Synthesis of potentially anti-inflammatory IPL576,092-contignasterol and IPL576,092-manoalide hybrids

Article abstract of DOI:10.1016/j.tet.2004.04.084

The synthesis of two potentially anti-inflammatory steroidal hybrid compounds has been accomplished through a 16- and 17-step sequence, respectively, starting from commercially available androst-5-en-3β-ol-17- one. The synthetic strategies are based both

Full text of DOI:10.1016/j.tet.2004.04.084

Tetrahedron 60 (2004) 5587–5593
Synthesis of potentially anti-inflammatory
IPL576,092-contignasterol and IPL576,092-manoalide hybrids
a
a
b
c
Irene Izzo,a Elvira Avallone, Carmela Della Monica, Agostino Casapullo, Maria Amigo
,
*
and Francesco De Riccardisa
,
*
aDipartimento di Chimica, University of Salerno, via S. Allende, Baronissi I-84081 (SA), Italy
bDipartimento di Scienze Farmaceutiche, University of Salerno via Ponte Don Melillo, I-84084 Fisciano (SA), Italy
´ ´ ´
Departamento de Farmacologia, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andres Estelles s/n,
c
46100 Burjassot, Valencia, Spain
Received 27 February 2004; revised 2 April 2004; accepted 29 April 2004
Abstract—The synthesis of two potentially anti-inflammatory steroidal hybrid compounds has been accomplished through a 16- and 17-step
sequence, respectively, starting from commercially available androst-5-en-3b-ol-17-one. The synthetic strategies are based both on
stereoselective side chains elaboration and high yielding functional group transformations.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
marine sesterterpene isolated from the sponge Luffariella
variabilis,3 new perspectives have been opened in the treat-
ment of inflammation-related pathological disorders.
Normal inflammation, a defense reaction caused by tissue
damage or injury and characterized by redness, heat,
swelling, and pain, is a highly regulated process. The
inflammatory response, also involved in asthma and allergy,
is induced by a localized release of histamine, leukotrienes,
prostaglandins and cytokines.
Recently, a novel and promising approach in drug
discovery, towards the development of new lead substances,
has emerged. It consists in the combination of parts of
structurally different naturally occurring bioactive products
to yield hybrid structures that can, in principle, exceed the
activities of their parent compounds.4 From this perspective
and as part of a broad program in the steroid area, we
designed and synthesized the hybrid entities 4 and 5, linking
the IPL576,092 trihydroxylated tetracyclic nucleus to the
contignasterol’s (17R,20S,22S,24S)-lactol5 and manoalide
g-hydroxybutenolide side chains, respectively. It is the
preparation of these two new compounds that is reported
here.
With the recent discovery of the anti-inflammatory potential
of contignasterol (1),1 a polyhydroxysteroid isolated from
the sponge Petrosia contignata, IPL576,092 (2),2
synthetic trihydroxypregnene, and manoalide (3), a
a
Keywords: IPL576,092; Contignasterol; Manoalide; Steroids; Anti-
inflammatory compounds.
2. Results and discussion
*
Corresponding authors. Tel.: þ39089965230; fax: þ39089965296;
e-mail addresses: dericca@unisa.it; iizzo@unisa.it
Two important steps required to generate target compounds
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.04.084

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I. Izzo et al. / Tetrahedron 60 (2004) 5587–5593
4 and 5 are: the synthesis of the fully protected common
intermediate (Z)-6a,7b-(diacetoxy)-3b-[(tert-(butyldi-
phenysilyl)-oxy]-5a-pregn-17(20)-ene (6), and a highly
reliable stereoselective method for the side chain pre-
cursor’s introduction.
2.1. Synthesis of IPL576,092-contignasterol hybrid
Sterol 6 and preformed contignasterol’s side chain inter-
mediate 7 were conveniently prepared from commercially
available androst-5-en-3b-ol-17-one (8), in 11 steps and
26% overall yield6 and (S)-carvone (9), in five steps and
48% overall yield,7 respectively.
Scheme 1. (a) KOH, MeOH, 12 h, 70%; (b) H2, Pt2/C, AcOEt, 3 h, 90%;
(c) DIBAL-H, CH2Cl2, 278 8C, 1.5 h, quant.; (d) HF/pyridine, 0 8C, 3 days,
88%.
contaminant, to give pure 13 (Scheme 1). Highly stereo-
selective catalytic D16-hydrogenation7 and subsequent
DIBAL-H mediated reduction, furnished the lactol 15.
Final HF-induced desilylation of the tert-(butyldiphenyl-
silyl) protecting group on C-3, provided the requested
3b,6a,7b-trihydroxy lactol 4 in good overall yield from
intermediate 10.13
The two precursors 6 and 7 were linked through a
stereochemically controlled Me2AlCl-mediated carbonyl-
ene reaction.8 Although several ene reactions of linear
aliphatic aldehydes have been reported,8 we have encoun-
tered serious difficulties for the convergent coupling
between 6 and 7. The Lewis base character of the tetracyclic
nucleus oxygenated substituents present in 6 and a probable
7b-acetoxy induced D ring unfavorable steric effect, could
be the reasons of the low yields. The best results9 were
achieved using a two-fold excess of the Lewis acid relative
to the aldehyde 710 and afforded an inseparable mixture of
diasteroimers 10 and 1111 (4:1 ratio in 66% overall yield,
based on recovered starting material 6). The excess of the
Me2AlCl acted as nucleophile, inducing the formation of
the C-5 epimeric lactone 12.
2.2. Synthesis of IPL576,092-manoalide hybrid
With the aim to combine IPL576,092 tetracyclic 3b,6a,7b-
trihydroxylated nucleus with the g-hydroxy butenolide
manoalide3/luffariellolide14 pharmacophore, we prepared
the electrophilic aldehyde 17, through a two-step sequence,
from fully protected 6, and exposed it to 3-furyllithium15
(Scheme 2). The (R) stereochemistry of the major epimer 18
was assigned by comparison with the results observed for
various alkyllithium or Grignard addition to this aldehyde,
where the preferential formation of the Cram adduct is well
documented.16
Scheme 2. (a) BF3·OEt2, (CH2O)n, CH2Cl2, 278 8C!230 8C, 0.5 h,
90%;6 (b) PDC, CH2Cl2, 2 h, 69%; (c) 3-bromofuran, n-BuLi, 278 8C,
15 min then 17, 278 8C, 1 h, 18: 57%, 19: 28%; (d) Ac2O, Py, 12 h, 20:
95%, 21: quant.
The stereochemical assignment was confirmed in two
independent ways. It was empirically deduced by com-
paring the magnitude of the vicinal coupling of the JH20–H22
in the acetylated derivatives 20 and 21. It is known,8 in fact,
that the H-20/H-22 vicinal coupling constant should be
smaller (,8 Hz) for the stereochemical arrangement present
in 20 (20S,22R), and larger (,10 Hz) for that present in
the epimer 21 (20S,22S). The observed JH20-H22 coupling
constants for 20 and 21 were 8.6 Hz and 10.7 Hz,
respectively.
In order to suppress the formation of byproducts, we turned
our attention to the non-alkylating BF3·OEt2 Lewis acid.12
Unfortunately, in this case, we observed the formation of a
mixture of unidentified compounds, with no trace of the
desired adduct 10.
The next problem to be faced was transformation of
precursor 10 to the requested target 4. To this end the
(20S,22S,24S)-D16-lactone 10, as a mixture with the (22R)-
epimer 11, was hydrolyzed and separated from its

I. Izzo et al. / Tetrahedron 60 (2004) 5587–5593
5589
The (22R) configuration of 18 was also confirmed using the
modified Mosher’s esters method,17 through comparison of
the 1H NMR spectra of a-methoxy-a-(trifluoromethyl)-
phenylacetic (MTPA) derivatives 22 and 23, easily achieved
from (þ)-(S)-MTPA-Cl and (2)-(R)-MTPA-Cl, respec-
tively.18
(15 mL), Me2AlCl (1 M in hexane, 6.72 mL, 6.72 mmol)
was added at 278 8C. The resulting mixture was warmed to
220 8C over 5 h and then stirred at 220 8C overnight. The
reaction was quenched with MeOH/H2O (18.0 mL, 1:1) at
278 8C. The aqueous layer was extracted with CH2Cl2
(3£20 mL) and the combined organic phases were succes-
sively washed with 1% aqueous HCl, saturated aqueous
NaHCO3, brine and then dried over Na2SO4. Removal of
solvent in vacuo gave the crude containing 10 and 11 in a
4:1 ratio [1H NMR analysis (CDCl3, 400 MHz) d: 5.34
(0.8H, bs, H-16), 5.41 (0.2H, bs, H0-16)], which was purified
by flash chromatography (20–80% diethyl ether in
petroleum ether) to furnish a mixture of 10 and 11
(0.168 g, 20% overall yield, 66%, based on recovered
starting material), of 6 (0.483 g, 70%) and 12 (variable
amounts).
Once ascertained the C-22 configuration of adducts 18 and
19, we first desilylated at C-3 (Scheme 3) and then at C-6,
C-7 deacetylated furyl alcohol 18. Fully deprotected tetrol
24 was then submitted to a rose Bengal mediated
photoxidation to give target g-hydroxy butenolide 5.19
Compounds 10–11. Rf¼0.47 (30% ethyl acetate in petro-
leum ether). HR-ESMS: m/z 797.4817 (calcd 797.4813 for
C49H69O7Si).
The activity of hybrid 5 has been tested on human synovial
sPLA2-IIA, exerting a 63% of inhibition at 100 mM.20
Additionally, this compound on lipopolysaccharide stimu-
lated human monocytes was able to reduce nitric oxide and
PGE2 (two important mediators of the inflammatory
process) at 10 mM (64 and 72%, respectively).21
Compound 12. Rf¼0.65 (30% ethyl acetate in petroleum
1
ether). H NMR (CDCl3, 300 MHz) d: 0.84 (6H, d over-
lapped, J¼7.0 Hz, –CH(CH3)2), 1.30 (1.5H, d, J¼6.2 Hz,
–CHCH3), 1.31 (1.5H, d, J¼6.2 Hz, –CHCH3), 1.41–1.90
(4H, m, CHCH2CH–, CH2CHCH2–, (CH3)2CH–, over-
lapped), 2.07 (0.5H, dd, J¼17.7, 10.5 Hz, –CHHCOO),
2.17 (0.5H, dd, J¼15.8, 11.1 Hz, –CHHCOO), 2.45 (0.5H,
dd, J¼15.8, 5.5 Hz, –CHHCOO), 2.58 (0.5H, dd, J¼17.7,
6.3 Hz, –CHHCOO), 4.32 (0.5H, m, –CHOCO), 4.41
(0.5H, m, –CHOCO). 13C NMR (CDCl3, 75 MHz) d:19.0,
19.1, 19.2 (£2), 21.0, 21.8, 32.1 (£2), 32.6, 33.0, 33.4, 34.0,
35.0, 37.8, 73.8, 76.7, 172.0, 173.3. HR-ESMS: m/z
157.1237 (calcd 157.1229 for C9H17O2).
Scheme 3. (a) (i) TBAF (1 M in THF), THF, 3 h; (ii) K2CO3, MeOH 12 h,
69% (two steps); (b) O2/hn, rose Bengal, i-Pr2EtN, CH2Cl2, 278 8C, 2 h,
40%.
3. Conclusions
4.2.2. Compound 13. Compounds 10 and 11 (0.115 g,
0.144 mmol) were dissolved in a 5% solution of KOH in
MeOH (5.0 mL) and allowed to react overnight at room
temperature. The reaction mixture was acidified with 2 M
HCl to pH¼1 at 0 8C. The solvents were concentrated in
vacuo, to remove the excess MeOH, and the aqueous layer
was extracted with ethyl acetate (3£10 mL). The combined
organic phases were dried over Na2SO4, filtered and
concentrated in vacuo to give a crude, which was flash
chromatographed (10–30% diethyl ether in chloroform) to
give pure 13 (0.072 g, 70%) as a white amorphous solid and
an inseparable mixture of 13 and its C-22 (R)-epimer
(0.031 g).
In conclusion, we have reported the synthesis of two new
IPL576,092-contignasterol and IPL576,092-manoalide
analogs in 29 and 10% overall yields, respectively, starting
from easily available (Z)-6a,7b-(diacetoxy)-3b-[(tert-(butyl-
diphenysilyl)-oxy]-5a-pregn-17(20)-ene (6).
4. Experimental
4.1. General methods
The NMR spectra were recorded at room temperature on
a Bruker DRX 400 spectrometer (1H at 400 MHz, 13C at
100 MHz) or on Bruker DRX 300 spectrometer (1H at
300 MHz, 13C at 75 MHz). Chemical shifts are reported
relative to the residual solvent peak (CHCl3: d¼7.26,
13CDCl3: d¼77.0, CD2HOD: d¼3.31, 13CD3OD: d¼49.0).
Instruments used to obtain physical data and experimental
conditions for the reactions and chromatography were the
same as described in the preceding paper.7
Compound 13. Rf¼0.33 (30% diethyl ether in chloroform,
double migration). IR (CHCl3) nmax (cm21) 3400, 2959,
2929, 2853, 1728, 1471, 1428, 1375, 1219, 1110, 1077, 772,
703. [a]D¼þ15.0 (c 1.0, CHCl3). 1H NMR (CDCl3,
400 MHz) d: 0.77 (3H, s, CH3-18), 0.872 (3H, s, CH3-19),
0.873 (6H, d, J¼6.3 Hz, (CH3)2CH–), 1.05 (9H, s,
(CH3)3CSi–), 1.14 (3H, d, J¼6.7 Hz, CH3-21), 2.14 (1H,
dd, J¼17.7, 9.9 Hz, H-28), 2.64 (1H, dd, J¼17.7, 6.6 Hz,
H0-28), 3.04 (1H, dd, J¼9.3, 8.9 Hz, H-6 or H-7), 3.21 (1H,
dd, J¼10.6, 8.9 Hz, H-7 or H-6), 3.56 (1H, m, H-3), 4.22
(1H, t-like, J¼8.9 Hz, H-22), 7.34–7.43 (6H, m, C6H5–),
7.67 (4H, m, C6H5–). 13C NMR (CDCl3, 100 MHz) d: 13.6,
16.1, 18.3, 18.9, 19.1, 19.3, 20.9, 27.0 (£3), 31.1, 31.3, 32.2,
32.4, 33.7, 34.0, 34.6, 35.9, 37.1, 37.7, 38.0, 39.7, 47.9,
4.2. Procedures for the synthesis of compounds 4, 10–15,
described in paragraph 2.1
4.2.1. Compounds 10 and 11. To a solution of 6 (0.694 g,
1.05 mmol) and 7 (0.582 g, 3.38 mmol) in dry CH2Cl2

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I. Izzo et al. / Tetrahedron 60 (2004) 5587–5593
48.0, 52.3, 56.0, 72.5, 74.8, 80.2, 83.5, 124.1, 127.4 (£2),
127.5 (£2), 129.4, 129.5, 134.6, 134.8, 135.8 (£4), 155.6,
172.3. HR-ESMS: m/z 713.4615 (calcd 713.4601 for
C45H65O5Si).
Compound 4. Rf¼0.17 (10% methanol in dichloromethane).
1H NMR (CDCl3, 400 MHz) d: 0.65 (3H, bs, CH3-18),
0.83–0.96 (6H, m, CH3-19, CH3-21 and (CH3)2CH–
overlapped), 3.03 (1H, m, H-6 or H-7), 3.18 (1H, m, H-7
or H-6), 3.40 (0.5H, bd, J¼8.3 Hz, H-22), 3.60 (1H, m,
H-3), 4.01 (0.5H, bd, J¼10 Hz, Hax-29), 4.63 (0.5H, bd,
J¼8.0 Hz, H-22), 5.31 (0.5H, bs, Heq-29). HR-ESMS: m/z
479.3749 (calcd 479.3736 for C29H51O5).
4.2.3. Compound 14. To a solution of 13 (0.042 g,
0.059 mmol) in ethyl acetate (3.0 mL), a catalytic amount
of 5% Pt/C (0.008 g) was added. The flask was evacuated
(50 Torr) and flushed three times with hydrogen. The
reaction mixture was then vigorously stirred under hydrogen
for 3 h at room temperature. It was filtered through a pad
of Celitew and concentrated in vacuo to afford 14 (0.038 g,
90%) which was used in the next step without further
purification.
4.3. Procedures for the synthesis of compounds 5, 17–24,
described in paragraph 2.2
4.3.1. Compound 17. To a solution of 166 (0.202 g,
˚
0.29 mmol) in dry CH2Cl2 (10 mL), 4 A molecular sieves
(0.20 g) and PDC (0.22 g, 0.59 mmol) were added. The
mixture was stirred at room temperature for 3 h, then diluted
with diethyl ether (10 mL) and allowed to stir for additional
45 min. Filtration through pad of silica gel (particle size
0.063–0.200 mm) and CaSO4 (10% in weight) afforded a
solution which was concentrated in vacuo. The residue was
flash-chromatographed (10–20% ethyl acetate in petroleum
ether) to afford 17 (0.140 g, 69%) as a pale yellow oil.
Compound 14. Rf¼0.68 (50% ethyl acetate in petroleum
ether). [a]D¼þ15.2 (c 1.1, CHCl3). 1H NMR (CDCl3,
400 MHz) d: 0.65 (3H, s, CH3-18), 0.82 (3H, s, CH3-19),
0.90 (6H, bd, J¼6.3 Hz, (CH3)2CH–), 0.95 (3H, d, J¼
6.5 Hz, CH3-21), 1.04 (9H, s, (CH3)3CSi–), 2.09 (1H, dd,
J¼17.7, 10.8 Hz, H-28), 2.64 (1H, dd, J¼17.7, 6.6 Hz,
H0-28), 3.01 (1H, dd, J¼9.3, 8.9 Hz, H-6 or H-7), 3.18 (1H,
dd, J¼10.6, 8.9 Hz, H-7 or H-6), 3.54 (1H, m, H-3), 4.33
(1H, bd, J¼11.2 Hz, H-22), 7.34–7.43 (6H, m, C6H5–),
7.67 (4H, m, C6H5–). 13C NMR (CDCl3, 100 MHz) d:
12.0, 12.6, 13.6, 19.1, 19.2, 19.3, 21.2, 26.5, 27.0 (£3),
28.0, 30.2, 31.2, 32.2, 32.4, 34.1, 35.5, 37.2, 38.0, 39.5,
40.3, 40.8, 43.2, 47.5, 50.6, 51.8, 55.5, 72.5, 74.8, 80.5,
82.5, 127.4 (£4), 129.4 (£2), 134.5, 134.8, 135.7 (£4),
172.7. HR-ESMS: m/z 715.4747 (calcd 715.4758 for
C45H67O5Si).
Compound 17. Rf¼0.44 (20% ethyl acetate in petroleum
ether). [a]D¼þ17.0 (c 1.0, CHCl3). 1H NMR (CDCl3,
400 MHz) d: 0.75 (3H, s, CH3-18), 0.95 (3H, s, CH3-19),
1.02 (9H, s, (CH3)3CSi–), 1.15 (3H, d, J¼6.7 Hz, CH3-21),
1.87 (3H, s, CH3CO), 1.97 (3H, s, CH3CO), 2.97 (1H, bq,
J¼5.4 Hz, H-20), 3.54 (1H, m, H-3), 4.68 (1H, dd, J¼11.0,
9.4 Hz, H-6 or H-7), 4.77 (1H, dd, J¼9.9, 9.4 Hz, H-7 or
H-6), 5.42 (1H, bs, H-16), 7.37 (6H, m, C6H5–), 7.63
(4H, m, C6H5–), 9.39 (1H, d, J¼2.3 Hz, H-22). 13C NMR
(CDCl3, 100 MHz) d: 13.0, 14.0, 15.5, 18.7, 20.3, 20.5,
21.1, 26.6 (£3), 30.7, 31.8 (£2), 31.9, 33.6, 35.6, 36.5, 37.6,
45.4, 46.0, 51.6, 54.3, 71.6, 73.8, 77.2, 126.9, 127.1 (£4),
129.2 (£2), 134.1, 134.2, 135.4 (£4), 150.3, 170.3, 170.4,
200.5. HR-ESMS: m/z 685.3931 (calcd 685.3924 for
C42H57O6Si).
4.2.4. Compound 15. To a solution of 14 (0.033 g,
0.046 mmol) in dry CH2Cl2 (2.0 mL), DIBAL-H (1 M in
CH2Cl2, 0.230 mL, 0.230 mmol) was added at –78 8C. The
reaction mixture was stirred at –78 8C for 90 min, then
quenched with MeOH/H2O (1.0 mL, 1:1) at –78 8C and
stirred at room temperature for 20 min. Filtration through a
pad of Celitew and concentration in vacuo afforded to a
residue which was purified by flash chromatography
(30–50% ethyl acetate in petroleum ether) to give 15
(0.033 g, quant.) as a white amorphous solid.
4.3.2. Compounds 18 and 19. To a solution of 3-bromo-
furan (0.065 mL, 0.72 mmol) in dry THF (2.5 mL), n-BuLi
(1.6 M in hexane, 0.32 mL, 0.51 mmol) was added at
278 8C. After being stirred for 30 min, to the resulting
mixture was added a solution of 17 (0.071 g, 0.10 mmol) in
dry THF (2.5 mL) and the mixture was stirred for 30 min at
278 8C. The mixture was neutralized with saturated
aqueous NH4Cl, concentrated in vacuo, to remove the
excess THF, and extracted with diethyl ether. The organic
phase was dried over Na2SO4, filtered and concentrated in
vacuo. The residue was flash-chromatographed (15–40% of
ethyl ether in petroleum ether) to give 18 (0.036 g, 57%) and
19 (0.018 g, 28%) as oils.
Compound 15. Rf¼0.66 (50% ethyl acetate in petroleum
ether). 1H NMR (CDCl3, 400 MHz) d: 0.64 (3H, s, CH3-18),
0.83 (3H, s, CH3-19), 0.86–0.96 (9H, m, (CH3)2CH–,
CH3-21 overlapped), 1.05 (9H, s, (CH3)3CSi–), 3.01 (1H,
dd, J¼9.3, 8.9 Hz, H-6 or H-7), 3.18 (1H, dd, J¼10.6,
8.9 Hz, H-7 or H-6), 3.41 (0.6H, bd, J¼10.3 Hz, H-22), 3.54
(1H, m, H-3), 3.99 (0.4H, bd, J¼11.1 Hz, H-22), 4.62 (0.6H,
bd, J¼8.9 Hz, Hax-29), 5.34 (0.4H, bs, Heq-29), 7.34–7.41
(6H, m, C6H5–), 7.67 (4H, m, C6H5–). HR-ESMS: m/z
717.4917 (calcd 717.4914 for C45H69O5Si).
Compound 18. Rf¼0.50 (30% ethyl acetate in petroleum
ether). IR (CHCl3) nmax (cm21) 2936, 2856, 1743, 1376,
1251, 1110, 1081, 1029, 703. [a]D¼þ8.4 (c 1.6, CHCl3). 1H
NMR (CDCl3, 400 MHz) d: 0.65 (3H, s, CH3-18), 0.94 (3H,
s, CH3-19), 1.01 (3H, d, J¼6.7 Hz, CH3-21), 1.02 (9H, s,
(CH3)3CSi–), 1.87 (3H, s, CH3CO), 1.96 (3H, s, CH3CO),
2.40 (1H, m, H-20), 3.52 (1H, m, H-3), 4.66 (1H, dd, J¼
11.0, 9.4 Hz, H-6 or H-7), 4.74 (1H, d, J¼5.6 Hz, H-22),
4.78 (1H, dd, J¼9.9, 9.4 Hz, H-6 or H-7), 5.46 (1H, bs,
H-16), 6.31 (1H, bs, H-40), 7.38, (8H, m, C6H5–, H-20 and
4.2.5. Compound 4. To a solution of 15 (0.044 g,
0.061 mmol) in dry pyridine (0.600 mL), 70% HF in
pyridine (0.073 mL) was added at 0 8C. The reaction
mixture was stirred for 3 days at 0 8C and then diluted
with CHCl3. The solvents were removed under a N2 stream
and the residue was flash-chromatographed (5–20%
methanol in a 0.1% solution of triethylamine in dichloro-
methane) to afford 4 (0.026 g, 88%) as a white amorphous
solid.

I. Izzo et al. / Tetrahedron 60 (2004) 5587–5593
5591
H-50 overlapped), 7.62 (4H, m, C6H5–). 13C NMR (CDCl3,
100 MHz) d: 13.3, 15.5, 15.9, 19.1, 20.7, 20.9, 21.4, 26.9
(£3), 31.1, 32.0, 32.1, 34.2, 36.0, 36.8, 37.8, 38.8, 46.4,
47.7, 52.0, 55.2, 69.3, 72.0, 74.3, 77.6, 108.7, 124.7, 127.5
(£4), 127.8, 129.5 (£2), 134.5, 134.6, 135.7 (£4), 139.4,
142.8, 156.0, 170.6, 170.8. HR-ESMS: m/z 753.4179 (calcd
753.4187 for C46H61O7Si).
(£4), 141.9, 149.0, 153.7, 170.0, 170.6, 170.8. HR-ESMS:
m/z 795.4281 (calcd 795.4292 for C48H63O8Si).
4.3.4. Compounds 22 and 23. To a solution of 18 (0.010 g,
0.013 mmol) in dry pyridine (0.150 mL), (S)-MTPA-Cl (or
(R)-MTPA-Cl 0.007 mL) was added at 0 8C. The resulting
mixture was stirred 1 h, concentrated under a N2 stream and
flash-chromatographed (30% of diethyl ether in petroleum
ether) to give 22 (or 23) in quantitative yield.
Compound 19. Rf¼0.35 (30% ethyl acetate in petroleum
ether). [a]D¼þ40.7 (c 0.2, CHCl3). 1H NMR (CDCl3,
400 MHz) d: 0.37 (3H, s, CH3-18), 0.91 (3H, s, CH3-19),
1.02 (9H, s, (CH3)3CSi–), 1.15 (3H, d, J¼6.7 Hz, CH3-21),
1.86 (3H, s, CH3CO), 1.95 (3H, s, CH3CO), 2.74 (1H, m,
H-20), 3.51 (1H, m, H-3), 4.65 (1H, dd, J¼11.0, 9.4 Hz, H-6
or H-7), 4.75 (2H, m, H-7 or H-6 and H-22 overlapped),
5.41 (1H, bs, H-16), 6.31 (1H, bs, H-40), 7.27–7.40 (8H, m,
C6H5–, H-20 and H-50 overlapped), 7.62 (4H, m, C6H5–).
13C NMR (CDCl3, 100 MHz) d: 13.3, 15.0, 18.9, 19.0, 20.7,
20.9, 21.4, 26.9 (£3), 31.1, 32.0, 32.1, 34.2, 35.9, 36.8, 36.9,
37.0, 37.8, 46.3, 48.0, 52.0, 54.6, 68.9, 72.0, 74.2, 77.5,
113.7, 123.6, 127.5 (£4), 129.5 (£2), 134.4, 134.5, 135.7
(£4), 141.5, 152.0, 155.0, 170.6, 170.8. HR-ESMS: m/z
753.4191 (calcd 753.4187 for C46H61O7Si).
Compound 22. 1H NMR (CDCl3, 400 MHz) d: 0.40 (3H, s,
CH3-18), 0.91 (3H, s, CH3-19), 0.93 (3H, d, J¼6.7 Hz,
CH3-21), 1.02 (9H, s, (CH3)3CSi–), 1.86 (3H, s, CH3CO),
1.94 (3H, s, CH3CO), 2.53 (1H, m, H-20), 3.40 (4H, m, H-3
and CH3O– overlapped), 4.62 (1H, dd, J¼11.0, 9.4 Hz, H-6
or H-7), 4.75 (1H, dd, J¼9.9, 9.4 Hz, H-7 or H-6), 5.28 (1H,
bs, H-16), 5.95 (1H, d, J¼9.6 Hz, H-22), 6.31 (1H, bs, H-40),
7.31–7.41 (8H, m, C6H5–, H-20 and H-50 overlapped), 7.63
(4H, m, C6H5–). LR-ESMS: m/z 969.9 (calcd 969.5 for
C56H68F3O9Si).
Compound 23. 1H NMR (CDCl3, 400 MHz) d: 0.40 (3H, s,
CH3-18), 0.91 (3H, s, CH3-19), 1.02 (9H, s, (CH3)3CSi–),
1.09 (3H, d, J¼6.7 Hz, CH3-21), 1.85 (3H, s, CH3CO), 1.95
(3H, s, CH3CO), 2.53 (1H, m, H-20), 3.49 (4H, m, H-3 and
CH3O– overlapped), 4.63 (1H, dd, J¼11.0, 9.4 Hz, H-6 or
H-7), 4.75 (1H, dd, J¼9.9, 9.4 Hz, H-7 or H-6), 5.33 (1H,
bs, H-16), 5.90 (1H, d, J¼8.8 Hz, H-22), 6.14 (1H, bs, H-40),
7.31–7.41 (8H, m, C6H5–, H-20 and H-50 overlapped), 7.63
(4H, m, C6H5–). LR-ESMS: m/z 969.1 (calcd 969.5 for
C56H68F3O9Si).
4.3.3. Compounds 20 and 21. To a solution of 18 (or 19)
(0.010 g, 0.013 mmol) in dry pyridine (0.100 mL), Ac2O
(0.040 mL) was added. The resulting mixture was stirred
overnight, concentrated under a N2 stream and flash-
chromatographed (10–20% of ethyl acetate in petroleum
ether) to give 20 (0.010 g, 95%) or 21 (0.011 g, quant.) as
oils.
Compound 20. Rf¼0.59 (50% diethyl ether in petroleum
ether). [a]D¼þ4.8 (c 0.7, CHCl3). 1H NMR (CDCl3,
400 MHz) d: 0.45 (3H, s, CH3-18), 0.92 (3H, s, CH3-19),
1.02 (9H, s, (CH3)3CSi–), 1.06 (3H, d, J¼6.7 Hz, CH3-21),
1.86 (3H, s, CH3CO), 1.95 (3H, s, CH3CO), 2.05 (3H, s,
CH3CO), 2.47 (1H, m, H-20), 3.51 (1H, m, H-3), 4.64 (1H,
dd, J¼11.0, 9.4 Hz, H-6 or H-7), 4.76 (1H, dd, J¼9.9,
9.4 Hz, H-7 or H-6), 5.33 (1H, bs, H-16), 5.78 (1H, d,
J¼8.6 Hz, H-22), 6.27 (1H, bs, H-40), 7.27–7.40 (8H, m,
C6H5–, H-20 and H-50 overlapped), 7.62 (4H, m, –C6H5).
13C NMR (CDCl3, 100 MHz) d: 13.3, 15.3, 15.9, 19.1, 20.7,
20.9, 21.2, 27.0 (£3), 29.7, 31.2 (£2), 32.2 (£2), 34.4, 36.0,
36.9, 37.3, 37.9, 46.4, 48.0, 52.1, 54.8, 71.7, 72.0, 74.3,
109.2, 124.1, 124.5, 127.5 (£4), 129.5 (£2), 134.7 (£2),
135.7 (£4), 140.5, 142.6, 154.6, 170.3, 170.6, 170.8.
HR-ESMS: m/z 795.4285 (calcd 795.4292 for C48H63O8Si).
4.3.5. Compound 24. To a solution of 18 (0.170 g,
0.23 mmol) in dry THF (3.0 mL), tetra-butylammonium
fluoride (TBAF, 1 M in THF, 0.92 mL, 0.92 mmol) was
added. The mixture was stirred at room temperature for 3 h,
then diluted with H2O, concentrated in vacuo, to remove the
excess THF, and extracted with diethyl ether. The organic
phase was dried over Na2SO4, filtered and concentrated in
vacuo. To a solution of the residue in MeOH (2.0 mL),
K2CO3 (0.010 g, 0.072 mmol) was added. The mixture was
stirred at reflux overnight and then quenched with CHCl3
(3.0 mL). Solvents were concentrated in vacuo to the half
of their volume. The procedure (CHCl3 addition and con-
centration) was repeated several times to precipitate the
carbonate. The solution was then filtrate through a pad
of Celitew, concentrated in vacuo and the residue was
flash-chromatographed (2–30% MeOH in CH2Cl2) to
give 24 (0.066 g; 69% for two steps) as a white amorphous
solid.
Compound 21. Rf¼0.69 (30% ethyl acetate in petroleum
ether). [a]D¼þ34.8 (c¼0.2, CHCl3). 1H NMR (CDCl3,
400 MHz) d: 0.30 (3H, s, CH3-18), 0.91 (3H, s, CH3-19),
1.02 (9H, s, (CH3)3CSi–), 1.08 (3H, d, J¼6.7 Hz, CH3-21),
1.85 (3H, s, CH3CO), 1.95 (3H, s, CH3CO), 2.07 (3H, s,
CH3CO), 2.79 (1H, m, H-20), 3.50 (1H, m, H-3), 4.63 (1H,
dd, J¼11.0, 9.4 Hz, H-6 or H-7), 4.75 (1H, dd, J¼9.9,
9.4 Hz, H-7 or H-6), 5.47 (1H, bs, H-16), 5.89 (1H, d, J¼
10.7 Hz, H-22), 6.31 0 (1H, bs, H-40), 7.34–7.41 (8H, m,
C6H5–, H-20 and H-5 overlapped), 7.62 (4H, m, C6H5–).
13C NMR (CDCl3, 100 MHz) d: 13.3, 14.9, 19.1, 19.4, 20.7,
20.8, 20.9, 21.4, 26.9 (£3), 29.8, 32.1 (£2), 34.2, 34.8, 35.9,
36.8, 37.8, 46.4, 48.1, 52.0, 54.4, 69.8, 72.0, 74.2, 77.5,
113.9, 124.0 (£2), 127.5 (£4), 129.5 (£2), 134.6 (£2), 135.7
Compound 24. Rf¼0.74 (25% methanol in chloroform).
[a]D¼þ32.6 (c 0.8, CDCl3). 1H NMR (CDCl3, 400 MHz) d:
0.70 (3H, s, CH3-18), 0.89 (3H, s, CH3-19), 1.04 (9H, s,
(CH3)3CSi–), 1.06 (3H, d, J¼6.7 Hz, CH3-21), 2.46 (1H, m,
H-20), 3.13 (1H, dd, J¼11.0, 9.4 Hz, H-6 or H-7), 3.28 (1H,
dd, J¼9.9, 9.4 Hz, H-7 or H-6) 3.58 (1H, m, H-3), 4.79 (1H,
d, J¼5.6 Hz, H-22), 5.60 (1H, bs, H-16), 6.33 (1H, bs, H-40),
7.35 (2H, bs, H-20 and H-50). 13C NMR (CD3OD, 100 MHz)
d: 14.0, 15.9, 18.5, 18.8, 22.4, 31.9, 33.3, 35.1, 36.2, 37.0,
38.5, 40.6, 41.2, 49.8, 54.0, 58.1, 71.0, 71.9, 75.9, 81.1,
110.4, 125.3, 130.3, 141.0, 143.7, 157.6. HR-ESMS: m/z
431.2787 (calcd 431.2797 for C26H39O5).

5592
I. Izzo et al. / Tetrahedron 60 (2004) 5587–5593
´
Sodano, G.; Terencio, M. C.; Paya, M.; Alcaraz, M. J. Curr.
Med. Chem. 1999, 6, 415–431, and references cited therein.
4. See: Tietze, L. F.; Bell, H. P.; Chandrasekhar, S. Angew.
Chem. Int. Ed. 2003, 42, 3996–4028, and references cited
therein.
4.3.6. Compound 5. To a solution of 24 (0.020 g,
0.048 mmol) in dry CH2Cl2 (2.0 mL) and THF (1.0 mL),
diisopropylethylamine (0.85 mL) and rose Bengal (0.001 g)
were added. The mixture was irradiated at 278 8C with a
200-W tungsten incandescent lamp for 2 h. The reaction
mixture was allowed to warm to 20 8C and concentrated in
vacuo. The crude residue was purified by HPLC (Vydac C18
analytical column; 10–95% of CH3CN and 0.1% TFA in
H2O and 0.1% TFA) to give 5 in (0.009 g, 40%) as a white
amorphous solid.
5. We have recently reported the synthesis of a series of model
compounds containing the four possible C-22/C-24 stereo-
isomers Izzo, I.; Pironti, V.; Della Monica, C.; Sodano, G.; De
Riccardis, F. Tetrahedron Lett. 2001, 42, 8977–8980, and
proposed a (22S,24S) configuration for the natural product.
After ten years from its first isolation, Andersen completed
contignasterol’s structural elucidation and assigned as (22R,
24R) the configurations of the side chain’s stereogenic centres
Yang, L.; Andersen, R. J. J. Nat. Prod. 2002, 65, 1924–1926,
Considering still in doubt the assignment of the C-22/C-24
stereogenic centres, we decided to synthesize a (17R,20S,22S,
24S)-side chain. It must be stressed that side chain seems to be
quite important for contingasterol’s activity. Infact, contig-
nasterol’s reduction product 1a did not inhibit histamine relase
from rat mast cells (Ref. 1b), suggesting that either the 15-keto
group and/or the hemiacetal is necessary for the inhibition of
histamine relase. The authors represent 1a as having the trans
C/D ring junction, yet it is doubtful that the reduction
conditions would cause epimerization of the 14b-hydrogen
Compound 5. Rf¼0.60 (25% methanol in chloroform).
[a]D¼þ46.9 (c 0.8, CH3OH). 1H NMR (CD3OD, 400 MHz)
d: 0.89 (3H, s, CH3-18), 0.94 (3H, s, CH3-19), 1.06 (3H, d,
J¼6.7 Hz, CH3-21), 2.49 (1H, m, H-20), 3.04 (1H, dd,
J¼11.0, 9.4 Hz, H-6 or H-7), 3.18 (1H, dd, J¼9.9, 9.4 Hz,
H-7 or H-6) 3.51 (1H, m, H-3), 4.60 (1H, bd, J¼3.8 Hz,
H-22), 5.69 (1H, bs, H-16), 6.12 (1H, bs, CvCHCO), 6.40
(1H, bs, HOCHO–). HR-ESMS: m/z 463.2690 (calcd
463.2696 for C26H39O7).
Acknowledgements
This work has been supported by the MIUR (‘Sostanze
`
naturali ed analoghi sintetici con attivita antitumorale’) and
by the Universita degli Studi di Salerno. LEO Pharma staff
`
´
of Denmark and Prof. Miguel Paya (Departamento de
Farmacologia, Facultad de Farmacia, Universidad de
Valencia, Spain) are gratefully acknowledged for the in
vitro anti-inflammatory tests.
.
6. Di Filippo, M.; Izzo, I.; Savignano, L.; Tecilla, P.; De
Riccardis, F. Tetrahedron 2003, 59, 1711–1717.
7. Izzo, I.; Della Monica, C.; Bifulco, G.; De Riccardis, F.
Tetrahedron 2004, 60, preceding paper. See doi: S0040-
4020(04)00658-1.
References and notes
8. Houston, T. A.; Tanaka, Y.; Koreeda, M. J. Org. Chem. 1993,
58, 4287–4292.
1. (a) Burgoyne, D. L.; Andersen, R. J. J. Org. Chem. 1992, 57,
525–535. (b) Takei, M.; Burgoyne, D. L.; Andersen;, R. J.
J. Pharm. Sci. 1994, 83, 1234–1235. (c) Andersen, R. J.;
Allen, T. M.; Burgoyne, D. L. PCT Int. Appl. Patent No:
WO94/14451, 1994.. (d) Bramley, A. M.; Langlands, J. M.;
Jones, A. K.; Burgoyne, D. L.; Ya, L.; Andersen, R. J.; Salari,
H. Br. J. Pharmacol. 1995, 115, 1433–1438. (e) More recently
contignasterol has been patented for the treatment of dry eyes:
Yanni, J. M.; Gamache, D. A. (Alcon, Inc., Switz.): PCT Int.
Appl. Patent No: WO02/94284, 2002.
9. No substantial improvements were observed varying the
reagents’ amounts (Me2AlCl: 6.4 equiv.!12.4 equiv.; 7:
3.2 equiv.!6.4 equiv.), reaction times (6 h!12 h) and tem-
peratures (278 8C!220 8C; 278 8C!0 8C; 278 8C!rt).
10. It is known that a lower ratio between Lewis acid and aldehyde
induces an Oppenauer-type oxidation of the secondary alcohol
present in the adduct (see Scheme II of Ref. 8).
11. The C-22 stereochemistries were assigned on strenght of
comparative analysis with our previous results. See: Izzo, I.;
Pironti, V.; Della Monica, C.; Sodano, G.; De Riccardis, F.
Tetrahedron Lett. 2001, 42, 8977–8980.
2. Shen, Y.; Burgoyne, D. L. J. Org. Chem. 2002, 67,
3908–3910, IPL-576,092 caused an 80% inhibition of the
inflammatory response, at an oral dose of 5 mg/kg, whereas
contignasterol, in the same model, caused a 60% inhibition of
the response at an oral dose of 50 mg/kg. In the in vitro studies,
IPL-576,092 showed a significant reduction in the release of
mediators of inflammation, including tumor necrosis factor-a
(TNF-a), hexosaminidase, prostaglandin-D2 (PGD2), and inter-
leukin 5 (IL5). IPL576,092 successfully completed Phase II
‘Allergen Challenge’ trial in April 2002 as a potential novel
oral anti-asthma therapy (in collaboration with Aventis
Pharma). The study confirmed the anti-inflammatory activity
of IPL576,092 in asthmatic subjects. IPL576,092 is now being
investigated for inflammatory diseases of the skin and eye
(visit: www.inflazyme.com).
12. A problem with BF3·OEt2 catalyzed ene reaction is that the
alcohol–Lewis acid complex, produced in the reaction, forms
a strong protic acid capable of protonating the double bond of
the ene adduct or the alkene. When Me2AlCl is used as Lewis
acid, the proton associated to the alcohol–Me2AlCl adduct is
quenched by the basic methyl aluminium substituent to yield
methane and an inert aluminium alkoxide which does not
undergo side reactions. See: (a) Snider, B. B.; Rodini, D. J.;
Kirk, T. C.; Cordova, R. J. Am. Chem. Soc. 1982, 104,
555–563. (b) Snider, B. B. Acc. Chem. Res. 1980, 13,
426–432.
13. Preliminary in vitro studies on the antiinflammatory activity of
compound 4 were performed at LEO Pharma research
laboratories of Denmark. These tests did not show any
3. (a) de Silva, E. D.; Scheuer, P. J. Tetrahedron Lett. 1980, 42,
1611–1614. (b) Soriente, A.; De Rosa, M.; Scettri, A.;

I. Izzo et al. / Tetrahedron 60 (2004) 5587–5593
5593
significant reduction in the release of TNF-a, IL-1, two
common inflammation activators. The assays followed the
procedures reported on: Ottosen, E. R.; Sørensen, M. D.;
17. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
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18. Dd¼dS2dR (CDCl3, 400 MHz). H3-21: Dd¼þ0.16 ppm; H-
16: Dd¼þ0.05 ppm; H-40(furane): Dd¼20.16 ppm.
¨
Bjorkling, F.; Skak-Nielsen, T.; Fjording, M. S.; Aaes, H.;
Binderup, L. J. Med. Chem. 2003, 5651–5662. In vivo tests
are currently in progress at LEO Pharma (Denmark).
14. Albizati, K. F.; Holman, T.; Faulkner, D. J.; Glaser, K. B.;
Jacobs, R. S. Experientia 1987, 43, 949–950.
19. Spectroscopic data of the g-hydroxy butenolide mojety are
consistent with those reported on literature. See: Kernan,
M. R.; Faulkner, D. J. J. Am. Chem. Soc. 1988, 53, 2773–2776.
20. Garcia Pastor, P.; Randazzo, A.; Gomez-Paloma, L.; Alcaraz,
M. J.; Paya, M. J. Pharmacol. Exp. Ther. 1999, 289, 166–172.
21. Posadas, I.; De Rosa, S.; Terencio, M. C.; Paya, M.; Alcaraz,
M. J. Br. J. Pharmacol. 2003, 138, 1571–1579.
15. Ihara, M.; Suzuki, S.; Taniguchi, N.; Fukumoto, K. J. Chem
Soc. Perkin. Trans. 1 1993, 2251–2258.
16. (a) Tsubuki, M.; Kanai, K.; Keino, K.; Kakinuma, N.; Honda,
T. J. Org. Chem. 1992, 57, 2930–2934. (b) Bartlett, P. A.
Tetrahedron 1980, 36, 1–72.