First total synthesis of the marine steroid alkaloid plakinamine B

Article abstract of DOI:10.1016/j.tet.2013.11.065

The first total synthesis of the marine steroid alkaloid plakinamine B (1) was accomplished in seven steps starting from known aldehyde 3. Key steps in this synthesis are the attachment of a vinylpyridine side chain by an aldol reaction, a chemoselective reduction of a pyridinium salt to a vinyl tetrahydropyridine, and the introduction of the 3α-methylamino group under Mitsunobu conditions. Plakinamine B and some of its precursors with amino or pyridinium side chains show significant antimicrobial activities.

Full text of DOI:10.1016/j.tet.2013.11.065

Tetrahedron 70 (2014) 1084e1090
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
First total synthesis of the marine steroid alkaloid plakinamine B
*
Markus Gans, Franz Bracher
€
€
€
€
Department fur Pharmazie, Zentrum fur Pharmaforschung, Ludwig-Maximilians-Universitat Munchen, Butenandtstr. 5-13, D-81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of the marine steroid alkaloid plakinamine B (1) was accomplished in seven steps
starting from known aldehyde 3. Key steps in this synthesis are the attachment of a vinylpyridine side
chain by an aldol reaction, a chemoselective reduction of a pyridinium salt to a vinyl tetrahydropyridine,
and the introduction of the 3a-methylamino group under Mitsunobu conditions. Plakinamine B and some
of its precursors with amino or pyridinium side chains show significant antimicrobial activities.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 27 September 2013
Received in revised form 16 November 2013
Accepted 19 November 2013
Available online 22 November 2013
Keywords:
Plakinamine B
Alkaloid
Azasteroid
Tetrahydropyridine
Antimicrobial activity
1. Introduction
dihydropyrrole, pyrrolidine, pyrrolidone) in the side chain, plakin-
amine L contains a branched acyclic aminoalkyl side chain,9i in the
Azasteroids having the amino group either as a part of the tet-
racyclic steroid core or in the side chain, or as a substituent attached
to the ring system, have attracted considerable interest due to their
significant biological activities.1,2 A subgroup of these alkaloids
contains both an amino (or oligoamino) substituent at C-3 in ring A,
and an amino group in the side chain attached to ring D. The first
representatives of this subgroup were isolated mainly from the
plant families Apocynaceae and Buxaceae.1,3,4 Representatives of
this class of alkaloids have been shown to exhibit antimicrobial,2,3
cytotoxic, antiulcer, acetyl-, and butyrylcholine esterase-in-
hibitory,3 and antiestrogenic activity.5 The alkaloid irehdiamine A is
able to kink DNA,6 and the bis-quaternary alkaloid malouetine
dichloride as well as the semisynthetic funtumine guanylhy-
drazone stabilize DNA G-quadruplexes.7
plakinamine I reported by Ridley and Faulkner9f the pyrrolidine ring
is part of a rigidized hexacyclic ring system.
Fig. 1. Structures of the alkaloids plakinamine A (1) and plakinamine B (2).
Plakinamines A and B showed antibacterial and antifungal ac-
tivities in preliminary screenings,8 for the related alkaloids anti-
microbial, cytotoxic, immunomodulatory, anti-HIV, DNA-, and
RNA-cleaving,9 as well as acetylcholine esterase-inhibitory activi-
ties10 have been reported.
Recently, related alkaloids named cortistatins, containing an
expanded ring B and showing potent anti-angiogenic activities,
were isolated from Corticium sp.11,12 Easily accessible anti-
angiogenic compounds inspired by the complex cortistatins, and
having high structural similarity to plakinamine B, have been re-
ported by the Corey group subsequently.13
In 1984, Rosser and Faulkner isolated two steroidal alkaloids
named plakinamine A (1) and plakinamine B (2) (Fig. 1), containing
3a-amino groups as well as N-heterocyclic substituents in the side
chain, from the Micronesian sponge Plakina sp.8 Later on, a consid-
erable number of related azasteroids (plakinamines C-L, lokyster-
olamines A and B, and others) has been isolated from sponges of the
Corticium genus,9 whereby the name plakinamine I was assigned to
two different alkaloids.9f,g These alkaloids contain either
a- or b-
configured primary, secondary or tertiary amino substituents at C-3,
and various heterocyclic ring systems (tetrahydropyridine,
As part of our ongoing research on the synthesis and biological
evaluation of steroids,14 azasteroids,15e19 and azasecosteroids,20,21
we worked out the first total synthesis of plakinamine B (2).
* Corresponding author. Tel.: þ49 89 2180 77301; fax: þ49 89 2180 77802; e-mail
address: Franz.Bracher@cup.uni-muenchen.de (F. Bracher).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.11.065

M. Gans, F. Bracher / Tetrahedron 70 (2014) 1084e1090
1085
2. Results and discussion
2.1. Synthesis
First, we intended to perform the conversion at the pyridine
moiety. N-Methylation of 5 with methyl iodide gave the pyr-
idinium salt 6 in high yield. Surprisingly, reduction of 6 under
standard conditions (NaBH4 in 1 M ethanoliceaqueous NaOH24
)
As a key intermediate we selected the known
D
7-steroidal al-
gave a mixture of two products in almost equal amounts. NMR
analysis of the mixture indicated that one of the products was the
desired vinyl tetrahydropyridine 7, while the other product looked
most likely to be the 4-alkyl tetrahydropyridine 8. Obviously,
NaBH4 in alkaline medium did not exclusively reduce the pyr-
idinium salt to the tetrahydropyridine, but also reduced in part the
vinyl group at the pyridinium ring. Since the products 7 and 8
could not be separated on a preparative scale, we performed nu-
merous variations of the reduction conditions (other complex
hydrides, pH values, stoichiometry, solvents, and temperature).
Finally, we found that reduction with NaBH4 in neutral solution
(90% EtOH) gave exclusively the desired vinyl tetrahydropyridine 7
in 83% yield (Scheme 1).
dehyde 3, which we had already utilized in previous in-
vestigations.15,17 This compound is easily available from ergosteryl
acetate by regio- and stereoselective hydrogenation of the C5eC6
double bond using Raney nickel W2 in the presence of 4-
dimethylaminobenzaldehyde,22 followed by selective ozonolysis
of the side chain double bond.23 The 3
a-amino group was to be
built up from the sterol by an amination reaction comprising an
inversion at C-3. For the construction of the 1,2,5,6-
tetrahydropyridine moiety we selected a NaBH4 reduction of an
appropriate N-methylpyridinium intermediate, as described pre-
viously by Reimann et al. for 3,4-disubstituted pyridinium salts.24
First attempts to build up a vinylpyridine side chain from al-
Scheme 1. Introduction of the vinyl tetrahydropyridine side chain.
dehyde 3 by Wittig olefination with an appropriate pyridylmethyl
triphenylphosphonium salt, failed. Finally, we could introduce the
pyridine substituent using an aldol reaction. For this purpose 3,4-
lutidine was regioselectively deprotonated at the 4-methyl group
with LDA,25 whereas metalation with n-butyllithium in the non-
complexing solvent hexane would result in metalation and/or ad-
dition reactions at C-6.26 Trapping of the lithiated 3,4-lutidine with
aldehyde 3 gave an epimeric mixture of the aldol products 4a. As
by-product we obtained the epimeric 3-hydroxy compounds 4b,
obviously arising from attack of excess lithiated 3,4-lutidine at the
ester group. Alcohol 4a was conveniently dehydrated to the vinyl-
pyridine 5 by treatment with mesyl chloride, triethylamine, and
catalytic amounts of 4-dimethylaminopyridine.27 The yield of this
reaction could be enhanced significantly by the addition of DBU.28
Product 5 was obtained exclusively in E-configuration, as can be
seen from the coupling constant (J¼15.7 Hz) of the olefinic protons
in the 1H NMR spectrum.
In order to perform the presumptive final part of the total
synthesis, the 3 -acetoxy group was to be converted to the 3a-
b
methylamino group under inversion. A limited number of related
conversions has been described in literature. Reductive amination
of 3-ketosteroids appeared not applicable, since it is known to give,
depending on the substrates and reaction conditions, either mix-
tures of
a
- and
b b-
-configured amines29,30 or pure undesired 3
amines.31e33
3
b-Hydroxysteroids have been converted to primary
3a-aminosteroids either via mesylation, conversion to the azide,
and reduction8,34 or via Mitsunobu reaction using azide13,35 or
phthalimide.36 Selective monomethylation of the primary to the
corresponding secondary N-methylamine requires two more
steps,8 making all of these approaches unattractive.
In a first attempt the 3
NaOH, and the resulting 3
b
-acetoxy group of 7 was hydrolyzed with
-alcohol 9 was converted to the corre-
b
sponding mesylate under standard conditions.8 But all attempts to
convert this mesylate directly to the secondary amine plakinamine
B (1) with methylamine under various reaction conditions, failed.
An alternative method for the synthesis of secondary amines
from alcohols under Mitsunobu conditions, utilizing the 2-
nitrobenzenesulfonyl residue as both activating and protecting
Having this building block in hands, two more synthetic oper-
ations had to be done: conversion of the pyridine ring to a N-methyl
1,2,5,6-tetrahydropyridine, and replacement of the 3-acetoxy group
by a methylamino group under inversion of the configuration.

1086
M. Gans, F. Bracher / Tetrahedron 70 (2014) 1084e1090
group, has been developed by Fukuyama et al.,37 and applied to the
introduction of a methylamino group starting from a primary al-
cohol by Ichikawa et al.38 later. We investigated this methodology
in a model reaction first. Thus, 5,6-dihydroergosterol (10) was
reacted with N-methyl-2-nitrobenzenesulfonamide under Mitsu-
nobu conditions. Sulfonamide 11 was obtained under inversion at
C-3, and cleavage of the sulfonamide group could be accomplished
smoothly under mild conditions by treatment with thiophenol/
give the alkaloid plakinamine B (2). The NMR data of this product
were identical to those reported for the natural product.
2.2. Biological activities
Since antibacterial and antifungal activity (1 strain each) had
been reported for plakinamine B (2),8 we subjected this alkaloid
and the intermediates described above to a screening for antibac-
terial and antifungal activities in an agar diffusion assay (Table 1).
37
Cs2CO3 to give the 3-aminosterol 12 (Scheme 2).
Scheme 2. Model reaction for the introduction of the 3a-methylamino group.
Surprisingly, this methodology could not be applied to the
conversion of the intermediate 9. Although the starting material
was completely consumed upon treatment with N-methyl 2-
nitrobenzenesulfonamide, Ph3P, and DEAD, we could not detect
the desired product with a N-methyl sulfonamide group at C-3. The
failure of this conversion might be due to undesired 4þ2 cycload-
dition of diethyl azodicarboxylate (dienophile) with the vinyl tet-
The results clearly indicate that plakinamine B is the by far most
active compound in this series. Its antibacterial activity is compa-
rable to that of the reference drug tetracycline, its antifungal activity
even superior to that of clotrimazole. Compounds 7 and 9 with
a tetrahydropyridine side chain showed slight antifungal activity,
and the pyridinium salts 6 and 15 exhibit slight antibacterial and
antifungal activities, which may be due to their detergent-like
properties.19 All other compounds were devoid of antimicrobial
activity.
rahydropyridine moiety (diene) in the steroid side chain of 9.39
A
related cycloaddition of a vinylhexahydroindene has been reported
by us recently.18
Consequently, the sequence of the crucial steps had to be
inverted (Scheme 3). Ester 5, still containing the pyridine ring, was
hydrolyzed to the alcohol 13, which was further converted, in the
Mitsunobu reaction described above, to the sulfonamide 14 under
inversion at C-3. Further on, reaction with methyl iodide gave the
pyridinium salt 15, and subsequent reduction with NaBH4 in 90%
EtOH led to the tetrahydropyridine 16. In the final step the N-sul-
fonyl group was removed by reaction with thiophenol/Cs2CO3 to
2.3. Conclusion
In conclusion, we have developed the first total synthesis of the
steroid alkaloid plakinamine B (2) in seven-steps (8% overall yield)
starting from known aldehyde 3. In contrast to its precursors, pla-
kinamine B shows high antimicrobial activities. Work is in progress
to evaluate the mechanisms of the antimicrobial actions recorded
here.
Scheme 3. Total synthesis of plakinamine B (2).

M. Gans, F. Bracher / Tetrahedron 70 (2014) 1084e1090
1087
Table 1
give, in this order of elution, 900 mg (23%) recovered starting
material 3, 2.1 g (41%; 53% based on recovered starting material) of
4a, and 850 mg (18%) of 3-hydroxy product 4b.
Results of the agar diffusion assay for antibacterial and antifungal activity a
Bacteriab
Fungic
Analytical data of 4a: white solid, Mp 211 ꢁC. IR (KBr):
n
Compound
E. coli
Pseud.
antim.
Staph.
hom.
Yarr.
lipol.
C. glabr.
Asp. niger
(cmꢀ1)¼3246, 2949, 2868, 1728, 1599, 1446, 1379, 1242, 1163, 1095,
1032, 897, 818, 607, 525. 1H NMR (CDCl3):
d
(ppm)¼8.33 (s, 1H, 20-
2
4a
4b
5
6
7
9
11
12
13
14
15
14
dd
d
12
d
d
d
7
d
d
d
d
d
d
6
13
d
d
d
10
d
d
d
d
d
d
7
19
d
d
d
12
6
18
d
d
d
11
6
8
Hþd, J¼4.9 Hz, 1H, 60-H), 7.09 (d, J¼4.9 Hz, 1H, 50-H), 5.16 (m, 1H,
7-H), 4.70 (m, 1H, 3-H), 3.94 (m, 1H, 22-H), 2.82 (m, 1H, 23-HH),
2.63 (m, 1H, 23-HH), 2.30 (s, 3H, 30-CH3), 2.03 (s, 3H, CH3C]O),
2.02 (m, 1H, 12-HH), 1.98e1.20 (m, 20 H), 1.13 (m, 1H, 4-HH), 1.06
(d, J¼6.0 Hz, 3H, 21-H), 0.81 (s, 3H, 19-H), 0.54 (s, 3H, 18-H). 13C
d
d
d
7
d
10
d
d
d
d
d
d
d
d
d
10
n.d.
d
7
7
NMR (CDCl3):
d
(ppm)¼170.7 (C]O), 150.6 (C-20), 147.3 (C-40),
d
d
d
d
d
11
d
12
n.d.
d
d
d
d
10
d
11
n.d.
147.1 (C-60), 139.1 (C-8), 132.2 (C-30), 124.7 (C-50), 117.6 (C-7), 73.4
(C-3), 72.8 (C-22), 54.8 (C-14), 52.5 (C-17), 49.2 (C-9), 43.3 (C-13),
41.7 (C-20), 40.0 (C-5), 39.5 (C-12), 38.8 (C-23), 36.8 (C-4), 34.2 (C-
10), 33.8 (C-1), 29.5 (C-6), 27.6 (C-16), 27.5 (C-2), 22.9 (C-15), 21.5
(C-11), 21.4 (CH3C]O), 16.4 (300-CH3), 12.9 (C-19), 12.2 (C-21), 11.8
(C-18). MS (CI): m/z (%)¼480 (20) [Mþþ1], 420 (28), 136 (60), 107
(100). Anal. Calcd for C31H45NO3 (479.71) C, 77.62; H, 9.46; N 2.92.
Found C, 77.43; H, 9.52; N, 2.64.
d
d
d
9
d
n.d.e
16
16
d
n.d.
30
d
n.d.
14
clotrimazole
tetracycline
a
Determined with 50 mg substance per disc, diameters of zones of inhibition in
mm, all experiments performed in triplicate.
b
E. coli¼Escherichia coli (gram-negative), Pseud. antim.¼Pseudomonas anti-
microbia (gram-negative), Staph. hom.¼Staphylococcus hominis (gram-positive).
3.1.2. (3b)-3,22-Dihydroxy-23-(3-methylpyridin-4-yl)-24-nor-5a-
c
Yarr. lipol.¼Yarrowia lipolytica (yeast), C. glabr.¼Candida glabrata (yeast), Asp.
chol-7-ene (mixture of diastereomers) (4b). Obtained as a by-
niger¼Aspergillus niger (mold).
product in the reaction described above; white solid, Mp 231 ꢁC.
d
d¼inactive.
e
IR (KBr):
n
(cmꢀ1)¼3398, 2937, 2871, 1726, 1601, 1446, 1381, 1261,
n.d.¼not determined.
1039, 822, 748, 702. 1H NMR CDCl3:
d
(ppm)¼8.27 (s, 1H, 20-Hþd,
3. Experimental section
3.1. General
J¼5.2 Hz, 1H, 60-H), 7.11 (d, J¼5.2 Hz, 1H, 50-H), 5.14 (m, 1H, 7-H),
3.90 (m, 1H, 22-H), 3.57 (m, 1H, 3-H), 2.82 (m, 1H, 23-HH), 2.59 (m,
1H, 23-HH), 2.28 (s, 3H, 30-CH3), 2.01 (m, 1H, 12-HH), 1.96e1.20 (m,
21 H), 1.04 (d, J¼6.4 Hz, 3H, 21-Hþm, 1H, 4-HH), 0.77 (s, 3H, 19-H),
All solvents used were of HPLC grade or p.a. grade and/or pu-
rified according to standard procedures. Chemical reagents were
purchased from Sigma Aldrich (Schnelldorf, Germany) and Acros
(Geel, Belgium). IR measurements were performed with a Jasco FT-
IR-410 spectrometer. All melting points were determined by open
0.52 (s, 3H, 18-H). 13C NMR (CDCl3):
d
(ppm)¼150.2 (C-20), 147.8 (C-
40), 146.8 (C-60), 139.2 (C-8), 132.4 (C-30), 124.8 (C-50), 117.8 (C-7),
72,7 (C-22), 70.9 (C-3), 54.9 (C-14), 52.5 (C-17), 49.4 (C-9), 43.3 (C-
13), 41.7 (C-20), 40.2 (C-5), 39.5 (C-12), 38.8 (C-23), 37.9 (C-1), 37.1
(C-4), 34.2 (C-10), 31.4 (C-6), 29.6 (C-16), 27.6 (C-2), 22.9 (C-15), 21.5
(C-11), 16.5 (30-CH3), 13.1 (C-19), 12.2 (C-21), 11.8 (C-18). MS (CI): m/
z (%)¼438 (100) [Mþþ1], 420 (58), 150 (50), 108 (70). Anal. Calcd for
€
tube capillary method on a Buchi melting point B-450 apparatus
and are uncorrected. NMR spectra were recorded on Jeol JNMR-GX
400 (400 MHz) spectrometer with tetramethylsilane as internal
standard and chemical shifts are reported in parts per million
(ppm). J values are given in Hertz. Multiplicities are abbreviated as
follows: s¼singlet, d¼doublet, t¼triplet, m¼multiplet. Signal as-
signments were carried out based on 1H, 13C, HMBC, HMQC, and
COSY spectra. Mass spectra (MS) were run by atmospheric pressure
chemical ionization (APCI) on an AB Sciex API 2000 LC/MS/MS
system or by chemical impact (CI) using a Hewlett Packard 5989A
mass spectrometer with 59980B Particle Beam LC/MS Interface.
HRMS were performed by electron impact (EI) at 70 eV on a Jeol
GCmate II or on a Finnigan MAT 95 spectrometer. Optical rotations
were determined on a Perkin Elmer 241 MC. Chromatographic
purification of products was performed by using flash column
chromatography (FCC) on Merck silica gel 60 as stationary phase.
C29H43NO2 (437.67) C, 79.59; H, 9.90; N 3.20. Found C, 79.32; H,
9.93; N, 3.07.
3.1.3. (3b)-Acetoxy-23-(3-methylpyridin-4-yl)-24-nor-5a,22E-chola-
7,22-diene (5). To a stirred solution of 4a (1.90 g, 3.96 mmol) in
20 mL chloroform was added 1.20 g (12.0 mmol) triethylamine. The
mixture was cooled to 0 ꢁC and treated with 0.69 g (6.0 mmol)
methanesulfonyl chloride. After 1 h stirring at room temperature
1.80 g (12.0 mmol) DBU was added and the mixture was refluxed
for 72 h. Then water (20 mL) was added to the cooled mixture and it
was left to stir for another hour, after which it was extracted with
chloroform (3ꢂ15 mL). The combined organic extracts were dried
(Na2SO4), the solvent was evaporated and the residue was purified
by flash column chromatography (ethyl acetate/hexane/methanol,
1:1:0.1) to give 1.40 g (77%) of 5 as a white solid, Mp 146 ꢁC. IR
3.1.1. (3
b
)-Acetoxy-22-hydroxy-23-(3-methylpyridin-4-yl)-24-nor-
(KBr):
n
(cmꢀ1)¼2947, 2870, 1728, 1639, 1591, 1547, 1446, 1404,
5
a-chol-7-ene (mixture of diastereomers) (4a). To a stirred solution
1379, 1242, 1093, 1030, 972, 895, 856, 820, 733. 1H NMR (CDCl3):
of 2.0 g (20 mmol) diisopropylamine in 60 mL of anhydrous THF
was added dropwise 12 mL (20 mmol) n-butyllithium (1. 6M in
hexane). After 15 min the solution was treated during 5 min with
a solution of 2.2 g (20 mmol) 3,4-lutidine in 20 mL of anhydrous
THF. The mixture was stirred for 1 h and the resulting intense red
solution was cooled to ꢀ78 ꢁC, then treated with a solution of 4.00 g
(10.7 mmol) of the aldehyde 3 in 20 mL anhydrous THF added
during 5 min. After 5 more min the now light yellow suspension
was poured into 120 mL of water and extracted with 3ꢂ60 mL
diethyl ether. The combined organic layers were dried with Na2SO4
and evaporated to dryness. The residue was purified by flash col-
umn chromatography (ethyl acetate/hexane/methanol, 1:1:0.1) to
d
(ppm)¼8.33 (s, 1H, 20-Hþd, J¼5.2 Hz, 1H, 60-H), 7.23 (d, J¼5.2 Hz,
1H, 50-H), 6.42 (d, J¼15.7 Hz, 1H, 23-H), 6.17 (dd, J1¼15.7 Hz,
J2¼9.0 Hz, 1H, 22-H), 5.16 (m, 1H, 7-H), 4.70 (m, 1H, 3-H), 2.30 (m,
1H, 20-H), 2.29 (s, 3H, 30-CH3), 2.06 (m, 1H, 12-HH), 2.03 (s, 3H,
CH3C]O),1.90e1.20 (m, 18 H), 1.15 (d, J¼6.7 Hz, 3H, 21-Hþm, 1H, 4-
HH), 0.82 (s, 3H, 19-H), 0.61 (s, 3H, 18-H). 13C NMR (CDCl3):
d
(ppm)¼170.7 (C]O), 150.8 (C-20), 147.2 (C-60), 144.5 (C-40), 142.9
(C-22), 139.1 (C-8), 130.0 (C-30), 123.0 (C-23), 119.3 (C-50), 117.7 (C-
7), 73.4 (C-3), 55.5 (C-17), 54.9 (C-14), 49.2 (C-9), 43.6 (C-13), 41.2
(C-20), 40.0 (C-5), 39.4 (C-12), 36.8 (C-4), 34.2 (C-10), 33.8 (C-1),
29.5 (C-6), 28.0 (C-16), 27.5 (C-2), 22.9 (C-15), 21.5 (CH3C]O), 21.4
(C-11), 20.4 (C-21),16.6 (30-CH3),13.0 (C-19),12.2 (C-18). MS (CI): m/

1088
M. Gans, F. Bracher / Tetrahedron 70 (2014) 1084e1090
z (%)¼462 (100) [Mþþ1], 420 (52). Anal. Calcd for C31H43NO2
give 34 mg (78%) of 9 as a white solid, Mp 189 ꢁC (decomp.). IR
(461.69) C, 80.65; H, 9.39; N, 3.03. Found C, 81.05; H, 9.82; N, 2.53.
(KBr):
n
(cmꢀ1)¼1607, 1519, 1480, 1253, 834. 1H NMR (CDCl3):
[
a]
20 þ18.2 (c 1.0, CHCl3).
d
(ppm)¼6.34 (d, J¼15.5 Hz, 1H, 23-H), 5.45 (dd, J1¼15.5 Hz,
D
J2¼8.7 Hz, 1H, 22-H), 5.14 (m, 1H, 7-H), 3.55 (m, 1H, 3-H), 2.86 (m,
2H, 200-H), 2.51 (m, 2H, 60-H), 2.33 (s, 3H, 10-CH3), 2.25 (m, 2H, 50-H),
2.13 (m, 1H, 20-H), 2.03 (m, 1H, 12-HH), 1.85e1.53 (m, 11 H), 1.75 (s,
3H, 30-CH3), 1.48 (m, 2H, 11-HHþ15-HH), 1.38 (m, 3H, 5-Hþ6-
HHþ15-HH), 1.26 (m, 3H, 1-HHþ17-Hþ12-HH), 1.06 (d, J¼6.7, 3H,
21-Hþm, 1H, 4-HH), 0.79 (s, 3H, 19-H), 0.56 (s, 3H, 18-H). 13C NMR
3.1.4. 4-[(3b)-Acetoxy-24-nor-5a,22E-chola-7,22-dien-23-yl]-1,3-
dimethylpyridinium iodide (6). 0.92 g (2.0 mmol) of the pyridine 5
was dissolved in a small amount of acetone, treated with 0.57 g
(4.0 mmol) methyl iodide and refluxed for 1 h. After cooling the
precipitated methiodide was collected by filtration and recrystal-
lized from ethanol/diethyl ether to give 1.1 g (91%) of 6 as yellow
(CDCl3):
d
(ppm)¼139.4 (C-8), 134.7 (C-22), 127.6/126.0 (C-30/C-40),
crystals, Mp 220 ꢁC. IR (KBr):
n
(cmꢀ1)¼2947, 2870, 1734, 1630,
124.8 (C-23), 117.6 (C-7), 70.8 (C-3), 60.5 (C-20), 56.2 (C-17), 55.1 (C-
14), 52.4 (C-60), 49.5 (C-9), 45.7 (10-CH3), 43.4 (C-13), 40.9 (C-20),
40.3 (C-5), 39.5 (C-12), 38.0 (C-1), 37.2 (C-4), 34.3 (C-10), 31.6 (C-6),
29.7 (C-16), 28.2 (C-2), 26.6 (C-50), 22.9 (C-15), 21.6 (C-11), 20.8 (C-
21), 16.5 (30-CH3), 13.0 (C-19), 12.1 (C-18). MS (CI): m/z (%)¼438
1504, 1473, 1446, 1365, 1323, 1244, 1161, 1093, 1032, 972, 897. 1H
NMR (DMSO-d6):
d
(ppm)¼8.77 (s,1H, 2-H), 8.67 (d, J¼6.6 Hz,1H, 6-
H), 8.16 (d, J¼6.6 Hz, 1H, 5-H), 6.88 (dd, J1¼15.6 Hz, J2¼9.1 Hz, 1H,
220-H), 6.71 (d, J¼15.6 Hz,1H, 230-H), 5.13 (m, 1H, 70-H), 4.58 (m, 1H,
30-H), 4.20 (s, 3H, 1-CH3), 2.45 (m, 1H, 200-H), 2.41 (s, 3H, 3-CH3),
2.02 (m, 1H, 120-HH), 1.98 (s, 3H, CH3C]O), 1.88e1.25 (m, 18 H), 1.15
(d, J¼6.7 Hz, 3H, 210-H), 1.12 (m, 1H, 4-HH), 0.77 (s, 3H, 190-H), 0.58
(100) [Mþþ1], 420 (82), 164 (98). Anal. Calcd for C30H47NO (437.72)
20
C, 82.32; H, 10.82; N, 3.20. Found C, 79.81; H, 10.31; N, 2.94. [a]
D
þ12.6 (c 1.0, CHCl3).
(s, 3H, 180-H). 13C NMR (DMSO-d6):
d
(ppm)¼170.4 (C]O), 152.3 (C-
4), 151.3 (C-220), 145.7 (C-2), 142.7 (C-6), 139.4 (C-80), 134.3 (C-3),
122.4 (C-5), 121.6 (C-230), 117.8 (C-70), 73.2 (C-30), 55.0 (C-170), 54.8
(C-140), 49.1 (C-90), 47.2 (1-CH3), 43.9 (C-30), 41.5 (C-200), 40.3 (C-50),
39.2 (C-120), 36.7 (C-40), 34.3 (C-100), 34.0 (C-10), 29.5 (C-60), 27.8 (C-
160), 27.7 (C-20), 23.2 (C-150), 21.7 (CH3C]O), 21.6 (C-110), 20.2 (C-
210), 16.8 (3-CH3), 13.3 (C-190), 12.6 (C-180). MS (EI): m/z (%)¼475 (8)
[MþꢀHI], 461 (55) [MþꢀCH3I], 313 (30), 174 (56), 107 (100). Anal.
3.1.7. N-[(3a)-Ergosta-7,22E-dien-3-yl]-N-methyl-2-nitrobenzene-
sulfonamide (11). 0.22 g (1.0 mmol) N-methyl-2-nitrobenzene-
sulfonamide and 0.39 g (1.5 mmol) triphenylphosphane were dis-
solved in 5 mL of anhydrous THF. The solution was stirred under
nitrogen and 0.20 g (0.50 mmol) 5,6-dihydroergosterol22 was
added, followed by 0.26 g (1.5 mmol) DEAD. The mixture was
stirred at room temperature for 3 h, concentrated in vacuo and the
residue was purified by flash column chromatography (hexane/
ethyl acetate, 2:1) to give 160 mg (53%) of 11 as a yellow solid, Mp
Calcd for C32H46INO2 (603.63) C, 63.67; H, 7.68; N, 2.32. Found C,
64.66; H, 7.77; N, 2.21. [
20
a
]
þ46 (c 0.25, MeOH).
D
159 ꢁC. IR (KBr):
n
(cmꢀ1)¼2954, 2871, 1543, 1371, 1346, 1321, 1215,
3.1.5. (3
yl)-24-nor-5
b
)-Acetoxy-23-(1,3-dimethyl-1,2,5,6-tetrahydropyridin-4-
,22E-chola-7,22-diene (7). 0.60 g (1.0 mmol) of the
1159, 1959, 970, 949, 922, 852, 781, 762, 737, 661, 590, 579, 532. 1H
a
NMR (CDCl3):
d
(ppm)¼8.02 (d, J¼8.9 Hz, 1H, 3-H), 7.66 (m, 2H, 4-
methiodide 6 was dissolved in 30 mL ethanol (90%). The solution
was cooled to 0 ꢁC, and 0.11 g (2.9 mmol) NaBH4 in 5 mL ethanol
(90%) was added slowly. After stirring for 2 h at 0 ꢁC and 24 h at
room temperature the excess of NaBH4 was destroyed by adding
2 M HCl, then the mixture was alkalized with 2 M NaOH and
extracted with diethyl ether (3ꢂ20 mL). The combined organic
layers were dried with Na2SO4 and the solvent was evaporated. The
residue was purified by flash column chromatography (ethyl ace-
tate/hexane/EtNMe2, 1:1:0.1) to give 400 mg (83%) of 7 as a white
Hþ5-H), 7.62 (d, J¼9.0 Hz, 1H, 6-H), 5.20 (m, 3H, 70-Hþ220-Hþ230-
H), 4.08 (m,1H, 30-H), 3.05 (s, 3H, NeCH3), 2.02 (m, 2H,120-HHþ200-
H), 1.92e1.33 (m, 18H), 1.25 (m, 3H, 120-HHþ160-HHþ170-H), 1.01 (d,
J¼6.7 Hz, 3H, 210-H), 0.90 (d, J¼6.9 Hz, 3H, 280-H), 0.83 (d, J¼6.3 Hz,
3H, 260-H), 0.81 (d, J¼6.5 Hz, 3H, 270-H), 0.74 (s, 3H, 190-H), 0.54 (s,
3H, 180-H). 13C NMR (CDCl3):
d
(ppm)¼148.2 (C-2), 139.7 (C-80),
135.7 (C-220), 133.5 (C-1), 133.3 (C-5), 132.0 (C-230), 131.6 (C-4),
131.0 (C-3), 124.2 (C-6), 117.4 (C-70), 56.0 (C-170), 55.3 (C-140), 51.5
(C-30), 49.8 (C-90), 43.5 (C-130), 42.9 (C-240), 40.6 (C-200), 39.5 (C-
120), 37.3 (C-50), 34.7 (C-10), 33.9 (C-100), 33.2/33.1 (C-250/NeCH3),
32.0 (C-40), 29.8 (C-60), 28.2 (C-160), 25.9 (C-20), 22.9 (C-150), 21.4 (C-
110), 21.2 (C-210), 20.0 (C-260), 19.7 (C-270),þ17.7 (C-280), 13.2 (C-190),
12.2 (C-180). MS (CI): m/z (%)¼597 (5) [M þ1], 412 (84), 381 (60),
solid, Mp 144 ꢁC (decomp.). IR (KBr):
n
(cmꢀ1)¼2945, 2871, 1732,
1647, 1446, 1379, 1242, 1161, 1093, 1032, 966, 899, 845, 756. 1H NMR
(CDCl3):
d
(ppm)¼6.32 (d, J¼15.5 Hz,1H, 23-H), 5.41 (dd, J1¼15.5 Hz,
J2¼9.0 Hz, 1H, 22-H), 5.13 (m, 1H, 7-H), 4.68 (m, 1H, 3-H), 2.88 (m,
2H, 20-H), 2.51 (m, 2H, 60-H), 2.31 (s, 3H, 10-CH3), 2.24 (m, 2H, 50-H),
2.11 (m, 1H, 20-H), 2.01 (s, 3H, CH3C]O), 2.00 (m, 1H, 12-HH),
1.90e1.20 (m, 18H), 1.70 (s, 3H, 30-H), 1.15 (m, 1H, 4-HH), 1.03 (d,
J¼6.7 Hz, 3H, 21-H), 0.80 (s, 3H, 19-H), 0.54 (s, 3H, 18-H). 13C NMR
295 (40), 125 (100). Anal. Calcd for C35H52N2O4S (596.88) C, 70.43;
H, 8.78; N, 4.69. Found C, 70.37; H, 8.62; N, 4.74. [
CHCl3).
20
a]
þ2.1 (c 1.0,
D
(CDCl3):
d
(ppm)¼170.7 (C]O), 139.4 (C-8), 134.7 (C-22), 127.6/
3.1.8. (3a)-Methylaminoergosta-7,22E-diene (12). 0.12 g (0.20 mmol)
125.9 (C-30/C-40), 124.8 (C-23), 117.4 (C-7), 73.5 (C-3), 60.5 (C-20),
56.0 (C-17), 55.0 (C-14), 52.4 (C-60), 49.2 (C-9), 45.7 (100-CH3), 43.4
(C-13), 41.0 (C-20), 40.0 (C-5), 39.5 (C-12), 36.8 (C-4), 34.2 (C-10),
33.8 (C-1), 29.5 (C-6), 28.1 (C-16), 27.5 (C-2), 26.5 (C-50), 22.9 (C-15),
21.5 (CH3C]O), 21.4 (C-11), 21.0 (C-21), 16.5 (30-CH3), 12.9 (C-19),
12.2 (C-18). MS (EI): m/z (%)¼479 (38) [Mþ], 464 (62), 313 (24), 252
(42), 110 (100), 84 (87). Anal. Calcd for C32H49NO2 (479.75) C, 80.12;
of the sulfonamide 11 was dissolved in 5 mL of acetonitrile,
then 28 mg (0.25 mmol) thiophenol and 0.20 g (0.60 mmol)
cesium carbonate was added, and the mixture was stirred at
room temperature for 12 h. After filtration the solvent was
removed and the residue was purified by flash column chromatog-
raphy (ethyl acetate/hexane/EtNMe2, 1:1:0.1) to give 65 mg (79%) of
12 as a white solid, Mp 102 ꢁC. IR (KBr):
n
(cmꢀ1)¼3429, 2951, 2787,
H, 10.29; N, 2.92. Found C, 80.23; H, 10.75; N, 2.70. [
CHCl3).
a]
20 þ13.0 (c 0.5,
1456, 1367, 1275, 1250, 1203, 1169, 1124, 1099, 1045, 1022, 997, 966,
D
939, 847, 827, 798, 746, 667.1H NMR (CDCl3):
d
(ppm)¼5.17 (m, 3H, C-
7þC-22þC-23), 2.72 (m, 1H, 3-H), 2.38 (s, 3H, NeCH3), 2.01 (m, 2H,
20-Hþ12-HH), 1.85 (m, 1H, 24-H), 1.82e1.16 (m, 21H), 1.01 (d,
J¼6.5 Hz, 3H, 21-H), 0.91 (d, J¼6.6 Hz, 3H, 28-H), 0.84 (d, J¼6.5 Hz,
3H, 27-H), 0.82 (d, J¼6.4 Hz, 3H, 26-H), 0.80 (s, 3H,19-H), 0.55 (s, 3H,
3.1.6. (3
yl)-24-nor-5
b
)-Hydroxy-23-(1,3-dimethyl-1,2,5,6-tetrahydropyridin-4-
,22E-chola-7,22-diene (9). 48 mg (0.10 mmol) of the
a
ester 7 was dissolved in 5 mL THF, treated with 5 mL 6 M NaOH and
refluxed for 12 h. After cooling and extraction with diethyl ether
(3ꢂ5 mL) the combined organic layers were dried with Na2SO4 and
the solvent was evaporated. The residue was purified by flash col-
umn chromatography (ethyl acetate/hexane/EtNMe2, 2:1:0.1) to
18-H). 13C NMR (CDCl3):
23),117.6 (C-7), 56.0 (C-17), 55.2 (C-14), 54.2 (C-3), 49.7 (C-9), 43.4 (C-
13), 42.8 (C-24), 40.5 (C-20), 39.5 (C-12), 35.1 (C-5), 34.9 (C-10), 34.1
(NeCH3), 33.1 (C-25), 32.5/32.4 (C-1/C-4), 29.7 (C-6), 28.1 (C-16),
d
(ppm)¼139.5 (C-8), 135.7 (C-22), 131.8 (C-

M. Gans, F. Bracher / Tetrahedron 70 (2014) 1084e1090
1089
25.5 (C-2), 22.9 (C-15), 21.3 (C-11), 21.2 (C-21), 20.0 (C-26), 19.6 (C-
filtration and recrystallized from ethanol/diethyl ether to give
27), 17.6 (C-28), 12.4 (C-19), 12.1 (C-18). MS (EI): m/z (%)¼411 (68)
290 mg (94%) of 15 as yellow crystals, Mp 210 ꢁC. IR (KBr):
n
[Mþ], 396 (50), 354 (36), 286 (24), 70 (100). Anal. Calcd For C29H49
N
(cmꢀ1)¼2933, 1709, 1626, 1543, 1504, 1473, 1371, 1333, 1227, 1159,
(411.72) C, 84.60; H, 12.00; N, 3.40. Found C, 83.85; H, 11.91; N, 3.27.
1122, 1057, 978, 922, 850, 781, 760, 729, 661, 642, 579, 534. 1H NMR
[a]
20 ꢀ2.2 (c 1.0, CHCl3).
(DMSO-d6):
d
(ppm)¼8.76 (s, 1H, 2-H), 8.67 (d, J¼6.6 Hz, 1H, 6-H),
D
8.15 (d, J¼6.6 Hz,1H, 5-H), 7.97 (m, 2H, 300-Hþ600-H), 7.86 (m, 2H, 400-
Hþ500-H), 6.89 (dd, J1¼15.7 Hz, J2¼9.0 Hz, 1H, 220-H), 6.70 (d,
J¼15.7 Hz,1H, 230-H), 5.12 (m,1H, 70-H), 4.20 (s, 3H,1-CH3), 3.95 (m,
1H, 30-H), 2.98 (s, 3H, SO2NeCH3), 2.45 (m,1H, 200-H), 2.41 (s, 3H, 3-
CH3), 2.02 (m, 1H, 120-HH), 1.90e1.20 (m, 19H), 1.13 (d, J¼6.8 Hz, 3H,
210-H), 0.71 (s, 3H, 190-H), 0.57 (s, 3H, 180-H). 13C NMR (DMSO-d6):
3.1.9. (3b)-Hydroxy-23-(3-methylpyridin-4-yl)-24-nor-5a,22E-
chola-7,22-diene (13). 0.12 g (0.26 mmol) of the ester 5 was dis-
solved in 10 mL THF, treated with 10 mL 6 M NaOH and refluxed for
12 h. After cooling and extraction with diethyl ether (3ꢂ10 mL) the
combined organic layers were dried with Na2SO4 and the solvent
was evaporated. The residue was purified by flash column chro-
matography (ethyl acetate/hexane/methanol, 2:1:0.1) to give 95 mg
d
(ppm)¼151.7 (C-4), 150.7 (C-220), 147.5 (C-200), 145.0 (C-2), 142.1
(C-6), 138.8 (C-80), 134.3 (C-500), 133.7 (C-3), 132.3 (C-400), 131.4 (C-
100), 129.7 (C-300), 124.1 (C-600), 121.7 (C-5), 120.9 (C-230), 117.3 (C-70),
54.4 (C-170), 54.3 (C-140), 51.0 (C-30), 48.6 (C-90), 46.6 (1-CH3), 43.3
(C-130), 40.8 (C-200), 38.6 (C-120), 36.3 (C-50), 33.7 (C-10), 33.3 (C-
100), 33.0 (SO2NeCH3), 31.1 (C-40), 29.0 (C-60), 27.1 (C-160), 25.2 (C-
20), 22.4 (C-150), 20.7 (C-110), 19.6 (C-210), 16.1 (3-CH3), 12.7 (C-190),
11.9 (C-180). MS (ESI): m/z (%)¼632 (24) [Mþþ1ꢀHI], 191 (20), 107
(100). Anal. Calcd for C37H50N3O4SI (759.79) C, 58.49; H, 6.63; N,
(87%) of 13 as a white solid, Mp 180 ꢁC. IR (KBr):
n
(cmꢀ1)¼3423,
3205, 2929, 2871, 1732, 1645, 1597, 1448, 1454, 1406, 1379, 1306,
1248, 1194, 1159, 1130, 1097, 1055, 968, 847, 818, 741. 1H NMR
(CDCl3):
d
(ppm)¼8.33 (s, 1H, 20-Hþd, J¼5.1 Hz, 1H, 60-H), 7.24 (d,
J¼5.1 Hz, 1H, 50-H), 6.42 (d, J¼15.8 Hz, 1H, 23-H), 6.17 (dd,
J1¼15.8 Hz, J2¼9.0 Hz,1H, 22-H), 5.17 (m,1H, 7-H), 3.60 (m,1H, 3-H),
2.32 (m, 1H, 20-H), 2.30 (s, 3H, 30-CH3), 2.04 (m, 1H, 12-HH),
1.90e1.20 (m, 19H), 1.15 (d, J¼6.6 Hz, 3H, 21-H), 1.09 (m, 1H, 4-HH),
5.53. Found C, 58.21; H, 6.96; N, 5.21. [
a
]
20 þ49.0 (c 0.5, MeOH).
D
0.81 (s, 3H, 19-H), 0.61 (s, 3H, 18-H). 13C NMR (CDCl3):
d
(ppm)¼
151.0 (C-20), 147.4 (C-600), 144.4 (C-40), 142.7 (C-22), 139.2 (C-8),
129.9 (C-30), 123.0 (C-23), 119.2 (C-50), 117.8 (C-7), 70.9 (C-3), 55.5
(C-17), 55.0 (C-14), 49.4 (C-9), 43.6 (C-13), 41.2 (C-20), 40.2 (C-5),
39.4 (C-12), 38.0 (C-1), 37.1 (C-4), 34.2 (C-10), 31.5 (C-6), 29.6 (C-16),
28.1 (C-2), 22.9 (C-15), 21.5 (C-11), 20.4 (C-21),16.6 (30-CH3),13.1 (C-
19), 12.2 (C-18). MS (EI): m/z (%)¼419 (24) [Mþ], 271 (78), 147 (54),
3.1.12. N-[(3
a)-23-(1,3-Dimethyl-1,2,5,6-tetrahydropyridin-4-yl)-24-
n o r - 5 , 2 2 E - c h o l a - 7 , 2 2 - d i e n - 3 - y l ] - N - m e t h y l - 2 -
a
nitrobenzenesulfonamide (16). 0.38 g (0.50 mmol) of the methio-
dide 15 was dissolved in 15 mL ethanol (90%). The mixture was
cooled to 0 ꢁC and 57 mg (1.5 mmol) NaBH4 in 2 mL ethanol (90%)
was added slowly. After stirring for 2 h at 0 ꢁC and 24 h at room
temperature the excess of NaBH4 was destroyed by adding 2 M HCl.
Then the mixture was alkalized with 2 M NaOH and extracted with
diethyl ether (3ꢂ10 mL). The combined organic layers were dried
with Na2SO4 and the solvent was evaporated. The residue was
purified by flash column chromatography (ethyl acetate/hexane/
EtNMe2, 1:1:0.1) to give 230 mg (72%) of 16 as a yellow solid, mp
107 (100). Anal. Calcd for C29H41NO (419.66) C, 83.00; H, 9.85; N,
20
3.34. Found C, 82.62; H, 9.88; N, 3.16. [
a
]
þ26.9 (c 1.0, CHCl3).
D
3.1.10. N-Methyl-N-[(3
chola-7,22-dien-3-yl]-2-nitrobenzenesulfonamide
(4.0 mmol) N-methyl-2-nitrobenzenesulfonamide and 1.56
a
)-23-(3-methylpyridin-4-yl)-24-nor-5
a
,22E-
(14). 0.88
g
g
(6.00 mmol) triphenylphosphane were dissolved in 20 mL of an-
hydrous THF. The solution was stirred under nitrogen and 0.84 g
(2.0 mmol) of the alcohol 13 was added, followed by 1.04 g
(6.00 mmol) DEAD. The mixture was stirred at room temperature
for 3 h, concentrated in vacuo and the residue was purified by flash
column chromatography (ethyl acetate/hexane, 2:1) to give 740 mg
79 ꢁC. IR (KBr):
n
(cmꢀ1)¼2931, 1545, 1458, 1371, 1346, 1292, 1217,
1159, 1124, 1059, 964, 922, 850, 779, 759, 740, 727, 661, 642, 579,
532. 1H NMR (CDCl3):
d
(ppm)¼8.02 (d, J¼9.5 Hz, 1H, 3-H), 7.67 (m,
2H, 4-Hþ5-H), 7.61 (d, J¼9.5 Hz, 1H, 6-H), 6.34 (d, J¼15.5 Hz, 1H,
230-H), 5.42 (dd, J1¼15.5 Hz, J2¼9.0 Hz,1H, 220-H), 5.14 (m,1H, 70-H),
4.07 (m, 1H, 30-H), 3.04 (s, 3H, SO2NeCH3), 2.86 (m, 2H, 200-H), 2.51
(m, 2H, 600-H), 2.33 (s, 3H, 100-CH3), 2.25 (m, 2H, 500-H), 2.13 (m, 1H,
200-H), 2.05 (m, 1H,120-HH),1.90e1.10 (m,19H),1.71 (s, 3H, 300-CH3),
1.05 (d, J¼6.5 Hz, 3H, 210-H), 0.75 (s, 3H, 190-H), 0.56 (s, 3H, 180-H).
(60%) of 14 as a yellow solid, Mp 84 ꢁC. IR (KBr):
n
(cmꢀ1)¼2933,
1736, 1643, 1590, 1545, 1446, 1406, 1371, 1219, 1159, 1124, 1059, 972,
922, 850, 779, 760, 741, 727, 661, 642, 579, 534. 1H NMR (CDCl3):
d
(ppm)¼8.32 (s, 1H, 200-Hþd, J¼5.1 Hz, 1H, 600-H), 8.02 (d, J¼9.2 Hz,
13C NMR (CDCl3):
d
(ppm)¼148.1 (C-2), 139.6 (C-80), 134.7 (C-220),
1H, 3-H), 7.66 (m, 2H, 4-Hþ5-H), 7.61 (d, J¼9.3 Hz, 1H, 6-H), 7.22 (d,
J¼5.1 Hz, 1H, 500-H), 6.41 (d, J¼15.7 Hz, 1H, 230-H), 6.16 (dd,
J1¼15.7 Hz, J2¼9.0 Hz, 1H, 220-H), 5.15 (m, 1H, 70-H), 4.06 (m, 1H, 30-
H), 3.03 (s, 3H, NeCH3), 2.30 (m, 1H, 200-H), 2.28 (s, 3H, 300-CH3),
2.03 (m, 1-H, 120-HH), 1.90e1.20 (m, 19H) 1.14 (d, J¼6.6 Hz, 3H, 210-
H), 0.76 (s, 3H, 190-H), 0.59 (s, 3H, 180-H). 13C NMR (CDCl3):
133.3 (C-1), 133.2 (C-5), 131.5 (C-4), 131.0 (C-3), 127.8 (C-400), 126.2
(C-300), 124.9 (C-230), 124.2 (C-6), 117.5 (C-70), 60.5 (C-200), 56.0 (C-
170), 55.2 (C-140), 52.4 (C-600), 51.5 (C-30), 49.7 (C-90), 45.7 (100-CH3),
43.5 (C-130), 41.0 (C-200), 39.4 (C-120), 37.2 (C-50), 34.7 (C-10), 33.8
(C-10), 33.1 (SO2NeCH3), 31.9 (C-40), 29.7 (C-60), 28.1 (C-160), 26.6
(C-500), 25.9 (C-20), 22.8 (C-150), 21.3 (C-110), 21.0 (C-210), 16.5 (300-
CH3), 13.1 (C-190), 12.2 (C-180). MS (EI): m/z (%)¼635 (3) [Mþ], 448
(30), 433 (47), 122 (41), 110 (53), 85 (100). Anal. Calcd for
d
(ppm)¼151.0 (C-200), 148.1 (C-2), 147.4 (C-600), 144.4 (C-400), 142.6
(C-220), 139.3 (C-80), 133.4 (C-1), 133.3 (C-5), 131.5 (C-4), 131.0 (C-3),
129.8 (C-300),124.2 (C-6),123.1 (C-230),119.2 (C-500),117.7 (C-70), 55.4
(C-170), 55.0 (C-140), 51.4 (C-30), 49.6 (C-90), 43.7 (C-130), 41.2 (C-200),
39.4 (C-120), 37.2 (C-50), 34.6 (C-10), 33.8 (C-100), 33.1 (NeCH3), 31.9
(C-40), 29.7 (C-60), 28.0 (C-160), 25.9 (C-20), 22.8 (C-150), 21.3 (C-110),
20.3 (C-210), 16.6 (300-CH3), 13.1 (C-190), 12.2 (C-180). MS (EI): m/z
(%)¼615 (5) [Mþ], 577 (64), 402 (44),120 (62),174 (44),147 (76),107
(100). Anal. Calcd for C36H47N3O4S (617.86) C, 69.98; H, 7.67; N,
C
37H53N3O4S (635.92) C, 69.89; H, 8.40; N, 6.61. Found C, 70.23; H,
20
8.69; N, 6.04. [
a
]
D
þ23.0 (c 0.5, CHCl3).
3.1.13. (3
methylamino-24-nor-5
a
)-23-(1,3-Dimethyl-1,2,5,6-tetrahydropyridin-4-yl)-3-
,22E-chola-7,22-diene (plakinamine B)
a
(2). 100 mg (0.157 mmol) of the sulfonamide 16 was dissolved in
5 mL of acetonitrile. 56 mg (0.50 mmol) thiophenol and 0.30 g
(0.90 mmol) cesium carbonate were added and the mixture was
stirred at room temperature for 24 h. After filtration the solvent was
removed and the residue was purified by flash column chroma-
tography (ethyl acetate/hexane/EtNMe2, 1:1:0.1) to give 50 mg
6.80. Found C, 69.59; H, 7.92; N, 6.59. [
a]
20 þ39.2 (c 0.5, CHCl3).
D
3.1.11. 4-[(3
a
)-(N-Methyl-2-nitrobenzolsulfamoyl)-24-nor-5a,22E-
chola-7,22-dien-23-yl]-1,3-dimethylpyridinium iodide (15). 250 mg
(0.404 mmol) of the pyridine 14 was dissolved in a small amount of
acetone, treated with 0.14 g (1.0 mmol) methyl iodide, and refluxed
for 1 h. After cooling the precipitated methiodide was collected by
(71%) of 2 as a viscous yellow oil. IR (KBr):
n
(cmꢀ1)¼3419, 2925,
2852, 2783, 1442, 1379, 1292, 1269, 1200, 1124, 962, 847, 721, 542.
1H NMR (CDCl3):
d
(ppm)¼6.34 (d, J¼15.6 Hz, 1H, 23-H), 5.42 (dd,

1090
M. Gans, F. Bracher / Tetrahedron 70 (2014) 1084e1090
J1¼15.6, J2¼8.8 Hz, 1H, 22-H), 5.16 (m, 1H, 7-H), 2.85 (m, 2H, 20-H),
2.74 (m, 1H, 3-H), 2.50 (m, 2H, 60-H), 2.37 (s, 3H, NHeCH3), 2.33 (s,
3H, 10-CH3), 2.25 (m, 2H, 50-H), 2.13 (m, 1H, 20-H), 2.01 (m, 1H, 12-
HH), 1.90e1.10 (m, 20H), 1.71 (s, 3H, 30-CH3), 1.05 (d, J¼6.4 Hz, 3H,
21-H), 0.80 (s, 3H, 19-H), 0.56 (s, 3H, 18-H). 13C NMR (CDCl3):
F.; Debitus, C.; Esposito, G.; Iuvone, T. J. Nat. Prod. 2002, 65, 1206e1209; (f)
Ridley, C. P.; Faulkner, D. J. J. Nat. Prod. 2003, 66, 1536e1539; (g) Zampella, A.;
D’Orsi, R.; Sepe, V.; De Marino, S.; Borbone, N.; Valentin, A.; Debitus, C.; Zollo,
F.; D’Auria, M. V. Eur. J. Org. Chem. 2005, 4359e4363; (h) Langjae, R.; Bussar-
awit, S.; Yuenyongsawad, S.; Ingkanian, K.; Plubrukarn, A. Steroids 2007, 72,
682e685; (i) Aknin, M.; Rudi, A.; Kashman, Y.; Vacelet, J.; Gaydou, E. M. Nat.
Prod. Commun. 2010, 5, 33e34.
d
(ppm)¼139.4 (C-8), 134.8 (C-22), 127.6/126.0 (C-30/C-40), 124.8 (C-
10. Khan, I.; Samad, A.; Khan, A. Z.; Habtemariam, S.; Badshah, S.; Abdullah, S. M.;
Ullah, N.; Khan, A.; Zia-Ul-Haq, M. Pharm. Biol. 2013, 51, 722e727.
11. Aoki, S.; Watanabe, Y.; Sanagawa, M.; Setiawan, A.; Kotoku, N.; Kobayashi, N. J.
Am. Chem. Soc. 2006, 128, 3148e3149.
23), 117.7 (C-7), 60.5 (C-20), 56.0 (C-17), 55.1 (C-14), 54.2 (C-3), 52.4
(C-60), 49.7 (C-9), 45.7 (100-CH3), 43.5 (C-13), 41.0 (C-20), 39.5 (C-12),
35.1 (C-5), 34.9 (C-10), 34.2 (NHeCH3), 32.5/32.4 (C-1/C-4), 29.7 (C-
6), 28.1 (C-16), 26.5 (C-50), 25.5 (C-2), 22.9 (C-15), 21.2 (C-11), 21.0
(C-21), 16.5 (30-CH3), 12.4 (C-19), 12.2 (C-18). MS (EI): m/z (%)¼450
(74) [Mþ], 435 (100), 277 (23), 84 (83). HRMS (EI): Calcd for
12. Chen, D. Y.; Tseng, C. C. Org. Biomol. Chem. 2010, 8, 2900e2911.
€
13. Czako, B.; Kurti, I.; Mammoto, A.; Ingber, D. E.; Corey, E. J. J. Am. Chem. Soc. 2009,
131, 9014e9019.
14. Giera, M.; Bracher, F. Sci. Pharm. 2008, 76, 599e604.
€
15. Giera, M.; Renard, D.; Plossl, F.; Bracher, F. Steroids 2008, 73, 299e308.
C
31H50N2: 450.3974, found 450.3989. [
a]
20 þ21.5 (c 1.0, MeOH, lit.8
€
16. Giera, M.; Plossl, F.; Bracher, F. Steroids 2007, 72, 633e642.
D
€
17. Renard, D.; Perruchon, J.; Giera, M.; Muller, J.; Bracher, F. Bioorg. Med. Chem.
þ29 (dihydrochloride)).
2009, 17, 8123e8137.
18. Mayer, C. D.; Bracher, F. Eur. J. Med. Chem. 2011, 46, 3227e3236.
The agar diffusion assay was performed as described by us
recently.18
19. Krojer, M.; Bracher, F. Sci. Pharm. 2013, 81, 329e338.
€
20. Lange, S.; Keller, M.; Muller, C.; Oliaro-Bosso, S.; Balliano, G.; Bracher, F. Eur. J.
Med. Chem. 2013, 63, 758e764.
€
€
21. Konig, M.; Muller, C.; Bracher, F. Bioorg. Med. Chem. 2013, 21, 1925e1943.
22. Tadros, W.; Boulos, A. L. Helv. Chim. Acta 1975, 58, 668e671.
23. Sakai, K.; Tsuda, K. Chem. Pharm. Bull. 1963, 11, 529e530.
24. Reimann, E.; Speckbacher, J.; Lotter, H. Arch. Pharm. (Weinheim, Ger.) 1987, 320,
385e393.
Conflict of interest
The authors declare no conflict of interest.
References and notes
25. Kaiser, E. M.; Bartling, G. J.; Thomas, W. R.; Nichols, S. B.; Nash, D. R. J. Org.
Chem. 1973, 38, 71e75.
26. Kaminski, T.; Gros, P.; Fort, Y. Eur. J. Org. Chem. 2003, 3855e3860.
27. Yadav, J. S.; Mysorekar, S. V. Synth. Commun. 1989, 19, 1057e1060.
28. Oediger, H.; Muller, F.; Eiter, H. Synthesis 1972, 591e598.
1. Atta-ur-Rahman; Choudhary, M. I. In The Alkaloids, Chemistry and Pharmacology;
Cordell, G. A., Ed.; Academic Press: New York, 1998; Vol. 50, pp 61e108.
2. Burbiel, J.; Bracher, F. Steroids 2003, 68, 587e594.
3. Devkota, K. P.; Lenta, B. N.; Fokou, P. A.; Sewald, N. Nat. Prod. Rep. 2008, 25,
612e630.
€
29. Jones, S. R.; Kinney, W. A.; Zhang, X.; Jones, L. M.; Selinsky, B. S. Steroids 1996,
61, 565e571.
30. Shu, Y.; Jones, S. R.; Kinney, W. A.; Selinsky, B. S. Steroids 2002, 67, 291e304.
31. Moriarty, R. M.; Tuladhar, S. M.; Guo, L.; Wehrli, S. Tetrahedron Lett. 1994, 35,
8103e8106.
4. Sun, Y.; Yan, Y.-X.; Chen, J.-C.; Lu, L.; Zhang, X.-M.; Li, Y.; Qiu, M.-H. Steroids
2010, 75, 818e824.
32. Moriarty, R. M.; Enache, L. A.; Kinney, W. A.; Allen, C. S.; Canary, J. W.; Tuladhar,
S. M.; Guo, L. Tetrahedron Lett. 1995, 36, 5139e5142.
33. Weis, A. L.; Bakos, T.; Alferiev, I.; Zhang, X.; Shao, B.; Kinney, W. A. Tetrahedron
Lett. 1999, 40, 4863e4864.
34. Matyas, L.; Kasal, A.; Babot Riera, Z.; Sunol, C. E. Collect. Czech. Chem. Commun.
2004, 69, 1506e1516.
5. Chang, L. C.; Bhat, K. P. L.; Pisha, E.; Kennely, E. J.; Fong, H. H. S.; Pezzuto, J. M.;
Kinghorn, A. D. J. Nat. Prod. 1998, 61, 1257e1262.
6. Dattagupta, N.; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4286e4290.
7. Brassard, B.; Gomez, D.; De Cian, A.; Paterski, R.; Montagnac, A.; Qui, K.-H.;
Temime-Smaali, N.; Trentsaux, C.; Mergny, J.-L.; Gueritte, F.; Riou, J.-F. Mol.
Pharmacol. 2007, 72, 631e640.
8. Rosser, R. M.; Faulkner, D. J. J. Org. Chem. 1984, 49, 5157e5160.
9. (a) Jurek, J.; Scheuer, P. J.; Kelly-Borges, M. J. Nat. Prod. 1994, 57, 1004e1007; (b)
De Marino, S.; Zollo, F.; Iorizzi, M.; Debitus, C. Tetrahedron Lett. 1998, 39,
7611e7614; (c) De Marino, S.; Iorizzi, M.; Zollo, F.; Roussakis, C.; Debitus, C. Eur.
J. Org. Chem. 1999, 697e701; (d) Lee, H.-S.; Seo, Y.; Rho, J.-R.; Shin, J.; Paul, V. J. J.
Nat. Prod. 2001, 64, 1474e1476; (e) Borbone, N.; De Marino, S.; Iorizzi, M.; Zollo,
35. Torelli, V.; Nedelec, L. US 4,376,734, 1984.
36. Enhsen, A.; Glombik, H.; Kramer, W.; Wess, G. EP 0614908 A2, 1994.
37. Fukuyama, T.; Jow, C. K.; Cheung, M. Tetrahedron Lett. 1995, 36, 6373e6374.
38. Ichikawa, Y.; Hirata, K.; Ohbayashi, M.; Isobe, M. Chem.dEur. J. 2004, 10,
3241e3251.
39. Gillis, B. T.; Beck, P. E. J. Org. Chem. 1962, 27, 1947e1951.