Synthesis and evaluation of verticipyrone analogues as mitochondrial complex I inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.03.070

Verticipyrone has recently been isolated from the culture broth of Verticillium sp. and shown to inhibit NADH fumarate reductase, as well as NADH oxidoreductase (complex I) of the mitochondrial electron transport chain. In order to assess the structural elements in verticipyrone essential for complex I inhibitor, 15 structural analogues were prepared and analyzed for their effects on mitochondrial NADH oxidoreductase and NADH oxidase activities. Also measured were the abilities of several of the analogues to inhibit respiration as judged by a shift to glycolysis, and to inhibit the growth of several mammalian cell lines. The nature of the pyrone ring was shown to be important to potency of inhibition, as was the length and nature of substituents in the side chain of the analogues.

Full text of DOI:10.1016/j.bmc.2010.03.070

Bioorganic & Medicinal Chemistry 18 (2010) 3481–3493
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of verticipyrone analogues as mitochondrial complex
I inhibitors
Simon J. Leiris, Omar M. Khdour, Zachary J. Segerman, Krystal S. Tsosie, Jean-Charles Chapuis,
*
Sidney M. Hecht
Center for BioEnergetics, The Biodesign Institute, and Department of Chemistry, Arizona State University, Tempe, AZ 85287, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Verticipyrone has recently been isolated from the culture broth of Verticillium sp. and shown to inhibit
NADH fumarate reductase, as well as NADH oxidoreductase (complex I) of the mitochondrial electron
transport chain. In order to assess the structural elements in verticipyrone essential for complex I inhib-
itor, 15 structural analogues were prepared and analyzed for their effects on mitochondrial NADH oxido-
reductase and NADH oxidase activities. Also measured were the abilities of several of the analogues to
inhibit respiration as judged by a shift to glycolysis, and to inhibit the growth of several mammalian cell
lines. The nature of the pyrone ring was shown to be important to potency of inhibition, as was the length
and nature of substituents in the side chain of the analogues.
Received 10 February 2010
Revised 24 March 2010
Accepted 26 March 2010
Available online 31 March 2010
Keywords:
Verticipyrone analogues
Mitochondrial complex I
NADH-ubiquinone oxidoreductase
NADH oxidase
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
of respiration as measured by a shift to glycolysis, and (iv) evalua-
tion of the cytotoxicities of key compounds in several mammalian
Recently, verticipyrone (Fig. 1) was isolated from the culture
broth of the fungus Verticillium sp. FKI-1083 as an inhibitor of
NADH fumarate reductase and complex I (NADH oxidoreductase)
of the mitochondrial electron transport chain.1 The fully substi-
cell lines.
The verticipyrone analogues exhibited a range of potencies as
inhibitors of the mitochondrial respiratory chain. Modification of
the 6-methoxy group of the -pyrone ring with a methyl group
did not drastically affect the complex I inhibitory potency. How-
ever, alterations of the -pyrone moiety to a -hydroxy- -pyrone
reduced the inhibitory potency dramatically. The position and ste-
reochemistry of the hydroxyl group on the side chain did not sig-
nificantly affect the potency of the prepared analogues, but
introduction of a biphenyl ether moiety led to more potent ana-
logues than those containing alkyl chains. Reported herein is the
preparation, biochemical and biological characterization of these
interesting new mitochondrial respiratory chain inhibitors. In
addition to providing compounds of utility for probing mitochon-
drial complex function, the findings bear relevance to the design
of exogenous complex I substrates.
c
tuted c-pyrone core and the unsaturated side chain of verticipy-
rone exhibit structural similarities both with ubiquinone as well
as piericidin, the most potent inhibitor of complex I (Fig. 1). Omura
and co-workers reported the total synthesis of verticipyrone and
analogues which inhibited NADH fumarate reductase from Ascariis
suum with IC50 values in the subnanomolar range.2
Given the interesting biological profile of verticipyrone and its
analogues,2 a variety of new verticipyrone analogues were envi-
sioned and prepared to investigate the structural elements of the
natural product responsible for its effects on mitochondrial elec-
tron transport. The structures of the new analogues were designed
to differ from verticipyrone analogue 1 in several respects, and in-
cluded modification of the pyrone moiety and aliphatic side chain
(Fig. 2).
c
c
a
2. Results and discussion
2.1. Chemistry
The analogues were studied to define their effects on the mito-
chondrial electron transport chain. Specific effects investigated in-
cluded (i) complex I function, as measured by NADH-ubiquinone
oxidoreductase, (ii) function of complexes I, III and IV, as measured
by NADH oxidase, (iii) evaluation of the consequences of inhibition
The targeted pyrones were prepared first. Tetramethyl-c-pyr-
one (2) was prepared in 46% yield following a literature procedure
by treating 3-pentanone with polyphosphoric acid and acetic acid
(Scheme 1).3 Known b-ketoester 3 was obtained by intermolecular
Claisen reaction of ethyl propionate in the presence of KOtBu.4 By
* Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S.M. Hecht).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.070

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S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
O
O
MeO
MeO
N
MeO
MeO
10
OH
OH
O
piericidin A1
ubiquinone
MeO
O
O
MeO
OH
O
verticipyrone
1
Figure 1. Structures of ubiquinone (coenzyme Q10), piericidin A1, verticipyrone and previously synthesized analogue 1.
O
O
OH
OH
O
O
6
2
O
O
O
O
O
OH
O
O
O
O
1
OH
H
N
(CH2)7
(CH2)2
OH
OH
OH
Figure 2. Structural modifications of verticipyrone, shown by individual structural domains.
PPA, AcOH
O
130 °C
46%
O
O
O
2
1) LDA (nBuLi + NH(iPr)2)
THF, 0 °C
2) N-acetylimidazole
THF, -78 °C
O
O
O
O
O
KOtBu,80 °C
61%
OEt
OEt
OEt
3
O
O
O
O
DBU
CaCO3, (MeO)2SO2
acetone, 50 °C
benzene
66% (2 steps)
43%
O
OH
5
4
Scheme 1. Synthesis of pyrones 2, 4 and 5.
using an approach similar to that used for the reported synthesis of
iromycin,5 3 was first acetylated using LDA and N-acetylimidazole,
affording the unstable intermediate diketo ester; treatment with
methoxy-c-pyrone (5) by selective O-methylation of 4 was accom-
plished by using dimethyl sulfate. According to the method of Hos-
okawa et al., treatment of 4 with dimethyl sulfate in presence of a
large excess of CaCO3 in acetone gave desired methylated pyrone 5
after four days of reaction at 50 °C.6 This compound was separated
DBU afforded the expected c-hydroxy-a-pyrone 4 via cyclization
of the intermediate (Scheme 1). This scheme was shorter and more
convenient operationally than a published route,2 although pro-
from its isomeric
c-methoxy-a-pyrone by chromatography on a
ceeding in lower yield. The preparation of the corresponding
a-
silica gel column.

S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
3483
With pyrones 2, 4 and 5 in hand, the synthetic approach envi-
sioned to obtain the tertiary alcohol analogues involved regioselec-
tive opening of suitable racemic, (R) and (S) epoxides by anions
derived from the pyrones. In support of this strategy, Zhang et al.
have recently reported regioselective alkylation of hydroxypyrones
by using nBuLi in a mixture of THF and HMPA.7 This report also
established the feasibility of epoxide opening by this method. Fur-
ther, Shimamura et al. found that the C2 methyl group of meth-
oxypyrone 5 can be regioselectively deprotonated by using either
LiHMDS or LDA.2 It was thought that tetramethyl pyrone 2 should
exhibit the same reactivity, presuming the use of stoichiometric
amounts of base to avoid the formation of disubstituted secondary
products. According to this strategy, the racemic epoxide 6 was
first prepared by treatment of 2-decanone with trimethylsulfoxo-
nium iodide and NaH in DMSO (Scheme 2) following a literature
procedure.8 The 2-decanone also provided access to the corre-
sponding 2-methyl-1-decene (7) in almost quantitative yield by
means of a Wittig reaction using triphenylmethylphosphonium io-
dide and nBuLi (Scheme 2).9 Stereoselective Sharpless dihydroxyla-
associated with the use of LiHMDS as a base, permitted the forma-
tion of coupling products 15, 16 and 17 in low (23–32%) yields
(Scheme 3). The same reaction conditions were applied successfully
to tetramethylpyrone
2 by using stoichiometric amounts of
LiHMDS (Scheme 3). After purification, the expected coupling prod-
ucts 18, 19 and 20 were obtained in low (16–25%) yields as well.
Another synthetic challenge involved the preparation of a
droxy- -pyridone derivative of coupling product 12. Initially, it was
thought that the synthesis of suitably protected -hydroxy- -pyri-
done as well as -methoxy- -pyridone would provide a route to
c-hy-
a
c
a
c
a
fully elaborated pyridone analogues via coupling with epoxides
and pyridone deprotection. This synthetic approach was abandoned
after many unsuccessful coupling attempts. It was then decided to
synthesize the pyridone moiety after alkyl epoxide coupling of the
c-hydroxy-a-pyrone 4. Accordingly, c-hydroxy-a-pyridone deriva-
tive 21 was then prepared by treatment of 12 with NH4OH in a
sealed tube (Scheme 3). However, after purification by preparative
thin layer chromatography, the compound proved to be rather
unstable. Therefore, freshly purified pyridone 21 was utilized imme-
diately for biochemical and biological assays, as well as for charac-
terization by MALDI mass spectrometry and 1H NMR spectroscopy.
The strong inhibitory activity of analogue 1 toward NADH
fumarate reductase prompted the preparation of this compound
and structural analogues to permit their more detailed study.
Treatment of methoxypyrone 5 with decanal in presence of LDA
gave the expected product 1 in 34% yield (Scheme 4). Study of
the influence of side chain length was enabled by preparing a sim-
ilar compound with a shorter side chain. Accordingly, the coupling
conditions used with decanal were then applied successfully with
valeraldehyde, affording desired analogue 22, albeit in low (18%)
yield (Scheme 4).
tion of 7 by the use of ADmix b and
a afforded the expected (R) and
(S) diols 8 and 9, respectively.10 The selective activation of the
primary alcohols was achieved by conversion to the respective
mesylates,11 the latter of which were treated with 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU)12 to give the corresponding (R) and
(S)-epoxides 10 and 11 in ꢀ75% yields (Scheme 2).
The regioselective opening of epoxides 6, 10 and 11 by
xy-
c-hydro-
a-pyrone 4 using conditions reported by Zhang et al. led to the
expected coupling products (Scheme 3).7 Compounds 12, 13 and
14 were obtained in this fashion, albeit in modest yields (25–
52%), probably due to the low reactivity of the epoxides, as well
as the need for careful purification of the formed product mixtures.
However, opening of the epoxides was observed to be regioselec-
tive; the undesired isomers were not formed. Similar coupling reac-
tions with methoxypyrone 5 proved to be more difficult. In fact,
after formation of the carbanion of 5 by treatment with LiHMDS
or LDA in THF at ꢁ78 °C, no coupling was observed following addi-
tion of the epoxides. The addition of known epoxide activating
agents such as LiCl or LiOTf also produced disappointing results. Fi-
nally, extensive investigation revealed that the addition of BF3ꢂEt2O,
Another class of analogues was prepared keeping the nature of
the pyrone ring (a-methoxy-c-pyrone) as well as the position of
the hydroxyl group on the side chain unchanged. The structural
modification of these compounds involved the end of the side chain
with the incorporation of phenyl, phenoxy and 4-phenylphenoxy
groups. Access to the first envisioned analogue was achieved by
Swern oxidation of commercially available 7-phenylheptanol, lead-
ing almost quantitatively to corresponding aldehyde 23 (Scheme 5),
NaH, (CH3)3S(I)O
THF:DMSO
78%
O
O
O
6
7
Ph3PMeI, nBuLi
THF, 0 °C - 25 °C
96%
1) MsCl, pyridine
CH2Cl2, 0 °C - 25 °C
ADmix-beta
2) DBU, CH2Cl2
74%
(R)
(R)
1:1 tBuOH-H2O, 0 °C
O
HO
HO
96%
10
11
8
7
1) MsCl, pyridine
CH2Cl2, 0 °C - 25 °C
ADmix-alpha
2) DBU, CH2Cl2
76%
(S)
(S)
1:1 tBuOH-H2O, 0 °C
HO
O
HO
89%
9
7
Scheme 2. Preparation of epoxides 6, 10 and 11.

3484
S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
nBuLi, 6, 10, 11
6:1 THF-HMPA, -78 °C to 25 °C
O
O
O
O
OH
12 (rac.)
52%
OH
OH
13 (R)
14 (S)
25%
27%
4
LiHMDS, BF3OEt2, 6, 10, 11
O
O
O
O
O
THF, -78 °C to 25 °C
OH
15 (rac.)
27%
O
16 (R)
23%
5
17 (S) 32%
LiHMDS, BF3OEt2, 6, 10, 11
O
O
O
THF, -78 °C to 25 °C
OH
18 (rac.)
16%
25%
20 (S) 23%
O
19 (R)
2
H
NH4OH
EtOH, 80°C, sealed tube
O
O
O
N
OH
21
OH
27%
12
OH
OH
Scheme 3. Synthesis of pyrone analogues 12–20 and pyridone analogue 21.
LDA, decanal
THF, -78 °C
O
O
O
O
O
O
OH
34%
O
O
O
1
5
LDA, valeraldehyde
THF, -78 °C
O
OH
18%
O
O
22
5
Scheme 4. Synthesis of 1 and analogue 22.
followed by coupling to 5 using the conditions described above (cf.
Schemes 4 and 6). Coupling product 28 was obtained in 44% yield
after purification (Scheme 6). 6-Phenoxy-1-hexanol (24) was pro-
duced in good yield, following the literature procedure involving
a nucleophilic substitution of 6-bromo-1-hexanol by phenol in
presence of K2CO3 (Scheme 5).13 6-Phenoxyhexanol was converted
to unstable aldehyde 25 in 94% yield (Scheme 5). The latter was
condensed with 5, affording verticipyrone analogue 29 in 35% yield
(Scheme 6). In the same manner, alcohol 26 was obtained in 57%
yield from 4-phenylphenol and oxidized to corresponding aldehyde
27 in 96% yield (Scheme 5). The coupling reaction afforded verti-
cipyrone analogue 30 in 46% yield (Scheme 6).
I (NADH: ubiquinone oxidoreductase) to complex III (ubiquinol:
cytochrome c oxidoreductase) by coenzyme Q10 and then from
complex III to complex IV (cytochrome c oxidase) by the peripheral
membrane protein cytochrome c. Nine of the 15 assayed pyrone
analogues (compounds 1, 15–20, 28 and 30), were found to be po-
tent inhibitors of the integrated electron transfer chain (NADH oxi-
dase) activity in beef heart submitochondrial particles (SMPs) with
IC50 values less than 75 nM (Table 1). Compound 30 was found to be
the most potent inhibitor with an IC50 value of 35.9 nM. Compound
22 did not inhibit mitochondrial respiratory chain function,
undoubtedly reflecting its very short alkyl side chain. These results
support the conclusion that the aliphatic chain plays an important
role in the interaction of the verticipyrone analogues with complex
I and possibly other components of the respiratory chain. In addi-
tion, the potent inhibitory activity of analogue 30 demonstrates
that the phenylphenoxy group promotes particularly effective
2.2. Inhibition of NADH oxidase and NADH-ubiquinone
oxidoreductase
The NADH oxidase and NADH-ubiquinone oxidoreductase activ-
ities of compounds 1, 12–22, 28, 29 and 30 were measured. NADH
oxidase activity reflects electron transfer in the respiratory chain
from complex I to IV; electrons are first transported from complex
interaction with the respiratory chain. The a-methoxy-c-pyrones
(1, 15–17, 28, 29 and 30) as well as the trimethylpyrone analogues
(18–20) showed minimal differences as inhibitors of mitochondrial
respiratory chain function (Table 1).

S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
3485
(COCl)2, DMSO, NEt3
CH2Cl2, -78 °C
H
HO
93%
O
23
(COCl)2, DMSO, NEt3
CH2Cl2, -78 °C
6-bromo-1-hexanol, K2CO3
DMF, 80 °C
HO
H
HO
O
O
94%
91%
O
24
25
(COCl)2, DMSO, NEt3
CH2Cl2, -78 °C
6-bromo-1-hexanol, K2CO3
DMF, 80 °C
HO
H
HO
O
O
96%
57%
O
26
Scheme 5. Synthesis of intermediate aldehydes 23, 25 and 27.
27
23
LDA,
O
O
O
O
O
O
O
THF, -78 °C
OH
44%
O
O
O
O
5
28
LDA, 25
O
THF, -78 °C
O
O
OH
35%
O
5
29
LDA, 27
O
O
O
O
THF, -78 °C
OH
46%
O
5
30
Scheme 6. Synthesis of verticipyrone analogues 28, 29 and 30.
In three cases (12 vs 13 and 14; 15 vs 16 and 17; 18 vs 19 and
20), the inhibitory properties of compounds racemic at the carbon
atom bearing the tertiary OH group were compared with those of
each of the enantiomers. As is clear from Table 1, no dramatic dif-
ferences were noted, although there was a possible tendency for
the racemates to be slightly less potent than the individual enanti-
omers. Perhaps more significantly, for those compounds with rea-
sonable inhibitory potency, the two enantiomers had quite similar
potencies, indicating a lack of effect of stereochemistry at the ter-
tiary OH center.
Interestingly, analogue 21 had more than 10-fold greater po-
tency than 12 as an inhibitor of NADH-ubiquinone oxidoreductase,
suggesting that the interactions of 21 with the mitochondrial
respiratory complex may differ from those of the other analogues
in some respects. Inhibition of complex I was measured directly
by measuring NADH-ubiquinone oxidoreductase activity in pres-
ence of a lipid-soluble exogenous ubiquinone-1 (Q1) through com-
plex I, as well as complex III inhibitor antimycin A and complex IV
inhibitor cyanide. It is interesting that for those compounds exhib-
iting reasonably potent inhibitory activity, IC50 values for the
NADH oxidase activity were lower with respect to those of the
NADH-ubiquinone oxidoreductase activity (Table 1). Such relative
extents of inhibitory potencies have been noted previously for
other complex I inhibitors.1,14,15 The substrate concentration was
higher in Q1-dependent NADH oxidation than in the integrated
NADH oxidase assay (which contained only ꢀ0.1–0.2
lM
Q1016,17), consistent with the interpretation that these inhibitors
compete with ubiquinone for binding to complex I.
2.3. Induction of lactate production
More than 70 years ago, Warburg observed that cancer cells fre-
quently exhibit increased glycolysis and depend significantly on
this metabolic pathway for the generation of ATP to meet their en-
ergy needs.18 The compromised ability of cancer cells to generate
ATP through oxidative phosphorylation forces the cells to increase
glycolysis to maintain their energy supply, thus rendering cancer
cells more highly dependent on this metabolic pathway for sur-
vival.19 Since glycolysis in the absence of mitochondrial function
yields two ATP and two lactate molecules per glucose catabolized,
it follows that the incremental lactate production arising from
inhibition of respiration is proportional to the extent of inhibition
of mitochondrial ATP production, assuming that the Pasteur ef-
fect20 compensates for respiratory inhibition in proportion to the
extent of inhibition. We have used this principle (the Pasteur ef-
fect) to evaluate known mitochondrial toxins, such as the
Dlac-

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S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
Table 1
Table 2
Inhibitory activities toward mitochondrial complex Ia of the described verticipyrone
analogues
Lactate production by MCF-7 human breast cancer cells incubated for 3 h in the
presence of some of the verticipyrone analogues
b
a
Compound
IC50
Compound (
lM)
IC50
(lM)
Incremental lactate production (lM)
NADH oxidase (nM)
NADH-ubiquinone (Q1)
oxidoreductase (nM)
1
12
13
0.90
4.9
8.6
1365
1175
692
1
12
13
14
15
16
17
18
19
20
21
22
28
29
30
Rotenone
64.9 1.7
13,590 500
14,740 460
10,670 690
52.6 1.0
45.9 1.7
43.2 2.2
55.0 1.7
50.8 2.3
47.5 3.4
4290 130
>20,000
230.9 5.7
16,410 240
19,020 450
13,300 1050
151.1 4.6
133.1 3.2
126.2 2.2
180.2 8.8
145.4 5.9
135.3 4.6
1250 96
>25,000
14
15
16
17
18
22
28
30
16.6
891
0.38
0.30
0.18
0.94
>20
0.88
0.34
1745
2229
1852
1099
419
1135
1513
1520
Antimycin A
<0.15
a
IC50 values were calculated as the dose which produced a 50% inhibition of the
maximum lactate production measured.
72.5 1.2
218.9 8.5
35.9 1.1
4.36 0.07
257.1 13.1
595 35.8
101.6 4.0
Nt
in presence (mitochondrial inhibition) and absence (basal condi-
tion) of antimycin A represent an amount of energy which corre-
lates well with that produced by the mitochondria prior to the
respiratory chain inhibition. Figure 3 shows lactate production by
semi-confluent MCF-7 cells exposed to varying concentrations of
several verticipyrone analogues for 3 h. Lactate production, which
is low under basal conditions, was strongly stimulated after incu-
bation with some of the verticipyrone analogues (1, 17, and 30)
and weakly stimulated by others (14, 22). In order to compare
these compounds, the concentration required to obtain half maxi-
mal lactate production (IC50) was calculated (Table 2). The potency
of the tested compounds in inducing a shift to glycolysis was in
reasonable agreement with the trend for mitochondrial complex
I inhibition (cf. Tables 1 and 2).
a
Performed using bovine heart submitochondrial particles (SMP).
The molar concentration required for half-maximal inhibition of the control.
b
Values are mean SD of three independent experiments. NADH-ubiquinone oxi-
doreductase was determined with ubiquinone 1 (Q1) as the ubiquinone substrate.
Control activity was approximately 0.75–0.9 l
mol minꢁ1 mgꢁ1 for NADH oxidase
and 0.65–0.75
determined.
l
mol minꢁ1 mgꢁ1 for NADH-ubiquinone oxidoreductase. Nt, not
200
180
160
140
120
100
80
2.4. Cytotoxicity toward mammalian cell lines
Table 3 shows the IC50 values for several verticipyrone ana-
logues against cultured human and mouse cell lines. The cytotox-
60
icity of the
(IC50 >31 M) in all tested cell lines, in parallel with their lack of
significant inhibition of mitochondrial complex I. On the other
hand, the cytotoxicity of the -methoxy- -pyrones (1, 16, 17, 28
c-hydroxy-a-pyrone analogues (12–14) was minimal
40
l
20
0
0.08
0.4
2
10
50
a
c
and 30) were in the low micromolar range. Interestingly, trim-
ethylpyrone 18 exhibited no cytotoxicity against the cultured cells.
In contrast with the effects of the verticipyrone analogues on
lactate production, the data in Table 3 reflect less correlation be-
tween the cytotoxicity data and the effects of individual com-
pounds on mitochondrial complex I. This may reflect factors such
as differences in cellular uptake and metabolism for the individual
compounds, especially where the analogues (e.g., for 18) have
unexpectedly low cytotoxic potency. Alternatively, it may result
Concentration (µg / mL)
Figure 3. Lactate production by MCF-7 cells exposed to compound 1 ( ), 14 ( ), 17
), 22 ( ), or 30 ( ) for 3 h. Data shown are from a typical experiment, repeated at
least three times, and each point represents the mean SD of three determinations.
(
acetogenins,14 as well as the verticipyrones that are the focus of
this study. While the basal lactate levels reflect glycolytic ATP,
the difference in the lactate concentrations (D-lactate) measured
Table 3
Inhibition values (IC50
, l
M) of several verticipyrone analogues determined by MTT assay using human and mouse cell linesa
Compound
MCF-7
MCF-10A
CRL-2365
CRL-2366
A549
3LL
1
12
13
14
16
17
18
28
30
23.2 0.6
>31
>31
9.9 0.3
>31
>31
29.9 0.6
>31
>31
>31
>31
>31
>31
2.7 0.3
3.3 1.5
>31
>27
19.5 0.7
20.7 0.6
>31
>31
22.2
>31
>31
>31
0.39 0.07
0.34 0.05
>31
5
>31
>31
>31
>31
3.3 0.3
3.3 0.3
>31
>27
17.4 0.9
5.3 0.3
5.6 0.3
>31
15.1 0.8
9.2 0.2
3.3 2.4
2.4 0.3
>31
>27
12.4 0.9
5.3 0.1
4.1 0.3
>31
>27
17.0 0.9
17.3 1.7
13.5 0.2
a
Cell Type: MCF-7 (breast carcinoma); MCF-10A (‘normal’ human breast); CRL-2365 (brain glioblastoma); CRL-2366 (DNA repair deficient brain glioblastoma); A549 (lung
carcinoma); 3LL (mouse Lewis lung carcinoma). DMSO was used as vehicle in the testing.

S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
3487
from the action of some of the analogues at cellular loci in addition
to mitochondrial complex I (e.g., for 16 and 17).
onate and 2.75 g (24.5 mmol) of powdered KOtBu was heated at
80 °C for 3 h. The reaction mixture was cooled and neutralized
with 1 N HCl. The mixture was extracted with portions of ethyl
ether. The combined organic phase was dried over anhydrous
MgSO4, filtered and concentrated under diminished pressure to af-
ford a crude oil. The residue was purified by chromatography on a
silica gel column (20 ꢃ 2 cm). Elution with 7:1 hexanes/ethyl ace-
tate gave 3 as a colorless oil: yield 2.40 g (61%); silica gel TLC Rf
0.62 (2:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 0.98 (t, 3H,
J = 7.2 Hz), 1.18 (t, 3H, J = 7.2 Hz,), 1.25 (d, 3H, J = 7.2 Hz), 2.47
(m, 2H), 3.45 (q, 1H, J = 14.1, 7.2 Hz) and 4.09 (q, 2H, J = 13.8,
7.2 Hz).
3. Conclusions
The preparation and study of several analogues of verticipyrone
was accomplished during the course of this study. The biochemical
studies of analogues 12–21 showed that the nature of the pyrone
ring could be very important to the potency of mitochondrial com-
plex I inhibition, but the hydroxyl group at the C3 position of the
side chain was not. The
and 30) and trimethyl-
ences in their inhibitory potency toward mitochondrial complex I.
In comparison, the cytotoxicity of the -methoxy- -pyrones (1,
16, 17, 28 and 30) tended to exhibit somewhat greater cytotoxicity
toward cultured mammalian cells than trimethyl- -pyrone 18. Ver-
a-methoxy-c-pyrones (1, 15–17, 28, 29
c-pyrones (18–20) showed limited differ-
4.1.3. 4-Hydroxy-3,5,6-trimethyl-2-pyrone (4)2
a
c
To a solution containing 5.75 mL (4.13 g, 40.8 mmol) of iPr2NH
in 80 mL of THF at 0 °C was added dropwise 25.0 mL (40.6 mmol)
of nBuLi (1.6 M in hexane). The reaction mixture was stirred at
0 °C for 30 min. A solution containing 2.47 g (15.6 mmol) of 3 in
5 mL of THF was then added dropwise. The reaction mixture was
stirred at 0 °C for 1 h and then cooled to ꢁ78 °C. A solution of
2.60 g (23.4 mmol) of N-acetylimidazole in 30 mL of THF was then
added dropwise. The reaction mixture was stirred at ꢁ78 °C for 2 h,
quenched with satd aq NH4Cl and extracted with portions of ethyl
acetate. The combined organic phase was washed with water and
brine, dried over anhydrous MgSO4, filtered and concentrated un-
der diminished pressure to afford the crude diketoester (3.00 g)
which was used immediately for the next step due to its instability.
To a solution containing 3.00 g (14.9 mmol) of diketoester in
50 mL of benzene was added 2.70 mL (17.9 mmol) of 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU). The reaction mixture was heated
at reflux for 10 h. After cooling to room temperature, the mixture
was poured into 30 mL of cooled (0 °C) satd aq NaHCO3. The aque-
ous layer was washed with portions of Et2O. The aqueous layer was
then acidified to pH 2 with 12 N aqueous HCl. The mixture was ex-
tracted with portions of Et2O. The combined organic extract was
dried over anhydrous MgSO4, filtered and concentrated under
diminished pressure to afford a crude residue. The residue was
purified by chromatography on a silica gel column (10 ꢃ 2 cm).
Elution with chloroform gave 4 as a colorless solid: yield 1.35 g
(57% over two steps); silica gel TLC Rf 0.21 (10:1 chloroform/meth-
anol); 1H NMR (CDCl3) d 1.96 (s, 3H), 1.99 (s, 3H) and 2.21 (s, 3H);
13C NMR (CDCl3) d 8.6, 10.0, 17.1, 98.4, 108.1, 166.4, 155.2 and
167.2.
c
ticipyrone analogue 22 showed no inhibitory activity compared to
1, underscoring the importance of the length and lipophilic nature
of the side chain. While the present compounds were designed as
inhibitors of complex I function, the findings regarding optimal side
chain structure for complex I binding may guide efforts to identify
exogenous complex I substrates of potential utility for augmenting
the function of the mitochondrial electron transport chain.
4. Experimental
4.1. Chemistry
Chemicals and solvents were of reagent grade and were used
without further purification. Anhydrous grade solvents were pur-
chased from VWR. All reactions involving air or moisture sensitive
reagents or intermediates were performed under an argon atmo-
sphere. Flash chromatography was carried out using Silicycle
200–400 mesh silica gel. Analytical TLC was carried out using
0.25 mm EM Silica Gel 60 F250 plates that were visualized by irra-
diation (254 nm) or by staining with p-anisaldehyde stain. 1H and
13C NMR spectra were obtained using Gemini 300, Inova 400, Inova
500 and VNMRS 800 MHz Varian instruments. Chemical shifts
were reported in parts per million (ppm, d) referenced to the resid-
ual 1H resonance of the solvent (CDCl3, 7.26 ppm). 13C spectra were
referenced to the residual 13C resonance of the solvent (CDCl3,
77 ppm). Splitting patterns were designated as follow: s, singlet;
br, broad; d, doublet; dd, doublet of doublets; t, triplet; q, quartet;
m, multiplet. High resolution mass spectra were obtained at the
Ohio State University Mass Spectrometry Facility.
4.1.4. 2-Methoxy-3,5,6-trimethyl-4-pyrone (5)2
Compound 5 was prepared according to the method of Hosoka-
wa et al.6 To a solution containing 365 mg (2.37 mmol) of 4 and
1.20 g (12.0 mmol) of CaCO3 in 15 mL of acetone was added
0.27 mL (2.81 mmol) of (MeO)2SO2. The reaction mixture was
heated to 50 °C and stirred for 4 days, cooled to room temperature
and filtered. Excess solvent was removed under diminished pres-
sure to afford a crude residue. The residue was purified by chroma-
4.1.1. 2,3,5,6-Tetramethyl-4-pyrone (2)2
Compound 2 was prepared according to the method of Letsinger
and Jamison.3 To a mixture of 195 g of polyphosphoric acid and
120 mL of acetic acid was added 4.90 mL (4.00 g, 46.4 mmol) of
3-pentanone. The resulting solution was heated at 130 °C with vig-
orous stirring for 8 h. The resulting dark black solution was cooled
to 0 °C and 500 mL of water was added. The mixture was extracted
with portions of ethyl ether. The combined organic phase was
washed with 10% aq KOH, dried over anhydrous MgSO4 and con-
centrated under diminished pressure to afford a crude residue.
The residue was purified by chromatography on a silica gel column
(40 ꢃ 10 cm). Elution with chloroform gave 2 as a yellow solid:
yield 3.25 g (46%); silica gel TLC Rf 0.63 (10:1 chloroform/metha-
nol); 1H NMR (CDCl3) d 1.86 (s, 6H) and 2.18 (s, 6H); 13C NMR
(CDCl3) d 9.8, 17.5, 118.5, 159.9 and 179.2.
tography on
a
silica gel column (15 ꢃ 3 cm). Elution with
chloroform gave 5 as a white solid: yield 172 mg (43%); silica gel
TLC Rf 0.39 (10:1 chloroform/methanol); 1H NMR (CDCl3) d 1.82
(s, 3H), 1.90 (s, 3H), 2.24 (s, 3H) and 3.92 (s, 3H); 13C NMR (CDCl3)
d 6.8, 10.0, 16.8, 55.3, 99.4, 118.4, 154.8, 162.1 and 180.8.
4.1.5. (rac) 1-Epoxy-2-methyldecane (6)
To a suspension of 485 mg (12.1 mmol) of NaH (60%) in a mix-
ture of 4 mL of THF and 3 mL of DMSO was added dropwise a solu-
tion containing 2.66 g (12.1 mmol) of trimethylsulfoxonium iodide
in 35 mL of DMSO while keeping the reaction temperature be-
tween 6 °C and 10 °C. The reaction mixture was stirred at 6 °C for
15 min and a solution containing 2.01 g (1.65 g, 10.5 mmol) of 2-
decanone in 5 mL of DMSO was added dropwise over a period of
4.1.2. Ethyl 2-methyl-3-oxo-pentanoate (3)2
Compound 3 was prepared according to the method of Yoshiz-
awa et al.4 A solution containing 3.56 g (34.8 mmol) of ethyl propi-

3488
S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
5 min. The reaction mixture was stirred at room temperature over-
night and 20 mL of hexane and 30 mL of semi-satd aq NH4Cl were
added successively. The reaction mixture was extracted with por-
tions of hexanes. The combined organic phase was dried over
anhydrous MgSO4, filtered and concentrated under diminished
pressure to afford a crude oil. The residue was purified by chroma-
tography on a silica gel column (15 ꢃ 2 cm). Elution with 12:1 hex-
anes/ethyl ether gave 6 as a colorless oil: yield 1.41 g (78%); silica
for 6 h and 1.93 g (15.3 mmol) of sodium sulfide was added. The
reaction mixture was allowed to warm to room temperature and
was stirred for 1 h. Twenty milliliters of CH2Cl2 and 40 mL of water
were then added successively and the reaction mixture was ex-
tracted with portions of CH2Cl2. The combined organic phase was
dried over anhydrous MgSO4, filtered and concentrated under
diminished pressure to afford a crude oil. The residue was purified
by chromatography on a silica gel column (8 ꢃ 2 cm). Step gradient
elution with 4:1 hexanes/ethyl acetate?2:1 hexanes/ethyl acetate
gel TLC Rf 0.52 (8:1 hexanes/ethyl ether); ½a D22
ꢄ
+1.2 (c 0.36, CHCl3);
1H NMR (CDCl3) d 0.87 (t, 3H, J = 7.1 Hz), 1.27 (m, 10H), 1.29 (s, 3H),
1.37 (m, 2H), 1.46 (m, 1H), 1.58 (m, 1H) and 2.57 (dd, 2H, J = 13,
4.5 Hz); 13C NMR (CDCl3) d 14.3, 21.1, 22.9, 25.5, 29.4, 29.7, 29.9,
32.1, 36.9, 54.1 and 57.3.
gave 9 as a colorless oil: yield 217 mg (89%); ½a D22
ꢄ
+5.9 (c 0.19,
CHCl3); silica gel TLC Rf 0.24 (6:3 hexanes/ethyl acetate); 1H NMR
(CDCl3) d 0.88 (t, 3H, J = 7 Hz), 1.16 (s, 3H), 1.29 (m, 12H), 1.49
(m, 2H), 2.05–2.38 (2 br s, 2H) and 3.43 (dd, 2H, J = 20.0,
11.2 Hz); 13C NMR (CDCl3) d 14.3, 22.8, 23.4, 23.9, 29.5, 29.8,
30.4, 32.1, 38.9, 70.0 and 73.2; mass spectrum (ESI), m/z
211.1673 (M+Na)+ (C11H24O2Na requires m/z 211.1674).
4.1.6. 2-Methyl-dec-1-ene (7)
Compound 7 was prepared according the method of Farhat
et al.21 To a solution containing 3.60 g (8.96 mmol) of methyltri-
phenylphosphonium iodide in 30 mL of THF at 0 °C was added
dropwise 5.60 mL (8.96 mmol) of nBuLi (1.6 M in hexane). The
reaction mixture was stirred at room temperature for 1 h, cooled
to 0 °C and a solution containing 1.00 mL (824 mg, 5.28 mmol) of
2-decanone in 5 mL of THF was added dropwise. The reaction mix-
ture was stirred at 0 °C for 15 min and then at room temperature
for 8 h. The solution was diluted with 50 mL of hexanes and filtered
through a pad of silica gel which was washed with 100 mL of 1:1
hexanes/ethyl ether. The filtrate was concentrated carefully under
diminished pressure to afford a crude oil. The residue was purified
by chromatography on a silica gel column (15 ꢃ 2 cm). Elution
with 20:1 hexanes/ethyl ether gave 7 as a colorless oil: yield
700 mg (86%); silica gel TLC Rf 0.89 (8:1 hexanes/ethyl ether); 1H
NMR (CDCl3) d 0.90 (t, 3H, J = 7.1 Hz), 1.30 (m, 10H), 1.44 (m,
2H), 1.72 (s, 3H), 2.01 (t, 2H, J = 7.5 Hz) and 4.69 (d, 2H,
J = 10.5 Hz); 13C NMR (CDCl3) d 13.1, 22.2, 22.7, 27.6, 29.3, 29.4,
29.5, 31.9, 37.8, 103.4 and 146.2.
4.1.9. (R)-1-Epoxy-2-methyldecane (10)
To a solution containing 127 mg (0.67 mmol) of 8 in 1.50 mL of
CH2Cl2 at 0 °C was added 136
lL (133 mg, 1.69 mmol) of pyridine.
The reaction mixture was stirred at 0 °C for 5 min and 80.0
l
L
(1.15 mmol) of mesyl chloride (MsCl) was added dropwise. The
reaction mixture was stirred at room temperature for 1 h,
quenched with satd aq NaHCO3, and extracted with portions of
CH2Cl2. The combined organic phase was washed with brine, dried
over anhydrous MgSO4, filtered and partially concentrated to a vol-
ume of ꢀ3 mL under diminished pressure.
To the solution containing the crude mesylate was added at
room temperature 1.31 mL (8.76 mmol) of 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU). The reaction mixture was stirred
for 10 h, diluted with 7 mL of CH2Cl2, quenched with satd aq NH4Cl
and extracted with portions of CH2Cl2. The combined organic phase
was washed with brine, dried over anhydrous MgSO4, filtered and
concentrated under diminished pressure to afford a crude oil. The
residue was purified by chromatography on a silica gel column
(5 ꢃ 1 cm). Elution with 17:1 hexanes/ethyl ether gave epoxide
10 as a colorless oil: yield 85.0 mg (74% over two steps); silica
4.1.7. (R)-2-Hydroxy-2-methyldecanol (8)
A 250-mL three necked round-bottomed flask equipped with a
mechanical stirrer was charged with 13.0 mL of a 1:1 mixture of
tBuOH/H2O and 1.80 g of ADmix-b. The resulting mixture was stir-
red vigorously at room temperature for 30 min and then cooled to
0 °C. A precipitate appeared and 200 mg (1.30 mmol) of 7 was
added in one portion. The resulting mixture was stirred vigorously
at 0 °C for 6 h and 1.93 g (15.3 mmol) of sodium sulfide was added.
The reaction mixture was allowed to warm to room temperature
and was stirred for 1 h. Twenty milliliters of CH2Cl2 and 40 mL of
water were then added successively and the reaction mixture
was extracted with portions of CH2Cl2. The combined organic
phase was dried over anhydrous MgSO4, filtered and concentrated
under diminished pressure to afford a crude oil. The residue was
purified by chromatography on a silica gel column (8 ꢃ 2 cm). Step
gradient elution with 4:1 hexanes/ethyl acetate?2:1 hexanes/
gel TLC Rf 0.52 (8:1 hexanes/ethyl ether); ½a D22
ꢄ
+5.2 (c 1.28, CHCl3);
1H NMR (CDCl3) d 0.87 (t, 3H, J = 7 Hz), 1.27 (m, 10H), 1.29 (s, 3H),
1.37 (m, 2H), 1.46 (m, 1H), 1.58 (m, 1H) and 2.57 (dd, 2H, J = 13.1,
4.5 Hz); 13C NMR (CDCl3) d 14.3, 21.1, 22.9, 25.5, 29.4, 29.7, 29.9,
32.1, 36.9, 54.1 and 57.3; mass spectrum (MALDI-TOF), m/z 171.2
(M)+ (theoretical m/z 171.3).
4.1.10. (S)-1-Epoxy-2-methyldecane (11)
To a solution containing 145 mg (0.77 mmol) of 9 in 2 mL of
CH2Cl2 at 0 °C was added 155
lL (151 mg, 1.69 mmol) of pyridine.
The reaction mixture was stirred at 0 °C for 5 min and 90.0
l
L
(1.17 mmol) of mesyl chloride (MsCl) was added dropwise. The
reaction mixture was stirred at room temperature for 1 h,
quenched with satd aq NaHCO3, and extracted with portions of
CH2Cl2. The combined organic phase was washed with brine, dried
over anhydrous MgSO4, filtered and partially concentrated to a vol-
ume of ꢀ3 mL under diminished pressure.
ethyl acetate gave 8 as a colorless oil: yield 235 mg (96%); ½a D22
ꢄ
ꢁ6.7 (c 0.48, CHCl3); silica gel TLC Rf 0.24 (6:3 hexanes/ethyl ace-
tate); 1H NMR (CDCl3) d 0.88 (t, 3H, J = 7 Hz), 1.16 (s, 3H), 1.29
(m, 12H), 1.49 (m, 2H), 2.05–2.38 (2 br s, 2H) and 3.43 (dd, 2H,
J = 20, 11 Hz); 13C NMR (CDCl3) d 14.3, 22.8, 23.4, 23.9, 29.5, 29.8,
30.4, 32.1, 38.9, 70.0 and 73.2; mass spectrum (ESI), m/z
211.1666 (M+Na)+ (C11H24O2Na requires m/z 211.1674).
To the solution containing the crude mesylate was added at
room temperature 1.50 mL (10.0 mmol) of DBU. The reaction mix-
ture was stirred for 10 h, diluted with 10 mL of CH2Cl2, quenched
with satd aq NH4Cl and extracted with portions of CH2Cl2. The
combined organic phase was washed with brine, dried over anhy-
drous MgSO4, filtered and concentrated under diminished pressure
to afford a crude oil. The residue was purified by chromatography
on a silica gel column (5 ꢃ 1 cm). Elution with 17:1 hexanes/ethyl
ether gave epoxide 11 as a colorless oil: yield 100 mg (76% over
4.1.8. (S)-2-Hydroxy-2-methyldecanol (9)
A 250-mL three necked round-bottomed flask equipped with a
mechanical stirrer was charged with 13.0 mL of 1:1 tBuOH/H2O
and 1.80 g of ADmix-a. The resulting mixture was stirred vigor-
ously at room temperature for 30 min and then cooled to 0 °C. A
precipitate appeared and 200 mg (1.30 mmol) of 7 was added in
one portion. The resulting mixture was stirred vigorously at 0 °C
two steps); silica gel TLC Rf 0.52 (8:1 hexanes/ethyl ether); ½a D22
ꢄ
ꢁ4.9 (c 0.95, CHCl3); 1H NMR (CDCl3) d 0.87 (t, 3H, J = 7 Hz), 1.27
(m, 10H), 1.29 (s, 3H), 1.37 (m, 2H), 1.46 (m, 1H), 1.58 (m, 1H)

S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
3489
and 2.57 (dd, 2H, J = 13.1, 4.5 Hz); 13C NMR (CDCl3) d 14.3, 21.1,
22.9, 25.5, 29.4, 29.7, 29.9, 32.1, 36.9, 54.1 and 57.3; mass spec-
trum, m/z 170.9 (M)+ (theoretical m/z 171.3).
with 3:1 hexanes/ethyl acetate?1:1 hexane/ethyl acetate gave
14 as a colorless oil: yield 17.0 mg (27%); silica gel TLC Rf 0.28
(9:1 chloroform/methanol); ½a D22
ꢄ
ꢁ11.4 (c 0.14, CHCl3); 1H NMR
(CDCl3) d 0.86 (t, 3H, J = 6.5 Hz), 1.18 (s, 3H), 1.20–1.31 (m, 12H),
1.46 (m, 2H), 1.73 (m, 2H), 1.75 (m, 2H), 1.96 (s, 3H), 1.97 (s, 3H)
and 2.58 (m, 2H); 13C NMR (CDCl3) d 8.7, 9.9, 14.3, 22.9, 24.2,
25.7, 26.7, 29.5, 30.4, 32.1, 39.0, 42.4, 72.7, 98.4, 105.0, 159.5,
165.0 and 166.3; mass spectrum (ESI), m/z 347.2200 (M+Na)+
(C19H32O4Na requires m/z 347.2198).
4.1.11. (rac)-6-[3-Hydroxy-3-methylundecyl]-3,5-dimethyl-4-
hydroxypyran-2-one (12)
To a solution containing 41.0 mg (0.266 mmol) of 4 in 1 mL of
6:1 THF/HMPA at ꢁ78 °C was added 382
lL (0.612 mmol) of nBuLi
(1.6 M in hexanes). The reaction mixture was stirred at ꢁ78 °C for
1 h and 90.0 mg (0.532 mmol) of neat racemic epoxide 6 was
added. The reaction mixture was stirred at ꢁ78 °C for 1 h and
was then allowed to warm slowly to room temperature and stirred
overnight. The reaction mixture was quenched with 1 N HCl and
extracted with portions of EtOAc. The combined organic phase
was washed with brine, dried over anhydrous MgSO4, filtered
and concentrated under diminished pressure to afford a crude
oil. The residue was purified by chromatography on a silica gel col-
umn (6 ꢃ 1.5 cm). Step gradient elution with 3:1 hexanes/ethyl
acetate?1:1 hexanes/ethyl acetate gave 12 as a colorless oil: yield
45.0 mg (52%); silica gel TLC Rf 0.28 (9:1 chloroform/methanol);
4.1.14. (rac)-2-[3-Hydroxy-3-methylundecyl]-6-methoxy-3,5-
dimethyl-4H-pyran-4-one (15)
To a solution containing 11.1 mg (0.065 mmol) of 5 in 0.40 mL
of THF at ꢁ78 °C was added dropwise 131
lL (0.131 mmol) of
1 M LiHMDS solution. The resulting mixture was stirred at
ꢁ78 °C for 1 h and 23.0 mg (0.131 mmol) of neat racemic epoxide
6 was added followed by 18.0
lL (0.131 mmol) of BF3ꢂEt2O. The
reaction mixture was stirred at ꢁ78 °C for 1 h and was then al-
lowed to warm slowly to room temperature and stirred overnight.
The reaction mixture was quenched with satd aq NH4Cl and ex-
tracted with portions of EtOAc. The combined organic phase was
washed with brine, dried over anhydrous MgSO4, filtered and con-
centrated under diminished pressure to afford a crude oil. The res-
idue was purified by chromatography on a silica gel column
(4 ꢃ 1 cm). Elution with 30:1 chloroform/methanol gave 15 as a
colorless oil: yield 6.02 mg (27%); silica gel TLC Rf 0.42 (9:1 chloro-
½
a 2D2 +1.5 (c 0.14, CHCl3); 1H NMR (CDCl3)
d 0.86 (t, 3H,
ꢄ
J = 6.5 Hz), 1.18 (s, 3H), 1.20–1.31 (m, 12H), 1.46 (m, 2H), 1.73
(m, 2H), 1.75 (m, 2H), 1.96 (s, 3H), 1.97 (s, 3H) and 2.58 (m, 2H);
13C NMR (CDCl3) d 8.7, 9.9, 14.3, 22.9, 24.2, 25.7, 26.7, 29.5, 29.8,
30.4, 32.1, 39.0, 42.4, 72.7, 98.4, 105.0, 159.5, 165.0 and 166.3;
mass spectrum (ESI), m/z 347.2196 (M+Na)+ (C19H32O4Na requires
m/z 347.2198).
form/methanol); ½a D22
ꢄ
+2.1 (c 0.14, CHCl3); 1H NMR (CDCl3) d 0.87
(t, 3H, J = 7 Hz), 1.23 (s, 3H), 1.26–1.37 (m, 12H), 1.49 (m, 2H),
1.75 (m, 2H), 1.84 (s, 3H), 1.94 (s, 3H), 2.66 (m, 2H) and 3.94 (s,
3H); 13C NMR (CDCl3) d 6.9, 9.8, 14.1, 22.6, 24.0, 25.6, 26.8, 29.2,
29.6, 30.1, 31.9, 38.4, 42.0, 55.3, 72.1, 99.4, 118.0, 158.5, 162.1
and 181.0; mass spectrum (ESI), m/z 361.2356 (M+Na)+
(C20H34O4Na requires m/z 361.2355).
4.1.12. (R)-6-[3-Hydroxy-3-methylundecyl]-3,5-dimethyl-4-
hydroxypyran-2-one (13)
To a solution containing 30.0 mg (0.194 mmol) of 4 in 1 mL of a
6:1 mixture of THF/HMPA at ꢁ78 °C was added 279
lL
(0.448 mmol) of nBuLi (1.6 M in hexanes). The reaction mixture
was stirred at ꢁ78 °C for 1 h and a solution containing 66.0 mg
(0.388 mmol) of (R)-epoxide 10 in 0.5 mL of THF was added. The
reaction mixture was stirred at ꢁ78 °C for 1 h and was then al-
lowed to warm slowly to room temperature and stirred overnight.
The reaction was quenched with 1 M HCl and extracted with por-
tions of EtOAc. The combined organic phase was washed with
brine, dried over anhydrous MgSO4, filtered and concentrated un-
der diminished pressure to afford a crude oil. The residue was puri-
fied by chromatography on a silica gel column (6 ꢃ 1 cm). Elution
with 3:1 hexanes/ethyl acetate?1:1 hexanes/ethyl acetate gave
13 as a colorless oil: yield 16.0 mg (25%); silica gel TLC Rf 0.28
4.1.15. (R)-2-[3-Hydroxy-3-methylundecyl]-6-methoxy-3,5-
dimethyl-4H-pyran-4-one (16)
To a solution containing 35.1 mg (0.208 mmol) of 5 in 1 mL of
THF at ꢁ78 °C was added dropwise 416
lL (0.416 mmol) of 1 M
LiHMDS solution. The reaction mixture was stirred at ꢁ78 °C for
1 h and a solution containing 50.0 mg (0.416 mmol) of (R)-epox-
ide 10 in 0.5 mL of THF was added followed by 52.0 lL
(0.416 mmol) of BF3ꢂEt2O. The reaction mixture was stirred at
ꢁ78 °C for 1 h and was then allowed to warm slowly to room
temperature and stirred overnight. The reaction mixture was
quenched with satd aq NH4Cl and extracted with portions of
EtOAc. The combined organic phase was washed with brine, dried
over anhydrous MgSO4, filtered and concentrated under dimin-
ished pressure to afford a crude oil. The residue was purified by
chromatography on a silica gel column (5 ꢃ 1.5 cm). Elution with
(9:1 chloroform/methanol); ½a D22
ꢄ
+12.1 (c 0.14, CHCl3) 1H NMR
(CDCl3) d 0.86 (t, 3H, J = 6.5 Hz), 1.18 (s, 3H), 1.20–1.31 (m, 12H),
1.46 (m, 2H), 1.73 (m, 2H), 1.75 (m, 2H), 1.96 (s, 3H), 1.97 (s, 3H)
and 2.58 (m, 2H); 13C NMR (CDCl3) d 8.7, 9.9, 14.3, 22.9, 24.2,
25.7, 26.7, 29.5, 29.8, 30.4, 32.1, 39.0, 42.4, 72.7, 98.4, 105.0,
159.5, 165.0 and 166.3; mass spectrum (ESI), m/z 347.2186
(M+Na)+ (C19H32O4Na requires m/z 347.2198).
30:1 chloroform/methanol gave 16 as
10.2 mg (23%); silica gel TLC Rf 0.42 (9:1 chloroform/methanol);
+13.2 (c 0.12, CHCl3); 1H NMR (CDCl3) d 0.87 (t, 3H,
a colorless oil: yield
½ ꢄ
a 2D2
4.1.13. (S)-6-[3-Hydroxy-3-methylundecyl]-3,5-dimethyl-4-
hydroxypyran-2-one (14)
J = 7 Hz), 1.23 (s, 3H), 1.26–1.37 (m, 12H), 1.49 (m, 2H), 1.75
(m, 2H), 1.84 (s, 3H), 1.94 (s, 3H), 2.66 (m, 2H) and 3.94 (s,
3H); 13C NMR (CDCl3) d 6.9, 9.8, 14.1, 22.6, 24.0, 25.6, 26.8,
29.2, 29.6, 30.1, 31.9, 38.4, 42.0, 55.3, 72.1, 99.4, 118.0, 158.5,
162.1 and 181.0; mass spectrum (ESI), m/z 361.2353 (M+Na)+
(C20H34O4Na requires m/z 361.2355).
To a solution containing 30 mg (0.194 mmol) of 4 in 1 mL of a
6:1 mixture of THF/HMPA at ꢁ78 °C was added 279
lL
(0.448 mmol) of nBuLi (1.6 M in hexanes). The reaction mixture
was stirred at ꢁ78 °C for 1 h and a solution containing 66.0 mg
(0.388 mmol) of (S)-epoxide 11 in 0.5 mL of THF was added. The
reaction mixture was stirred at ꢁ78 °C for 1 h and was then al-
lowed to warm slowly to room temperature and stirred overnight.
The reaction was quenched with 1 M HCl and extracted with por-
tions of EtOAc. The combined organic phase was washed with
brine, dried over anhydrous MgSO4, filtered and concentrated un-
der diminished pressure to afford a crude oil. The residue was puri-
fied by chromatography on a silica gel column (6 ꢃ 1 cm). Elution
4.1.16. (S)-2-[3-Hydroxy-3-methylundecyl]-6-methoxy-3,5-
dimethyl-4H-pyran-4-one (17)
To a solution containing 25.1 mg (0.148 mmol) of 5 in 1 mL of
THF at ꢁ78 °C was added dropwise 300
lL (0.298 mmol) of 1 M
LiHMDS solution. The reaction mixture was stirred at ꢁ78 °C
for 1 h and a solution containing 36.0 mg (0.298 mmol) of (S)-
epoxide 11 in 0.5 mL of THF was added, followed by 40.0 lL

3490
S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
(0.298 mmol) of BF3ꢂEt2O. The reaction mixture was stirred at
ꢁ78 °C for 1 h and was then allowed to warm slowly to room
temperature and stirred overnight. The reaction mixture was
quenched with satd aq NH4Cl and extracted with portions of
EtOAc. The combined organic phase was washed with brine,
dried over anhydrous MgSO4, filtered and concentrated under
diminished pressure to afford a crude oil. The residue was puri-
fied by chromatography on a silica gel column (5 ꢃ 1 cm). Elution
with 30:1 chloroform/methanol gave 17 as a colorless oil: yield
16.3 g (32%); silica gel TLC Rf 0.42 (9:1 chloroform/methanol);
4.1.19. (S)-2-[3-Hydroxy-3-methylundecyl]-3,5,6-trimethyl-4H-
pyran-4-one (20)
To a solution containing 12.5 mg (0.074 mmol) of 2 in 0.5 mL of
THF at ꢁ78 °C was added dropwise 150
lL (0.150 mmol) of 1 M
LiHMDS solution. The reaction mixture was stirred at ꢁ78 °C for
1 h and a solution containing 18.0 mg (0.150 mmol) of (S)-epoxide
11 in 0.5 mL of THF was added followed by 20.0 lL (0.150 mmol) of
BF3ꢂEt2O. The reaction mixture was stirred at ꢁ78 °C for 1 h and
was then allowed to warm slowly to room temperature and stirred
overnight. The reaction mixture was quenched with satd aq NH4Cl
and extracted with portions of ethyl acetate. The combined organic
phase was washed with brine, dried over anhydrous MgSO4, fil-
tered and concentrated under diminished pressure to afford a
crude residue. The residue was purified by chromatography on a
silica gel column (5 ꢃ 1 cm). Elution with 30:1 chloroform/metha-
nol gave 20 as a colorless oil: yield 5.5 mg (23%); silica gel TLC Rf
½
a 2D2
ꢄ
ꢁ9.6 (c 0.21, CHCl3); 1H NMR (CDCl3)
d 0.87 (t, 3H,
J = 7.1 Hz), 1.23 (s, 3H), 1.26–1.37 (m, 12H), 1.49 (m, 2H), 1.75
(m, 2H), 1.84 (s, 3H), 1.94 (s, 3H), 2.66 (m, 2H) and 3.94 (s,
3H); 13C NMR (CDCl3) d 6.9, 9.8, 14.1, 22.6, 24.0, 25.6, 26.8,
29.2, 29.6, 30.1, 31.9, 38.4, 42.0, 55.3, 72.1, 99.4, 118.0, 158.5,
162.1 and 181.0; mass spectrum (ESI), m/z 361.2357 (M+Na)+
(C20H34O4Na requires m/z 361.2355).
0.51 (9:1 chloroform/methanol); ½a D22
ꢄ
ꢁ11.9 (c 0.29, CHCl3); 1H
NMR (CDCl3) d 0.88 (t, 3H, J = 6.5 Hz), 1.21 (s, 3H), 1.23–1.35 (m,
12H), 1.49 (m, 2H), 1.74 (m, 2H), 1.93 (s, 3H), 1.95 (s, 3H), 2.26
(s, 3H) and 2.64 (m, 2H); 13C NMR (CDCl3) d 9.9, 10.2, 14.3,17.9,
22.9, 24.2, 26.4, 26.9, 29.5, 29.8, 30.4, 32.1, 38.9, 42.3, 72.4,
118.5, 119.0, 160.4, 164.0 and 179.8; mass spectrum (ESI), m/z
345.2399 (M+Na)+ (C20H34O3Na requires m/z 345.2406).
4.1.17. (rac)-2-[3-Hydroxy-3-methylundecyl]-3,5,6-trimethyl-
4H-pyran-4-one (18)
To a solution containing 30.6 mg (0.197 mmol) of 2 in 1 mL of
THF at ꢁ78 °C was added dropwise 406
lL (0.394 mmol) of 1 M
LiHMDS solution. The reaction mixture was stirred at ꢁ78 °C for
1 h and 34.0 mg (0.394 mmol) of neat racemic epoxide 6 was
added followed by 53.0
l
L (0.394 mmol) of BF3ꢂEt2O. The reaction
4.1.20. (rac)-6-[3-Hydroxy-3-methylundecyl]-3,5-dimethyl-4-
hydroxypyridin-2-one (21)
mixture was stirred at ꢁ78 °C for 1 h and was then allowed to
warm slowly to room temperature and stirred overnight. The reac-
tion mixture was quenched with satd aq NH4Cl and extracted with
portions of EtOAc. The combined organic phase was washed with
brine, dried over anhydrous MgSO4, filtered and concentrated un-
der diminished pressure to afford a crude oil. The residue was puri-
fied by chromatography on a silica gel column (7 ꢃ 2 cm). Elution
with 40:1 chloroform/methanol gave 18 as a colorless oil: yield
10.7 mg (16%); silica gel TLC Rf 0.51 (9:1 chloroform/methanol);
To a solution containing 23.4 mg (0.071 mmol) of 12 in 0.5 mL
of EtOH was added 4 mL of NH4OH. The reaction mixture was
heated to 70 °C in a sealed tube for 4 days, cooled to 0 °C, neutral-
ized to pH 7 with 1 N HCl and extracted with portions of ethyl ace-
tate. The combined organic phase was dried over anhydrous
MgSO4, filtered and concentrated under diminished pressure to af-
ford a crude oil. The residue was purified by preparative thin layer
chromatography. Elution with 9:1 chloroform/methanol gave 21 as
a yellow oil: yield 6.21 mg (27%); silica gel TLC Rf 0.23 (9:1 chloro-
½
a 2D2 +1.6 (c 0.23, CHCl3); 1H NMR (CDCl3)
d 0.88 (t, 3H,
ꢄ
J = 6.5 Hz), 1.21 (s, 3H), 1.23–1.35 (m, 12H), 1.49 (m, 2H), 1.74
(m, 2H), 1.93 (s, 3H), 1.95 (s, 3H), 2.26 (s, 3H) and 2.64 (m, 2H);
13C NMR (CDCl3) d 9.9, 10.2, 14.3, 17.9, 22.9, 24.2, 26.4, 26.9,
29.5, 29.8, 30.4, 32.1, 38.9, 42.3, 72.4, 118.5, 119.0, 160.4, 164.0
and 179.8; mass spectrum (ESI), m/z 345.2402 (M+Na)+
(C20H34O3Na requires m/z 345.2406).
form/methanol); ½a D22
ꢄ
+1.1 (c 0.09, CHCl3); 1H NMR (CDCl3) d 0.86
(t, 3H, J = 6.5 Hz), 1.12–1.42 (m, 12H), 1.23 (s, 3H), 1.58 (s, 3H),
1.75 (m, 2H), 1.87 (s, 3H), 2.11 (m, 2H), 2.58 (m, 2H), 3.62 (br s,
1H) and 8.7 (br s, 1H); mass spectrum (ESI), m/z 346.2353
(M+Na)+ (C19H33NO3Na requires m/z 346.2358).
4.1.21. 2-(2-Hydroxyundecyl)-6-methoxy-3,5-dimethyl-4H-
pyran-4-one (1)2
4.1.18. (R)-2-[3-Hydroxy-3-methylundecyl]-3,5,6-trimethyl-4H-
pyran-4-one (19)
To a solution containing 23.1 mg (0.136 mmol) of 5 in 2 mL of
To a solution containing 17.4 mg (0.104 mmol) of 2 in 0.5 mL
THF at ꢁ78 °C was added dropwise 258
0.79 M LDA solution in THF. The reaction mixture was stirred at
ꢁ78 °C for 1 h and solution containing 256 (212 mg,
lL (0.204 mmol) of
of THF at ꢁ78 °C was added dropwise 208
lL (0.208 mmol) of
1 M LiHMDS solution. The reaction mixture was stirred at
ꢁ78 °C for 1 h and a solution containing 25.0 mg (0.208 mmol)
of (R)-epoxide 10 in 0.5 mL of THF was added followed by
a
lL
1.36 mmol) of decanal in 1 mL of THF was added. The reaction mix-
ture was stirred at ꢁ78 °C for 3 h, quenched with satd aq NH4Cl
and extracted with portions of ethyl acetate. The combined organic
phase was washed with brine, dried over anhydrous MgSO4, fil-
tered and concentrated under diminished pressure to afford a
crude oil. The residue was purified by chromatography on a silica
gel column (5 ꢃ 1 cm). Elution with 30:1 chloroform/methanol
gave 1 as a colorless oil: yield 14.9 mg (34%); silica gel TLC Rf
0.46 (9:1 chloroform/methanol); 1H NMR (CDCl3) d 0.83 (t, 3H,
J = 6.8 Hz), 1.26–1.37 (m, 14H), 1.40 (m, 2H), 1.77 (s, 3H), 1.92 (s,
3H), 2.70 (m, 2H), 3.95 (s, 3H) and 3.99 (m, 1H); 13C NMR (CDCl3)
d 6.7, 10.2, 14.1, 22.6, 25.7, 29.3, 29.5, 29.6, 31.8, 37.4, 39.2, 55.3,
69.8, 99.3, 119.8, 156.3, 162.3 and 180.9.
26.0
l
L (0.208 mmol) of BF3ꢂEt2O. The reaction mixture was stir-
red at ꢁ78 °C for 1 h and was then allowed to warm slowly to
room temperature and stirred overnight. The reaction mixture
was quenched with satd aq NH4Cl and extracted with portions
of EtOAc. The combined organic phase was washed with brine,
dried over anhydrous MgSO4, filtered and concentrated under
diminished pressure to afford a crude residue. The residue was
purified by chromatography on a silica gel column (5 ꢃ 1.5 cm).
Elution with 40:1 chloroform/methanol gave 19 as a colorless
oil: yield 8.3 mg (25%); silica gel TLC Rf 0.51 (9:1 chloroform/
methanol); ½a 2D2
ꢄ
+8.7 (c 0.16, CHCl3); 1H NMR (CDCl3) d 0.88 (t,
3H, J = 6.5 Hz), 1.21 (s, 3H), 1.23–1.35 (m, 12H), 1.49 (m, 2H),
1.74 (m, 2H), 1.93 (s, 3H), 1.95 (s, 3H), 2.26 (s, 3H) and 2.64
(m, 2H); 13C NMR (CDCl3) d 9.9, 10.2, 14.3, 17.9, 22.9, 24.2,
26.4, 26.9, 29.5, 29.8, 30.4, 32.1, 38.9, 42.3, 72.4, 118.5, 119.0,
160.4, 164.0 and 179.8; mass spectrum (ESI), m/z 345.2402
(M+Na)+ (C20H34O3Na requires m/z 345.2406).
4.1.22. 2-(2-Hydroxyhexyl)-6-methoxy-3,5-dimethyl-4H-pyran-
4-one (22)
To a solution containing 23.2 mg (0.136 mmol) of 5 in 2 mL of
THF at ꢁ78 °C was added dropwise 258
lL (0.204 mmol) of
0.79 M LDA solution in THF. The reaction mixture was stirred at

S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
3491
ꢁ78 °C for 1 h and
a
solution containing 145
l
L
(117 mg,
temperature, quenched with satd aq NH4Cl and extracted with por-
tions of CH2Cl2. The combined organic phase was washed with
brine, dried over anhydrous MgSO4, filtered and concentrated un-
der diminished pressure to afford a crude oil. The residue was puri-
fied by chromatography on a silica gel column (10 ꢃ 2 cm). Elution
with 6:1 hexanes/ethyl acetate gave aldehyde 25 as a yellow oil:
yield 142 mg (94%); silica gel TLC Rf 0.57 (4:1 hexanes/ethyl ace-
tate); 1H NMR (CDCl3) d 1.45–1.89 (m, 6H), 2.47 (m, 2H), 3.86
(m, 2H), 6.90 (m, 3H), 7.27 (m, 2H) and 9.77 (t, 1H, J = 1.6 Hz);
13C NMR (CDCl3) d 25.7, 29.1, 33.4, 43.8, 67.4, 114.4, 114.5, 120.5,
129.3, 129.5, 158.9 and 202.8.
1.36 mmol) of valeraldehyde in 1 mL of THF was added. The reac-
tion mixture was stirred at ꢁ78 °C for 3 h, quenched with satd aq
NH4Cl and extracted with portions of ethyl acetate. The combined
organic phase was washed with brine, dried over anhydrous
MgSO4, filtered and concentrated under diminished pressure to af-
ford a crude oil. The residue was purified by chromatography on a
silica gel column (5 ꢃ 1 cm). Elution with 50:1 chloroform/metha-
nol gave 22 as a colorless oil: yield 7.21 mg (18%); silica gel TLC Rf
0.43 (9:1 chloroform/methanol); 1H NMR (CDCl3) d 0.89 (t, 3H,
J = 7.1 Hz), 1.36 (m, 2H), 1.53 (m, 2H), 1.67 (m, 2H), 1.79 (s, 3H),
1.93 (s, 3H), 2.51 (br s, 1H), 2.73 (m, 2H), 3.95 (s, 3H) and 3.99
(m, 1H); 13C NMR (CDCl3) d 6.8, 10.2, 14.1, 22.6, 27.8, 33.8, 37.1,
39.1, 69.9, 99.4, 119.9, 155.9, 162.3 and 180.9; mass spectrum
(ESI), m/z 277.1414 (M+Na)+ (C14H22O4Na requires m/z 277.1416).
4.1.26. 6-[(4-Phenyl)-phenoxy]-1-hexanol (26)
To a solution containing 563 mg (3.31 mmol) of 4-phenylphenol
and 216 lL (300 mg, 1.65 mmol) of 6-bromo-1-hexanol in 10 mL of
DMF was added 462 mg (3.34 mmol) of K2CO3. The reaction mix-
ture was heated to 80 °C and stirred for 2 h. The reaction mixture
was diluted in ethyl acetate and washed with water and brine.
The combined organic phase was dried over anhydrous MgSO4, fil-
tered and concentrated under diminished pressure to afford a
crude colorless solid. The residue was purified by chromatography
on a silica gel column (12 ꢃ 2 cm). Step gradient elution with 6:1
hexanes/ethyl acetate?2:1 hexanes/ethyl acetate gave alcohol 26
as a colorless solid: yield 258 mg (57%); silica gel TLC Rf 0.15 (4:1
hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.42–1.57 (m, 4H),
1.62 (m, 2H), 1.83 (m, 2H), 3.67 (t, 2H, J = 6.4 Hz), 4.01 (t, 2H,
J = 6.4 Hz), 6.96 (m, 2H), 7.30 (m, 1H), 7.43 (t, 2H, J = 7.2 Hz) and
7.55 (m, 4H); 13C NMR (CDCl3) d 25.6, 25.9, 29.3, 32.7, 62.9, 67.9,
114.7, 126.8, 128.0, 128.2, 128.6, 133.6, 140.8 and 158.6; mass
spectrum (ESI), m/z 293.1515 (M+Na)+ (C18H22O2Na requires m/z
293.1517).
4.1.23. 7-Phenylheptanal (23)
To a solution containing 257
chloride in 7 mL of CH2Cl2 at ꢁ78 °C was added dropwise 287
(316 mg, 4.00 mmol) of DMSO. The reaction mixture was stirred
at ꢁ78 °C for 30 min and a solution containing 300 L (288 mg,
lL (381 mg, 3.00 mmol) of oxalyl
lL
l
1.50 mmol) of 7-phenylheptanol in 2 mL of CH2Cl2 was added
dropwise. The reaction mixture was stirred at ꢁ78 °C for 1 h and
2.09 mL (1.51 g, 15.0 mmol) of NEt3 was added dropwise. The reac-
tion mixture was stirred at ꢁ78 °C for 45 min at which time it was
allowed to warm to room temperature, quenched with satd aq
NH4Cl and extracted with portions of CH2Cl2. The combined organ-
ic phase was washed with brine, dried over anhydrous MgSO4, fil-
tered and concentrated under diminished pressure to afford a
crude oil. The residue was purified by chromatography on a silica
gel column (10 ꢃ 1 cm). Elution with 7:1 hexanes/ethyl acetate
gave aldehyde 23 as a yellow oil: yield 267 mg (93%); silica gel
TLC Rf 0.62 (4:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.37
(m, 4H), 1.64 (m, 4H), 2.42 (m, 2H), 2.62 (t, 2H, J = 7.6 Hz), 7.18
(m, 3H), 7.29 (m, 2H) and 9.76 (t, 1H, J = 1.6 Hz); 13C NMR (CDCl3)
d 22.0, 29.0, 31.3, 35.9, 43.8, 125.7, 128.3, 128.4, 128.5, 142.6 and
202.8.
4.1.27. 6-[(4-Phenyl)phenoxy]-1-hexanal (27)
To a solution containing 154
lL (228 mg, 1.80 mmol) of oxalyl
chloride in 5 mL of CH2Cl2 at ꢁ78 °C was added dropwise 174
l
L
(191 mg, 2.43 mmol) of DMSO. The reaction mixture was stirred
at ꢁ78 °C for 30 min and a solution containing 244 mg (0.90 mmol)
of 26 in 5 mL of CH2Cl2 was added dropwise. The reaction mixture
was stirred at ꢁ78 °C for 1 h and 1.31 mL (9.00 mmol) of NEt3 was
added dropwise. The reaction mixture was stirred at ꢁ78 °C for
45 min at which time it was allowed to warm to room tempera-
ture, quenched with satd aq NH4Cl and extracted with portions
of CH2Cl2. The combined organic phase was washed with brine,
dried over anhydrous MgSO4, filtered and concentrated under
diminished pressure to afford a crude colorless solid. The residue
4.1.24. 6-Phenoxy-1-hexanol (24)
Compound 24 was prepared according the method of Suzuki
et al.13 To a solution containing 312 mg (3.31 mmol) of phenol
and 216 lL (300 mg, 1.65 mmol) of 6-bromo-1-hexanol in 7 mL
of DMF was added 462 mg (3.34 mmol) of K2CO3. The reaction
mixture was heated to 80 °C and stirred for 2 h. The reaction mix-
ture was diluted in ethyl acetate and washed with water and brine.
The combined organic phase was dried over anhydrous MgSO4, fil-
tered and concentrated under diminished pressure to afford a
crude colorless solid. The residue was purified by chromatography
on a silica gel column (12 ꢃ 3 cm). Elution with 3:1 hexanes/ethyl
acetate gave alcohol 24 as a colorless solid: yield 293 mg (91%); sil-
ica gel TLC Rf 0.15 (4:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d
1.38–1.75 (m, 6H), 1.85 (m, 2H), 2.80 (br s, 1H), 3.61 (t, 2H,
J = 6.8 Hz), 3.94 (t, 2H, J = 6.4 Hz), 6.91 (m, 3H) and 7.29 (m, 2H);
13C NMR (CDCl3) d 25.6, 29.3, 32.7, 33.9, 62.4, 67.9, 114.4, 114.5,
120.6, 129.3, 129.5 and 159.0; mass spectrum (ESI), m/z
217.1201 (M+Na)+ (C12H18O2Na requires m/z 217.1204).
was purified by chromatography on
a silica gel column
(10 ꢃ 1 cm). Elution with 7:1 hexanes/ethyl acetate gave aldehyde
27 as a colorless solid: yield 235 mg (96%); silica gel TLC Rf 0.48
(4:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.55 (m, 2H), 1.73
(m, 2H), 1.84 (m, 2H), 2.48 (m, 2H), 4.01 (t, 2H, J = 6.4 Hz), 6.96
(m, 2H), 7.31 (m, 1H), 7.42 (t, 2H, J = 7.2 Hz), 7.55 (m, 4H) and
9.79 (t, 1H, J = 1.6 Hz); 13C NMR (CDCl3) d 21.8, 25.7, 29.1, 43.8,
67.6, 114.7, 126.8, 128.0, 128.2, 128.6, 133.7, 140.8, 158.7 and
202.5.
4.1.28. 2-(2-Hydroxy-8-phenyloctyl)-6-methoxy-3,5-dimethyl-
4H-pyran-4-one (28)
4.1.25. 7-Phenoxy-1-hexanal (25)
To a solution containing 20.3 mg (0.119 mmol) of 5 in 2 mL of
To a solution containing 133
chloride in 5 mL of CH2Cl2 at ꢁ78 °C was added dropwise 149
l
L (197 mg, 1.54 mmol) of oxalyl
THF at ꢁ78 °C was added dropwise 226
lL (0.178 mmol) of
lL
0.79 M LDA solution in THF. The reaction mixture was stirred at
ꢁ78 °C for 1 h and a solution containing 114 mg (0.595 mmol) of
23 in 1 mL of THF was added. The reaction mixture was stirred
at ꢁ78 °C for 3 h, quenched with satd aq NH4Cl and extracted with
portions of ethyl acetate. The combined organic phase was washed
with brine, dried over anhydrous MgSO4, filtered and concentrated
under diminished pressure to afford a crude oil. The residue was
(164 mg, 2.08 mmol) of DMSO. The reaction mixture was stirred
at ꢁ78 °C for 30 min and a solution containing 151 mg (0.77 mmol)
of 24 in 2 mL of CH2Cl2 was added dropwise. The reaction mixture
was stirred at ꢁ78 °C for 1 h and 1.07 mL (777 mg, 7.70 mmol) of
NEt3 was added dropwise. The reaction mixture was stirred at
ꢁ78 °C for 45 min at which time it was allowed to warm to room

3492
S. J. Leiris et al. / Bioorg. Med. Chem. 18 (2010) 3481–3493
purified by chromatography on a silica gel column (7 ꢃ 2 cm). Elu-
tion with 50:1 chloroform/methanol gave 28 as a colorless oil:
yield 19.3 mg (44%); silica gel TLC Rf 0.44 (9:1 chloroform/metha-
nol); 1H NMR (CDCl3) d 1.35 (m, 5H), 1.53 (m, 3H), 1.61 (m, 2H),
1.78 (s, 3H), 1.92 (s, 3H), 2.60 (m, 2H), 2.70 (m, 2H), 3.95 (s, 3H),
3.98 (m, 1H), 7.16 (m, 3H) and 7.28 (m, 2H); 13C NMR (CDCl3) d
6.7, 10.3, 25.6, 29.4, 31.4, 35.9, 37.4, 39.2, 55.3, 69.8, 99.3, 119.8,
128.3, 128.4, 128.5, 142.7, 156.0, 162.3 and 180.9; mass spectrum
(ESI), m/z 381.2045 (M+Na)+ (C22H30O4Na requires m/z 381.2042).
tion (
te] = [(
l
D
g/mL) was calculated from the following formula: [lacta-
OD363) (10f)(90)]/9.1 where f is the dilution factor used
to bring the lactate concentration within the measurable range of
the lactate titration curve, 90 represents the molecular weight of
lactic acid and 9.1 is the extinction coefficient (mMꢁ1 cmꢁ1) of
the reaction at 363 nm. IC50 values were calculated as the dose
which produced a 50% inhibition of the maximum lactate produc-
tion measured.
4.2.2. Cell culture and MTT assay
4.1.29. 2-(2-Hydroxy-7-phenoxyheptyl)-6-methoxy-3,5-
dimethyl-4H-pyran-4-one (29)
MCF-7, A-549, and 3LL cells were cultured using RPMI-1640
medium, whereas CRL-2365 and CRL-2366 were maintained in
DMEM medium, all supplemented with 10% fetal bovine serum,
To a solution containing 23.2 mg (0.135 mmol) of 5 in 2 mL of
THF at ꢁ78 °C was added dropwise 253
l
L (0.200 mmol) of
penicillin G (100 U/mL), and streptomycin (100 lg/mL). MCF-10A
0.79 M LDA solution in THF. The reaction mixture was stirred at
ꢁ78 °C for 1 h and a solution containing 261 mg (1.35 mmol) of
25 in 1 mL of THF was added. The reaction mixture was stirred
at ꢁ78 °C for 3 h, quenched with satd aq NH4Cl and extracted with
portions of ethyl acetate. The combined organic phase was washed
with brine, dried over anhydrous MgSO4, filtered and concentrated
under diminished pressure to afford a crude oil. The residue was
purified by chromatography on a silica gel column (8 ꢃ 2 cm). Elu-
tion with 50:1 chloroform/methanol gave 29 as a colorless oil:
yield 17.8 mg (35%); silica gel TLC Rf 0.46 (9:1 chloroform/metha-
nol); 1H NMR (CDCl3) d 1.56–1.63 (m, 6H), 1.78 (s, 3H), 1.84 (m,
2H), 1.93 (s, 3H), 2.73 (m, 2H), 3.95 (s, 3H), 3.96 (m, 2H), 3.98
(m, 1H), 6.89 (m, 3H) and 7.27 (m, 2H); 13C NMR (CDCl3) d 6.7,
10.3, 29.2, 37.2, 39.3, 55.8, 67.6, 68.6, 99.3, 114.4, 119.9, 120.6,
129.5, 155.9, 159.3, 162.3 and 180.9; mass spectrum (ESI), m/z
383.1837 (M+Na)+ (C21H28O5Na requires m/z 383.1834).
cells were cultured in Mammary Epithelial Growth Medium
(MEGM, Lonza) according to the manufacturer’s protocol.
In vitro inhibition of human cancer cell growth was assessed
using the standard MTT assay, essentially as describe.23
4.2.3. Mitochondrial complex I activity
Beef heart mitochondria were obtained by a large-scale proce-
dure.24 Inverted submitochondrial particles (SMP) were prepared
by the method of Matsuno-Yagi and Hatefi,25 and stored in a buffer
containing 0.25 M sucrose and 10 mM Tris–HCl, pH 7.4, at ꢁ80 °C.
Inhibitory effects of verticipyrone analogues on bovine heart mito-
chondrial complex I (NADH oxidase and NADH: ubiquinone oxido-
reductase) were evaluated by modification of a method described
previously.26 Stock solutions (2 mg/mL in ethanol) of verticipyrone
analogues were prepared and kept in the dark at ꢁ80 °C. Maximal
ethanol concentration never exceeded 2% and had no influence on
the control enzymatic activity. Beef heart SMP were diluted to
4.1.30. 2-[2-Hydroxy-7-(4-phenyl)phenoxyheptyl]-6-methoxy-
3,5-dimethyl-4H-pyran-4-one (30)
To a solution containing 20.4 mg (0.119 mmol) of 5 in 2 mL of
THF at ꢁ78 °C was added dropwise 226
0.5 mg/mL, and treated with 300 lV NADH to activate complex
I.27 The enzymatic activities were assayed at 30 °C and monitored
spectrophotometrically with a Molecular Devices SPECTRA Max-
M5 (340 nm, e
6.22 mMꢁ1 cmꢁ1). NADH oxidase activity was deter-
lL (0.178 mmol) of
0.79 M LDA solution in THF. The reaction mixture was stirred at
ꢁ78 °C for 1 h and a solution containing 159 mg (0.595 mmol) of
27 in 2 mL of THF was added. The reaction mixture was stirred at
ꢁ78 °C for 3 h, quenched with satd aq NH4Cl and extracted with
portions of ethyl acetate. The combined organic phase was washed
with brine, dried over anhydrous MgSO4, filtered and concentrated
under diminished pressure to afford a crude colorless solid. The res-
idue was purified by chromatography on a silica gel column
(8 ꢃ 2 cm). Elution with 50:1 chloroform/methanol gave 30 as an
oil: yield 23.8 mg (46%); silica gel TLC Rf 0.44 (9:1 chloroform/meth-
anol); 1H NMR (CDCl3) d 1.44–1.67 (m, 6H), 1.78 (s, 3H), 1.83 (m,
2H), 1.93 (s, 3H), 2.71 (m, 2H), 3.95 (s, 3H), 3.97 (m, 1H), 4.01 (t,
2H, J = 6.4 Hz), 6.96 (m, 2H), 7.29 (m, 1H), 7.41 (t, 2H, J = 7.2 Hz)
and 7.53 (m, 4H); 13C NMR (CDCl3) d 6.7, 10.3, 25.6, 26.1, 29.2,
37.3, 39.3, 55.3, 67.8, 69.7, 99.3, 114.7, 119.8, 126.8, 128.0, 128.2,
128.6, 133.6, 140.8, 156.0, 158.6, 162.3 and 180.9; mass spectrum
(ESI), m/z 459.2151 (M+Na)+ (C27H32O5Na requires m/z 459.2147).
mined in a reaction medium (2.5 mL) containing 50 mM Hepes, pH
7.5, containing 5 mM MgCl2. The final amount of mitochondrial
protein was 30 lg. The reaction was initiated by adding 50 lM
NADH after the pre-equilibration of SMP with inhibitor for 5 min.
The initial rates were calculated from the linear portion of the
traces. The inhibition of NADH-Q1 oxidoreductase activity was also
determined under the same experimental conditions except that
the reaction medium (2.5 mL) contained 0.25 M sucrose, 1 mM
MgCl2, 2
lM antimycin A, 2 mM KCN, 50 lM ubiquinone Q1 and
50 mM phosphate buffer, pH 7.4.
IC50 values were taken as the final compound concentrations in
the assay cuvette that yielded 50% inhibition of the enzymatic
activity. When ubiquinone-1 (Q1) was used as a substrate, the rates
in the presence of 1 lV rotenone were subtracted (nonphysiologi-
cal electron transfer) (ꢀ15%).28 Verticipyrone analogues needed to
reach a greater concentration to achieve full inhibition, a slower
decay of the complex I and NADH oxidase activities beyond the
IC50. Maximal inhibition of NADH oxidation was not complete,
even at a relatively high concentration of inhibitor (data not
shown).
4.2. Biochemical and biological evaluation
4.2.1. Lactate assay
Using an adaptation of the method of Everse,22 mitochondrial
toxins were assessed using MCF-7 human breast carcinoma cells.
Cells were plated at 2 ꢃ 105 cells per well in tissue culture treated
12-well plates and incubated for 24 h (resulting in about 50% cell
confluence). The wells were rinsed with a pre-warmed solution
of Krebs-Ringer buffer (Sigma) and the compounds were then
added at the appropriate concentrations in 0.5 mL of buffer and
incubated for 3 h in comparison with controls containing the same
amount of buffer. The supernatant was recovered and the variation
Acknowledgment
KST was supported by NIH Research Grant R25GM071798,
awarded by the National Institute of General Medical Sciences.
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