.15 (dd, J 20.0 and 10.4, 2H), 2.89 (septet, J 10.4,
H), 2.65 (dd, J 20.0 and 10.4, 2H), 2.51 (d, J 10.4, 2H).
d (CDCl , 75 MHz) 173.4 (C), 142.6 (C), 126.3 (CH), 124.4
3
C
(
CH), 51.5 (CH ), 39.7 (CH ), 38.9 (CH ), 36.0 (CH). n
3
2
2
max
ꢂ1
KBr/cm 3022, 2926, 1739, 1436, 1199, 1151, 742. m/z (EI,
þꢁ
þꢁ
7
0 eV) 190 (M , 20 %), 116 (100), 84(60).
white, crystalline solid, mp 194–2008C (Found: M , 197.0836.

The Pharmacophore of Salvileucalin B
1683
þꢁ
C H NO requires: M , 197.0841). dH (CDCl , 300 MHz)
1
6
of the resulting light-yellow oil to flash chromatography (silica,
3 : 2 v/v pentane/diethyl ether elution) gave, after concentration
of the appropriate fractions (R 0.6 in 65 : 35 v/v hexane/ethyl
3
11
3
.36 (m, 2H), 6.21 (m, 2H), 2.31 (m, 3H), 2.16 (br s, 4H). dC
CDCl , 75 MHz) 199.8 (C), 127.1 (CH), 123.4 (CH), 112.2 (C),
(
3
F
5
KBr/cm 3050, 2979, 2953, 2873, 2238, 1698, 1447, 1430,
0.4 (CH ), 43.2 (CH ), 36.2 (C), 29.3 (C), 25.1 (CH). n
acetate), the title alcohol 11 (33 mg, 16 %) as a white, crystalline
þꢁ
2
ꢂ1
2
max
solid, mp 54–588C (Found: M , 334.0489. C H F O S
1
4 13 3 4
þꢁ
1
7
1
407, 1395, 1336, 1311, 1292, 1255, 1166, 1143, 1035, 972,
þꢁ
requires: M , 334.0487). dH (CDCl , 300 MHz) 6.09
3
93. m/z (EI, 70 eV) 197 (M , 70 %), 153 (100), 141 (58),
28 (75), 115 (55).
(m, 2H), 6.02 (m, 3H), 3.63 (d, J 5.3, 2H), 2.45 (m, 1H), 1.66
(dd, J 16.4 and 4.8, 2H), 1.47 (d, J 16.4, 2H) (signal due to OH
group proton not observed). dC (CDCl , 75 MHz) 143.9 (C),
3
1
5
25.2 (CH), 122.9 (CH), 118.4 (q, JC,F 320, CF ), 115.7 (CH),
3
Compound 9
5.8 (CH ), 37.5 (C), 33.2 (CH ), 26.5 (CH), 23.1 (C). n
2
ꢂ1
2
max
A solution of compound 8 (200 mg, 1.01 mmol) in DCM
3mL) was added to THF (20 mL) and the resulting magnetically
KBr/cm 3436, 3036, 2929, 2859, 1653, 1418, 1247, 1208,
þꢁ
(
1
(
139, 1009, 971, 892, 809. m/z (EI, 70 eV) 334 (M , 95 %), 201
20), 183 (45), 155 (85), 129 (100), 128 (77), 116 (74), 115 (85).
stirred mixture cooled to ꢂ788C and then treated, dropwise, with
LiHMDS (1.42 mL of a 1 M solution in THF, 1.42 mmol). After
a further 0.33 h N-phenyl triflimide (471 mg, 1.32 mmol) was
added in one portion then the reaction mixture allowed to warm
to 188C and stirred at this temperature for 16 h before being
concentrated under reduced pressure to give a light-yellow oil.
Subjection of this material to flash chromatography (silica,
7 : 3 - 1 : 9 v/v pentane/diethyl ether gradient elution) and
concentration of the appropriate fractions (R 0.5 in 65 : 35 v/v
hexane/ethyl acetate) gave the enol triflate 9 (310 mg, 93 % at
Compound 2
A magnetically stirred solution of alcohol 11 (44 mg,
0.13 mmol) in THF (2.5 mL) was treated with Pd(PPh )
3 4
(15 mg, 0.0132 mmol, 10 mol %), LiCl (11 mg, 0.264 mmol),
and triethylamine (73 mL, 0.526 mmol). The reaction flask was
evacuated and then refilled with carbon monoxide three times
before being stirred under an atmosphere of carbon monoxide at
188C for 4 h. After this time the reaction mixture was sparged
with nitrogen and then concentrated under reduced pressure.
Subjection of the resulting light-yellow oil to flash chromatog-
raphy (silica, 7 : 3 v/v pentane/diethyl ether) gave, after concen-
F
9
tion) (Found: M , 329.0338. C H F NO S requires: M
5 % conversion) as white flakes, mp 798C (with decomposi-
þꢁ
þꢁ
,
1
4
10
3
3
3
2
29.0333). d (CDCl , 400 MHz) 6.33 (m, 2H), 6.06 (m, 3H),
H 3
.67 (m, 1H), 1.88 (dd, J 16.8 and 6.4, 2H), 1.46 (d, J 16.8, 2H).
d (CDCl , 100 MHz) 137.2 (C), 127.0 (CH), 120.9 (CH), 118.4
tration of the appropriate fractions (R 0.6 in 65 : 35 v/v hexane/
F
C
3
(
quartet, JC–F 321), 115.1 (CH), 112.0 (C), 43.0 (C), 31.5 (CH2),
6.6 (CH), 15.9 (C). d (CDCl , 400 MHz) ꢂ73.0 (s, 3F). n
ethyl acetate), the title lactone 2 (25 mg, 89 %) as colourless,
þꢁ
2
KBr/cm
crystals, mp 105–1108C (Found: M , 212.0840. C14
(CDCl
H
O
12
2
F
3
max
ꢂ1
þꢁ
2933, 2237, 1656, 1425, 1205, 1140, 964. m/z
þꢁ
requires: M , 212.0837). d
H
3
, 400 MHz) 7.07
(
1
EI, 70 eV) 329 (M , 60 %), 196 (56), 168 (100), 153 (55),
41 (93), 115 (48).
(d, J 6.8, 1H), 6.14 (m, 2H), 5.92 (m, 2H), 3.89 (s, 2H), 2.90
(m, 1H), 1.74 (dd, J 11.6 and 4.4, 2H), 1.25 (d, J 11.6, 2H). d
C
(CDCl , 100 MHz) 169.5 (C), 132.3 (CH), 125.2 (C), 123.6
3
(
2
1
CH), 122.1 (CH), 67.1 (CH ), 37.8 (CH), 30.8 (C), 30.2 (CH ),
2
Compound 11
2
ꢂ1
2.7 (C). nmax KBr/cm 2949, 2927, 1755, 1652, 1234, 1072,
040, 1005, 750. m/z (EI, 70 eV) 212 (M , 90 %), 197 (50), 183
Step i. Under protection from light, a magnetically stirred
solution of nitrile 9 (200 mg, 0.61 mmol) in DCM (5 mL) was
cooled to ꢂ408C and then treated, dropwise, with DIBAL-H
þꢁ
(65), 167 (92), 155 (100), 153 (75), 152 (65), 115 (62).
(
1.2 mL of a 1 M solution in hexane, 1.22 mmol). The ensuing
Compounds 12–14
mixture was stirred at this temperature for 1 h before being
treated with ethyl acetate (0.5 mL) and then allowed to warm to
A magnetically stirred solution of lactone 2 (12 mg,
1
88C and quenched with HCl (1 mL of a 1 M aqueous solution),
0.06 mmol) in ammoniacal methanol (2 mL) maintained under
a nitrogen atmosphere at 188C was treated with Raney-cobalt
(spatula tip full of catalyst maintained as a slurry in MeOH/
a process that resulted in the development of a bright-yellow
colouration in the organic phase which thickened over time. The
reaction mixture was stirred vigorously for ,0.33 h and then the
separated organic phase was concentrated under a stream of
nitrogen. The resulting yellow oil was subjected to flash chro-
matography (silica, 4 : 1 v/v pentane/diethyl ether elution) and
H O). The flask containing the ensuing mixture was then
2
flushed three times with hydrogen before a balloon of hydrogen
was attached to it. After 0.5 h the reaction mixture was flushed
with nitrogen and then filtered through Celite and the filtrate
concentrated under reduced pressure to give a clear, colourless
oil. Subjection of this material to flash chromatography
(silica, 4 : 1 v/v pentane/diethyl ether elution) gave three
fractions, A, B, and C.
concentration of the appropriate fractions (R 0.9 in 65 : 35 v/v
F
hexane/ethyl acetate) gave a bright-yellow oil that was immedi-
ately resubjected to treatment with DIBAl-H as specified
immediately below.
Step ii. A magnetically stirred solution of the bright-yellow
oil obtained in step i in DCM (5 mL) was cooled to ꢂ788C and
then treated dropwise with DIBAl-H (1.8 mL of a 1 M solution
in DCM) and the resulting mixture allowed to warm to 188C over
Concentration of fraction A (R 0.6 in 65 : 35 v/v hexane/
F
ethyl acetate) gave compound 12 (0.6 mg, 5 %) as a white,
þꢁ
crystalline solid (Found: M , 214.0993. C14
þꢁ
H
O
requires:
14
2
M , 214.0994). d (CDCl , 400 MHz) 6.99 (d, J 9.6, 1H),
H 3
5.97 (m, 1H), 5.82 (m, 1H), 4.41 (d, J 12.8, 1H), 4.13 (d, J 12.8,
1
6 h before being treated with ethyl acetate (0.5 mL) and then
HCl (1 mL of a 1 M aqueous solution). The resulting viscous
mixture was diluted with DCM (5 mL) and then stirred vigor-
ously for 0.5 h. The separated aqueous phase was extracted with
DCM (2 ꢀ 4 mL) and the combined organic phases were then
1H), 2.94 (m, 1H), 2.27 (m, 1H), 1.99 (m, 1H), 1.90–1.40
(complex m, 4H), 1.00 (m, 2H). d
(C), 134.7 (CH), 126.7 (CH), 126.5 (C), 124.2 (CH), 67.6 (CH
(CDCl
, 100 MHz) 169.6
),
), 28.8 (C), 28.0 (C),
C
3
2
38.4 (C), 33.7 (CH), 33.4 (CH
), 30.2 (CH
ꢂ1
2
2
washed with water (1 ꢀ 5 mL) before bring dried (MgSO ),
22.9 (CH
1436, 1359, 1239, 1052, 1020, 1009. m/z (EI, 70 eV) 214 (M
), 18.9 (CH
2
2
). nmax KBr/cm 2926, 2854, 1758, 1653,
4
þꢁ
filtered, and concentrated under reduced pressure. Subjection
,

1
684
N. Heinrich et al.
þꢁ
1
(
00 %), 199 (10), 186 (32), 169 (22), 155 (26), 141 (28), 129
29), 128 (30), 115 (31), 91 (31).
crystalline solid, mp 85–898C (Found: M , 218.1307.
C H O requires: M , 218.1307). dH (CDCl , 400 MHz)
þꢁ
1
4
18
2
3
Concentration of fraction B (R 0.6 in 65 : 35 v/v hexane/
F
4.75 (m, 2H), 2.58 (m, 1H), 2.40 (m, 1H), 2.24 (dd, J 11.6 and
6.0, 1H), 2.08 (m, 1H), 2.00–1.80 (complex m, 2H), 1.75–1.05
(complex m, 10H). d (CDCl , 100 MHz) 174.8 (C), 172.3 (C),
ethyl acetate) gave compound 13 (5 mg, 40 %) as a white,
þꢁ
crystalline solid, mp 138–1408C (Found: M , 214.0992.
C H O requires: M , 214.0994). dH (CDCl , 400 MHz)
C
3
þꢁ
123.2 (C), 69.1 (CH ), 46.4 (CH), 44.1 (C), 38.7 (CH ), 35.3
2 2
1
4
14
2
3
6
2
4
1
.93 (d, J 6.8, 1H), 5.66 (m, 2H), 4.09 (s, 2H), 2.87 (m, 1H),
.56 (d, J 16.8, 2H), 2.30 (dm, J 16.8, 2H), 1.66 (dd, J 11.6 and
.8, 2H), 0.96 (d, J 11.6, 2H). d (CDCl , 100 MHz) 169.7 (C),
(CH ), 32.5 (CH ), 32.0 (CH), 31.7 (CH ), 28.5 (CH ), 25.5
2 2 2 2
ꢂ1
(CH ), 20.9 (CH ). n
KBr/cm 2924, 2850, 1769, 1667,
1432, 1383, 1342, 1299, 1195, 1046, 1018, 1007. m/z (EI, 70 eV)
2
2
max
C
3
þꢁ
33.7 (CH), 126.3 (C), 124.6 (CH), 67.1 (CH ), 32.9 (CH ), 32.7
2
218 (M , 70 %), 189 (15), 121 (100), 91 (30).
2
ꢂ1
(
1
CH), 31.6 (C), 25.3 (C), 23.6 (CH ). n
KBr/cm 2923,
756, 1652, 1431, 1239, 1146, 1084, 1052, 1004. m/z (EI, 70 eV)
2
max
Compounds 16 and 17
þꢁ
2
(
14 (M , 100 %), 199 (22), 186 (39), 185 (40), 155 (29), 141
38), 129 (59), 116 (52), 115 (57), 91 (67), 84 (60), 79 (52).
Using the same protocol as defined immediately above, a
solution of lactone 2 (12 mg, 0.0565 mmol) in ethyl acetate
(2 mL) containing 10 % palladium on carbon (2 mg) was sub-
jected to hydrogenation at 188C for 0.33 h. The crude reaction
mixture was then subjected to flash chromatography (silica,
45 : 1 v/v pentane/diethyl ether elution) and thus afforded two
fractions, A and B.
Concentration of fraction C (R 0.7 in 65 : 35 v/v hexane/
F
ethyl acetate) gave compound 14 (6 mg, 50 %) as a white,
þꢁ
crystalline solid, mp 95–998C (Found: M , 216.1149.
C H O requires: M , 216.1150). dH (CDCl , 400 MHz)
þꢁ
1
4
16
2
3
5
1
4
.57 (d, J 2.4, 2H), 4.02 (dd, J 10.4 and 0.8, 1H), 3.98 (d, J
0.4, 1H), 2.89 (dd, J 11.6 and 7.9, 1H), 2.45–2.05 (complex m,
H), 1.90–1.51 (complex m, 7H). d (CDCl , 100 MHz) 178.6
C
Concentration of fraction A (R 0.7 in 65 : 35 v/v hexane/
F
3
ethyl acetate) gave compound 16 (2 mg, 11 %) as a white,
crystalline solid that was identical, in all respects, with the
material obtained by the method defined immediately above.
(C), 124.6 (CH), 124.5 (CH), 67.7 (CH ), 39.3 (CH ), 36.8
2 2
(CH ), 35.9 (CH), 29.8 (C), 29.7 (CH), 27.9 (CH ), 26.6 (CH ),
2
2
2
ꢂ1
26.3 (C), 25.8 (C), 25.5 (CH ). n KBr/cm 2851, 1778,
1436, 1365, 1189, 1152, 1078, 1032, 986. m/z (EI, 70 eV) 216
2
max
Concentration of fraction B (R 0.5(7) in 65 : 35 v/v hexane/
F
ethyl acetate) gave compound 17 (7 mg, 54 %) as a white,
crystalline solid that was identical, in all respects, with the
material obtained by the method defined immediately above.
þꢁ
(M
, 100 %), 158 (22), 143 (28), 129 (42), 117 (42), 91 (75).
Compounds 15–17
A magnetically stirred solution of lactone 2 (12 mg,
.06 mmol) in ethyl acetate (2 mL) maintained at 188C was
Compound 18
0
A magnetically stirred solution of triflate 9 (80 mg,
treated with 5 % rhodium on alumina (3 mg). The reaction
vessel containing the resulting mixture was flushed three times
with hydrogen and then maintained under a balloon of this gas
for 0.33 h before being flushed with nitrogen and then filtered
through Celite. The filtrate was concentrated under reduced
pressure to give a clear, colourless oil that was subjected to flash
column chromatography (silica, 4 : 1 v/v pentane/diethyl ether)
thus giving three fractions, A, B, and C.
0
(
0
.243 mmol) in THF (3.0 mL) was treated with Pd(PPh3)4
28 mg, 0.024 mmol, 10 mol %), triethylamine (135 mL,
.97 mmol), and methanol (0.79 mL, 19.4 mmol). The reaction
flask was evacuated and then refilled with carbon monoxide
three times. The reaction mixture was then left stirring under an
atmosphere of carbon monoxide at 188C for 3 h before being
sparged with nitrogen and then concentrated under reduced
pressure. Subjection of the resulting light-yellow oil to flash
chromatography (silica, 1 : 1 v/v pentane/diethyl ether) gave,
Concentration of fraction A (R 0.6 in 65 : 35 v/v hexane/
F
ethyl acetate) gave compound 15 (3 mg, 25 %) as a white,
þ
after concentration of the appropriate fractions (R 0.3 in 65 : 35
F
crystalline solid, mp 85–908C (Found: [M þ H] , 217.1231.
þ
v/v hexane/ethyl acetate), the title ester 18 (54 mg, 93 %) as
þꢁ
C H O requires: [M þ H] , 217.1229).
d
00 MHz) 6.92 (d, J 7.2, 1H), 4.37 (s, 2H), 2.78 (m, 1H), 1.93
(CDCl ,
3
1
4
17
2
H
colourless crystals, mp 127–1308C (Found: M , 239.0946.
C H NO requires: M , 239.0946). dH (CDCl , 400 MHz)
4
(
1
(
þꢁ
1
5
13
2
3
m, 2H), 1.75–0.80 (complex m, 10 H). d (CDCl , 100 MHz)
C
3
7
.10 (d, J 8.0, 1H), 6.30 (dd, J 7.6 and 2.8, 2H), 5.98 (dd, J 7.6
69.8 (C), 133.7 (CH), 126.3 (C), 66.7 (CH ), 34.2 (CH ), 33.1
2 2
and 2.4, 2H), 3.80 (s, 3H), 2.63 (m, 1H), 1.86 (dd, J 12.4 and 5.2,
2H), 1.41 (d, J 12.4, 2H). d (CDCl , 100 MHz) 164.9 (C), 137.5
(CH), 126.8 (CH), 122.2 (C), 121.2 (CH), 114.4 (C), 52.1 (CH ),
CH), 31.6 (C), 24.5 (C), 22.0 (CH ), 21.9 (CH ). n
2
2
max
ꢂ1
C
3
KBr/cm 2929, 2853, 1761, 1657, 1450, 1364, 1235, 1072,
þ
3
1
(
056, 1038, 1010. m/z (ESI, þve) 239 ([M þ Na] , 97 %), 217
ꢂ1
þ
41.9 (C), 30.9 (CH ), 28.0 (CH), 12.9 (C). n KBr/cm 3041,
2
max
[M þ H] , 100).
2
996, 2927, 2950, 2849, 2232, 1724, 1619, 1438, 1367, 1267,
þꢁ
Concentration of fraction B (R 0.7 in 65 : 35 v/v hexane/
F
1191, 1159, 1131, 1096, 1040, 970. m/z (EI, 70 eV) 239 (M
30 %), 205 (30), 180 (100), 153 (40), 152 (27), 115 (20).
,
ethyl acetate) gave compound 16 (6 mg, 50 %) as a white,
þꢁ
crystalline solid, mp 44–468C (Found: M , 218.1312.
C H O requires: M , 218.1307). dH (CDCl , 400 MHz)
þꢁ
1
4
18
2
3
Compounds 19–21
4
.29 (d, J 9.2, 1H), 4.17 (dd, J 9.2 and 1.2, 1H), 2.86 (dd,
J 11.6 and 8.0, 1H), 2.04 (m, 1H), 1.80–1.05 (complex m, 13H),
.78 (m, 1H). d (CDCl , 100 MHz) 178.6 (C), 67.7 (CH ), 40.5
A magnetically stirred solution of compound 18 (40 mg,
0.17 mmol) in ammoniacal methanol (2.0 mL) maintained at
188C was treated with Raney-cobalt (spatula tip full of catalyst
maintained as a slurry in aqueous methanol). The reaction vessel
containing the resulting mixture was flushed three times with
hydrogen and then maintained under a balloon of this gas for 4 h.
The reaction mixture was then flushed with nitrogen before
being filtered through Celite and the filtrate was concentrated
under reduced pressure to give a clear, colourless oil. Subjection
0
(
2
(
1
(
C
3
2
CH ), 37.8 (CH ), 36.4 (CH), 30.8 (C), 30.1 (CH), 27.6 (CH ),
2 2 2
6.4 (C), 25.8 (C), 25.0 (CH ), 24.2 (CH ), 22.2 (CH ), 21.5
2 2 2
ꢂ1
CH ). n
KBr/cm 2930, 2850, 1775, 1447, 1361, 1175,
þꢁ
2
max
145, 1075, 1027, 988. m/z (EI, 70 eV) 218 (M , 100 %), 145
20), 131 (25), 91 (45).
Concentration of fraction C (R 0.5(7) in 65 : 35 v/v hexane/
F
ethyl acetate) gave compound 17 (1 mg, 8 %) as a white,

The Pharmacophore of Salvileucalin B
1685
of this material to flash column chromatography (silica, 3 : 7 v/v
pentane/diethyl ether) gave three fractions, A, B and C.
Concentration of fraction A (R 0.5) gave compound 19
Data for Compound 21
C H O , M 241.29, T 200 K, monoclinic, space group
1
5 15 2
F
˚
C2/c, Z 8, a 20.7142(5), b 7.7593(1), c 15.8461(4) A;
(
(
15 mg, 37 %) as a white, crystalline solid, mp 130–1348C
˚
3
b 105.3732(12)8; V 2455.78(9) A , D 1.305 g cm , 2816
ꢂ3
x
þꢁ
þꢁ
,
Found:
M
,
241.1103. C H NO
15
requires:
M
1
5
2
unique data (2ymax 558), R 0.050 [for 2348 reflections with
I . 2.0s(I)]; Rw 0.139 (all data), S 1.02.
2
1
41.1103). d (CDCl , 400 MHz) 7.06 (d, J 8.0, 1H), 6.09 (m,
H
H), 5.95 (m, 1H), 3.78 (s, 3H), 2.76 (m, 1H), 2.40–2.02
3
(
1
1
complex m, 4H), 1.74 (m, 2H), 1.21 (m, 2H). dC (CDCl3,
Structure Determination
00 MHz) 165.0 (C), 140.2 (CH), 139.8 (C), 132.4 (CH),
21.6 (CH), 117.6 (C), 52.0 (CH ), 33.8 (CH ), 33.7 (C), 33.6
Images were measured on a Nonius Kappa CCD diffractometer
˚
[13]
3
2
(MoKa, graphite monochromator, l 0.71073 A) and data
(C), 31.6 (CH), 30.8 (CH ), 29.5 (C), 22.4 (CH ), 20.2 (CH ).
nmax KBr/cm 3031, 2927, 2850, 2230, 1716, 1616, 1433,
1
2
2 2 2
ꢂ1
extracted using the DENZO package. Structure solution was
[14]
by direct methods (SIR92). The structures of compounds 2, 8,
367, 1263, 1189, 1156, 1127, 1098, 850, 760. m/z (EI, 70 eV)
þꢁ
17, and 21 were refined using the CRYSTALS program
[
41 (M , 65 %), 226 (100), 209 (35), 182 (70).
Concentration of fraction B (R 0.4) gave compound 20
15]
package.
Atomic coordinates, bond lengths and angles, and
F
displacement parameters have been deposited at the
Cambridge Crystallographic Data Centre (CCDC nos. 883755–
(
(
10 mg, 25 %) as a white, crystalline solid, mp 150–1548C
Found:
þꢁ
þꢁ
,
M
,
241.1101. C H NO
1
requires:
M
5
15
2
8
83758 for compounds 2, 8, 17, and 21, respectively). These
2
41.1103). dH (CDCl , 400 MHz) 7.06 (d, J 7.6, 1H), 5.75
3
data can be obtained free-of-charge via www.ccdc.cam.ac.uk/
data_request/cif.
(s, 2H), 3.79 (s, 3H), 2.70 (m, 1H), 2.59 (s, 4H), 1.72 (dd,
J 12.4 and 5.2, 2H), 1.25 (d, J 12.4, 2H). d (CDCl , 100 MHz)
C
3
1
(
65.1 (C), 139.8 (CH), 124.0 (C), 123.6 (CH), 116.5 (C), 52.0
CH ), 34.1 (CH ), 31.6 (C), 31.2 (CH), 23.8 (CH ), 23.6 (C).
Biological Studies
Cytotoxicity Assays
3
2
2
ꢂ1
nmax KBr/cm 3035, 2995, 2923, 2834, 2229, 1724, 1618,
1
440, 1269, 1269, 1210, 1189, 1161, 1122, 1096, 766. m/z
þꢁ
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay was used to evaluate the cytotoxicity of
(
1
EI, 70 eV) 241 (M , 100 %), 226 (50), 209 (55), 208 (60),
80 (55), 115 (35), 67 (50).
Concentration of fraction C (R 0.6) gave compound 21
compounds 2 and 12–17 against the relevant cancer cell line.
This assay was modified slightly from that previously
F
(
(
2 mg, 5 %) as a pale-yellow, crystalline solid, mp 133–1348C
Found: M , 241.1102. C H NO requires: M , 241.1103).
[16]
described.
Briefly, cells (2000 per well in 180 mL of RPMI
þꢁ
þꢁ
1
5
15
2
1640 supplemented with 10 % fetal bovine serum (FBS) for
SW620 and SW620 Ad300, 3000 or 5000 per well in 180 mL of
Dulbecco’s modied Eagle’s medium (DMEM) supplemented
with 10 % FBS for KB-3-1 and KB-V1, respectively) were
seeded evenly in a 96-well micro-plate, and the plate was
d (CDCl , 400 MHz) 7.35 (d, J 6.8, 1H), 6.06 (m, 1H), 5.89
H
3
(br s, 1H), 5.78 (m, 1H), 3.76 (s, 3H), 3.69 (s, 1H), 2.95–2.83
(m, 2H), 2.60 (dd, J 17.0 and 6.5, 1H), 2.46 (d, J 17.0, 1H), 2.26
(m, 1H), 2.16 (m, 1H), 1.60 (dd, J 11.9 and 3.8, 1H). d (CDCl ,
C 3
1
(
00 MHz) 165.4 (C), 147.8 (CH), 143.0 (C), 126.0 (CH), 124.1
C), 122.4 (CH), 120.1 (CH), 118.6 (C), 52.1 (CH ), 41.6 (C),
incubated for 18 h (378C; 5 % CO ) to allow cells to attach.
2
3
The compounds to be tested were dissolved in 5 % DMSO (v/v)
and diluted from 1 mM to 300 nM. Aliquots (20 mL) of each
dilution (or of 5 % aqueous DMSO for control wells) were added
to the plate in duplicate. After 68 h of incubation (378C; 5 %
3
cm 3041, 2951, 2867, 2233, 1718, 1644, 1435, 1361, 1285,
9.1 (2 ꢀ CH ), 35.0 (CH), 34.1 (CH), 32.7 (CH ). n
KBr/
max
2
2
ꢂ1
þꢁ
1247, 1112, 977. m/z (EI, 70 eV) 241 (M , 40 %), 182 (50),
125 (48), 116 (62), 117 (80), 91 (100).
CO ), a solution of MTT (Sigma, USA) in phosphate-buffered
2
saline (PBS) was added to each well to a final concentration of
ꢂ1
0
.4 mg mL and the plate was incubated for a further 4 h (378C;
Crystallographic Studies
5
and precipitated formazan crystals were dissolved in DMSO
% CO ). After that time, the medium was carefully aspirated
2
Data for Compound 2
C H O , M 212.25, T 200 K, triclinic, space group P ꢀꢁ , Z 2,
1
4 12 2
(100 mL per well). Finally, the absorbance of each well was
˚
˚
a 8.0384(2), b 8.2320(2), c 8.7349(2) A; a 79.3581(14)8,
measured at 570 nm using a PowerWave XS Microplate Reader
from Bio-Tek Instruments Inc. (Vinooski, VT). The IC50 value
was calculated as the concentration of the compound required
for 50 % inhibition of the cancer cells using Prism 5.0 from
GraphPad Software Inc. (La Jolla, CA). Results are presented in
the Supplementary Material.
3
b80.7333(17)8, g65.1725(14)8;V513.30(2) A , D 1.373 g cm
ꢂ3
,
x
2
995 unique data (2ymax 608), R 0.040 [for 2512 reflections
with I . 2.0s(I)]; Rw 0.101 (all data), S 0.98.
Data for Compound 8
C H NO, M 197.24, T 200 K, triclinic, space group P ꢀꢁ , Z 2,
1
3 11
˚
a 6.7916(3), b 8.2851(3), c 9.0747(3) A; a 75.690(3)8,
Flow Cytometry (Calcein AM Assay)
3
b 83.678(2)8, g 88.747(2)8; V 491.76(3) A , D 1.332 g cm
ꢂ3
˚
,
236 unique data (2ymax 558), R 0.040 [for 1812 reflections with
x
The flow cytometry assay was a modification of that
[17]
2
described previously.
(
Thus, cells which overexpress P-gp
SW620 Ad300) were harvested with trypsin and resuspended
I . 2.0s(I)]; Rw 0.098 (all data), S 0.98.
in completed medium to give a final concentration of 50 ꢀ
Data for Compound 17
4
ꢂ1
1
0 cells mL and pre-incubated with 20 mM solutions of the
C H O , M 218.30, T 200 K, monoclinic, space group
1
synthetic analogues or verapamil for 0.5 h at 378C in 5 % CO2.
Cells were then incubated with 2.5 mM calcein AM for 1 h before
being washed twice with cold PBS. Samples were analysed on a
BD FACSCanto II flow cytometer (Becton Dickinson, San
Jose, CA). Calcein fluorescence was detected with a 488 nm
4 18 2
˚
P2 /c, Z 4, a 6.6120(2), b 11.4845(5), c 14.9989(6) A;
1
3
b 90.017(2)8; V 1138.95(8) A , D 1.273 g cm , 2558 unique
ꢂ3
˚
x
TM
data (2ymax 558), R 0.039 [for 2206 reflections with I . 2.0s(I)];
Rw 0.102 (all data), S 1.00.

1
686
N. Heinrich et al.
argon laser and a 530 nm band-pass filter. Data were analysed by
FCSexpress 3 (De Novo Software, Los Angeles, CA). For each
sample, 10 000 events were collected. Inhibition was evaluated
using the following equation: FAR (fluorescence arbitrary
ratio) ¼ calcein fluorescence intensity (Geo mean) in the pres-
ence of the synthetic analogues at 20 mM/calcein fluorescence
intensity (Geo mean) in the presence of PBS. The positive
control was a 20 mM solution of verapamil which displayed a
FAR of 43.5.
(b) Also see: S. Levin, R. R. Nani, S. E. Reisman, Org. Lett. 2010, 12,
780. doi:10.1021/OL902848K
[
7] G. M. Ksander, R. deJesus, A. Yuan, C. Fink, M. Moskal, E. Carlson,
P. Kukkola, N. Bilci, E. Wallace, A. Neubert, D. Feldman,
T. Mogelesky, K. Poirier, M. Jeune, R. Steele, J. Wasvery, Z. Stephan,
E. Cahill, R. Webb, A. Navarrete, W. Lee, J. Gibson, N. Alexander,
H. Sharif, A. Hospattankar, J. Med. Chem. 2001, 44, 4677.
doi:10.1021/JM010294E
[
8] Z. Puterov a´ , A. Andicsov a´ , D. V e´ gh, Tetrahedron 2008, 64, 11262.
doi:10.1016/J.TET.2008.09.032
[
9] (a) E. D. Goddard-Borger, R. V. Stick, Org. Lett. 2007, 9, 3797.
doi:10.1021/OL701581G
Supplementary Material
(
b) Also see: N. Fischer, E. D. Goddard-Borger, R. Greiner, T. M.
The X-ray crystal structures for compounds 8, 17, and 21,
1
cytotoxicity results for compounds 2 and 12–17, and H or
C NMR spectra of compounds 2, 4–9, and 11–21 are available
on the Journal’s website.
Klap o¨ tke, B. W. Skelton, J. Stierstorfer, J. Org. Chem. 2012, 77, 1760.
doi:10.1021/JO202264R
1
3
[
10] (a) For related examples of cyclopropane ring cleavage reactions see:
M. T. Wuesthoff, B. Rickborn, J. Org. Chem. 1968, 33, 1311.
doi:10.1021/JO01267A111
Acknowledgements
(
b) F. Zutterman, A. Krief, J. Org. Chem. 1983, 48, 1135. doi:10.1021/
The authors thank the Australian Research Council and the Institute of
Advanced Studies for generous financial support. Drs S. Bates and R. Robey
JO00155A052
[
11] W. C. Still, M. Kahn, A. Mitra, J. Org. Chem. 1978, 43, 2923.
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12] A. B. Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen,
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OM9503712
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276: Macromolecular Crystallography, Part A (Eds C. W. Carter, Jr,
R. M. Sweet) 1997, pp. 307–326 (Academic Press: New York, NY).
(NCI) are gratefully acknowledged for providing the SW620 and SW620
Ad300 cell lines and Dr M. Gottesman (NCI) is thanked for providing the
KB-3-1 and KB-V1 cell lines.
[
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