Synthesis of new alkylaminooxysterols with potent cell differentiating activities: Identification of leads for the treatment of cancer and neurodegenerative diseases

Article abstract of DOI:10.1021/jm901063e

We describe here the syntheses and the biological properties of new alkylaminooxysterols. Compounds were synthesized through the trans-diaxial aminolysis of 5,6-α-epoxysterols with various natural amines including histamine, putrescine, spermidine, or spermine. The regioselective synthesis of these 16 new 5α-hydroxyl-6β-aminoalkylsterols is presented. Compounds were first screened for dendrite outgrowth and cytotoxicity in vitro, and two leads were selected and further characterized. 5α-Hydroxy-6β-[2- (1Himidazol-4-yl)ethylamino]cholestan-3β-ol, called dendrogenin A, induced growth control, differentiation, and the death of tumor cell lines representative of various cancers including metastatic melanoma and breast cancer. 5α-Hydroxy-6β-[3-(4-aminobutylamino)propylamino]cholest-7-en- 3β-ol, called dendrogenin B, induced neurite outgrowth on various cell lines, neuronal differentiation in pluripotent cells, and survival of normal neurones at nanomolar concentrations. In summary, we report that two new alkylaminooxysterols, dendrogenin A and dendrogenin B, are the first members of a class of compounds that induce cell differentiation at nanomolar concentrations and represent promising new leads for the treatment of cancer or neurodegenerative diseases.

Full text of DOI:10.1021/jm901063e

J. Med. Chem. 2009, 52, 7765–7777 7765
DOI: 10.1021/jm901063e
Synthesis of New Alkylaminooxysterols with Potent Cell Differentiating Activities: Identification of
Leads for the Treatment of Cancer and Neurodegenerative Diseases
†
,†
ꢀ §
Philippe de Medina,†,‡ Michael R. Paillasse,†,‡ Bruno Payre, Sandrine Silvente-Poirot, and Marc Poirot*
†
‡
Equipe “Metabolisme, Oncogenese et Differenciation Cellulaire”, Inserm U563 and AFFICHEM and Centre de Microscopie Electronique
§
ꢀ
Applique a la Biologie, Faculte de Medicine de Rangueil, Universite Paul Sabatier, Toulouse, France
ꢁ
ꢀ
ꢀ ꢁ
ꢀ
ꢀ
ꢀ
Received July 17, 2009
We describe here the syntheses and the biological properties of new alkylaminooxysterols. Compounds
were synthesized through the trans-diaxial aminolysis of 5,6-R-epoxysterols with various natural amines
including histamine, putrescine, spermidine, or spermine. The regioselective synthesis of these 16 new
5R-hydroxyl-6β-aminoalkylsterols is presented. Compounds were first screened for dendrite outgrowth
and cytotoxicity in vitro, and two leads were selected and further characterized. 5R-Hydroxy-6β-[2-(1H-
imidazol-4-yl)ethylamino]cholestan-3β-ol, called dendrogenin A, induced growth control, differentia-
tion, and the death of tumor cell lines representative of various cancers including metastatic melanoma
and breast cancer. 5R-Hydroxy-6β-[3-(4-aminobutylamino)propylamino]cholest-7-en-3β-ol, called
dendrogenin B, induced neurite outgrowth on various cell lines, neuronal differentiation in pluripotent
cells, and survival of normal neurones at nanomolar concentrations. In summary, we report that two
new alkylaminooxysterols, dendrogenin A and dendrogenin B, are the first members of a class of
compounds that induce cell differentiation at nanomolar concentrations and represent promising new
leads for the treatment of cancer or neurodegenerative diseases.
The microsomal antiestrogen binding site (AEBSa) is a
nanomolar target for the antitumor and chemopreventive
drug tamoxifen (Tam) that was first described 30 years ago.1
The AEBS was shown to be different from the estrogen
receptor R which is known as the primary target of Tam for
the hormonotherapy of breast cancers.2,3 Pharmacological
studies established that the AEBS bound a number of drugs
belonging to various pharmacological classes in addition
to Tam, including selective estrogen receptor modulators, in-
hibitors of cholesterol biosynthesis, and selectively binds diphe-
nylmethane derivatives of tamoxifen such as PBPE and
tesmilifene.4-9 We and others established that the AEBS was
involved in the cytotoxicity induced by its cognate ligands.6,7,9-15
One selective AEBS ligand, tesmilifene, was developed for the
treatment of breast and prostate cancers despite the lack of a
clear understanding of its molecular mechanism of action at that
time.16-21 The molecular characterization and the identification
of the AEBS established that it was a multienzyme complex
involved in cholesterol metabolism.4,22-25 We established that in
mammalian cells, the AEBS subunits involved in Tam binding
are the 3β-hydroxysterol-Δ8-Δ7-isomerase (D8D7I) and the 3β-
hydroxysterol-Δ7-reductase (DHCR7)4 and we recently showed
that it contained the cholesterol-5,6-epoxide hydrolase (ChEH)
(de Medina et al., unpublished results), which catalyzes the
hydration of 5,6-epoxysterols such as 5,6R- and 5,6β-epoxy-
cholestan-3β-ol (CEs). Post-lanosterol cholesterol biosynthesis
enzymes such as D8D7I and DHCR7 are involved in genetic
syndromes26 in which organ developments are impaired sug-
gesting a possible link between the AEBS/ChEH complex and
cell differentiation. This led us to show that the AEBS con-
trolled the differentiation and death of breast cancer cells
through the modulation of cholesterol metabolism and the
production of sterol autoxidation products such as CEs.10,27,28
These results raised the possibility that defects in these enzymes
may affect the catalytic properties of the ChEH and the
metabolism of CEs, which opens up the possibility of the
involvement of CEs or their metabolites in mammalian devel-
opmental programs. CEs in the plasma of healthy human
subjects occurred at levels of 1-100 nM.29 At these concentra-
tions, we showed that CEs did not induce tumor cell differ-
entiation (reported in the present paper). Interestingly, the
AEBS was earlier described as a binding site for histamine,30
suggesting that CE and histamine may be concentrated at the
level of the AEBS. The spatial proximity between CE bearing a
putative electrophilic oxirane group (epoxide) and histamine
that contains nucleophile nitrogen groups led us to hypothesize
a possible proximity induced catalysis31 of a ring-opening
reaction of CEs with histamine. In that case, the reaction would
give 6-histaminylcholestan-3,5-diols (HiCD) that might be
active to induce cell differentiation. In the present study, we
reported the synthesis of HiCD and the synthesis of the product
of aminolysis of 5,6R-epoxysteroids with various natural
amines. The synthesized compounds were tested for differentia-
tion of mammalian cells.
*To whom correspondence should be addressed. Phone: 33 (5) 61 42
46 48. Fax: 33 (5) 61 42 46 31. E-mail: Marc_Poirot@hotmail.com.
a Abbreviations: AEBS, antiestrogen binding site; Tam, tamoxifen;
PBPE, N-pyrrolidino-2-[4-(benzyl)phenoxy]ethanamine HCl; D8D7I,
3
3β-hydroxysterol-Δ8-Δ7-isomerase; DHCR7, 3β-hydroxysterol-Δ7-
reductase; ChEH, cholesterol-5,6-epoxide hydrolase; CE, 5,6-epoxycho-
lestan-3β-ol; HiCD, 6-histaminylcholestan-3,5-diols; DDA, dendrogen-
in A; DDB, dendrogenin B; DHA, docosahexaenoic acid; ATRA,
all-trans-retinoic acid; BDNF, brain derived neurotrophic factor.
r
2009 American Chemical Society
Published on Web 09/22/2009
pubs.acs.org/jmc

7766 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Chart 1. Epoxidation and Aminolysis of Steroids
de Medina et al.
Chemistry
backbone (15) induced a drastic diminution of dendrite out-
growth and 10-fold reduction in cytotoxicity compared with
14. The addition of an ethyl group on C24 of the isooctyl side
chain (16) has no incidence on the biological activities com-
pared with 14, showing that the increase in steric hindrance of
the isooctyl side chain of the sterol backbone is not deleterious
for activity. Compound 17 is a regioisomer of 14 in which
histamine was grafted on by its imidazole ring. 17 was less
potent in inducing dendrite outgrowth on U937 and P19 cells
and had a 10-fold decrease in cytotoxicity compared with 14
showing that regioisomers 14 and 17 were not equipotent.
Acetylation of the hydroxyl in C3 of ring B of the steroid
backbone (18) did not change the biological properties of 14,
whereas the replacement of the acetyl group by a butyryl
group (19) led to a strong decrease of both the dendritogenesis
and cytotoxicic potencies on the two tested cell lines. This
indicates that the presence of a bulky substituent on the C3
position decreases the biological activities. The elimination of
the isooctyl side chain and its replacement by a ketone group
(20) abolished the dendrite outgrowth properties and de-
creased the cytotoxicity compared with 14. Replacing hista-
mine by 1,4-diaminobutane (putrescine) (21) on 14 decreased
dendrite outgrowth potencies and cytotoxicity. Compound 21
has a stronger potency to induce dendrite outgrowth on P19
cells than on U937 cells. This tendency was more pronounced
in the presence of a double bond between C7 and C8 on ring B
of the steroid backbone (22). Compounds obtained by the
aminolysis of 1 or 2 with spermidine grafted through the two
possible primary amines gave compounds 23, 24, 25, and 26.
These compounds showed weak potencies to stimulate den-
drite outgrowth on U937 cells but high potencies to stimulate
dendrite outgrowth on p19 cells with no cytotoxicity on both
cell lines, the most potent being 24. Compound 27 is a
spermidine conjugate, but in this case spermidine was con-
jugated by the secondary amine. 27 was highly efficient at
inducing dendrite outgrowth on both U937 and P19 cells but,
as observed for the histamine and putrescine conjugates, was
cytotoxic. This showed that regiosiomers 24, 26, and 27 were
not equipotent for the induction of dendrite outgrowth on
both cell lines. Coupling spermine to epoxide 1 or 2 gave
compounds 28 and 29 that were potent inducers of dendrite
outgrowth on P19 cells. Compounds 28 and 29 were not
cytotoxic and weakly efficient in inducing dendrite outgrowth
on U937 cells. From these studies, it can be concluded that the
products of conjugation through epoxide ring opening with
biogenic amines gave products with potent capacities to
induce dendrite outgrowth at nanomolar concentrations.
Two families of compounds have been delineated depending
on the biogenic amines that were grafted: (1) histamine
derivatives (14, 16, 19) that were potent inducers of dendrite
outgrowth on U937 and P19 cells and were cytotoxic; (2)
spermidine derivatives (24 and 25) that induced selectively
dendrite outgrowth on P19 cells without being cytotoxic.
Sterol-5,6-epoxides (1-6) were synthesized as described
previously (Chart 1)32,33 and gave a 75:25 mixture of R- and
β-epoxisterol isomers. R-Epoxysterols 1-6 were purified by
recrystallization and used for coupling with alkylamines,
using 3 equiv of LiClO4 in ethyl alcohol as solvent,34 and
afforded the corresponding 6β-aminoalkyloxysterols through
trans diaxial opening of the oxirane ring (Table 1). No
reactions were obtained under these conditions with the
β-CE. The absence of reactivity of β-CE can be explained by
the impossibility of a trans diaxial opening with the amine in
the transition state. New alkylaminooxysterols varied in their
steroid structures by the presence or the absence of a double
bond in the C8-C7 position, the esterification of the 3β
hydroxyl by an acetyl or a butyryl group, and the presence
of an ethyl group on the C24 position. Histamine, putrescine,
spermidine (SPD), and spermine were used for the aminolysis
of sterol epoxides. SPD contains three different nucleophilic
groups; thus, in order to avoid mixtures of regioisomers, the
synthesis of three protected spermidines were prepared as
described in Scheme 1 and used as reported above to obtain
the expected 6β-aminoalkylsterols. The deprotection of the
amines was done with trifluoroacetic to obtain the expected
free amines (23-27). The molecular structures of synthesized
compounds are presented in Table 1. The compounds were
then tested for biological applications.
Biology
Induction of Dendrite Outgrowth and Cytotoxicity on
U-937 and P19 Cells. U937 is a monocytic-like cell line that
can undergodifferentiationintomacrophages upon treatment
with 100 nM phorbol myristyl acetate for 1 day,35,36and then a
treatment with IL-4 and GM-CSF induced a dendrite exten-
sion (Table 1). P19 is a pluripotent mouse carcinoembryonic
cell line that can be differentiated into neurons with micro-
molar concentrations of all-trans-retinoic acid (ATRA). The
main characteristic of this effect is the appearance and the
extension of dendrites.37,38 As a preliminary screening, com-
pounds were tested for dendrite outgrowth stimulation on
U937 and P19 cells, which allows a rapid morphological
assessment of cell differentiation. Compound 14 corresponds
to the product of aminolysis of R-CE with histamine by its
aminoethyl side chain. 14 was a potent inducer of dendrite
outgrowth on cells. Modification of cell morphology was
observed on 40-50% of adherent cells of U937 and
65-70% of P19 cells. In both cases the length of the dendrites
was greater than 200 μm after 48 h of treatment. We have
investigated the impact of treatment with the synthesized
compounds on U937 and P19 cell morphology and have
measured their cytotoxicity. 14 was cytotoxic with IC50 of
0.8 and 2.4 μM in U937 and P19 cells, respectively. The
presence of a double bond on C7-C8 on ring B of the steroid

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7767
Table 1. Effect of Compounds on Dendrite Outgrowth of Lymphoid U937 Cells and Carcinoembryonic P19 Cellsa
U937
P19
dendrite
outgrowth
cytotoxicity,
IC50 (μM)
dendrite
outgrowth
cytotoxicity,
IC50 (μM)
compd
ATRA
R1
R2
R3
Z
-
þ
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
IL4 þ GMCSF
cholesterol
R-CE
H
H
H
H
H
H
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
H
β-CE
2
H
H
Δ7,8
-
3
C2H5
H
4
CH3CO
n-C3H5CO H
-
-
-
-
-
-
-
-
5
6
histamine
H
-
-
-
-
-
-
-
H
H
-
-
-
-
-
-
spermidine
spermine
putrescine
R-CE þ histamine
R-CE þ spermidine
-
-
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
-NH(CH2)2-(1H-imidazol-5-yl)
-NH(CH2)2-(1H-imidazol-5-yl)
þþþþ
0.8 ( 0.2
7.6 ( 0.4
1.1 ( 0.1
7.7 ( 0.6
0.9 ( 0.1
8.2 ( 0.6
9.8 ( 0.7
2.1 ( 0.2
2.6 ( 0.2
-
þþþþ
2.4 ( 0.2
5.3 ( 0.5
2.5 ( 0.3
8.9 ( 0.4
2.5 ( 0.2
9.4 ( 0.6
7.3 ( 0.5
2.6 ( 0.2
2.8 ( 0.2
-
H
Δ7,8
-
þ
þþ
H
C2H5 -NH(CH2)2-(1H-imidazol-5-yl)
þþþþ
þþþþ
þ
H
CH3CO
nC3H7CO
H
H
H
-
H
H
H
H
H
H
H
H
H
-N-2-aminoethyl-(1H-imidazol-1-yl)
-NH(CH2)2-(1H-imidazol-5-yl)
-NH(CH2)2-(1H-imidazol-5-yl)
-NH(CH2)2-(1H-imidazol-5-yl)
-HN(CH2)4NH2
-
-
-
-
þ
þþþþ
-
þþþþ
-
H
H
H
H
H
H
H
H
H
H
-
-
-
þþ
þþ
þ
þþþ
þþþþ
þþþþ
þþþþþ
þþþþ
þþþþ
þþþþ
þþþ
þþþ
-HN(CH2)4NH2
Δ7,8
-
-HN(CH2)3NH(CH2)4NH2
-HN(CH2)3NH(CH2)4NH2
-HN(CH2)4NH(CH2)3NH2
-HN(CH2)4NH(CH2)3NH2
2HN(CH2)3-N((CH2)4NH2
-HN(CH2)3HN(CH2)4NH(CH2)3NH2
Δ7,8
-
þ
-
-
-
-
-
-
þ
Δ7,8
Δ7,8
-
þ
þ
7.5 ( 0.6
-
-
8.9 ( 0.7
-
-
þ
-HN(CH2)3HN(CH2)4NH(CH2)3NH2 Δ7,8
þ
a Z is the status of the C8-C7 bond (single or double). Δ7,8: double bond between C8 and C7 of the steroid backbone. IC50: concentration of a
compound required to induce 50% of the cytotoxicity. Cells were treated for 48 h with 100 ng/ml IL-4 and GM-CSF. Dendrite outgrowth was measured
using a light microscope after 48 h of incubation of cells with 100 nM of each indicated compounds. -: dendrites are not detectable on cells. þ: the length
of dendrites is <5 μm. þþ: the length of dendrites is between 5 and 10 μm. þþþ: the length of dendrites is between 10 and 50 μm. þþþþ: the length of
dendrites is between 50 and 100 μm, or the length of dendrites is >100 μm. þþþþþ: the length of dendrites is >100 μm, and a dendritic contact network
is observable. Cell death was determined by the trypan blue exclusion test.
Scheme 1. Protection of Spermidinea
a Reagents and conditions. A: (a) MeOH, 0 °C, then room temp, overnight, 26-47%; (b) Boc2O, DCM, 0 °C, then room temp, 4 h, 82-85%;
(c) LiAlH4/Et2O, 0 °C, then room temp, 1 h, 65-82%. B: (a) BocON, THF, 0 °C, then room temp, overnight, 39%.
The potencies of these new alkylaminooxysterols were pecu-
liar to their structure because treatment of cells with reactant
alone (sterol epoxides and amines) or in association was
unable to induce dendrite outgrowth on a precursor of a

7768 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
de Medina et al.
Figure 1. DDA (14) induced growth control and characteristics of differentiation in MCF-7 cells, a human breast adenocarcinoma cell
line. (A) Cell cycle distribution of MCF-7 cells treated with solvent vehicle (control) or 1 μM DDA for 48 h. Cell cycle distribution was
measured as described in the Experimental Section by FACS flow analysis using a Becton Dickinson FACS system. (B) Staining of neutral
lipids with oil red O (ORO) in MCF-7 cells treated with solvent vehicle (control) or 1 μM DDA for 48 h. Morphologic and biochemical
changes were evaluated by light microscopy (ꢀ40) of ORO stained cells counterstained with Mayer’s hematoxylin as described in
the Experimental Section. (C) Quantification of triglycerides present in MCF-7 cells treated for 48 h with solvent vehicle (control), 1 μM
DDA, or 5 μM tamoxifen (Tam) was carried out as described in the Experimental Section. (D) Quantification of milk fat globulin expressed in
MCF-7 cells treated for 48 h with solvent vehicle (control), 1 μM DDA, or 5 μM tamoxifen (Tam) was carried out as described in the
Experimental Section.
Chart 2. Structure of Dendrogenin A
Chart 3. Structure of Dendrogenin B
stimulated the accumulation of neutral lipid containing in-
tracytoplasmic vesicles stained in red with oil red O. We
determined that the nature of the neutral lipids that accumu-
lated in cells were triacyl glycerols (Figure 1), the major lipids
found in milk, and that DDA stimulated the expression of
milk fat globulin which is one of the major proteins found in
milk (Figure 1). Altogether, these data established that DDA
induced differentiation in MCF-7 cells. Interestingly, the
concentrations of tamoxifen and PBPE that were required
to obtain a similar effect as 1 μM DDA were 5 and 20 μM,
showing that DDA was much more active. We next evaluated
the impact of DDA on the differentiation of human melanoma
cells (SK-Mel-28). DDA induced cell cycle arrest in the
G0-G1 phase (Figure 2A). DDA dramatically changed in the
cell morphology from fibroblastic (Figure 2B, panel 1) to
dendritic that resembled normal melanocytes (Figure 2B,
neuron and they were not cytotoxic (up to 20 μM) (Table 1).
This indicated that these new alkylaminooxysterols displayed
specific effects. Compounds 14 and 24 were further character-
ized for their pharmacological properties because they were
the most potent compounds of their series and were named
dendrogenin A (DDA) (Chart 2) and dendrogenin B (DDB)
(Chart 3), respectively, because of their ability to stimulate
dendrite outgrowth.
DDA Induces Cell Differentiation. Since we showed that
DDA induced characteristics of differentiation in U937 and
P19, we studied its effects on differentiation of other tumor
cell lines. We have investigated the effects of DDA on the
differentiationofhumanbreastcancercells(MCF-7). Figure1
shows that DDA induced the arrest of cells in the G0-G1
phase of the cell cycle, modified the cell morphology, and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7769
Figure 2. DDA (14) induced growth control and characteristics of differentiation in SK-Mel-28 cells, a human melanoma cell line. (A) Cell
cycle distribution of SK-Mel-28 cells treated with solvent vehicle (control) or 1 μM DDA for 48 h. Cell cycle distribution was measured as
described in the Experimental Section by FACS flow analysis using a Becton Dickinson FACS system. (B) Morphologic changes of SK-Mel-28
treated with solvent vehicle (1) or with 1 μM DDA for 48 h (2) were evaluated by light microscopy (ꢀ40) as described in the Experimental
Section. (C) Electron micrographs of SK-Mel-28 cells treated with solvent vehicle (1) or 1 μM DDA for 48 h (2, 3). After fixation of the cells
and embedding in Epon 812, ultrathin sections of the cells were prepared, stained with uranyl acetate and lead citrate, and examined in a
Hitachi electron microscope: N, nucleus; C, cytoplasm; bars, 1.25 μm for panel 1, 0.5 μm for panel 2, and 0.1 μm for panel 3. Arrows indicate
the presence of types III and IV melanosomes. (D) Melanogenesis in SK-Mel-28 cells treated with solvent vehicle (control), 1 μM DDA, or
5 μM ATRA for 48 h. Melanogenesis was measured in cell lysates by measuring the absorbance at 405 nm as described in the Experimental
Section.
Table 2. Effect of DDA on the Induction of Cytotoxicity on a Panel of
Tumor Cell Linesa
panel 2). Ultrastructure analysis by electron microscopy
revealed the presence of type IV melanosomes in the DDA-
cell
cell type
IC50 (μM)
treated cells (Figure 2C, panels 2 and 3), and biochemical
studies confirmed that DDA stimulated melanogenesis at
lower concentrations than ATRA (Figure 2D). Altogether
these data established that DDA induced differentiation of
SK-Mel-28 cells toward melanocytes and also that at higher
concentrations, DDA killed MCF-7 and SK-Mel-28 cells.
DDA shows promising properties in vitro as a potent inducer
of tumor cell differentiation.
DDA Is Cytotoxic against Various Tumor Cell Lines.
Having shown that DDA induced cell differentiation and
death in tumor cell lines of different origins as described
above, we measured the cytotoxicity of DDA (14) on a panel
of tumor cell lines representative of different cancers. We
report in Table 2 that DDA was active against every cell line
tested in the micromolar range. It is noted that these tumor
cells of human and mouse origins were sensitive to DDA in
the hundreds of nanomolar to the micromolar range. This
effect was specific to DDA in our series because its regioi-
somer (17) was only weakly cytotoxic and DDB was not
toxic to these cells up to 20 μM for 72 h. DDA shows
promising properties in vitro as a potent inducer of tumor
cell death.
MCF-7
TSA
breast carcinoma, human
breast carcinoma, mouse
melanoma, human
2.31 ( 0.2
1.28 ( 0.2
2.12 ( 0.2
0.68 ( 0.1
2.56 ( 0.6
1.80 ( 0.4
2.24 ( 0.5
1.35 ( 0.5
2.32 ( 0.2
0.74 ( 0.2
0.79 ( 0.3
1.32 ( 0.4
5.53 ( 0.8
0.89 ( 0.2
0.42 ( 0.1
SK-MEL-28
B16F10
A549
melanoma, mouse
lung carcinoma, human
embryonal carcinoma, mouse
neuroblastoma, human
neuroblastoma, human
neuroblastoma, mouse
glioma, human
P19
SK-N-SH
SH-SY5Y
Neuro2A
U87
U937
NB4
monocytic leukemia, human
promyelocytic leukemia, human
acute myeloid leukemia, human
colon carcinoma, human
intestinal carcinoma, human
KG1
HCT-8
SW620
a Cells were incubated with increasing concentrations of DDA ran-
ging from 10 nM to 20 μM for 72 h, and cytotoxicity was quantified by
trypan blue exclusion tests (see Experimental Section).
showed that DDB induced the expression of MAP2A
(Figure 3B) and β3-tubulin (Figure 3C) in dendrites, indicat-
ing that the dendrites may be neurites. The neuronal pheno-
type of P19 cells treated with DDB was confirmed by showing
that DDA stimulated the expression of the N-methyl-D-
aspartate receptor (NMDAR1) (Figure 3C) which is a char-
acteristic of P19 differentiation into neurons.39 Thus, DDB
induced P19 cells to differentiate into neurons and the den-
drite extensions were neurites.
Effect of DDB on Neuritogenesis, Neuron Differentiation,
and Neuron Survival. Given that DDB induced dendrite
outgrowth on P19 cells (Table 1), the differentiation of P19
cells was further investigated phenotypically. Figure 3 shows
that 48 h of exposure of P19 cells to 10 nM DDB produced
dendrite expansion and the appearance of a network with
contact between cells (Figure 3A). Immunocytochemistry
using antibodies directed against MAP2A and β3-tubulin
We next investigated whether the stimulation of neurite
outgrowth could be observed on a set of tumor cell lines of

7770 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
de Medina et al.
Figure 3. DDB induce the differentiation of the P19 carcino embryonic cells into neurons. Cells were treated with solvent vehicle (1) or 10 nM
DDB (2) for 48 h and (A) analyzed by light microscopy for morphology or (B) analyzed by immunocytochemistry for the expression of MAP2A
and (C) the expression of β3-tubulin. (D) The expression of NMDAR1 was shown by Western blotting after extraction of the protein from
treated cells.
Figure 4. DDB induced neurite outgrowth on cells from various
cell lines. Cells were treated for 48 h with 100 nM DDB. Neurite
Figure 5. DDB promotes mouse purified motoneuron survival.
Solvent vehicle, 100 ng/mL BDNF, or 10 nM DDB was added to
the culture immediately after seeding. Cells were counted at days 2,
3, 4, and 6. Results are expressed as a percentage of surviving
motoneurons.
outgrowth was quantified by measuring the absorbance (A562
reading of crystal violet extracted from stained isolated neurites.
)
neuronal and human origins. Figure 4 shows that DDB
induced neurite outgrowth not only on P19 cells but also
on human (SH-SY5Y, SK-NS-H) and mouse (Neuro2A)
neuroblastoma and human glioma (U87) cells. No toxicity
was observed up to 20 μM with DDB. Under the same
conditions, 2-5 μM ATRA and 40-60 μM docosahexaenoic
acid (DHA) were required to produce an effect comparable
to that obtained with DDB. ATRA and DHA are nonpepti-
dic compounds known to induce neurite outgrowth.40 This
established that DDB acts as a neurotrophin on both mouse
and human cells at very low concentrations.
The effect of DDB on the survival of purified mouse
motoneurons was tested, and as can be seen in Figure 5,
DDB stimulated motoneuron survival. A time course study
was performed and showed that DDB was extremely potent
and the effect challenged peptidic neurotrophin BDNF.
DDB shows promising properties in vitro as a potent inducer
of neurite outgrowth, neuron differentiation, and neuron
survival that justify further investigations into its potential to
treat neurodegenerative diseases and for neuroregenerative
therapy.
of cell differentiation and confirmed this hypothesis through a
series of in vitro experiments. Compounds were first tested for
their ability to stimulate dendrite outgrowth and for their
cytotoxicity on two different cell lines (U937 and P19).
Structure-activity studies were done by synthesizing various
5,6-epoxysterols and -steroids that were conjugated to differ-
ent biogenic amines, and protected forms of SPD were
synthesized to obtain selective regioisomers. Two families of
compounds were obtained on the basis of their biological
properties. The first class included compounds containing a
sterol backbone and a histamine (grafted by the primary amine)
or a putrescine on C6. These compounds were potent inducers
of dendrite outgrowth and were cytotoxic. The second class
included sterols containing or not containing a double bond at
C7-C8 and SPD or SPM grafted through a primary amine.
These compounds induced neurite outgrowth on P19 cells
without cytotoxicity but did not induce dendrite outgrowth
on monocytic U937 cells. From these compounds, the most
potent compounds of each series were selected and tested more
specifically. Dendrogenin A (DDA) (14) induced differentia-
tion and cell death in the human breast adenocarcinoma cell
line MCF-7 and the human metastatic melanoma SK-Mel-28.
Dendrogenin A (DDA) (14) was tested for cytotoxicity on
tumor cell lines representative of different cancers and showed
Conclusion
We describe here the synthesis of new alkylaminooxyster-
ols. We hypothesized that these compounds may be inductors

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7771
a high potency to kill tumor cells (at nanomolar to sub-
micromolar concentrations). Dendrogenin B (DDB) (24)
induced neurite outgrowth on different cell lines of neuronal
origin, more specifically inducing the differentiation of plur-
ipotent embryonic carcinoma stem cells (P19) into neurons at
nanomolar concentrations. Finally we showed that DDB
helped motoneurons to survive. Altogether, these data showed
that dendrogenin A and dendrogenin B are new alkylaminoox-
ysterols with high potency in in vitro models which deserve
further investigations on preclinical models of cancer and
neurodegenerative diseases. Moreover, since these compounds
were synthesized on the basis of a hypothesis of a metabolic
transformation of 5,6-epoxysterols with natural amines, the
question that remains to be answered is whether the active
alkylaminooxysterols synthesized here are natural metabolites.
Studies are ongoing in our laboratory to detect these com-
pounds in mammalian tissues.
(400 MHz, CDCl3) 5.45 (1H, d, J = 4.0 Hz), 3.84 (1H, m), 3.02
(1H, d, J = 4.1 Hz), 1.03 (3H, s), 0.92 (3H, d, J = 6.4 Hz), 0.88
(3H, d, J = 2.0 Hz), 0.87 (3H, d, J = 2.0 Hz), 0.55 (3H, s); NMR
δC (100 MHz, CDCl3) 148.29, 114.94, 68.60, 67.17, 56.05, 55.12,
54.77, 42.46, 41.56, 39.99, 39.69, 38.95, 36.33, 36.25, 35.14, 33.68,
31.39, 28.21, 27.95, 24.08, 23.41, 23.03, 22.76, 21.15, 19.01, 16.63,
12.01; m/z 401.6 [M þ H]þ, 418.6 [M þ NH4]þ.
5,6r-Epoxystigmastan-3β-ol (3). Recrystallization of the crude
product in acetone/water (9:1) gave the R-epoxide as white
needles (0.54 g, 54%): mp 109-110 °C (lit. 109-110 °C41);
[R]D -40 (lit. -4041); IR (CCl4), 3620, 3438, 2940, 2870, 1466,
1377, 1089, 1037, 966 cm-1; Rf[EtOAc] = 0.65; NMR δH
(300 MHz, CDCl3) 4.00-3.80 (1H, m), 2.89 (1H, d, J =
4.4 Hz), 2.06 (1H, m), 2.00-1.75 (4H, m), 1.70-0.90 (28H, m),
0.89(3H, d, J = 6.6Hz), 0.84 (3H, t, J = 7.2Hz), 0.82(3H, d, J =
6.4 Hz), 0.80 (3H, d, J = 6.6 Hz), 0.61 (3H, s);NMR δC (75 MHz,
CDCl3) 68.74, 65.67, 59.29, 56.85, 55.78, 45.81, 43.45, 42.55,
42.33, 39.87, 39.40, 36.13, 34.85, 33.89, 32.40, 31.10, 30.16, 29.89,
29.13, 28.82, 28.09, 26.89, 26.08, 24.05, 23.04, 20.64, 19.82,
19.02, 18.69, 15.92, 11.96, 11.85; m/z 431.4 [M þ H]þ, 448.4
[M þ NH4]þ.
Experimental Section
5,6r-Epoxycholestan-3β-acetate (4). Evaporation of the sol-
vent gave a white solid corresponding to a 70:30 mixture of R- 4
and β-epoxide (66%). The mixture was used without further
purification for aminolysis: mp 112 °C; Rf[EtOAc] = 0.6; NMR
Chemical Synthesis, Materials, and Instrumentation. Reagents
were purchased from Sigma-Aldrich (St. Louis, MO). The
progress of the reactions was monitored by TLC using Merck
silica gel 60 F254 (0.040-0.063 mm) with detection with 50%
methanolic H2SO4 and 0.2% ethanolic ninhydrin. Melting
points were determined with a Kofler apparatus and are un-
corrected. 1H NMR and 13C NMR spectra were recorded on a
Bruker spectrometer AC250, AC300, or AC400. Solutions were
prepared in deuteriochloroform (CDCl3), deuterated methanol
(CD3OD), or deuterated dimethyl sulfoxide (DMSO-d6) with
TMS as internal standard. ESI-MS and CI-MS spectra were
obtained with a quadrupolar NERMAG R10-10 spectrometer.
Infrared (IR) spectra were recorded on a Perkin-Elmer IR-881
spectrometer. Optical rotations were measured with a Perkin-
Elmer 241 polarimeter. Microanalyses were carried at the Ecole
Nationale Superieure de Chimie at Paul Sabatier University in
Toulouse (France), and all the values were within (0.3% of the
calculated compositions. High resolution mass spectrometry
(HRMS) was performed with a Waters LCT spectrometer,
and the observed mass yields referred to purified products and
are not optimized. All moisture-sensitive reactions were carried
out under an argon atmosphere using oven-dried glassware and
anhydrous solvents. All the organic layers were dried using
anhydrous magnesium sulfate. All test compounds showed
g95% purity.
δ
H (300 MHz, CDCl3) 4.97 (1H, m), 2.90 (1H, d, J = 4.2 Hz),
2.03 (3H, s), 1.09 (3H, s), 0.92 (3H, d, J = 6.6 Hz), 0.89 (3H, d,
J = 1.5 Hz), 0.86 (3H, d, J = 1.5 Hz), 0.63 (3H, s); NMR δC
(75 MHz, CDCl3) 170.17, 71.38, 63.55, 59.14, 56.77, 56.17,
50.96, 42.23, 42.27, 39.49, 37.99, 36.66, 36.12, 35.72, 34.98,
32.46, 32.13, 29.86, 28.76, 28.06, 27.22, 24.14, 22.81, 22.55,
21.92, 18.67, 17.02, 15.85, 12.20; m/z 445 [M þ H]þ, 462
[M þ NH4]þ.
5,6r-Epoxycholestan-3β-butyrate (5). Evaporation of the sol-
vent gave a white solid corresponding to a 70:30 mixture of R-5
and β-epoxide (64%). The mixture was used without further
purification for aminolysis: mp 86 °C; Rf[EtOAc] = 0.9; NMR
δ
H (300 MHz, CDCl3) 4.96 (1H, m), 2.88 (1H, d, J = 4.2 Hz),
2.23 (3H, m), 2.12 (1H, m), 1.07 (3H, s), 0.60 (3H, s); NMR
δC (75 MHz, CDCl3) 172.85, 71.12, 65.24, 59.87, 59.19, 56.80,
42.46, 42.35, 39.52, 39.40, 38.10, 36.52, 36.15, 35.79, 35.05,
32.16, 29.89, 28.79, 28.10, 28.03, 27.30, 24.07, 23.87, 22.84,
22.58, 20.61, 18.64, 18.54, 15.89, 13.64, 11.87; m/z 473
[M þ H]þ, 490 [M þ NH4]þ.
5,6r-Epoxyandrostan-3β,17β-diol (6). The crude product was
purified by column chromatography [EtOAc] to give 6 as a white
powder (0.58 g, 55%): Rf[EtOAc] = 0.6; NMR δH (300 MHz,
CDCl3) 3.76 (1H, m), 3.56 (1H, t, J = 5.6 Hz), 2.95 (1H, d, J =
4.5 Hz), 1.13 (3H, s), 0.72 (3H, s); NMR δC (75 MHz, CDCl3)
80.79, 67.76, 66.03, 59.24, 51.69, 43.03, 42.55, 39.21, 36.22,
34.73, 32,33, 30.44, 39.81, 29.09, 28.15, 22.72, 20.04, 14.90,
10.13; m/z 307.6 [M þ H]þ, 329.6 [M þ Na]þ.
General Procedure for the 5,6-Epoxidation. m-Chloroperben-
zoic acid (mCPBA) (4.25 mmol) in methylene chloride (10 mL)
was added dropwise to a solution of the sterol or the steroid
(2.5 mmol) in methylene chloride (25 mL) over a period of 1 min
at room temperature. The reaction mixture was stirred over
20 min and then washed with aqueous sodium sulfite (10%) and
sodium hydrogenocarbonate (5%) and dried over anhydrous
sodium carbonate. The solvents were evaporated under reduced
pressure to give the crude product.
N4-(2-Cyanoethyl)-1,4-diaminobutane(7). Acrylonitrile (1.19 g,
22.5 mmol) was added to a stirred solution of 1,4-diaminobutane
(1.32 g, 15 mmol) in methanol (35 mL) at 0 °C. The mixture was
stirred at room temperature overnight. The solvent was evapo-
rated and the resulting oil purified by column chromatography
[isopropylamine/MeOH/CHCl3, 1:5:15] to give compound 7 as a
colorless oil (1.01 g, 47%): Rf[isopropylamine/MeOH/CHCl3,
1:5:15] = 0.5; NMR δH (250 MHz, CDCl3) 2.92 (2H, t, J =
6.5 Hz), 2.70 (2H, t, J = 6.5 Hz), 2.65 (2H, t, J = 6.5 Hz),
2.52 (2H, t, J = 6.5Hz), 1.45 (4H, m); NMRδC (75MHz, CDCl3)
118.94, 49.22, 45.24, 42.20, 31.51, 27.57, 18.93; m/z 142.2
[M þ H]þ.
5,6r-Epoxycholestan-3β-ol (1). Recrystallization of the crude
product in acetone/water (9:1) gave the R-epoxide 1 as white
needles (0.58 g, 58%): mp 140-141 °C (lit. 142 °C); Rf[EtOAc] =
0.54; NMR δH (300 MHz, CDCl3) 3.84 (1H, m), 2.83 (1H, d,
J = 8.6 Hz), 0.99 (3H, s), 0.83 (3H, d, J = 6.5 Hz), 0.81 (3H, d,
J = 1.5 Hz), 0.78 (3H, d, J = 1.5 Hz), 0.54 (3H, s); NMR δC
(75 MHz, CDCl3) 68.68, 65.76, 59.33, 56.85, 55.87, 42.57, 42.34,
39.86, 39.49, 39.41, 36.14, 35.76, 34.86, 32.41, 31.06, 29.89,
28.82, 28.07, 27.99, 24.05, 23.84, 22.81, 22.55, 20.64, 18.64,
15.93, 11.86; m/z 403.6 [M þ H]þ, 420.6 [M þ NH4]þ.
N4,N8-Di-tert-butyloxycarbonyl-N4-(2-cyanoethyl)-1,4-dia-
minobutane (8). A solution of di-tert-butyl dicarbonate
(895 mg, 4.1 mmol) in CH2Cl2 (3 mL) was added dropwise
to solution of 7 (283 mg, 2 mmol) in CH2Cl2 (3 mL) at 0 °C.
The mixture was stirred at room temperature for 4 h. Water
(20 mL) was added, and the mixture was extracted with
5,6r-Epoxycholest-7-en-3β-ol(2). Recrystallization of the crude
product in acetone produced white needles of the R-epoxide (0.7 g,
70%): mp 146-148 °C (lit. 144-146 °C33); [R]D -113 (lit. -112)
(c 0.59, CHCl3);33) IR CHCl3 3620, 3440, 2955, 2875, 1643,
1465, 1382, 1038, 898, 868 cm-1. Rf[EtOAc] = 0.64; NMR δH

7772 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
de Medina et al.
CH2Cl2. The solvent was evaporated and the residue was
purified by column chromatography [MeOH/CHCl3, 1:3] to
give compound 8 as a colorless oil (560 mg, 82%): Rf[MeOH/
CHCl3, 1:3] = 0.39; NMR δH (250 MHz, CDCl3) 3.44 (2H, t,
J = 6.8 Hz), 3.26 (2H, t, J = 6.8 Hz), 3.12 (2H, m), 2.60 (2H,
m), 1.54-1.42 (4H, m), 1.44 (9H, s), 1.42 (9H, s); NMR δC
(75 MHz, CDCl3) 156.01, 155.41, 118.41, 80.47, 79.18, 47.24,
43.44, 40.03, 28.40, 27.37, 25.99, 16.96; m/z 342.4 [M þ H]þ.
N4,N8-Di-tert-butyloxycarbonylspermidine (9). To a stirred
solution of lithium aluminum hydride (7.12 mmol) in Et2O
(100 mL) at 0 °C, compound 8 (608 mg, 1.78 mmol) in Et2O
(50 mL) was added dropwise over a 30 min period. After 1 h, a 1
M aqueous solution of sodium hydroxide was added and the
precipitate filtered. The organic phase was extracted, washed
with brine, and dried over anhydrous sodium carbonate. The
solvent was evaporated to give compound 9 as a colorless oil
(400 mg, 65%): Rf[isopropylamine/MeOH/CHCl3, 1:5:15] =
0.60; NMR δH (400 MHz, CDCl3) 3.13 (6H, m), 2.70 (2H, t, J =
6.8 Hz), 1.66-1.26 (6H, m), 1.46 (9H, s), 1.44 (9H, s); NMR δC
(100 MHz, CDCl3) 156.21, 155.61, 79.65, 79.01, 46.74, 43.20,
40.44, 39.3, 31.80, 28.63, 27.64, 25.80; m/z 346.4 [M þ H]þ.
N3-(2-Cyanopropyl)-1,3-diaminopropane (10). Compound 10
was prepared as described above for compound 7, using
1,3-diaminopropane (2 g, 2.25 mL, 27 mmol), 4-bromobutyro-
nitrile (4.2 g, 29 mmol), and other reagents scaled accordingly.
Evaporation of the solvent gave an oil that was purified by chro-
matography [isopropylamine/MeOH/CHCl3, 1:5:15] to give
compound 10 as a colorless oil (991 mg, 26%): Rf[isopropyl-
amine/MeOH/CHCl3, 1:5:15] = 0.32; NMR δH (250 MHz,
CDCl3) 2.82 (2H, t, J = 6.8 Hz), 2.72 (2H, t, J = 6.8 Hz),
2.63 (2H, t, J = 6.5 Hz), 2.46 (2H, t, J = 6.5 Hz), 1.52 (4H, m);
NMR δC (75 MHz, CDCl3) 118.91, 49.20, 45.34, 42.22, 31.48,
27.56, 19.00; m/z 142.3 [M þ H]þ.
General Procedure for the Aminolysis of Epoxides. To a
solution of the epoxide (1-6) (0.25 mmol) in anhydrous ethanol
(3 mL) was added lithium perchlorate (0.75 mmol) and the
amine (histamine, Nω-acetylhistamine, putrescine, 9, 12, or 13)
as a free base (1 mmol) dissolved in anhydrous ethanol (1 mL).
The reaction mixture was stirred and refluxed for 4 days and
periodically monitored by TLC (eluted with the appropriate
solvent system). The solvent was evaporated, and the residue
was diluted in ethyl acetate, washed three times with water, and
dried. The solvent was then removed and the crude product
purified by either column chromatography or reverse phase
HPLC. Column chromatography was done at atmospheric
˚
pressure, using 60 A silica (Aldrich) deactivated by mixing with
2.5% v/v triethylamine for 1 h and using a gradient of MeOH in
EtOAc for elution.
5r-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol
(14). Compound 1 and histamine were used as reactants and treated
as described in the general procedure. The crude product of the
reaction was purified by column chromatography to give com-
pound 14 as a white powder (69 mg, 54%). Compound 14 was
further purified by RP-HPLC using an acetonitrile gradient (40% B
for 8 min, then to 100% B in 20 min; A is 95:5 water/acetonitrile,
0.1% TFA; B is 95:5 acetonitrile/water 0.1% TFA) with a retention
20
time of 18 min. The purity was g98%. [R]D -13.0 (EtOH);
Rf[MeOH/NH3, 28%, 8:2] = 0.82; NMR δH (400 MHz, MeOD)
7.64 (1H, s), 6.90 (1H, s), 4.03 (1H, m), 3.10 (1H, m), 2.81 (3H, m),
2.52 (1H, m), 2.52 (1H, dt), 1.09 (3H, s), 0.94 (3H, d, J = 6.5 Hz),
0.90 (3H, d, J = 1.2 Hz), 0.89 (3H, d, J = 1.2 Hz), 0.72 (3H, s);
NMR δC (100 MHz, MeOD) 135.75, 130.70, 118.06, 75.82, 67.10,
64.90, 56.47, 55.96, 49.35, 45.58, 42.78, 40.63, 40.23, 39.51, 38.30,
36.18, 35.94, 32.74, 30.45, 30.40, 29.47, 28.14, 27.96, 25.85, 23.99,
23.77, 22.03, 21.78, 18.06, 16.56, 11.56; HRMS m/z 514.7373
(calculated for C32H56N3O2 514.7376).
N1,N3-Di-tert-butyloxycarbonyl-N3-(3-cyanopropyl)-1,3-dia-
minopropane (11). 11 was prepared as compound 8 above, using
compound 10 (353 mg, 2.5 mmol) and other reagents scaled
accordingly. Evaporation of the solvent gave an oil that was
purified by column chromatography [MeOH/CHCl3, 1:3] to
give compound 11 as a colorless oil (725 mg, 85%): Rf[MeOH/
CHCl3, 1:3] = 0.42; NMR δH (400 MHz, CDCl3) 3.31 (4H, m),
3.12 (2H, m), 2.37 (2H, t, J = 7.2 Hz), 1.90 (2H, m), 1.70 (2H,
m), 1.49 (9H, s), 1.45 (9H, s); NMR δC (100 MHz, CDCl3)
146.95, 145.41, 120.02, 80.56, 79.46, 45.77, 37.70, 28.62, 27.61,
23.20, 14.95; m/z 342.4 [M þ H]þ.
5r-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholest-7-en-
3β-ol(15). Compound 2 and histamine wereusedasreactants and
treated as described in the general procedure. The crude product
of the reaction was purified by column chromatography to give
20
compound 15 as a white powder (66 mg, 52%): [R]D -45.3
(EtOH); Rf[MeOH/NH3, 28%, 8:2] = 0.82; NMR δH (400 MHz,
MeOD) 8.83 (1H, s), 7.52 (1H, s), 5.44 (1H, d, J = 4.0 Hz), 4.1
(1H, m), 3.62-3.50 (2H, m), 3.40 (1H, m), 3.37-3.31 (2H, m),
1.13 (3H, s), 1.01 (3H, d, J = 6.0 Hz), 0.93 (3H, d, J = 0.7 Hz),
0.91 (d, J = 0.7 Hz), 0.69 (3H, s); NMR δC (100 MHz, MeOD)
135.58, 130.70, 118.09, 110.10, 75.78, 67.64, 64.70, 56.19, 54.42,
47.92, 44.51, 40.30, 38.09, 33.50, 31.51, 24.10, 22.93, 22.15, 19.30,
18.13, 12.65; HRMS m/z 512.4205 (calculated for C32H54N3O2
512.4211).
N1,N4-Di-tert-butyloxycarbonylspermidine (12). 12 was pre-
pared as compound 9 above, using 11 (830 mg, 2.43 mmol) and
other reagents scaled accordingly. Evaporation of the solvent gave
compound 12 as a colorless oil (690 mg, 82%). Rf[isopropylamine/
MeOH/CHCl3, 1:5:15] = 0.60; NMR δH (400 MHz, CDCl3)
3.26-3.12 (6H, m), 2.72 (2H, t, J = 6.8 Hz), 1.56-1.41 (6H, m),
1.47 (9H, s), 1.44 (9H, s); NMR δC (100 MHz, CDCl3) 156.21,
155.61, 79.65, 79.01, 47.05, 43.60, 42.07, 37.72, 31.05, 28.82, 28.66,
26.22; m/z 346.3 [M þ H]þ.
5r-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]stigmastan-
3β-ol (16). 3 and histamine were used as reactants and treated as
described in the general procedure. The crude product was
purified by column chromatography to give compound 16 as a
white powder (61 mg, 45%): Rf[MeOH/NH3, 28%, 8:2] = 0.82;
NMR δH (300 MHz, MeOD) 8.64 (1H, s), 7.40 (1H, s), 4.11 (1H,
m), 3.55 (1H, dt, J = 6.0, 3.0), 3.40 (1H, m), 3.30-3.19 (2H, m),
3.03 (1H, d, J = 6.6 Hz), 1.16 (3H, s), 0.79 (3H, s); NMR δC
(75 MHz, MeOD) 118.67, 73.67, 66.17, 64.77, 55.99, 55.57,
45.88, 44.65, 42.67, 39.79, 39.39, 38.77, 37.85, 36.01, 32.26,
31.99, 31.36, 30.11, 29.93, 28.98, 27.21, 23.51, 22.75, 22.31,
20.86, 20.72, 19.16, 18.79, 17.99, 17.85, 15.09, 11.33; HRMS
m/z 542.4670 (calculated for C34H60N3O2 542.4680).
5r-Hydroxy-6β-[4-(2-Aminoethyl)imidazol-1-yl]cholestan-3β-ol
(17). Compound 1 and Nω-acetylhistamine were used as reactants
and treated as described in the general procedure. The crude
product of the reaction was purified by column chromatography
to give the N-acetylated-form of compound 17 as a white powder
(39 mg, 28%): mp 170-172 °C; [R]D20 -11.7 (EtOH); Rf[AcOEt/
MeOH, 1:1] = 0.64; NMR δH (300 MHz, MeOD) 7.68 (1H, br),
7.05 (1H, br), 4.05 (1H, m), 3.98 (1H, d, J = 6.0 Hz), 3.42 (2H, t,
J = 7.1 Hz), 2.74 (2H, t, J = 7.1 Hz), 1.19 (3H, s), 0.99 (3H, d, J =
6.6 Hz), 0.93 (3H, br), 0.91 (3H, br), 0.83 (3H, s), 0.75 (3H, s);
N1,N8-Di-tert-butyloxycarbonylspermidine (13). 2-(tert-Butoxy-
carbonyloxyimino)-2-phenylacetonitrile (508 mg, 2.7 mmol) in
anhydrous THF (5 mL) was added dropwise to a stirred solution
of spermine (150 mg, 1.03 mmol) in anhydrous THF (20 mL) at
0°C. The mixture was stirred and allowed to rise to room temperature
overnight. The solvent was then removed. The crude product was
diluted in ethyl acetate (20 mL), extracted with 5% aqueous sodium
hydroxide solution (3 times) and then water (3 times), and dried. The
solvent was evaporated and the resulting oil was purified by chroma-
tography [EtOAc/MeOH, 1:1] to give compound 13 as a light-yellow
oil (213 mg, 60%). Further recrystallization from light petroleum
gave the product as white crystals (139 mg, 39%): mp 84-85 °C (lit.
85.5-86.5 °C); Rf[EtOAc/aceton/acetic acid/water, 5:3:1:1] = 0.56;
NMR δH (400 MHz, CDCl3) 3.16-3.02 (4H, m), 2.60 (2H, t, J =
6.6 Hz), 2.54 (2H, t, J = 6.6 Hz), 1.59 (2H, m), 1.45 (4H, t, J = 6.6
Hz), 1.37 (18H, s); NMR δC (100 MHz, CDCl3) 156.32, 79.16, 49.64,
47.95, 40.64, 39.44, 30.08, 28.65, 28.04, 27.58; m/z 346.4 [M þ H]þ.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7773
NMR δC (75 MHz, MeOD) 171.78, 137.69, 137.09, 117.25, 75.24,
66.63, 62.16, 56.25, 26.18, 44.79, 42.31, 41.69, 39.96, 39.34, 39.08,
38.02, 36.01, 35.76, 62.86, 32.57, 30.38, 30.12, 28.02, 27.79, 27.38,
23.72, 23.61, 21.87, 21.62, 21.26, 20.93, 17.87, 15.02, 11.48; m/z
556.45 [M þ H]þ. The intermediate was then diluted in a mixture
of ethanol (10 mL) and 1 N HCl (2 mL) and refluxed for 10 h. The
solvent was then removed in vacuo, and the crude product was
diluted in EtOAc, washed with brine, and dried. Removal of the
solvent gave a yellow solid which was subsequently purified by
column chromatography to give 17 as a white powder (21 mg,
58%): Rf[MeOH/NH3, 28%, 8:2] = 0.72; NMR δH (300 MHz,
MeOD) 9.04 (1H, s), 7.60 (1H, s), 4.18 (1H, d, J = 6.0 Hz), 4.08
(1H, m), 3.30 (2H, t, J = 7.1 Hz), 3.15 (2H, t, J = 7.1 Hz), 0.98
(3H, d, J = 6.3 Hz), 0.91 (3H, br), 0.89 (3H, br), 0.85 (3H, s), 0.76
(3H, s); NMR δC (75 MHz, MeOD) 129.20, 128.87, 121.32, 74.56,
66.33, 64.82, 56.31, 56.11, 44.54, 42.66, 40.86, 39.94, 39.28, 38.24,
37.71, 35.93, 32.55, 32.08, 30.02, 29.70, 27.93, 27.75, 23.55, 23.40,
22.32, 21.79, 21.54, 20.84, 17.79, 16.96, 15.24, 11.51; HRMS m/z
514.7357 (calculated for C32H56N3O2 514.7376).
5r-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-
acetate (18). Compound 4 and histamine were used as reactants
and treated as described in the general procedure. The crude
product of the reaction was purified by column chromatography
togive compound 18 as a whitepowder (55mg, 40%): Rf [MeOH/
NH3, 28%, 8:2] = 0.84; NMR δH (300 MHz, MeOD) 7.57 (1H,
s), 6.85 (1H, s), 5.17 (1H, m), 3.15-3.10 (1H, m), 2.99-2.92 (1H,
m), 2.82 (2H, t, J = 6.3 Hz), 2.62 (1H, m), 1.91 (3H, s), 1.04 (3H,
s), 0.84 (3H, d, J = 6.5 Hz), 0.79 (3H, d, J = 1.2 Hz), 0.77 (3H, d,
J = 1.2 Hz), 0.64 (3H, s); NMR δC (75 MHz, MeOD) 169.17,
136.05, 134.81, 114.75, 74.97, 71.25, 64.85, 56.19, 55.62, 49.55,
46.76, 42.63, 40.16, 39.92, 39.29, 38.03 35.95, 35.72, 32.40, 30.15,
28.66, 28.21, 27.90, 27.75, 23.92, 23.64, 23.55, 21.82, 21.57, 20.81,
19.93, 17.84, 15.99, 11.44; HRMS m/z 546.4459 (calculated for
C34H58N3O3 556.4473).
5r-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-
butyrate (19). Compound 5 and histamine were used as reactants
and treated as described in the general procedure. The crude
product of the reaction was purified by column chromatography
to give compound 19 as a white powder (61 mg, 42%): Rf[EtOAc/
MeOH, 8:2] = 0.43; NMR δH (300 MHz, MeOD) 7.45 (1H, s),
6.73 (1H, s), 5.12 (1H, m), 2.91 (1H, m), 2.71-2.65 (3H, m), 2.35
(1H, m), 2.18 (2H, t, J = 7.0 Hz), 1.08 (3H, s), 0.89 (3H, d, J =
6.5 Hz), 0.80 (3H, d, J = 1.2 Hz), 0.78 (3H, d, J = 1.2 Hz), 0.61
(3H, s); HRMS m/z 584.4771 (calculated for C36H62N3O3
584.4786).
5r-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]androstan-
3β,17-diol (20). Compound 6 and histamine were used as reac-
tants and treated as described in the general procedure. The crude
product of the reaction was purified by column chromatography
to give compound 20 as a white powder (44 mg, 42%): Rf[MeOH/
NH3 28% 8:2] = 0.77; NMR δH (300 MHz, MeOD) 4.09 (1H,
m), 3.62 (1H, m), 3.49 (2H, m), 3.03 (2H, t, J = 6.8 Hz), 2.91 (1H,
m), 1.18 (3H, s), 0.82 (3H, s); NMR δC (75 MHz, MeOD) 136.58,
129.11, 113.53, 80.83, 74.04, 66.32, 34.91, 50.10, 49.93, 45.10,
43.05, 39.95, 38.12, 37.40, 36.59, 32.37, 30.20, 30.11, 29.20, 26.96,
22.66, 20.42, 15.68, 10.64. HRMS m/z 418.3068 (calculated for
C24H40N3O3 418.3064).
5r-Hydroxy-6β-(4-aminobutylamino)cholest-7-en-3β-ol (22).
Compound 2 and 1,4-diaminobutane were used as reactants
and treated as described in the general procedure. The crude
product was purified by column chromatography to give com-
pound 22 as a white powder (62 mg, 51%): [R]D20 -43.6 (EtOH);
Rf[MeOH/NH3, 28%, 8:2] = 0.36; NMR δH (300 MHz, MeOD)
5.40 (1H, br), 4.01 (1H, m), 2.93-2.82 (1H, m), 2.85 (2H, t, J =
7.2 Hz), 2.67-2.57 (2H, m), 1.03 (3H, s), 0.99 (3H, d, J =
6.3 Hz), 0.92 (3H, d, J = 0.9 Hz), 0.90 (3H, d, J = 0.9 Hz), 0.64
(3H, s); NMR δC (75 MHz, MeOD) 139.78, 117.55, 76.54, 64.04,
54.42, 49.59, 43.31, 43.14, 40.13, 39.99, 39.51, 39.31, 37.07,
31.12, 35.90, 32.80, 30.43, 27.78, 27.63, 27.60, 27.19, 23.59,
22.75, 21.84, 21.63, 24.56, 18.04, 11.16; HRMS m/z 489.4401
(calculated for C31H57N2O2 489.4415).
5r-Hydroxy-6β-[3-(4-aminobutylamino)propylamino]cholestan-
3β-ol (23). Compounds 1 and 9 were used as reactants and treated
as described in the general procedure. The crude product was
purified by column chromatography to give the intermediate as
a yellow powder (36 mg, 20%): Rf[EtOAc] = 0.51; NMR δH
(300 MHz, MeOD) 4.06 (1H, m), 3.35-3.23 (7H, m), 3.08 (2H, t,
J = 6.8 Hz), 1.50 (9H, s), 1.46 (9H, s), 1.16 (3H, s), 0.96 (3H, d,
J = 6.6 Hz), 0.92 (3H, d, J = 1.2 Hz), 0.90 (3H, d, J = 1.2 Hz),
0.75 (3H, s); NMR δC (75 MHz, MeOD) 157.17, 154.71, 79.74,
78.45, 66.95, 65.75, 56.28, 55.90, 46.89, 46.21, 45.43, 42.59, 40.49,
40.06, 39.63, 39.31, 38.14, 35.97, 35.75, 32.28, 30.22, 29.39,
27.95, 27.77, 27.43, 27.42, 27.01, 25.62, 25.14, 23.90, 23.56,
21.81, 21.56, 20.88, 17.83, 16.22, 11.37; m/z 748.80 [M þ H]þ,
770.85 [M þ Na]þ. The intermediate was then diluted in a DCM/
TFA (1:1 v/v) mixture and stirred at room temperature for 1 h.
The reaction was quenched with 5 mL water. The aqueous layer
was basified with 5% aqueous sodium hydrogenocarbonate solu-
tion and extracted with butanol and the organic layer dried.
Evaporation of the solvent gave compound 23 as a yellow powder
(83 mg, 71%): Rf[isopropanol/NH3, 28%/water, 7:3:1] = 0.62;
NMR δH (300 MHz, CDCl3) 4.10 (1H, m), 3.18-2.98 (8H, m),
1.12 (3H, s), 0.96 (3H, d, J = 6.6 Hz), 0.92 (3H, d, J = 0.9 Hz),
0.89 (3H, d, J = 0.9 Hz), 0.76 (3H, s); NMR δC (75 MHz, CDCl3)
73,66, 66.28, 64.36, 56.13, 55.69, 46.91, 46.03, 44.64, 44.47, 42.67,
39.88, 39.29, 38.55, 37.76, 35.94, 35.71, 34.39, 32.05, 30.05, 29.81,
27.89, 27.75, 27.23, 24.11, 23.55, 23.43, 22.73, 21.95, 21.82, 21.57,
20.75, 17.82, 14.92, 11.30; HRMS m/z 548.5141 (calculated for
C34H66N3O2 548.5150).
5r-Hydroxy-6β-[3-(4-aminobutylamino)propylamino]cholest-
7-en-3β-ol (24). Compounds 2 and 9 were used as reactants and
treated as described in the general procedure. The crude product
was purified by column chromatography to get the intermediate
as a yellow powder (142 mg, 76%): Rf[EtOAc] = 0.38; NMR δH
(300 MHz, MeOD) 5.40 (1H, br), 3.35-3.22 (4H, m), 3.07 (2H,
t, J = 6.8 Hz), 2.85 (1H, m), 2.54 (2H, t, J = 6.6 Hz), 1.49 (9H,
s), 1.46 (9H, s), 1.04 (3H, s), 0.99 (3H, d, J = 6.0 Hz), 0.92 (3H, d,
J = 0.9 Hz), 0.90 (3H, d, J = 0.9 Hz), 0.64 (3H, s); NMR δC
(75 MHz, MeOD) 157.15, 156.06, 139.74, 117.70, 79.44, 78.42,
76.58, 67.19, 64.13, 56.14, 54.44, 46.62, 45.19, 44.75, 43.42,
43.34, 43.17, 40.36, 39.97, 39.65, 39.32, 37.08, 36.12, 35.91,
32.88, 30.41, 27.79, 27.63, 27.46, 27.00, 23.60, 22.79, 21.85,
21.65, 21.61, 18.05, 17.55, 11.24; m/z 746.95 [M þ H]þ. The
intermediate was then treated as described for 23 above to give
the desired product as a light-yellow powder (91 mg, 64%).
Compound 24 was further purified by RP-HPLC using an
acetonitrile gradient (25-100% B in 60 min; A is 95:5 water/
acetonitrile, 0.1% TFA; B is 95:5 acetonitrile/water) with a
5r-Hydroxy-6β-(4-aminobutylamino)cholestan-3β-ol (21).
Compound 1 and 1,4-diaminobutane were used as reactants
and treated as described in the general procedure. The crude
product was purified by column chromatography to give com-
pound 21 as a white powder (43 mg, 35%): Rf[MeOH/NH3, 28%,
8:2] = 0.45; NMR δH (300 MHz, CDCl3) 3.99 (1H, m), 2.81 (2H,
t, J = 7.5 Hz), 2.67 (1H, m), 2.43 (1H, m), 2.34 (1H, m), 1.06 (3H,
s), 0.87 (3H, d, J = 6.3 Hz), 0.83 (3H, d, J = 0.9 Hz), 0.81 (3H, d,
J = 0.9 Hz), 0.66 (3H, s); NMR δC (75 MHz, CDCl3) 67.52,
63.77, 58.34, 56.41, 55.79, 47.87, 45.66, 42.75, 41.29, 40.66, 40.02,
39.53, 38.46, 36.24, 35.92, 33.01, 30.64, 30.41, 29.80, 28.29, 28.10,
27.91, 27.10, 24.37, 22.88, 22.47, 21.18, 18.65, 17.47, 12.27;
HRMS m/z 491.4577 (calculated for C31H59N2O2 491.4571).
20
retention time of 33 min. The purity was g98%. [R]D -34.2
(EtOH); Rf[isopropanol/NH3 28%/water, 7:3:1] = 0.62; NMR
δ
H (300 MHz, CDCl3) acidic form 5.44 (1H, d, J = 4.2 Hz), 4.11
(1H, m), 3.69 (1H, d, J = 4.2 Hz), 3.40 (2H, m), 3.26-3.15 (6H,
m), 1.14 (3H, s), 1.03 (3H, d, J = 6.0 Hz), 0.96 (3H, d, J =
0.6 Hz), 0.93 (3H, d, J = 0.6 Hz), 0.73 (3H, s); NMR δC
(75 MHz, CDCl3) 149.13, 109.00, 73.83, 66.69, 62.77, 56.06,
54.84, 46.99, 45.39, 44.61, 43.76, 43.29, 43.15, 39.27, 38.66,
36.68, 36.00, 35.80, 32.20, 31.36, 30.07, 27.76, 27.48, 24.19,

7774 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
de Medina et al.
23.53, 22.84, 22.25, 21.80, 21.55, 21.42, 17.95, 16.83, 11.34;
HRMS m/z 546.4990 (calculated for C34H64N3O2 546.4993).
5r-Hydroxy-6β-[4-(3-aminopropylamino)butylamino]cholestan-
3β-ol (25). Compounds 1 and 12 were used as reactants and treated
as described in the general procedure. The crude product was
purified by column chromatography to give the intermediate as
a yellow powder (116 mg, 62%); Rf[EtOAc] = 0.54; NMR δH
(300 MHz, MeOD) 4.06 (1H, br), 3.34 (3H, m), 3.25 (4H, m), 3.06
(2H, t, J = 6.8 Hz), 1.49 (9H, s), 1.47 (9H, s), 1.15 (3H, s), 0.96
(3H, d, J = 6.6 Hz), 0.92 (3H, d, J = 0.9 Hz), 0.90 (3H, d, J =
0.9 Hz), 0.75 (3H, s); NMR δC (75 MHz, MeOD) 157.08, 156.06,
79.60, 78.57, 75.82, 66.97, 64.52, 56.29, 55.90, 49.01, 46.44, 45.43,
44.76, 44.21, 42.58, 40.41, 40.07, 39.31, 38.14, 37.56, 35.98, 35.75,
32.46, 30.29, 30.24, 37.96, 27.77, 27.43, 27.42, 23.90, 23.57,
21.83, 21.58, 20.89, 17.85, 16.29, 11.35; m/z 748.80 [M þ H]þ,
770.85 [M þ Na]þ. The intermediate was then treated as described
for compound 23 above to give compound 25 as a light-yellow
powder (23 mg, 64%): Rf[isopropanol/NH3 28%/water, 7:3:1] =
0.61; NMR δH (300 MHz, CDCl3) 4.04 (1H, m), 3.40-3.26 (7H,
m), 3.10 (2H, t, J = 6.8 Hz), 1.16 (3H, s), 0.97 (3H, d, J = 6.8 Hz),
0.92 (3H, d, J = 0.6 Hz), 0.90 (3H, d, J = 0.9 Hz), 0.75 (3H, s);
NMR δC (75 MHz, CDCl3) 72,99, 67.08, 64.39, 56.30, 55.49,
46.81, 44.74, 44.42, 42.69, 39.99, 39.32, 38.35, 37.69, 35.94, 35.62,
34.67, 32.14, 30.17, 29.97, 27.89, 27.65, 27.22, 24.06, 23.66, 23.39,
22.73, 21.95, 21.82, 21.57, 17.82, 14.92, 11.30; HRMS m/z
548.5139 (calculated for C34H64N3O2 548.5150).
was further purified by RP-HPLC as described for compound 24
and presents a retention time of 26 min: Rf[isopropanol/NH3
28%/water, 7:3:1] = 0.63; NMR δH (300 MHz, CDCl3) acidic
form 5.13 (1H, d, J = 3.9 Hz), 3.96 (1H, m), 3.51 (1H, d, J =
4.0 Hz), 3.40 (4H, m), 3.24 (4H, m), 1.10 (3H, s), 0.94 (3H, d, J =
6.4 Hz), 0.83 (6H, d, J = 0.6 Hz), 0.57 (3H, s);NMR δC (75 MHz,
MeOD) 149.12, 109.00, 73.83, 66.39, 62.87, 62.76, 56.05, 54.83,
45.39, 44.61, 43.75, 43.14, 39.26, 39.13, 38.66, 36.67, 35.99, 35.80,
32.20, 30.07, 27.76, 27.47, 24.18, 23.53, 22.84, 22.61, 22.24, 21.79,
21.54, 21.41, 17.95, 16.84, 11.34; HRMS m/z 546.5006 (calculated
for C34H64N3O2 546.4993).
5r-Hydroxy-6β-N-{3-[4-(3-Aminopropylamino)butylamino]-
propylamino}cholestan-3β-ol (28). Compound 1 and N,N0-bis-
(3-aminopropyl)butane-1,4-diamine were used as reactants
and treated as described in the general procedure. The crude
product was purified by column chromatography to give the
intermediate as a yellow powder (48 mg, 32%). Compound 28
was further purified by RP-HPLC using a acetonitrile gradient
(0% B for 10 min, then to 100% B in 60 min; A is 95:5 water/
acetonitrile, 0.1% TFA; B is 95:5 acetonitrile/water) with a
retention time of 45 min: Rf[isopropanol/NH3 28%/water,
7:3:1] = 0.37; NMR δH (300 MHz, MeOD) 4.11 (1H, m),
3.39-3.23 (2H, m), 3.19-3.06 (10H, m), 2.91 (1H, d, J =
1.5 Hz), 1.11 (3H, s), 0.97 (3H, d, J = 6.6 Hz), 0.92 (3H, d, J =
0.9 Hz), 0.90 (3H, d, J = 0.9 Hz), 0.77 (3H, s); NMR δC
(75 MHz, MeOD) 73.67, 66.19, 64.76, 56.06, 55.58, 45.88,
44.65, 44.65, 42.67, 39.79, 39.37, 37.85, 31.99, 31.36, 30.11,
29.92, 28.98, 27.21, 22.31, 20.80, 20.73, 19.16, 17.85, 11.33;
HRMS m/z 605.5709 (calculated for C37H73N4O2 605.5728).
5r-Hydroxy-6β-N-{3-[4-(3-Aminopropylamino)butylamino]-
propylamino}cholest-7-en-3β-ol (29). Compound 2 and N,N0-
bis(3-aminopropyl)butane-1,4-diamine were used as reactants
and treated as described in the general procedure. The crude
product was purified by column chromatography to give the
intermediate as a yellow powder (57 mg, 38%). Compound 29
was further purified by RP-HPLC as described for 1 h and
presents retention time of 45 min: Rf[isopropanol/NH3 28%/
water, 7:3:1] = 0.37; NMR δH (300 MHz, MeOD) 5.34 (1H,
br), 4.05 (1H, m), 3.38-3.21 (2H, m), 3.18-3.03 (11H, m), 1.06
(3H, s), 0.97 (3H, d, J = 6.0 Hz), 0.90 (3H, d, J = 0.6 Hz), 0.87
(3H, d, J = 0.9 Hz), 0.65 (3H, s); NMR δC (75 MHz, MeOD)
150.89, 110.13, 75.18, 67.67, 64.61, 58.37, 57.46, 56.21, 48.33,
48.26, 46.88, 45.97, 45.88, 45.18, 44.55, 40.70, 38.10, 37.85,
37.43, 37.23, 33.53, 31.55, 29.20, 28.86, 25.42, 24.96, 24.25,
23.96, 23.72, 23.22, 22.97, 22.85, 19.37, 18.42, 18.14, 12.68;
HRMS m/z 603.5552 (calculated for C37H71N4O2 603.5572).
Cell Culture. All cell lines used in this study were from the ATCC
with the exception of TS/A (murine mammary adenocarcinoma)
which was kindly provided by P. Lollini (Bologna, Italy). A549
(human lung pulmonary cancer cells), HT29 (human colon
adenocarcinoma), MCF-7 (human breast ductal cells), TS/A
(murine mammary adenocarcinoma), SW 620 (mouse colon cancer
cells), and HCT-8 (human colon adenocarcinoma) cells were grown
in RPMI 1640 medium supplemented with 1.2 mM glutamine, 5%
fetal bovine serum, penicillin, and streptomycin (50 units/mL).
U937 (human myeloid lymphoma), SH-SY5Y (human neu-
roblastoma), and SK-N-SH (human neuroblastoma) were grown
in RPMI 1640 medium supplemented with 1.2 mM glutamine, 10%
fetal bovine serum, penicillin, and streptomycin (50 units/mL). U87
(human glioblastoma), Neuro2A (mouse neuroblastoma), B16-
F10 (mouse melanoma), SK-Mel-28 (human melanoma), NB4
(myeloid lymphoma), and KG1 (myeloid lymphoma) cells were
grown in DMEM medium supplemented with 3.2 mM glutamine,
10% fetal bovine serum (FBS), penicillin, and streptomycin (50
units/mL). Cells were incubated in a humidified atmosphere with
5% CO2 at 37 °C.
5r-Hydroxy-6β-[4-(3-aminopropylamino)butylamino]cholest-
7-en-3β-ol (26). Compounds 2 and 12 were used as reactants and
treated as described in the general procedure. The crude product
was purified by column chromatography to give the intermedi-
ate as a yellow powder (90 mg, 48%): Rf[EtOAc] = 0.38; NMR
δ
H (300 MHz, MeOD) 5.39 (1H, br), 4.06 (1H, m), 3.25 (4H, m),
3.07 (2H, t, J = 6.8 Hz), 2.62 (1H, m), 2.05 (2H, m), 1.49 (9H, s),
1.47 (9H, s), 1.15 (3H, s), 0.96 (3H, d, J = 6.3 Hz), 0.92 (3H, d,
J = 0.9 Hz), 0.90 (3H, d, J = 0.9 Hz), 0.75 (3H, s); NMR δC
(75 MHz, MeOD) 157.06, 156.06, 79.41, 78.59, 75.83, 66.98,
64.54, 56.30, 55.90, 49.02, 46.45, 45.43, 44.69, 42.59, 40.42,
40.08, 39.32, 38.15, 37.53, 35.99, 35.76, 32.48, 30.30, 31.25,
27.98, 27.77, 27.45, 27.44, 23.91, 23.58, 21.85, 21.61, 20.91,
17.88, 16.32, 11.38; m/z 746.95 [M þ H]þ. The intermediate was
then treated as described for compound 23 above to give
compound 26 as a light-yellow powder (85 mg, 94%). It was
further purified by RP-HPLC as described for compound 24
and presents a retention time of 32 min: [R]D20(EtOH) -33.5;
Rf[isopropanol/NH3 28%/water, 7:3:1] = 0.63; NMR δH (300
MHz, CDCl3) 5.40 (1H, br), 4.07 (1H, m), 3.76-3.46 (2H, m),
3.40 (1H, m), 3.21 (t, J = 6.8 Hz, 2H), 3.15 (t, J = 6.9 Hz, 2H),
3.04 (t, J = 6.8 Hz, 2H), 1.11 (s, 3H), 0.99 (d, J = 6 Hz, 3H), 0.92
(d, J = 0.9 Hz, 3H), 0.90 (d, J = 0.9 Hz, 3H), 0.69 (s, 3H); NMR
δC (75 MHz, MeOD) 149.12, 109.00, 73.83, 66.39, 62.87, 62.76,
56.05, 54.83, 45.39, 44.61, 43.75, 43.14, 39.26, 39.13, 38.66,
36.67, 35.99, 35.80, 32.20, 30.07, 27.76, 27.47, 24.18, 23.53,
22.84, 22.61, 22.24, 21.79, 21.54, 21.41, 17.95, 16.84, 11.34;
HRMS m/z 546.4999 (calculated for C34H64N3O2 546.4993).
5r-Hydroxy-6β-[(4-aminobutyl)(3-aminopropyl)amino]cholest-
7-en-3β-ol (27). Compounds 2 and 13 were used as reactants and
treated as described in the general procedure. The crude product
was purified by column chromatography to give the intermediate
as a yellow powder (87 mg, 47%): Rf[EtOAc/MeOH, 3:1] = 0.90;
NMR δH (300 MHz, MeOD) 5.37 (1H, br), 4.04 (1H, m),
3.33-3.17 (6H, m), 2.82 (1H, m), 2.10 (2H, m), 1.49 (9H, s),
1.47 (9H, s), 1.13 (3H, s), 0.94 (3H, d, J = 6.3 Hz), 0.90 (3H, d,
J = 0.9 Hz), 0.88 (3H, d, J = 0.9 Hz), 0.73 (3H, s); NMR δC
(75 MHz, CDCl3) 157.06, 156.06, 79.41, 78.59, 73.83, 66.39,
62.87, 62.76, 56.05, 54.83, 49.02, 46.45, 45.43, 44.69, 42.59,
40.42, 40.08, 39.32, 38.15, 37.53, 35.99, 35.76, 32.48, 30.30,
31.25, 27.98, 27.77, 27.45, 27.44, 23.91, 23.58, 21.85, 21.61,
20.91, 17.88, 16.32, 11.38;m/z 746.85 [M þ H]þ. The intermediate
was then treated as described for compound 23 above to give the
product as a light-yellow powder (62 mg, 71%). Compound 27
Cell Death Assays. Cells were seeded in RPMI with 5% FBS
into 12-well plates at 30 000 cells/well. The cells were then
treated with solvent vehicle (0.1% ethanol), 40 μM PBPE, or
10 μM Tx for 3 days. Treated cells were incubated in the presence

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7775
or in the absence of 2.5 μg/mL cycloheximide, 2.5 pg/mL
actinomycin D, 100 μM z-VAD-fmk, 100 μM z-DEVD-fmk,
500 μM vitamin E, 1 mM N-acetylcysteine, 500 μM butylated
hydroxytoluene, 10 mM 3-MA, or 50 nM Baf A1. Cell death was
determined by trypan blue exclusion assay. Cells were scraped
and resuspended in trypan blue solution (0.25% w/v in PBS) and
counted in a Malassez cell under a light microscope.
Dendrite Outgrowth on U937. Cells were suspended at 1.1 ꢀ
106 cells/mL in RPMI medium supplemented with 10% of heat
inactivated serum and cultured in 12-well plates with 10 ng/mL
of PMA (Sigma) for 48 h. Adherent cells were washed with
complete medium and then were treated with 100 ng/mL IL-4
and 100 ng/mL GM-CSF or with indicated compounds. Cul-
tured cells were observed by phase-contrast microscopy for
evidence of increasing size, formation of clusters, and neurite
outgrowth. At the end of culture, cell count was measured on an
automated cell counter (Sysmex, Milton Keynes, U.K.). Cyto-
centrifuge preparations of unmanipulated and cultured blasts
were stained with Giemsa. Examination of these cytospin pre-
parations allowed assessment of viability and maturation. Cells
that had increased in size, acquired copious gray cytoplasm
without cytoplasmic granules or vacuoles, and developed long
cytoplasmic processes were defined as mature leukemic DCs
morphologically.
with a cresyl violet solution. Cell bodies were removed from the
top of the membrane, and the dye was extracted from the
neurites and quantified at 562 nm on a spectrophotometer.43
Melanin Measurement. Melanin measurement was done as
previously described.44 Briefly, cells were treated for 24 h with
increasing concentrations of DDA, then were collected, washed
with PBS, and centrifuged for 5 min at 1200 rpm. Pellets
(corresponding to 2 ꢀ 106 cells) were solubilized in 200 μL of
1 M NaOH and incubated for 1 h at 80 °C. The melanin content
was measured spectrophotometrically at 405 nm on a Labsys-
tems FluoroSkan Ascent FL microplate fluorimeter.
Oil Red O and Staining Procedures. Cells were grown on glass
coverslips and treated with drugs for 72 h and then fixed with
3.7% paraformaldehyde for 1 h at room temperature followed
by washing twice with PBS (Euromedex) and stained with oil red
O in 60% (v/v) isopropyl alcohol and hematoxylin. Quantifica-
tion of lipid accumulation was achieved by extracting oil red O
from the stained cells with isopropyl alcohol and measuring the
extinction of the extract at 510 nm. The value obtained using a
control culture was subtracted from the resulting values. The oil
red O absorbance was corrected by costaining DNA with SYBR
green dye (Molecular Probes) and quantified on a Labsystems
FluoroSkan Ascent FL microplate fluorimeter. The cell number
was determined from a standard curve.
Phenotypic Characterization by Immunocytochemistry. P19
cells were seeded into six-wells culture plates (10 000 cells/well)
and treated for 24 h with the solvent vehicle or indicated
compounds. Cultured cells were fixed for 10 min in 4% paraf-
ormaldehyde, washed with PBS, and permeabilized with PBS
1% BSA and 0.05% saponin for 10 min. Cells were saturated
with PBS 3% BSA for 30 min at room temperature. Cells were
then incubated with mouse polyclonal antibody to mouse
MAP2A (Chemicon, MAB3418, 1/100), β3-tubulin (Sigma,
T8660, 1/200), in PBS 1% BSA, 0.05% saponin for 1 h at room
temperature. Slides were washed with PBS 0.1% BSA and
0.05% saponin and incubated for 1 h with TRITC-conjugated
secondary antibody. The glass coverslips were mounted onto
slides using fluorsafe (Calbiochem) as mounting medium. Cells
were observed by fluorescence microscopy using a Zeiss LSM
510 microscope (Zeiss, Gottingen, Germany). Staining of MCF-
7 cells for milk fat globulin was performed exactly as previously
described using monoclonal antihuman milk fat globulin
(MFG) from (Chemicon, ABA4087).28
Cell Cycle Analysis. Cell cycle analysis was performed by
flow cytometry. Cells were plated onto 100 mm plates at a
density of 250 000 cells and then treated with solvent vehicle or
the indicated compounds for 24 and 48 h. Cells were trypsi-
nized and washed once with phosphate-buffered saline (PBS),
fixed in 70% ethanol, and stored at 4 °C for subsequent cell
cycle analysis. Fixed cells were washed with PBS and incubated
with PBS containing 100 μg/mL RNaseA for 30 min at 37 °C,
then stained with 0.25% Tween-20 and 50 μg/mL propidium
iodide (PI) for 30 min at 37 °C in the dark. The DNA content of
1 ꢀ 106 stained cells was analyzed by flow cytometry. The
fractions of cells in the sub-G1, G0/G1, S, and G2/M phases
were calculated using Cell Quest software (Becton Dickinson,
Mansfield, MA).
Western Blotting. Cells were plated onto 100 mm plates at a
density of 250000 and then treated with indicated compounds for
48 h. Cells were lysed with 50 mM Tris, pH 7.4, 150 mM NaCl,
0.1% SDS, 1% deoxycholic acid, 1% Triton X-100, 1 mM
PMSF, 1 mM EDTA, and a cocktail of protease and phosphatase
inhibitors (Roche). For the detection of NMDAR1, proteins
were separated on 7.5% SDS-PAGE gels, electrotransferred
onto polyvinylidene difluoride membranes, and incubated over-
night at 4 °C with the mouse antimouse NMDAR1 (1:1000) or
the mouse antihuman glyceraldehyde 3-phosphate dehydrogen-
ase (1:1000). Visualization was achieved with an Enhanced
Chemiluminescence Plus kit (Amersham Biosciences), and fluor-
escence was measured by either autoradiography or using a
PhosphorImager (Storm 840, Amersham Biosciences).
Proliferation Assays. Cells were seeded in the appropriate
medium into 12-well plates at 30 000 per well. The cells were then
treated for 3 days with increasing test drug concentrations
(10 nM to 10 μM). The drugs and medium were changed after
48 h. Cells were harvested by trypsinization and counted on a
Coulter counter. Experiments were repeated in triplicate.
Transmission Electron Microscopy. Cells were fixed with 2%
glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4) for
1 h and washed with the Sorensen phosphate buffer (0.1 M) for
12 h. The cells were then postfixed with 1% OsO4 in Sorensen
phosphate buffer (0.05 M Sorensen phosphate, 0.25 M glucose,
1% OsO4) for 1 h. The cells were then washed twice with distilled
water and prestained with an aqueous solution of 2% uranyl
acetate for 12 h. Samples were then treated exactly as previously
described.45
Biostatistical Analysis. Values are the mean ( SE of three
independent experiments each carried out in duplicate. Statis-
tical analysis was carried out using a Student’s t test for unpaired
variables. 1 and 11 in the figures refer to statistical probabilities
(P) of <0.001 and <0.0001, respectively, compared with con-
trol cells that received the solvent vehicle alone.
Triglyceride Quantification. MCF-7 cells (5 million) were
treated for 48 h with the solvent vehicle or tested compounds.
Cells were then washed three times in PBS, and lipids were
extracted by liquid extraction according to the method of Bligh
and Dyer.42 Triglycerides analyses were done according to
previously published procedures.28
Assay for Neurite Protein. Cells were plated on inserts with a
3 μm pore membrane at their base coated with poly-D-lysine.
The inserts were then placed into 24-well plates. During the 24 h
incubation in the presence of the test compounds, neurons
extended their neurites through the microporous membrane to
the lower chamber. Membrane inserts were then removed,
neurons were fixed in methanol, and the neurites were stained
Acknowledgment. This work was supported by an internal
ꢁ
grant from the “INSERM”, the “Ministere de la Recherche”
through the GenHomme network”, the “Conseil Regional
ꢀ ꢀ
Midi-Pyrenees”, and the European Commission FP6 Integrated
Project EUROHEAR.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.

7776 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
de Medina et al.
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