Synthesis of spermidinylcholestanol and spermidinylcholesterol, squalamine analogues

Article abstract of DOI:10.1016/j.tet.2004.09.055

Several novel squalamine-related polyaminosterols are reported. The synthesis of 7α-N-[3N-(4-aminobutyl)aminopropyl]aminocholestanol I, 6α-N-[3N-(4-aminobutyl)aminopropyl]aminocholestanol II, 7α and 7β-N-[3N-(4-aminobutyl)aminopropyl]aminocholesterol (III and IV), was accomplished from cholesterol, they provide the first examples in which spermidine is introduced in the B steroidal ring. These molecules showed comparable antibacteria and fungi activities to squalamine, and were cytotoxic on a human non-small cell bronchopulmonary carcinoma line (NSCLC-N6). Therefore, these molecules with antibiotic and cytotoxic activities are promising for immune-compromised patients in cancer chemotherapy. Graphical Abstract.

Full text of DOI:10.1016/j.tet.2004.09.055

Tetrahedron 60 (2004) 11477–11486
Synthesis of spermidinylcholestanol and spermidinylcholesterol,
squalamine analogues
a
Bassima Choucair, Michel Dherbomez, Cristos Roussakis and La ¨ı la El Kihel
b
c
a,†,*
a
Universit e´ de Caen, Cyceron, LRA, Av. Becquerel, 14000 Caen, France
IUT de La Rochelle, Rue Fran c¸ ois de Vaux de Fauletier, 17000 La Rochelle, France
Facult e´ de Pharmacie de Nantes, Laboratoire de Pharmacologie Marine, 44035 Nantes cedex, France
b
c
Received 12 July 2004; revised 9 September 2004; accepted 17 September 2004
Available online 13 October 2004
Abstract—Several novel squalamine-related polyaminosterols are reported. The synthesis of 7a-N-[3N-(4-aminobutyl)aminopropyl]ami-
nocholestanol I, 6a-N-[3N-(4-aminobutyl)aminopropyl]aminocholestanol II, 7a and 7b-N-[3N-(4-aminobutyl)aminopropyl]aminocholes-
terol (III and IV), was accomplished from cholesterol, they provide the first examples in which spermidine is introduced in the B steroidal
ring. These molecules showed comparable antibacteria and fungi activities to squalamine, and were cytotoxic on a human non-small cell
bronchopulmonary carcinoma line (NSCLC-N6). Therefore, these molecules with antibiotic and cytotoxic activities are promising for
immune-compromised patients in cancer chemotherapy.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
genic agent, preventing the formation of new blood vessels
in developing malignant tumors. Squalamine also inhibits
3
Squalamine is a novel aminosterol isolated from the tissues
of the dog fish shark, Squalus acanthias. This compound has
an unusual chemical structure, its a 5a-cholestane with 7a-
tumor growth in multiple animal models and is currently in
phase II clinical trial for treatment of advanced non-small
cell lung cancer. The combination of squalamine and
4
1
hydroxy, 3b-spermidinyl and 24R-sulfate groups (Fig. 1).
cisplatin has shown high activity against human cancer
cells. More recently, seven new aminosterols related to
5
Squalamine was the first example of a natural product which
is a stero ¨ı dal polyamine (spermidine). Squalamine exhibits
antimicrobial activity against a wide variety of micro-
organisms (Gram negative bacteria, Gram positive bacteria
squalamine were discovered from the liver of the dog fish
shark S. acanthias. These compounds exhibited a relatively
invariant cholestane skeleton with a trans A/B ring junction,
with spermidine or spermine attached equatoriallly at C3.
The side chain had several variations: The sterol side chain
had a hydroxyl or a hydroxymethyl group at the C24
position, and a sulfate group at either the C24 or C26
position. One of these aminosterols was dessulfated, but had
a ketone function at C24 and a double bond D25–26, while
another had a hydroxyl group at the C12 position. These
compounds had a broad spectrum of antimicrobial activity
2
and fungi). Its also demonstrates promise as an antiangio-
6
comparable to squalamine.
However, only trace amounts of these molecules were
present in the different tissues of the shark. Chemical
syntheses of squalamine has been accomplished from the
Figure 1. Squalamine.
7
expensive starting materials, 3b-acetoxy-5-cholenic acid,
5
materials used include stigmasterol,
8
-cholenic acid and desmosterol. Inexpensive starting
9
Keywords: Squalamine; Aminosterol; Polyaminosterol; Antibiotic;
10–12
methyl 3-keto-5a-
chenodeoxy cholonate, 3-keto-23,24-bis-norchol-4-en-
Antiangiogenic.
1
3
*
Corresponding author. Tel.: C33 2 31 93 64 58; fax: C33 2 31 93 34 73;
e-mail: elkihel@pharmacie.unicaen.fr
1
2-ol and methyl chenodeoxycholonate. Fourteen to
4
15
2
†
seventeen steps are needed to synthesize this compound, but
these syntheses are accomplished with a low overall yield
(1.9–19%). For these reasons, several squalamine analogues
Present address: Centre d’Etude et de Recherche sur le M e´ dicament de
Normandie, UFR des Sciences Pharmaceutiques, 5 rue Vaub e´ nard 14032
Caen cedex, France.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.09.055

11478
B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
were synthesized. Only one analogue has been described
1
conserved and the asymmetric polyamine (spermidine) was
introduced at the C7 position. Therefore, the synthesis steps
were minimized. 7a-Polyaminocholestanol (I), 6a-polya-
minocholestanol (II) 7a- and 7b-polyaminocholesterols (III
and IV) were synthesized (Scheme 1).
6–17
with a spermidine moiety at the side chain,
analogues have spermidine attached at the C3 position.
but the other
1
8–19
Selinsky’s groups has synthesized squalamine analogues
with a 24R,S-hydroxy or 24R,S-amino, with or without the
7
-hydroxy group. Their aim was to explore the relationship
between the functional groups and antimicrobial activity.
Some of these analogues exhibit antimicrobial activity
comparable to squalamine.
2. Results and discussion
2
0
7
a-N-[3N-(4-Aminobutyl)aminopropyl]aminocholestanol I
Biological studies of squalamine and analogues led to the
following conclusions. The sterol side chain could be
dessulfated. The 7a-hydroxyl group could be suppressed.
The structure of the polyamine on the steroid could be varied.
The steroid can be have other functions on the side chain.
was prepared from cholesterol. As depicted in Scheme 2, the
hydroxyl group of cholesterol was protected with a
tetrahydropyranyl group 1 (99%). Oxidation with chromium
trioxide–pyridine complex afforded the allylic ketone 2 in
6
2% yield. Selective reduction of the D5 double bond under
Birsh conditions, with lithium/ammonia at K78 8C, gave
the A/B trans junction in 82% yield (compound 3). Alcohol
functionality of this compound was deprotected to give
compound 4. The intermediate 5 was obtained in 84% yield
via a reductive amination of ketone 4 with 3N-(tertio-
butylcarbonyl)aminopropane utilizing sodium cyanoboro-
hydride (pH 5–6 was ajusted with acetic acid) as the
reducing agent. Probably due to steric hinderance, only the
In this way, our approach was to develop new analogues of
squalamine. Squalamine has a spermidine unit at C3 and a
hydroxyl at the C7 position of the steroidal skeleton. We
describe analogues with spermidine at C7 and a hydroxyl
group at C3 position. These analogues were synthesized
from cholesterol, an inexpensive starting material. The
hydroxyl group at the C3 position of this steroid was
Scheme 1. Analogues of squalamine.
Scheme 2. Synthesis of 7a-spermidinylcholestanol. Reagents and conditions: (a) CrO
(d) NH
3
-2py, CH
–CN, DMF, 60 8C; (g) LiAlH
2
Cl
2
, RT; (b) Li/NH
3
, THF, K78 8C; (c) PPTS, ethanol, reflux;
, NiCl , 6H O THF, reflux.
2
(CH
2
)
3
NHBOC, NaBH
3
CN, AcOH pH 5–6; (e) TFA, CH
2
Cl
2
; (f) Br–(CH
2
)
3
4
2
2

B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
11479
Scheme 3. Synthesis of 6a-spermidinylcholestanol. Reagents and conditions: (a) CH
d) LiAlH , THF, reflux; (e) acrylonitrile, MeOH; (f) LiAlH /NiCl ,6H O, THF, reflux; (g) Br–(CH
reflux.
3
COOH, HNO
3
; (b) CH
3
COOH/Zn, reflux; (c) HONH
2
.HCl, py, reflux;
–CN, DMF, 60 8C; (h) LiAlH /NiCl , 6H O, THF,
(
4
4
2
2
2
)
3
4 2 2
a epimer was observed. The amine function was depro-
tected 6 (79%) and alkylated by 4-bromobutyronitrile to
give compound 7 in 51% yield. Nitrile reduction of 7 was
achieved with lithium aluminium hydride in presence of
nickel chloride hexahydrate giving the polyaminosterol I in
was treated with high density (dZ1.53) nitric acid in
anhydrous acetic acid to give the 6-nitro derivative 9 in 74%
yield. Ketone 10 was treated with zinc in acetic acid.
Oxime derivative 11 was obtained from the ketone by
reaction with hydroxylamine hydrochloride in pyridine and
reduced by lithium aluminium hydride in tetrahydrofuran, to
3
0% yield.
6
a-aminocholestanol (12). Probably due to steric hinder-
6a-N-[3N-(4-Aminobutyl)aminopropyl]aminocholestanol
II was prepared as shown in Scheme 3 cholesteryl acetate
ance, only the a epimer was observed. Acrylonitrile
was reacted in methanol with aminosterol (12) giving the
Scheme 4. Synthesis of 7a- and 7b-spermidinylcholesterol. Reagents and conditions. (a) Lead IV acetate, (CH
CH ]CH–CN, MeOH; (d) LiAlH ,/NiCl , 6H O, THF, reflux; (e) Br–(CH ) –CN, DMF, 60 8C, 72 h, (f) LiAlH
3
)
3
SiN
3
, CH ; (b) LiAlH
2
Cl
2
4
, THF reflux; (c)
2
4
2
2
2
3
4
,/NiCl
, 6H O, THF, reflux.
2 2

11480
B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
6
a-N(2-cyanoethyl)aminocholestanol (13). The nitrile
function was reduced by lithium aluminium hydride
compound 14) and the amine obtained was alkylated by
-bromobutyronitrile to give compound 15 in 51% yield.
antiproliferative properties on this cell line (IC !
50
3.3 mg/mL; significative activity when IC !10 mg/mL).
5
0
(
4
Nitrile reduction of 15 was achieved with lithium
aluminium hydride in presence of nickel chloride hexa-
hydrate giving the polyaminosterol II in 35% yield.
3. Conclusion
There are two a key elements in our synthesis of squalamine
analogues. Its the first example, to our knowledge, in which
spermidine is introduced into the B steroid ring. Cholesterol
was chosen as the starting material in the synthesis of these
squalamine analogues. An inexpensive material allowing
preparation of significant quantities of material following a
multistep synthetic process. Analogues I and II were
prepared in seven and eight steps, respectively. Aminoster-
ols (III) and (IV) were easily prepared from epimeric
mixture, 7a,b-aminocholesterol in six steps.
The polyaminosterols III and IV were synthesized ster-
oselectively from 7a and 7b-aminocholesterol as a key
2
1
intermediate . A convenient novel synthetic route was
developped. Polyaminocholesterols (III) and (IV) were
easily prepared from epimeric mixture, 7a,b-aminocholes-
terol. This epimeric mixture was obtained in two steps from
cholesteryl acetate (Scheme 4): cholesterol was acetylated
by using acetic anhydride and pyridine to give the acetate 8
(
90%), and the azido group was introduced directly by
The results of these studies clearly show the antiprolifera-
tive effect on the cloned NSCLC-N6 cell line and the
antimicrobial activity against a broad spectrum of microor-
ganisms. 7a- and 7b-aminospermidinylcholesterol were
active against Gram negative bacteria. No activity with
analogues I and II was observed on Gram negative bacteria.
The stereochemistry of spermidinyl moiety at the steroid
seemed to have small affect on antimicrobial activity.
trimethylsilyl azide action in presence of lead IV acetate on
allylic position C-7 of cholesteryl acetate. a/b Epimeric
mixture 16 (77%a and 23%b) was obtained in 68% yield.
Reduction of epimeric azides 16 was acheived with lithium
aluminium hydride to give amine 17. Acrylonitrile was
reacted in methanol with amine epimeric mixture 17
required the 7a- and 7b-N(2-cyanoethyl)aminocholesterol
(
18a and 18b) easily separated by chromatography on silica
gel column. Each nitrile function was reduced by lithium
aluminium hydride in presence of nickel chloride hexahy-
drate (19a and 19b) and the amine obtained was alkylated
by 4-bromobutyronitrile to give compounds 20a and 20b.
Finally, each nitrile was reduced with lithium aluminium
hydride in presence of nickel chloride hexahydrate to give
squalamine analogue III in 46% yield and IV in 48% yield.
Our results suggest that introduction of spermidine at B
steroid ring is a key target of biological activites for
polyaminosterols. Therefore, these molecules with anti-
biotic and cytotoxic properties are promising for immune-
compromised patients in cancer chemotherapy.
4. Experimental
Minimum inhibitory concentrations (MIC) of these amino-
sterols (I, II, III and IV) were determined against fungi
4
.1. General procedure
(
(
S. cerevisiaie and C. albicans), and both Gram-positive
S. aureus and E. hirae) and Gram-negative (E. coli)
All melting points are uncorrected The IR spectra were
recorded on a Perkin Elmer 1600 FT-IR spectrometer.
bacteria (Table 1).
1
13
Nuclear magnetic resonance ( H NMR and C NMR)
spectra were obtained on a Brucker DPX 250 MHz or a Jeol
JNM 400 MHz. The chemical shift values (parts per
million) are relative to tetramethylsilane as solvent
reference and coupling constant (J) values are expressed
in Hertz. The mass spectra were recorded Jeol-GC mate
(GC–MS system). The optical rotations were measured with
Perkin Elmer 343 polarimeter. Thin-layer chromatography
was run on Merck silica gel 60 F254. Developed plates were
The activity on C. albicans was compared to clinical
references products (Amphotericin B, Econazole, Nystatin
and 5-fluorocytosine, Table 2).
The antiproliferative activity of these squalamine analogues
was studied in vitro on a human non-small cell broncho-
pulmonary (NSCLC-N6). The cytotoxicity determinations
(
inhibitory concentration: IC ) showed clearly strong
50
Table 1. Determination of MIC (mg/mL) values after 48 h incubation. NI: no inhibition with MIC O50 mg/mL
Strain
S. cerevisiaie ATCC28383
C. albicans CIP1180-79
S. aureus CIP 54127
E. hirae CIP 5855
E. coli 54127
Analogue I
Analogue II
Analogue III
Analogue IV
Squalamine
6.25
1.56
3.12
3.12
—
25
6.25
3.12
3.12
6.25
1–2
6.25
1.56
3.12
6.25
—
NI
NI
6.25
3.12
1–2
6.25
6.25
3.12
4–8
Table 2. Determination of MIC (mg/mL) values after 48 h incubation
Compounds
Amp B
0.4
Econazole
1.5
Nystatin
6
5-Fc
O50
I
II
III
IV
C. albicans
CIP1180-79
25
6.25
12.5
3.12

B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
11481
visualized by upon spraying with sulfuric acid/ethanol 2:8
and heating.
evaporate at room temperature, and the residue was dissoved
in ethyl ether (30 mL) and washed with 0.1 N HCl (25 mL)
and water (25 mL). The organic layer was dried over sodium
sulfate and evaporated. The crude product was purified by
chromatography on silica gel column (hexane/ethyl acetate
4
1
.1.1. 3b-[(Tetrahydropyran-2R,S-yl)oxy]cholest-5-ene
. To a solution of cholesterol (10 g, 24 mmol) in methylene
chloride (50 mL) was added 3,4-dihydropyrane freshly
distilled (3.18 mL, 36 mmol) and pyridinium para-toluene
sulfonate (0.59 g, 2.4 mmol). The mixture was stirred at
room temperature and under nitrogen atmosphere for 1 h
8:2). The desired product 3 (2.89 g, 82%) was obtained as a
white solid. IR: n: 2940 (CH
(CDCl
1
), 1707 (C]O ketone). H NMR
, 250 MHz), d: 0.65 (s, 3H, Me 18), 0.86 (d, 6H, JZ
2
3
6.5 Hz, Me 27 and Me 26), 0.90 (d, 3H, JZ6.4 Hz, Me 21),
0.93 (s, 3H, Me 19), 3.46–3.48 (m, 1H, H-6 of THP), 3.57–
0
3.59 (m, 1H, H3a), 3.88–3.90 (m, 1H, H-6 of THP), 4.68
3
0 min. The solution was washed with water (50 mL), and
0
the organic layer was dried over sodium sulfate and
evaporated to give the crude product, which was purified
by chromatography on silica gel column (hexane/ethyl
acetate 9.5:0.5) to afford compound 1 as a white solid
0
13
(br s, 1H, H-2 of THP). C NMR (CDCl ): d: 12.1, 18.8,
3
19.7, 21.7, 22.7, 22.9, 23.8, 25.0, 25.4, 26.8, 28.1, 28.5, 33.2,
34.8, 35.1, 35.7, 36.2, 36.5, 39.6, 42.5, 44.2, 45.1, 45.7, 48.9,
52.1, 56.1, 63.0, 77.6, 96.5, 211.5. MS-EI: m/zZ486
(
11.29 g, 99%).
C%
C%
(24%, M ), 385 (10%, M Ktetrahydropyranyl), 367
(32%, 385 –H O).
1
H NMR (CDCl , 250 MHz), d: 0.68 (s, 3H, Me 18), 0.86 (d,
3
2
6
Me 21), 0.97 (s, 3H, Me 19), 3.49–3.54 (m, 2H, H-6 of
H, JZ6.5 Hz, Me 27 and Me 26), 0.92 (d, 3H, JZ6.5 Hz,
0
THP), 3.92 (m, 1H, H3a), 4.71 (br s, 1H, H-2 of THP), 5.33
4.1.4. 7-Oxo-cholestan-3b-ol 4. Pyridinium para-toluene
sulfonate (0.6, 2.4 mmol) was added to a solution of
compound 3 (1.2 g, 2.4 mmol) in ethanol (25 mL) and
heated under reflux for 12 h. The solvent was evaporated
and the residue was dissolved in ethyl ether/ethyl acetate
(5:5). The mixture was washed with brine, dried over
sodium sulfate and evaporated to give the crude product,
which was purified by chromatography (hexane/methylene
chloride 5:5) and crystallized from ethanol/ethyl ether (5:5)
to afford product 4 (0.89 g, 85%) as a white solid. Mp
0
1
3
(
td, 1H, JZ1.01, 6.8 Hz, H6). C NMR (CDCl ): d: 12.1,
3
1
2
4
8.7, 19.4, 19.7, 21.2, 22.7, 23.9, 24.1, 25.4, 28.1, 28.3,
9.6, 31.2, 32.0, 32.1, 35.9, 36.2, 37.5, 38.0, 39.1, 39.6,
0.0, 42.4, 50.3, 56.0, 56.8, 63.0, 77.3, 96.8, 122.0, 140.5.
C%
C%
MS-EI: m/zZ470 (10%, M ), 386 (14%), M Ktetra-
hydropyranyl), 368 (28%, 386 –H O), 85 (100%).
2
4
5
.1.2. 3b-[(Tetrahydropyran-2R,S-yl)oxy]-7-oxo-cholest-
-ene 2. Pyridine (16.2 mL, 200 mmol) was added to a
140 8C. IR: n: 3419 (OH alcohol), 2948 (CH
), 1706 (C]O
ketone). H NMR (CDCl , 250 MHz), d: 0.65 (s, 3H, Me
3
2
1
solution of chromium anhydride (10.61 g, 106 mmol) in
methylene chloride (100 mL). The solution was stirred at
room temperature and under nitrogen atmosphere for 2 h.
The protected cholesterol 1 (5 g, 10 mmol) was added, and
the mixture was stirred for 12 h. The solution was filtered,
18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26), 0.89 (d, 3H,
JZ6.4 Hz, Me 21), 0.91 (s, 3H, Me 19), 3.57–3.59 (m, 1H,
1
H3a). C NMR (CDCl ): d: 12.1, 19.1, 22.2, 22.7, 24.1,
3
3
25.3, 28.3, 28.7, 29.7, 31.3, 36.0, 36.3, 36.4, 36.5, 38.2,
39.1, 39.8, 42.8, 46.4, 47.2, 49.2, 50.3, 55.4, 55.6, 70.9,
washed with 0.1 N HCl (100 mL), 5% NaHCO (100 mL)
3
C%
and with water 100 mL). The organic layer was dried over
sodium sulfate and evaporated to give the crude product,
which was purified by chromatography on silica gel column
212.5. MS-EI: m/zZ402 (18%, M ), 385 (100%,
C%
%
K OH).
M
(
6
hexane/ethyl acetate 8:2) to afford pure compounds 2 (3 g,
2%). IR: n: 2944 (CH ), 1673 (C]O allylic ketone), 1651
4.1.5. 7a-N-(3N-tert-Butoxycarbonyl-aminopropyl)ami-
nocholestan-3b-ol 5. To a solution of 3N-(tertiobutylcar-
bonyl)aminopropane (0.921 g, 5.29 mmol) and compound 4
(1.29 g, 2.64 mmol) in anhydrous methanol (10 mL) was
added sodium borohydride (0.25 g, 3.96 mmol) and the pH
was ajusted with acetic acid to 5–6. The mixture was stirred
at room temperature and under argon atmosphere for 48 h.
The solution was treated with 0.1 N HCl (30 mL), 5%
2
1
(
C]C alkene). H NMR(CDCl , 250 MHz), d: 0.68 (s, 3H,
3
Me 18), 0.85 (d, 6H, JZ6.5 Hz, Me 27 and Me 26), 0.90 (d,
3
2
H, JZ6.5 Hz, Me 21), 0.93 (s, 3H, Me 19), 3.45–3.49 (m,
0
H, H-6 of THP), 3.55–3.64 (m, 1H, H3a), 4.72 (br s, 1H,
0
13
H-2 of THP), 5.68 (d, 1H, JZ1.02 Hz, H6). C NMR
CDCl ): d: 12.3, 17.7, 19.2, 20.2, 21.5, 22.7, 24.1, 25.7,
(
3
2
3
1
6.7, 28.1, 28.3, 28.9, 29.7, 31.5, 36.0, 36.5, 38.8, 39.1,
9.8, 40.4, 43.4, 45.; 50.3, 55.1, 63.2, 75.3, 97.4, 126.4,
65.9, 202.5. MS-EI: m/zZ400 (19%, M Ktetrahydro-
NaHCO (30 mL) and extracted with methylene chloride.
3
The organic layer was washed with, brine and dried over
sodium sulfate. The solution was evaporated and the crude
product was purified by chromatography (ethyl acetate/
hexane 5:5 and methylene chloride/methanol 9:1) to afford
C%
pyranyl), 344 (75%), 85 (100%).
1
4
.1.3. 3b-[(Tetrahydropyran-2R,S-yl)oxy]cholestan-7-
product 5 (1.467 g, 84%) as amorphous solid. H NMR
(CDCl , 250 MHz), d: 0.65 (s, 3H, Me 18), 0.84 (d, 6H, JZ
one 3. Tetrahydrofuran (10 mL) was cooled to K78 8C
with dry ice/acetone, and ammonia was then collected to a
total volume of approximately 20 mL. Lithium wire (0.2 g,
3
6.5 Hz, Me 27 and Me 26), 0.91 (d, 3H, JZ6.4 Hz, Me 21),
1.02 (s, 3H, Me 19), 1.45 (s, 9H, HN–CO –C(CH ) ), 2.48
2
3 3
28 mmol) was added in small pieces to the solution with
vigorous stirring. A deep blue solution was obtained after the
(m, 1H, St–HN–CH H –(CH ) –NH–), 2.57 (m, 1H, H7b
0
0
of a epimer), 2.72 (m, 1H, HN–CH H –(CH ) –NH–), 3.21
a a 2 2
a a 2 2
lithium was completely dissolved. Ketone 2 (3.5 g,
.22 mmol) was dissolved in THF (50 mL) and added to
(t, 2H, JZ5.1 Hz, –CH –NH–BOC), 3.63 (m, 1H, H3a),
2
13
7
5.80 (br s, 1H, HN–CO –C(CH ) , D O exchange).
C
2
3 3
2
the flask in a steady stream from an addition funnel. The
solution was stirred for 40 min and then quenched by the
addition of ammonium chloride and ethanol unitil the blue
coloration dissipated. The ammonia was allowed to
NMR (CDCl ): d: 12.1, 15.1, 18.8, 21.8, 22.7, 23.9, 26.3,
3
27.1, 28.1, 28.2, 28.3, 29.7, 33.6, 33.8, 35.2, 35.9, 36.3,
37.1, 39.5, 39.6, 39.8, 41.0, 42.9, 43.0, 46.5, 47.6, 54.4,
56.3, 61.9, 71.6, 78.8, 155.9. MS-EI: m/zZ560 (25%,

11482
B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
C%
C%
), 416 (25%, M KCH ]CH–NHBoc), 402 (64%,
M
4
Na SO .10H O, filtered through a pad of celite, and washed
2 4 2
2
% C
16 – CH ), 387 (22%, St ), 293 (100%).
with ethyl ether/n butanol (6:4). Removal of the solvent in
vacuo and purification by flash chromatography (dichlor-
omethane/methanol/ammoniaque 8:1.5:0.5) to afford a pure
3
4
.1.6. 7a-N-(3N-Aminopropyl)aminocholestan-3b-ol 6.
1
To a solution of compound 5 (1.55 g, 2.75 mmol) in
methylene chloride (20 mL) was added dropwise trifluor-
oacetic acid (1.92 mL, 24.8 mmol). The mixture was stirred
at room temperature for 3 h. The mixture was treated with
% NaHCO (20 mL) and washed with water (20 mL). The
3
organic layer was dried over sodium sulfate and evaporated.
The crude product was purified by chromatography
methylene chloride/methanol/ammoniaque 8:1:1) to give
product 6 (1.0 g, 79%) as amorphous solid. H NMR
CDCl , 250 MHz), d: 0.65 (s, 3H, Me 18), 0.85 (d, 6H, JZ
.5 Hz, Me 27 and Me 26), 0.91 (d, 3H, JZ6.4 Hz, Me 21),
.02 (s, 3H, Me 19), 2.63 (m, 1H, H7b of a epimer), 2.74 (m,
0
H, St–HN–CH Ha –(CH ) –NH ), 2.87 (m, 1H, St–HN–
2 2 2
product I as amorphous solid (0.15 g, 30%). H NMR
(CDCl
JZ6.5 Hz, Me 27 and Me 26), 0.91 (d, 3H, JZ6.5 Hz, Me
21), 1.03 (s, 3H, Me 19), 2.44 (m, 1H, St–HN–CH
(CH –NH–), 2.60 (m, 1H, H7b of a epimer), 2.67 (m, 1H,
St–HN–CH –(CH –NH–), 2.81 (t, 2H, JZ5.7 Hz,
–HN–(CH –CH –NH ), 3.11–3.23 (t, 4H, J Z9.5 Hz,
Z7.6 Hz, St–HN–(CH –CH –NH– and –NH–CH
(CH –NH ), 3.58 (m, 1H, H3a), 7.15 (br s, 2H, –NH2,
O exchange), 8.10 (br s, 1H, –NH–, D O exchange), 8.32
O exchange). C NMR (CDCl ): d:
, 250 MHz): d: 0.68 (s, 3H, Me 18), 0.85 (d, 6H,
3
H –
0
a a
5
)
2 2
H
0
)
2 2
a
a
)
2
3
2
2
1
(
J
2
2
)
2
2
–
2
1
)
2
3
2
(
D
2
2
3
1
3
6
1
1
(br s, 1H, –NH–, D
2
3
12.1, 15.2, 18.8, 21.8, 22.7, 23.9, 25.6, 25.7, 26.3, 28.1,
28.2, 29.5, 29.8, 33.6, 33.8, 35.2, 35.9, 36.3, 37.1, 39.6,
39.7, 40.0, 41.0, 42.9, 43.0, 46.1, 46.6, 47.6, 47.7, 54.2,
a
CH H –(CH ) –NH ), 3.25 (t, 2H, JZ7.5 Hz, –HN–
0
a
a 2 2 2
(
St–NH–(CH ) , D O exchange). C NMR (CDCl ): d:
CH ) –CH –NH ), 3.66 (m, 1H, H3a), 8.91 (br s, 1H,
56.3, 62.0, 71.6. MS-EI: m/z)Z473 (3%, St–NH–(CH
2
)
3
–
2
2
2
2
1
3
C
C
NH–CH
NH–CH
(5%, 387 –H
), 430 (5%, St–NH–CH –CH ), 416 (50%, St–
2 2
2
2
2
3
2
C
C
C
1
3
3
2.1, 15.2, 19.0, 21.5, 22.7, 24.0, 26.3, 28.1, 28.3, 31.7,
3.6, 33.8, 36.0, 36.2, 36.4, 36.9, 37.7, 38.2, 39.2, 39.7,
9.9, 42.9, 43.2, 46.6, 47.6, 51.1, 56.3, 61.9, 71.4. MS-EI:
2
), 402 (100%, St–NH ), 387 (8%, St ), 369
O). Anal. Calcd for C34 O: C 76.77; H
12.32; N 7.90. Found: C 76.75; H 12.39; N 7.92.
H N
65 3
2
C%
%
C%
C
m/zZ443 (16%, M K CH ), 428 (12%, M K CH –
3
2
C
C
NH ), 416 (47%, St–NH–CH ), 402 (7%, St–NH ), 387
4.1.9. 3b-Acetoxy-6-nitro cholest-5-ene 9. To a solution of
nitric acid (9.6 mL, 197 mmol) in acetic acid (36 mL) was
added dropwise cholesteryl acetate (12.5 g, 29 mmol). The
mixture was stirred for 2 h. The ice was added to the solution.
The product was filtered, washed with water and crystallized
2
2
C
100%, St ).
(
4
.1.7. 7a-N-[3N-(3-Cyanopropyl)aminopropyl]amino-
cholestan-3b-ol 7. To a solution of amine 6 (0.330 g,
.71 mmol) in DMF (5 mL) was added sodium hydrogeno-
0
from ethanol to afford product 9 (10.3 g, 74%) as a yellow
solid. Mp 110 8C. H NMR (CDCl , 250 MHz), d: 0.68
1
carbonate (0.09 g, 1.07 mmol). After stirring at room
temperature for 15 min, 4-bromobutyronitrile (0.14 mL,
3
(s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
0.91 (d, 3H, JZ6.5 Hz, Me 21), 1.03 (s, 3H, Me 19), 2.17
1
.43 mmol) was added and the mixture was heated at 60 8C
1
–CO), 4.6–4.68 (m, 1H, H3a). C NMR
3
for 36 h. The solution was diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine,
dried over sodium sulfate and evaporated. The residue was
purified by chromatography (hexane/ethyl acetate 5:5)
(s, 3H, CH
(CDCl ): d: 11.9, 18.7, 19.2, 21.0, 22.7, 23.2, 23.9, 24.4,
3
3
27.8, 28.1, 28.3, 30.4, 30.5, 33.7, 35.4, 35.9, 36.2, 38.7, 39.6,
39.8, 42.1, 51.8, 55.8, 56.4, 72.8, 130.2, 131.3, 171.2. MS-EI:
C%
C%
afforded product 7 (0.190 g, 51%) as a oil. IR: n: 2932
m/zZ473 (27%, M ), 426 (20%, M KHNO ), 398 (46%,
2
C%
1
CH ), 2254 (CN). H NMR (CDCl , 250 MHz): d: 0.65
(
(
M
K(CH COOHC.CH )), 383 (100%).
3 3
2
3
s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
.91 (d, 3H, JZ6.5 Hz, Me 21), 1.1 (s, 3H, Me 19), 1.19
t, 2H, JZ6.7 Hz, –NH–CH –CH –CH –CN), 2.47–2.53
0
4.1.10. 3b-Acetoxy-cholestan-6-one 10. To a solution of
compound 9 (0.890 g, 1.87 mmol) in acetic acid (20 mL)
was added zinc (2 g, 30 mmol) and the solution was refluxed
for 4 h 30 min. The solution was filtered, diluted with water
and extracted with methylene chloride. The organic layer
was dried over sodium sulfate and evaporated. The crude
product was purified by chromatography (Hexane/ethyl
acetate 9.8:0.2) and crystallized from ethanol (0.60 g, 72%).
Mp 136 8C.
(
(
2
2
2
t, 4H, J Z7.06 Hz, J Z7.13 Hz, –CH –CN and –NH–
1
2
2
CH –(CH ) –CN), 2.60 (ddd, 1H, J7b–8Z4.3 Hz, J7b–6
Z
2
2 2
5
Hz, H7b of a epimer), 2.68 (m, 1H, St–HN–CH H –
0
a a
(
CH ) –NH–), 2.82 (m, 1H, St–HN–CH H –(CH ) –NH–),
0
2 2 a a 2 2
3
.38 (m, 1H, H3a), 3.75 (t, 2H, JZ5.9 Hz, St–HN–(CH ) –
2 2
13
CH –NH–), 8.98 (br s, 1H, St–NH–, D O exchange).
C
2
2
NMR (CDCl ): d: 12.0, 14.1, 15.2, 18.8, 21.5, 22.7, 24.0,
3
2
3
5
4.6, 25.6, 26.3, 28.1, 28.4, 31.6, 36.0, 36.2, 36.4, 36.9,
7.6, 38.0, 39.1, 39.6, 39.8, 42.9, 43.0, 43.2, 46.5, 51.0,
1
IR: n: 1730 (C]O/ester), 1718 (C]O/ketone). H NMR
4.4, 56.3, 56.5, 60.5, 71.3, 120.0. MS-EI: m/zZ487
(CDCl , 250 MHz), d: 0.66 (s, 3H, Me 18), 0.76 (d, 6H, JZ
3
C
2
(2%, St–NH–(CH ) –NH–CH –CH ), 416 (46%, St–NH–
CH ), 402 (7%, St–NH ), 387 (8%, StC), 369 (5%, 386 –
H O), 169 (100%).
2
6.5 Hz, Me 27 and Me 26), 0.90 (d, 3H, JZ6.5 Hz, Me 21),
2
3
2
C
2
C
0.95 (s, 3H, Me 19), 2.02 (s, 3H, CH –COO), 4.67 (m, 1H,
3
1
3
H3a). C NMR (CDCl ): d: 12.3, 13.4, 19.0, 21.7, 21.8,
3
2
39.2, 39.8, 41.3, 43.2, 43.3, 47.0, 54.1, 56.4, 56.8, 73.2,
4.1, 24.3, 26.4, 27.2, 28.3, 28.4, 36.0, 36.4, 36.7, 38.3,
4
.1.8. 7a-N-[3N-(4-Aminobutyl)-3-aminopropyl]amino-
cholestan-3b-ol I. A solution of nitrile 7 (0.500 g,
.9 mmol) in dry THF (5 mL) was added dropwise to a
suspension of lithium aluminium hydride (0.21 g,
C%
170.0, 210.7. MS-EI: m/z)Z444 (25%, M ), 384 (100%,
C%
%
KCH COOH), 369 (27%, 384 – CH ).
3 3
0
M
5
0
.6 mmol) and nickel chloride hexahydrate (0.213 g,
.9 mmol) in dry THF (20 mL) under argon. The mixture
4.1.11. 3b-Acetoxy-6N-hydroxyiminocholestane 11.
Ketone 10 was added to a solution of hydroxylamine
chloride (0.751 g, 10 mmol) in pyridine (8 mL). The
was refluxed for 6 h. The reaction mixture quenched with

B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
11483
mixture was refluxed under argon atmosphere for 5 h. The
solution was diluted with water and extracted with
methylene chloride. The organic layer was treated with
4.1.14. 6a-N-(3N-Aminopropyl)aminocholestanol 14.
A solution of nitrile 11a (0.40 g, 0.87 mmol) in dry THF
(5 mL) was added dropwise to a suspension of lithium
aluminium hydride (0.19 g, 5.25 mmol) and nickel hexahy-
drate (0.206, 0.87 mmol) in dry THF (10 mL) under argon.
The mixture was refluxed for 2 h. The reaction mixture
quenched with Na SO $10H O, filtered through a pad of
celite, and washed with ethyl ether/n-butanol (6:4).
Removal of the solvent in vacuo and purification by flash
chromatography (methylene chloride/methanol/ammonia-
0
.1 N HCl (15 mL), 5% NaHCO (15 mL) and washed with
3
water. The solution was dried over sodium sulfate and
evaporated. The product was purified by chromatography
(
7
hexane/ethyl acetate 9:1) to afford product 11 (2.38 g,
6%) as a white solid. IR: n: 2950 (O–H alcohol), 1734
2
4
2
1
(
2
C]O/ester), 1712 (C]N–OH). H NMR (CDCl3,
50 MHz), d: 0.65 (s, 3H, Me 18), 0.85 (d, 6H, JZ6.5 Hz,
Me 27 and Me 26), 0.91 (d, 3H, JZ6.5 Hz, Me 21), 0.99
s, 3H, Me 19), 2.02 (s, 3H, CH –COO), 3.32 (dd, 1H, JZ
que 7:2.5:0.5) to afford a pure product 12a (0.18 g, 41%) as
amorphous solid. H NMR (CDCl , 250 MHz), d: 0.68
1
(
3
3
9
O–H). C NMR (CDCl ): d: 12.4, 18.9, 19.0, 21.7, 22.9,
.0 Hz, C –H), 4.67 (m, 1H, H3a), 7.73 (br s, 1H, –C]N–
(s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
0.91 (d, 3H, JZ6.5 Hz, Me 21), 1.04 (s, 3H, Me 19), 2.70
5
1
3
3
2
3
7
4.2, 24.4, 24.5, 24.8, 27.4, 27.9, 28.3, 28.5, 30.0, 36.1,
6.2, 36.3, 36.5, 39.3, 39.8, 40.0, 43.3, 49.7, 56.5, 57.0,
(m, 2H, St–NH–CH –CH –), 2.74 (m, 1H, St–NH–CH H –
0
2 2 a a
CH –), 2.80 (m, 1H, H6b of a epimer), 2.87 (m, 1H,
2
0
St–HN–CH H –(CH ) –), 3.00 (t, 2H, JZ12 Hz, St–HN–
a a 2 2
13
(CH ) – CH –NH ), 3.66 (m, 1H, H3a). C NMR (CDCl3):
2 2 2 2
C%
3.6, 159.9, 171.0. MS-EI: m/zZ459 (11%, M ), 442
C%
%
C%
(
(
17%, M K OH), 399 (79%, M KCH COOH), 384
3
%
100%, 399 – CH ).
d: 12.1, 15.3, 18.7, 20.9, 22.7, 23.9, 24.1, 28.1, 28.2, 30.3,
3
3
4
4
3.4, 33.6, 34.1, 35.0, 35.8, 36.2, 36.5, 39.6, 40.0, 40.6,
4.4, 45.6, 49.5, 52.0, 56.3, 56.9, 60.2, 73.1. MS-EI: m/zZ
4
.1.12. 6a-Aminocholestanol 12. Under nitrogen atmos-
phere, solution of 11 (1.50 g, 3.27 mmol) in tetrahydrofuran
8 mL) was added dropwise over 10 min to a stirred
suspension of lithium aluminium hydride (0.744 g,
9 mmol) in tetrahydrofuran (20 mL) at 0 8C. The mixture
C% C%
60 (M , 13%), 443 (12%, M KNH ), 402 (100%,
3
C
St–NH ).
(
4
.1.15. 6a-N-[3N-(3-Cyanopropyl)aminopropyl]amino-
cholestanol 15. To a solution of amine 14 (0.270 g,
.580 mmol in DMF (4 mL) was added sodium hydro-
1
was refluxed for 4 h, after which it was cooled and quenched
by careful addition of saturated aqueous sodium sulfate. The
solution was filtered, dried and evaporated under reduced
pressure. The crude product was purified by column
chromatography (methylene chloride/methanol/triethyl-
amine (8:1.8:0.2) crystallized from ethanol/ethyl ether
0
genocarbonate (0.073 g, 0.871 mmol). After stirring at room
temperature for 35 min, 4-bromobutyronitrile (0.115 mL,
1.161 mmol) was added and the mixture was heated at 60 8C
for 36 h. The solution was diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine,
dried over sodium sulfate and evaporated. The residue was
purified by chromatography (hexane/ethyl acetate 6:4)
(8:2) to give amine 12 (0.900 g, 70%) as a white solid.
Mp 127–128 8C. IR: n: 3500–3000 (OH alcohol and NH
2
1
amine). H NMR (CDCl , 250 MHz), d: 0.69 (s, 3H, Me 18),
3
afforded product 15 (0.180 g, 59%) as a oil. IR: n: 3500–
3
0
2
.86 (d, 6H, Me 27 and Me 26), 0.91 (d, 3H, JZ6.5 Hz, Me
1
000 (OH alcohol and NH amine); 2254 (nitrile). H NMR
1), 1.02 (s, 3H, Me 19), 2.32 (br s, 2H, NH , D O
2
2
(CDCl , 250 MHz), d: 0.65 (s, 3H, Me 18), 0.85 (d, 6H, JZ
3
exchange), 2.95 (dd, 1H, H6b of a epimer, 3.64 (m, 1H,
H3a). C NMR (CDCl ): d: 12.5, 16.9, 19.0, 21.4, 22.9,
1
3
6.5 Hz, Me 27 and Me 26), 0.90 (d, 3H, JZ6.5 Hz, Me 21),
.08 (s, 3H, Me 19), 2.49 (t, 2H, JZ7.2 Hz, –HN–(CH ) –
3
1
2
2
2
3
4.2, 24.6, 28.4, 28.5, 30.0, 30.4, 31.8, 35.9, 36.1, 36.3,
6.5, 39.5, 39.9, 40.3, 43.0, 47.0, 52.4, 54.7, 56.4, 56.7. MS-
CH –CN), 2.59 (td, 1H, J Z6.1 Hz, J Z3.9 Hz, H6b of a
2
1
2
H –(CH ) –NH), 2.85
0
a 2 2
C%
C%
epimer), 2.64 (m, 1H, St–HN–CH
(
a
EI: m/zZ403 (16%, M ), 386 (24%, M KNH ), 248
3
m, 1H, St–HN–CH H –(CH ) –NH), 3.44 (m, 1H, H3a),
0
a
a
2 2
(
100%).
3
.61 (t, 2H, JZ5.7 Hz, –HN–CH –(CH ) –CN), 4.30 (t, 2H,
2 2 2
JZ6.2 Hz, St–NH–(CH ) –CH –NH–), 8.69 (br s, 1H, St–
2
1
2
3
2
4
.1.13. 6a-N-(2N-Cyanoethyl)aminocholestanol 13. A
solution of amine 12 (0.72 g, 1.8 mmol) and acrylonitrile
1.1 mL, 16 mmol) in methanol (10 mL) was stirred at room
NH–, D O exchange). C NMR (CDCl ): d: 12.5, 15.4;
2 3
16.7, 19.0; 20.9, 22.9, 23.2, 24.1, 24.6, 25.6, 28.4, 28.2,
30.8, 33.4, 33.6, 35.0, 35.8, 36.0, 36.5, 39.9, 43.0, 44.8,
46.1, 46.6, 48.3, 49.5, 52.0, 56.3, 56.9, 60.2, 72.2, 120.3.
(
temperature for 24 h. The solvent was evaporated giving the
crude product which was purified by chromatography
(hexane/ethyl acetate 8:2) and crystallized from ethanol/
ethyl ether (8:2) to afford (0.760 g, 93%) of nitrile 13 (b) as
C
MS-EI: m/zZ487 (20%, St–NH–(CH ) –NH–CH –CH ),
2 3 2 2
C
473 (4%, St–NH–(CH
CH
2
)
3
–NH–CH
), 416 (29%, St–NH–
2
2
C
C
), 402 (14%, St–NH ), 315 (100%).
a white solid. MpZ136 8C. IR: n: 3391–3000 (OH alcohol
1
and NH amine), 2248 (nitrile). H NMR (CDCl , 250 MHz),
4.1.16. 6a-N-[3N-(4-Aminobutyl)aminopropyl]amino-
cholestanol II. Nitrile 15 (0.070 g, 0.132 mmol) was
reduced in the same manner as 7 to yield (0.025 g,
3
d: 0.68 (s, 3H, Me 18), 0.84 (dd, 6H, J Z6.6 Hz, J Z
1
2
0
1
2
.8 Hz, Me 27 and Me 26), 0.91 (d, 3H, JZ6.5 Hz, Me 21),
1
.04 (s, 3H, Me 19), 2.44 (m, 2H, –HN–CH –CH –CN),
.62 (m, 1H, H6b of a epimer), 2.74 (m, 1H, St–HN–
–CH –CN), 2.92 (m, 1H, St–HN–CH H –CH2–
35%). H NMR (CDCl , 250 MHz): d: 0.67 (s, 3H, Me
3
2
2
18), 0.84 (d, 6H, JZ6.6 Hz, Me 27 and Me 26), 0.91 (d, 3H,
JZ6.5 Hz, Me 21), 0.95 (s, 3H, Me 19), 2.45 (m, 1H, St–
CH CH
a
0
0
a
a
2
a
1
3
CN), 3.6–3.7 (m, 1H, H3a). C NMR (CDCl ): d: 11.6,
3
HN–CH H –(CH ) –NH–), 2.54 (m, 1H, H6b of a epimer),
0
a a 2 2
1
3
5
4
8.6, 18.7, 19.7, 22.5, 22.8, 23.9, 28.0, 28.2, 31.4, 35.8,
6.2, 36.9, 37.4, 39.5, 42.1, 43.0, 43.8, 49.5, 52.8, 55.8,
8.2, 58.3, 58.9, 59.0, 59.3, 59.9, 71.6, 118.9. MS-EI: m/zZ
2.69 (m, 1H, St–HN–CH H –(CH ) –NH–), 2.79 (t, 2H,
0
a a 2 2
JZ6.3 Hz, –HN–(CH ) –CH –NH ), 3.15 (t, 2H, JZ
2
3
2
2
9.2 Hz, St–HN–(CH ) –CH –NH–), 3.62 (m, 1H, H3a),
7.12 (br s, 2H, –NH2, D O exchange), 7.89 (br s, 1H, –NH–,
2
2
2
2
C%
C
56 (12%, M ), 416 (30%, St–NH–CH ), 301 (100%).
2

11484
B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
1
3
D O exchange), 8.25 (br s, 1H, –NH–, D O exchange).
C
NMR (CDCl ): d: 12.0, 16.2, 18.6, 21.0, 22.5, 22.8, 23.8,
4.1.19. 7a-N-(2-Cyanoethyl)aminocholesterol 18a and
7b-N-(2-cyanoethyl)aminocholesterol 18b. A solution of
amine 17 (0.714 g, 1.77 mmol) and acrylonitrile (0.8 mL,
15 mmol) in methanol (5 mL) was stirred at room
temperature for 24 h. The solvent was evaporated giving
the crude product which was purified by chromatography
(hexane/ethyl acetate) to afford 0.5 g (66%) of 18a and 18b,
respectively, 70% (a) 30% (b) as amorphous solid.
2
2
3
2
3
5
4.4, 24.8, 28.0, 28.2, 30.4, 30.5, 31.6, 35.6, 35.8, 36.0,
6.1, 38.7, 38.9, 39.5, 40.0, 42.5, 42.6, 47.3; 47.8, 48.9,
0.6, 51.1, 54.8, 56.3, 59.2, 71.9. MS-EI: m/z (%)Z473
(4%, St–NH–(CH ) –NH–CH ), 444 (5%, St–NH–(CH ) –
2 3 2 2 2
CH ), 430 (10%, St–NH–CH –CH ), 416 (40%, St–NH–
C
C
C
2
2
C
2
C
C
CH ), 402 (100%, St–NH ), 387 (72%, St ). Anal. Calcd
2
for C H N O: C 76.77; H 12.32; N 7.90. Found: C 76.72;
3
4 65 3
H 12.38; N 7.93.
Compound 18a. IR: n: 3481–3314 (OH alcohol and NH
amine); 2254 (CN). H NMR (CDCl , 250 MHz): d: 0.68
1
3
4
.1.17. 3b-Acetyl-7a,b-azidocholesterol 16. Trimethylsi-
(s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
0.91 (d, 3H, JZ6.5 Hz, Me 21), 1.05 (s, 3H, Me 19), 2.40
lyl azide (15 mL, 104.9 mmol) was added dropwise to a
solution of cholesteryl acetate (4.5, 10.5 mmol) and lead (IV)
acetate (9.45 g, 21 mmol) in methylene chloride (40 mL).
The mixture was stirred for 2 h. the solution was diluted with
water and the precipitate lead (II) azide was removed by
fitration, and decomposed with sodium nitrite/dilute hydro-
chloric acid. The organic layer was dried over sodium sulfate
and evaporated. The crude product was purified by
chromatography (hexane/ethyl acetate 8:2) to give 3.4 g
(68% of epimeric mixture: a epimer 77% and b epimer 23%)
of 16 as colorless oil. IR: n: 2103 (N azide) and 1727 (C]O
ester). H NMR (CDCl , 250 MHz): d Z0.67 (s, 3H,
1
(m, 2H, St–NH–CH
7.25 Hz, J7b–8Z4.2 Hz, H7b of a epimer), 2.77 (m, 1H,
–CH –CN), 3.08 (m,1H, St–NH–CH H 00
–
a
H –CH –CN), 2.69 (dd, 1H, J7b–6Z
a 2
0
a
St–NH–CH
CH –CN), 3.49 (m, 1H, H3a), 5.55 (dd, 1H, J6–7bZ5 Hz,
6–4!1 Hz, H6 of a epimer). C NMR (CDCl
H
0
a
a
2
a
2
1
3
J
): d: 12.1,
3
19.1, 19.4, 19.8, 22.9, 23.2, 24.1, 26.7, 28.1, 28.3, 29.9,
35.9, 36.3, 37.1, 39.6, 39.8, 41.0, 42.9, 43.0, 44.2, 47.6,
54.4, 56.3, 56.4, 60.2, 71.6, 119.2, 124.0, 143.5. MS-EI:
C%
C
m/zZ454 (22%, M ), 385 (100%, St ).
3
1
1
Compound 18b. IR: n: 2934 (CH
(CDCl , 250 MHz): d: 0.68 (s, 3H, Me 18), 0.86 (d, 6H, JZ
6.5 Hz, Me 27 and Me 26), 0.93 (d, 3H, JZ6.5 Hz, Me 21),
1.02 (s, 3H, Me 19), 2.39 (m, 2H, St–NH–CH –CH –CN),
–CH –CN), 2.77 (ddd, 1H, J
), 2249 (CN). H NMR
3
H
2
8-Me), 0.87 (dd, 6H, JZ6.6, 1.92 Hz, CH(CH ) ), 0.92
3
3
2
(
d, 3H, JZ6.6 Hz, 21-Me), 1.03 (s, 3H, 19-Me), 2.04 (s, 3H,
CH –COO), 3.27 (ddd, 0.23H, J7a–8Z8.5 Hz, J7a–6Z1 Hz
2
2
3
2.66 (m, 1H, St–NH–CH
H
a
0
and J7a–4Z1.5 Hz, H-7a of b epimer), 3.5 (ddd, 0.77H,
J7b–8Z4.5 Hz, J7b–6Z5 Hz and J7b–4Z1.5 Hz, H-7b of a
epimer), 4.66 (m, 1H, 3-H), 5.30 (dd, 0.23H, J6–7a!1 Hz and
J6–4Z1 Hz, 6-H of bepimer), 5.56 (dd, 0.77H, J6–7bZ5.1 Hz
a
2
7a–
Z8.3 Hz, J7a–6Z1.2 Hz, H7a of b epimer), 2.91 (m,1H,
8
St–NH–CH
J
H –CH –CN), 3.46 (m, 1H, H3a), 5.21 (d, 1H,
0
a 2
13
a
6–7aZ3 Hz, H6 of b epimer). C NMR (CDCl ): d: 13.7,
3
1
3
and J6–4Z1.6 Hz, 6-H of a epimer). C NMR (CDCl ): d:
18.6, 18.8, 20.8, 21.3, 22.7, 24.3, 25.1, 28.0, 29.7, 33.4,
37.4, 37.7, 37.9, 38.5, 39.7, 40.7, 41.2, 43.7, 45.0, 51.3,
54.2, 54.8, 58.5, 73.1, 120.6, 125.5, 144.7. MS-EI: m/zZ
3
1
3
7
2.4, 12.7, 19.0, 21.8, 22.9, 23.2, 24.2, 24.5, 28.0, 28.4, 28.5,
6.1, 36.2, 36.5, 39.2, 39.9, 40.0, 43.2, 49.7, 54.6, 54.3, 58.1,
C%
C%
C
2.3, 120.2, 149.3, 171.0. MS-EI: m/zZ469 (5%, M ), 441
454 (14%, M ), 385 (100%, St ).
C%
(
100%, M KN2).
4
.1.20. 7a-N-(3-Aminopropyl)aminocholesterol 19a.
4
.1.18. 7a,b-Aminocholesterol 17. Under nitrogen atmos-
phere, solution of 16 (1 g, 2.13 mmol) in tetrahydrofuran
10 mL) was added dropwise over 10 min to a stirred
A solution of nitrile 18a (0.2 g, 0.44 mmol) in dry THF
(5 mL) was added dropwise to a suspension of lithium
aluminium hydride (0.14 g, 2.64 mmol) and nickel chloride
hexahydrate (0.105 g, 0.44 mmol) in dry THF (15 mL) under
argon. The mixture was refluxed for 1 h. The reaction
(
suspension of lithium aluminium hydride (1 g, 2.13 mmol) in
tetrahydrofuran (20 mL) at 0 8C. The mixture was refluxed
for 4 h, after which it was cooled and quenched by careful
addition of saturated aqueous sodium sulfate. The solution
was filtered, dried and evaporated under reduced pressure.
The crude product was purified by column chromatography
mixture quenched with Na SO .10H O, filtered through a
2 4 2
pad of celite, and washed with ethyl ether/n butanol (6:4).
Removal of the solvent in vacuo and purification by
flash chromatography (methylene chloride/methanol/
(
give 7a,b-aminocholesterol (0.93 g, 70%) as amorphous
methylene chloride/methanol/ammoniaque 8:1.8:0.2) to
ammoniaque 7:2:1) to afford a pure product 19a (0.4 g,
1
69%) as amorphous solid. IR: n: 3434 (NH
(CDCl
JZ6.5 Hz, Me 27 and Me 26), 0.91 (d, 3H, JZ6.5 Hz, Me
amine). H NMR
2
white solid. IR: n: 3445–3000 (OH alcohol and NH amine).
2
, 250 MHz): d: 0.66 (s, 3H, Me 18), 0.86 (d, 6H,
3
1
H NMR (CDCl , 400 MHz), d: 0.69 (s, 3H, Me 18), 0.86
3
21), 0.99 (s, 3H, Me 19), 2.17 (m, 1H, St–NH–CH
(CH –NH ), 2.30 (m, 2H, St–NH–CH –CH –CH –NH
2.55 (m, 1H, St–NH–CH
H –
0
(
d, 6H, JZ6.5 Hz, Me 27 and Me 26), 0.92 (d, 3H, JZ6.5 Hz,
a
a
Me 21), 1.05 (s, 3H, Me 19), 3.49 (m, 1H, H3),, 3.54 (ddd, 1H,
J7a–8Z8.5 Hz, J7a–6Z1.2 Hz, J7a–4Z1.5 Hz, H7a of b
epimer), 3.58 (ddd, 1H, J7b–8Z4.8 Hz, J7b–6Z5.3 Hz,
J7b–4Z1.2 Hz, H7b of a epimer), 4.62 (br s, 2H, NH , D O
2
)
2
2
2
2
2
),
2
H
0
–(CH ) –NH ), 2.74 (m, 1H,
H7b of a epimer), 2.81 (t, 2H, JZ5.29 Hz, St–NH–(CH
CH –NH ), 3.56 (m, 1H, H3a), 5.66 (dd, 1H, J6–7b
4.85 Hz, J6–4!1 Hz, H6 of a epimer). C NMR (CDCl ): d:
3
a
a
2 2
2
) –
2 2
Z
2
2
2
2
1
3
exchange), 5.55 (dd, 1H, J6–7bZ5.2 Hz, J6–4!1 Hz, H6 of a
epimer), 5.26 (dd, 1H, J6–7aZ1.2 Hz, J6–4!1 Hz, H6 of b
12.0, 19.0, 19.7, 21.0, 22.9, 23.1, 24.2, 27.0, 28.3, 31.6, 34.6,
35.9, 36.5, 37.0, 37.4, 39.6, 39.8, 40.0, 42.1, 42.4, 42.6, 43.6,
47.6, 54.3, 56.3, 61.4, 71.2, 125.3, 142.9.
1
epimer). C NMR (CDCl ): d: 12.1, 15.2, 18.8, 21.8, 22.7,
3
3
2
4
1
3.9, 26.2, 28.1, 28.3, 29.8, 35.9, 36.2, 37.1, 37.4, 39.6, 39.8,
1.8, 42.9, 43.5, 46.5, 52.2, 49.6; 53.5; 56.3, 71.6, 128.6,
C%
C%
41.0. MS-EI: m/zZ401 (71%, M ), 384 (4%, M
K
4.1.21. 7b-N-(3-Aminopropyl)aminocholesterol 19b.
Nitrile 18b (0.250 g, 0.55 mmol) was reduced in the same
manner as 18a to yield (0.180 g, 72%) of product 19b as
NH ), 351 (29%), 289 (100%). Anal. Calcd for C H NO
(
3
27 47
401.68): C 80.7, H 11.7, N 3.5. Found: C 80.3, H 11.3, N 3.2.

B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
11485
amorphous solid. IR: n: 3500–3430 (OH alcohol and NH
amine), 2961 (CH ). H NMR (CDCl , 250 MHz): d: 0.68
1H, –NH–, D O exchange), 8.44 (br s, 1H, –NH–, D O
2 2
2
1
13
exchange). C NMR (CDCl ): d: 12.1, 18.6, 18.8, 21.7,
2
3
3
(
0
(
s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
.90 (d, 3H, JZ6.5 Hz, Me 21), 1.03 (s, 3H, Me 19), 2.63
m, 1H, St–HN–CH Ha –(CH ) –NH –), 2.74 (m, 1H, H7a
22.7, 23.9, 25.4, 25.6, 26.3, 28.1, 28.2, 29.5, 31.7 35.9, 36.3,
37.0, 37.4, 39.6, 39.7, 40.0, 40.7, 40.8, 43.0, 46.1, 46.6,
46.8, 47.6, 54.2, 56.3, 65.4, 71.5, 122.2, 139. MS-EI (m/z)Z
0
a
2 2
2
C
of b epimer), 2. 87 (m, 1H, St–NH–CH Ha
a
3
0
–(CH ) –NH ),
2 2
471 (3%, St–NH–(CH ) –NH–CH ), 442 (2%, St–NH–
2
2 3
2
C
C
C
.25 (t, 2H, JZ5.8 Hz, St–NH–(CH ) –CH –NH ), 3.66
(CH ) –CH ), 400 (100%, St–NH ), 385 (24%, St ). [a]
2 2 2 D
K418 (CZ0.2 M in MeOH). Anal. Calcd for C H N O:
2
2
2
2
1
3
20
( C
NMR (CDCl ): d: 13.7, 18.6, 18.8, 20.8, 21.3, 22.7, 24.3,
m, 1H, H3a), 5.63 (br s, 1H, St–NH–, D O exchange).
2
34 63 3
3
C 77.35, H 11.66, N 7.96. Found: C 77.29, H 11.69, N 7.95.
2
4
1
5.1, 28.0, 29.7, 33.4, 33.8, 37.4, 37.7, 37.9, 38.5, 39.7,
0.6, 40.7, 41.2, 43.7, 45.6, 51.3, 54.2, 59.0, 58.5, 73.1,
20.6, 122.8, 139.0.
4.1.25. 7b-N-[3N-(4-Aminobutyl)aminopropyl]amino-
cholesterol IV. Compound IV (0.040 g, 0.78 mmol) was
prepared in the same manner as I to yield (0.02 g, 48%) as
amorphous solid. H NMR (CDCl , 250 MHz): d: 0.69
4
.1.22. 7a-N-[3N-(3-Cyanopropyl)aminopropyl]amino-
1
3
cholesterol 20a. Compound 20a was obtained from amine
9a (0.46 g, 1 mmol) as described in the preparation of
compound 7 to yield (0.22 g, 42%) as a oil. IR: n: 2935
(s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
1
0
.90 (d, 3H, JZ6.5 Hz, Me 21), 0.93 (s, 3H, Me 19), 2.37
0
–(CH ) –), 2.42–2.47 (m, 2H, –HN–
1
(m, 1H, St–NH–CH
(
a
H
a
2 2
(
CH ), 2249 (CN). H NMR (CDCl , 250 MHz): d: 0.68 (s,
2 3
CH ) –CH –NH and –HN–CH –(CH ) –NH ), 2.70
2 3 2 2 2 2 3 2
3
H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26), 0.92
(m, 1H, St–NH–CH H
a
0
–(CH ) –), 2.75 (ddd, 1H, J7b–8Z
a
2 2
(
d, 3H, JZ6.5 Hz, Me 21), 0.99 (s, 3H, Me 19), 2.40 (t, 2H,
4
8
.3 Hz, J7b–6Z5 Hz, H7a of b epimer), 2.79 (t, 2H, JZ
.3 Hz, St–NH–(CH ) –CH –NH–), 3.5 (m, 1H, H3a), 5.30
JZ7.7 Hz, –CH –CN), 2.50 (m, 1H, St–NH–CH H
(CH ) –NH–), 2.62 (ddd, 1H, J7b–8Z4.3 Hz, J7b–6Z5 Hz,
2 2
H7b of a epimer), 2.95 (m, 1H, St–NH–CH Ha –(CH ) –
NH–), 3.5 (m, 1H, H3a), 5.62 (dd, 1H, J6–7bZ1.8 Hz,
J6–4!1 Hz, H6 of a epimer). C NMR (CDCl ): d: 12.0,
0
–
a a
2
2
2
2
(d, 1H, JZ3.0 Hz, H6 of b epimer), 7.19 (br s, 2H, –NH2,
0
a
2 2
D O exchange), 8.13 (br s, 1H, –NH–, D O exchange), 8.39
2
2
13
1
3
(br s, 1H, –NH–, D O exchange). C NMR (CDCl ): d:
2 3
3
12.1, 18.6, 18.8, 21.7, 22.7, 23.9, 25.4, 25.6, 26.3 28.1, 28.2,
1
2
4
1
5.2, 19.0, 19.1, 21.2, 22.9, 23.2, 24.1, 24.3, 27.2, 28.1,
8.3, 36.2, 36.6, 37.3, 37.7, 37.8, 39.4, 39.7, 40.7, 40.8,
3.0, 44.5, 46.1, 46.3, 46.8, 53.4, 56.3, 65.4, 72.0, 119.9,
21.1, 143.0. MS-EI: m/zZ471 (6%, St–NH–(CH ) –NH–
2
4
1
CH
9.5, 31.7, 35.9, 36.3, 37.0, 37.4, 39.6, 39.7, 40.0, 40.7,
0.8, 43.0, 46.1, 46.6, 46.8, 47.6, 54.2, 56.3, 59.0, 71.5,
22.8, 139.0. MS-EI: m/zZ471 (1%, St–NH–(CH ) –NH–
2
3
2
3
C
C
C
20
C
2
C
2
2
), 400 (33%, St–NH ), 385 (100%, St ). [a]
D
C418
O: C
CH ), 428 (16%, St–NH–CH –CH ), 400 (12%,
2
C C
St–NH ), 385 (100%, St ).
(CZ0.2 M in MeOH). Anal. Calcd for C34
H
63
N
7.35, H 11.66, N 7.96. Found: C 77.27, H 11.71, N 7.94.
3
7
4.1.23. 7b-N-[3N-(3-Cyanopropyl)aminopropyl]amino-
cholesterol 20b. Amine 19b (0.180 g, 0.40 mmol) was
alkylated in the same manner as 19a to yield 0.10 g (50%) of
product 20b as a oil. IR: n: 2935 (CH ), 2249 (CN). H NMR
1
2
(
CDCl , 250 MHz): d: 0.68 (s, 3H, Me 18), 0.86 (d, 6H, JZ
References and notes
3
6
.5 Hz, Me 27 and Me 26), 0.90 (d, 3H, JZ6.5 Hz, Me 21),
.93 (s, 3H, Me 19), 2.45 (t, 2H, JZ7.0 Hz, St–HN–(CH ) –
0
NH–(CH ) –CH –CN), 2.53 (m, 1H, St–NH–CH H –CH2–
1. Moore, K. S.; Wehrli, S.; Roder, H.; Rogers, M.; Forrest, J. N.;
MacCrimmon, D.; Zasloff, M. Proc. Natl Acad. Sci. U.S.A.
1993, 90, 1354–1358.
2
3
0
2
2
2
a
a
)
epimer), 2.86 (m, 1H, St–NH–CH Ha –CH –), 3.53 (m, 1H,
, 2.74 (ddd, 1H, J7a–8Z8.7 Hz, J7a–6Z1.2 Hz, H7a of b
0
2. Wehrli, S. L.; Moore, K. S.; Roder, H.; Durell, S.; Zasloff, M.
Steroids 1993, 58, 370–378.
a
2
1
3
H3a), 5.31 (d, 1H, J6–7aZ3.0 Hz, H6 of b epimer).
C
NMR (CDCl ): d: 12.4, 15.4, 19.1, 19.4, 21.7, 22.9, 24.1,
3. Sills, A. K. Jr.; Williams, J. I.; Tyler, B. M.; Epstein, D. S.;
Sipos, E. P.; Davis, J. D.; McLane, M. P.; Pitchford, S.;
Cheshire, K.; Gannon, F. H.; Kinney, W. A.; Chao, T. L.;
Donowitz, M.; Laterra, J.; Zasloff, M.; Brem, H. Cancer Res.
1998, 58, 2784–2792.
3
2
3
6
6.8, 27.0, 28.4, 28.8, 28.9, 30.0, 32.0, 36.1, 36.4, 36.5,
7.4, 39.8 40.1, 42.4, 43.6, 44.6, 46.8, 48.3, 48.8, 55.7, 57.2,
0.4, 71.8, 120.2, 124.6, 142.6. MS-EI: m/zZ471 (3%, St–
C
C
NH–(CH ) –NH–CH ), 428 (26%, St–NH–CH –CH );
4
2
3
2
2
2
C
00 (21%, St–NH ).
4. Williams, J. I.; Weitman, S.; Gonzalez, C. M.; Jundt, C. H.;
Marty, J.; Stringer, S. D.; Holroyd, K. J.; Mc Lane, M. P.;
Chen, Q.; Zasloff, M.; Von Hoff, D. D. Clin. Cancer Res. 2001,
7, 724–733.
4.1.24. 7a-N-[3N-(4-Aminobutyl)aminopropyl]amino-
cholesterol III. Compound III (0.09 g, 0.16 mmol) was
prepared in the same manner as I to yield (0.04 g, 46%) as
amorphous solid. H NMR (CDCl , 250 MHz): d: 0.68
5. Li, D.; Williams, J. I.; Pietras, R.-J. Oncogene 2002, 21,
2805–2814.
1
3
(
0
(
s, 3H, Me 18), 0.86 (d, 6H, JZ6.5 Hz, Me 27 and Me 26),
.91 (d, 3H, JZ6.5 Hz, Me 21), 0.99 (s, 3H, Me 19), 2.66
m, 1H, St–NH–CH Ha –(CH ) –NH–), 2.74 (ddd, 1H,
6. Rao, M. N.; Shinnar, A. E.; Noecker, L. A.; Chao, T. L.;
Feibush, B.; Snyder, B.; Sharkansky, I.; Sarkahian, A.;
Zhang, X.; Jones, S. R.; Kinney, W. A.; Zasloff, M. J. Nat.
Prod. 2000, 63, 631–635.
0
a
2 2
J7b–8Z4.3 Hz, J7b–6Z5.0 Hz, H7b of a epimer), 2.80
(
St–NH–CH H
t 2H, JZ5.4 Hz, –HN–(CH ) –CH –NH ), 2.92 (m, 1H,
7. Moriarty, R. M.; Tuladhar, S. M.; Guo, L.; Wehrli, S.
Tetrahedron Lett. 1994, 35(44), 8103–8106.
2
3
2
2
0
–(CH ) –NH–), 3.00–3.48 (m, 4H, St–NH–
a
a
2 2
(
CH ) –CH –NH– and NH–CH –(CH ) –NH ), 3.63 (m,
2
H, H3a), 5.66 (dd, 1H, J6–7bZ1.8 Hz, J6–4!1.0 Hz, H6 of
8. Pechulis, A. D.; Bellevue, F. H. III; Christopher, L.; Cioffi,
C. L.; Trapp, S. G.; Fojtik, J. P.; McKitty, A. A.; Kinney,
W. A.; Frye, L. L. J. Org. Chem. 1995, 60, 5121–5126.
2
2
2
2 3
2
1
a epimer), 7.23 (br s, 2H, –NH2, D O exchange), 8.15 (br s,
2

11486
B. Choucair et al. / Tetrahedron 60 (2004) 11477–11486
9
. Okumura, K.; Nakamura, Y.; Takeuchi, S.; Kato, I.; Fujimoto,
Y.; Ikekawa, N. Chem. Pharm. Bull. 2003, 51(10), 1177–1182.
0. Rao, M. N.; McGuigan, M. A.; Zhang, X.; Shaked, Z.; Kinney,
15. Zhou, X. D.; Cai, F.; Zhou, W. S. Tetrahedron 2002, 58,
10293–10299.
1
16. Kikuchi, K.; Bernard, E. M.; Sadownik, A.; Regen, S. L.;
Armstrong, D. Antimicrob. Agents Chemother. 1997, 41(7),
1433–1438.
W. A.; Bulliard, M.; Laboue, B.; Lee, N. E. J. Org. Chem.
1997, 62, 4541–4545.
1
1. Jones, S. R.; Selinsky, B. S.; Rao, M. N.; Zhang, X.; Kinney,
W. A.; Tham, F. S. J. Org. Chem. 1998, 63, 3786–3789.
2. Zhang, X.; Rao, M. N.; Jones, S. R.; Shao, B.; Feibush, P.;
McGuigan, M.; Tzodikov, N.; Feibush, B.; Sharkansky, I.;
Snyder, B.; Mallis, L. M.; Sarkahian, A.; Wilder, S.; Turse,
J. E.; Kinney, W. A. J. Org. Chem. 1998, 63, 8599–8603.
3. Zhou, X.-D.; Cai, F.; Zhou, W.-S. Tetrahedron Lett. 2001, 42,
17. Chitkul, B.; Atrash, B.; Bradley, M. Tetrahrdron Lett. 2001,
42, 6214–6221.
1
18. Kim, H. S.; Choi, B. S.; Kwon, K. C.; Lee, S. O.; Kwak, H. J.;
Lee, C. H. Bioorg. Med. Chem. 2000, 8, 2059–2065.
19. Khabnadideh, S.; Tan, C. L.; Croft, S. L.; Kendrick, H.;
Yardley, V.; Gilbert, I. H. Bioorg. Med. Chem. Lett. 2000, 10,
1237–1239.
1
1
2537–2539.
20. Shu, Y.; Jones, S. R.; Kinney, W. A.; Selinsky, B. S. Steroids
2002, 67, 291–304.
4. Kinney, W. A.; Zhang, X. H.; Williams, J. I.; Jhonston, S.;
Michalak, L. S.; DeshPande, M.; Dostal, L.; Rosazza, J. P. N.
Org. Lett. 2000, 2(19), 2921–2922.
21. Choucair, B.; Dherbomez, M.; Roussakis, C.; El Kihel, L.
Bioorg. Med. Chem. Lett. 2004, 14, 4213–4216.