Concise syntheses and biological activities of ganomycin I and fornicin A

Article abstract of DOI:10.1002/ejoc.201301269

The first enantioselective syntheses of ganomycin I, a meroterpenoid isolated from the Vietnamese mushroom Ganoderma colossum, and the related meroterpenoid fornicin A were accomplished. Our methodology for the total syntheses of these compounds featured the construction of the butenolide moiety by asymmetric dihydroxylation followed by Julia-Kocienski type olefin formation and ring-closing metathesis reactions. The absolute configurations of the two natural products were determined by comparisons of specific rotation. A cell-based assay of the synthetic compounds with transfected human embryonic kidney 293 tetoff (E-PR293) cells indicated that ganomycin I possesses cytotoxicity and fornicin A possesses weak anti-HIV-1 protease activity without cytotoxicity.

Full text of DOI:10.1002/ejoc.201301269

FULL PAPER
DOI: 10.1002/ejoc.201301269
Concise Syntheses and Biological Activities of Ganomycin I and Fornicin A
Arata Yajima,*[a] Shota Urao,[a] Ryo Katsuta,[a] and Tomoo Nukada[a]
Keywords: Natural products / Terpenoids / Configuration determination / Asymmetric synthesis / Ring-closing metathesis
The first enantioselective syntheses of ganomycin I,
a
closing metathesis reactions. The absolute configurations of
the two natural products were determined by comparisons of
specific rotation. A cell-based assay of the synthetic com-
pounds with transfected human embryonic kidney 293 tet-
off (E-PR293) cells indicated that ganomycin I possesses cyto-
toxicity and fornicin A possesses weak anti-HIV-1 protease
activity without cytotoxicity.
meroterpenoid isolated from the Vietnamese mushroom
Ganoderma colossum, and the related meroterpenoid for-
nicin A were accomplished. Our methodology for the total
syntheses of these compounds featured the construction of
the butenolide moiety by asymmetric dihydroxylation fol-
lowed by Julia–Kocienski type olefin formation and ring-
of the other hydroquinones remain undetermined. The re-
lated natural product clavilactone B (4),[8] isolated from the
culture broth of the basidiomycete Clitocybe clavipes, pos-
sesses the opposite absolute configuration to that of gano-
mycin I in its lactone moiety (Figure 1).[9] This difference is
of interest for confirming the absolute configuration of nat-
ural ganomycin I, and the promising biological activities of
ganomycin I motivated us to synthesize it as part of our
meroterpenoid-synthesis project.[10] Furthermore, because
fornicin A has not been reported to have HIV-1 protease
inhibitory activity, we were also interested in clarifying
whether fornicin A exhibited this activity. This paper de-
scribes the first total syntheses of ganomycin I and for-
nicin A and asseses their biological activities.
Introduction
Various prenylated hydroquinones and quinones play im-
portant roles in the life systems of a range of organisms.
For example, menaquinones and ubiquinones or the corre-
sponding reduced form are essential coenzymes in the redox
systems of microorganisms,[1,2] and menaquinones also play
multiple roles in mammalian cells.[3,4] Recently, bioactive
secondary metabolites containing prenylated hydro-
quinones or quinones have been isolated from a wide vari-
ety of microorganisms. Considering their structural similar-
ity with menaquinones and ubiquinones, these secondary
metabolites might be synthesized by similar biosynthetic
pathways, namely, alkylation of a hydroquinone derivative
with prenyl pylophosphate followed by modifications, such
as oxidation of the side chains.[2] Two structurally related
compounds, ganomycin I (1)[5] and fornicin A (2),[6] are
meroterpenoids consisting of prenylated hydroquinones
with a γ-butylolactone moiety in their side chains (Fig-
ure 1). Ganomycin I was isolated from the Vietnamese
mushroom Ganoderma colossum as an HIV-1 protease in-
hibitor by Hattori and co-workers in 2009,[5] and fornicin A
was isolated from the Chinese mushroom G. fornicatum as
a cytotoxic agent against human larynx carcinoma (Hep-2)
cells by Che and co-workers in 2006.[6] The genus of the
basidiomycete Ganoderma sp. contains many prenylated
hydroquinones, such as ganomycins A and B[7] and fornic-
ins B and C.[6] Although the absolute configuration of gan-
omycin I was proposed as R on the basis of the CD spec-
trum of the natural product,[5] the absolute configurations
Figure 1. Structures of ganomycin I, fornicin A, and related natural
products.
[a] Department of Fermentation Science, Faculty of Applied
Bioscience, Tokyo University of Agriculture (NODAI),
Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502, Japan
E-mail: ayaji@nodai.ac.jp
Results and Discussion
Synthetic Plan
http://www.nodai.ac.jp/fer/original/shigen
The retrosynthetic analysis of ganomycin I and for-
nicin A is outlined in Scheme 1. We planned to construct
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301269.
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A. Yajima, S. Urao, R. Katsuta, T. Nukada
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the 3-alkyl-5-aryl-γ-butenolide portion of the compounds son of its specific rotation with literature data.[12a] Although
by ring-closing metathesis (RCM) of A. This strategy would Julia olefination was effective for constructing the vin-
enable us to obtain the various synthetic analogues of the ylbenzyl alcohol moiety of 6, these conditions could not be
ganomycin family because wide varieties of the vinylbenzyl applied to the synthesis of ganomycin I. Decomposition of
alcohol segment (B) and the α-methylenecarboxylic acid the methoxymethyl (MOM) protective groups of the hydro-
segment (C) could be obtained. The structurally related nat- quinone portion occurred during the preparation of the
ural products, such as an indole alkaloid from the plant corresponding phenyl sulfide intermediate, and deprotec-
Raputia simulans 3[11] and clavilactones 4[8] (Figure 1), tion was difficult when using more tolerant protective
could also be synthesized by employing this strategy. There groups, such as methyl or benzyl ether, instead of MOM
have been previous reports concerning the preparation of ether in the final stage of the synthesis. Thus, we selected
optically active vinylbenzyl alcohols that have been the 2-benzothiazolyl (BT) sulfide derivative 11 as an inter-
achieved by employing such strategies as enzymatic resolu- mediate. The two hydroxy groups of commercially available
tion,[12] Sharpless asymmetric epoxidation (AE)[13] or dihy- 2,5-dihydroxybenzaldehyde (7) were protected as MOM
droxylation (AD),[14] asymmetric allylic substitution,[15] and ethers, and Horner–Wadsworth–Emmons homologation
asymmetric alkenylation.[16] We selected AD to introduce followed by reduction of the resulting ester group by diiso-
the asymmetric center of the target compounds because of butylaluminium hydride (DIBAL-H) gave (E)-allylic
its high stereoselectivity and simple methodology. Although alcohol 10. The hydroxyl group was substituted with a 2-
Schneider and Kazmaier reported the synthesis of optically thiobenzothiazolyl group under Mitsunobu conditions to
active 1-vinylbenzyl alcohol from ethyl cinnamate in five produce BT sulfide 11. The double bond of 11 was dihy-
steps through AD,[14] we focused on establishing a more droxylated with AD-mix-α to give the optically active diol
straightforward transformation for vinylbenzyl alcohol by 12, and the sulfide was oxidized with mCPBA to give sulf-
employing mild reaction conditions, such as Julia– one 13. In contrast, 13 could not be prepared when AD
Kocienski type olefin formation. The precursor for Julia–
Kocienski type olefination, D, would be prepared from the
corresponding allylic sulfide, E, by AD.
Scheme 1. Retrosynthetic analysis of ganomycin I and fornicin A.
Synthesis of the Optically Active Segment
Scheme 2. Synthesis of optically active 14. Reagents and conditions:
The synthesis of the key intermediate 14 is summarized
(a) AD-mix-β, MeSO2NH2, tBuOH, H2O, 0 °C, 66%; (b) mCPBA,
in Scheme 2. In our preliminary studies, cinnamyl phenyl
CH2Cl2, 78%; (c) Na(Hg), Na2HPO4, THF, MeOH, –15 °C, 77%;
sulfide (5)[17] was converted into optically pure (er = 99:1)
(d) MOMCl, K2CO3, acetone, 0 °C to room temp., 98%; (e) (EtO)2-
POCH2CO2Et, NaH, THF, 0 °C to room temp., 96%; (f) DIBAL-
H, CH2Cl2, hexane, –78 to 0 °C, 99%; (g) 2-mercaptobenzo-
thiazole, Ph3P, diethyl azodicarboxylate (DEAD), THF, 0 °C to
room temp., 89%; (h) AD-mix-α, MeSO2NH2, tBuOH, H2O, 0 °C,
85%; (i) mCPBA, CH2Cl2, –78 °C, 92%; (j) DBU, CH2Cl2, –10 °C,
(S)-(–)-1-vinylbenzyl alcohol (6) by employing a three-step
operation: AD, oxidation of the sulfide into the correspond-
ing sulfone with m-chloroperbenzoic acid (mCPBA), and
Julia olefination with sodium amalgam. The absolute con-
figuration of the product was confirmed as S by a compari- 80%; (k) (NH4)6Mo7O24·4H2O, H2O2, MeOH.
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Syntheses and Biological Activities of Ganomycin I and Fornicin A
was conducted after converting 11 into the corresponding AD-mix-β. Because the specific rotation of the synthetic R
sulfone 15. Smiles rearrangement of β-hydroxysulfone 13 isomer {[α]2D6 = +33.8 (c = 0.10, MeOH)} was in good
under mild conditions {treatment with 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU)[18] at low temperature} gave (R)-
14 in good yield. The enantiomeric ratio of the resulting
vinylbenzyl alcohol 14 was determined as 98:2 by HPLC
analysis. The absolute configuration of 14 was estimated by
analogy with 6.
Completion of the Synthesis of Ganomycin I
With the key vinylbenzyl alcohol in hand, the prenyl side
chain segment was synthesized (Scheme 3). Geranyl chlor-
ide, prepared from 16, was alkylated with lithiated β-
methallyl alcohol to afford known alcohol 17,[19] and a two-
step oxidation of 17 gave the corresponding carboxylic acid
19. After optimization of the conditions of the acylation of
14 with 19, Yamaguchi esterification gave a satisfactory
yield of 20. Contrary to our expectation, the key step, RCM
of 20, was somewhat difficult. When widely used commer-
cially available catalysts, such as Grubbs catalysts or modi-
fied Grubbs catalysts, were used in this reaction,[20] only a
complex mixture was obtained (Table 1, Figure 2). This was
caused by uncontrolled metathesis reactions of the poly-ole-
fin side chain of 20. Fortunately, the reaction proceeded
smoothly without formation of the undesired side product
when freshly recrystallized Grubbs first generation catalyst
was used at room temperature. The best reaction conditions
gave the desired compound 21 in 80% yield (er Ͼ 97:3)
when two portions of 5 mol-% catalyst were loaded into the
Scheme 3. Synthesis of ganomycin I. Reagents and conditions:
(a) Ph3P, CCl4, reflux; (b) β-methallyl alcohol, nBuLi, TMEDA,
reaction medium. The reaction temperature was an impor-
tant factor because a complex mixture was obtained at ele-
Et2O, –78 °C to room temp., then geranyl chloride; (c) MnO2, hex-
vated temperatures, even when recrystallized Grubbs first
generation catalyst was used. The two MOM ethers of 21
were then hydrolyzed with pTsOH to give (R)-ganomycin I.
The overall yield was 32.4% in ten steps starting from 2,5-
dihydroxybenzaldehyde. The (S)-ganomycin I was also syn-
ane, 75% (three steps); (d) NaClO2, NaH2PO4, 2-methyl-2-butene,
tBuOH, H2O, 0 °C to room temp., 83%; (e) Cl3C6H2COCl,
DIPEA, DMAP, toluene, then (R)-14, 92% based on 14; (f) Grubbs
1st catalyst, CH2Cl2, 80%; (g) TsOH, EtOH, 85%. TMEDA =
tetramethylethylenediamine, DIPEA
= diisopropylethylamine,
DMAP 4-(dimethylamino)pyridine, Grubbs 1st catalyst
=
=
thesized from (S)-12, which was prepared through AD with [(PCy3)2Cl2Ru=CHPh].
Table 1. Optimization of reaction conditions for the ring-closing metathesis.[a]
Entry
Catalyst[b]
Temp. [°C]
Yield [%][c]
[e]
1
2
3
4
5
6
7
Hoveyda–Grubbs 2nd
Grubbs 1st[f]
Grubbs 2nd
Zhan 1C
Zhan 1B
Piers–Grubbs 1st
Piers–Grubbs 2nd
room temp. to 40[d]
room temp.
–
80
[e]
room temp. to 40[d]
room temp. to 40[d]
room temp. to 40[d]
40
–
[e]
–
[e]
–
21
30
40
[a] Reaction conditions (unless otherwise specified): catalyst (20 mol-%), CH2Cl2 (0.03–0.04 m). [b] Freshly recrystallised reagent was used.
[c] Isolated yield. [d] Reaction proceeded very slowly at room temperature. [e] Complex mixture. [f] Two portions of 5 mol-% catalysts
were used.
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A. Yajima, S. Urao, R. Katsuta, T. Nukada
FULL PAPER
agreement with that of the reported natural product {[α]2D3 magnitude of the specific rotation of the natural product
= +36 (c = 0.10, MeOH)},[5] the absolute configuration of {[α]2D3 = +9.52 (c = 0.32, MeOH)} was considerably smaller
the natural product was confirmed as R, as previously as- than that of the synthetic product 2 {[α]2D6 = +25.4 (c =
signed by Hattori.
0.32, MeOH)},[6] the absolute configuration of the natural
product was concluded to be R on the basis of the sign of
rotation, which was the same as ganomycin I. The differ-
ence might be due to the presence of unknown impurities
in the sample of the natural fornicin A.
Biological Studies
We then investigated the anti-HIV-1 protease activity of
the synthetic compounds. Although Hattori performed the
enzyme assay with commercially available recombinant
HIV-1 protease,[5] we conducted cell-based assays with
transfected human embryonic kidney 293 tet-off (E-PR293)
cells[22] to clarify in detail the biological activity of the com-
pounds (Figure 3). However, we did not observe anti-HIV-
1 protease activity with the enantiomers of ganomycin I in
this assay system because the compounds showed cytotoxi-
city against the cells at the tested concentrations. In con-
trast, fornicin A showed very weak anti-HIV-1 protease ac-
tivity on the basis of the WST-1 assay,[23] without cyto-
toxicity. The activity of fornicin A increased in a dose-de-
pendent manner at concentrations Ͼ 12.5 μg/mL, but the
activity was considerably weaker than that of the positive
control, saquinavir (SQV).
Figure 2. Structures of catalysts for ring-closing metathesis.
Synthesis of Fornicin A
Fornicin A (2) was synthesized in a manner similar to
that described above, starting from acid segment 23 pre-
pared from known aldehyde 22[21] (Scheme 4). Although the
Figure 3. Inhibitory effects of the synthetic compounds against
HIV-1 protease. The inhibitory effects of samples were calculated
as the relative rate of WST-1 formazan production compared to
that of doxycycline (Dox)-treated cells. Each value represents
meanϮstandard deviation of duplicate determinations. SQV =
saquinavir, PMSF = phenylmethanesulfonyl fluoride, R = (R)-
ganomycin I, S = (S)-ganomycin I, F = fornicin A, DMSO = di-
methyl sulfoxide.
Conclusions
We have achieved the first total syntheses of ganomycin I
and fornicin A by employing asymmetric dihydroxylation
followed by Julia–Kocienski type olefin formation and ring-
closing metathesis as the key steps. The developed synthetic
method is applicable to the synthesis of other meroterpeno-
ids, such as clavilactones. The proposed absolute configura-
tion of ganomycin I was confirmed as R, and the absolute
configuration of fornicin A was also determined as R. Be-
Scheme 4. Synthesis of fornicin A. Reagents and conditions: (a)
NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH, H2O, 0 °C to room
temp., 89%; (b) Cl3C6H2COCl, DIPEA, DMAP, toluene, then (R)-
14, 83%; (c) Grubbs 1st catalyst, CH2Cl2, 78%; (d) TsOH, EtOH,
79%.
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Syntheses and Biological Activities of Ganomycin I and Fornicin A
56.1, 60.3, 95.0, 95.1, 115.4, 116.3, 119.1, 119.6, 125.0, 139.3, 151.1,
152.0, 167.1 ppm. C15H12O6 (288.26): calcd. C 60.80, H 6.80; found
C 60.75, H 6.58.
cause the cell-based HIV-1 protease inhibitory activities of
the synthetic compounds indicated that ganomycin I is
cytotoxic against E-PR293 cells, it would be difficult to de-
velop ganomycin I for use as an anti-HIV therapeutic agent.
Although fornicin A was reported to be cytotoxic against
(E)-3-[2,5-Bis(methoxymethoxy)phenyl]prop-2-en-1-ol (10): A solu-
tion of 9 (58.0 mg, 196 μmol) in anhydrous CH2Cl2 (2 mL) was
Hep-2 cells,[6] this compound exhibited weak anti-HIV-1 cooled to –78 °C, then DIBAL-H (1.0 m in hexane, 588 μL,
588 μmol) was added. The mixture was stirred for 1 h at 0 °C, then
saturated aqueous potassium sodium tartrate solution (5 mL) was
added and the mixture was stirred for 1 h. The mixture was ex-
tracted with diethyl ether, then the combined organic layers were
washed with brine, dried with Na2SO4, and concentrated. The resi-
due was purified by column chromatography (hexane/ethyl acetate,
6:1) to give 10 (49.1 mg, 99%) as a colorless oil. IR (liquid film):
protease activity without cytotoxicity against E-PR293
cells.
Experimental Section
General Methods: Optical rotations were measured with a Jasco P-
2100 polarimeter. IR spectra were measured with a Jasco IR-4100
spectrometer. 1H NMR spectra were recorded with a Jeol ECX400
(400 MHz) spectrometer using CDCl3 (δ = 7.26 ppm), [D6]acetone
(δ = 2.05 ppm), or CD3OD (δ = 3.30 ppm) as internal standard.
13C NMR spectra were recorded with a Jeol ECX400 (100 MHz)
spectrometer using CDCl3 (δ = 77.0 ppm), [D6]acetone (δ =
29.8 ppm), or CD3OD (δ = 49.0 ppm) as internal standard. Ele-
mental compositions were analyzed with a J-Science MICRO-
CORDER JM10 apparatus. High-resolution ESI-MS data were re-
corded with a Thermo Fisher Exactive mass spectrometer. Column
chromatography was performed with Wakogel-C200 silica gel. All
air- or moisture-sensitive reactions were conducted under an argon
atmosphere. Dried solvents for reactions (THF, CH2Cl2, and tolu-
ene) were purchased from Wako Pure Chemical Industries, Ltd.
ν
max = 3430, 2952, 2902, 2827, 1493, 1402, 1280, 1221, 1192, 1152,
˜
1078, 1008, 922 cm–1. H NMR (400 MHz, CDCl3): δ = 3.48 (s, 3
H), 3.49 (s, 3 H), 4.34 (dd, J = 1.6, 5.7 Hz, 2 H), 5.13 (s, 2 H), 5.14
(s, 2 H), 6.37 (dt, J = 16.0, 5.7 Hz, 1 H), 6.89 (dd, J = 3.0, 8.7 Hz,
1 H), 6.94 (t, J = 1.6 Hz, 1 H), 7.03 (d, J = 8.7 Hz, 1 H), 7.11 (d,
J = 3.0 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 55.8,
56.1, 63.9, 95.0, 95.4, 114.5, 116.4, 116.7, 125.4, 127.6, 129.8, 149.6,
152.2 ppm. C13H18O5 (254.28): calcd. C 61.40, H 7.14; found C
61.54, H 7.40.
1
(E)-2-{3Ј-[2ЈЈ,5ЈЈ-Bis(methoxymethoxy)phenyl]prop-2-enylthio}-1,3-
benzothiazole (11): Diethylazodicarboxylate (DEAD; 40% in tolu-
ene, 2.32 mL, 5.89 mmol) was added to a solution of 10 (1.50 g,
5.89 mmol), triphenylphosphine (1.59 g, 5.89 mmol), and 2-mer-
captbenzothiazole (1.01 g, 5.89 mmol) in anhydrous THF (20 mL)
at 0 °C. After stirring at room temperature for 10 h, the reaction
was quenched by the addition of saturated aqueous NaHCO3 solu-
tion (30 mL). The reaction mixture was extracted with ethyl acetate
and the combined organic layers were washed with water and brine,
dried with Na2SO4, and concentrated. The residue was purified by
flash column chromatography on silica gel (hexane/ethyl acetate,
2,5-Bis(methoxymethoxy)benzaldehyde (8): A solution of 2,5-di-
hydroxybenzaldehyde (7; 400 mg, 2.90 mmol) and K2CO3 (4.09 g,
29.0 mmol) in acetone (60 mL) was cooled to 0 °C, and chlo-
romethyl methyl ether (689 μL, 8.70 mmol) was added. The re-
sulting mixture was stirred at room temperature for 10 h, then di-
luted with water and extracted with ethyl acetate. The combined
organic layer was washed with water and brine, and dried with
Na2SO4. After concentration in vacuo, the residue was purified by
column chromatography (hexane/ethyl acetate, 3:1) to give 8
20:1) to give 11 (2.12 g, 89%) as a yellow oil. IR (liquid film): ν
˜
max
= 2965, 2921, 2854, 1644, 1585, 1504, 1453, 1201, 1055 cm–1. 1H
NMR (400 MHz, CDCl3): δ = 3.43 (s, 3 H), 3.46 (s, 3 H), 4.20 (dd,
J = 1.1, 7.3 Hz, 2 H), 5.09 (s, 2 H), 5.11 (s, 2 H), 6.38 (dt, J = 16.0,
7.3 Hz, 1 H), 6.88 (dd, J = 3.0, 9.0 Hz, 1 H), 7.00 (d, J = 9.0 Hz,
1 H), 7.02 (d, J = 16.0 Hz, 1 H), 7.11 (d, J = 3.0 Hz, 1 H), 7.29
(ddd, J = 1.2, 8.0, 8.0 Hz, 1 H), 7.42 (ddd, J = 1.2, 8.0, 8.0 Hz, 1
H), 7.76 (br. d, J = 8.0 Hz, 1 H), 7.90 (d, J = 8.0 Hz, 1 H) ppm.
13C NMR (100 MHz, CDCl3): δ = 36.4, 55.9, 56.1, 95.0, 95.4,
114.6, 116.5, 116.9, 120.9, 121.6, 124.2, 124.6, 126.0, 127.3, 128.8,
135.4, 149.5, 152.2, 153.2, 166.2 ppm. C20H21NO4S2 (403.51):
calcd. C 59.53, H 5.25, N 3.47; found C 59.37, H 5.25, N 3.75.
(642 mg, 98%) as a colorless oil. IR (liquid film): ν
= 2955,
˜
max
2904, 2828, 1686, 1610, 1493, 1429, 1390, 1274, 1219, 1201, 1153,
1
1080, 1008, 924 cm–1. H NMR (400 MHz, CDCl3): δ = 3.47 (s, 3
H), 3.52 (s, 3 H), 5.15 (s, 2 H), 5.25 (s, 2 H), 7.17 (d, J = 8.9 Hz,
1 H), 7.23 (dd, J = 3.0, 8.9 Hz, 1 H), 7.49 (d, J = 3.0 Hz, 1 H),
10.45 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 55.9, 56.3,
94.8, 95.1, 114.5, 116.8, 124.7, 126.0, 151.9, 154.9, 189.3 ppm.
C11H14O5 (226.23): calcd. C 58.40, H 6.24; found C 58.28, H 6.32.
(E)-3-[2,5-Bis(methoxymethoxy)phenyl]prop-2-enoate (9): To a sus-
pension of NaH (60%, 19.2 mg, 400 μmol) in anhydrous THF (2ЈR,3ЈS)-2-{3Ј-[2ЈЈ,5ЈЈ-Bis(methoxymethoxy)phenyl]-2Ј,3Ј-di-
(3 mL), triethyl phosphonoacetate (82.4 μL, 400 μmol) was added
at 0 °C, and the resulting mixture was stirred for 30 min. A solution
of 8 (45.2 mg, 200 μmol) in THF (2 mL) was then added dropwise
to the reaction mixture, which was stirred for 1 h at 0 °C. After
completion of the reaction, saturated aqueous NH4Cl solution was
added, and the aqueous layer was extracted with diethyl ether. The
combined organic layers were washed with brine, dried with
Na2SO4, and concentrated. The residue was purified by column
chromatography (hexane/ethyl acetate, 2:1) to give 9 (58.0 mg,
hydroxyprop-2-enylthio}-1,3-benzothiazole (12): To a solution of 11
(1.50 g, 3.72 mmol) in tBuOH/water (1:1, 40 mL) were added Me-
SO2NH2 (35.2 mg, 370 μmol) and AD-mix-α (5.20 g) at 0 °C. After
stirring for 12 h, the reaction was quenched with saturated aqueous
Na2S2O3 (20 mL). After stirring for 30 min, the mixture was ex-
tracted with ethyl acetate and the combined organic layers were
washed with water and brine, dried with Na2SO4, filtered, and con-
centrated. The residue was purified by column chromatograph
(hexane/ethyl acetate, 1:1) to give (2ЈR,3ЈS)-12 (1.38 g, 85%) as a
white solid. [α]2D6 = +0.52 (c = 0.10, CHCl3); m.p. 72–74 °C. IR
96%) as a colorless oil. IR (liquid film): ν
= 2955, 2903, 2827,
˜
max
1713, 1633, 1495, 1400, 1367, 1319, 1282, 1154, 1080, 994 cm–1. 1H
NMR (400 MHz, CDCl3): δ = 1.34 (t, J = 7.3 Hz, 1 H), 3.48 (s, 3
H), 3.50 (s, 3 H), 4.26 (q, J = 7.3 Hz, 2 H), 5.13 (s, 2 H), 5.19 (s,
2 H), 6.47 (d, J = 16.0 Hz, 1 H), 7.02 (dd, J = 3.0, 8.9 Hz, 1 H),
(KBr): νmax = 3531, 3454, 3002, 2974, 2927, 2892, 2827, 1493, 1398,
˜
1
1314, 1270, 1221, 1150, 1074, 1051, 1002, 933, 917 cm–1. H NMR
(400 MHz, CDCl3): δ = 1.4–1.8 (br., 2 H), 3.40 (dd, J = 6.4,
14.6 Hz, 1 H), 3.43 (s, 3 H), 3.48 (s, 3 H), 3.62 (dd, J = 5.5, 14.6 Hz,
7.08 (d, J = 8.9 Hz, 1 H), 7.23 (d, J = 3.0 Hz, 1 H), 8.00 (d, J = 2 H), 4.15 (dd, J = 5.5, 11.5 Hz, 2 H), 5.12–5.15 (m, 5 H), 6.93
16.0 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.2, 55.8,
(dd, J = 3.2, 9.2 Hz, 1 H), 7.03 (d, J = 9.2 Hz, 1 H), 7.25 (d, J =
Eur. J. Org. Chem. 2014, 731–738
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
735

A. Yajima, S. Urao, R. Katsuta, T. Nukada
3.2 Hz, 1 H), 7.32 (ddd, J = 1.1, 8.0, 8.0 Hz, 1 H), 7.43 (ddd, J = H 7.14; found C 61.15, H 6.85. The er of (S)-14 was estimated to
FULL PAPER
1.1, 8.0, 8.0 Hz, 1 H), 7.74 (d, J = 8.0 Hz, 1 H), 7.84 (d, J = 8.0 Hz,
1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 36.6, 56.0, 56.2, 69.7,
74.5, 95.07, 95.14, 115.2, 116.2, 116.3, 121.1, 121.2, 124.7, 126.4,
130.5, 135.2, 149.1, 152.1, 152.3, 168.8 ppm. C20H23NO6S2
(437.52): calcd. C 54.90, H 5.30, N 3.20; found C 55.06, H 5.57, N
3.41.
be 98:2 on the basis of HPLC analysis.
(E)-6,9-Dimethyl-2-methylideneundeca-5,9-dienal (17): To a solution
of geraniol 16 (4.00 g, 32.4 mmol) in carbon tetrachloride (50 mL)
was added triphenylphosphine (10.2 g, 38.9 mmol). The reaction
mixture was heated to reflux for 1 h, then cooled to 0 °C, and hex-
ane (20 mL) was added. After stirring for an additional 5 min, the
(2ЈS,3ЈR)-12: In the same manner as that described above, precipitated triphenylphosphine oxide was filtered off. The solvent
(2ЈS,3ЈR)-isomer (710 mg, 82 %) was obtained starting from 11
(800 mg) with AD-mix-β. [α]2D6 = –0.67 (c = 0.10, CHCl3).
C20H23NO6S2 (437.52): calcd. C 54.90, H 5.30, N 3.20; found C
54.67, H 5.13, N 3.14.
was removed in vacuo to afford crude geranyl chloride.
At –78 °C, N,N,NЈ,NЈ-tetramethylethylenediamine (TMEDA;
19.5 mL, 130 mmol) was added dropwise to nBuLi (2.68 m in hex-
ane, 48.5 mL, 130 mmol), resulting in the formation of a white pre-
cipitate. After stirring for 10 min, β-methallyl alcohol (6.82 mL,
81.0 mmol) was added dropwise, then anhydrous diethyl ether
(2ЈR,3ЈS)-2-{3Ј-[2ЈЈ,5ЈЈ-Bis(methoxymethoxy)phenyl]-2Ј,3Ј-di-
hydroxyprop-2-enylsulfonyl}-1,3-benzothiazole (13): To a solution of
(2ЈR,3ЈS)-12 (500 mg, 1.14 mmol) in CH2Cl2 (10 mL) was added (80 mL) was added. The cooling bath was removed and stirring
m-CPBA (70%, 570 mg, 2.28 mmol) at –78 °C. After stirring for was continued for 22 h at room temperature, during which time a
1 h, the mixture was poured into saturated aqueous NaHCO3 solu-
tion (10 mL) then extracted with CH2Cl2. The combined organic
layers were washed with water and brine, dried with Na2SO4, fil-
tered, and concentrated. The residue was purified by column
chromatography (hexane/ethyl acetate, 1:1) to give (2ЈR,3ЈS)-13
(535 mg, 92%) as a white solid. [α]2D6 = +4.4 (c = 0.10, CHCl3);
dark-orange gum was observed in the reaction mixture. Sub-
sequently, the reaction mixture was cooled to –78 °C and the crude
geranyl chloride prepared above in diethyl ether (10 mL) was
added. The cooling bath was removed and the resultant yellow mix-
ture was warmed to room temperature and stirred for 1 h. The
mixture was cooled to 0 °C and quenched with 1 n HCl (100 mL).
The aqueous phase was extracted with diethyl ether and the com-
m.p. 80–82 °C. IR (KBr): ν
= 3530, 3455, 2967, 2935, 2898,
˜
max
2832, 1487, 1398, 1314, 1145, 1080, 1021, 926 cm–1 1H NMR bined organic layers were washed with water and brine, dried with
.
(400 MHz, CDCl3): δ = 2.99 (d, J = 5.6 Hz, 1 H), 3.35 (br. s, 1 H),
3.40 (s, 3 H), 3.44 (s, 3 H), 3.65 (dd, J = 2.3, 15.1 Hz, 1 H), 3.87
(dd, J = 9.6, 15.1 Hz, 1 H), 4.55 (m, 1 H), 4.93 (dd, J = 5.5, 5.5 Hz,
1 H), 5.05–5.13 (m, 4 H), 6.93 (dd, J = 3.2, 9.2 Hz, 1 H), 7.03 (d,
J = 9.2 Hz, 1 H), 7.04 (d, J = 3.2 Hz, 1 H), 7.63 (m, 2 H), 8.00
Na2SO4, filtered and concentrated. The residue was purified by col-
umn chromatography (hexane/ethyl acetate, 10:1) to give a mixture
of 17 and remaining β-methallyl alcohol.
To the mixture of alcohols in hexane (60 mL) was added activated
MnO2 (14.4 g, 162 mmol) and the mixture was stirred for 6 h at
room temperature. The mixture was filtered through a pad of Ce-
lite, and the filtrate was concentrated. The residue was purified by
column chromatography (hexane/ethyl acetate, 10:1) to give 18
(5.01 g, 75% in three steps from 16) as a colorless oil. IR (liquid
(dd, J = 0.9, 7.8 Hz, 1 H), 8.18 (dd, J = 0.9, 7.8 Hz, 1 H) ppm. 13
C
NMR (100 MHz, CDCl3): δ = 55.9, 56.3, 58.5, 69.7, 71.6, 94.9,
95.0, 115.4, 116.0, 116.9, 122.3, 125.4, 127.7, 128.1, 129.1, 136.7,
149.0, 152.3, 152.4, 166.5 ppm. C20H23NO8S2 (469.52): calcd. C
51.16, H 4.94, N 2.98; found C 51.31, H 5.09, N 3.17.
film): ν
= 2915, 1696, 1451, 1380, 942 cm–1 1H NMR
.
˜
max
(2ЈS,3ЈR)-13: In the same manner as that described above, (400 MHz, CDCl3): δ = 1.58 (s, 3 H), 1.60 (s, 3 H), 1.67 (d, J =
(2ЈS,3ЈR)-isomer (39.9 mg, 85 %) was obtained starting from
0.9 Hz, 3 H), 1.98 (t, J = 7.8 Hz, 2 H), 2.06 (dt, J = 7.8, 7.8 Hz, 2
H), 2.16 (dt, J = 7.8, 7.8 Hz, 2 H), 2.28 (t, J = 7.8 Hz, 2 H), 5.05–
5.13 (m, 2 H), 5.98 (s, 1 H), 6.25 (d, J = 0.9 Hz, 1 H), 9.54 (s, 1
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 16.0, 17.7, 25.7, 26.0,
26.6, 27.9, 39.6, 123.1, 124.2, 131.3, 134.2, 136.1, 149.8, 194.7 ppm.
C14H22O (206.33): calcd. C 81.50, H 10.75; found C 81.31, H 10.53.
(2ЈS,3ЈR)-12 (44.0 mg). [α]2D6
=
–4.9 (c 0.10, CHCl3).
=
C20H23NO8S2 (469.52): calcd. C 51.16, H 4.94, N 2.98; found C
51.28, H 4.79, N 2.79.
(R)-1-[2Ј,5Ј-Bis(methoxymethoxy)phenyl]prop-2-en-1-ol (14): To a
solution of (2ЈR,3ЈS)-13 (100 mg, 213 μmol) in anhydrous CH2Cl2
(10 mL) was added DBU (63.7 μL, 426 μmol) at 0 °C, and the reac-
tion mixture was stirred for 1 h. Evaporation of the solvent gave
a crude mixture, which was purified by column chromatography
(hexane/ethyl acetate, 4:1) to give (R)-14 (67.7 mg, 80%) as a color-
(E)-6,9-Dimethyl-2-methylideneundeca-5,9-dienoic Acid (19): To a
stirred solution of 18 (850 mg, 4.12 mmol) and 2-methyl-2-butene
(4.90 mL, 41.2 mmol) in tBuOH (20 mL) and water (10 mL) were
added a solution of NaH2PO4 (2.54 g, 20.9 mmol) in water (5 mL)
and a solution of NaClO2 (80 %, 1.14 g, 12.4 mmol) in water
(5 mL) at room temperature. After stirring for 1 h, the mixture was
less oil. [α]2D6 = +114 (c = 0.10, CHCl ). IR (liquid film): ν
=
˜
3
max
3462, 2953, 2902, 2827, 1495, 1441, 1404, 1190, 1152, 1078, 1007,
1
923 cm–1. H NMR (400 MHz, CDCl3): δ = 3.47 (s, 3 H), 3.48 (s, diluted with brine, and the aqueous layer was extracted with
3 H), 5.12 (s, 2 H), 5.16 (s, 2 H), 5.19 (dt, J = 10.5, 1.4 Hz), 5.34 CHCl3. The combined organic layers were dried with Na2SO4, fil-
(dt, J = 17.4, 1.4 Hz, 1 H), 5.42 (br. d, J = 5.5 Hz, 1 H), 6.10 (ddd, tered and concentrated. The residue was purified by column
J = 5.5, 10.5, 17.4 Hz, 1 H), 6.93 (dd, J = 3.2, 8.7 Hz, 1 H), 7.03
(d, J = 8.7 Hz, 1 H), 7.04 (d, J = 3.2 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 55.9, 56.2, 70.9, 95.0, 95.2, 114.7, 115.78,
115.84, 116.1, 132.9, 139.3, 149.4, 152.3 ppm. C13H18O5 (254.28):
calcd. C 61.40, H 7.14; found C 61.66, H 7.30. The er of (R)-14
was estimated to be 98:2 on the basis of HPLC analysis
{CHIRALPAK IA; 4.6 mmϫ25 cm; hexane/EtOH, 9:1; flow rate:
0.9 mL/min; detection: 254 nm; tR = 12.1 [(R)-14], 10.7 [(S)-
14] min}.
chromatography (hexane/ethyl acetate, 10:1) to give 19 (760 mg,
83%) as a colorless oil. IR (liquid film): ν = 3200–3000, 2969,
˜
max
2917, 1696, 1629, 1442, 1304, 1223, 949 cm–1. 1H NMR (400 MHz,
CDCl3): δ = 1.60 (s, 6 H), 1.78 (d, J = 0.9 Hz, 3 H), 1.98 (t, J =
7.8 Hz, 2 H), 2.06 (dt, J = 7.8, 7.8 Hz, 2 H), 2.20 (dt, J = 7.8,
7.8 Hz, 2 H), 2.35 (t, J = 7.8 Hz, 2 H), 5.05–5.15 (m, 2 H), 5.65 (d,
J = 0.9 Hz, 1 H), 6.27 (d, J = 1.4 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 16.0, 17.7, 25.7, 26.68, 26.74, 31.6, 39.7,
123.1, 124.3, 127.3, 131.3, 136.1, 139.7, 173.0 ppm. C14H22O2
(222.33): calcd. C 75.63, H 9.97; found C 75.87, H 10.11.
(S)-14: In the same manner as that described above, the (S)-isomer
(41.7 mg, 77 %) was obtained by starting from (2ЈS,3ЈR)-14 (1ЈR,5E)-1Ј-[2ЈЈ,5ЈЈ-Bis(methoxymethoxy)phenyl]prop-2-en-1-yl
(100 mg). [α]2D6 = –109 (c = 0.10, CHCl3). C13H18O5: calcd. C 61.40,
6,10-Dimethyl-2-methylideneundeca-5,9-dienoate (20): To a stirred
736
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 731–738

Syntheses and Biological Activities of Ganomycin I and Fornicin A
solution of 19 (35.1 mg, 158 μmol) in toluene (2 mL) were added
diisopropylethylamine (DIPEA; 110 μL, 631 μmol), Cl3C6H2COCl
(100 μL, 631 μmol), and 4-(dimethylamino)pyridine (DMAP;
154 mg, 1.26 mmol). To the mixture was added (R)-14 (40.1 mg,
124 μmol) in toluene (2 mL) and the resulting mixture was diluted
with toluene (2 mL) and stirred for 8 h. The mixture was diluted
with benzene (10 mL) and saturated aqueous NaHCO3 solution
(15 mL), the layers were separated, and the aqueous layer was ex-
tracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried with Na2SO4, filtered, and con-
centrated. The residue was purified by column chromatography
tracted with CHCl3. The combined organic layers were dried with
Na2SO4, filtered and concentrated. The residue was purified by
flash column chromatography (hexane/ethyl acetate, 3:1) to give
(R)-1 (10.7 mg, 85%) as a yellow oil. [α]2D6 = +33.8 (c = 0.10,
MeOH) [ref.[5] [α]2D3 = +36 (c = 0.10, MeOH)]. IR (liquid film): ν
˜
max
= 3358, 2921, 1740, 1504, 1453, 1299, 1201, 1055, 984, 665 cm–1.
1H NMR (400 MHz, CD3OD): δ = 1.55 (s, 6 H), 1.64 (s, 3 H),
1.95–2.10 (m, 4 H), 2.24–2.40 (m, 4 H), 5.04 (br. t, J = 7.0 Hz, 1
H), 5.11 (br. t, J = 6.8 Hz, 1 H), 6.21 (d, J = 1.4 Hz, 1 H), 6.45 (d,
J = 2.7 Hz, 1 H), 6.59 (dd, J = 2.7, 8.7 Hz, 1 H), 6.66 (d, J =
8.7 Hz, 1 H), 7.33 (d, J = 1.4 Hz, 1 H) ppm. 13C NMR (100 MHz,
(hexane/ethyl acetate, 10:1) to give (1ЈR,5E)-20 (67.0 mg, 92%) as CD3OD): δ = 16.2, 17.8, 25.9, 26.2, 26.9, 27.7, 40.8, 79.8, 113.2,
a colorless oil. [α]2D6 = +0.39 (c = 0.10, CHCl3). IR (liquid film):
117.15, 117.17, 172.8, 123.5, 124.0, 125.4, 132.2, 133.0, 137.8,
148.9, 151.1, 151.5, 176.8 ppm. HRMS (ESI): calcd. for C21H27O4
[M + H]+ 343.1904; found 343.1897.
1
ν
˜
= 2913, 1719, 1632, 1499, 1154, 1080, 1011, 924 cm–1. H
max
NMR (400 MHz, CDCl3): δ = 1.57 (s, 3 H), 1.60 (s, 3 H), 1.68 (d,
J = 0.9 Hz, 3 H), 1.96 (m, 2 H), 2.04 (dt, J = 7.3, 7.3 Hz, 2 H),
2.18 (dt, J = 7.8, 7.8 Hz, 2 H), 2.37 (t, J = 7.3 Hz, 2 H), 3.46 (s, 3
H), 3.48 (s, 3 H), 5.06–5.16 (m, 6 H), 5.20 (dt, J = 10.5, 1.4 Hz),
5.28 (dt, J = 17.4, 1.4 Hz, 1 H), 5.56 (dd, J = 1.4, 1.4 Hz, 1 H),
6.04 (ddd, J = 5.5, 10.1, 16.9 Hz, 1 H), 6.24 (d, J = 1.4 Hz, 1 H),
6.69 (dt, J = 5.5, 1.4 Hz, 1 H), 6.94 (dd, J = 2.7, 8.7 Hz, 1 H), 7.04
(d, J = 8.7 Hz, 1 H), 7.06 (d, J = 2.7 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 16.0, 17.7, 25.7, 26.7, 26.8, 32.1, 39.7, 55.9,
56.0, 70.7, 95.0, 95.2, 115.6, 115.8, 116.1, 116.4, 123.2, 124.3, 125.1,
129.6, 131.3, 135.8, 136.0, 140.5, 149.1, 152.2, 166.0 ppm.
C27H38O6 (458.59): calcd. C 70.71, H 8.35; found C 70.45, H 8.23.
(S)-1: In the same manner as that described above, the (S)-isomer
(9.8 mg, 85%) was obtained starting from (5S,3ЈЈE)-21 (14.5 mg).
[α]2D6 = –33.4 (c = 0.10, MeOH). HRMS (ESI): calcd. for C21H27O4
[M + H]+ 343.1904; found 343.1897.
6-Dimethyl-2-methylidenehept-5-enoic Acid (23): To a stirred solu-
tion of 22[21] (1.50 g, 10.9 mmol) and 2-methyl-2-butene (12.8 mL,
109 mmol) in tBuOH (40 mL) and water (20 mL) were added a
solution of NaH2PO4 (6.60 g, 54.3 mmol) in water (10 mL) and a
solution of NaClO2 (80%, 3.68 g, 32.6 mmol) in water (10 mL) at
room temperature. After stirring for 1 h, the mixture was diluted
with brine and the aqueous layer was extracted with CHCl3. The
combined organic layers were dried with Na2SO4, filtered and con-
centrated. The residue was purified by column chromatography
(hexane/ethyl acetate, 10:1) to give 23 (1.49 g, 89%) as a colorless
(1ЈS,5E)-20: In the same manner as that described above, the (1ЈS)-
isomer (60.1 mg, 88 %) was obtained starting from (S)-14
(38.0 mg). [α]2D6 = –0.43 (c = 0.10, CHCl3). C27H38O6 (458.59):
calcd. C 70.71, H 8.35; found C 70.42, H 8.08.
oil. IR (liquid film): ν
= 3500–3000, 2970, 2927, 1696, 1630,
˜
max
(5R,3ЈЈE)-5-[2Ј,5Ј-Bis(methoxymethoxyphenyl)]-3-(4ЈЈ,8ЈЈ-dimeth-
1444, 1306, 1225, 950 cm–1. 1H NMR (400 MHz, CDCl3): δ = 1.60
ylnona-3ЈЈ,7ЈЈ-dien-1ЈЈ-yl)-2(5H)-furanone (21): A solution of (s, 3 H), 1.69 (d, J = 0.9 Hz, 3 H), 2.20 (dt, J = 7.3, 7.3 Hz, 2 H),
(1ЈR,5E)-20 (15.1 mg, 32.9 μmol) and [(PCy3)2Cl2Ru=CHPh]
(Grubbs 1st gen. catalyst; 1.4 mg, 1.6 μmol) in degassed CH2Cl2
(2 mL) was stirred for 5 h at room temperature. Then additional
[(PCy3)2Cl2Ru=CHPh] (1.4 mg, 1.6 μmol) was added. After stirring
for 3 h, the solvent was evaporated and the residue was purified by
flash column chromatography (hexane/ethyl acetate, 10:1) to give
(5R,3ЈЈE)-21 (11.3 mg, 80%) as a yellow oil. [α]2D6 = +16.6 (c = 0.10,
2.35 (t, J = 7.3 Hz, 2 H), 5.10 (m, 1 H), 5.64 (dd, J = 1.3, 1.3 Hz,
1 H), 6.27 (d, J = 0.9 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 17.7, 25.6, 26.9, 31.6, 123.2, 127.3, 132.5, 139.7, 173.0 ppm.
C9H14O2 (154.21): calcd. C 70.10, H 9.16; found C 70.32, H 9.03.
(R)-[2,5-Bis(methoxymethoxy)phenyl]prop-2-en-1-yl 6-Methyl-2-
methylidenehept-5-enoate (24): In the same manner as that de-
scribed for the preparation of 20, 24 (38.5 mg, 83%) was obtained
as a colorless oil starting from (R)-14 (30.4 mg) and 23 (18.5 mg).
CHCl ). IR (liquid film): ν
= 2909, 1766, 1501, 1280, 1154,
˜
3
max
1001, 924 cm–1. 1H NMR (400 MHz, CDCl3): δ = 1.58 (s, 6 H),
1.63 (d, J = 0.9 Hz, 3 H), 1.93–2.08 (m, 4 H), 2.27 (dt, J = 6.9,
6.9 Hz, 2 H), 2.36 (br. t, J = 6.9 Hz, 2 H), 3.44 (s, 3 H), 3.49 (s, 3
H), 5.04–5.14 (m, 4 H), 5.17 (d, J = 6.9 Hz, 1 H), 5.19 (d, J =
6.9 Hz, 1 H), 6.20 (d, J = 1.8 Hz, 1 H), 6.84 (d, J = 3.2 Hz, 1 H),
6.97 (dd, J = 3.2, 9.2 Hz, 1 H), 7.06 (d, J = 9.2 Hz, 1 H), 7.16 (dd,
J = 1.8, 1.8 Hz, 1 H) ppm. 13C NMR (100 MHz, CD3OD): δ =
16.2, 17.8, 25.9, 26.2, 26.9, 27.7, 40.8, 56.1, 56.5, 79.6, 96.1, 96.4,
115.7, 117.0, 118.4, 124.0, 125.3, 133.6, 137.8, 150.6, 153.7,
176.4 ppm. C25H34O6 (430.54): calcd. C 69.74, H 7.96; found C
69.94, H 7.72. The er of (5R)-21 was estimated to be Ͼ 97:3 on
the basis of HPLC analysis {CHIRALPAK IC; 4.6 mmϫ25 cm;
hexane/CH2Cl2, 7:3; flow rate: 0.9 mL/min; 5 °C, detection:
254 nm; tR = 22.6 [(5R)-21], 20.2 [(5S)-21] min}.
[α]2D6 = –10.6 (c = 0.10, CHCl ). IR (liquid film): νmax = 2928, 2826,
˜
3
1719, 1632, 1498, 1153, 1079, 1008, 924 cm–1. 1H NMR (400 MHz,
CDCl3): δ = 1.57 (s, 3 H), 1.67 (d, J = 0.9 Hz, 3 H), 2.17 (dt, J =
7.5, 7.5 Hz, 2 H), 2.36 (t, J = 7.5 Hz, 2 H), 3.47 (s, 3 H), 3.48 (s, 3
H), 5.10 (d, J = 7.5 Hz, 1 H), 5.11 (d, J = 7.5 Hz, 1 H), 5.16 (s, 2
H), 5.18 (dt, J = 10.5, 1.4 Hz, 1 H), 5.28 (dt, J = 1.4, 16.9 Hz, 1
H), 5.56 (d, J = 1.4 Hz, 1 H), 6.04 (ddd, J = 5.5, 10.5, 17.0 Hz, 1
H), 6.25 (d, J = 1.4 Hz, 1 H), 6.69 (dt, J = 5.5, 1.4 Hz, 1 H), 6.94
(dd, J = 3.2, 8.7 Hz, 1 H), 7.04 (d, J = 8.7 Hz, 1 H), 7.07 (d, J =
3.2 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 17.7, 25.6,
27.0, 32.0, 55.9, 56.0, 70.7, 95.0, 95.2, 115.6, 115.8, 116.1, 116.4,
123.3, 125.1, 129.6, 132.2, 135.7, 140.5, 149.1, 152.2, 166.0 ppm.
C22H30O6 (390.48): calcd. C 67.67, H 7.74; found C 67.72, H 7.90.
(R)-5-[2Ј,5Ј-Bis(methoxymethoxyphenyl)]-3-(4ЈЈ-methylnona-3ЈЈ-en-
1ЈЈ-yl)-2(5H)-furanone (25): In the same manner as that described
for the preparation of 21, 25 (18.5 mg, 78%) was obtained as a
yellow oil starting from 24 (25.6 mg). [α]2D6 = –1.8 (c = 0.10, CHCl3).
(5S,3ЈЈE)-21: In the same manner as that described above, the (5S)-
isomer (12.9 mg, 85 %) was obtained starting from (1ЈS,5E)-20
(16.2 mg). [α]2D6 = –16.2 (c = 0.10, CHCl3). C25H34O6 (430.54):
calcd. C 69.74, H 7.96; found C 69.99, H 7.67.
IR (liquid film): νmax = 2930, 1762, 1497, 1193, 1153, 1080, 1000,
˜
1
(R)-Ganomycin I (1): To a solution of (5R,3ЈЈE)-21 (15.9 mg,
36.9 μmol) in ethanol (1 mL) was added p-toluenesulfonic acid
925 cm–1. H NMR (400 MHz, CDCl3): δ = 1.57 (s, 3 H), 1.62 (s,
3 H), 2.27 (dt, J = 6.9, 6.9 Hz, 2 H), 2.37 (t, J = 6.9 Hz, 2 H), 3.45
(12.7 mg, 73.9 μmol). After stirring at room temperature for 18 h, (s, 3 H), 3.50 (s, 3 H), 5.09 (s, 2 H), 5.10 (m, 1 H), 5.19 (d, J =
the mixture was diluted with brine, and the aqueous layer was ex- 10.1 Hz, 1 H), 6.21 (d, J = 1.4 Hz, 1 H), 6.84 (d, J = 3.2 Hz, 1 H),
Eur. J. Org. Chem. 2014, 731–738
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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737

A. Yajima, S. Urao, R. Katsuta, T. Nukada
FULL PAPER
6.97 (dd, J = 3.2, 9.2 Hz, 1 H), 7.07 (d, J = 9.2 Hz, 1 H), 7.16 (d,
J = 1.4 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 17.7,
25.4, 25.6, 25.9, 56.0, 56.2, 95.1, 95.2, 115.0, 115.5, 117.2, 122.8,
125.7, 133.0, 147.7, 149.3, 152.3, 153.7, 174.1 ppm. C20H26O6
(362.42): calcd. C 66.28, H 7.23; found C 66.37, H 7.04.
Acknowledgments
Mr. Hideaki Inoue is thanked for his assistance with synthesis.
(R)-Fornicin A (2): In the same manner as that described for the
preparation of 1, 2 (6.1 mg, 79%) was obtained as a yellow oil
starting from 25 (10.2 mg). [α]2D6 = +25.4 (c = 0.32, MeOH) [ref.[6]
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[α]2D3 = +9.52 (c = 0.32, MeOH)]. IR (liquid film): ν
= 3379,
˜
max
2921, 1734, 1505, 1453, 1299, 1199, 1055, 984 cm–1. 1H NMR
(400 MHz, [D6]acetone): δ = 1.58 (s, 3 H), 1.66 (s, 3 H), 2.25–2.35
(m, 4 H), 5.13 (m, 1 H), 6.21 (d, J = 1.4 Hz, 1 H), 6.54 (d, J =
2.7 Hz, 1 H), 6.66 (dd, J = 2.7, 8.7 Hz, 1 H), 6.78 (d, J = 8.7 Hz,
1 H), 7.36 (dd, J = 1.4, 1.4 Hz, 1 H), 7.79 (br. s, 1 H), 8.25 (br. s,
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26.7, 78.2, 113.2, 116.8, 117.1, 123.9, 124.0, 132.9, 133.2, 148.2,
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Cells: E-PR293 cells derived from human embryonic kidney 293
tet-off cells (tTA-expressing cell line) were established as described
previously.[22] Cells were maintained in Dulbecco’s modified Eagle
medium (DMEM, Wako, Japan) supplemented with 10% fetal bo-
vine serum (FBS), 100 units/mL penicillin, 100 ng/mL streptomycin
(Nacalai Tesque, Japan), and 100 ng/mL doxycycline (Dox, Clon-
tech, Japan).
[9]
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Drugs: Saquinavir (SQV, Roche, Japan) and phenylmethanesulfonyl
fluoride (PMSF, Sigma–Aldrich, Japan) were used as positive and
negative control, respectively. The reference compounds were di-
luted to 10 μm with DMEM-10% FBS and then twofold serial di-
lutions were prepared on 96-well plate. The tested samples [(R)-1,
(S)-1, and (R)-2] were dissolved in dimethyl sulfoxide (DMSO) and
the concentrations were adjusted to 100 mg/mL. Dissolved samples
were sealed and stored at –20 °C until use. For cell-based assays,
100 mg/mL of samples were diluted to 200 μg/mL with DMEM-
10% FBS and then twofold serial dilutions were prepared as well
as the reference compounds.
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E-PR293 Cell-Based Assay to Evaluate the Inhibitory Effects
against HIV-1 Protease: For the cell-based assay, E-PR293 cells
were collected by trypsinization and washed with serum-free me-
dium three times to remove doxycycline (Dox). Cells were resus-
pended in DMEM-10%FBS without Dox and then 2.5ϫ104 cells
were seeded into each well on 96-well plate that contained twofold
serial dilutions of reference compounds or samples. Each ad-
ditional volume of cells and drugs was made equal (50 μL + 50 μL).
After 48 h, the expression of EGFP was detected by fluorescence
microscopy. The growth of the cells was measured by a WST-1
assay, as described previously.[23] Briefly, the cells were incubated
with 0.5 mm WST-1 solution containing 20 mm 1-methoxy PMS
(Dojindo, Japan) at 37 °C for 1 h. The production of WST-1
formazan was measured with a microplate reader (Infinite M200,
TECAN, Japan) at 450–650 nm. The inhibitory effects of samples
were calculated as the relative rate of WST-1 formazan production
compared to that of the Dox-treated cells.
Supporting Information (see footnote on the first page of this arti-
cle): Copies of the 1H and 13C NMR spectra for all new com-
pounds.
Received: August 23, 2013
Published Online: November 26, 2013
738
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Eur. J. Org. Chem. 2014, 731–738