Synthesis and comparison of the antiinflammatory activity of manoalide and cacospongionolide B analogues

Article abstract of DOI:10.1021/jm980027h

We have synthesized analogues of two naturally occurring antiinflammatory marine compounds, manoalide and cacospongionolide B, containing a pyranofuranone moiety which is considered the pharmacophoric group. The two compounds, and hence their analogues, d

Full text of DOI:10.1021/jm980027h

3232
J . Med. Chem. 1998, 41, 3232-3238
Syn th esis a n d Com p a r ison of th e An tiin fla m m a tor y Activity of Ma n oa lid e a n d
Ca cosp on gion olid e B An a logu es
Margherita De Rosa,† Silvana Giordano,† Arrigo Scettri,† Guido Sodano,*,† Annunziata Soriente,†
Pablo Garc´ıa Pastor,‡ Maria J . Alcaraz,‡ and Miguel Paya´*,‡
Dipartimento di Chimica, Universita` di Salerno, Via S. Allende, 84081 Baronissi (SA), Italy, and Department of
Pharmacology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andre´s Estelle´s s/ n, 46100 Burjassot, Valencia, Spain
Received J anuary 15, 1998
We have synthesized analogues of two naturally occurring antiinflammatory marine compounds,
manoalide and cacospongionolide B, containing a pyranofuranone moiety which is considered
the pharmacophoric group. The two compounds, and hence their analogues, differ in the
presence or absence in the dihydropyran ring of an hemiacetal function which was considered
essential to irreversibly inactivate phospholipase A2 (PLA2). The two series of compounds were
tested for their inhibitory effects on secretory PLA2 belonging to the groups I, II, and III, and
the activities were found to be similar in both series, irrespective of the presence or absence of
the additional hemiacetal function. In addition, the PLA2 inhibitory activity increases with
the increasing hydrophobic character of the side chain linked to the pyranofuranone moiety.
The most active compounds, FCA and FMA, carry a farnesyl residue linked to the pyrano-
furanone substructure. The most potent PLA2 inhibitor, FMA, was tested in the mouse
carrageenan paw edema at the oral dose of 10 mg/kg and showed an activity similar to the
reference antiinflammatory drug, indomethacin.
In tr od u ction
structure-activity relationship studies.4-10 All of the
models propose that the rings of the pyranofuranone
moiety open to generate R,â-unsaturated carbonyl com-
pounds which could react in different ways with lysine
residues of PLA2. In particular, it was proposed that
the aldehyde group arising from the hemiacetal function
in the dihydropyran ring was essential to irreversibly
inactivate PLA2.5,7,11,12
On the other hand, cacospongionolide B (2), which
lacks the hemiacetal function in the pyran ring, showed
potent inhibitory activity on recombinant human syn-
ovial PLA2 in vitro similar to that of manoalide, while
a lower inhibitory activity was shown on other secretory
enzymes such as bee venom PLA2 and the enzyme
present in exudates from zymosan-injected rat air
pouch.13,14
Marine invertebrates have proved to be a valuable
source of antiinflammatory compounds which are potent
inhibitors of phospholipase A2 (PLA2).1
Among these new antiinflammatory compounds, a
class of biologically active sesterterpenes containing a
pyranofuranone substructure has been isolated from soft
sponges. These sesterterpenes can be grouped in two
subclasses, exemplified by manoalide2 (1) and cacospon-
gionolide B3 (2), differing from each other by the
We report here the synthesis of manoalide and
cacospongionolide B analogues and the comparison of
the PLA2 inhibitory activity of both series in order to
obtain additional information on structure-activity
relationship, particularly (i) on the role of the hemiac-
etal function of the pyran ring and (ii) on the influence
of the hydrophobic side chain in both series of com-
pounds.
Ch em istr y
substitution in the dihydropyran ring.
We have recently reported15 a new synthetic approach
to pyranofuranones, which are considered the poten-
tially pharmacophoric groups of these sesterterpenes.
The procedure has been used for the synthesis of the
methyl analogues of both manoalide and cacospongiono-
lide B (MMA, 11a and MCA, 12a ).
Since the synthetic sequence involves an alkylation
step in which a side chain could be linked to the
pyranofuranone moiety, the procedure is amenable to
the synthesis of many analogues and to the synthesis
Manoalide (1), which is by far the most studied
compound, has been shown to irreversibly inhibit PLA2
with the corresponding modification of a specific number
of its lysine residues. Although the mechanism of
inactivation has not been elucidated yet, differents
models have been proposed mainly on the basis of
* Authors to whom correspondence should be addressed.
† University of Salerno.
‡ University of Valencia.
S0022-2623(98)00027-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/21/1998

Manoalide and Cacospongionolide B Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3233
Sch em e 1
of manoalide and cacospongionolide B. The synthetic
sequence, for which we disclose here full experimental
details, is reported in Scheme 1.
belonging to the manoalide class of compounds for which
no biological data have been reported.
As far as the synthetic sequence is concerned, it
should be noted that the synthetic steps between
compounds 5 and 8 were generally performed without
isolation of the intermediates 6 and 7, except for the
isolation of 6d in the course of the synthesis of FMA
(11d ) and FCA (12d ). In addition, compounds 9 and
9a -d were unstable. In particular 9 largely decom-
posed even if stored at -20 °C overnight, and therefore,
it was reacted in the subsequent step immediately after
its preparation.
The analogues were prepared by following the previ-
ously outlined procedure,15 varying the alkyl halide in
the alkylation step of the intermediate 5 (Scheme 1).
When 5 was subjected to the subsequent reaction
sequence without any alkylation step, the unsubstituted
analogues 11 (MA ) manoalide analogue) and 12 (CA
) cacospongionolide B analogue) were obtained in 8.5%
and 5% total yields. Conversely, the manoalide ana-
logues 11a -d and the cacospongionolide B analogues
P h a r m a cologica l Resu lts
A. En zym e In h ibitor y Activity. The two series of
compounds were tested for their inhibitory effects on
secretory PLA2 belonging to the groups I (Naja naja
venom and porcine pancreatic enzymes), II (human
synovial recombinant enzyme), and III (bee venom
enzyme). The results are reported in Table 1. The
farnesyl analogues FMA (11d ) and FCA (12d ) were the
most effective compounds and inhibited preferentially
bee venom and human synovial PLA2. The IC50 of FCA
was in the µM range, similar to that of manoalide,
whereas FMA showed a higher potency.
Figures 1 and 2 illustrate the influence of FCA and
FMA on the velocity of the enzymatic reaction as a
function of enzyme concentration for bee venom PLA2.
The results were similar with both compounds. The
regression line for FCA- or FMA-treated samples was
12a -d were obtained in a 9.7, 3.2, 5.0, and 2.1% and
15.4, 1.2, 3.0, and 1.4% total yields, respectively, by
using methyl iodide, crotyl bromide, benzyl bromide, and
farnesyl bromide in the alkylation step. The selection
of the readily available alkyl halides was dictated by
the consideration of their high reactivity coupled with
the need of alkyl chains of variable hydrophobicity. In
particular, farnesyl bromide was selected because it
allowed the preparation of the farnesyl manoalide
analogue (FMA, 11d ) which is the lower homologue of
thorectolide (13),16 a naturally occurring sesterterpene

3234 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Ta ble 1. Effect of Compounds on Secretory PLA2 Activitiesa
De Rosa et al.
human synovial
bee venom
% I (10 µM) IC50(µM)
N. naja venom
% I (10 µM)
pancreas
% I (10 µM)
% I (10 µM) IC50(µM)
MA (11)
CA (12)
5.2 ( 2.0
10.1 ( 3.0
3.0 ( 1.5
1.4 ( 0.7
1.8 ( 1.7
7.1 ( 3.2
7.1 ( 1.3
7.6 ( 2.7
33.6 ( 2.2c
17.7 ( 0.7b
17.0 ( 1.7b
1.0 ( 1.0
0.2 ( 0.2
18.9 ( 3.1b
42.3 ( 2.2c
3.2 ( 1.9
NDd
0.7 ( 0.4
9.3 ( 2.6
ND
ND
ND
ND
ND
ND
ND
ND
0.9
ND
ND
ND
ND
ND
ND
ND
0.5
MMA (11a )
MCA (12a )
CMA(11b)
CCA (12b)
BMA (11c)
BCA (12c)
FMA (11d )
FCA (12d )
manoalide
6.8 ( 1.6
14.9 ( 2.8
8.0 ( 2.2
5.6 ( 1.3
0.1 ( 0.0
15.1 ( 2.7
17.6 ( 1.9
16.1 ( 2.2
17.4 ( 2.1
39.8 ( 1.0c
30.0 ( 2.0c
32.3 ( 2.7c
21.2 ( 5.7
24.8 ( 4.5
11.9 ( 3.7
15.5 ( 1.4
74.6 ( 0.9c
63.1 ( 1.0c
93.2 ( 0.2c
24.8 ( 5.8
40.1 ( 7.3c
16.1 ( 7.5
30.3 ( 3.6
84.2 ( 2.4c
71.8 ( 3.6c
62.5 ( 3.8c
4.2
3.9
1.7
7.5
a
b
d
Means ( SEM (n ) 6). P < 0.05. c P < 0.01. ND ) not determined for those compounds which do not reach 50% inhibition at
10 µM.
Ta ble 2. Inhibition of Bee Venom PLA2 by FCA, FMA, Manoalide, and Cacospongionolide B over a Range of Preincubation Times
and Irreversibility of Inhibitiona,d
% inhibition for postincubation treatment
preincubation
time, min
% irreversibility
of inhibition
compound
buffer
hydroxylamine
FCA
FCA
FCA
5
15
60
5
15
60
5
15
60
5
15
60
58.5 ( 1.5
58.7 ( 0.5
65.3 ( 0.3
95.4 ( 2.5
95.4 ( 2.5
98.4 ( 1.3
80.8 ( 2.5
80.9 ( 1.9
75.2 ( 1.2
42.4 ( 1.3
44.2 ( 2.4
47.3 ( 2.0
50.1 ( 2.1
51.1 ( 2.5
65.1 ( 0.3
81.9 ( 1.8
89.4 ( 2.8
99.3 ( 0.4
39.1 ( 2.2
56.9 ( 1.7
71.5 ( 1.3
21.0 ( 3.2
28.0 ( 0.9
43.1 ( 1.8
85.6b
87.1
99.7
85.8
93.7
cacospongionolide B
cacospongionolide B
cacospongionolide B
FMA
FMA
FMA
manoalide
manoalide
manoalide
100.0
48.4c
70.3c
95.1
49.5c
63.3c
91.1
a
b
d
Means ( SEM (n ) 6). P < 0.05 c P < 0.01. Final concentration of FCA, FMA, manoalide, and cacospongionolide B at 10, 5, 10,
and 50 µM, respectively.
F igu r e 2. Effect of FMA (0.5 µM)) on bee venom PLA2 activity
as a function of enzyme concentration. Results show means (
SEM for n ) 6: Squares (enzyme control); circles (enzyme +
0.5 µM FMA).
F igu r e 1. Effect of FCA (1 µM) on bee venom PLA2 activity
as a function of enzyme concentration. Results show means (
SEM for n ) 6: squares (enzyme control); triangles (enzyme
+ 1 µM FCA). Data for Figures 1 and 2 have been obtained
using different pools of substrate.
this effect was not evidenced after 1 h of incubation of
drug-treated enzyme solutions.
shifted to the right of control values at a given velocity,
without a significant difference in the slopes, which
suggests an irreversible inhibition17 as has been re-
ported for manoalide.18 Moreover, the reversibility of
enzyme inhibition after treatment with hydroxylamine9
was also tested. Table 2 shows the inhibition profile of
bee venom PLA2, preincubated with FCA and FMA and
with manoalide and cacospongionolide B, after treat-
ment with hydroxylamine. It can be observed that only
in the early stages is there a partial recovery of enzyme
activity, the reversibility being more effective for
manoalide and its analogue FMA. On the other hand,
None of the compounds tested inhibited cytosolic PLA2
activity from the human monocytic cell line U937 in a
significant way (data not shown). Thus, the active
compounds FCA and FMA showed a selectivity for
secretory PLA2.
B. An tiin fla m m a tor y Activity. The most potent
PLA2 inhibitor, FMA (11d ), was tested in the mouse
carrageenan paw edema at the oral dose of 10 mg/kg.
An inhibitory effect on paw edema was observed 3 and
5 h after carrageenan administration, with percentages
of inhibition of 42.9 ( 6.6 (P < 0.01) and 43.2 ( 7.5 (P

Manoalide and Cacospongionolide B Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3235
(δ) scale; for the spectra in CDCl3, the CHCl3 signal was used
< 0.01), respectively. At the same dose, the reference
antiinflammatory drug, indomethacin, inhibited edema
by 40.0 ( 5.8% 3 h after carrageenan (P < 0.01) and by
42.3 ( 4.1% at the 5 h determination (P<0.01).
1
as internal standard (δ 7.26 H, δ 77.0 13C); for the spectra in
CD3OD, the MeOH signal was used as internal standard (δ
1
3.34 H, δ 49.0 13C). J values are given in Hz. Mass spectra
were taken on a VG TRIO 2000 instrument. The molecular
ion peak (M+) is reported; when the molecular ion peak is
undetectable, the first fragmentation ion peak is listed. All
of the analogues reported here show an abundant M+ - 1 ion.
Column chromatographic separations were carried out using
Silica gel 60 (70-230 mesh and 230-400 mesh, Merck).
Hexane was dried by distillation from P2O5; i-Pr2NH from
CaH2; Et2O, CH2Cl2, and toluene were freshly distilled from
CaH2 under an argon atmosphere from CaH2. THF was
distilled under argon and then from sodium benzophenone.
Syn th esis of F a r n esyl An a logu es F MA (11d ) a n d F CA
(12d ). 1,3-Bis(tr im eth ylsiloxy)-1-m eth oxybu ta -1,3-d ien e
(3).19 To a solution of diisopropylamine (0.9 mL, 6.39 mmol)
in dry THF (22 mL) under argon was added at 0 °C 4.3 mL of
n-BuLi (1.6 M in hexane). The reaction mixture was cooled
to -78 °C, and methyl-3-trimethylsiloxybut-2-enoate (1.06 mL,
5.31 mmol) was added. The solution was stirred for 3 min
and then quenched with trimethylchlorosilane (1.08 mL, 8.55
mmol). After 10 min, the solvent was removed under reduced
pressure, and the residue was washed and filtered with cold
dry hexane. The hexane was removed from the filtrate in
vacuo to yield 1.24 g of 3 (90% yield).
Discu ssion
Our results confirm that the pyranofuranone part
interacts with PLA2 enzymes and that the hydrophobic
region of the molecule facilitates this interaction as
previously noted.8 Nevertheless, this moiety of the
molecule can be either linear, as in FMA (11d ) and FCA
(12d ), or include a cyclic part (manoalide and cacospon-
gionolide B), but a certain degree of lipophilicity is
necessary since analogues with a shorter lateral chain
present a drastic reduction in activity.
Several reports on the structure-activity relationship
of manoalide indicate that the most likely initial reac-
tion between manoalide and PLA2 is the formation of a
Schiff base between a lysine residue and the aldehyde
generated upon opening of the manoalide γ-lactone
ring.8-10 Hydroxylamine methodology9 has been used
to cleave Schiff bases, resulting in a partial recovery of
the enzyme activity. In this respect, it should be noted
that the presence of the second masked aldehyde group
in the pyran ring was judged important for the complete
inactivation of PLA2 and for the irreversibility of the
biological effect.5,9 However, these conclusions were
reached by comparing the bioactivity of manoalide with
that of analogues in which the dihydropyran ring was
opened, the nature of the interaction of the pyran ring
with PLA2 remaining undefined.9 From our results
(Table 2), it can be deduced that the recovery of enzyme
activity after hydroxylamine treatment appears only in
the early stages and is more prominent for those
compounds which have two masked aldehyde groups
such as manoalide and FMA. The differences observed
within the values of the percentage of reversal inhibition
with those previously reported for manoalide9 can be
strictly attributed to the experimental conditions used
in the hydroxylamine methodology.
The comparison reported here (Table 1) of the activi-
ties of analogues having the same side chain and
differing only in the presence or absence of the hemi-
acetal function in the dihydropyran ring shows that the
hemiacetal function is not essential for PLA2 inhibition
nor for the irreversibilitry of enzyme inhibition. It may
eventually increase the inhibitory potency as in the case
of FCA and FMA. The comparison of the PLA2 inhibi-
tory profile of these analogues shows a certain selectiv-
ity for secretory PLA2 enzymes belonging to groups II
and III (CA, CCA, FMA and FCA). The interaction of
the most active compounds (FCA and FMA) with the
enzymatic protein appears to be irreversible.
1H NMR (CDCl3) δ 4.48 (s, 1H), 4.15 (d, J ) 1.4 Hz, 1H),
3.93 (d, J ) 1.4 Hz, 1H), 3.55 (s, 3H), 0.21 (s, 9H), 0.23 (s,
9H).
5-F u r a n -3-yl-5-h yd r oxy-3-oxo-p en ta n oic Acid Meth yl
Ester (5). To a solution of 3 (2.5 g, 10 mmol) in 58 mL of dry
Et2O under argon at -78 °C were added 3-furylaldehyde (1.44
g, 15 mmol) and BF3Et2O (1.42 g, 10 mmol). The reaction
mixture was stirred for 1 h in the dark, saturated NaHCO3
was slowly added, and the mixture was allowed to warm to
room temperature and extracted with Et2O. The extract was
dried (MgSO4) and concentrated to give an oil, which was
column chromatographed (CHCl3/Et2O, 96:4) to give 5 (1.7 g,
82%) as a pale yellow oil.
1H NMR (CDCl3) δ 7.32 (s, 2H) and 6.33 (s, 1H) (furan
protons), 5.07 (dd, J ) 3.9, 8.3 Hz, 1H, H-5), 3.66 (s, 3H, OCH3),
3.46 (s, 2H, H-2), 2.96 (dd, J ) 17.1, 8.3 Hz, 1H, H-4a), 2.83
(dd, J ) 17.4, 3.9 Hz, 1H, H-4b).
13C NMR (CDCl3) δ 202.5, 167.2, 143.4, 139.0, 127.2, 108.3,
62.8, 52.5, 50.2, 49.5. Anal. C, H.
2-(3-F u r a n -3-yl-3-h yd r oxy-p r op ion yl)-5,9,13-tr im eth yl-
tetr a d eca -4,8,12-tr ien oic Acid Meth yl Ester (6d ). A mix-
ture of 5 (0.71 g, 3.34 mmol), anhydrous THF (3 mL), and
commercial LiOH monohydrate (0.14 g, 3.34 mmol) was
stirred, in
a round-bottom flask equipped with a reflux
condenser, at about 55 °C for 15 min.20 After the reaction
mixture was cooled to 40 °C, farnesyl bromide (0.71 g, 5.01
mmol) was added, and the resulting mixture was stirred for 3
h at 40 °C. The reaction mixture was allowed to cool to room
temperature, HCl 0.1 N was added until neutrality, and the
mixture was extracted with Et2O. The organic layer was dried
over anhydrous Na2SO4 and evaporated under vacuum. Col-
umn chromatography (CHCl3/Et2O, 98:2) gave 529 mg of 6d
(70%) as a diastereomeric mixture (13C NMR analysis).
1H NMR (CDCl3) δ 7.39 (s, 2H) and 6.37 (s, 1H) (furan
protons), 5.14-5.02 (m, 4H, H-4, H-8, H-12, overlapped with
the carbinol proton), 3.71 (s, 3H, OCH3), 3.51 (t, J ) 7.4 Hz,
1H, H-2), 2.97 (dd, J ) 8.8, 15.7 Hz, 1H, H-2a propionyl
moiety), 2.90 (dd, J ) 3.4, 15.7 Hz, 1H, H-2b propionyl moiety),
2.5 (t, J ) 7.3 Hz, 2H, H-3), 2.01 (m, 8H, H-6, H-7, H-10, H-11),
1.70 (s, 3H, CH3), 1.65 (s, 3H, CH3), 1.57 (s, 6H, 2 × CH3).
13C NMR (CDCl3) δ 205.2, 169.5, 143.3, 139.0, 138.9, 135.2,
131.3, 127.2, 124.2, 123.7, 119.2, 108.4, 62.9 and 62.8 (1:1,
diastereomeric carbinol carbons), 59.3, 52.4, 49.4, 39.6, 26.8,
26.75, 26.67, 26.4, 25.6, 17.6, 16.1, 15.9. Anal. C, H.
Interestingly, the antiinflammatory activity of FMA
after oral administration to mice is similar to that of
indomethacin, suggesting that this class of compounds
could be a model for the development of new antiin-
flammatory agents.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Optical rotations were
measured at the sodium D line (589 nm) at room temperature
with a J ASCO DIP 1000 polarimeter. NMR spectra were
recorded on a Bruker AM 250 (250.13 MHz for 1H and 62.89
MHz for 13C) spectrometer. Chemicals shifts are given in ppm
6-F u r a n -3-yl-3-(3,7,11-tr im eth yl-d od eca -2,6,10-tr ien yl)-
5,6-d ih yd r o-p yr a n -2-on e (8d ). A solution of Zn(BH4) in
2
Et2O (56 mL, 0.2 M) was added to a solution of 6d (1.14 g, 2.7
mmol) in anhydrous Et2O (40 mL), under N2 at 0 °C.21 After

3236 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
De Rosa et al.
the reaction mixture was stirred for 1 h at 0 °C, water was
added, and stirring was continued for 30 min. The mixture
was acidified with 0.1 N HCl and extracted with Et2O. The
ethereal extract was dried (Na2SO4) and, after evaporation of
the solvent, the reduction product was dissolved in 0.1 N
NaOH (150 mL) and stirred for 3 h at 40 °C. The solution
was then allowed to cool to room temperature and acidified
with 2 N H2SO4. Extraction with Et2O, drying of the ethereal
extract over anhydrous Na2SO4, and evaporation of the solvent
afforded the crude hydroxyacid 7d (862 mg).
To a solution of 7d (862 mg) in CH2Cl2 (3 mL) were added,
acetic anhydride (1.1 g) and pyridine (1.7 mL), and the solution
was stirred overnight at room temperature. The reaction
mixture was then diluted with Et2O (10 mL), and 1 N HCl
(10 mL) was added. The ethereal layer was washed with
saturated NaHCO3 and brine. Drying (Na2SO4) and evapora-
tion gave crude acetylated lactone (759 mg) which was dis-
solved in CHCl3 (5 mL), and DBU (three drops) was added.
The solution was allowed to stand at room temperature for 2
h and then acidified with 2 N HCl and extracted with Et2O.
The ethereal layer was washed with H2O and dried (Na2SO4).
The solvent was removed under vacuum, and the product was
chromatographed (SiO2, CHCl3) to afford 8d (248 mg, 0.674
mmol, 35.7% from 6d ).
of dodecatrienyl moiety), 4.50 (dd, J ) 4.1, 9.6 Hz, 1H, H-2),
4.19 (d, J ) 15.6 Hz, 1H, H-6a), 4.10 (d, J ) 15.6 Hz, 1H, H-6b),
2.63 (d, J ) 7.1 Hz, 2H, H-1 of dodecatrienyl moiety), 2.30 (m,
2H, H-3), 2.07 (m, 8H, H-4, H-5, H-8, H-9 of dodecatrienyl
moiety), 1.68 (s, 3H, CH3), 1.60 (s, 9H, 3 × CH3).
13C NMR (CDCl3) δ 143.3, 139.5, 124.6, 124.3, 120.9, 117.8,
68.9, 68.5, 39.9, 31.8, 31.6, 27.0, 26.7, 25.9, 18.0, 16.2. Anal.
C, H.
5-H yd r oxy-4-{6-h yd r oxy-5-(3,7,11-t r im et h yl-d od eca -
2,6,10-t r ien yl)-3,6-d ih yd r o-2H -p yr a n -2-yl}-5H -fu r a n -2-
on e (F MA, 11d ). To a solution of 9d (22 mg, 0.064 mmol) in
dry CH2Cl2 (6 mL) was added N,N-diisopropylethylamine (49
µL), and oxygen gas was bubbled through the solution for 10
min.23 Thereafter, polymer-bound rose bengal catalyst (3.3 mg,
15% by weight) was added. The solution was stirred at -78
°C under an atmosphere of oxygen and irradiated with a 500-W
tungsten incandescent lamp for 6 h. The reaction mixture was
allowed to warm to room temperature, the photosensitizer was
removed by filtration, and the CH2Cl2 solution was washed
with aqueous NaH2PO4 buffer (1 M, pH ) 4.3). The organic
extracts were dried over anhydrous Na2SO4, and the solvent
was evaporated in vacuo. The reaction mixture was purified
by chromatography (silica gel, CHCl3) to obtain the γ-hydroxy-
butenolide FMA (11d , 9 mg, 38% yield).
1H NMR (CDCl3) δ 7.47, 7.40, and 6.44 (s, 1H each, furan
protons), 6.57 (m, 1H, H-4), 5.36 (dd, J ) 4.9, 10.8 Hz, 1H,
H-6), 5.20-5.05 (m, 3H, H-2, H-6, H-10 of dodecatrienyl
moiety), 3.02 (bs, 2H, H-1 of dodecatrienyl moiety), 2.59 (m,
2H, H-5), 2.07 (m, 8H, H-4, H-5, H-8, H-9 of dodecatrienyl
moiety), 1.66 (s, 3H, CH3 ), 1.61(s, 3H, CH3), 1.59 (s, 6H, 2 ×
CH3). Anal. C, H.
6-F u r a n -3-yl-3-(3,7,11-tr im eth yl-d od eca -2,6,10-tr ien yl)-
5,6-d ih yd r o-2H-p yr a n -2-ol (9d ). To a toluene solution (0.3
M) of unsaturated lactone 8d (248 mg, 0.674 mmol) cooled to
-78 °C was added DIBAL (1 M in CH2Cl2, 0.7 mL, 0.674 mmol)
dropwise.22 The solution was stirred until TLC analysis judged
the reaction to be complete. The mixture was then poured
into a rapidly stirred mixture of ice (2.2 g) and acetic acid (0.7
mL), CHCl3 (5.0 mL) was added, and the mixture was stirred
vigorously for 10 min. After that time, another portion (9 mL)
of CHCl3 was added, and vigorous stirring continued until two
distinct layers formed when the stirring was stopped (40 min).
The mixture was extracted with CHCl3, and the organic layer
was washed with bicarbonate and brine and dried over
anhydrous Na2SO4. After the solvent was removed under
reduced pressure, the residue was purified by column chro-
matography (SiO2). Elution with CHCl3 afforded 102.2 mg
(0.28 mmol) of 9d (41%).
1H NMR (CDCl3) δ 6.14 (s, 1H, H-5), 6.07 (s, 1H, H-3), 5.68
(m, 1H, H-4 of pyran ring), 5.29 (s, 1H, H-6 of pyran ring),
5.19-5.10 (m, 3H, H-2, H-6, H-10 of dodecatrienyl moiety),
4.87 (dd, J ) 4.1, 9.8 Hz, 1H, H-2 of pyran ring), 2.79 (d, J )
7.1 Hz, 2H, H-1 of dodecatrienyl moiety), 2.25 (m, 2H, H-3 of
pyran ring), 2.09-2.02 (m, 8H, H-4, H-5, H-8, H-9 of dodec-
atrienyl moiety), 1.70-1.61 (s, 12H, 4 × CH3).
13C NMR (CDCl3) δ pyranofuranone moiety 170.2 (C-2 of
γ-hydroxybutenolide ring), 168.4 (C-4 of γ-hydroxybutenolide
ring), 137.7 (C-5 of pyran ring), 120.6 (C-4 of pyran ring), 116.9
(C-3 of γ-hydroxybutenolide ring), 97.9 (C-5 of γ-hydroxy-
butenolide ring), 91.3 (C-6 of pyran ring), 62.9 (C-2 of pyran
ring), 29.1 (C-3 of pyran ring), farnesyl moiety 138.1, 135.1,
131.3, 124.3, 123.9, 119.9, 39.6, 31.0, 26.7, 26.5, 25.7, 18.1, 16.0.
MS m/z 384 (M+ - H2O). Anal. C, H.
5-Hydr oxy-4-{5-(3,7,11-tr im eth yl-dodeca-2,6,10-tr ien yl)-
3,6-d ih yd r o-2H-p yr a n -2-yl}-5H-fu r a n -2-on e (F CA, 12d ).
To a solution of 10d (11 mg, 0.034 mmol) in dry CH2Cl2 (2.7
mL) was added N,N-diisopropylethylamine (26 µL, 4.3 equiv),
and oxygen gas was bubbled through the solution for 10 min.23
Thereafter, polymer-bound rose bengal catalyst was added (1.6
mg, 15% by weight). The solution was stirred at -78 °C under
an atmosphere of oxygen and irradiated with a 500-watt
tungsten incandescent lamp for 6 h. The reaction mixture was
allowed to warm to room temperature, the photosensitizer was
removed by filtration, and the CH2Cl2 solution was washed
with aqueous NaH2PO4 buffer (1 M, pH ) 4.3). The organic
extracts were dried over anhydrous Na2SO4, and the solvent
was evaporated under a vacuum. The reaction mixture was
purified by chromatography (silica gel, CHCl3) to obtain the
γ-hydroxybutenolide FCA (12d 6.3 mg, 52% yield).
1H NMR (CDCl3) δ 7.47, 7.40 and 6.44 (s, 1H each, furan
protons), 5.70 (m, 1H, H-4), 5.29 (d, J ) 1.1 Hz, 1H, H-2), 5.22-
5.10 (m, 3H, H-2, H-6, H-10 of dodecatrienyl moiety), 4.97 (dd,
J ) 3.8, 10.9 Hz, 1H, H-6), 2.80 (d, J ) 7.2 Hz, 2H, H-1 of
dodecatrienyl moiety), 2.10 (m, 2H, H-5), 2.06 (m, 8H, H-4,
H-5, H-8, H-9 of dodecatrienyl moiety), 1.68 (s, 3H, CH3), 1.64
(s, 3H, CH3), 1.60 (s, 6H, 2 × CH3).
13C NMR (CDCl3) δ 143.2, 139.4, 137.7, 136.4, 135.2, 131.3,
126.5, 124.4, 124.0, 122.0, 120.6, 108.9, 91.8, 61.8, 39.7, 31.6,
31.3, 26.7, 26.5, 25.7, 17.7, 16.0. Anal. C, H.
1H NMR (CDCl3) δ 6.14 (s, 1H, H-5), 6.05 (s, 1H, H-3), 5.55
(m, 1H, H-4 of pyran ring), 5.11-5.09 (m, 3H, H-2, H-6, H-10
of dodecatrienyl moiety), 4.41 (bt, H-2 of pyran ring), 4.15 (m,
2H, H-6 of pyran ring), 2.63 (d, J ) 7.1 Hz, 2H, H-1 of
dodecatrienyl moiety), 2.28 (m, 2H, H-3 of pyran ring), 2.08-
1.97 (m, 8H, H-4, H-5, H-8, H-9 of dodecatrienyl moiety), 1.68-
1.60 (s, 12H, 4 × CH3).
2-F u r a n -3-yl-5-(3,7,11-tr im eth yl-d od eca -2,6,10-tr ien yl)-
3,6-d ih yd r o-2H-p yr a n (10d ). A dry methylene chloride (0.9
mL) solution of the crude lactol (47.2 mg, 0.14 mmol) and
triethylsilane22 (24.4 mg, 0.21 mmol) was cooled under argon
at -78 °C, BF3Et2O (18 mL, 0.15 mmol) was added, and the
solution was stirred until TLC indicated the disappearance of
lactol and then quenched by the addition of saturated NaHCO3
(1 mL). The mixture was warmed to room temperature with
vigorous stirring and was extracted with Et2O. The organic
layer was washed with saturated NaHCO3 and brine. The
combined organic layers were dried over Na2SO4, and the
solvent was evaporated, affording an oil which was purified
by chromatography (silica gel, petroleum ether/ether, 99:1)
yielding 11 mg of 10d (48% yield).
13C NMR (CDCl3) δ pyranofuranone moiety 171.6 (C-2 of
γ-hydroxybutenolide ring), 168.4 (C-4 of γ-hydroxybutenolide
ring), 137.6 (C-5 of pyran ring), 120.2 (C-4 of pyran ring), 117.3
(C-3 of γ-hydroxybutenolide ring), 96.9 (C-5 of γ-hydroxy-
butenolide ring), 68.3 (C-6 of pyran ring), 62.9 (C-2 of pyran
ring), 29.6 (C-3 of pyran ring), farnesyl moiety 135.2, 131.4,
124.3, 123.9, 120.2, 118.3, 39.6, 31.4, 26.7, 26.4, 25.7, 17.7, 16.0.
MS m/z 386 (M+). Anal. C, H.
Syn th eses of th e Oth er An a logu es (MA, CA, CMA,
CCA, BMA, BCA). The syntheses of the other analogues have
been performed as for FMA and FCA, giving rise to the
following compounds.
1H NMR (CDCl3) δ 7.41, 7.39, and 6.42 (s, 1H each, furan
protons), 5.57 (m, 1H, H-4), 5.18-5.09 (m, 3H, H-2, H-6, H-10

Manoalide and Cacospongionolide B Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3237
5-H yd r oxy-4-(6-h yd r oxy-3,6-d ih yd r o-2H -p yr a n -2-yl)-
5H-fu r a n -2-on e (MA, 11): H NMR (CD3OD) δ 6.11 (s, 1H,
butenolide ring), 69.4 (C-2 of pyran ring), 68.0 (C-6 of pyran
ring), 29.1 (C-3 of pyran ring), side chain 138.2, 129.1, 128.5,
126.4, 39.7. MS m/z 272 (M+). Anal. C, H.
1
H-5), 6.03-6.08 (m, 1H, H-4 of pyran ring), 5.99 (s, 1H, H-3),
5.82 (dd, 1H, J ) 2.1 Hz, 10.0 Hz, H-5 of pyran ring), 5.41 (d,
1H, J ) 2.5 Hz, H-6 of pyran ring), 4.87 (br m, 1H, H-2 of
pyran ring), 2.23 (br m, 2H, H-3 of pyran ring).
13C NMR (CD3OD) δ 172.3 (C-2 of γ -hydroxybutenolide
ring), 170.8 (C-4 of γ -hydroxybutenolide ring), 128.7 (C-4 of
pyran ring), 126.8 (C-5 of pyran ring), 117.8 (C-3), 99.3 (C-5
of γ -hydroxybutenolide ring), 96.5 (C-6 of pyran ring), 65.3
(C-2 of pyran ring), 30.0 (C-3 of pyran ring). MS m/z 180 (M+
- H2O). Anal. C, H.
Biologica l Assa ys. Assa y of Secr etor y P LA2. This
activity was assayed by using a modification of the method of
Franson et al.24 N. naja venom, bee venom, and porcine
pancreatic and human recombinant synovial enzymes were
diluted in 10 µL of 100 mM Tris-HCl, 1 mM CaCl2 buffer, pH
7.5. Enzymes were preincubated at 37 °C for 5 min with 2.5
µL of test compound solution or its vehicle in a final volume
of 250 µL. Incubation proceeded for 15 min in the presence of
10 µL of autoclaved oleate-labeled membranes and was
terminated by addition of 100 µL ice-cold solution of 0.25%
BSA in saline to a final concentration of 0.07% w/v. After
centrifugation at 2500g for 10 min at 4 °C, the radioactivity
in the supernatants was determined by liquid scintillation
counting. Results were expressed as percentages of inhibition,
and IC50 values were determined by plotting log concentration
versus inhibition values in the range from 10 to 90% inhibition.
Inhibition/reactivation studies with hydroxylamine hydrochlo-
ride9 were performed by incubating bee venom PLA2 at 41 °C
with FCA, FMA, manoalide, and cacospongionolide B or with
methanol (control) in Tris buffer (100 mM Tris-HCl, 1 mM
CaCl2 buffer, pH 7.5) over a range of different preincubation
times, using those inhibitory concentrations of drugs which
produce submaximal (90-95%) effect in order to appreciate
recoveries. The control and drug-treated enzyme solutions
were then diluted 2-fold with Tris buffer or with hydroxy-
lamine hydrochloride in Tris buffer (final concentration of
hydroxylamine 50 mM, pH 7.5). The solutions were incubated
for 2 h at 41 °C, and then the PLA2 activity was measured
(final concentration of FCA, FMA, manoalide, and cacospon-
gionolide B under assay conditions was 10, 5, 10, and 50 µM,
respectively).
Assa y of Cytosolic P LA2. Cytosolic PLA2 activity was
measured as the release of radiolabeled arachidonic acid
according to the method of Clark et al.25 using cytosolic
fractions of human monocytic U937 cells as the source of
enzyme and 1-palmitoyl-2-[14C] arachidonyl-sn-glycero-3-phos-
phocholine (57.0 mCi/mmol, 2 × 106 cpm) as the substrate.
Test compounds were dissolved in methanol and added to the
reaction mixture just before the addition of the enzyme
solution. The final concentration of methanol in the reaction
mixture was less than 1%, which showed no effect on the
enzyme activity. The reaction was stopped after a 60 min
incubation period at 37 °C by adding with 0.5 mL of isopropyl
alcohol/heptane/0.5 M H2SO4 (10:5:1). Heptane (0.7 mL) and
water (0.2 mL) were then added, and the solution was
vigorously mixed for 15 s. The heptane phase was mixed with
100 mg of silica gel 60 (Merck, 70-230 mesh) and centrifuged,
and the radioactivity in each supernatant was measured.26
Ca r r a geen a n Ed em a . Swelling was induced following a
modification of the technique of Sugishita et al.27 Female
Swiss mice (20-25 g) were fasted for 12 h with free access to
water. Drugs or vehicle (ethanol, Tween 80, distilled water:
5:5:90, v/v/v) was administered orally (0.5 mL) 1 h before
injection of carrageenan (0.05 mL; 3% w/v in saline) into the
subplantar area of the right hind paws of the animals in groups
of six. The volumes of injected and contralateral paws were
measured at 1, 3, and 5 h after induction of edema by using a
plethysmometer (Ugo Basile, Comerio, Italy). The volume of
edema was calculated for each animal as the difference
between the carrageenan-injected and contralateral paw vol-
ume. The antiinflammatory effect was expressed as the
percentage of inhibition compared with that of the control
group.
5-H yd r oxy-4-(3,6-d ih yd r o-2H -p yr a n -2-yl)-5H -fu r a n -2-
1
on e (CA, 12): H NMR (CDCl3) δ 6.17 (s, H-5), 6.06 (s, H-3),
5.75-5.84 (m, 2H, H-4 and H-5 of pyran ring), 4.42 (dd, J )
5.0, 9.0 Hz H-2 of pyran ring), 4.28 (br s, 2H, H-6 of pyran
ring), 2.23 (br m, H-3 of pyran ring).
13C NMR δ (CDCl3) δ 170.2 (C-2 of γ -hydroxybutenolide
ring), 168.3 (C-4 of γ -hydroxybutenolide ring), 126.3 (C-4 of
pyran ring), 122.8 (C-5 of pyran ring), 117.8 (C-3), 97.3 (C-5
of γ -hydroxybutenolide ring), 69.3 (C-6 of pyran ring), 65.9
(C-2 of pyran ring), 29.7 (C-3 of pyran ring). MS m/z 182 (M+).
Anal. C, H.
4-(5-Bu t-2-en yl-6-h yd r oxy-3,6-d ih yd r o-2H-p yr a n -2-yl)-
5-h yd r oxy-5H-fu r a n -2-on e (CMA, 11b): 1H NMR (CDCl3)
δ 6.14 (s, 1H, H-5), 6.06 (s, 1H, H-3), 5.69 (m, 1H, H-4 of pyran
ring), 5.50-5.40 (m, 2H, H-2, H-3 of butenyl moiety), 5.28 (s,
1H, H-6 of pyran ring), 4.85 (bd, H-2 of pyran ring), 2.76 (bs,
2H, H-1 of butenyl moiety), 2.26 (m, 2H, H-3 of pyran ring),
1.67 (d, J ) 6.0 Hz, 3H, CH3 of butenyl moiety).
13C NMR (CDCl3) δ pyranofuranone moiety 171.6 (C-2 of
γ-hydroxybutenolide ring), 168.4 (C-4 of γ-hydroxybutenolide
ring), 136.7 (C-5 of pyran ring), 121.4 (C-4 of pyran ring), 117.5
(C-3 of γ-hydroxybutenolide ring), 98.0 (C-5 of γ-hydroxy-
butenolide ring), 91.1 (C-6 of pyran ring), 62.9 (C-2 of pyran
ring), 30.0 (C-3 of pyran ring), side chain 127.9, 127.2, 36.2,
17.9. MS m/z 251 (M+ - 1). Anal. C, H.
4-(5-Bu t-2-en yl-3,6-d ih yd r o-2H-p yr a n -2-yl)-5-h yd r oxy-
5H-fu r a n -2-on e (CCA, 12b): 1H NMR (CDCl3) δ 6.14 (s, 1H,
H-5), 6.05 (s, 1H, H-3), 5.57 (m, 1H, H-4 of pyran ring), 5.53-
5.31 (m, 2H, H-2, H-3 of butenyl moiety), 4.42 (bs, 1H, H-2 of
pyran ring), 4.15 (m, 2H, H-6 of pyran ring), 2.60 (d, J ) 6.0
Hz, 2H, H-1 of butenyl moiety), 2.28 (m, 2H, H-3 of pyran ring),
1.67 (d, J ) 6.0 Hz, 3H, CH3 of butenyl moiety).
13C NMR (CDCl3) δ pyranofuranone moiety 171.6 (C-2 of
γ-hydroxybutenolide ring), 168.4 (C-4 of γ-hydroxybutenolide
ring), 136.7 (C-5 of pyran ring), 121.4 (C-4 of pyran ring), 117.5
(C-3 of γ-hydroxybutenolide ring), 98.0 (C-5 of γ-hydroxy-
butenolide ring), 68.1 (C-6 of pyran ring), 62.9 (C-2 of pyran
ring), 30.3 (C-3 of pyran ring), side chain 127.9, 127.2, 36.2,
17.8. MS m/z 236 (M+). Anal. C, H.
4-(5-Ben zyl-6-h yd r oxy-3,6-d ih yd r o-2H -p yr a n -2-yl)-5-
h yd r oxy-5H-fu r a n -2-on e (BMA, 11c): 1H NMR (CDCl3) δ
7.31-7.16 (m, 5H, phenyl), 6.11 (s, 1H, H-5), 6.02 (s, 1H, H-3),
5.61 (m, 1H, H-4 of pyran ring), 5.18 (s, 1H, H-6 of pyran ring),
4.86 (bd, 1H, H-2 of pyran ring), 3.41 (ABq, 2H, J ) 15.5 Hz,
ArCH2), 2.24 (m, 2H, H-3 of pyran ring).
13C NMR (CDCl3) δ pyranofuranone moiety 171.6 (C-2 of
γ-hydroxybutenolide ring), 168.4 (C-4 of γ-hydroxybutenolide
ring), 137.1 (C-5 of pyran ring), 122.6 (C-4 of pyran ring), 117.5
(C-3 of γ-hydroxybutenolide ring), 98.1 (C-5 of γ-hydroxy-
butenolide ring), 90.7 (C-6 of pyran ring), 62.8 (C-2 of pyran
ring), 28.9 (C-3 of pyran ring), side chain 138.2, 129.1, 128.5,
126.4, 38.1. MS m/z 270 (M+ - H2O). Anal. C, H.
4-(5-Ben zyl-3,6-d ih yd r o-2H-p yr a n -2-yl)-5-h yd r oxy-5H-
1
fu r a n -2-on e (BCA, 12c): H NMR (CDCl3) δ 7.33-7.15 (m,
Sta tistica l An a lysis. The level of statistical significance
was determined by analysis of variance (ANOVA) followed by
Dunnett’s t-test for multiple comparisons.
5H, phenyl), 6.15 (s, 1H, H-5), 6.03 (s, 1H, H-3), 5.58 (m, 1H,
H-4 of pyran ring), 4.41 (bt, 1H, H-2 of pyran ring), 4.12 (ABq,
2H, J ) 11.8 Hz, H-6 of pyran ring), 3.26 (ABq, 2H, J ) 15.5
Hz, ArCH2), 2.30 (m, 2H, H-3 of pyran ring).
Ack n ow led gm en t. This work was supported by the
European Commission, MAST-III program (contract no.
MAS3-CT95-0032), by MURST (40%) and CNR (Italian
side), and by grant SAF95-1046 CICYT (Spanish side).
13C NMR (CDCl3) δ pyranofuranone moiety 171.0 (C-2 of
γ-hydroxybutenolide ring), 167.8 (C-4 of γ -hydroxybutenolide
ring), 136.6 (C-5 of pyran ring), 118.5 (C-4 of pyran ring), 117.6
(C-3 of γ -hydroxybutenolide ring), 97.7 (C-5 of γ -hydroxy-

3238 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
De Rosa et al.
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