Synthesis of the F11334's from o-prenylated phenols: ΜM inhibitors of neutral sphingomyelinase (N-SMase)

Article abstract of DOI:10.1016/S0040-4020(02)00391-5

F11334A₁, F11334A₂, F11334A₃, and their corresponding resorcinol analogs were synthesized along with F11334B₁, and F11263. In the course of these synthetic studies, several conditions for manipulating (2,3-propanediol) and (2,3-epoxypropyl) o-substituted phenols were developed as well as a variety of conditions for cleavage of aryl O-t-butyl carbonates. From enzyme assays it appears that the hydroquinone nucleus is the essential structural necessary for N-SMase inhibition. Furthermore, it appears that this family of compounds is comprised of irreversible inhibitors.

Full text of DOI:10.1016/S0040-4020(02)00391-5

TETRAHEDRON
Pergamon
Tetrahedron 58 22002) 4559±4565
Synthesis of the F11334's from o-prenylated phenols:
mM inhibitors of neutral sphingomyelinase ꢀN-SMase)
a
Christopher C. Lindsey, Consuelo Gomez-Dõaz, Jose M. Villalba and Thomas R. R. Pettus
b
b,p
a,p
Â
Â
Â
aDepartment of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
bDepartment of Biologia Celular, Fisiologia e Inmunologia, Campus Rabanales, Edi®cio C6, 3aPlanta,
Universidad de Cordoba, 14014 Cordoba, Spain
Received 10 January 2002; accepted 1 April 2002
AbstractÐF11334A1, F11334A2, F11334A3, and their corresponding resorcinol analogs were synthesized along with F11334B1, and
F11263. In the course of these synthetic studies, several conditions for manipulating 22,3-propanediol) and 22,3-epoxypropyl) o-substituted
phenols were developed as well as a variety of conditions for cleavage of aryl O-t-butyl carbonates. From enzyme assays it appears that the
hydroquinone nucleus is the essential structural necessary for N-SMase inhibition. Furthermore, it appears that this family of compounds is
comprised of irreversible inhibitors. q 2002 Published by Elsevier Science Ltd.
Recently, small molecules that serve as inhibitors for
sphingomyelinase have become of interest from a thera-
peutic standpoint.1 Sphingomyelinases are a subclass of
phospholipase C enzymes responsible for the cleavage of
sphingomyelin to ceramide and phosphocholine; an event
believed essential for triggering cell apoptosis. Two
mammalian sphingomyelinases are known. Acidic sphingo-
myelinase 2A-SMase), located in lysosomes, is stimulated
by cytostatic levels of FADD and interleukin 1.2 Neutral
sphingomyelinase 2N-SMase), found in the plasma
membrane, is stimulated by TNFR1/p55TNF activation of
the FAN protein and requires Mg21 for activity.3 It is
thought that inhibitors of sphingomyelinase may prove
useful for the treatment of a variety of maladies4 including
herpes,5 stroke,6 and myocardial infarction.7 One potentially
useful application for N-SMase inhibitors is stopping the
progression of HIV to AIDS. Apoptotic cell death, regarded
as the principal mechanism for depletion of T-cells, is
believed to be distinct from the mechanism of
HIV infection and replication.8 Some suspect that the HIV
proteins, gp120, gp160, and HIV-tat interact with the
Fas/FasL, TRAIL, and TNFR1/p55TNF receptors to
accelerate apoptotic cell death of T-lymphocytes via acti-
vation of neutral sphingomyelinase 2N-SMase).9 Activation
of N-SMase, in turn, stimulates caspase production leading
to T-cell apoptosis.10 Interestingly, long-term survivors of
HIV who have refused all treatment and remained healthy
and immunocompetent for 10 or more years, have been
shown to maintain lower than normal ceramide levels.11
This has led some to deduce that therapies directed at
down-modulating ceramide should slow the progression of
HIV by reducing lymphocyte apoptosis even if viral repli-
cation is unaffected by this course of action.12
By far the simplest inhibitors reported to be speci®c for
N-SMase are the F11334's 21a±3a, 4, 5),1e and F11263
26)1f 2Fig. 1). These compounds are produced by the fungus
Acremonium murorum. For compounds 1a±3a and 4±6
Ogita reported IC50 values ranging from 0.8±200 mg/mL
215±942 mM) against N-SMase that was harvested from
rat brain microsomes. The increased potency of hydro-
quinone derivative 1a relative to the cyclic ethers 2a and
3a was ascribed to free hydroxyl groups. In view of the
activity of F11263 26), we concluded that the bio-activity
of 1a±3a and 4, 5 resulted from the ability of these entities
to oxidize to the corresponding p-quinone. In order to con®rm
this suspicion, we set out to construct 1a±3a, 4±6, and their
corresponding resorcinol analogs 1b±3b using a common
synthetic sequence and then compare the inhibitory activity
between the resorcinol and hydroquinone derivatives.
Recently, we reported a very ef®cient three-pot construction
of o-prenylated phenols from the corresponding salicyl-
aldehydes.13a,b This procedure was used to construct 7a
and 7b in 63% and 60% overall yield from the respective
salicylaldehydes. Upon oxidation with 1,1-dimethyl-
dioxirane 2DMDO) 22.0 equiv.), these respective o-prenyl-
ated phenols 7a and 7b produce the corresponding epoxides
8a and 8b in excellent yield 2Scheme 1). In principle, this
epoxidation is amenable to enantioselectivity incorporating
Shi's method,14 however, it is believed that racemic
compounds would prove adequate for this survey.
Keywords: dihydroxylation; quinines; phenols; epoxidation; aryl O-t-butyl
carbonate deprotection; neutral sphingomyelinase.
Corresponding authors. Tel.: 11-805-893-5687; fax: 11-805-893-5690
p
The epoxides 8a and 8b, both of which were unstable
0040±4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020202)00391-5

4560
C. C. Lindsey et al. / Tetrahedron 58 02002) 4559±4565
Scheme 1. Synthesis of F11334A1 21), F11334A2 22), F11334A3 23), F11334B1 24) and F11263 26).
Figure 1. F11334's with respective inhibition of N-Smase. The IC50's for 1a±3a and 4±6 were reported by Ogita.1e,f

C. C. Lindsey et al. / Tetrahedron 58 02002) 4559±4565
4561
Table 1. Calculated IC50's for 1a and 1b regarding N-Smase from micro-
somes of rat brain
ate, which undergoes BOC cleavage most likely as a result
of the imidazole that is liberated during the course of the
reaction.16 Oxidation of the resulting hydroquinone 10
20.1 M in CH2Cl2) with 2,3-dichloro-5,6-dicyanobenzo-
quinone 2DDQ) 21.1 equiv.) and subsequent base promoted
opening of the cyclic carbonate 20.05 M in benzene)
triggered by re¯uxing with N,N-diisopropylethylamine
2DIPEA) 212.0 equiv.) provides the quinone 6. Reduction
of F11263 26) 20.5 M in 1/1 acetone and H2O) with excess
of a 0.5 M aqueous solution of sodium dithionite produces
F11334B1 24) in 53% from the cyclic carbonate 10.
Compound
Inhibition without pre-
incubation 2mg/mL)
Inhibition with pre-
incubation 2mg/mL)
1a
1b
46.6
.300.0
34.4
.300.0
Increased inhibition with pre-incubation suggests the compound acts in an
irreversible reaction with the enzyme.
towards chromatography, could be opened to exclusively
yield either the benzohydropyran or benzohydrofuran
nucleus. For example, exposure of either the resorcinol or
hydroquinone epoxide 20.1 M in methylene chloride) to
camphorsulphonic acid 2CSA) 22.0 equiv.) results in a
[6-endo-trig]-closure affording the respective benzohydro-
pyran. Presumably treatment with acid leads to a cation
intermediate which undergoes cyclization. Prolonged
contact with CSA results in subsequent cleavage of the
aryl O-t-butyl carbonate 2aryl-OBOC) to afford 3a or 3b,
respectively. Alternatively, an exclusive [5-exo-tet]-cycli-
zation leading to the corresponding benzohydrofuran is
executed on either of these epoxides by exposure of the
respective substrate 20.1 M in MeOH) to LiOH 22.0 equiv).
Presumably treatment with base leads to the corresponding
o-22,3-epoxypropyl)phenoxide, which undergoes a nucleo-
philic cyclization.15 Prolonged contact with LiOH results in
subsequent cleavage of the aryl O-t-butyl carbonate and
affords 2a and 2b, respectively. Our attempts to open type
8 epoxides to their corresponding diols failed. Efforts to
convert various o-22,3-epoxypropyl)phenols to their corre-
sponding o-23-hydroxyprop-1-ene)phenols by eliminatory
opening were also unsuccessful. We therefore devised a
different sequence to address the synthesis of 1a, 1b, 4, and 6.
1. Neutral-sphingomyelinase inhibition
We suspected that the biological activity of 1a, 4, and 5
results from their ability to easily oxidize to the correspond-
ing para-quinones. This conclusion is suggested by the
potency of F11263 26) and the lack thereof for 2a and
3a.1f However, it is surprising that these small molecules
display even moderate amounts of activity, since all other
inhibitors with comparable activity such as CoQ10,17
scyphostatin,18 and manumycin1h are substantially more
complex with large lipophyllic regions.
Inhibitory assays were conducted on 1a and 1b with
N-SMase isolated from the plasma membranes 2PM) of rat
livers as well as the microsomes of rats brain. Assays with
1a and 1b were carried out with and without pre-incubation.
Activity without pre-incubation suggests the ability of the
inhibitor to compete with the sphingomyelin substrate at the
active site of the enzyme, whereas increased inhibition with
pre-incubation suggests irreversible inhibition.
The IC50 for 1a and 1b regarding N-SMase isolated from
brain microsomes are shown in Table 1. Our studies con®rm
that F11334A1 21a) is an inhibitor of N-SMase [68% inhi-
bition at 63.67 mg/mL]. This is within the experimental
error of the activity observed with scyphostatin and manu-
mycin.1h Furthermore, the 25% increased activity of 1a
[93% inhibition at 63.67 mg/mL] upon pre-incubation,
suggests that 1a is an irreversible inhibitor. The resorcinol
derivative 1b, on the other hand, shows very little if any
inhibition of N-SMase. This observation therefore supports
the idea that the activity of 1a requires oxidation to the
corresponding p-quinone, a dif®cult proposition in the
case of the resorcinol compound 1b.
Independent dihydroxylation of the o-prenylated phenols 7a
and 7b is accomplished in t-BuOH 20.26 M) with osmium
tetroxide 2OsO4) 20.05 equiv.), and N-methylmorpholine
N-oxide 2NMO) 21.1 equiv). Yields of the respective
o-22,3-propanediol)-phenols 9a and 9b are essentially
quantitative. Exposing either the hydroquinone or resorcinol
diol 20.05 M in CH3NO2) to ZnBr2 25 equiv.) cleaves the
remaining aryl O-t-butyl carbonate furnishing the respective
tetrol 1a and 1b in yields ranging from 80 to 95%. On the
other hand re¯uxing 9b 20.1 M in benzene) with N,N0-car-
bonyldiimidazole 2CDI) 24 equiv.) affords a cyclic carbon-
F11334A1
No Pre-
The results shown in Fig. 2 depict a more precise picture of
this family of compounds as inhibitors of apoptotic sig-
naling. These tests were performed using the liver
plasma membrane rather than brain microsomes.19 In our
experience, puri®ed plasma membranes prove more reliable
as assays for apoptotic signal transduction because the
presence of the A-SMase is minimal.19 Moreover, the par-
ticipation of N-SMases obtained from brain homogenates
rich in the Golgi apparatus and Endoplasmic Reticulum
has been questioned in regards to their role in apoptotic
signaling and the sphingomyelin cycle.20 Compound 1a
displayed greater potency towards N-SMase isolated
from this source. The IC50 for 1a was calculated to
be 15.6 mg/mL. With pre-incubation the IC50 value fell
by approximately 25% 2more active) to 11.9 mg/mL,
incubation
1000
800
600
400
200
Preincubation
No Preincubation
IC50 =
15.0 ug/mL
Preincubation
IC50 =
11.0 ug/mL
0
0
10
20
Inhibitor Concentration
ug/mL
Figure 2. Inhibition of PM N-SMase with 1a at concentrations of 0.2, 2.1,
6.4, 21.2 mg/mL. A negative slope indicates increasing inhibition at
higher concentrations of the inhibitor. Counts per minute 2CPM). 488.0
and 419.0 cpm equals 50% inhibition without and with pre-incubation,
respectively.

4562
C. C. Lindsey et al. / Tetrahedron 58 02002) 4559±4565
suggesting again that 1a acts as an irreversible competitive
inhibitor.
homogenates were centrifuged for 15 min at 1000g to
remove nuclei and cell debris. Membranous material
contained in supernatants was recovered by ultracentrifu-
gation at 105,000g for 90 min and resuspended in 50 mM
Tris±HCl, pH 7.6 containing 1 mM phenylmethylsulfonyl-
¯uoride and 20 mg/mL each of chymostatin, leupeptin,
antipain and pepstatin A. Brain membranes were stored at
2808C until needed.
In conclusion, a common prenylated intermediate is
converted into the constituents of the F11334 family of
natural products as well as some of their resorcinol analogs.
A key feature is the application of basic, acidic, and neutral
conditions for cleaving aryl O-t-butyl carbonates concur-
rently with other synthetic operations. F11334A1 21a) is
con®rmed to be a micromolar inhibitor of N-SMase, while
its resorcinol counterpart 1b shows no activity. The
increased activity of 1a upon pre-incubation suggests that
it is an irreversible inhibitor. The plasma membrane appears
to provide N-SMase that is more readily inhibited by 1a than
N-SMase isolated from brain microsomes. From these
experiments we conclude that the F11334 family of natural
products offer greater structural simplicity and comparable
biological activity to signi®cantly more complex molecules
such as scyphostatin and manumycin A, thereby demon-
strating that a large lipophyllic region is not necessary for
N-SMase inhibition.
2.3. N-SMase assay
For the determination of the N-SMase activity, the potential
inhibitors were dissolved in ethanol and aliquots containing
the required amounts for the assays dried under a nitrogen
stream in the dark, redissolved in 40 mL of assay buffer
275 mM Tris±HCl, pH 7.4 containing 0.05% Triton X-100
and 5 mM MgCl2) and mixed with 10 mL of membrane
sample 230±35 mg protein). Together with controls, the
probes were pre-incubated for 0 and 30 min at 378C. After
addition of 10 nmol [14C]sphingomyelin 2ca. 50,000 cpm) in
50 mL of assay buffer, the reaction was proceeded for
another 30 min. The reaction was stopped by adding
750 mL chloroform/methanol 21:1, v/v). After addition of
200 mL water, lipids were extracted and the radioactivity
of the polar upper phase, containing [14C]phosphoryl-
choline, was determined by scintillation counting. As 1a
showed a loss of activity in the presence of a thiol, the
assays were carried out in a buffer free of dithiothreitol
and b-mercaptoethanol.
2. Experimental
2.1. General information
All the reagents were newly purchased or freshly prepared as
stipulated. All column chromatography was conducted using
60 microns silica gel with the indicated solvent systems.
2.4. Epoxidation
2.2. Preparation of biological samples
To a ¯ask containing the neat o-prenylated phenol, 7a or
7b13 2280.0 mg, 1.00 mmol) was added a solution of DMDO
220.0 mL, 0.1 M in acetone, 2.00 mmol, 2.0 equiv.) in a
dropwise fashion. After the reaction was stirred for 10 min
at room temperature, the solution was subsequently dried
with sodium sulfate and concentrated to give 8a or 8b.
Analytical pure sample could not be obtained; however
the corresponding NMR results for 8a and 8b are described
below.
The preparation of membranes used as a source for
N-SMase assays was carried out analogous to the method
described by Hannun21 and modi®ed with respect to his
more recent publications.22
Preparation of liver plasma membrane N-SMase. A crude
membrane fraction was obtained from rat liver homogenates
by differential centrifugation. Highly enriched plasma
membranes were then puri®ed from crude fractions by the
two-phase partition method with a phase system composed
of 6.4% Dextran T 500, 6.4% PEG 3350, 0.25 M sucrose
and 5 mM potassium phosphate buffer, pH 7.8.23
Membranes were then washed by centrifugation, resus-
pended in 50 mM Tris±HCl, pH 7.6 containing 1 mM
phenylmethylsulfonyl¯uoride and 20 mg/mL each of
chymostatin, leupeptin, antipain and pepstatin A, and stored
at 2808C until needed. Plasma membrane purity was
checked by marker enzymes as described.24
2.4.1. Compound 8a. 1H NMR [CDCl3, 400 MHz] d 6.97±
6.88 2m, 3H), 3.07 2dd, 1H, J1ˆ9.7 Hz, J2ˆ2.7 Hz), 2.91
2dd, 1H, J1ˆ14.8 Hz, J2ˆ2.9 Hz), 2.81 2dd, 1H, J1ˆ
14.8 Hz, J2ˆ9.7 Hz), 1.56 2s, 9H), 1.50 2s, 3H), 1.37 2s,
3H); 13C NMR [CDCl3, 100.6 MHz] d 153.5, 152.6,
144.4, 125.3, 123.2, 121.3, 117.8, 83.2, 65.3, 61.4, 32.0,
27.9, 24.9, 19.0.
1
2.4.2. Compound 8b. H NMR [CDCl3, 400 MHz] d 7.08
2d, 1H, Jˆ8.2 Hz), 6.75 2d, 1H, Jˆ2.4 Hz), 6.68 2dd, 1H,
J1ˆ8.2 Hz, J2ˆ2.4 Hz), 3.04 2dd, 1H, J1ˆ9.9 Hz, J2ˆ
2.7 Hz), 2.94 2dd, 1H, J1ˆ14.8 Hz, J2ˆ2.7 Hz), 2.78 2dd,
1H, J1ˆ14.8 Hz, J2ˆ9.9 Hz), 1.56 2s, 9H), 1.49 2s, 3H), 1.37
2s, 3H); 13C NMR [CDCl3, 100.6 MHz] d 156.6, 152.1,
151.3, 131.0, 122.1, 113.4, 110.7, 83.7, 65.5, 61.5, 31.7,
27.9, 24.9, 19.0.
Preparation of brain membranes. A membranous fraction
was obtained from rat brains as described by Liu et al.21
Frozen brains were brie¯y thawed and the cerebella and
brain stem removed. The remainder of the brain tissue
was homogenized with the aid of a motor-driven Te¯on
pestle glass homogenizer in ®ve volumes of 50 mM
Tris±HCl, pH 7.4 containing 2 mM EDTA, 5 mM EGTA,
1 mM sodium orthovanadate, 10 mM B-glycerolphosphate,
1 mM sodium ¯uoride, 1 mM sodium molybdate, 1 mM
phenylmethylsulfonyl¯uoride and 20 mg/mL each of
chymostatin, leupeptin, antipain and pepstatin A. Crude
2.5. Benzohydropyran formation/acidic cleavage of aryl
O-t-butyl carbonate
To a solution of the epoxide 8a or 8b, 263.6 mg, 0.229 mmol,

C. C. Lindsey et al. / Tetrahedron 58 02002) 4559±4565
4563
0.1 M in CH2Cl2) was added the camphorsulfonic acid
20.106 g, 0.458 mmol, 2.0 equiv.) and the resulting mixture
was allowed to stir for 2 days at room temperature. The
mixture was concentrated, re-dissolved in ethyl acetate,
washed with water and brine, dried with sodium sulfate
and condensed. Chromatography with silica gel 285:15
EtOAc/pet. ether) furnished 3a or 3b.
25.9, 25.6; IR [CH2Cl2, nmax cm21] 3584, 2978, 2935, 2854,
1701, 1493, 1260; MS 2EI) m/z 491 249.58), 136 281.10),
123 238.22), 107 225.47), 59 258.62), 43 2100.00); HRMS
2EI) m/z calcd for C11H14O3 194.094294, found
194.093577.1e
2.6.2. res-F11334A2 ꢀ2b). 285:15 EtOAc/pet. ether). A yield
of 92% was deduced using DMF as an internal comparing
the methine proton signal at 6.81 ppm with the aldehyde
proton at 7.96 ppm using 1H NMR. White solid.
2.5.1. Compound F11334A3 ꢀ3a). All spectra was found to
be identical to those previously reported. A yield of 65%
was deduced using DMF as an internal comparing the
methine proton signal at 3.70 ppm with the methyl signal
at 2.90 ppm using 1H NMR. White solid. Mpˆ154±1558C;
1H NMR [CD3OD, 400 MHz] d 6.62 2s, 1H), 6.56 2d, 1H,
Jˆ 8.5 Hz), 6.52 2dd, 1H, J1ˆ8.5 Hz, J2ˆ2.5 Hz), 3.70 2dd,
1H, J1ˆ7.5, Hz, J2ˆ5.5 Hz), 2.93 2dd, 1H, J1ˆ16.5 Hz,
J2ˆ5.5 Hz), 1.29 23H, s), 1.20 23H, s); 13C NMR [CDCl3,
100.6 MHz] d 152.2, 147.8, 122.4, 118.9, 116.9, 116.1,
78.0, 71.2, 32.9, 26.3, 21.4; IR [CH2Cl2, nmax cm21] 3586,
3000, 2932, 2857, 1703, 1497, 1278, 1256; MS 2EI) m/z 194
270.05), 161 227.42), 136 221.61), 123 2100.00), 71 246.14);
HRMS 2EI) m/z calcd for C11H14O3 194.094294, found
194.094078.
1
Mpˆ139±1418C; H NMR [CD3OD, 400 MHz] d 6.81 2d,
1H, Jˆ8.1 Hz), 6.22 2dd, 1H, J1ˆ8.1 Hz, J2ˆ2.2 Hz), 6.17
2d, 1H, Jˆ1.8 Hz), 4.52 2t, 1H, Jˆ8.8 Hz), 3.01 2d, 2H,
Jˆ8.8 Hz), 1.20 2s, 3H), 1.17 2s, 3H); 13C NMR [CD3OD,
100.6 MHz] d 162.5, 158.8, 125.9, 119.1, 108.2, 98.1, 91.2,
72.7, 31.1, 25.4, 25.2; IR [CH2Cl2, nmax cm21] 2583, 3043,
2977, 1620, 1499, 1460; MS 2EI) m/z 136 217.29), 101
229.47), 85 2100.00); HRMS 2EI) m/z calcd for C11H14O3
194.094294, found 194.094058.
2.7. Dihydroxylation
A solution of the respective o-prenylated phenol 7a or 7b,
20.0379 g, 0.138 mmol, 0.258 M in t-butanol, 1.9 M in
water) was combined with NMO 20.0205 g., 0.158 mmol,
1.14 equiv.). A catalytic amount of OsO4 20.036 mL,
0.076 M in t-BuOH, 0.02 equiv.) was added and the solution
was allowed to stir for 12 h at room temperature. Sodium
sul®te 20.034 g, 0.28 mmol, 2.0 equiv.) was then added to
the solution and allowed to stir for 0.5 h. This solution was
then extracted with ethyl acetate 2three times), washed with
brine, dried with sodium sulfate, and concentrated. Chroma-
tography 275:25 EtOAc/pet. ether) with silica gel furnished
9a or 9b.
2.5.2. res-F11334A3 ꢀ3b). A yield of 60% was deduced
using DMF as an internal comparing the methine proton
signal at 3.70 ppm with the aldehyde proton at 7.96 ppm
1
1
using H NMR. White solid. Mpˆ149±1508C; H NMR
[CD3OD, 400 MHz] d 6.84 2d, 1H, Jˆ8.2 Hz), 6.30 2dd,
1H, J1ˆ8.2 Hz, J2ˆ2.4 Hz), 6.18 2d, 1H, Jˆ2.4 Hz), 3.70
2dd, 1H, J1ˆ7.7 Hz, J2ˆ5.5 Hz), 2.89 2dd, 1H, J1ˆ16.1 Hz,
J2ˆ5.3 Hz), 2.61 2dd, 1H, J1ˆ16.1 Hz, J2ˆ7.7 Hz), 1.31 2s,
3H), 1.22 2s, 3H); 13C NMR [CDCl3, 100.6 MHz] d 155.7,
153.7, 131.0 110.8, 108.8, 104.0, 77.1, 70.0, 30.0, 24.9,
22.6; IR [CH2Cl2, nmax cm21] 3583, 2978, 2935, 1734,
1624, 1598, 1508, 1452, 1371; MS 2EI) m/z 136 27.53),
124 215.19), 123 2100.0), 43 216.61); HRMS 2EI) m/z
calcd for C11H14O3 195.102120, found 195.101500.
2.7.1. Compound 9a. A yield of 93% was deduced using
DMF as an internal comparing the methine proton signal at
3.46 ppm with the methyl proton signal at 2.99 ppm using
1
1H NMR. White solid. Mpˆ40±428C; H NMR [CDCl3,
2.6. Benzohydrofuran formation/basiccleavage of aryl
O-t-butyl carbonate
400 MHz] d 8.22 2bs, 1OH), 6.88 2dd, 1H, J1ˆ8.6 Hz,
J2ˆ2.7 Hz), 6.85 2d, 1H, J1ˆ2.7 Hz), 6.75 2d, 1H,
Jˆ8.6 Hz), 4.06 2bs, 1OH), 3.46 2d, 1H, Jˆ9.7 Hz), 2.73
2dd, 1H, J1ˆ14.3 Hz, J2ˆ10.3 Hz), 2.59 2dd, 1H,
J1ˆ14.3 Hz, J2ˆ1.5 Hz), 1.71 2bs, OH), 1.55 2s, 9H), 1.24
2s, 3H), 1.20 2s, 3H); 13C NMR [CDCl3, 100.6 MHz] d
153.6, 153.1, 144.1, 127.3, 123.7, 120.9, 117.5, 83.9, 80.5,
73.2, 34.1, 27.9, 26.0, 23.5; IR [CH2Cl2, nmax cm21] 3313,
3043, 2977, 1755, 1598, 1496, 1372, 1241; MS 2EI) m/z 123
212.85), 85 214.77), 71 215.28), 59 227.82), 57 2100.00), 42
239.27); HRMS 2EI) m/z C16H24O6 312.155946, found
312.156532.
To a solution of the epoxide 8a or 8b 279.1 mg, 0.269 mmol,
0.05 M in MeOH) was added LiOH´H2O 20.0401 g,
0.957 mmol, 3.56 equiv.) at room temperature and the
resulting mixture was allowed to stir for 2 days. The reac-
tion was diluted with ethyl acetate and washed with aqueous
0.1N HCl. The aqueous layer was then extracted three times
with ethyl acetate. The combined organic layers were then
washed with brine, dried with sodium sulfate, and concen-
trated. Chromatography with silica gel 285:15 EtOAc/pet.
ether) furnished 2a or 2b.
2.7.2. Compond 9b. A yield .99% was deduced employing
DMF as an internal standard comparing the methine proton
signal at 2.60 ppm with the methyl proton signal at
2.6.1. F11334A2 ꢀ2a). A yield of 65% was deduced using
DMF as an internal comparing the methine proton signal at
1
4.50 ppm with the aldehyde proton at 7.96 ppm using H
1
2.99 ppm using H NMR. White solid. Mpˆ124±1268C;
1
NMR. White solid. Mpˆ137±1388C; H NMR [CD3OD,
1H NMR [CDCl3, 400 MHz] d 8.39 2bs, OH), 7.023 2d,
1H, Jˆ8.2 Hz), 6.76 2d, 1H, Jˆ2.4 Hz), 6.68 2dd, 1H,
J1ˆ8.2 Hz, J2ˆ2.4 Hz), 3.74 2bs, OH), 3.67 2dd, 1H,
J1ˆ10.3 Hz, J2ˆ1.6 Hz), 2.82 2dd, 1H, J1ˆ10.1 Hz,
J2ˆ14.6 Hz), 2.60 2dd, 1H, J1ˆ13.0 Hz, J2ˆ14.3 Hz),
1.70 2bs, OH), 1.56 2s, 9H), 1.31 2s, 3H), 1.29 2s, 3H); 13C
NMR [CDCl3, 100.6 MHz] d 153.7, 153.1, 144.3, 127.2,
400 MHz] d 6.62 2br s, 1H), 6.52 2br d, 1H, Jˆ8.5 Hz),
6.48 2dd, 1H, J1ˆ8.5 Hz, J2ˆ2.5 Hz), 4.50 2dd, 1H,
J1ˆ9.5 Hz, J2ˆ8.5 Hz), 3.12 2dd, J1ˆ15.5 Hz, J2ˆ
8.5 Hz) 3.06 2dd, 1H, J1ˆ15.5 Hz, J2ˆ9.5 Hz), 1.22 2s,
3H), 1.20 2s, 3H); 13C NMR [CD3OD, 100.6 MHz] d
155.0, 152.6, 129.7, 115.3, 113.5, 110.2, 90.7, 73.0, 32.5,

4564
C. C. Lindsey et al. / Tetrahedron 58 02002) 4559±4565
123.7, 121.0, 117.8, 83.8, 80.8, 73.3, 34.3, 28.0, 26.4, 23.4;
IR [CH2Cl2, nmax cm21] 3270, 3022, 2977, 2934, 1755,
1623, 1595, 1507, 1460; MS 2EI) m/z 124 210.97), 123
233.78), 59 225.44), 57 2100.00), 43 225.54); HRMS 2EI)
m/z calcd for C16H24O6 312.158063, found 312.157289.
1089; MS 2EI) m/z 238 212.90), 176 214.99), 161 2100),
123 288.38), 107 18), 77 223), 68 234); HRMS 2EI) m/z
mass calcd for C12H14O5 238.084124, found 238.084958.
2.8.4. Oxidation of hydroquinone and elimination of
carbonate. To a solution of 10 20.0946 g, 0.397 mmol,
0.1 M in methylene chloride) was added DDQ 20.0944 g,
0.416 mmol, 1.05 equiv.) at room temperature. After 10 min
the reaction was quenched with water, extracted three times
with methylene chloride, washed with brine, dried with
sodium sulfate, and concentrated. Chromatography with
silica gel 265:35 EtOAc/pet. ether) furnished a yellow
solid. A solution of the resulting quinone 20.01629 g,
0.06896 mmol, 0.01 M in benzene) was stirred with di-
isopropylethylamine 20.134 g, 1.03 mmol, 15.0 equiv.) at
re¯ux for 48 h. The reaction was quenched with 0.1N
HCl, extracted three times with ethyl acetate, washed with
brine once, dried with sodium sulfate, and concentrated.
Analytically pure sample could not be obtained; however
the corresponding NMR results are given. 1H NMR
[2CD3)2CO, 400 MHz] d 6.91±6.86 2m, 2H), 6.69 2d, 1H,
Jˆ8.6 Hz), 6.66 2m, 1H) 6.33 2d, 1H, Jˆ16.0 Hz), 1.34 2s,
6H).1f
2.8. Zincbromide cleavage of aryl O-t-butyl carbonate
ZnBr2 236.0 mg, 0.16 mmol, 5.0 equiv.) was added to a
solution of 9a or 9b 29.9 mg, 0.032 mmol, 0.05 M in nitro-
methane) and allowed to stir at room temperature
overnight. The reaction was ®ltered through a plug of
celite, diluted with ethyl acetate, washed with water and
brine,
dried
with
Na2SO4
and
concentrated.
Chromatography with silica gel 21:1 EtOAc/pet. ether)
furnished 1a or 1b.
2.8.1. F11334A1 ꢀ1a). The yield was computed to be .99%
by 1H NMR employing DMF as the internal standard
comparing the methine proton signal at 2.60 ppm with the
methyl proton signal at 2.99 ppm. Isolated yield, 57%. Red
1
solid. Mpˆ298C; H NMR [CD3OD, 400 MHz] d 6.62 2d,
1H, Jˆ3.0 Hz), 6.50 2dd, 1H, J1ˆ8.5 Hz, J2ˆ3.0 Hz), 6.63
2d, H, Jˆ8.5 Hz), 3.60 2dd, 1H, J1ˆ10.0 Hz, J2ˆ2.0 Hz),
2.80 2dd, 1H, J1ˆ13.5 Hz, J2ˆ2.0 Hz), 2.60 2dd, 1H, J1ˆ
13.5 Hz, J2ˆ10.0 Hz), 1.23 2s, 6H); 13C NMR [CD3OD,
100.6 MHz] d 151.8, 150.1, 129.6, 119.3, 117.7, 115.2,
81.2, 74.3, 34.8, 26.2, 25.4; IR [CH2Cl2, nmax cm21] 3577,
2971, 2927, 2854, 1701, 1497, 1271; MS 2EI) m/z 152
265.24), 124 226.08), 123 2100.00), 122 223.71), 59
296.71), 43 249.11); HRMS 2EI) m/z calcd for C11H16O4
212.104859, found 212.105328.1e
2.8.5. Reduction of quinone. To a solution of F11263 26)
20.0133 g, 0.0690 mmol, 0.1 M in acetone) was added
sodium hydrosul®te 20.241 g, 1.38 mmol, 20.0 equiv.,
0.5 M in water) and the mixture was allowed stir at room
temperature for 5 min. The reaction was quenched with
0.1 M HCl, extracted three time with ethyl acetate, washed
with brine, dried with sodium sulfate and concentrated.
Chromatography using silica gel 225:75) EtOAc/pet. ether
furnished F11334B1 as a yellow oil. The yield was
1
computed to be 53% by H NMR employing DMF as the
2.8.2. res-F11334A1 ꢀ1b). Isolated yield, 59%. Brown oil.
1H NMR [CD3OD, 400 MHz] d 6.88 2d, 1H, Jˆ8.1 Hz),
6.26 2d, 1H, Jˆ2.4 Hz), 6.22 2dd, 1H, J1ˆ8.1 Hz, J2ˆ
2.4 Hz), 3.35 2dd, 1H, J1ˆ11.4 Hz, J2ˆ2.0 Hz), 2.80 2dd,
1H, J1ˆ14.3 Hz, J2ˆ2.0 Hz), 2.50 2dd, 1H, J1ˆ14.3 Hz,
J2ˆ10.3 Hz), 1.22 2s, 6H); 13C NMR [CD3OD, 100.6
MHz] d 158.1, 157.7, 132.8, 119.3, 107.9, 104.1,
internal standard comparing the methine signal at 6.55 ppm
with the methine proton signal of the aldehyde at 7.96 ppm.
1H NMR [2CD3)2CO, 400 MHz] d 6.90 2d, 1H, Jˆ2.9 Hz),
6.88 2d, 1H, Jˆ16.0 Hz), 6.69 2d, 1H, Jˆ8.6 Hz), 6.55 2dd,
1H, J1ˆ8.4 Hz, J2ˆ2.9 Hz), 6.33 2d, 1H, Jˆ16 Hz), 1.34 2s,
6H); 13C NMR [CD3OD, 100.6 MHz] d 151.4, 149.2, 138.3,
126.4, 122.7, 117.7, 116.2, 113.5, 71.8, 30.1; IR [CH2Cl2,
nmax cm21] 3584, 3066, 2971, 2927, 2861, 1500, 1446,
1332, 1296, 1276, 1254; MS 2EI) m/z 176 217.81), 161
2100.0), 42 212.15); HRMS 2EI) m/z mass calcd for
C11H13O3 194.094294, found 194.095008.1e
81.0, 74.0, 33.7, 25.8, 25.2; IR [CH2Cl2, nmax cm21
]
3627, 2949, 2839, 1730, 1595, 1511, 1296; MS 2EI) m/z
212 29.43), 123 2100), 107 214.35), 59 241.75); HRMS
2EI) m/z mass calcd for C11H16O4 212.104859, found
212.105715.
2.8.3. Cyclic carbonate formation and OBOC cleavage.
A dry ¯ask charged with compound 9a 20.02 g, 0.06 mmol,
0.1 M in benzene) and the carbonyldiimidazole 20.05 g,
0.3 mmol, 5.0 equiv.) was stirred at re¯ux for 12 h. The
reaction was quenched with 0.1N HCl, extracted three
times with ethyl acetate, washed once with brine, dried
with sodium sulfate, and concentrated. Chromatography
with silica gel 225:75 EtOAc/pet. ether) furnished 10 as a
white solid. Isolated yield, 66%. Mpˆ159±1608C; 1H NMR
[CD3OD, 400 MHz] d 6.64±6.62 2m, 2H), 6.54 2dd, 1H,
J1ˆ8.6 Hz, J2ˆ2.9 Hz), 4.75 2dd, 1H, J1ˆ8.8 Hz, J2ˆ
4.9 Hz), 3.01 2dd, 1H, J1ˆ14.1 Hz, J2ˆ4.9 Hz), 2.83 2dd,
Acknowledgements
T. R. R. P. and C. C. L. are grateful for support from the
University of California Cancer Research Coordinating
Committee in the form of two awards 219990641) and
2SB010064) along with additional ®nancial support from
NSF 2CHE-9971211), NSF CAREER 20135031), PRF
2PRF34986-G1), NIH 2GM64831), the University wide
AIDS Initiative 2K00-SB-039) and Research Corporation
2R10296). We are also thankful for the support of
our M.S. facility funded in part by DAAD19-00-1-0026.
J. M. V. recognizes the support of PB98-0329-CO2-02,
1H, J1ˆ14.1 Hz, J2ˆ8.8 Hz), 1.47 2s, 3H), 1.41 2s, 3H); 13
C
NMR [CD3OD, 100.6 MHz] d 156.5, 151.3, 149.6, 124.4,
118.8, 116.9, 115.8, 86.3, 86.2, 31.4, 26.1, 21.4; IR [CH2Cl2,
nmax cm21] 3586, 3404, 3091, 3036, 2962, 2928, 2856,
1793, 1605, 1506, 1449, 1378, 1269, 1261, 1176, 1123,
Â
1FD97-0457-C02-02 2Spanish Ministerio de Educacion y
Cultura) CVI-276 2Junta de Andalucõa) and C. G. D.
acknowledges stipend funding from 1FD97-0457-C02-02.
Â

C. C. Lindsey et al. / Tetrahedron 58 02002) 4559±4565
4565
96, 1438±1442. 2c) Sloand, E. M.; Young, N. S.; Kumar, P.;
Weichold, F. F.; Sato, T.; Maciejewski, J. P. Blood 1997, 89,
1357±1363.
References
1. For examples of N-SMase inhibitors, see: 2a) Tanaka, M.;
Nara, F.; Suzuki-Konagai, K.; Hosoya, T.; Ogita, T. J. Am.
Chem. Soc. 1997, 119, 7871±7872. 2b) Arenz, C.; Giannis, A.
Angew. Chem., Int. Ed. 2000, 39, 1440±1442. 2c) Uchida, R.;
Tomoda, H.; Dong, Y.; Omura, S. J. Antibiot. 1999, 52, 572±
574. 2d) Hakogi, T.; Monden, Y.; Iwama, S.; Katsumura, S.
Org. Lett. 2000, 17, 2627±2629. 2e) Tanaka, M.; Nara, F.;
Yamasato, Y.; Ono, Y.; Ogita, T. J. Antibiot. 1999, 52,
827±830. 2f) Ogura, Y.; Nara, F.; Hosoya, T. Jpn. Kokai
Tokkyo Koho, JP08053387 A2 19960227, 1996. 2g) Arenz,
C.; Giannis, A. Eur. J. Org. Chem. 2001, 1, 137±140.
2h) Arenz, C.; Thutewohl, M.; Block, O.; Waldmann, H.;
Altenbach, H.-J.; Giannis, A. ChemBioChem 2001, 2, 141±
143. 2i) Arenz, C.; Giannis, A. Angew. Chem., Int. Ed. 2000,
39, 1440±1442.
10. Ghafourifar, P.; Klein, S. D.; Schucht, O.; Schenk, U.;
Pruschy, M.; Rocha, S.; Richter, C. J. Biol. Chem. 1999,
274, 6080±6084.
11. De Simone, C.; Grazia, C. M.; Roncaioli, P.; Moretti, S.;
Famularo, G.; Alesse, E. Immunol. Today 1996, 17, 48.
12. 2a) Claus, R.; Russwurm, S.; Meisner, M.; Kinscherf, R.;
Deigner, H. P. Curr. Drug Targets 2000, 1, 185±205.
2b) Moretti, S.; Alesse, E.; Di Marzio, L.; Zazzeroni, F.;
Ruggeri, B.; Marcellini, S.; Famularo, G.; Steinberg, S. M.;
Boschini, A.; Cifone, M. G.; De Simone, C. Blood 1998, 91,
3817±3824.
13. 2a) Jones, R. M.; Van De Water, R. W.; Lindsey, C. C.;
Hoarau, C.; Ung, T.; Pettus, T. R. R. J. Org. Chem. 2001,
66, 3435±3441. 2b) Van De Water, R. W.; Magdziak, D. J.;
Chau, J. N.; Pettus, T. R. R. J. Am. Chem. Soc. 2000, 122,
6502±6503.
2. 2a) Hofmeister, R.; Wiegmann, K.; Korherr, C.; Bernardo, K.;
Kronke, M.; Falk, W. J. Biol. Chem. 1997, 272, 27730±27736.
2b) Schwandner, R.; Wiegmann, K.; Bernardo, K.; Kreder, D.;
Kronke, M. J. Biol. Chem. 1998, 273, 5916±5922.
3. 2a) Nalivaeva, N. N.; Rybakina, E. G.; Pivanovich, I. Y.;
Kozinets, I. A.; Shanin, S. N.; Bartfai, T. Cytokine 2000, 12,
229±232. 2b) Adam-Klages, S.; Adam, D.; Wiegmann, K.;
Struve, S.; Kolanus, W.; Schneider-Mergener, J.; Kroenke,
M. Cell 1996, 86, 937±947. 2c) Adam, D.; Wiegmann, K.;
Adam-Klages, S.; Ruff, A.; Kroenke, M. J. Biol. Chem.
1996, 271, 14617±14622.
14. Wang, Z.-X.; Tu, T.; Frohn, M.; Zhang, J.-R.; Shi, Y. J. Am.
Chem. Soc. 1997, 119, 11224±11235.
15. 2a) De Bernardi, M.; Vidari, G.; Finzi, P. V. Tetrahedron
1992, 48, 7331±7344. 2b) Capon, B.; Thomson, J. W.
J. Chem. Soc., Perkin Trans. 2 1977 27), 917±918.
16. Kutney, J. P.; Rateliffe, A. H. Synth. Commun. 1975, 5, 47±52.
17. 2a) Fernandez-Ayala, D. J. M.; Martin, S. F.; Barroso, M. P.;
Gomez-Diaz, C.; Villalba, J. M.; Rodriguez-Aguilera, J. C.;
Lopez-Lluch, G.; Navas, P. Antioxid. Redox. Signal. 2000, 2
22), 263±275. 2b) Lopez-Lluch, G.; Barroso, M. P.; Martin,
S. F.; Fernandez-Ayala, D. J. M.; Gomez-Diaz, C.; Villalba,
J. M.; Navas, P. Biofactors 1999, 9 22±4), 171±177.
18. Nara, F.; Tanaka, M.; Hosoya, T.; Suzuki-Konagai, K.; Ogita,
T. J. Antibiot. 1999, 52, 525±530.
4. Saikumar, P.; Dong, Z.; Mikhailov, V.; Denton, M.;
Weinberg, J. M.; Venkatachalam, M. A. Am. J. Med. 1999,
107, 489±506.
5. Amtmann, E.; Zoller, M.; Schilling, G. Drugs Exp. Clin. Res.
2000, 26, 57±65.
19. Martin, S. F.; Navarro, F.; Forthoffer, N.; Navas, P.; Villalba,
J. M. J. Bioenerg. Biomembr. 2001, 33, 143±153.
20. 2a) Sawai, H.; Domae, N.; Nagan, N.; Hannun, Y. A. J. Biol.
Chem. 1999, 274, 38131±38139. 2b) Tepper, A. D.; Ruurs, P.;
Borst, J.; van Blitterswijk, W. J. BBRC 2001, 280, 634.
21. Liu, B.; Hannun, Y. A. J. Biol. Chem. 1997, 272, 16281±
16287.
6. Yu, Z. F.; Nikolova-Karakashian, M.; Zhou, D.; Cheng, G.;
Schuchman, E. H.; Mattson, M. P. J. Mol. Neurosci. 2000, 15,
85±97.
7. Hernandez, O. M.; Discher, D. J.; Bishopric, N. H.; Webster,
K. A. Circ. Res. 2000, 86, 198±204.
8. 2a) Gougeon, M.-L. Handb. Exp. Pharmacol. 2000, 142, 127±
150. Apoptosis and its modulation by drugs. 2b) Famularo, G.;
De Simone, C.; Marcellini, S. Med. Hyp. 1997, 48, 423±429.
2c) Gougeon, M. L.; Montagnier, L. Ann. N.Y. Acad. Sci. 1999,
887, 199±212.
22. Liu, B.; Hassler, D. F.; Smith, G. K.; Weaver, K.; Hannun,
Y. A. J. Biol. Chem. 1998, 273, 34472±34479.
23. Navarro, F.; Navas, P.; Burgess, J. R.; Bello, R. I.; de Cabo,
R.; Arroyo, A.; Villalba, J. M. FASEB J. 1998, 1665±1673.
24. Navas, P.; Mowack, D. D.; Morre, D. J. Cancer Res. 1989, 49,
2147±2156.
9. 2a) Bartz, S. R.; Emerman, M. J. Virol. 1999, 73, 1956±1963.
2b) Ullrich, C. K.; Groopman, J. E.; Ganju, R. K. Blood 2000,