Synthetic studies of neoclerodane diterpenoids from Salvia splendens and evaluation of opioid receptor affinity

Article abstract of DOI:10.1016/j.tet.2008.08.043

Salvinorin A (1), a neoclerodane diterpene from the hallucinogenic mint Salvia divinorum, is the only known non-nitrogenous and specific κ-opioid agonist. Several structural congeners of 1 isolated from Salvia splendens (2-8) together with a series of semisynthetic derivatives (9-24), some of which possess a pyrazoline structural moiety (9, 19-22), have been tested for affinity at human μ, δ, and κ opioid receptors. None of these compounds showed high affinity binding to these receptors. However, 10 showed modest affinity for κ receptors suggesting that other natural neoclerodanes from different Salvia species may possess opioid affinity.

Full text of DOI:10.1016/j.tet.2008.08.043

Tetrahedron 64 (2008) 10041–10048
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthetic studies of neoclerodane diterpenoids from Salvia
splendens and evaluation of opioid receptor affinity
,
b
,
c
Gianfranco Fontana a, Giuseppe Savona a , Benjamın Rodrıguez , Christina M. Dersch ,
*
*
´
´
Richard B. Rothman c, Thomas E. Prisinzano d
a
`
`
Dipartimento di Chimica Organica ‘E. Paterno’, Universita degli Studi di Palermo, Parco d’Orleans 2, I-90128 Palermo, Italy
b
´
´
Instituto de Qu´ımica Organica, Consejo Superior de Investigaciones Cientıficas (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain
c Clinical Psychopharmacology Section, IRP, NIDA, NIH, DHHS, Baltimore, MD 21224, USA
d Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2008
Received in revised form 28 July 2008
Accepted 14 August 2008
Available online 16 August 2008
Salvinorin A (1), a neoclerodane diterpene from the hallucinogenic mint Salvia divinorum, is the only
known non-nitrogenous and specific
Salvia splendens (2–8) together with a series of semisynthetic derivatives (9–24), some of which possess
a pyrazoline structural moiety (9, 19–22), have been tested for affinity at human , and opioid re-
ceptors. None of these compounds showed high affinity binding to these receptors. However, 10 showed
modest affinity for receptors suggesting that other natural neoclerodanes from different Salvia species
may possess opioid affinity.
k-opioid agonist. Several structural congeners of 1 isolated from
m
,
d
k
k
Keywords:
Salvia splendens
Opioid receptors
Ó 2008 Elsevier Ltd. All rights reserved.
Neoclerodane diterpenes
Semisynthetic derivatives
15
1. Introduction
O
O
O
14
16
Salvinorin A (1), one of the neoclerodane diterpenes of Salvia
12
R2
AcO
H
´
divinorum Epling & Jativa (Labiatae), is a highly unusual drug that
O
O
R1
1
O
11
20
O
H
H
H
17
O
H
exerts its hallucinogenic effects as a potent and selective agonist of
AcO
O
O
10
the k
-opioid receptor.1 In the past few years, intensive research on
8
4
6
the pharmacology and chemical transformations of salvinorin A
have been made,1–6 and its biosynthetic pathway has been eluci-
dated7 and its asymmetric total synthesis has been achieved.8
Recently,9 some of us have reinvestigated the diterpene con-
stituents of Salvia splendens Sellow ex Roem. & Schult isolating four
new neoclerodanes (salvisplendins A–D, 4, 6, 7, and 8, respectively)
together with the artifact 9, that results from 8 by treatment of the
crude extract with diazomethane,9 and salviarin (2),10 splendidin
(3),11 and splenolides A and B (5),12 all of them previously reported
as constituents of this species.9–13
Taking into account the structural similarity between salvinorin
A (1) and the diterpenes found in S. splendens (2–9), we decided to
test these substances and two of their available derivatives (10 and
11)9 in order to explore whether such compounds may be useful as
neuropharmacological agents.
19
18
O
O
CO2Me
O
O
2 R1 = R2 = H
1
6
3 R1 = R2 = OAc
4 R1 = H, R2 = OH
5 R1 = H, R2 = OAc
O
O
O
OAc
H
H
H
H
O
O
RO
RO
3'
OR
N
N
O
O
O
O
O
O
9
R = H
8
R = H
11 R = Ac
7
R = H
10 R = Ac
* Corresponding authors. Tel.: þ39 091 597193; fax: þ39 091 596825 (G.S.); tel.:
þ34 91 5622900; fax: þ34 91 5644853 (B.R.).
Moreover, we have now obtained several new semisynthetic
E-mail addresses: giussav@unipa.it (G. Savona), iqor107@iqog.csic.es (B.
´
derivatives (12–24) starting from the more abundant constituents
Rodrıguez).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.043

10042
G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
of S. splendens (2, 3, 5, 7, and 9),9,13 and these compounds were also
assayed as potential agonists at the opioid receptors. Three cler-
odanes of S. splendens, salviarin (2), splendidin (3), and splenolide B
(5), have recently been tested for affinity at opioid receptors and
none of these diterpenes showed significant binding to any of the
opioid receptor subtypes.14 This work, however, demonstrates the
potential utility of evaluating other neoclerodanes for their in-
teraction with opioid receptors.
lactone in these compounds. Moreover, the HMBC spectrum of
14 showed correlation between the carbonyl carbon of the ac-
etate (d 170.0) and a methine proton (d 5.91, H-12), which in
turn correlated with the C-13, C-14, and C-16 furanic carbons (
d
119.7, 109.9, and 141.9, respectively), thus confirming the pro-
posed structures. NOE experiments on 13 and 14 established
that the hydrogens at C-8 and C-11 were
respectively, since irradiation at Me-20 produced, among others,
strong NOE enhancement in the signal of H-8
þ6.5% in 14), whereas irradiation at 4.58 and 4.49 (H-11
and 14, respectively) caused NOEs in the H-1 and H-10
a- and b-oriented,
a
(þ4.9% in 13 and
d
b
in 13
2. Results and discussion
b
b
pro-
As reported previously,1 salvinorin A (1) with the natural (
b)
configuration at C-8 shows higher affinity and efficacy at the k-
tons (þ8.1 and þ8.4% in 13; þ8.1 and þ11.5% in 14, respectively)
and not in Me-20.
Splendidin (3) also reacts in a similar way by treatment with
Na2CO3 in MeOH giving 15 (C20H22O7) (Scheme 1). The HMBC
spectra of 15 and its diacetyl derivative 16 (C24H26O9) showed the
same correlations that those observed for 13 and 14, and NOE ex-
opioid receptor. Hence, our first objective was to epimerize the
C-8 asymmetric center of salviarin, splendidin, and splenolide B
(2, 3, and 5, respectively). Treatment of 2 with K2CO3 in MeOH
yielded 8-epi-salviarin (12) (Scheme 1), a derivative, which had
´
periments were also in agreement with an
their H-8 and H-11 protons, respectively.
a- and b-orientation for
been obtained by Rodrıguez-Hahn and co-workers using simi-
lar reaction conditions.15 The complete assignment of the 1H
NMR spectrum together with other spectroscopic data of 12
have not been reported previously, and they are now included
The epimerization of the C-8 position of salviarin (2) under
basic conditions via enolate formation, followed by protonation
from the opposite face1,5 gave compound 12. In the case of 3 and 5,
however, no epimerization was observed and this may be
explained by an initial deacetylation at C-11, followed by a trans-
lactonization to the more stable cis fused 17,11-g-lactones (15 and
13, respectively), in which epimerization at C-8 via an enolate is
less favored.
Preparation of 8-epi-splenolide B (17) and 8-epi-splendidin (18)
was achieved as follows (Scheme 1). Treatment of 5 with 1,8-
diazobicyclo[5.4.0]undec-7-ene (DBU) in CH2Cl2 solution for 72 h at
room temperature yielded 17 (40%) together with minor quantities
of 4 (15%) and 13 (10%), whereas identical treatment of 3 in anhy-
drous CH2Cl2 for 24 h at room temperature gave 18 (70% yield) and
in Section 4. The
by NOE experiments because irradiation at
strong NOE enhancements in the H-6
protons (þ4.7, þ5.1, and þ15.3% NOE enhancement, re-
b
-configuration16 of H-8 in 12 was supported
d
2.62 (H-8) caused
b
,
H-10 and H-12b
b
,
spectively). This NOE behavior differs from that of the H-8
a
epimer 2.13 In addition, the coupling constant J8 ¼11.9 Hz
b,7a
and J8 ¼3.4 Hz in 12, as compared with those of
2
b,7b
(J8
b
¼4.0 Hz and J8
¼2.8 Hz),13 further supported the
a
,7
a
a,7b
-configuration of H-8.
When 5 was treated with K2CO3 in MeOH under the same
conditions that those described for (see Section 4), only
compound 13 (C20H22O6) was obtained and it was transformed
into a monoacetyl derivative (14, C22H24O7) after treatment with
Ac2O–pyridine (Scheme 1). The 1H and 13C NMR spectra of 13
and 14 showed the presence of a 17,11-
17,12- -lactone in 5. In particular, the HMBC correlations ob-
served for 13 and 14 between the H-11 proton and the C-17
2
starting material (3, 15%). Like in 12, the b-orientation of H-8 in 17
and 18 was strongly supported by NOE experiments and by the
observed vicinal J(H,H) couplings of the H-8 methine and H-7
methylene protons (see Section 4).
g-lactone instead of the
d
Treatment of salvisplendin C (7) with an ethereal solution of
diazomethane9,17,18 gave the pyrazoline derivative 19 (Scheme 2).
carbonyl carbon strongly support the presence of a 17,11-g-
O
O
R2
12
R2
O
H
R1
H
O
R1
11
H
H
H
O
H
O
8
O
O
O
O
12 R1 = R2 = H
13 R1 = H, R2 = OH
14 R1 = H, R2 = OAc
15 R1 = R2 = OH
17 R1 = H, R2 = OAc
18 R1 = R2 = OAc
d
d
16 R1 = R2 = OAc
23 R1 = H, R2 = OCS-Im
24 R1 = R2 = H
e
a
b
b
Salviarin (2)
8-epi-salviarin (12)
8-epi-splendidin (17)
8-epi-splenolide B(18)
a
a
17,11−γ
17,11−γ
-lactone (15)
Splendidin (3)
-lactone (13)
Splenolide B(5)
c
12-1H-imidazole-1-carbothioate (23)
Scheme 1. Reagents and conditions: (a) K2CO3/MeOH, rt, 4 h 65% (12), 73% (13), 58% (15); (b) DBU (10 equiv), CH2Cl2, rt, 72 h 40% (17), 24 h 70% (18); (c) CDI, DMAP (cat.), CH2Cl2,
reflux, 2 h, 80%; (d) Ac2O/Py 1:2, rt, quant.; (e) n-Bu3SnH, AIBN (cat.), toluene, reflux, 6 h, 45%.

G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
10043
O
4. Experimental
4.1. General
O
O
OAc
H
OAc
H
H
O
H
H
RO
3'
Melting points were determined on a Kofler block and are un-
corrected. Optical rotations were measured on a Perkin–Elmer
241 MC polarimeter. IR spectra were obtained on a Perkin–Elmer
Spectrum One spectrophotometer. 1H and 13C NMR spectra were
recorded in CDCl3 solution, except for 13 and 15 (methanol-d4), on
a Varian INOVA 400 spectrometer at 400 and 100 MHz, re-
spectively. Chemical shifts are reported in the
erenced to residual CHCl3 7.25) or methanol-d4
for protons and to the solvent signals (dCDCl 77.00, dCD OD 49.00) for
carbons. All the assignments for protons and carbons were in
agreement with 2D COSY, gHSQC, gHMBC, and 1D NOESY spectra.
Mass spectra were registered in the positive EI (70 eV) mode on
a Hewlett–Packard 5973 instrument. Elemental analyses were
conducted on a LECO CHNS-932 apparatus. Merck Si gel 7734 (70–
230 mesh) deactivated with 15% (w/v) of water was used for gravity
column chromatography and Si gel Merck LiChroprep 15–25/25–
a
3'
OR
b
OH
N
N
N
O
N
O
O
O
O
O
9
R = H
21 R = Ac
22 R = COPh
19 R = H
20 R = Ac
Salvisplendin C (7)
c
d
d
scale and are ref-
(d
(d
3.30) signals
Scheme 2. Reagents and conditions: (a) CH2N2, Et2O, 0 ꢁC, 1 h, 73%; (b) Ac2O/Py 1:2,
40 ꢁC, one week, 55%; (c) Ac2O/Py 1:2, rt, overnight, quant.; (d) PhCOCl (10 equiv),
DMAP (cat.), CH2Cl2/Py 2:1, 0 ꢁC to rt, overnight, 50%.
3
3
The regio- and stereochemistry of the pyrazoline moiety of 19 are
identical to those of the already described derivative 9,9 as it was
evidenced by comparison of their 1H and 13C NMR spectra (see Ref.
9 and Section 4).19
The acetyl derivatives 2019 and 21 and the benzoate 22 were
prepared by standard procedures (Scheme 2) and their structural
assignments were confirmed by extensive spectroscopic studies
(see Section 4).
40 mm 1/1 was used for flash chromatography (elution under 0.7 psi
of Ar). Merck 5554 Kieselgel 60 F254 sheets were used for thin-layer
chromatographic analysis. Petroleum ether (bp 50–70 ꢁC) was used
for column chromatography.
Finally, compound 24 was obtained from 13 by initial formation
of the 12-1H-imidazole-1-carbothioate (23) followed by reaction of
this intermediate with n-Bu3SnH (Scheme 1).
Compounds 2–22 and 24 were then evaluated for affinity at
opioid receptors. These analogues were screened to gain
a greater understanding into the role of the orientation of the
4.2. Compounds for biological assays
Samples of compounds 2–11 and starting materials (2, 3, 5, 7,
and 9) for obtaining the new derivatives 12–24 were available from
a previous work.9
furan ring plays in the high affinity and selectivity of 1 for
k
4.3. General procedure for reaction of compounds 2, 3, and 5
with potassium carbonate–methanol
receptors. Several pyrazolines were also evaluated based on
a previous report, which suggested that this ring system may
potentially impart CNS depressant activity and/or anti-in-
flammatory activity.20 Unfortunately, none of these compounds
showed high affinity binding to human opioid receptors
(Ki>10,000 nM). However, diterpene 10 showed weak affinity for
The starting material (0.5 mmol) was treated with 5 mL of
a saturated solution of K2CO3 in MeOH. The resulting solution was
stirred for 4 h at room temperature and then neutralized by adding
2 N HCl and extracted with 5 portions (10 mL each) of 2:1 CHCl3–
EtOH. The organic phase was dried over Na2SO4 and the solvent was
evaporated in vacuo. The crude of reaction was purified by flash
chromatography (FC). Elution with 3:1 EtOAc–petroleum ether
gave 12 (110 mg, 65% yield) starting from salviarin (2). Elution with
99:1 CHCl3–MeOH gave 13 (130 mg, 73%) from splenolide B (5).
Elution with 39:1 CHCl3–MeOH gave 15 (108 mg, 58%) from
splendidin (3).
k
opioid receptors (Ki¼6910ꢀ570 nM). These results are not
surprising given the differences in this series of agents com-
pared to 1. Some potential reasons for the lack of affinity
compared to 1 are (a) the absence of a ketone at C-1; (b) the
lack of a C-2 substituent; (c) the inverted stereochemistry at C-
8; (d) the inverted stereochemistry at C-12; and (e) the in-
corporation of the C-4 carbomethoxy group into an additional
ring. To begin to address possible explanations, several ana-
logues were prepared. It was envisioned that inversion of the C-
8 stereochemistry would result in enhanced affinity at opioid
receptors. This structural change, however, did not increase the
affinity of 3 (18) or 4 (17) for opioid receptors. These changes,
however, are not parallel to the salvinorin A series. Given the
previous SAR for 1 that the C-2 position has profound effects on
affinity for opioid receptors,21 21 and 22 were synthesized from
9. Interestingly, introduction of an acetyl group (21) did not
4.3.1. Compound 12 (8-epi-salviarin)
Colorless needles (EtOAc–petroleum ether); mp 232–235 ꢁC;
18
18
[a
]
ꢂ49.5 (c 0.105, CHCl3); lit.15 mp 233–235 ꢁC; [
a
]
ꢂ48 (c 0.1,
D
D
CHCl3); Rf¼0.55 (silica gel, 3:2 EtOAc–petroleum ether); IR (KBr)
nmax 3152, 3134, 2951, 1792, 1759, 1728, 1507, 1450, 1287, 1221, 1180,
1156, 1032, 993, 875, 808, 735, 702, 602 cmꢂ1 1H NMR (400 MHz,
;
CDCl3)
d
7.45 (1H, dd, J16,14¼0.9 Hz, J16,15¼1.8 Hz, H-16), 7.42 (1H, t,
J15,14¼J15,16¼1.8 Hz, H-15), 6.40 (1H, dd, J14,15¼1.8 Hz, J14,16¼0.9 Hz,
H-14), 6.00 (1H, ddt, J2,1 ¼J2,4 ¼2.4 Hz, J2,1 ¼5.1 Hz, J2,3¼9.9 Hz, H-
increase affinity for
k opioid receptors and the addition of
a benzoyl group (22) did not increase affinity for
m
opioid
a
b
b
2), 5.62 (1H, dtd, J3,1 ¼J3,4 ¼2.8 Hz, J3,1 ¼1.4 Hz, J3,2¼9.9 Hz, H-3),
receptors.
a
b
b
5.35 (1H, dd, J12
¼11.1 Hz, J12
¼6.4 Hz, H-12b), 4.18 (1H, dd,
b,11a
b,11b
J19a,6 ¼1.8 Hz, J19a,19b¼9.1 Hz, pro-S H-19a), 4.14 (1H, d,
b
3. Conclusion
J19b,19a¼9.9 Hz, pro-R H-19b), 2.80 (1H, tdd, J4 ¼J4 ¼2.8 Hz,
b
,1
a
b,3
J4 ¼2.5 Hz, J4 ¼2.4 Hz, H-4
b
), 2.62 (1H, dd, J8 ¼11.9 Hz,
b
,1
b
b
,2
b,7a
The data collected in this work indicate that the previous
structure–activity relationships (SAR) may not be applicable to
these neoclerodanes of general structure 9. They also suggest that
these two series are not binding in an identical manner and that the
orientation of the furan ring is a key interaction in the mode of
binding of neoclerodane diterpenes at opioid receptors.
J8 ¼3.4 Hz, H-8
b
), 2.15 (2H, m, H-1
b
and H-7
), 2.10 (1H, dd,
), 2.06 (1H, m, H-1 ), 1.88
¼11.1 Hz, H-11 ), 1.87 (1H, dd,
), 1.85 (1H, dddd, J7 ¼3.8 Hz,
b), 2.11 (1H, dt,
b,7b
J6 ¼13.8 Hz, J6 ¼J6 ¼3.8 Hz, H-6
a
a
,6
b
a
,7
a
a,7b
J11
¼14.0 Hz, J11
¼6.4 Hz, H-11
b
a
b
,11
a
b,12b
(1H, dd, J11
¼14.0 Hz, J11
a
a
,11
b
a,12b
J10 ¼11.8 Hz, J10 ¼4.7 Hz, H-10
b
b
,1a
b
,1
b
a,6a
J7 ¼12.1 Hz, J7 ¼14.8 Hz, J7 ¼11.9 Hz, H-7
a), 1.35 (1H, dddd,
a
,6
b
a
,7
b
a,8b

10044
G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
J6 ¼13.8 Hz, J6 ¼12.1 Hz, J6 ¼3.9 Hz, J6
¼1.8 Hz, H-6
b
),
C-3), 110.8 (d, C-14), 88.7 (d, C-11), 70.8 (t, C-19), 68.0 (d, C-12), 66.7
(d, C-1), 53.8 (d, C-4), 47.8 (d, C-8), 46.8 (d, C-10), 44.7 (s, C-9), 42.9
(s, C-5), 31.9 (t, C-6), 17.7 (q, C-20), 17.3 (t, C-7); EIMS m/z 374 [M]þ
(3), 356 (7), 278 (61), 259 (25), 203 (33), 159 (20), 145 (41), 135 (83),
117 (38), 97 (100), 91 (42), 81 (15), 69 (14). Anal.: C 64.27%, H 6.02%;
calcd for C20H22O7: C 64.16%, H 5.92%.
b
,6
a
b
,7
a
b,7
b
b,19a
0.87 (3H, s, Me-20); these assignments are in agreement with the
partial 1H NMR data previously reported15 for 8-epi-salviarin; 13C
NMR (100 MHz, CDCl3)
d 175.5 (s, C-18), 173.3 (s, C-17), 143.9 (d, C-
15),139.6 (d, C-16),129.2 (d, C-2),124.1 (s, C-13),121.1 (d, C-3),108.4
(d, C-14), 70.1 (t, C-19), 69.9 (d, C-12), 52.3 (d, C-4), 47.5 (d, C-8), 47.3
(d, C-10), 43.3 (t, C-11), 41.6 (s, C-5), 36.1 (s C-9), 34.9 (t, C-6), 22.3 (t,
C-1), 20.4 (q, C-20), 18.5 (t, C-7); EIMS m/z 342 [M]þ (64), 327 (4),
314 (2), 298 (17), 283 (18), 231 (40), 218 (20), 159 (41), 145 (39), 131
(55), 121 (45), 117 (47), 105 (46), 94 (100), 91 (89), 79 (25), 65 (13),
55 (11). Anal.: C 70.08%, H 6.59%; calcd for C20H22O5: C 70.16%, H
6.48%.
4.4. 8-epi-Splenolide B (17) from splenolide B (5)
To a solution of 5 (50 mg, 0.125 mmol) in CH2Cl2 (0.5 mL), 170 mL
(173 mg, 1.14 mmol) of DBU were added in one portion via syringe.
The solution was left under Ar for 72 h at room temperature. Then
the reaction mixture was quenched by addition of 1 N HCl (1 mL)
and was extracted with CHCl3 (4ꢃ5 mL). The collected organic
phase was washed with brine and dried over Na2SO4. Removal of
the solvent by distillation at reduced pressure followed by FC (1–5%
acetone–CH2Cl2 in gradient) successively yielded 17 (20 mg, 40%), 4
(7 mg, 15%), and 13 (4 mg, 10%).
4.3.2. Compound 13
18
Colorless needles (EtOAc); mp 254–256 ꢁC; [
a]
ꢂ59.8 (c 0.194,
D
Me2CO); Rf¼0.42 (silica gel, 95:5 CHCl3–MeOH); IR (KBr) nmax 3488,
3138, 3110, 3070, 2942, 2864, 2580, 1765, 1599, 1502, 1445, 1386,
1165, 1023, 873, 840, 738, 699, 602 cmꢂ1 1H NMR (400 MHz,
;
CD3OD)
d
7.42 (1H, dt, J16,12¼J16,14¼0.9 Hz, J16,15¼1.8 Hz, H-16), 7.41
Compound 17: colorless rectangular plates (EtOAc–petroleum
18
(1H, t, J15,14¼J15,16¼1.8 Hz, H-15), 6.43 (1H, dd, J14,15¼1.8 Hz,
ether); mp 201–203 ꢁC; [
a
]
ꢂ84.4 (c 0.263, CHCl3); Rf¼0.35 (silica
D
J14,16¼0.9 Hz, H-14), 6.03 (1H, ddt, J2,1 ¼J2,4 ¼2.2 Hz, J2,1 ¼5.9 Hz,
gel, 5% acetone–CH2Cl2); IR (KBr) nmax 3138, 3036, 2918, 1765, 1740,
a
b
b
J2,3¼10.0 Hz, H-2), 5.52 (1H, dtd, J3,1 ¼J3,4 ¼2.9 Hz, J3,1 ¼1.2 Hz,
1730, 1503, 1385, 1249, 1237, 1190, 1176, 1153, 1024, 1011, 967, 876,
a
b
b
J3,2¼10.0 Hz, H-3), 4.91 (1H, dd, J12,11 ¼4.1 Hz, J12,16¼0.9 Hz, H-12),
798, 702, 599 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.51 (1H, ddd,
b
4.38 (1H, d, J11 ¼4.1 Hz, H-11
b
), 4.25 (1H, d, J19a,19b¼9.2 Hz, pro-R
J16,12 ¼1.8 Hz, J16,14¼0.9 Hz, J16,15¼1.9 Hz, H-16), 7.42 (1H, t,
b
,12
b
H-19a), 4.20 (1H, dd, J19b,6 ¼1.9 Hz, J19b,19a¼9.2 Hz, pro-S H-19b),
J15,14¼J15,16¼1.9 Hz, H-15), 6.55 (1H, dd, J14,15¼1.9 Hz, J14,16¼0.9 Hz,
H-14), 5.94 (1H, ddt, J2,1 ¼J2,4 ¼2.5 Hz, J2,1 ¼5.8 Hz, J2,3¼10.0 Hz, H-
b
2.73 (2H, m, H-4
b
and H-8
a
), 2.18 (1H, ddddd, J1 ¼18.4 Hz,
b
,1
a
a
b
b
J1 ¼5.9 Hz, J1 ¼1.2 Hz, J1 ¼2.4 Hz, J1
¼4.6 Hz, H-1
b
), 2.05
2), 5.60 (1H, dtd, J3,1 ¼J3,4 ¼2.7 Hz, J3,1 ¼1.4 Hz, J3,2¼10.0 Hz, H-3),
b
,2
b
,3
b
,4
b
b,10
b
a
b
b
(1H, ddddd, J1 ¼18.4 Hz, J1 ¼2.2 Hz, J1 ¼2.9 Hz, J1 ¼2.8 Hz,
5.30 (1H, dd, J12
¼2.3 Hz, J12 ¼1.8 Hz, H-12
b
), 5.12 (1H, d,
), 4.13 (2H, br s, H2-19), 2.81 (1H, tdd,
J4 ¼J4 ¼2.7 Hz, J4 ¼2.4 Hz, J4 ¼2.5 Hz, H-4 ), 2.78 (1H, dd,
), 2.27 (1H, dtd, J7 ¼3.5 Hz,
a
,1
b
a
,2
a
,3
a,4
b
b
,11
a
b,16
J1
¼11.8 Hz, H-1
a
), 2.03 (1H, dddd, J7 ¼3.8 Hz, J7 ¼4.2 Hz,
J11
¼2.3 Hz, H-11
a
a
,10
b
b
,6
a
b
,6
b
a
,12
b
J7 ¼14.8 Hz, J7 ¼2.9 Hz, H-7
b
), 1.79 (1H, dddd, J7 ¼4.0 Hz,
b
b
,7
a
b
,8
a
a
,6
a
b
,1
a
b
,3
b,1b
b,2
J7 ¼13.9 Hz, J7 ¼14.8 Hz, J7 ¼5.6 Hz, H-7
a
), 1.73 (1H, dd,
J8 ¼12.3 Hz, J8 ¼3.9 Hz, H-8
b
a
,6
b
a
,7
b
a,8
a
b
,7
a
b
,7
b
b,6a
J10 ¼11.8 Hz, J10 ¼4.6 Hz, H-10
b
), 1.73 (1H, m, H-6 ), 1.20 (1H,
a
J7 ¼J7 ¼3.9 Hz, J7 ¼14.8 Hz, H-7
b
), 2.16 (3H, s, 11
b
-OAc),
),
), 1.90 (2H, m, H-
), 1.72 (1H, dddd, J7 ¼3.2 Hz, J7 ¼13.8 Hz, J7
b
,1
a
b,1b
b
,6
b
b
,8
b
b,7a
dddd, J6 ¼14.0 Hz, J6 ¼13.9 Hz, J6 ¼4.2 Hz, J6
¼1.9 Hz, H-
2.09 (1H, ddd, J6 ¼13.8 Hz, J6 ¼3.2 Hz, J6 ¼3.5 Hz, H-6
a
b,6
a
b,7
a
b
,7
b
b
,19b
a
,6
b
a
,7
a
a,7b
6
b
), 1.03 (3H, s, Me-20); 13C NMR (100 MHz, CD3OD)
d
179.1 (s, C-
2.04 (1H, dd, J10 ¼11.8 Hz, J10 ¼6.6 Hz, H-10
b
b
,1
a
b,1b
17), 177.5 (s, C-18), 144.2 (d, C-15), 140.8 (d, C-16), 130.5 (d, C-2),
126.3 (s, C-13), 121.0 (d, C-3), 109.6 (d, C-14), 87.0 (d, C-11), 70.0 (t,
C-19), 67.2 (d, C-12), 53.2 (d, C-4), 46.9 (d C-8), 44.1 (s, C-9), 41.8 (s,
C-5), 41.4 (d, C-10), 32.0 (t, C-6), 23.3 (t, C-1), 17.1 (q, C-20),17.0 (t, C-
7); EIMS m/z 358 [M]þ (30), 340 (20), 304 (35), 262 (100), 233 (23),
203 (6), 187 (5), 171 (4), 157 (14), 139 (38), 129 (13), 117 (16), 105
(14), 97 (29), 91 (28), 81 (8). Anal.: C 67.21%, H 6.29%; calcd for
1a
and H-1
b
¼
a,7b
a
,6a
a
,6
b
14.8 Hz, J7 ¼12.3 Hz, H-7
a
), 1.34 (1H, td, J6 ¼J6 ¼13.8 Hz,
a
,8
b
b,6
a
b,7a
J6 ¼3.9 Hz, H-6
b
), 0.76 (3H, s, Me-20); 13C NMR (100 MHz, CDCl3)
175.2 (s, C-18), 170.7 (s, C-17), 169.9 (s, 11-OCOCH3), 144.0 (d,
b,7b
d
C-15), 139.4 (d, C-16), 128.9 (d, C-2), 124.1 (s, C-13), 120.9 (d, C-3),
108.3 (d, C-14), 78.6 (d, C-12), 74.2 (d, C-11), 69.6 (t, C-19), 52.4 (d,
C-4), 43.2 (d, C-8), 40.9 (s, C-5), 39.4 (d, C-10), 39.2 (s, C-9), 34.7 (t,
C-6), 21.8 (t, C-1), 21.0 (q, 11-OCOCH3), 18.6 (t, C-7), 15.2 (q, C-20);
EIMS m/z 400 [M]þ (1), 358 (1), 340 (63), 325 (100), 312 (1), 297 (4),
244 (5), 189 (8), 143 (10), 129 (13), 117 (11), 105 (11), 95 (22), 91 (27),
81 (10), 55 (7). Anal.: C 66.08%, H 6.19%; calcd for C22H24O7: C
65.99%, H 6.04%.
C
20H22O6: C 67.02%, H 6.19%.
4.3.3. Compound 15
18
Colorless fine needles (MeOH); mp 260–262 ꢁC; [
a]
ꢂ101.5 (c
D
0.481, Me2CO); Rf¼0.20 (silica gel, 95:5 CHCl3–MeOH); IR (KBr) nmax
3492, 3443, 3140, 3039, 2945, 2585, 2550, 2430, 1774, 1759, 1500,
1371, 1296, 1170, 1020, 961, 875, 797, 702, 607 cmꢂ1
;
1H NMR
4.5. 8-epi-Splendidin (18) from splendidin (3)
(400 MHz, CD3OD)
d
7.51 (1H, dt, J16,12¼J16,14¼0.8 Hz, J16,15¼1.9 Hz,
H-16), 7.45 (1H, t, J15,14¼J15,16¼1.9 Hz, H-15), 6.49 (1H, dd,
To a solution of 3 (50 mg, 0.109 mmol) in anhydrous CH2Cl2
(0.5 mL), 150 mL (153 mg, 1.00 mmol) of DBU were added in one
portion via syringe. The solution was left under Ar for 24 h at room
temperature. Workup as described above for 17 followed by FC
(49:1 CH2Cl2–acetone as eluent) gave 18 (35 mg, 70%) and
unreacted 3 (7.5 mg, 15%).
J14,15¼1.9 Hz, J14,16¼0.8 Hz, H-14), 5.91 (1H, ddd, J2,1 ¼1.8 Hz,
a
J2,3¼10.0 Hz, J2,4 ¼2.6 Hz, H-2), 5.62 (1H, d, J11 ¼3.6 Hz, H-11
b
),
b
b,12
5.51 (1H, ddd, J3,1 ¼2.0 Hz, J3,2¼10.0 Hz, J3,4 ¼3.2 Hz, H-3), 4.97 (1H,
a
b
dd, J12,11 ¼3.6 Hz, J12,16¼0.8 Hz, H-12), 4.38 (1H, dddd, J1 ¼1.8 Hz,
b
a,2
J1 ¼2.0 Hz, J1 ¼3.2 Hz, J1
¼10.4 Hz, H-1
a), 4.35 (1H, d,
a
,3
a,4
b
a,10b
J19a,19b¼9.2 Hz, pro-R H-19a), 4.20 (1H, dd, J19b,6 ¼1.8 Hz,
Compound 18: colorless rectangular plates (EtOAc–n-pentane);
b
18
J19b,19a¼9.2 Hz, pro-S H-19b), 2.80 (1H, td, J4 ¼J4 ¼3.2 Hz,
mp 232–235 ꢁC; [
a
]
ꢂ142.1 (c 0.151, CHCl3); Rf¼0.30 (silica gel,
b,1
a
b,3
D
J4 ¼2.6 Hz, H-4
b
), 2.66 (1H, ddd, J8 ¼1.2 Hz, J8 ¼5.4 Hz,
49:1 CH2Cl2–acetone); IR (KBr) nmax 3139, 3109, 3015, 2922, 1771,
1734, 1370, 1238, 1175, 1160, 1026, 965, 876, 607 cmꢂ1 1H NMR
(400 MHz, CDCl3)
b
,2
a
,6
a
a,7a
J8 ¼2.4 Hz, H-8
a
), 1.93 (1H, dddd, J7 ¼2.7 Hz, J7 ¼4.0 Hz,
;
a
,7
b
b
,6a
b,6b
J7 ¼14.8 Hz, J7 ¼2.4 Hz, H-7
b
), 1.82 (1H, dddd, J7 ¼3.8 Hz,
d
7.47 (1H, dt, J16,12 ¼J16,14¼0.8 Hz, J16,15¼1.7 Hz,
b
,7
a
b
,8
a
a
,6
a
b
J7 ¼13.8 Hz, J7 ¼14.8 Hz, J7 ¼5.4 Hz, H-7
a
), 1.75 (1H, d,
H-16), 7.41 (1H, t, J15,14¼J15,16¼1.7 Hz, H-15), 6.56 (1H, dd,
a
,6
b
a
,7
b
a,8a
J10 ¼10.4 Hz, H-10
b
),1.63 (1H, dddd, J6 ¼14.0 Hz, J6 ¼3.8 Hz,
J14,15¼1.7 Hz, J14,16¼0.8 Hz, H-14), 5.71 (1H, ddd, J2,1 ¼2.8 Hz,
b
,1
a
a
,6
b
a
,7
a
a
J6 ¼2.7 Hz, J6 ¼1.2 Hz, H-6
a
), 1.21 (1H, dddd, J6 ¼14.0 Hz,
J2,3¼10.0 Hz, J2,4 ¼1.6 Hz, H-2), 5.69 (1H, dd, J3,2¼10.0 Hz,
a
,7
b
a
,8
a
b
,6
a
b
J6 ¼13.8 Hz, J6 ¼4.0 Hz, J6
¼1.8 Hz, H-6
b
), 1.20 (3H, s, Me-
180.5 (s, C-17), 177.5 (s, C-18),
144.3 (d, C-15),141.6 (d, C-16),137.6 (d, C-2),127.1 (s, C-13),120.7 (d,
J3,4 ¼1.6 Hz, H-3), 5.50 (1H, dd, J1 ¼2.8 Hz, J1 ¼1.6 Hz,
b
,7
a
b
,7
b
b
,19b
b
a
,2
a,4b
20); 13C NMR (100 MHz, CD3OD)
d
J1
H-12
¼10.8 Hz, H-1
a
), 5.40 (1H, dd, J12
¼0.4 Hz, J12 ¼0.8 Hz,
a
,10
b
b
,11
a
b,16
b
), 5.10 (1H, d, J11
¼0.4 Hz, H-11
a), 4.23 (1H, d,
a
,12b

G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
10045
J19a,19b¼9.2 Hz, pro-R H-19a), 4.18 (1H, dd, J19b,6 ¼1.7 Hz,
the reaction was chromatographed (Si gel 230–400 mesh column,
20 g, 4:1 petroleum ether–EtOAc as eluent) yielding 20 (13.5 mg,
55%).
b
J19b,19a¼9.2 Hz, pro-S H-19b), 2.89 (1H, q, J4 ¼J4 ¼J4 ¼1.6 Hz,
b
,1
a
b
,2
b,3
H-4
b
), 2.88 (1H, dd, J8 ¼12.6 Hz, J8 ¼3.8 Hz, H-8
b), 2.45 (1H, d,
b
,7
a
b,7b
J10 ¼10.8 Hz, H-10 ), 2.33 (1H, dddd, J7 ¼3.2 Hz, J7 ¼3.6 Hz,
b
b
,1
a
b
,6
a
b,6b
J7 ¼14.8 Hz, J7 ¼3.8 Hz, H-7
b
), 2.22 (3H, s, 11
b
-OAc), 2.10 (3H,
),
1.71 (1H, dddd, J7 ¼3.2 Hz, J7 ¼14.0 Hz, J7 ¼14.8 Hz,
4.7.1. Compound 14
b
,7
a
b,8b
s, 1
b
-OAc), 2.07 (1H, dt, J6 ¼13.8 Hz, J6 ¼J6 ¼3.2 Hz, H-6
a
Colorless prisms (EtOAc–petroleum ether); mp 130–132 ꢁC;
a
,6
b
a
,7a
a,7b
18
[
a
]
ꢂ30.6 (c 0.435, CHCl3); Rf¼0.40 (silica gel, 99:1 CH2Cl2–ace-
a
,6a
a
,6
b
a
,7
b
D
J7 ¼12.6 Hz, H-7
a
), 1.42 (1H, dddd, J6 ¼13.8 Hz, J6 ¼14.0 Hz,
tone); IR (KBr) nmax 3134, 3033, 2937, 2263, 1777, 1758, 1738, 1504,
1372, 1235, 1158, 1023, 962, 874, 799, 756, 741, 697, 603 cmꢂ1 1H
NMR (400 MHz, CDCl3)
a
,8
b
b
,6
a
b,7a
J6 ¼3.6 Hz, J6
¼1.7 Hz, H-6
b
), 0.80 (3H, s, Me-20); 13C NMR
173.8 (s, C-18), 170.6 (s, 1-OCOCH3), 170.3 (s, 11-
;
b
,7
b
b,19b
(100 MHz, CDCl3)
d
d
7.49 (1H, dd, J16,14¼0.7 Hz, J16,15¼1.8 Hz,
OCOCH3), 169.9 (s, C-17), 143.9 (d, C-15), 138.9 (d, C-16), 130.7 (d, C-
2), 124.9 (s, C-13), 122.4 (d, C-3), 108.0 (d, C-14), 79.2 (d, C-12), 76.2
(d, C-11), 69.8 (t, C-19), 68.2 (d, C-1), 52.2 (d, C-4), 43.1 (d, C-8), 42.0
(d, C-10), 41.7 (s, C-5), 39.2 (s, C-9), 34.2 (t, C-6), 21.4 (q, 1-OCOCH3),
21.2 (q, 11-OCOCH3), 18.2 (t, C-7), 15.1 (q, C-20); EIMS m/z 458 [M]þ
(1), 398 (39), 356 (100), 323 (52), 203 (27), 189 (30), 176 (18), 161
(10), 129 (10), 110 (8), 95 (20), 91 (12), 81 (10). Anal.: C 62.59%, H
5.89%; calcd for C24H26O9: C 62.88%, H 5.72%.
H-16), 7.41 (1H, t, J15,14¼J15,16¼1.8 Hz, H-15), 6.49 (1H, dd,
J14,15¼1.8 Hz, J14,16¼0.7 Hz, H-14), 5.99 (1H, ddt, J2,1 ¼J2,4 ¼2.4 Hz,
a
b
J2,1 ¼4.8 Hz, J2,3¼10.2 Hz, H-2), 5.91 (1H, d, J12,11 ¼3.1 Hz, H-12),
b
b
5.60 (1H, dddd, J3,1 ¼3.2 Hz, J3,1 ¼1.2 Hz, J3,2¼10.2 Hz, J3,4 ¼3.4 Hz,
a
b
b
H-3), 4.49 (1H, d, J11 ¼3.1 Hz, H-11
b
), 4.18 (1H, dd, J19a,6 ¼1.6 Hz,
b
,12
b
J19a,19b¼9.0 Hz, pro-S H-19a), 4.14 (1H, d, J19b,19a¼9.0 Hz, pro-R H-
19b), 2.73 (1H, tt, J4 ¼J4 ¼3.4 Hz, J4 ¼J4 ¼2.4 Hz, H-4
b),
b
,1
a
b
,3
b,1
b
b,2
2.20 (1H, dddd, J7 ¼3.8 Hz, J7 ¼4.0 Hz, J7 ¼14.5 Hz,
b,6
a
b
,6
b
b,7a
J7 ¼2.8 Hz, H-7 ), 2.14 (1H, ddddd, J1 ¼18.0 Hz, J1 ¼2.4 Hz,
b
b
,8
a
a
,1b
a,2
4.6. Preparation of compound 19 from salvisplendin C (7)
J1 ¼3.2 Hz, J1 ¼3.4 Hz, J1
¼11.8 Hz, H-1
a
), 2.10 (2H, m, H-1
b
a
,3
a
,4
b
a,10b
and H-8
a
), 2.07 (3H, s, 12-OAc), 1.77 (1H, dtd, J6 ¼14.0 Hz,
a,6b
A solution of 7 (100 mg, 0.257 mmol) in MeOH (1 mL) was
treated with an excess of CH2N2 in Et2O at 0 ꢁC. After 1 h, the
J6 ¼J6 ¼3.8 Hz, J6 ¼1.4 Hz, H-6
a
), 1.73 (1H, dd,
), 1.64 (1H, dddd,
),
a
,7
a
a,7
b
a,8a
J10 ¼11.8 Hz,
J10 ¼5.2 Hz,
H-10b
b
,1a
b,1b
solvent was removed in vacuo and pure pyrazoline derivative 19
J7 ¼3.8 Hz, J7 ¼14.0 Hz, J7 ¼14.5 Hz, J7 ¼5.4 Hz, H-7
a
a
,6
a
a
,6
b
a,7
b
a,8a
18
was recovered as an amorphous solid. [
a
]
ꢂ26.4 (c 0.106,
1.21 (1H, tdd, J6 ¼J6 ¼14.0 Hz, J6 ¼4.0 Hz, J6
¼1.6 Hz, H-
D
b,6
a
b,7
a
b
,7
b
b,19a
CHCl3); Rf¼0.30 (silica gel, 3:2 EtOAc–petroleum ether); IR (KBr)
nmax 3463, 3130, 2942, 1766, 1543, 1489, 1434, 1374, 1241, 1185,
6
b
), 1.08 (3H, s, Me-20); 13C NMR (100 MHz, CDCl3)
d
176.3 (s, C-
17), 175.1 (s, C-18), 170.0 (s, 12-OCOCH3), 144.0 (d, C-15), 141.9 (d,
C-16), 128.9 (d, C-2), 121.0 (d, C-3), 119.7 (s, C-13), 109.9 (d, C-14),
84.1 (d, C-11), 68.6 (t, C-19), 67.1 (d, C-12), 52.1 (d, C-4), 45.5 (d, C-
8), 42.7 (s, C-9), 40.9 (s, C-5), 40.8 (d, C-10), 31.0 (t, C-6), 22.7 (t, C-
1), 21.1 (q, 12-OCOCH3), 16.6 (q, C-20), 16.5 (t, C-7); EIMS m/z 400
[M]þ (2), 358 (30), 340 (19), 304 (32), 262 (100), 233 (24), 203 (7),
187 (6), 157 (16), 139 (35), 129 (14), 117 (18), 105 (14), 97 (28), 91
(29), 81 (8). Anal.: C 66.15%, H 6.01%; calcd for C22H24O7: C 65.99%,
H 6.04%.
1018, 969, 925, 875, 810, 755, 602 cmꢂ1 1H NMR (400 MHz,
;
CDCl3)
d
7.33 (1H, dd, J16,14¼1.0 Hz, J16,15¼1.8 Hz, H-16), 7.32 (1H, t,
J15,14¼J15,16¼1.8 Hz, H-15), 6.34 (1H, dd, J14,15¼1.8 Hz, J14,16¼1.0 Hz,
H-14), 5.68 (1H, dd, J12,11a¼6.7 Hz, J12,11b¼5.1 Hz, H-12), 5.45 (1H,
0b,30
a
d, J19a,19b¼9.0 Hz, pro-R H-19a), 4.63 (1H, d, J3
¼16.8 Hz, H-
30
4.23 (1H, dd, J3
b
), 4.27 (1H, dd, J19b,6 ¼2.0 Hz, J19b,19a¼9.0 Hz, pro-S H-19b),
b
¼6.0 Hz, J3
¼16.8 Hz, H-30
a
), 4.23 (1H, m, H-
0a,3
a
0a,30
b
7
6
H-1
b
b
), 2.74 (1H, ddd, J6 ¼14.9 Hz, J6 ¼3.7 Hz, J6 ¼2.0 Hz, H-
b
,6
a
b,7
b
b,19b
), 2.58 (1H, dd, J6 ¼14.9 Hz, J6 ¼2.6 Hz, H-6
a
), 2.29 (1H, m,
a
,6
b
a,7b
b
), 2.26 (1H, m, H-1
a
), 2.18 (1H, ddd, J3 ¼6.3 Hz,
4.7.2. Compound 16
a,2a
18
,30
a
D
J3 ¼13.1 Hz, J3
¼6.0 Hz, H-3
a
), 2.05 (1H, dd, J11a,11b¼15.8 Hz,
Amorphous, white powder; [
a]
ꢂ78.9 (c 0.503, CHCl3); Rf¼0.35
a
,2
b
a
J11a,12¼6.7 Hz, H-11a), 1.97 (3H, s, 11-OAc), 1.79 (1H, qd,
(silica gel, 99:1 CH2Cl2–acetone); IR (KBr) nmax 3146, 2947, 1781,
J8 ¼2.9 Hz, J8 ¼7.0 Hz, H-8
b
), 1.77 (1H, dd, J11b,11a¼15.8 Hz,
1740, 1503, 1373, 1234, 1168, 1024, 962, 915, 875, 800, 756,
b
,7
b
b,17
J11b,12¼5.1 Hz, H-11b), 1.67 (1H, dddd, J2 ¼2.7 Hz, J2 ¼3.1 Hz,
603 cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
7.47 (1H, dd, J16,14¼0.9 Hz,
a
,1
a
a,1b
J2 ¼13.4 Hz, J2 ¼6.3 Hz, H-2
a
), 1.37 (1H, dd, J10 ¼11.9 Hz,
J16,15¼1.8 Hz, H-16), 7.39 (1H, t, J15,14¼J15,16¼1.8 Hz, H-15), 6.42 (1H,
a
,2
b
a
,3
a
b,1a
J10 ¼2.5 Hz, H-10
b
), 1.16 (3H, d, J17,8 ¼7.0 Hz, Me-17), 0.88 (3H,
dd, J14,15¼1.8 Hz, J14,16¼0.9 Hz, H-14), 5.89 (1H, d, J12,11 ¼6.3 Hz, H-
b
,1
b
b
b
s, Me-20), 0.33 (1H, qd, J2 ¼J2 ¼J2 ¼13.1 Hz, J2 ¼4.5 Hz,
12), 5.75 (1H, ddd, J2,1 ¼2.0 Hz, J2,3¼10.0 Hz, J2,4 ¼2.6 Hz, H-2), 5.69
b
,1
a
b,2
a
b,3
a
b
,1b
a
b
H-2b
); 13C NMR (100 MHz, CDCl3)
d
170.7 (s, C-18), 169.9 (s, 12-
(1H, ddd, J3,1 ¼2.0 Hz, J3,2¼10.0 Hz, J3,4 ¼2.6 Hz, H-3), 5.65 (1H, ddt,
a
b
OCOCH3), 143.4 (d, C-15), 140.1 (d, C-16), 125.7 (s, C-13), 108.7 (d,
C-14), 95.7 (s, C-4), 81.8 (t, C-30), 72.7 (d, C-7), 71.2 (t, C-19), 64.5
(d, C-12), 45.5 (s, C-5), 44.7 (d, C-10), 42.4 (t, C-11), 40.3 (d, C-8),
39.4 (s, C-9), 38.2 (t, C-6), 35.4 (d, C-3), 28.9 (t, C-2), 21.2 (q, 11-
OCOCH3), 19.4 (q, C-20), 18.7 (t, C-1), 12.6 (q, C-17); EIMS m/z 430
[M]þ (3), 370 (1), 342 (2), 300 (4), 277 (6), 247 (19), 231 (15), 218
(33), 190 (35), 175 (29), 121 (28), 105 (28), 94 (100), 91 (34), 81
(27), 55 (21). Anal.: C 63.95%, H 7.19%, N 6.42%; calcd for
J1 ¼J1 ¼2.0 Hz, J1 ¼2.6 Hz, J1
¼10.8 Hz, H-1
a), 5.18 (1H, d,
a
,2
a
,3
a
,4
b
a,10b
J11 ¼6.3 Hz, H-11 ), 4.24 (1H, d, J19a,19b¼9.2 Hz, pro-R H-19a),
b
b
,12
4.21 (1H, dd, J19b,6 ¼1.6 Hz, J19b,19a¼9.2 Hz, pro-S H-19b), 2.81 (1H,
b
q, J4 ¼J4 ¼J4 ¼2.6 Hz, H-4 ), 2.32 (1H, ddd, J8 ¼1.2 Hz,
b
b,1
a
b,2
b
,3
a,6a
J8 ¼4.4 Hz, J8 ¼2.8 Hz, H-8
a), 2.17 (3H, s, 1
b-OAc), 2.11 (1H, d,
a
,7
a
a,7b
J10 ¼10.8 Hz, H-10
b
), 2.10 (1H, dddd, J7 ¼3.6 Hz, J7 ¼4.0 Hz,
b
,1a
b,6
a
b,6b
J7 ¼14.4 Hz, J7 ¼2.8 Hz, H-7
b
), 2.02 (3H, s, 12-OAc), 1.76 (1H,
dtd, J6 ¼13.8 Hz, J6 ¼J6 ¼3.6 Hz, J6 ¼1.2 Hz, H-6 ), 1.71
J7 ¼14.4 Hz,
b
,7
a
b,8a
a
a
,6
b
a,7
a
a
,7
b
a,8a
C
23H30N2O6: C 64.17%, H 7.02%, N 6.51%.
(1H,
dddd,
J7 ¼3.6 Hz,
J7 ¼13.6 Hz,
a
,6
a
a
,6
b
a,7b
J7 ¼4.4 Hz, H-7
a), 1.27 (1H, dddd, J6 ¼13.8 Hz, J6 ¼13.6 Hz,
a
,8
a
b
,6
a
b,7a
4.7. Procedure for the acetylation of compounds 9, 13, 15,
and 19
J6 ¼4.0 Hz, J6
¼1.6 Hz, H-6
,19b
b
), 1.11 (3H, s, Me-20); 13C NMR
175.8 (s, C-17), 173.6 (s, C-18), 169.8 (s, 1
b
,7
b
b
(100 MHz, CDCl3)
d
b-
OCOCH3),169.2 (s,12-OCOCH3),143.9 (d, C-15),142.0 (d, C-16),131.0
(d, C-2), 122.4 (d, C-3), 120.6 (s, C-13), 109.3 (d, C-14), 83.7 (d, C-11),
68.7 (t, C-19), 68.3 (d, C-1), 65.3 (d, C-12), 51.9 (d, C-4), 45.2 (d, C-8),
Treatment of 9 (20 mg, 0.052 mmol), 13 (50 mg, 0.140 mmol),
and 15 (50 mg, 0.134 mmol) with Ac2O–pyridine (1:2, 1 mL) for
24 h at room temperature followed by standard workup and puri-
fication by FC yielded quantitatively the acetyl derivatives 21 (19:1
CH2Cl2–acetone as eluent), 14 (elution with 32:1 CH2Cl2–acetone),
and 16 (elution with 19:1 CH2Cl2–acetone), respectively. Compound
19 (20 mg, 0.052 mmol) was treated with Ac2O–pyridine (1:2,
3 mL) for one week at 40 ꢁC. After standard workup the residue of
43.0 (d, C-10), 42.5 (s, C-9), 41.5 (s, C-5), 30.4 (t, C-6), 21.3 (q, 1b-
OCOCH3), 21.0 (q, 12-OCOCH3), 17.2 (q, C-20), 16.3 (t, C-7); EIMS m/z
458 [M]þ (1), 416 (3), 398 (100), 356 (85), 320 (20), 277 (17), 259
(57), 231 (17), 222 (17), 203 (28), 157 (20), 143 (44), 139 (48), 135
(55), 117 (26), 105 (14), 97 (420), 91 (28), 81 (14). Anal.: C 62.69%, H
5.84%; calcd for C24H26O9: C 62.88%, H 5.72%.

10046
G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
4.7.3. Compound 20
4.8. Benzoylation of compound 9 to give compound 22
20
Amorphous, white solid; [
a
]
ꢂ38.2 (c 0.204, CHCl3); Rf¼0.55
D
(silica gel, 3:2 EtOAc–petroleum ether); IR (KBr) nmax 3135, 2947,
1774, 1738, 1545, 1434, 1371, 1234, 1180, 1020, 976, 875, 603 cmꢂ1
1H NMR (400 MHz, CDCl3)
7.33 (2H, m, H-15 and H-16), 6.33
Benzoyl chloride (120
wise via syringe, at 0 ꢁC under Ar, to a solution of 40 mg
(0.103 mmol) of 9 and a little crystal of 4-DMAP in 750 L of dry 2:1
mL, 145 mg, 1.03 mmol) was added drop-
;
d
m
(1H, dd, J14,15¼2.0 Hz, J14,16¼1.0 Hz, H-14), 5.67 (1H, dd,
CH2Cl2–pyridine. The solution was warmed at room temperature
and left to stir for 24 h. Afterward, the solution was carefully poured
into a crushed-ice/NaHCO3 mixture (5 mL). Extraction with EtOAc
(4ꢃ5 mL) was followed by removal of residual pyridine by washing
with 6 N HCl and subsequent neutralization of the combined or-
ganic phase with an aqueous saturated solution of NaHCO3. Finally,
the organic phase was dried (Na2SO4) and the solvent was removed
by distillation in vacuo, giving a crude of reaction that was purified
J12,11a¼7.4 Hz, J12,11b¼4.7 Hz, H-12), 5.45 (1H, ddd, J7 ¼2.5 Hz,
b,6a
J7 ¼4.0 Hz, J7 ¼3.5 Hz, H-7 ), 4.99 (1H, d, J19a,19b¼9.3 Hz, pro-
b
b
,6
b
b,8b
¼17.0 Hz, H-30
b), 4.31 (1H, dd,
0b,30
a
R H-19a), 4.66 (1H, d, J3
J19b,6 ¼2.1 Hz, J19b,19a¼9.3 Hz, pro-S H-19b), 4.24 (1H, dd,
b
J3
¼6.0 Hz,
¼17.0 Hz,
H-30
¼2.1 Hz, H-6
a
),
2.86
), 2.54 (1H, dd,
), 2.20 (1H, ddd, J3 ¼6.6 Hz,
(1H,
ddd,
0a,3
a
0a,30
b
J3
J6 ¼15.6 Hz, J6 ¼4.0 Hz, J6
b
b
,6
a
b,7
b
b,19b
J6 ¼15.6 Hz, J6 ¼2.5 Hz, H-6
a
a
a
,6
b
a
,7
b
a
a,2a
a
,30
J3 ¼12.7 Hz, J3
a
¼6.0 Hz, H-3
), 2.12 (3H, s, 7
a-OAc), 2.07 (1H,
by FC (39:1 CH2Cl2–acetone as eluent) to give pure 22 (25 mg, 50%).
,2
b
20
dd, J11a,11b¼16.0 Hz, J11a,12¼7.4 Hz, H-11a), 1.97 (3H, s, 11-OAc), 1.95
(1H, qd, J8 ¼3.5 Hz, J8 ¼7.0 Hz, H-8 ), 1.78 (1H, dd,
J11b,11a¼16.0 Hz, J11b,12¼4.7 Hz, H-11b), 1.71 (1H, dddd,
),
Amorphous, white solid; [
a
]
D
ꢂ37.1 (c 0.197, CHCl3); Rf¼0.35 (silica
b
gel, 95:5 CH2Cl2–acetone); IR (KBr) nmax 3066, 2974, 2928, 1780,
b
,7
b
b,17
1719, 1602, 1451, 1376, 1272, 1176, 1153, 1116, 1026, 909, 874, 716,
J2 ¼2.3 Hz, J2 ¼3.5 Hz, J2 ¼13.5 Hz, J2 ¼6.6 Hz, H-2
a
600 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.36 (2H, m, H-15, and H-
a
,1
a
a,1
b
a,2
b
a,3a
1.42 (1H, dd, J10 ¼12.0 Hz, J10 ¼2.4 Hz, H-10
b
), 1.35 (1H,
16), 6.34 (1H, t, J14,15¼J14,16¼1.5 Hz, H-14), 5.01 (1H, d,
b
,1
a
b,1b
¼17.4 Hz, H-30
b
), 5.01 (1H, dd, J12
¼9.9 Hz, J12
¼7.1 Hz,
b,1a
¼
b
,2
a
b
,2
b
b
,10
b
0b,30
a
b
,11
a
b,11b
dddd, J1
b
13.0 Hz, J1 ¼3.5 Hz, J1 ¼4.0 Hz, J1
¼2.4 Hz, H-
J3
H-12
1
J1
), 1.26 (1H, dddd, J1 ¼13.0 Hz, J1 ¼2.3 Hz, J1 ¼12.6 Hz,
b
), 4.54 (1H, d, J19a,19b¼9.4 Hz, pro-R H-19a), 4.42 (1H, ddd,
a
,1
b
a
,2
a
a,2b
¼12.0 Hz, H-1
a
), 1.04 (3H, d, J17,8 ¼7.0 Hz, Me-17), 0.84 (3H,
J2 ¼11.3 Hz, J2 ¼4.3 Hz, J2 ¼10.3 Hz, H-2
b
), 4.39 (1H, dd,
a
,10
b
b
b,1a
b,1
b
b,3a
s, Me-20), 0.40 (1H, dddd, J2 ¼12.6 Hz, J2 ¼4.0 Hz,
J19b,6 ¼1.9 Hz, J19b,19a¼9.4 Hz, pro-S H-19b), 4.36 (1H, dd,
b,1
a
b,1b
b
); 13C NMR (100 MHz, CDCl3)
170.0 (s, C-18), 169.82 (s, 12-OCOCH3), 169.78 (s, 7-OCOCH3),
J3
¼6.1 Hz,
¼17.4 Hz,
H-30
a
),
2.81
(1H,
¼1.9 Hz, H-6
dddd,
),
), 2.31 (1H, dd,
and H-
b
,2
a
b
,3
a
0a,3
a
0a,30
b
J3
J2 ¼13.5 Hz, J2 ¼12.7 Hz, H-2
b
d
J6 ¼14.3 Hz, J6 ¼13.3 Hz, J6 ¼3.9 Hz, J6
b
b,6
a
b
,7
a
b,7
b
b
,19b
a,2
b
a
,30
a
143.5 (d, C-15), 140.1 (d, C-16), 125.6 (s, C-13), 108.6 (d, C-14), 95.2
(s, C-4), 81.8 (t, C-30), 73.4 (d, C-7), 70.6 (t, C-19), 64.4 (d, C-12),
45.3 (s, C-5), 44.4 (d, C-10), 42.0 (t, C-11), 39.4 (d, C-8), 39.3 (s, C-
9), 35.8 (t, C-6), 35.4 (d, C-3), 28.8 (t, C-2), 21.4 (q, 7-OCOCH3),
21.2 (q, 12-OCOCH3), 19.1 (q, C-20), 18.7 (t, C-1), 12.0 (q, C-17);
EIMS m/z 472 [M]þ (1), 430 (1), 412 (1), 402 (3), 342 (7), 260 (30),
249 (37), 231 (49), 185 (33), 172 (42), 157 (53), 119 (48), 111 (47),
94 (100), 81 (31), 55 (18). Anal.: C 63.63%, H 6.71%, N 5.79%; calcd
for C25H32N2O7: C 63.54%, H 6.83%, N 5.93%.
2.51 (1H, dd, J3 ¼10.3 Hz, J3
¼6.1 Hz, H-3
a
J11
¼13.2 Hz, J11
¼7.1 Hz, H-11
b
), 2.17 (2H, m, H-6
a
b
,11
a
b,12b
7
1
b
b
), 2.05 (1H, ddd, J1 ¼12.8 Hz, J1 ¼4.3 Hz, J1
¼2.1 Hz, H-
b
,1
a
b,2
b
b,10b
), 1.86 (1H, dd, J11
¼13.2 Hz, J11
¼9.9 Hz, H-11
a
), 1.81 (1H,
a
,11
b
a,12b
dd, J10 ¼12.9 Hz, J10 ¼2.1 Hz, H-10
b
), 1.78 (1H, ddd,
b,1a
b,1b
J7 ¼3.9 Hz, J7 ¼13.3 Hz, J7 ¼15.0 Hz, H-7
a
a
), 1.56 (1H, ddd,
), 1.20 (3H, s, Me-
a
,6
a
a
,6
b
a,7b
J1 ¼12.8 Hz, J1 ¼11.3 Hz, J1
¼12.9 Hz, H-1
a
,1
b
a
,2
b
a,10b
17), 0.85 (3H, s, Me-20), 2
a
-benzoate 8.02 (2H, dd, J¼8.3, 1.5 Hz, H-
200 and H-600), 7.61 (1H, tt, J¼8.0, 1.5 Hz, H-400), 7.47 (2H, br t,
J¼8.2 Hz, H-300 and H-500); 13C NMR (100 MHz, CDCl3)
d 169.4 (s, C-
4.7.4. Compound 21
18), 143.4 (d, C-15), 139.0 (d, C-16), 128.3 (s, C-13), 108.7 (d, C-14),
96.7 (s, C-4), 83.2 (s, C-8), 79.7 (t, C-30), 74.4 (d, C-2), 70.0 (d, C-12),
68.5 (t, C-19), 47.2 (s, C-9), 45.7 (s, C-5), 44.6 (t, C-11), 41.2 (d, C-3),
39.8 (d, C-10), 29.7 (t, C-7), 27.3 (t, C-1), 27.2 (t, C-6), 26.9 (q, C-17),
17.2 (q, C-20), 2-benzoate 165.9 (s, C-7), 133.7 (d, C-400), 129.6 (2d, C-
200 and C-600), 129.3 (s, C-100), 128.6 (2d, C-300 and C-500); EIMS m/z
490 [M]þ (1), 475 (1), 462 (3), 447 (5), 368 (2), 164 (8), 121 (21), 105
(100), 95 (10), 91 (9), 77 (32). Anal.: C 68.38%, H 6.41%, N 5.59%;
calcd for C28H30N2O6: C 68.56%, H 6.16%, N 5.71%.
20
Colorless fine needles (EtOAc–n-pentane); mp 222–224 ꢁC; [
a]
D
ꢂ54.6 (c 0.509, CHCl3); Rf¼0.42 (silica gel, 95:5 CH2Cl2–acetone); IR
(KBr) nmax 3127, 3018, 2966, 2928,1782,1741,1542,1500,1459,1373,
1240, 1202, 1151, 1045, 1025, 965, 910, 874, 797, 719, 600 cmꢂ1; 1H
NMR (400 MHz, CDCl3)
d
7.35 (1H, t, J15,14¼J15,16¼1.5 Hz, H-15), 7.34
(1H, dd, J16,14¼0.8 Hz, J16,15¼1.5 Hz, H-16), 6.32 (1H, dd,
J14,15¼1.5 Hz, J14,16¼0.8 Hz, H-14), 4.95 (1H, dd, J12
¼9.9 Hz,
,11
a
¼17.3 Hz, H-30b
b), 4.50 (1H,
b
,11
b
0b,30
a
), 4.91 (1H, d, J3
J12
¼7.1 Hz, H-12
b
d, J19a,19b¼9.4 Hz, pro-R H-19a), 4.32 (1H, dd, J19b,6 ¼2.0 Hz,
b
0a,3
a
J19b,19a¼9.4 Hz, pro-S H-19b), 4.30 (1H, dd, J3
¼6.1 Hz,
4.9. Preparation of compound 23 from compound 13
0a,30
b
b,1a
b,1b
J3
¼17.3 Hz, H-30
a
), 4.14 (1H, ddd, J2 ¼11.4 Hz, J2 ¼4.3 Hz,
J2 ¼10.4 Hz, H-2
b
), 2.76 (1H, dddd, J6 ¼14.0 Hz, J6 ¼13.4 Hz,
A
solution
of
13
(60 mg,
0.167 mmol), 1,10-thio-
b
,3
a
b,6
a
b,7a
J6 ¼3.3 Hz, J6
¼2.0 Hz, H-6
b
), 2.31 (1H, dd, J3 ¼10.4 Hz,
carbonyldiimidazole (100 mg, 0.561 mmol), and 4-DMAP (4 mg,
0.033 mmol) in dry CH2Cl2 (7 mL) was refluxed under Ar for 2 h.
Then, the solvent was distilled in vacuo and the solid residue was
b
,7
,30
b
b
,19b
a
,2
b
J3
¼6.1 Hz, H-3
a
), 2.25 (1H, dd, J11
¼13.2 Hz, J11
¼7.1 Hz,
a
a
b
,11
a
b
,12b
H-11
b), 2.12 (2H, m, H-6a and H-7b), 2.08 (3H, s, 2a-OAc), 1.89 (1H,
ddd, J1
12.7 Hz, J1 ¼4.3 Hz, J1
¼2.2 Hz, H-1
b
), 1.83 (1H, dd,
purified by FC (3:2 petroleum ether–EtOAc as eluent) to give pure
b,1
a
¼
b
,2
b
b,10b
20
J11
¼13.2 Hz, J11
¼9.9 Hz, H-11
a
), 1.75 (1H, ddd, J7 ¼3.8 Hz,
23 (62 mg, 80%). Vitreous, yellowish solid; [
a]
ꢂ9.5 (c 0.042,
a
,11
b
a
,12
b
a
,6
a
D
J7 ¼13.4 Hz, J7 ¼15.5 Hz, H-7
a
), 1.70 (1H, dd, J10 ¼13.0 Hz,
CHCl3); Rf¼0.35 (silica gel, 3:2 petroleum ether–EtOAc); IR (KBr)
nmax 3127, 3031, 2916, 1776, 1632, 1527, 1503, 1444, 1295, 1155, 1117,
1062, 1024, 964, 873, 794, 746, 664, 603 cmꢂ1; 1H NMR (400 MHz,
a
,6
b
a
,7
b
b,1a
J10 ¼2.2 Hz, H-10
b
a
), 1.42 (1H, ddd, J1 ¼12.7 Hz, J1 ¼11.4 Hz,
b
,1
b
a
,1
b
a,2b
J1
¼13.0 Hz, H-1
), 1.18 (3H, s, Me-17), 0.83 (3H, s, Me-20); 13C
170.4 (s, 2-OCOCH3), 169.3 (s, C-18), 143.3
a
,10
b
NMR (100 MHz, CDCl3)
d
CDCl3)
d
7.51 (1H, dd, J16,14¼0.9 Hz, J16,15¼1.9 Hz, H-16), 7.43 (1H, t,
(d, C-15),139.0 (d, C-16),128.2 (s, C-13),108.7 (d, C-14), 96.6 (s, C-4),
83.1 (s, C-8), 79.7 (t, C-30), 73.8 (d, C-2), 69.9 (d, C-12), 68.5 (t, C-19),
47.1 (s, C-9), 45.6 (s, C-5), 44.5 (t, C-11), 41.0 (d, C-3), 39.6 (d, C-10),
29.7 (t, C-7), 27.143 (2d, C-1 and C-6), 26.9 (q, C-17), 21.1 (q, 2-
OCOCH3), 17.2 (q, C-20); EIMS m/z 428 [M]þ (0.2), 413 (3), 400 (4),
385 (33), 368 (4), 358 (8), 340 (3), 325 (3), 309 (5), 269 (10), 229
(16), 164 (31), 121 (100), 105 (26), 95 (42), 91 (39), 81 (45), 55 (19).
Anal.: C 64.52%, H 6.71%, N 6.41%; calcd for C23H28N2O6: C 64.47%, H
6.59%, N 6.54%.
J15,14¼J15,16¼1.9 Hz, H-15), 6.44 (1H, dd, J14,15¼1.9 Hz, J14,16¼0.9 Hz,
H-14), 6.00 (1H, ddt, J2,1 ¼J2,4 ¼2.4 Hz, J2,1 ¼5.0 Hz, J2,3¼10.1 Hz, H-
a
b
b
2), 5.60 (1H, dddd, J3,1 ¼3.0 Hz, J3,1 ¼1.3 Hz, J3,2¼10.1 Hz,
a
b
J3,4 ¼3.4 Hz, H-3), 5.56 (1H, d, J12,11 ¼1.2 Hz, H-12), 4.66 (1H, d,
b
b
J11 ¼1.2 Hz, H-11
b
), 4.20 (1H, dd, J19a,6 ¼1.6 Hz, J19a,19b¼9.0 Hz,
b
,12
b
pro-S H-19a), 4.14 (1H, d, J19b,19a¼9.0 Hz, pro-R H-19b), 2.71 (1H,
ddt, J4 ¼2.8 Hz, J4 ¼J4 ¼2.4 Hz, J4 ¼3.4 Hz, H-4
b
), 2.18 (1H,
), 1.94 (1H, br dd,
), 1.75 (1H, br dt, J6 ¼14.0 Hz,
b
,1
a
b
,1
b
b
,2
b,3
m, H-1
a
), 2.14 (1H, m, H-1
b), 1.98 (1H, m, H-7
b
J8 ¼5.2 Hz, J8 ¼2.1 Hz, H-8
a
a
,7
a
a
,7
b
a,6b

G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
10047
J6 ¼J6 ¼3.2 Hz, H-6
a
), 1.69 (1H, dd, J10 ¼11.8 Hz,
under 95% air/5% CO2 at 37 ꢁC. Cell monolayers were harvested and
a
,7
a
a
,7
b
b,1a
J10 ¼5.0 Hz, H-10
b
b
), 1.59 (1H, dddd, J7 ¼3.2 Hz, J7 ¼13.5 Hz,
frozen in ꢂ80 ꢁC.
,1
b
a
,6
a
a,6b
J7 ¼14.4 Hz, J7 ¼5.2 Hz, H-7
a
), 1.75 (1H, br dt, J6 ¼14.0 Hz,
a
,7
b
a
,8
a
a,6b
J6 ¼J6 ¼3.2 Hz, H-6
a
), 1.23 (3H, s, Me-20), 1.18 (1H, dddd,
J6 ¼14.0 Hz, J6 ¼13.5 Hz, J6 ¼3.9 Hz, J6 ¼1.6 Hz, H-6 ),
1H-imidazole-1-carbothioate 7.69 (1H, s, H-20), 7.10 (2H, s, H-40 and
H-50); 13C NMR (100 MHz, CDCl3)
176.4 (s, C-17), 175.1 (s, C-18),
4.11.1. Opioid binding assays
a
,7
a
a,7b
b
We used either [125I]IOXY (6
b
-iodo-3,14-dihydroxy-17-cyclo-
b
,6
a
b,7
a
b,7
b
b,19a
propylmethyl-4,5
a
-epoxymorphinan) (SA¼2200 Ci/mmol) to label
d
m
,
d
and k D
binding sites [20] or [3H][ -Ala2-MePhe4,Gly-ol5]enke-
144.5 (d, C-15), 142.0 (d, C-16), 128.9 (d, C-2), 121.0 (d, C-3), 117.6 (s,
C-13), 110.7 (d, C-14), 85.6 (d, C-11), 68.5 (t, C-19), 52.2 (d, C-4), 47.3
(d, C-12), 46.1 (s, C-9), 44.1 (d, C-8), 41.3 (s, C-5), 41.1 (d, C-10), 31.0
(t, C-6), 22.8 (t, C-1), 16.9 (q, C-20), 16.6 (t, C-7), 1H-imidazole-1-
carbothioate 223.4 (þs, C]S), 134.9 (d, C-20), 132.0 (d, C-40), 121.9 (d,
C-50); EIMS m/z [M] absent, 453 [MꢂCH3]þ (1), 421 (2), 374 (29),
372 (25), 356 (6), 340 (80), 261 (72), 203 (22),157 (30), 145 (39), 129
(33), 113 (84), 108 (86), 91 (100), 81 (43), 76 (53), 65 (17), 53 (20).
Anal.: C 61.28%, H 5.02%, N 6.03%, S 6.91%; calcd for C24H24N2O6S: C
61.52%, H 5.16%, N 5.98%, S 6.84%.
phalin ([3H]DAMGO, SA¼44–48 Ci/mmol) to label MOP, [3H][
D-
Ala2,
D
-Leu5]enkephalin ([3H]DADLE, SA¼40–50 Ci/mmol) to label
DOP and [3H](ꢂ)-U69,593 (SA¼50 Ci/mmol) to label KOP binding
sites. On the day of the assay, cell pellets were thawed on ice for
15 min then homogenized with a polytron in 10 mL/pellet of ice-
cold 10 mM Tris–HCl, pH 7.4. Membranes were then centrifuged at
30,000ꢃg for 10 min, resuspended in 10 mL/pellet ice-cold 10 mM
Tris–HCl, pH 7.4 and again centrifuged 30,000ꢃg for 10 min.
Membranes were then resuspended in 25 ꢁC 50 mM Tris–HCl, pH
7.4 (w100 mL/pellet hMOP-CHO, 50 mL/pellet hDOP-CHO, and
120 mL/pellet hKOP-CHO). All assays took place in 50 mM Tris–
4.10. Preparation of compound 24 from compound 23
HCl, pH 7.4, with a protease inhibitor cocktail [bacitracin (100
mL), bestatin (10 g/mL), leupeptin (4 g/mL), and chymostatin
(2 g/mL)], in a final assay volume of 1.0 mL. All drug dilution
curves were made up with buffer containing 1 mg/mL BSA. Non-
specific binding was determined using 10 naloxone (for
mg/
m
m
A
solution of 23 (50 mg, 0.107 mmol), 150
mL
(162 mg,
m
0.535 mmol) of 96% n-Bu3SnH, and 2,20-azo-bis-isobutyronitrile
(AIBN, 3 mg, 0.018 mmol) in toluene (5 mL) was heated at reflux
under Ar for 6 h. Then, the mixture was evaporated to a little vol-
ume and partitioned between acetonitrile and n-hexane (5 mL
each). The acetonitrile layer was washed with n-hexane (4ꢃ5 mL)
to remove the residual tin compounds. The acetonitrile phase was
distilled in vacuo and the residue was purified by FC (39:1 acetone–
mM
[
125I]IOXY binding) or 20 levallorphan ([3H]DAMGO and
mM
[3H]DADLE) or 10
m
M (ꢂ)-U69,593 (for [3H]U69,593 binding).
[
125I]IOXY concentrations were w10 pM. The other [3H]radio-
ligands were used at w2 nM concentrations. Triplicate samples
were filtered with Brandell Cell Harvesters (Biomedical Research &
Development Inc., Gaithersburg, MD), over Whatman GF/B filters,
after a 2 h incubation at 25 ꢁC. For [125I]IOXY binding, the filters
were punched into 12ꢃ75 mm glass test tubes and counted in
a Micromedic gamma counter at 80% efficiency. For [3H]DAMGO,
[3H]DADLE, and [3H]-(ꢂ)U69,593 binding, the filters were
punched into 24-well plates to which was added 0.6 mL of LSC-
cocktail (Cytoscint). Samples were counted, after an overnight
extraction, in a Trilux liquid scintillation counter at 44% efficiency.
CH2Cl2 as eluent) to give 24 (16 mg, 45%). Colorless rectangular
20
plaques (EtOAc–n-pentane); mp 211–214 ꢁC; [
a]
ꢂ91.3 (c 0.196,
D
CHCl3); Rf¼0.50 (silica gel, 3:2 petroleum ether–EtOAc); IR (KBr)
nmax 3142, 3107, 2963, 2860, 1780, 1766, 1502, 1450, 1352, 1296,
1176, 1152, 1035, 966, 873, 785, 702, 602 cmꢂ1; 1H NMR (400 MHz,
CDCl3)
d
7.36 (1H, t, J15,14¼J15,16¼1.8 Hz, H-15), 7.31 (1H, ddd,
J16,12b¼1.0 Hz, J16,14¼0.8 Hz, J16,15¼1.8 Hz, H-16), 6.34 (1H, dd,
J14,15¼1.8 Hz, J14,16¼0.8 Hz, H-14), 5.97 (1H, ddt, J2,1 ¼J2,4 ¼2.4 Hz,
a
b
J2,1 ¼5.0 Hz, J2,3¼10.0 Hz, H-2), 5.60 (1H, dddd, J3,1 ¼3.1 Hz,
Opioid binding assays had w30 mg protein per assay tube. In-
hibition curves were generated by displacing a single concentra-
tion of radioligand by 10 concentrations of drug.
b
a
J3,1 ¼1.3 Hz, J3,2¼10.0 Hz, J3,4 ¼3.4 Hz, H-3), 4.35 (1H, dd,
b
b
J11
¼4.0 Hz, J11
¼9.2 Hz, H-11
b
), 4.19 (2H, s, H-19a and H-
b,12a
b,12b
19b), 2.74 (1H, tt, J4 ¼J4 ¼3.4 Hz, J4 ¼J4 ¼2.4 Hz, H-4
b), 2.71
b,1
a
b,3
b,1
b
b,2
(1H, br dd, J12a,11 ¼4.0 Hz, J12a,12b¼15.2 Hz, H-12a), 2.61 (1H, ddd,
4.11.2. [35S]GTP-
The [35S]-GTP-
g
-S binding assays
-S assays were conducted as described else-
b
J12b,11 ¼9.2 Hz, J12b,12a¼15.2 Hz, J12b,16¼1.0 Hz, H-12b), 2.54 (1H,
g
b
ddd, J8 ¼1.2 Hz, J8 ¼5.1 Hz, J8 ¼2.3 Hz, H-8
a
), 2.16 (1H,
where.22 In this description, buffer ‘A’ is 50 mM Tris–HCl, pH 7.4,
containing 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA and buffer ‘B’
is buffer A plus 1.67 mM DTT and 0.15% BSA. On the day of the assay,
cells were thawed on ice for 15 min and homogenized using
a
,6
a
a
,7
a
a,7b
dddd, J7 ¼3.8 Hz, J7 ¼4.0 Hz, J7 ¼14.8 Hz, J7 ¼2.3 Hz, H-
b
,6
a
b
,6
b
b
,7
a
b,8a
7
b
), 2.09 (1H, m, H-1
b
), 2.05 (1H, m, H-1
a
), 1.82 (1H, m, H-6
a), 1.79
(1H, dd, J10 ¼11.1 Hz, J10 ¼5.3 Hz, H-10
b
), 1.77 (1H, dddd,
b
,1
a
b,1b
J7 ¼4.1 Hz, J7 ¼13.6 Hz, J7 ¼14.8 Hz, J7 ¼5.1 Hz, H-7
a
b
),
),
a polytron in 50 mM Tris–HCl, pH 7.4, containing 4
m
g/mL leu-
g/mL
a
,6
a
a
,6b
a
,7
b
a,8a
1.27 (1H, ddd, J6 ¼14.2 Hz, J6 ¼13.6 Hz, J6 ¼4.0 Hz, H-6
peptin, 2 g/mL chymostatin, 10 g/mL bestatin, and 100 m
m
m
b,6
a
b,7
a
b
,7
b
1.03 (3H, s, Me-20); 13C NMR (100 MHz, CDCl3)
d
176.2 (s, C-17),
bacitracin. The homogenate was centrifuged at 30,000ꢃg for
175.3 (s, C-18), 143.2 (d, C-15), 140.2 (d, C-16), 128.8 (d, C-2), 121.1
(d, C-3), 119.7 (s, C-13), 111.3 (d, C-14), 83.8 (d, C-11), 68.7 (t, C-19),
52.1 (d, C-4), 45.6 (d, C-8), 42.9 (s, C-9), 41.0 (s, C-5), 39.8 (d, C-10),
31.2 (t, C-6), 25.9 (t, C-12), 22.7 (t, C-1), 17.2 (q, C-20), 16.7 (t, C-7);
EIMS m/z 342 [M]þ (56), 327 (1), 261 (100), 233 (7), 215 (7), 203 (8),
157 (24), 145 (24), 117 (28), 105 (27), 91 (57), 81 (34), 53 (17). Anal.:
C 70.51%, H 6.31%; calcd for C20H22O5: C 70.16%, H 6.48%.
10 min at 4 ꢁC, and the supernatant discarded. The membrane
pellets were resuspended in buffer B and used for [35S]GTP-
g
-S
-S binding was determined as described
previously. Briefly, test tubes received the following additions:
50 L buffer A plus 0.1% BSA, 50 L GDP in buffer A/0.1% BSA (final
concentration¼40 M), 50 L drug in buffer A/0.1% BSA, 50
35S]GTP-
-S in buffer A/0.1% BSA (final concentration¼50 pM), and
300 L of cell membranes (50 g of protein) in buffer B. The final
concentrations of reagents in the [35S]GTP-
-S binding assays were
50 mM Tris–HCl, pH 7.4, containing 100 mM NaCl, 10 mM MgCl2,
1 mM EDTA, 1 mM DTT, 40 M GDP, and 0.1% BSA. Incubations
proceeded for 3 h at 25 ꢁC. Nonspecific binding was determined
using GTP- -S (40 -S were sepa-
M). Bound and free [35S]-GTP-
binding assays. [35S]GTP-
g
m
m
m
m
mL
[
g
m
m
4.11. Biological assays
g
As described,22 the recombinant CHO cells (hMOP-CHO, hDOP-
CHO, and hKOP-CHO) were produced by stable transfection with
the respective human opioid receptor cDNA, and provided by Dr.
Larry Toll (SRI International, CA). The cells were grown on plastic
flasks in DMEM (90%) (hDOP-CHO and hKOP-CHO) or DMEM/F-12
(45%/45%) medium (hMOP-CHO) containing 10% FetalClone II
(HyClone) and Geneticin (G-418: 0.10–0.2 mg/mL) (Invitrogen)
m
g
m
g
rated by vacuum filtration through GF/B filters. The filters were
punched into 24-well plates to which was added 0.6 mL LSC-
cocktail (Cytoscint). Samples were counted, after an overnight ex-
traction, in a Trilux liquid scintillation counter at 27% efficiency.

10048
G. Fontana et al. / Tetrahedron 64 (2008) 10041–10048
4.11.3. Data analysis and statistics
National Institute on Drug Abuse. The content is the sole re-
sponsibility of the authors and does not necessarily represent the
official views of the National Institute on Drug Abuse or the Na-
tional Institutes of Health.
These methods are described elsewhere.22–24 For the [35S]GTP-
g
-S binding experiments, the percent stimulation of [35S]GTP-
g-S
binding was calculated according to the following formula: (SꢂB)/
Bꢃ100, where B is the basal level of [35S]GTP-
g-S binding and S is
the stimulated level of [35S]GTP-
g-S binding. Agonist dose–re-
References and notes
sponse curves (10 points/curve) are generated, and the data of
several experiments, typically 3, are pooled. The EC50 values (the
concentration that produces 50% maximal stimulation of [35S]GTP-
1. For a summary on the history and ethnopharmacology of Salvia divinorum, and
for the chemistry and pharmacology of salvinorin A, see: Harding, W. W.;
Tidgewell, K.; Byrd, N.; Cobb, H.; Dersch, C. M.; Butelman, E. R.; Rothman, R. B.;
Prisinzano, T. E. J. Med. Chem. 2005, 48, 4765–4771.
g-S binding) and Emax are determined using either the program
MLAB-PC (Civilized Software, Bethesda, MD), KaleidaGraph (Ver-
sion 3.6.4, Synergy Software, Reading, PA) or Prism 4.0 (GraphPad
Software, Inc., San Diego, CA). In most cases, the percent stimula-
tion is reported as a percent of the maximal stimulation as de-
termined with 1000 nM DAMGO, 500 nM SNC80 or 500 nM
(ꢂ)-U50,488 in the appropriate cell type. For determination of Ke
values using the ‘shift’ experimental design, agonist (DAMGO,
(ꢂ)-U50,488 or SNC80) dose–response curves are generated, using
the appropriate cell type, in the absence and presence (10 points/
curve) of a test agent. The data of several experiments, typically 3,
are pooled, and the Ke values were calculated according to the
equation: [Test Drug]/(EC50-2/EC50-1ꢂ1), where EC50-2 is the EC50
value in the presence of the test drug and EC50-1 is the value in the
absence of the test drug. For opioid binding experiments, the
pooled data of three experiments (typically 30 data points) were fit
to the two-parameter logistic equation for the best-fit estimates of
the IC50 and N values: Y¼100/(1þ([INHIBITOR]/IC50)N), where ‘Y’ is
the percent of control value. Ki values for test drugs are calculated
according to the standard equation: Ki¼IC50/(1þ[radioligand]/Kd]).
For the [125I]IOXY experiments, the concentration of [125I]IOXY was
far below its Kd values for MOP, KOP or DOP, so the IC50 values
equaled the Ki values. For the [3H]radioligands, the following Kd
values (nMꢀSD, n¼3) were used in the Ki calculation: [3H]DAMGO
(0.93ꢀ0.04), [3H]DADLE (1.9ꢀ0.3), and [3H](ꢂ)-U69,593 (11ꢀ0.6).
The corresponding Bmax values were (fmol/mg proteinꢀSD, n¼3)
[3H]DAMGO (1912ꢀ68), [3H]DADLE (3655ꢀ391), and [3H](ꢂ)-
U69,593 (3320ꢀ364).
2. Be´guin, C.; Richards, M. R.; Wang, Y.; Chen, Y.; Liu-Chen, L.-Y.; Ma, Z.; Lee, D. Y.
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J.-G.; Beguin, C.; Carlezon, W. A., Jr.; Cohen, B. Bioorg. Med. Chem. Lett. 2005, 15,
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a
- or
substituent is placed, respectively, below or above the plane of the formulas.
However, for clarity we use throughout the text the - or -nomenclature,
b-configuration should be described as ent-b or ent-a, indicating that the
a
b
indicating that a substituent is placed, respectively, below or above the plane of
the formulas depicted for the described substances.
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4.11.4. Drugs
19. It is of interest to note that the
a strong shielding effect on the H-2
respectively. Moreover, only one (H-30
pyrazoline part of 9 and 19–22 is coupled with the vicinal H-3
in these compounds, and in other 3 ,4 -pyrazoline derivatives of neoclerodan-
18,19-olides, the dihedral angle between H-30 is close to 90ꢁ (see
and H-3
b
-oriented pyrazoline ring of 19 and 20 causes
proton, which resonates at 0.33 and 0.40,
) of the 30-methylene protons of the
proton, because
Salvinorin
A related compounds were made up in 100%
b
d
DMSOþ14.3 M 2-mercaptoethanol to produce a 10 mM solution.
a
a
Compounds were then aliquoted into 50 mL volumes in microfuge
b
b
tubes and frozen at ꢂ80ꢁ C until the day of the assay. Any leftover
b
a
compound was discarded.
Rodrı´guez, B. Magn. Reson. Chem. 2001, 39, 150–154).
20. Krapcho, J.; Turk, C. F. J. Med. Chem. 1979, 22, 207–210.
21. Prisinzano, T. E.; Rothman, R. B. Chem. Rev. 2008, 108, 1732–1743.
22. Rothman, R. B.; Murphy, D. L.; Xu, H.; Godin, J. A.; Dersch, C. M.; Partilla, J. S.;
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801–810.
23. Xu, H.; Lu, Y. F.; Thomas, J. B.; Carroll, F. I.; Rice, K. C.; Rothman, R. B. Synapse
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Acknowledgements
This work was supported in part by funds from the Spanish
´
´
`
‘Comision Interministerial de Ciencia y Tecnologıa’ (CICYT), grant
No. CTQ2006-15279-C02-02, Italian ‘Universita degli Studi di
Palermo’, grant ‘Ex 60%-2005’, and R01DA018151 (to T.E.P.) from the
24. Xu, H.; Partilla, J. S.; Wang, X.; Rutherford, J. M.; Tidgewell, K.; Prisinzano, T. E.;
Bohn, L. M.; Rothman, R. B. Synapse 2007, 61, 166–175.