Aminosterols from the dogfish shark Squalus acanthias

Article abstract of DOI:10.1021/np990514f

Seven new aminosterols related to squalamine (8) were isolated from the liver of the dogfish shark Squalus acanthias. Their structures (1-7) were determined using spectroscopic methods, including 2D NMR and HRFABMS. These aminosterols possess a relatively invariant cholestane skeleton with a trans AB ring junction, a spermidine or spermine attached equatorially at C3, and a steroidal side-chain that may be sulfated. The structure of the lone spermine conjugate, 7 (MSI-1436), was confirmed by its synthesis from (5α,7α,24R)-7- hydroxy-3-ketocholestan-24-yl sulfate. Some members of this family of aminosterols exhibit a broad spectrum of antimicrobial activity comparable to squalamine.

Full text of DOI:10.1021/np990514f

J . Nat. Prod. 2000, 63, 631-635
631
Am in oster ols fr om th e Dogfish Sh a r k Squ a lu s a ca n th ia s
Meenakshi N. Rao,† Ann E. Shinnar,‡ Lincoln A. Noecker, Tessa L. Chao, Binyamin Feibush, Brad Snyder,
Ilya Sharkansky, Ani Sarkahian, Xuehai Zhang, Stephen R. J ones, William A. Kinney,*,§ and Michael Zasloff
Magainin Pharmaceuticals, Inc., 5110 Campus Drive, Plymouth Meeting, Pennsylvania 19462
Received October 15, 1999
Seven new aminosterols related to squalamine (8) were isolated from the liver of the dogfish shark Squalus
acanthias. Their structures (1-7) were determined using spectroscopic methods, including 2D NMR and
HRFABMS. These aminosterols possess a relatively invariant cholestane skeleton with a trans AB ring
junction, a spermidine or spermine attached equatorially at C3, and a steroidal side-chain that may be
sulfated. The structure of the lone spermine conjugate, 7 (MSI-1436), was confirmed by its synthesis
from (5R,7R,24R)-7-hydroxy-3-ketocholestan-24-yl sulfate. Some members of this family of aminosterols
exhibit a broad spectrum of antimicrobial activity comparable to squalamine.
In the search for novel host defense agents, squalamine
(8) (Figure 1) was identified as the first aminosterol from
the dogfish shark, Squalus acanthias (Squalidae), by its
antimicrobial activity.1,2 Squalamine also exhibits antian-
giogenic and antitumor properties3 and is currently in
Phase II clinical trials for the treatment of advanced
nonsmall cell lung cancer. Attempts to obtain large amounts
of squalamine from the dogfish shark resulted in the
discovery, isolation, and characterization of a family of
novel aminosterols. These aminosterols possess a relatively
invariant steroid skeleton with a trans AB ring junction,
a polyamine attached equatorially at C3, and a cholestane-
related side chain that may be sulfated.
F igu r e 1. Structure of squalamine (8).
Ta ble 1. MIC of Aminosterolsa
MIC (µg/mL)
P. aeruginosa C. albicans
S. aureus
E. coli
compound
(29213)
(25922)
(27853)
(90028)
1
2
3
4
5
6
7
8
8-16
256
128
128
16
8
16
1
4
256
32
128
16
16
16
4
128
16
32
32
32
2
Resu lts a n d Discu ssion
4-8
The original purification scheme for squalamine1 was
modified to make practical the processing of 40-kg batches
of dogfish liver. The process includes (a) an extraction into
water/acetic acid/ethanol/ammonium sulfate; (b) batch
adsorption and extraction from XAD resin; (c) adsorption
and elution from a cation exchange column (propylsulfonic
acid resin); and, finally, (d) reversed-phase separation by
HPLC (YMC-ODS column). Although squalamine is the
most abundant aminosterol found in dogfish shark liver
(400-800 mg, 0.001-0.002%), seven additional amino-
sterols (1-7) are found in amounts of 20-100 mg (0.00005-
0.00025%) for a typical shark liver preparation (40 kg).
Other minor aminosterols, found in trace quantities of <2
mg/40 kg shark liver, have not been fully characterized.
The aminosterols 1-7 reported here are water-soluble,
elute before squalamine, and are numbered in order of their
elution on preparative reversed-phase HPLC. These com-
pounds have a broad spectrum of antimicrobial activity
comparable to squalamine (8) (Table 1) and, in the case of
aminosterols 4-7, exhibit similar potency.
8
8-16
2
2
1
1
4
16
16
a
ATCC numbers in parentheses; squalamine (8) is included for
comparison, and data compare well with previously reported
antimicrobial data.1
by comparison with synthetic squalamine4,7 and synthetic
7. The synthetic 7 reported here was made from a 2.5-fold
excess of spermine and the stereochemically defined 3-keto
derivative used previously in the synthesis of 8.7 For the
other aminosterols with stereogenic centers at C-24, the
stereochemistry is presumed to be 24R. The stereochem-
istry of compounds 1-3 at C-25 is not yet established.
The steroid core of these compounds is minimally
substituted, with the polyamine spermidine found at C-3
in compounds 1-6 and 8. Substitution at the 3-equatorial
position is confirmed by large diaxial couplings to protons
identified as H-2ax and H-4ax. The polyamine region in 8
Full assignments of the proton and carbon signals for
these aminosterols (Figure 2, Tables 2 and 3) were obtained
1
and 2-6 (3.2-2.9 ppm) in the H NMR spectra integrates
for nine protons and includes the 3-axial methine proton
on the A-ring and the eight methylene protons adjacent to
nitrogen atoms in the polyamine. In 1, the polyamine
region is overlapped by signals from H-28 protons next to
a sulfur atom. Similarly, the polyamine spermine found in
7 integrates for 13 protons and includes the 3-axial methine
proton and the 12 methylene protons adjacent to nitrogen
1
by 1H-1H- and H-13C-correlated 2D spectroscopy (COSY,
HMQC, HMBC). The stereochemistry of C-20 and C-24 in
squalamine (8) and compound 7 was proven to be 20R,24R
* To whom correspondence should be addressed. Tel.: (215) 598-2923.
E-mail: kinney.cheshire@att.net.
† Current address: Bristol-Myers Squibb Company PRI, New Brunswick,
NJ 08903.
atoms. The H-1H COSY correlations between these down-
field proton signals and those for the interior methylene
protons found upfield are diagnostic for the attached
1
‡ Current address: Chemistry Department, Barnard College, New York,
NY 10027.
§ Current address: 16 Thompson Mill Rd., Newtown, PA 18940.
10.1021/np990514f CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/05/2000

632 J ournal of Natural Products, 2000, Vol. 63, No. 5
Rao et al.
The 13C NMR and DEPT-135 spectra of compound 2
revealed one methylene and two methine carbons substi-
tuted with an oxygen. In the HMBC spectrum both the
methine carbon at δ 74.2 and the methylene carbon at δ
71.7 gave correlations with the proton signals of the methyl
group (H-27) and methine (H-25) of the side chain. Ad-
ditional correlations were evident between the diaste-
reotopic protons on H-26 (δ 4.12, 3.96) and C-24, C-25 (δ
32.2), and C-27 (δ 14.4). Assignment of the sulfate to C-26
was based on the chemical shift data as well as the
fragmentation pattern in the FABMS, which produced a
[M + H - SO3]+ fragment from the primary aliphatic
sulfate.
1
Initial H-1H COSY studies of compound 3 were distin-
guished by the absence of through-bond connectivities
between positions 23 and 25, while 13C NMR showed a
characteristic carbonyl signal at δ 212.2. Upon interroga-
tion by HMBC, this resonance correlated with protons at
2.82 (H-25) and 2.66, 2.46 (2H, H-23a,b), consistent with a
C-24 keto functionality. Cross-peaks in the HMBC are also
seen between C-24 and δ 3.78 (m, 2H, H-26a,b) and 0.95 (d,
3H, H-27). Evidence for the position of the sulfate on C-26
included the downfield shift of the methylene carbon (δ
67.3) correlated with the multiplet at δ 3.78 on HMQC and
fragmentation of the primary sulfate on FABMS to give
an [M + H - SO3]+ ion.
The 13C NMR spectrum of compound 6 exhibited three
downfield carbon signals at δ 205.2, 145.9, and 125.9,
suggesting both a keto functionality and a double bond.
The multiplicities of the carbons at δ 145.9 and 125.9 were
found to be quaternary and methylene, respectively. Sup-
porting evidence for a double bond was the observation of
olefinic proton signals integrating for one proton each at δ
1
6.05 and 5.84. The H-1H COSY revealed that the down-
field H-27 methyl (δ 1.83, dd) shared cross-peaks with the
protons at δ 6.05 and 5.84. This evidence and additional
HMBC data placed the double bond between C-25 and
C-26. Only one sp3 carbon bearing an oxygen atom was
detected by 13C NMR, and it was identified as the typical
C-7 resonance on the sterol core. It is apparent that
compound 6 is related to 3 by the elimination of H2SO4. In
fact, samples of 3 were found to contain traces of 6 after
storage for several years at freezer temperatures. Com-
pound 3 is unique in containing the sulfate in a â-position
relative to a carbonyl, making it more prone to elimination
reactions than sulfates 2, 4, 5, 7, or 8. These aminosterol
sulfates were stable to similar isolation and storage condi-
tions.
F igu r e 2. Structures of major aminosterols isolated from dogfish
shark liver.
polyamine. Only aminosterol 4 has an additional oxygen-
bearing methine carbon signal at C-12, which correlates
with a single proton observed at δ 3.95. The lack of a large
diaxial coupling with adjacent protons on C-11 indicates
that the hydroxyl group is attached axially [J 11e,12 ) J 11a,12
≈ 4 Hz (COSY)]. Characteristic of all aminosterols reported
here is the broad singlet for H-7 found at δ 3.8 correlating
with a carbon methine signal at δ 68 in methanol-d4.
The structural diversity within this group of compounds
is observed primarily in the side chain. However, the side
chains of 4 and 7 are identical to that of 8, as seen by
comparison of their NMR data. For example, typical HMBC
experiments for these molecules gave correlations between
C-24 and H-25 and the terminal methyl groups H-26/H-
27, as well as between H-24 and C-23, C-25, C-26/C-27.
Compound 5 is 24-hydroxymethyl-substituted squalamine.
The 1H NMR spectrum displayed an AB quartet (δ 3.95)
that correlated with a methylene carbon signal at δ 72.5.
From HMBC data, cross-peaks were clearly seen between
these diastereotopic protons (H-28a,b) and the quaternary
carbon C-24 possessing the sulfate (δ 76.3), C-25 (δ 34.3),
and C-23 (δ 32.4). HRFABMS produced a fragment corre-
sponding to the desulfated ion [M + H - SO3]+. The origin
of this atypical sterol with 28 carbons may be the plant
sterol, campesterol.
Compound 1 has 1H and 13C NMR signals consistent
with a cysteine residue as well as a keto function, which
would result from cysteinylation of 6. Cysteinylation is a
well-described metabolic modification in nonsteroidal xe-
nobiotics.8,9
The biosynthetic origin of these compounds remains to
be elucidated. The structures clearly resemble the known
bile alcohols of the shark, which are frequently found as
sulfuric acid esters.10 However, the aminosterols are
uniquely distinguished from bile alcohols by the polyamine
modification. Although aminosterols of this family have not
been identified as yet in mammals, it should be noted that
closely related steroidal alcohols have been discovered,
which represent logical biosynthetic precursors to the
aminosterol class. Human astrocytes, for example, can
metabolically convert cholesterol to 24-hydroxycholesterol,
7R,25-dihydroxycholest-4-en-3-one, and other alcohols.11
Further conversion of 3-keto-hydroxycholestanes of this
type to aminosterols of the squalamine family would

Aminosterols from Dogfish Shark
J ournal of Natural Products, 2000, Vol. 63, No. 5 633
Ta ble 2. 1H NMR Spectral Data of Major Aminosterols (δ, ppm, in CD3OD, except as noted; J in Hz, in Parentheses)
compound
position
1
2
3a
4
5
6
7
8
1ax
1eq
2ax
2eq
3
4ax
4eq
5
6ax
6eq
7
8
9
1.12
1.86
1.56
1.96
3.13
1.43
1.61
1.74
1.53
1.45
3.80
1.44
1.23
1.35
1.54
1.13
1.98
1.43
1.14
1.86
1.58
2.00
3.14
1.43
1.65
1.76
1.54
1.46
3.80
1.40
1.25
1.34
1.54
1.18
2.00
1.43
0.98
1.70
1.44
1.83
3.04
1.31
1.49
1.62
1.37
1.28
3.61
1.29
1.17
1.20
1.44
1.05
1.88
1.33
1.18
1.83
1.56
2.04
3.16
1.42
1.65
1.75
1.54
1.46
3.81
1.47
1.59
1.15
1.87
1.59
2.02
3.13
1.43
1.64
1.75
1.55
1.48
3.80
1.44
1.28
1.37
1.55
1.20
2.01
1.45
1.12
1.86
1.56
1.96
3.12
1.42
1.63
1.74
1.53
1.45
3.80
1.41
1.23
1.34
1.54
1.14
1.99
1.47
1.14
1.86
1.59
2.01
3.12
1.44
1.65
1.75
1.55
1.46
3.80
1.40
1.27
1.36
1.55
1.16
2.00
1.44
1.15
1.85
1.60
2.03
3.11
1.44
1.64
1.75
1.54
1.46
3.79
1.44
1.29
1.37
1.56
1.18
2.00
1.44
11ax
11eq
12ax
12eq
14
1.58b
1.64b
3.95c br s
1.96
15a,b
16a,b
17
1.78, 1.14
1.89, 1.35
1.14
1.77, 1.12
1.93, 1.32
1.18
1.67, 0.98
1.77, 1.24
1.06
1.76, 1.11
1.90, 1.31
1.82
1.78, 1.13
1.96, 1.32
1.22
1.76, 1.11
1.89, 1.34
1.16
1.76, 1.12
1.92, 1.34
1.17
1.77, 1.11
1.92, 1.34
1.18
18
19
20
0.70 s, 3H
0.86 s, 3H
1.43
0.71 s, 3H
0.87 s, 3H
1.44
0.61 s, 3H
0.74 s, 3H
1.32
0.72 s, 3H
0.85 s, 3H
1.42
0.71 s, 3H
0.87 s, 3H
1.37
0.70 s, 3H
0.86 s, 3H
1.45
0.70 s, 3H
0.86 s, 3H
1.44
0.71 s, 3H
0.87 s, 3H
1.44
21
0.94 d, 3H
(6.9)
0.97 d, 3H
(6.4)
0.87 d, 3H
(6.2)
1.02 s, 3H
(6.4)
0.99 d, 3H
(6.6)
0.96 d, 3H
(6.6)
0.96 d, 3H
(6.3)
0.97 d, 3H
(6.5)
22a
22b
23a
23b
24
1.75
1.23
2.54
2.54
d
d
1.50
1.44
1.62
1.16
2.66
2.46
1.49
1.31
1.71
1.51
1.47
1.11
1.72
1.39
1.77
1.28
2.75
2.67
1.50
1.22
1.71
1.53
1.54
1.31
1.71
1.53
3.44
4.14
4.12
4.13
25
26
2.89
1.16 d, 3H
(6.9)
1.85
4.12, 3.96
2.82
3.78
2.08
0.96 d, 3H
(6.9)
1.88
0.96 d, 3H
(6.9)
2.05
0.95 d, 3H
(7.7)
2.06
0.96 d, 3H
(6.8)
6.05, 5.84
27
2.85, 2.62
0.99 d, 3H
(6.8)
0.95 d, 3H
(7.1)
0.93 d, 3H
(6.9)
0.95 d, 3H
(6.9)
1.83 dd, 3H
(1.3, 1.0)
0.94 d, 3H
(7.7)
0.94 d, 3H
(6.8)
28
29
30
1′
2′
3′
4′
5′
6′
7′
3.14, 2.97
3.96
d
3.15
2.11
3.15
3.08
1.80
1.75
2.98
3.95 ABq (9.7)
3.16
2.11
3.16
3.09
1.80
1.76
2.99
2.99
1.93
2.99
2.92
1.62
1.58
2.81
f
3.19e
2.16
3.16e
3.08
1.82
1.75
2.99
3.18
2.12
3.18
3.10
1.79
1.78
2.99
3.14
2.10
3.14
3.07
1.80
1.76
2.98
3.17
2.12
3.17
3.09
1.81
1.81
3.09
3.14
2.09
3.06
3.17
2.13
3.17
3.09
1.80
1.77
2.98
8′
9′
10′
a
c
d
In DMSO-d6. b,e Assignments within column may be interchanged. J 11e,12 ) J 11a,12 ≈ 4 Hz (COSY). Not observed. f NHR,â ) 8.73 s,
1H; 8.62 br s, 1H, NH2γ ) 7.79 s, 2H.
at 400 and 100 MHz, respectively, using MeOH-d4 or DMSO-
d6 with tetramethylsilane (1H NMR) and solvent peaks (13C
NMR, δ 49.15 and 39.51, respectively) as reference standard.
1H-1H DQF COSY and COSY-45, inverse-detected 1H-13C
HMQC, and HMBC experiments were performed using Bruker
software and conventional pulse sequences. Carbon multiplici-
ties were confirmed using DEPT experiments. FABMS were
measured on a VG Analytical ZAB 2SE spectrometer with
3-nitrobenzyl alcohol and PEG-600 as matrix (M-Scan, West
Chester, PA 19380). Negative ion ESIMS were measured on
a Micromass Autospec at the University of Iowa, Iowa City,
IA. Alternatively, laser desorption mass spectra (MALDI) were
obtained on a PerSeptive Biosystems Voyager instrument.
require the presence of an unknown enzyme that could
couple spermidine or spermine to the A ring of the steroid.
Although the aminosterols reported here possess anti-
biotic activity, their biological functions in the shark are
not well understood in every case. Compound 7 (MSI-1436),
however, has been studied extensively in vivo and has been
shown to induce profound appetite suppression and weight
loss in mammals, including mice, rats, dogs, and mon-
keys.12 It remains to be proven whether compound 7 is
responsible for the sporadic feeding behavior of the dogfish,
an animal that normally eats only once every two weeks.
Additional studies of biological and pharmacological activi-
ties of the other aminosterols described in this report are
in progress.
Fresh dogfish shark liver, normally a waste product, was
obtained from the commercial harvest of shark for edible
products at Seatrade Fishery, Portsmouth, NH.
Minimum inhibitory concentrations (MIC) for the bacteria
and yeast were determined by incubating (0.9-1.1) × 105 cfu/
mL of log-phase microbes in 0.5× trypticase soy broth with
increasing amounts of aminosterol in 96-well microtiter plates
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. 1H and 13C NMR
spectra were recorded at 300 K on a Bruker AC or AMX-400

634 J ournal of Natural Products, 2000, Vol. 63, No. 5
Rao et al.
Ta ble 3. 13C NMR Spectral Data of Major Aminosterols (δ,
Chromatographic purification of separate YMC column
fractions on a column of propylsulfonic acid resin (700 mL) as
described above resulted in homogeneous aminosterol com-
pounds. If the cation exchange eluates were not clear, the
solution was ultrafiltered (3-kD cutoff, Amicon, Woburn, MA)
prior to loading on a YMC-ODS (25 × 1 cm) or Dynamax (25
× 4 cm, Rainin, Woburn, MA) column. A gradient of CH3CN/
H2O containing 0.1% TFA eluted the aminosterols.
ppm, in CD3OD, except as noted)
compound
position
1
2
3a
4
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
37.7 37.8
26.0 26.1
59.0 59.1
32.2 31.9
38.7 38.7
37.7 37.8
68.4 68.4
41.1 41.1
46.9 46.9
36.8 36.9
22.1 22.2
41.0 41.1
43.8 43.9
51.8 51.8
24.6 24.7
29.3 29.6
57.6 58.0
12.4 12.6
11.5 11.7
36.9 37.4
19.1 19.5
30.8 33.3
40.1 32.2
35.8 37.6 37.9
24.1 25.9 26.1
55.9 59.2 59.2
37.7
26.1
59.0
37.7 37.9
25.9 26.1
59.0 59.2
30.1 32.1 32.1b 32.2c 32.0 32.1
36.4 38.7 39.7
36.5 37.7 37.8
65.3 68.4 68.4
39.1 41.2 41.2
44.7 40.4 46.8
35.1 36.5 37.0
20.4 29.8 22.3
39.1 73.8 41.1
41.9 47.5 43.9
49.9 43.5 51.8
23.0 24.3 24.8
27.5 28.8 29.6
55.5 48.3 57.7
11.6 13.1 12.6
10.6 11.4 11.7
34.7 37.2 38.0
18.3 18.3d 19.7
28.9 33.0 30.2
38.6
37.7
68.4
41.1
46.9
37.0
22.1
41.0
43.8
51.8
24.6
29.3
57.6
12.4
11.5
36.9
19.1
38.5 38.7
37.7 37.8
68.3 68.4
41.0 41.2
46.7 46.8
36.8 37.0
22.1 22.3
41.0 45.4
43.7 43.0
51.7 51.8
24.4 24.7
29.4 29.5
57.5 57.7
12.4 12.6
11.6 11.7
37.2 37.4
19.4 19.6
The isolated aminosterols were analyzed as their o-phthala-
ldehyde (OPA) (precolumn-OPA-treated) derivatives using a
YMC-ODS-AQ (120 Å, 250 × 4.6 mm) column with fluores-
cence detection. The mobile phase was a gradient (20-70%B,
30 min; 70-100%B, 5 min) of Buffer A (0.1% TFA in 90:10
H2O/CH3CN) and Buffer B (0.1% TFA in 10:90 H2O/CH3CN)
at 1 mL/min. Fluorescence excitation was at 230 nm and
detection at 455 nm.
The 1H and 13C NMR data for compounds 1-8 are displayed
in Tables 2 and 3. Additional analytical and experimental
information is described below.
Com p ou n d 1: white powder; HRFABMS m/z 665.5043 [M
+ H]+ (calcd for C37H69N4O4S 665.5039).
Com p ou n d 2: white powder; FABMS m/z 642 [M - H]-
(calcd for C34H64N3O6S 642.5), 565 [M + H - SO3]+; HRESIMS
m/z 642.4502 [M - H]- (calcd for C34H64N3O6S 642.4515).
Com p ou n d 3: white powder; HRFABMS m/z 562.4933 [M
+ H - SO3]+ (calcd for C34H64N3O3 fragment 562.4947); MALDI
MS 642 [M + H]+ (calcd for C34H64N3O6S 642).
32.3c 32.5 32.7
37.3 28.0 32.4b 35.5
28.2 28.3
86.5 86.6
32.0 32.2
18.4 18.6
18.3 18.4
216.3 74.2 212.2 86.3 76.3 205.2
47.5 40.7
35.5 71.7
17.1 14.4
34.2
45.3 32.3 34.3 145.9
67.3 18.4d 17.4 125.9
13.1 18.2d 17.4
72.5
17.9
Com p ou n d 4: white powder; HRFABMS m/z 644.4687 [M
+ H]+ (calcd for C34H66N3O6S 644.4672); anal. calcd for
54.5
171.7
C
34H65N3O6S/2TFA/3H2O; C 49.29%, H 7.95%, N 4.54%, S
42.9e 43.1f 40.7g 43.0h 43.1i
43.0j 42.9k 43.9l
24.7m 24.6n 24.6
46.1j 45.9k 46.0l
3.46%; found: C 49.17%, H 7.86%, N 4.53%, S 3.29%.
2′
3′
4′
5′
6′
7′
8′
9′
24.6 24.6
22.5 24.6 24.6
Com p ou n d 5: white powder; HRFABMS m/z 578.5264 [M
+ H - SO3]+ (calcd for C35H68N3O3 fragment 578.5260), MALDI
MS 658 [M + H]+ (calcd for C35H68N3O6S 658).
46.0e 46.1f 43.8g 46.0h 46.0i
48.9 48.5
24.4 24.4
25.7 25.7
40.1 40.2
46.0 48.4 48.4
22.5 24.2 24.3
24.0 25.9 25.7
38.1 40.1 40.2
48.5
48.2o 48.4
24.5m 24.2p 24.3
24.1p 25.7
48.2o 40.1
45.9
Com p ou n d 6: white powder; HRFABMS m/z 544.4823 [M
25.8
40.1
+ H]+ (calcd for C34H62N3O2 544.4842).
Com p ou n d 7: white powder; HRESIMS m/z 683.5130 [M
- H]- (calcd for C37H71N4O5S 683.5145); anal. calcd for
C37H72N4O5S/3TFA/H2O; C 49.42%, H 7.43%, N 5.36%, S
3.07%, F 16.36%; found: C 49.34%, H 7.35%, N 5.08%, S 2.76%,
F 16.44%.
25.4n
10′
37.9
a
b-p
In DMSO-d6.
Assignments within column may be inter-
changed.
Syn th esis of Com p ou n d 7. To a solution of spermine
hydrochloride (870 mg, 2.5 mmol) in CH3OH (15 mL) was
added sodium methoxide (0.5 M, 24.5 mL, 12 mmol) in CH3-
OH. Potassium (5R,7R,24R)-7-hydroxy-3-ketocholestan-24-yl
sulfate (536 mg, 1.0 mmol)7 was added and stirred overnight
at room temperature. The reaction mixture was cooled to -78
°C, treated with sodium borohydride (70 mg, 1.8 mmol), and
stirred for 3.5 h. After warming to room temperature, the
solvent was removed, and the solid material was dried in
vacuo. The solid was dissolved in H2O, acidified with trifluo-
roacetic acid to pH 2, and filtered on Celite. The solution was
applied to preparative reversed-phase HPLC and gave 7 (534
mg, 52%) as the trifluoroacetate salt. The 1H and 13C NMR
were in agreement with data obtained for the natural material.
A product (85 mg, 8.0%) that eluted after 7 was identified as
(Corning, Corning, NY) at 37 °C for 18-24 h.1 Initial sample
concentrations were 2 mg/mL in 250 mM of sodium acetate,
pH 6.6.
Extr a ction a n d Isola tion . Freshly ground dogfish shark
liver (20 kg) was suspended in a solution of 12% (v/v) acetic
acid (82 L) at 75 °C for 1 h. Ammonium sulfate (18 kg) and
95% ethanol (17 L) were added, followed by vigorous agitation
for 5 min. The suspension was allowed to stand for 6 days,
and the organic phase was discarded. After the aqueous phase
was filtered through cheesecloth, the solution from two such
20-kg batches containing the crude aminosterols was slowly
stirred with XAD-16 resin (5 kg, Supelco, Bellefonte, PA) for
20 h. The bulk resin was rinsed with H2O (10 L) and
resuspended in 70% EtOH (20 L) for 20 min.
The ethanolic extracts from 10 batches of XAD resin (200
L) were filtered through a 5-µm Polypure DCF filter (Gelman
Sciences, Ann Arbor, MI) and applied to a cation exchange
column of propylsulfonic acid resin (3 kg, J . T. Baker, Phil-
lipsburg, NJ ). The column was washed with 20% 2-propanol
followed by washing with 0.4 M potassium acetate in 10%
2-propanol until absorption at 254 nm was constant. The
aminosterols were then eluted with either 3.6 or 4.5 M
potassium acetate in 10% 2-propanol (15-30 L). The separate
eluates were diluted to 1 M salt concentrations with H2O and
further purified on a YMC-ODS (10 µm, 120 Å, 25 × 10 cm,
YMC, Wilmington, NC). The YMC column was washed with
0.1% TFA in H2O (40 L) and 0.1% TFA in 25% CH3CN (10 L).
Aminosterols were eluted with a gradient of 25-34% CH3CN
containing 0.1% TFA (470 mL/min, 0.5 L fractions) over 13
min, then 34-40% CH3CN containing 0.1% TFA (470 mL/min,
235-mL fractions) over 22 min.
1
the 3R-isomer of 7: H NMR (CD3OD) δ 4.13 (m, 1H, H-24),
3.80 (br s, 1H, H-7), 3.44 (m, 1H, H-3), 3.15-3.04 (m, 12H),
2.10-1.00 (m, 35H), 0.95, 0.95, 0.93 (ddd, 9H, H-21, H-26,
H-27), 0.86 (s, 3H, H-19), 0.70 (s, 3H, H-18); 13C NMR (CD3-
OD) δ 86.5 (C-24), 68.4 (C-7), 57.6, 56.8, 51.9, 48.3, 48.3, 46.8,
46.1, 46.0, 44.4, 43.8, 41.1, 41.0, 38.0, 37.3, 37.3, 37.2, 33.4,
32.9, 32.5, 32.1, 30.2, 29.4, 28.2, 25.5, 24.6, 24.3, 24.3, 24.1,
23.8, 21.8, 19.5, 18.5, 18.5, 12.5, 11.0.
Com p ou n d 8: white powder; for 1H and 13C NMR data in
DMSO-d6, see Wehrli et al.2
Ack n ow led gm en t. We wish to thank Steve Barndollar,
J eff Young, and their dedicated fishermen (Seatrade Fishery,
Portsmouth, NH) for providing the freshly harvested dogfish
livers, which served as the source of the aminosterols reported
here.

Aminosterols from Dogfish Shark
J ournal of Natural Products, 2000, Vol. 63, No. 5 635
Tham, F. S. J . Org. Chem. 1998, 63, 3786-3789.
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NP990514F