Synthetic atpenin analogs: Potent mitochondrial inhibitors of mammalian and fungal succinate-ubiquinone oxidoreductase

Article abstract of DOI:10.1016/j.bmcl.2010.01.066

Atpenins and harzianopyridone represent a unique class of penta-substituted pyridine-based natural products that are potent inhibitors of complex II (succinate-ubiquinone oxidoreductase) in the mitochondrial respiratory chain. These compounds block electron transfer in oxidative phosphorylation by inhibiting oxidation of succinate to fumarate and the coupled reduction of ubiquinone to ubiquinol. From our investigations of complex II inhibitors as potential agricultural fungicides, we report here on the synthesis and complex II inhibition for a series of synthetic atpenin analogs against both mammalian and fungal forms of the enzyme. Synthetic atpenin 2e provided optimum mammalian and fungal inhibition with slightly higher potency than natural occurring atpenin A5.

Full text of DOI:10.1016/j.bmcl.2010.01.066

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1665–1668
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic atpenin analogs: Potent mitochondrial inhibitors of mammalian
and fungal succinate-ubiquinone oxidoreductase
*
Thomas P. Selby , Kenneth A. Hughes, James J. Rauh, Wayne S. Hanna
DuPont Crop Protection, Stine-Haskell Research Center, 1094 Elkton Road, Newark, DE 19711, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Atpenins and harzianopyridone represent a unique class of penta-substituted pyridine-based natural
products that are potent inhibitors of complex II (succinate-ubiquinone oxidoreductase) in the mitochon-
drial respiratory chain. These compounds block electron transfer in oxidative phosphorylation by inhib-
iting oxidation of succinate to fumarate and the coupled reduction of ubiquinone to ubiquinol. From our
investigations of complex II inhibitors as potential agricultural fungicides, we report here on the synthe-
sis and complex II inhibition for a series of synthetic atpenin analogs against both mammalian and fungal
forms of the enzyme. Synthetic atpenin 2e provided optimum mammalian and fungal inhibition with
slightly higher potency than natural occurring atpenin A5.
Received 11 December 2009
Revised 8 January 2010
Accepted 11 January 2010
Available online 21 January 2010
Keywords:
Synthetic atpenin analogs
Mitochondrial electron transport
Succinate-ubiquinone oxidoreductase
Complex II inhibition
Ó 2010 Elsevier Ltd. All rights reserved.
Pentasubstituted pyridines
Succinate-ubiquinone oxidoreductase (SQR, complex II) plays a
critical role in ATP production by enabling electron transfer as an
integral component of mitochondrial respiration and the Krebs cy-
cle. SQR specifically catalyzes the oxidation of succinate to fuma-
rate with concomitant reduction of ubiquinone to ubiquinol
(Fig. 1). The enzyme is a four subunit membrane-bound dehydro-
genase that comprises a FAD-containing flavoprotein, a subunit
containing multiple iron-sulfur clusters and two smaller hydro-
phobic cytochrome b containing subunits that anchor the catalytic
portion to the mitochondrial membrane.1,2
Like atpenin A5, carboxin reportedly blocks binding of ubiquinone
to the active site.7,8 The carboxamide class of chemistry remains of
high interest to the agrochemical research community where on-
going efforts have given rise to new complex II-inhibiting broad-
Fumarate
Ubiquinol
Succinate
SQR
Fe-S
FAD
Cytochrome b
Ubiquinone
Harzianopyridone (initially isolated from Trichoderma harzia-
num and later obtained from the soil fungus Trichoderma sp.),
atpenin B and atpenin A5 (both isolated from Penicillium sp.) are
representative of the atpenin class of penta-substituted pyridine-
based natural products that were reported to be potent inhibitors
of SQR (Fig. 2).3 Harzianopyridone is generally represented in the
literature as the 4-hydroxy-2-pyridone tautomer whereas atpenins
B and A5 are usually shown as the 2,4-dihydroxypyridine tauto-
mer, assignments in both cases inferred from X-ray crystallo-
graphic structure analyses.4,5
Atpenins bind to the quinone-binding site (Q-site) of complex II
but in a slightly different fashion than that of the natural substrate
ubiquinone as evidenced by an X-ray crystal study where atpenin
A5 was co-crystallized with procaryotic SQR.6
The agricultural fungicides carboxin and boscalid are examples
of the carboxamide agrochemical class that also inhibit complex II.
Figure 1. SQR activity in mitochondrial respiration.
O
OH
O
OH
N
MeO
MeO
MeO
MeO
N
O
OH
H
Atpenin B
Harzianopyridone
O
Cl
O
OH
MeO
MeO
Cl
MeO
MeO
n
Me
N
OH
O
Atpenin A5
Ubiquinone
* Corresponding author. Tel.: +1 302 451 4560; fax: +1 302 366 5738.
E-mail address: thomas.p.selby@usa.dupont.com (T.P. Selby).
Figure 2. Structures of harzianopyridone, atpenins, and the natural substrate
ubiquinone.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.066

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T. P. Selby et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1665–1668
spectrum antifungal product candidates such as penthiopyrad and
bixafen.9,10
precursor to compounds of formula 1) via acylation on oxygen with
N,N-diisopropyl chloroformate in the presence of silver carbon-
ate.15 Electrophilic bromination of 4 (vs introduction via lithiation
by the method of Quéguiner et al.) provided a good yield of 3-bro-
mopyridine 5 that was allowed to undergo a ‘halogen dance’ in the
presence of LDA and a small amount of bromine as a concentrated
solution in THF to afford 2-bromopyridine 6. Metal–halogen ex-
change on 6 and trapping with trimethylborate followed by oxida-
tion in peracetic provided 2-pyridone 7. Alkylation on oxygen with
SEMCl and sodium hydride afforded the SEM protected pyridyl car-
bamate 8, which served as a precursor to atpenin analogs of for-
mula 2.
Preparation of keto 4-pyridones of formula 1 from pyridyl car-
bamate 4 is shown in Scheme 2. Lithiation of 4 with an excess of
butyl lithium followed by quenching with a series of aldehydes
gave alcohol adducts that underwent PCC oxidation to afford the
ketopyridyl carbamates 9. Cleavage of the carbamate protecting
group on 9 by heating in methanolic KOH afforded compounds of
formula 1 (R values listed in Table 1) following isolation upon
acidic workup.
Activity against filamentous fungi, that is, Trichophyton sp., has
been reported for atpenins with some in vivo activity reported for
harzianopyridone against agriculturally relevant pathogens.11,12
Although we found atpenin A5 to be an extremely potent inhibitor
of mammalian and fungal SQR (vide infra), only modest levels of
in vivo activity were observed in a fungal growth assay against sev-
eral pathogens of agricultural significance. However, our interest in
complex II as a biological target prompted us to explore a series of
synthetic atpenin analogs with the goal of improving in vivo effi-
cacy, preferably with higher selectivity for the fungal enzyme form.
Here, we report on the synthesis and inhibitory activity against
mammalian and fungal SQR (with observed fungicidal activity)
for a series of synthetic analogs, focusing on structures of formulae
1 and 2 (see Fig. 3).
Quéguiner et al. reported the first syntheses of racemic atpenin
B and harzianopyridone by a unique metalation route involving an
innovative ‘clockwise’ functionalization of a pyridine nucleus.13,14
By a modification of that method, we made a series of compounds
of formulae 1 and 2 from commercially available 2,3-dimethoxy-
pyridine (3).
Structurally closer to natural occurring atpenins, analogs of for-
mula 2 were made from 8 as outlined in Scheme 3. Lithiation of 8
Scheme 1 outlines the syntheses of key pyridine precursors to
both 1 and 2. Lithiation of 3 with 1.5 equiv of n-butyl lithium
(more than 1 equiv of base was used, presumably to overcome che-
lation by the methoxy groups of 3 as reported by Quéguiner et al.)
followed by low temperature addition of trimethylborate and sub-
sequent oxidation in peracetic acid afforded 2,3-dimethoxy-4-pyri-
done that was protected as the N,N-diisopropyl carbamate 4 (the
i-Pr2NOCO
MeO
O
c
a,b
R
1
4
MeO
N
9
Scheme 2. Reagents and conditions: (a) (i) 2.5 M n-BuLi (3.0 equiv), THF, À70 °C,
1 h; (ii) RCHO (3.5 equiv), THF, À70 °C, 1 h, 80–90%; (b) PCC (3.0 equiv), CH2Cl2,
molecular sieves, 25 °C, 2 h, 75–85%; (c) (i) 5 N KOH in MeOH, reflux, 2–3 h; (ii)10%
aq acetic acid quench, 25 °C, 35–60%.
O
O
O
OH
MeO
MeO
MeO
MeO
R
R
N
H
N
OH
1
2
Table 1
R = (un)substituted alkyl, aryl, alkenyl
Inhibitory activity of synthetic atpenins against mammalian and fungal complex II
Entry
R
Bovine IC50
(
lM) Septoria n. IC50 (lM)
Figure 3. Synthetic atpenin targets.
1a
1b
1c
1d
1e
1f
1g
1h
1i
2-Butyl
>30
>30
>30
6.56
0.454
3.40
>30
0.492
0.795
12.8
0.096
0.020
0.127
0.125
0.003
0.005
>30
>30
>30
>30
2.98
26.7
>30
4.08
2.75
20.7
2.58
0.122
1.78
0.234
0.004
0.051
2-Chlorophenyl
2-Phenoxyphenyl
3-Chlorophenyl
3-Phenoxyphenyl
4-Chlorophenyl
4-Methoxyphenyl
4-Phenoxyphenyl
4-Biphenyl
2-Naphthyl
2-Butyl
2-Hexyl
Cyclohexyl
i-Pr2NOCO
MeO
MeO
a,b
Br
c
MeO
N
MeO
N
4
3
1j
i-Pr2NOCO
MeO
i-Pr2NOCO
MeO
2a
2b
2c
2d
2e
2f
d
e
3-Heptyl
MeO
N
5
MeO
N
6
Br
CH(Me)(CH2)8Me
CH(Me)(CH2)2–
CH@C(Me)2
4-Chlorophenyl
3-Phenoxyphenyl
CH(Me)Ph
i-Pr2NOCO
MeO
i-Pr2NOCO
MeO
2g
2h
2i
0.326
0.112
0.008
0.008
0.630
1.27
>30
13.8
>30
>30
>30
f
2.33
0.299
0.024
>30
1.94
>30
13.95
>30
>30
2j
CH(Me)CH2-(4-t-BuPh)
CH(Ph)2
MeO
N
H
7
O
MeO
N
OSEM
2k
14
15
16
17
18
—
8
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
Atpenin A5
Scheme 1. Reagents and conditions: (a) (i) 2.5 M n-BuLi (1.5 equiv), THF,
À70 °C?0 °C, 1 h; (ii) trimethylborate (2.5 equiv), À70 °C to À50 °C; (iii) 32 wt %
MeCO3H in acetic acid (2 equiv), À50 °C?10 °C, 86%; (b) N,N-diisopropyl chloro-
formate (1.5 equiv), Ag2CO3 (1.5 equiv) toluene, 110 °C, 3 h, 72%; (c) Br2 (2.5 equiv),
CCl4, 0 °C?25 °C, 24 h, 82%; (d) LDA (3 equiv), Br2 (cat.), THF, À70 °C to À40 °C, 1 h,
75%; (e) (i) 2.5 M n-BuLi (1.5 equiv), THF, À70 °C; (ii) trimethylborate (2.5 equiv),
À70 °C; (iii) 32 wt % MeCO3H in acetic acid (2 equiv), À70 °C?10 °C, 66%; (f) SEMCl
(1.2 equiv), NaH (1.3 equiv), THF, 0 °C?25 °C, 1 h, 65%.
0.004
1.79
0.011
0.245
—
Boscalid
IC50 values represent the mean of triplicate determinations with a standard error of
less than 5%.

T. P. Selby et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1665–1668
1667
O
OR
N
i-Pr2NOCO
MeO
O
MeO
MeO
a (formation of 17)
c
a,b
R
2g
8
b (formation of 18)
OR
Cl
MeO
O
N
10
O
OSEM
17 R = COMe
18 R = SO2Me
MeO
MeO
d
R
2
Scheme 5. Reagents and conditions: (a) acetic anhydride neat, 25 °C, 25% (b)
methanesulfonyl chloride (3.5 equiv) 25 °C, i-Pr2EtN (5 equiv), THF, 25 °C, 30%.
N
H
OSEM
wheat glume blotch) sub-mitochondrial membrane assay. SQR
inhibition was measured in plate-based tests where succinic acid
served as the electron donor and the dye 2,6-dichlorophenol-indo-
phenol (DCIP) was used as the final electron acceptor. Electron
transfer from the succinate substrate to ubiquinone (with concom-
itant formation of ubiquinol) was measured spectrophotometri-
11
Scheme 3. Reagents and conditions: (a) (i) 2.5 M n-BuLi (3.0 equiv), THF, À70 °C,
1 h; (ii) RCHO (4.0 equiv), THF, À70 °C, 1 h, 50–65%; (b) PCC (3.0 equiv), CH2Cl2,
molecular sieves, 25 °C, 50–80%; (c) (i) 5 N KOH in MeOH, reflux, 2–3 h (ii) 10% aq
acetic acid quench, 35–55%; (d) concd HCl, MeOH, reflux, 1–2 h, 70–80%.
cally by ubiquinol-dependent reduction of DCIP.17 Table
1
with an excess of butyl lithium and quenching with aldehydes gave
alcohols that were oxidized with PCC to the ketopyridyl carba-
mates 10. Cleavage of the carbamate protecting group by heating
in methanolic KOH gave pyridones of formula 11 after acidic work-
up, generally in moderate yields. Heating 11 in acidic methanol re-
sulted in removal of the SEM group with isolation of fully
unprotected synthetic atpenins 2 (R values listed in Table 1).16
In Scheme 4, the preparation of several 4-chlorobenzoylpyri-
dones (14–16) is outlined where, in place of the atpenin hydroxy
group adjacent to the pyridine ring nitrogen, bromine, methoxy
and methyl groups were inserted.
Bromination of 6 provided 2,3-dibromopyridine 12 which was
selectively lithiated at the 3-position via metal halogen exchange.
Trapping with 4-chlorobenzaldehyde gave an alcohol intermediate
that was immediately oxidized with PCC to afford benzoylpyridine
13. Removal of the carbamate protecting group by heating in alco-
holic KOH followed by an acidic quench generated a mixture of 14
and 15 that were separated by chromatography. Treatment of 13
with AlMe3 resulted in displacement of the remaining bromine at
the 2-position with methyl where 16 was isolated upon de-protec-
tion in alcoholic KOH and acidic workup.
summarizes mammalian and fungal inhibitory data as micromolar
IC50 values.
Acyl pyridones of formula 1 where R = 2-butyl (1a), 2-chloro-
phenyl (1b), 2-phenoxyphenyl (1c), and 4-methoxyphenyl (1g)
did not provide significant mammalian or fungal SQR inhibition be-
low 30
phenyl (1f), and 2-naphthyl (1j) did afford mammalian IC50
values in the 3–13 M range but with fungal IC50 values greater
than 20 M. Pyridones where R = 3-phenoxyphenyl (1e), 4-phen-
oxyphenyl (1h), and 4-biphenyl (1i) had higher potency with
mammalian IC50 values slightly below 1 M and fungal IC50 values
in the 2-5 M range.
lM. Analogs where R = 3-chlorophenyl (1d), 4-chloro-
l
l
l
l
On the other hand, 3-acyl-2,4-dihydroxypyridines of formula 2
in most cases provided much higher levels of SQR inhibition with
the mammalian enzyme form consistently showing greater sensi-
tivity than the fungal form. Substituents where R = 2-butyl (2a)
and 2-hexyl (2b) gave mammalian IC50 values in the 0.02–0.1 lM
range but with fungal IC50 values 6–27-fold higher. Analogs where
R = cyclohexyl (2c), 3-heptyl (2d), 4-chlorophenyl (2g), 3-phenoxy-
phenyl (2h), and CH(Ph)2 (2k) were less active with both mamma-
lian and fungal IC50 values above 0.1 lM.
Synthesis of two bis-protected 3-benzoyl 2,4-dihydroxypyri-
dines is shown in Scheme 5. Heating the benzoyl-substituted
dihydroxypyridine 2g (compound 2 where R is 4-chlorophenyl)
in acetic anhydride gave the bis-acetylated adduct 17 and reaction
with methane sulfonyl chloride in the presence of Hunig’s base
gave the bis-sulfonylated 18.
Substituents where R = CH(Me)(CH2)8Me (2e), CH(Me)(CH2)2CH
@C(Me)2 (2f), CH(Me)Ph (2i), and CH(Me)CH2(4-t-butylphenyl)
(2j) provided the highest levels of inhibition with mammalian
IC50 values below 0.01
l
M. Although the fungal IC50 value for 2i
M, 2e, 2f, and 2j all had fungal IC50 values close
M. Optimum activity was attained with 2e which
had mammalian and fungal IC50 values of 0.003 and 0.004 lM,
was close to 0.3
to or below 0.05
l
l
Prepared compounds were evaluated for complex II activity in
both a mammalian (bovine heart) and fungal (Septoria nodorum,
respectively. Compound 2e was found to be slightly more active
than atpenin A5 which showed mammalian and fungal IC50 values
of 0.004 and 0.011
Surprisingly, 2-bromopyrimidinone 14 showed mammalian and
fungal inhibition in the IC50 range of 1–2 M whereas 2-methoxy-
pyrimidinone 15 had IC50 values above 30 M. In between the
activity of 14 and 15, 2-methyl pyrimidinone 16 had mammalian
and fungal IC50 values in the 13–14 M range.
Prior to obtaining data revealing 2g to be a poor inhibitor of fun-
gal SQR, a mammalian IC50 value of 0.326 M obtained early in this
program prompted investigation of the bis-acetylated and bis-sulf-
onylated dihydroxy pyrimidines 17 and 18 as pro-forms where
expression of in vivo activity might be improved.18 As expected,
IC50 values for both compounds were greater than 30 lM.
Although some of these synthetic atpenin analogs showed bet-
ter fungal inhibition of SQR than the commercial complex II inhib-
lM, respectively.
i-Pr2NOCO
i-Pr2NOCO
MeO
O
l
MeO
Br
Br
a
l
b,c
6
MeO
N
Cl
MeO
N
Br
l
12
13
O
O
l
MeO
MeO
d (formation of 14 &15)
or
R1
Cl
N
e (formation of 16)
H
14 R1 = Br
15 R1 = OMe
16 R1 = Me
iting fungicide boscalid (fungal IC50 = 0.245 lM), surprisingly,
Scheme 4. Reagents and conditions: (a) Br2, (5 equiv) CCl4, 0 °C?25 °C, 3 days,
35%; (b) (i) 2.5 M n-BuLi (1.1 equiv), THF, À70 °C; (ii) 4-chlorobenzaldehyde
(2.0 equiv), THF, À70 °C, 1 h; (iii) (c) PCC (2 equiv), CH2Cl2, molecular sieves,
25 °C, 80%; (d) (i) 5 N KOH in MeOH, reflux; (ii) 20% aq acetic acid quench, 25 °C,
35%; (e) (i) 2 M AlMe3 in hexane (2 equiv) Pd(PPh3)4 (5 mol %) DME, 70 °C, 80%); (ii)
5 N KOH in MeOH, reflux; (iii) 20% aq acetic acid quench, 65%.
substantial levels of in vivo activity against S. nodorum were not
observed for any of these atpenin analogs in both an antifungal
growth assay or whole plant assay at 200 ppm. Limited in vivo
activity on other agricultural pathogens was detected as well.

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T. P. Selby et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1665–1668
14. Trécourt, F.; Mallet, M.; Mongin, O.; Quéguiner, G. J. Heterocycl. Chem. 1995, 32,
In summary, a series of synthetic atpenin analogs, were pre-
1117.
pared via ‘clockwise’ functionalization of commercially available
2,3-dimethoxypyridine (3) via a modification of the synthetic
method of Quéguiner.13,14 Pyridones of formula 1 provided modest
levels of inhibition relative to atpenin A5 with some analogs show-
ing low micromolar affinity. On the other hand, dihydroxypyri-
dines of formula 2 gave much higher levels of mammalian and
fungal SQR inhibition with some compounds demonstrating low
nanomolar binding. The long-chain 3-alkanoyl (13 carbons) substi-
tuted dihydroxypyridine 2e provided optimum SQR inhibition and
was slightly more active than atpenin A5 on both mammalian and
fungal SQR. Evidently, the presence of stereochemical centers on
the 3-acyl atpenin side chain (as in the case of atpenin A5) is not
critical for high SQR potency. Unfortunately, similar to atpenin
A5, no synthetic analogs showed higher affinity for fungal versus
mammalian SQR or substantial levels of in vivo antifungal activity.
The strong likelihood that poor in vivo activity resulted from an is-
sue around fungal penetration (membrane/cell wall permeability),
translocation within the pathogen, metabolism or efflux precluded
our continued interest in atpenin analogs as potential agricultural
fungicides.
15. Quéguiner et al. reported that use of 2.2 equiv of n-butyl lithium gave optimum
results, presumably due to chelation of an equivalent of base by the methoxy
groups and ring nitrogen. However, we found that satisfactory product yields
were obtained with 1.5 equiv of n-butyl lithium.
16. All new compounds gave satisfactory spectral data consistent with their
structures. As a representative example of the general method for making keto
dihydroxypyridine analogs of formula 2, synthesis of 3-(4-chlorobenzoyl)-2,4-
dihydroxy-5,6-dimethoxypyridine (2g) as outlined in Scheme 3 (steps a–d) is
as follows: (step a) To a solution of 1 g (2.3 mmol) of 2,3-dimethoxy-6-(2-
trimethylsilylethoxymethoxy)-4-pyridyl
N,N-diisopropylcarbamate
(8)13
stirring in 15 mL of THF at À70 °C, n-butyllithium (2.5 M, 2.8 mL) was added
dropwise. After stirring 1 h at À70 °C, 4-chlorobenzaldehyde (1.15 g, 8.2 mmol)
in 5 mL of THF was added dropwise. The reaction stirred 2 h at À70 °C,
quenched with 8 mL of a 1:1:1 mixture of ethanol, water, and glacial acetic
acid, followed by extracting with dichloromethane. The separated organic layer
was dried over magnesium sulfate and evaporated in vacuo to give orange oil.
Purification by MPLC on silica gel (10–30% ethyl acetate in hexanes) yielded
0.92 g (70%) of the 3-(4-chlorophenyl)-methanol adduct of 8, isolated as a clear
oil. (step b) To a solution of this oil (0.75 g, 1.3 mmol) stirring in 10 mL of
dichloromethane over 0.5 g of ground molecular sieves at room temperature,
pyridium chlorochromate (0.91 g, 4.2 mmol) was added in portions over 1 min.
The reaction was stirred at ambient temperature for 2 h, diluted with 20 mL of
diethyl ether and filtered through a medium glass frit filter. Evaporating in
vacuo gave a brown oil that was purified by MPLC on silica gel (10–20% ethyl
acetate in hexanes) to afford 0.61 g (82%) of the benzoylpyridine 10 where
R = 4-chlorophenyl as a clear oil. 1H NMR (300 MHz), CDCl3): d 7.39 (s, 2H),
7.37 (s, 2H), 5.45 (s, 2H), 4.00 (s, 3H), 3.81 (s, 3H), 3.90–3.69 (m, 2H), 3.90–3.69
(m, 2H), 3.51–3.43 (m, 2H), 1.10 (dd J = 5.1, 6.2 Hz, 12H), 0.90–0.74 (m, 2H).
(steps c and d) A 0.54 g (0.95 mmol) sample of 10 was dissolved in 12 mL of 5 N
potassium hydroxide in methanol and heated at reflux for 1 h. After quenching
with 10 mL of 10% aqueous acetic acid followed by neutralization with
saturated sodium bicarbonate, the reaction mixture was extracted with ethyl
acetate. The separated organic layer was dried over magnesium sulfate and
concentrated in vacuo to afford 0.31 g of crude pyridone 11 where R = 4-
chlorophenyl, isolated as a yellow oil (NMR confirmed removal of the SEM
protecting group). The entire sample of 11 was heated in 10 mL of a 1:10
mixture of concentrated HCl and methanol at reflux for 1 h. The reaction
mixture was concentrated in vacuo, treated with saturated aqueous sodium
bicarbonate (20 mL) and extracted with dichloromethane. The separated
organic layer was dried over magnesium sulfate and evaporated in vacuo to
give a yellow solid. Purification by MPLC on silica gel (10–20% ethyl acetate in
hexanes) afforded 0.17 g of the dihydroxypyridine 2g (57%) as a yellow-tinted
solid; 1H NMR (300 MHz), CDCl3): d 7.51 (d, J = 8.6 Hz, 2H, ArH), 7.38 (d,
J = 8.6 Hz, 2H, ArH), 3.83 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C NMR (100 MHz,
acetone-d): d 53.608, 59.957, 99.675, 124.357, 127.946, 130.063, 137.013,
Acknowledgment
¯
The authors wish to thank Satoshi Omura from Kitasato Univer-
sity for kindly supplying a sample of atpenin A5.
References and notes
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;
¯
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mp 210–211 °C.
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¯
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17. The complex II assay was run in a 96-well microplate format using sub-
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ubiquinone as the electron acceptor. The reaction was initiated by addition
of succinate and allowed to proceed for 10 min. Reduction of the dye 2,6-
dichlorophenol-indophenol (DCIP) was monitored by absorbance at
k = 600 nm.
18. Investigating pro-forms of a much more active fungal SQR inhibitor such as 2e
where there might be a higher possibility of attaining significant in vivo
activity remains to be explored.
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¯
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