Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols

Article abstract of DOI:10.1016/j.bmc.2011.07.053

New derivatives of 1,4-dideoxy-1,4-imino-d-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-d-ribitol (13, IC ₅₀ ～2 μM) and its C₁₈

Full text of DOI:10.1016/j.bmc.2011.07.053

Bioorganic & Medicinal Chemistry 19 (2011) 7720–7727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols
Claudia Bello a, , Giovanna Dal Bello b, , Michele Cea c, , Aimable Nahimana d, Dominique Aubry d,
Anna Garuti c, Giulia Motta c, Eva Moran c, Floriana Fruscione e, Paolo Pronzato b, Francesco Grossi b,
Franco Patrone c, Alberto Ballestrero c, Marc Dupuis a, Bernard Sordat a, Kaspar Zimmermann f,
Jacqueline Loretan f, Markus Wartmann f, Michel A. Duchosal d, Alessio Nencioni c, Pierre Vogel a,
⇑
a Laboratory of Glycochemistry and Asymmetric Synthesis, Swiss Federal Institute of Technology (EPFL), Batochime, CH-1015 Lausanne, Switzerland
b National Cancer Research Institute, 16132 Genoa, Italy
c Department of Internal Medicine, University of Genoa, Italy
d Service and Central Laboratory of Hematology, University Hospital (CHUV), Lausanne, Switzerland
e Department of Experimental Medicine, University of Genoa, Italy
f Novartis, Institutes for Biomedical Research, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
New derivatives of 1,4-dideoxy-1,4-imino-
D
-ribitol have been prepared and evaluated for their cytotox-
Received 26 April 2011
Revised 21 July 2011
Accepted 23 July 2011
Available online 30 July 2011
icity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-
M) and its C18-analogues (IC50 <10 M) are cytotoxic toward SKBR3 (breast cancer)
M) growth of JURKAT cells.
D-
ribitol (13, IC50 ꢀ2
l
l
cells. 13 also inhibits (IC50 ꢀ8
l
Ó 2011 Published by Elsevier Ltd.
Keywords:
Breast cancer
Iminoalditols
JURKAT cells
Oleyl derivative
1. Introduction
anti-cancer activities most probably related to their
dase inhibitory activity.5,6
a-mannosi-
1,4-Dideoxy-1,4-imino-D-ribitol 1 is a natural product with
importantbiologicalactivities. It wasfirstisolated fromtherootbark
of Mulberry tree (Morus Alba L.).1 This compound has been studied in
some details because it is a mimic of furanosides and, consequently,
might interact with carbohydrate-processing enzymes.2 In addition
HO
OH
O
O
C18H37
P
MeO
NMe
OH
3
N
H
1
O O
2
to the glycosidase inhibitory activity, 1,4-dideoxy-1,4-imino-D-ribi-
tol strongly inhibits the activities of eukaryotic DNA polymerases
with almost no activity on prokaryotic DNA polymerases, nor on
DNA metabolic enzymes.3 These properties suggested a potential
clinical application of 1 as anti-HIV agent.4 This compound has been
proposed to be also an anti-neoplastic and anti-proliferative agent.
Unfortunately, the in vitro studies on cancer cell lines did not show
any cytotoxic activity, probably because of the high polarity of 1 that
hampers the compound to cross cell membranes.
HO
O
O
C
14H29
HO
HO
C8H17
(CH2)8
H
H
H
AcN
H2N
OH
O
3
4
Numerous 2-methyleneamino derivatives of 1 were synthe-
sized in order to increase lypophilicity and cell membrane perme-
ability. This has led to the discovery of compounds with promising
Derivatization of iminosugars with aliphatic side chains has
been extensively studied in order to improve their biological activ-
ity, lipophilicity and bioavailability.7 For instance N-alkylation of
1-deoxynojirimycin has led to compounds of therapeutic interest,
like N-butyl-1-deoxynojirimycin (Zavesca) that has been approved
as a drug for the treatment of Gaucher’s disease,8 or like N-nonyl-
1-deoxynojirimycin, that inhibits hepatitis B virus in cell based as-
⇑
Corresponding author.
E-mail address: pierre.vogel@epfl.ch (P. Vogel).
These authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.07.053

C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
7721
O
O
O
O
HO
O
O
H
H
c), d)
b)
O
a)
O
O
HO
H
O
O
O
HO
g)
O
N
HO
O
O
HO
e)
OH
O
Bn
O
8
6
7
5
O
O
O
O
O
O
O
f)
h)
OH
OH
O
OH
OH
OH
N
N
N
N
H
Boc
Boc
Bn
Boc
9
10
11
12
Scheme 1. Synthesis of 1,4-dideoxy-1,4-imino-
D-ribitol 12. (a) Acetone/DMP (5:1), p-TSAcat, rt, 99% yield; (b) (1) Red-Al (2 equiv), THF/toluene, 0 °C; (2) Rochelle’s salt (sat
aq. solution), rt, 62% yield; (c) MsCl (3 equiv), NEt3 (3.5 equiv), CH2Cl2, 0 °C; (d) Benzylamine, 65 °C, 2d, 77% yield (two steps); (e) AcOH, 55 °C, 50% conversion; (f). Pd(OH)2-
C
cat, H2, Boc2O (3 equiv), MeOH, rt, 73% yield; (g) NaIO4 (1.5 equiv), MeOH/H2O (4:1), 0 °C, 1 h, 90% yield; (h) NaBH4 (1.5 equiv), MeOH, 0 °C, 72% yield.
2. Results and discussion
O
O
HO
OH
2.1. Synthesis of pyrrolidine-fatty alcohol conjugates
a),b)
OH
O
N
Boc
N
H
8
N-tert-Butoxycarbonyl-1,4-dideoxy-2,3-O-isopropylidene-1,4-
D
-ribitol 12 was prepared by reduction of the aldehyde 11,16
7
imino-
synthesized in turn from
12
13
D-gulonic acid c-lactone 5 following a
modified procedure (Scheme 1) based on Fleet’s synthesis.17 The
modifications allowed us to perform the synthesis faster and in
better overall yield, thanks to faster work-up and purification of
the intermediates.
Scheme 2. Synthesis of oleyl pyrrolidine 13. Reagents and conditions: (a) RBr
(1.5 equiv), NaH (1.5 equiv), DMF, 60 °C, 40% yield; (b) TFA/H2O (4:1), 0 °C, 43%
yield.
The targeted compound 13 was obtained through SN2 displace-
ment of oleyl bromide with alcohol 12 followed by deprotection of
both the diol and amine groups by treatment with trifluoroacetic
acid and water (Scheme 2).18
HO
OH
O
O
a), b)
OR
OH
N
H
N
In order to study the role of the aliphatic side-chain in the
mechanism of action of this compound and to out-line a struc-
ture–activity relationship, we prepared a class of derivatives (14–
24, Scheme 3) containing the same pyrrolidine moiety but with
side chains being either fatty alkyl groups of different lengths
(14, 15, 16, 17, 18, 23, 24) and with various degrees of unsaturation
(19, 20), or containing aromatic groups like benzyl (21) or p-phe-
nylbenzyl (22) groups that are found in many drugs.19–21
These compounds were obtained via SN2 displacements by 12 of
the methanesulfonates or bromides derived from the corresponding
fatty alcohols, without solvent, in 50% aqueous solution of NaOH as a
base, and in the presence of tetrabutylammonium iodide as a phase
transfer catalyst.
Boc
12
14-22
a), c), b)
12
23, 24
Scheme 3. Synthesis of pyrrolidine derivatives 14–24 analogues of oleyl derivative
13. Reagents and conditions: (a) RBr or ROMs, Bu4NI, 50% aq NaOH; (b) TFA/H2O
(4:1), 0 °C; (c) Pd(OH)2/charcoal, H2/MeCN. Total yields obtained for 14–24 were
84%(14: R = pent-5-en-1-yl), 38% (15: R = n-C7H15), 26% (16: R = n-C10H21), 28% (17:
R = n-C14H29), 57% (18: R = palmitoleyl = cis-D9 C16H31), 32% (19: R = lino-
leyl = cis,cis-D9 D12 C18H33), 44% (20: R = linolenyl = cis,cis,cis-D9 D12 D15 C18H31),
, , ,
44% (21: R = benzyl), 40% (22: R = p-phenylbenzyl), 82% (23: R = pentyl), 75% (24:
R = octadecyl).
says.9
inhibitory activity toward intestinal isomaltase and their potency
was highly dependent on the alkyl chain length.10 N-Alkylated-
a-1-C-Alkyl-1-deoxynojirimycin derivatives have shown
2.2. Biological results
D
-
Compound 13 and its analogues were evaluated for their ability
to inhibit SKBR3 (HER2+ breast cancer) cell growth, using the MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium) assay, which determines the number
of metabolically active cells, after 72 h exposure. SKBR3 cells were
selected for these experiments based on preliminary data indicat-
ing that this cell line is highly susceptible to the activity of 13 and
related compounds. Namely the IC50 of these drugs is up to 10-
times lower in these cells as compared to other cancer cell lines
tested (including breast, lung, prostate cancer, and glioblastoma
cells).
fagosamine derivatives and N-alkyl-(2R,3S,4R,5S)-2-(hydroxy-
methyl)-5-methylpyrrolidine-3,4-diol derivatives with long alkyl
chains exhibited enhanced cytotoxic activities on a panel of cancer
cell lines compared to their non alkylated progenitors.11
We hypothesized that attaching another natural compound
having lipophilic character to the hydroxymethylene lateral chain
of the dihydroxypyrrolidine core could lead to new pyrrolidine
derivatives (e.g., 13) with potent anticancer activity. Our attention
was attracted by oleyl alcohol that exhibits weak in vitro cytotoxic
activity toward cancer cell lines.12 The oleyl moiety would make
the new compound more lipophilic, allowing cell penetration,
and could also be directly involved in its cytotoxic activity.
Oleyl pyrrolidine 13 killed almost all SKBR3 cells already at a
concentration of 6.25
l
M. An IC50 ꢀ2
lM can be estimated from
In addition, 1,4-dideoxy-1,4-imino-
D-ribitol imitates the polar
moieties of anti-tumor compounds such as Edelfosine (2),13 Jaspine
Figure 1. The results obtained with analogues 14–24 (Fig. 1) al-
lowed us to establish a structure–activity relationship as a function
of the lateral chain length. We observed that the cytotoxic activity
starts with a chain length of more than seven carbon atoms, as the
heptyl derivative 15 was not active while the decyl 16 already
B (3)14 or oleyl 2-acetamido-2-deoxy-
a-D-glucopyranosides (e.g.,
4),15 while the oleyl group would have the same role than the less
polar side-chain of the latter cytotoxic agents. This might lead to
novel anti-cancer agents.

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C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
Figure 1. Viability assays of compounds 13–23 (left) and 18–24 (right, 13 is used as reference) on SKBR3 breast cancer cell lines after 72 h exposure. Results are presented as
means of duplicate wells.
showed some activity, although much weaker than that of 13
(Fig. 1).
of acute T-cell lymphoblastic leukemia) (Fig. 2). Apoptotic cells
were quantified by staining with annexin-V and 7-ADD and flow
cytometric analysis. Cell viability was monitored using Trypan blue
assay.
The compounds have a similar effect on both solid and hemato-
logical malignancies: indeed, SKBR3 cells and JURKAT cells were
sensitive to both compounds, the most active being 13 (IC50
With 18 that has a chain length of sixteen carbon atoms the
cytotoxic activity was almost the same as that of the oleyl deriva-
tive 13 (Fig. 1). These results indicate that the anti-cancer activity
of the new compounds increase with the chain length (best activ-
ities with minimum 14 carbon atoms), in accordance with the
hypothesis that a higher lypophilicity would help the transport
through the cytoplasmic membrane or an incrustation of the
compound in the membrane.
ꢀ8
l
M, estimated from Fig. 2), while 22 killed all the cells only
at a concentration of 100
lM.
Compound 13 induced time-dependent cell death in JURKAT
The comparison between the results obtained with the oleyl
compound 13 and that with compounds bearing linoleyl 19, linole-
nyl 20 and octadecyl 24 pointed out that in the absence of unsat-
uration or in the presence of two or three double bonds the
cytotoxicity was slightly reduced (Fig. 1).This suggests that the
double bond could play a role, even if not determinant, in the can-
cer cell growth inhibitory activity of these compounds.
The presence of an aromatic moiety instead of the aliphatic side
chain lowered the cytotoxic activity: the benzyl derivative 21 did
not kill cancer cells even at high concentrations, while the p-phe-
nylbenzyl analogue 22 exhibited a moderate effect (Fig. 1) that is
cells, as shown in the time course analysis of cell death induced
by 10 lM of 13 on JURKAT cells (Fig. 3).
In a preliminary assay compound 13 was evaluated on normal
hematopoietic progenitorcells (HPC) and the results were compared
to those obtained toward JURKAT cells (Fig. 3, material and methods
ESI). This comparison disclosed that compound 13 inducescell death
in normal cells to a lower extent than in cancer cells, or, at least, that
the cell death is induced more slowly in normal cells than in cancer
cells. In fact, after 24 h of exposure to 10 lM of 13, the percentage of
cell death in JURKAT cells was 100 (Fig. 3A, fourth bar), 60% were in
the late apoptotic phase (black part of the histogram) and 40% in the
early apoptotic phase (white part of the histogram), whereas under
the same conditions the percentage of cell death was 60 in normal
cells (Fig. 3B, second bar), 20% of CD34+CD38low cells were in the
early apoptotic phase and 40% in the late apoptotic phase.
Further studies will be performed in order to confirm these
encouraging observations and to have a better insight into the selec-
tivity of compound 13 and its analogues.
in the same range of that displayed by the
a-mannosidase inhibi-
tors we described in a previous work.6
Contrary to 13 and its analogues 14–21 and 23–24 that do not
inhibit -mannosidase from jack beans (results not shown), 22
shows a moderate -mannosidase inhibitory activity. It is thus
possible that the weak cytotoxic activity of 22 arises from its
a
a
a
-
mannosidase inhibitory activity (as thought to be the case with
other dihydroxypyrrolidine derivatives we reported recently6).
This is not the case for 13.
The mechanism of action of our compounds has not been estab-
lished yet. As a-mannosidase from Jack bean was not inhibited by
In order to compare the effect of the drugs on solid and hema-
tological malignancies, the cytotoxicity of compounds 13 and 22
was also evaluated toward JURKAT cells (a well-established model
13 it is possible that human a-mannosidases, key enzymes of the
N-glycosidation pathway in cells,22 might not be the targets of
13 (and its O-alkyl derivatives).
Figure 2. Sensitivity of JURKAT cells toward compounds 22 (left) and 13 (right). The
percentage of early apoptotic cells (annexin V+7AADꢁ) are shown as white columns
and that of late apoptotic cells (annexin V+7AAD+) are shown as solid black
columns.
Figure 3. (A) 10 lM 13 induces cell death in JURKAT cells in a time dependent
manner and (B, measured after 24 h) in a less extent in normal hematopoietic
progenitor cells (HPC).

C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
7723
series of compounds are now in course and, together with the anal-
ysis of their toxicity/selectivity, will be discussed in a forthcoming
report.
3. Conclusions
In summary, we have prepared a library of new 5-O-alkyl and 5-
O-aryl 1,4-imino-1,4-dideoxy-D-ribitols. These compounds are ac-
tive as inhibitors of cancer cell growth; their activity depends of
the length of the alkyl side chain. The oleyl derivative 13 and its
C-18 analogues 19, 20 and 24 exhibited the most potent anti-can-
cer activity, with 13 being the most efficient in killing tumor cells.
These results indicate an active role of the side chain and of the
number and position of the insaturations in determining the bio-
logical properties of this class of compounds. Studies on the mech-
anism of action are now in course, in order to establish if they act
via the same pathway than Edelfosine 2, Jaspine B 3 or glycosides
of type 4.
Figure 4. Viability assays of oleyl alcohol on SKBR3 and on JURKAT cells and of 1,4-
dideoxy-1,4-imino-
wells.
D-ribitol 1 on SKBR3 cancer cells. Results are means of triplicata
Jurkat
PBMC
100
80
60
40
20
0
100
80
60
40
20
0
4. Experimental
4.1. General methods
0
10
0
10
All commercially available reagents (Fluka, Aldrich, Acros) were
used without further purification. Technical solvents were used for
extraction without any purification. For reactions requiring anh.
conditions, dry solvents were bought (Fluka) or filtered prior to
use (Innovative Technology). In absence of any particular notifica-
tion, experiments were carried out under argon atmosphere. Reac-
tions were monitored by thin layer chromatography (Merck silica
gel 60F254 plates). Revelation was carried out by UV light
(254 nm) and KMnO4 [MnO4 (3 g), K2CO3 (20 g, AcOH (0.25 ml)
and H2O (300 ml)] or Pancaldi [(NH4)6Mo4 (21 g), Ce(SO4)2 (1 g),
H2SO4 (31 ml) and H2O (470 ml)] reagents. Purifications were per-
formed by flash chromatography (Fluka silica gel 60, 230–400
mesh, 0.04–0.063 mm). 1H NMR spectra were recorded on Bruker
ARX-400 and DPX-400 spectrometers at 400 MHz, on Bruker
Avance-500 at 500 MHz and on Bruker AvanceII-800 at 800 MHz.
The signals of residual solvents were used as reference (MeOH-
d4: 3.32 ppm, CDCl3: 7.27 ppm). Coupling constants are given in
Hz. When necessary, the 1H-signal assignments were confirmed
by COSY-45 or by analogy with spectra of similar compounds.
13C NMR spectra were recorded on Bruker ARX-400 and DPX-400
spectrometers at 101 MHz, on Bruker Avance-500 at 125 MHz
and on Bruker Avance II-800 at 200 MHz. The signals of residual
solvents were used as reference (MeOH-d4: 49 ppm, CDCl3:
77 ppm). When necessary, the 13C-signals assignments were con-
firmed by HSQC spectra. IR spectra were recorded on Perkin Elmer
Paragon 1000 FT-IR spectrometer or on Perkin Elmer Spectrum One
FT-IR spectrometer. UV spectra were recorded on a Perkin Elmer
Lambda 10 UV/VIS spectrometer. Optical rotations were recorded
at 25 °C on a Jasco P-1020 polarimeter. Mass spectra: GC-IE/CI/MS
Concentration of 1/µM
Concentration of 1/µM
Figure 5. Sensitivity of JURKAT cells and peripheral blood mononuclear cells
toward compounds 1,4-dideoxy-1,4-imino- -ribitol 1. The percentage of early
D
apoptotic cells (annexin V+7AADꢁ) are shown as white columns and that of late
apoptotic cells (annexin V+7AAD+) are shown as solid black columns.
Preliminary assays showed that compound 13 is an effective
cell cycle modulator: the treatment of SKBR3 cells with 13 results
in inhibition of cell growth accompanied by enhanced accumula-
tion of cells in the G0/G1 phase of the cell cycle and reduction in
S phase. Higher concentrations of 13 led to cell demise with DNA
fragmentation and appearance of hypodyploid cell nuclei. The
cytotoxic activity of the drug was detected after 6–12 h of incubat-
ing and was initially characterized by extensive intracellular
vacuolization.
Compounds 13, 17–20 and 22 have been tested on 15 Ba/F3 cells
models whose proliferation depends on the engineered expression
of specific tyrosine kinases.23 None of the compounds reproducibly
achieved half-maximal suppression of cell proliferation (resazurin
sodium salt reducing ability, determined after 48 h of potential
drug incubation, 9 point IC50 curves determined in three indepen-
dent experiments at concentrations below 10 lM). Thus 13 and
analogues can be considered as devoid of relevant inhibitory activ-
ity against the respective kinases as assessed in the Ba/F3 model.
However, as the Ba/F3 model represents a cellular system, one
can deduce that compound 13 (as well as 17–20, 22) does not show
unspecific cytotoxity at concentrations lower than 10
Interestingly, we found that neither oleyl alcohol nor 1,4-dide-
oxy-1,4-imino- -ribitol alone exhibited significant cytotoxic activ-
ity in our cellular models (Figs. 4 and 5). Thus the activity of 13 and
its analogues appears to rely on both 1,4-dideoxy-1,4-imino- -ribi-
lM.
D
experiments were perfomed by Francisco Sepulveda with
a
1200L instrument (Varian) coupled to a GC CP-3800 (Varian).
ESI-TOF-MS experiments were performed on a Q-Tof Ultima mass
spectrometer (Waters) fitted with a standard Z-spray ion source
and operated in the positive ionization mode. MALDI-TOF-MS
experiments were performed with an AXIMA CFR-Plus instrument
(Shimadzu) operated in the positive reflectron ionization mode.
D
tol and amphiphilic tail, although, again, the reason for this
requirement remains to be determined.
With respect to the mechanism of action of 13 and its analogues
an important feature has to be taken into account: as they are
amphiphilic, they could have detergent activity,24 causing cell
death through membrane disruption.19b–f,25 Although not all solu-
ble amphiphiles have comparable detergent proprieties, we cannot
exclude at this stage that our compounds can have this activity.
Further studies have to be done in order to determine their deter-
gent power, as it has been done, for example, for Edelfosine.13 More
exhaustive studies, about the mechanism of action of these new
4.2. General procedure 1: SN2 reactions
Tetrabutylammonium iodide (TBI: 1–1.4 equiv) was added to R-
X (5.2 equiv or more)and the mixture was stirred at 25 °C for 20 min.
N-tert-Butoxycarbonyl-1,4-dideoxy-2,3-O-isopro
pylidene-1,4-

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C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
imino-
D
-ribitol 12 then NaOH (0.11–0.3 ml of a 50% aqueous solu-
(CH3CN/NH4OH 12: 1) gave 13 (0.021 g, 0.06 mmol, 43% yield) as a
tion, 4.5–5 equiv) were added and the solution was stirred for 5 h
at 25 °C (reaction monitored by TLC, petroleum ether/ethyl acetate
1:4). Ethyl acetate (5 ml) and water (5 ml) were added and the
phases were separated. The aqueous phase was extracted with
ethyl acetate (3 ꢂ 5 ml), the collected organic layers were washed
with brine (5 ml) and dried (MgSO4). Solvent evaporation in vacuo
and flash chromatography gave the pure product.
white foam.
+13; ½
25
577
25
435
25
½
a 2D5
ꢃ
aꢃ
+4; ½
a
ꢃ
+16; ½
aꢃ
+24 (c = 0.076, MeOH). IR
405
(neat): 3346, 2920, 2851, 1664, 1203, 1185, 1138, 1096, 850,
798, 723. 1H NMR (800 MHz, MeOH-d4, ol = oleyl): d 5.35 (m, 2H,
H–C(9) and H–C(10) ol) 4.27 (m, 1H, H–C(4)) 4.12 (m, 1H, H–
C(3)) 3.77 (dd, 2J = 10.5, 3J = 2.3, 1H, HH–C(6)) 3.66 (dd, 2J = 10.5,
3J = 6.4, 1H, HHC-(6)) 3.62 (m, 1H, H–C(2)) 3.59 (m, 1H, HHC(1)
ol) 3.53 (m, 1H, HHC(1) ol) 3.27 (dd, 2J = 14.6, 3J = 10.1, 1H,
HHC(5)) 3.23 (dd, 2J = 14.6, 3J = 7.4, 1H, HHC(5)) 2.06 (m, 4H, H2-
C(8) and H2-C(11) ol) 1.64 (m, 2H, H2C(2) ol) 1.35 (m, 22 H, C(2)-
(CH2)5-C(8) and C(11)-(CH2)6-C(18) ol) 0.93 (t, 3J = 7.1, 3H, CH3
ol). 13C NMR (200 MHz, MeOH-d4, ol = oleyl): d: 130.9 (C(9) ol)
130.8 (C(10) ol) 73.5 (C(3)) 72.8 (C(1) ol) 71.2 (C(4)) 68.6 (C(2))
62.3 (C(6)) 51.2 (C(5)) 33.1 (CH2 ol) 30.3 (CH2 ol) 30.8 (CH2 ol)
30.6 (CH2 ol) 30.6 (CH2 ol) 30.6 (CH2 ol) 30.4 (CH2 ol) 30.3 (CH2
ol) 28.1 (CH2 ol) 28.1 (CH2 ol) 27.2 (CH2 ol) 23.7 (CH2 ol) 14.4
C(18) ol). HR-MALDI-TOF-MS: calcld for C23H45NO3: 384.3477,
found 384.3473 ([M+H]+).
4.3. General procedure 2: deprotection of Boc and acetonides
A 0.3–0.5 M solution of the protected pyrrolidine-fatty alcohol
conjugate in cold trifluoroacetic acid was stirred for 10 min at
0 °C. Water (0.05 ml) was added and the solution was stirred at
0 °C for 10 min, then at room temperature until consumption of
the starting material (monitored by TLC, pertroleum ether/ ethyl
acetate 5:1 and CH2Cl2/MeOH 9:1 with 1% NH4OH 25% aq). The sol-
vent was evaporated in vacuo and the crude was neutralized with
25% aqueous solution of NH4OH (0.035 ml). Solvent evaporation
and flash chromatography gave the pure products.
4.3.2. 1,4-Dideoxy-5-O-(pent-5-en-1-yl)-1,4-imino-D-ribitol 14
General procedure 1. TBI: 0.137 g, 0.37 mmol, R–X: 1-bromo-5-
pentene (0.5 ml, 0.412 g, 1.41 mmol, 5.2 equiv), 12: 0.101 g,
0.37 mmol, NaOH: 0.15 ml. Pure N-tert-butoxycarbonyl-1,4-dide-
4.3.1. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-
ribitol 13
N-Benzyl-1,4-dideoxy-2,3-O-isopropylidene-1,4-imino-D-allitol
oxy-2,3-O-isopropylidene-5-O-(pent-5-en-1-yl)-1,4-imino-D-ribi-
926 (1.005 g, 3.42 mmol) was dissolved in dry methanol (35 ml),
Boc2O (2.378 g, 12.8 mmol, 2 equiv) was added and then
Pd(OH)2-C as catalyst under Argon atmosphere. The mixture was
stirred under H2 atmosphere for 3 h. The catalyst was filtered off
on a Celite pad and the product was purified by flash chromatog-
raphy (diethyl ether/petroleum ether 4: 1 to diethyl ether 100%)
tol was obtained (0.124 g, 0.35 mmol, 96% yield) as a colourless
oil.
Deprotection performed following the general procedure 2: pro-
tected 14: 0.072 g, 0.21 mmol, TFA: 0.45 ml. Pure 14 (0.037 g,
0.18 mmol, 88% yield) was obtained as a white foam after flash col-
umn chromatography on silica gel (CH2Cl2/MeOH 87:13, 1% NH3 aq).
25
577
25
435
25
405
½
a 2D5
ꢃ
+35; ½
a
ꢃ
+29; ½
aꢃ
+56; ½
a
ꢃ
+68 (c = 0.11, MeOH). IR
giving
dene-1,4-imino-
white solid.
N-tert-butyloxycarbonyl-1,4-dideoxy-2,3-O-isopropyli-
D
-allitol 1026 (0.474 g, 1.56 mmol, 46% yield) as
(neat): 3488, 3269, 2940, 2913, 2865, 2506, 1770, 1316, 1191,
1079, 873, 576. 1H NMR (400 MHz, MeOH-d4, pent = pentenyl): d
5.84 (ddt, 3J = 10.9, 3J = 6.7, 3J = 6.7, H–C(4) pent) 5.03 (ddd,
2J = 17.1, 3J = 3.5, 4J = 1.7, 1H, Htrans-C(5) pent) 4.96 (bd, 3J = 11.2,
1H, Hcis-C(5) pent) 4.05 (m, 1H, H–C(4)) 3.81 (dd, 2J = 7.1, 3J = 5.3,
1H, H–C(3)) 3.59 (dd, 2J = 9.8, 3J = 3.7, HH–C(6)) 3.48–3.52 (m, 3H,
H2-C(1) pent and HH–C(6)) 3.18 (dd, 2J = 12.0, 3J = 5.4, 1H, HH–
C(5)) 3.09 (m, 1H, H–C(2)) 2.81 (dd, 2J = 12.0, 3J = 3.9, 1H, HH–
C(5)) 2.13 (m, 2H, H2 -C(3) pent) 1.68 (m, 2H, H2-C(2) pent). 13C
NMR (101 MHz, MeOH-d4, pent = pentenyl): d 139.5 (d, 1J = 192,
C(4) pent) 115.2 (t, 1J = 154, C(5) pent) 74.6 (d, 1J = 144, C(4))
72.5 (d, 1J = 154, C(3)) 71.8 (t, 1J = 144, H2C-C(2)) 71.7 (t, 1J = 144,
C(1) pent) 63.3 (d, 1J = 138, C(2)) 52.4 (t, 1J = 139, C(5)) 31.41 (t,
1J = 145, C(2) pent) 30.0 (t, 1J = 125, C(3) pent). HR-ESI-TOF-MS:
calcd for C10H19NO3: 202.1438, found: 202.1434 ([M+H]+).
NaIO4 (0.53 g, 2.47 mmol, 2.8 equiv) was added to a solution of
10 (0.261 g, 0.86 mmol) in methanol/water (6.7 ml, 4:1) at 0 °C and
the solution was stirred at 0 °C for 1 h. The solution was poured
into water and ethyl acetate was added. The two phases were sep-
arated and the aqueous phase was extracted twice with ethyl ace-
tate. The combined organic phases were washed with brine, dried
with MgSO4 and the solvent was evaporated in vacuo giving pure
1117 (0.209 g, 0.77 mmol, 90% yield) that was used without any
further purification.
NaBH4 (0.031 g, 0.8 mmol, 1.6 equiv) was added portionwise to
a solution of aldehyde 11 (0.145 g, 0.5 mmol) in methanol (1.5 ml)
at 0 °C. The solution was stirred for 1 h at 0 °C, cold water was
added (0.5 ml) and then ethyl acetate (1 ml). The two phases were
separated, the aqueous phase was extracted twice with ethyl
acetate (2 ꢂ 0.5 ml) and the collected organic layers were washed
with brine, dried with MgSO4 and the solvent was evaporated in
vacuo to afford N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-isopro-
4.3.3. 1,4-Dideoxy-5-O-heptyl-1,4-imino-D-ribitol 15
General procedure 1. TBI: 0.164 g, 0.44 mmol, 1 equiv, R-X: hep-
tyl methanesulfonate (0.5 ml, excess), 12: 0.10 g, 0.37 mmol,
NaOH: 0.15 ml. Pure N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-
pylidene-1,4-imino-D
-ribitol 1217 (0.099 g, 0.36 mmol, 72% yield).
12 (0.099 g, 0.36 mmol) was dissolved in DMF (2 ml), NaH
(0.022 g (60% in oil), 1.5 equiv) and then oleyl bromide
(0.149 g, 0.45 mmol, 1.25 equiv) was added and the mixture
was stirred at room temperature for 1 h, then at 60 °C for
18 h. Methanol was added, then water (2 ml) and the solution
was extracted with diethyl ether (3 ꢂ 2 ml). The combined or-
ganic phases were washed successively with water and brine,
dried (MgSO4) and the solvent was evaporated in vacuo. After
flash chromatography (petroleum ether/AcOEt 7:1) N-tert-butox-
ycarbonyl-1,4-dideoxy-2,3-O-isopropylidene-5-O-[(9Z)-octadec-9-
en-1-yl]-1,4-imino-D-ribitol was obtained (0.075 g, 0.14 mmol,
40% yield) as a colourless oil.
Deprotection of Boc and acetonide protecting groups (TFA/H2O,
4:1 vol: vol, 0 °C for 3 h) and purification by flash chromatography
isopropylidene-5-O-heptyl-1,4-imino-
D
-ribitol
was
obtained
(0.070 g, 0.19 mmol, 51% yield) as a colourless oil after flash col-
umn chromatography on silica gel (petroleum ether/ethyl acetate
100:0 ? 95:5 ? 90:10). Deprotection performed following the
general procedure 2: protected 15: 0.070 g, 0.19 mmol, TFA:
0.45 ml. Pure 15 (0.033 g, 0.14 mmol, 75% yield) was obtained as
white foam after flash column chromatography on silica gel
CH2Cl2/MeOH 93:7, 1% NH3 aq).
25
577
25
435
25
405
½
a 2D5
ꢃ
+40; ½
a
ꢃ
+43; ½
aꢃ
+74; ½
a
ꢃ
+84 (c = 0.0605, MeOH) IR
(pure): 3484, 3267, 2917, 2870, 1081, 873, 658. 1H NMR (400 MHz,
MeOH-d4, hep = heptyl): d 4.04 (m, 1H, H–C(4)) 3.79 (dd, 3J = 5.4,
3J = 6.9, 1H, H–C(3)) 3.59 (dd, 3J = 3.6, 2J = 9.8, 1H, HH–C(6)) 3.46–
3.51 (m, 3H, H2-C(1) hep and HH–C(6)) 3.15 (dd, 3J = 5.4,
2J = 12.0, 1H, HH–C(5)) 3.08 (m, 1H, H–C(2)) 2.80 (dd, 3J = 4.0,

C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
7725
2J = 12.0, 1H, HH–C(5)) 1.59 (m, 2H, H2-C(2) hep) 1.32–1.39 (m, 8H,
4.3.6. 1,4-Dideoxy-5-O-[(9Z)-hexadec-9-en-1-yl]-1,4-imino-
ribitol 18
General procedure 1. TBI: 0.132 g, 0.36 mmol, 1.3 equiv, R-X: pal-
mitoleyl methanesulfonate (0.5 ml excess), 12: 0.074 g, 0.27 mmol,
NaOH: 0.11 ml. Pure N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-
D-
C(2)-(CH2)4-C(7) hep) 0.91 (t, 3J = 6.8, 3H, H3-C(7) hep). 13C NMR
(101 MHz, MeOH-d4): d 74.7 (d, 1J = 147, C(3)) 72.6 (t, 1J = 139,
C(6)) 72.5 (d, 1J = 149, C(4)) 71.8 (t, 1J = 142, C(1) hep) 63.3 (d,
1J = 140, C(2)) 52.4 (t, 1J = 140, C(5)) 33.0 (t, 1J = 124, C(2) hep)
30.7 (t, 1J = 123, C(3) hep) 30.3 (t, 1J = 124, C(4) hep) 29.2 (t,
1J = 126, C(5) hep) 23.7 (t, 1J = 124, C(6)hep) 14.4 (q, 1J = 124, C(7)
hep). HR-ESI-MS: calcd for C12H25NO3: 232.1907, found 232.1914
([M+H]+).
isopropylidene-5-O-[(9Z)-hexadec-9-en-1-yl]-1,4-imino-D-ribitol
was obtained (0.090 g, 0.18 mmol, 67% yield) as a colourless oil
(flash column chromatography: PE/AcOEt 7:1). Deprotection per-
formed following the general procedure 2: protected 18: 0.090 g,
0.18 mmol TFA: 0.34 ml. Pure 18 (0.054 g, 0.15 mmol, 84% yield)
was obtained as a white foam after flash column chromatography
4.3.4. 1,4-Dideoxy-5-O-decyl-1,4-imino-D-ribitol 16
25
on silica gel (CH2Cl2/MeOH 9:1, 1% NH3 aq). ½a D25
ꢃ
+21; ½
a
ꢃ
+50;
577
General procedure 1. TBI: 0.163 g, 0.44 mmol, 1.2 equiv, R–X: de-
cyl bromide (0.5 ml, excess), 12: 0.098 g, 0.36 mmol; NaOH: 0.2 ml.
Pure N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-isopropylidene-5-
O-decyl-1,4-imino-D-ribitol was obtained (0.050 g, 0.12 mmol,
34% yield) as a colourless oil (flash column chromatography: petro-
leum ether/ ethyl acetate 9:1).
25
435
25
405
½
aꢃ
+46; ½
a
ꢃ
+48 (c = 0.1160, CHCl3). IR (pure): 3313, 3257,
2913, 2848, 1080, 1034, 874. 1H NMR (400 MHz, CDCl3, pal = pal-
mitoleyl): d 5.44 (m, 2H, H–C(9) and H–C(10) pal) 4.14 (m, 1H, H–
C(4)) 3.89 (m, 1H, H–C(3)) 3.69 (br s 3H, 2 xH–O and H-N) 3.57
(dd, 2J = 9.7, 3J = 4.5, 1H, HHC(6)) 3.53 (dd, 2J = 9.7, 3J = 5.2, 1H,
HHC(6)) 3.44 (t, 3J = 6.7, 2H, H2C(1) pal) 3.24 (dd, 2J = 12.0,
3J = 5.1, 1H, HHC(5)) 3.17 (m, 1H, H–C(2)) 2.96 (dd, 2J = 9.7,
3J = 3.0, 1H, HHC(5)) 2.00 (m, 4H, H2C(8) and H2C(11) pal) 1.55
(m, 2H, H2C(2) pal) 1.28 (br s, 18H, C(2)-(CH2)5-C(8) and C(11)-
(CH2)4-C(16) pal) 0.88 (t, 3J = 6.8, 3H, H3C(16) pal). 13C NMR
(101 MHz, CDCl3, pal = palmitoleyl): d 129.9 (d, 1J = 153, C(9)
pal) 129.8 (d, 1J = 153, C(10) pal) 74.1 (d, 1J = 146, C(3)) 71.7 (t,
1J = 139, C(1) pal) 71.4 (d, 1J = 150, C(4)) 70.8 (t, 1J = 142, C(6))
62.1 (d, 1J = 138, C(2)) 51.8 (t, 1J = 138, C(5)) 31.7 (t, 1J = 127,
H2C pal) 29.7 (t, 1J = 125, H2C pal) 29.7 (t, 1J = 124, H2C pal) 29.5
(t, 1J = 124, H2C pal) 29.5 (t, 1J = 124, H2C pal) 29.4 (t, 1J = 124,
H2C pal) 29.3 (t, 1J = 124, H2C pal) 28.9 (t, 1J = 124, H2C pal) 27.2
(t, 1J = 124, H2C pal) 27.2 (t, 1J = 124, H2C pal) 26.1 (t, 1J = 125,
H2C pal) 22.6 (t, 1J = 124, H2C pal) 14.1 (q, 1J = 124, C(16) pal).
HR-ESI-TOF-MS: calcd for C21H41NO3: 356.3159, found 356.2715
([M+H]+).
Deprotection performed following the general procedure 2:
protected 16: 0.050 g, 0.12 mmol, TFA: 0.5 ml. Pure 16
(0.0252 g, 0.09 mmol, 77% yield) was obtained as a white foam
after flash column chromatography on silica gel (CH2Cl2/MeOH
9:1, 1% NH3 aq).
25
577
25
435
25
405
½
a 2D5
ꢃ
+31; ½
a
ꢃ
+23; ½
aꢃ
+57; ½
aꢃ
+77 (c = 0.0575, MeOH). IR
(pure): 3511, 3268, 2917, 2850, 1078, 982, 874. 1H NMR (400 MHz,
CDCl3, dec = decyl): d 4.12 (m, 1H, H–C(4)) 3.86 (m, 1H, H–C(3))
3.54 (m, 2H, H2-C-C(2)) 3.44 (t, 3J = 6.7, 2H, H2-C(1) dec) 3.28 (m,
2H, 2ꢂ H–O) 3.22 (dd, 2J = 12.0, 3J = 5.1, 1H, HH–C(5)) 3.11 (m,
1H, H–C(2)) 2.90 (dd, 2J = 12.0, 3J = 3.1, 1H, HH–C(5)) 1.55 (m, 2H,
H2-C(2) dec) 1.26 (br s 14H, C(2)-(CH2)7-C(10) dec) 0.88 (t,
3J = 6.7, 3H, H2-C(10) dec). 13C NMR (101 MHz, CDCl3): d 74.4 (d,
1J = 143, C(3)) 71.6 (t, 1J = 139, C(1) dec) 71.5 (d, 1J = 149, C(4))
71.2 (t, 1J = 139, H2C-C(2)) 62.2 (d, 1J = 139, C(2)) 52.0 (t, 1J = 139,
C(5)) 31.9 (t, 1J = 122, C(2) dec) 29.6 (t, 1J = 124, C(4) and C(3)
dec) 29.6 (t, 1J = 125, C(5) dec) 29.5 (t, 1J = 125, C(6) dec) 29.3 (t,
1J = 124, C(7) dec) 26.1 (t, 1J = 124, C(8) dec) 22.7 (t, 1J = 124, C(9)
dec) 14.1 (q, 1J = 124, C(10) dec). HR-ESI-TOF-MS: calcd for
4.3.7. 1,4-Dideoxy-5-O-[(9Z,12Z)-octadeca-9,12-dien-1-yl]-1,4-
imino-D-ribitol 19
General procedure 1. TBI: 0.153 g, 0.41 mmol, 1.2 equiv, R-X: lino-
leyl methanesulfonate (0.5 ml, excess), 12: 0.090 g, 0.33 mmol,
NaOH: 0.12 ml. Pure N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-iso-
C
15H31NO3: 274.2382, found 274.2381 ([M+H]+).
4.3.5. 1,4-Dideoxy-5-O-tetradecyl-1,4-imino- -ribitol 17
D
propylidene-5-O-[(9Z,12Z)-octadeca-9,12-dien-1-yl]-1,4-imino-D-
General procedure 1. TBI: 0.100 g, 0.27 mmol, 1 equiv, R-X: tetra-
decyl methanesulfonate (0.412 g, 1.41 mmol, 5.2 equiv), CH3CN
(0.5 ml) 12: 0.073 g, 0.27 mmol, NaOH: 0.25 ml. Pure N-tert-butox-
ycarbonyl-1,4-dideoxy-2,3-O-isopropylidene-5-O-tetradecyl-1,4-
ribitol was obtained (0.086 g, 0.17 mmol, 52% yield) as a colourless
oil (flash column chromatography: PE/AcOEt 7:1). Deprotection per-
formed following the general procedure 2: protected 19: 0.090 g,
0.18 mmol TFA: 0.34 ml. Pure 19 (0.039 g, 0.10 mmol, 61% yield)
imino-D-ribitol was obtained (0.046 g, 0.1 mmol, 37% yield) as a
was obtained as a white foam after flash column chromatography
25
on silica gel (CH2Cl2/MeOH 9:1, 1% NH3 aq). ½a D25
ꢃ
+35 ½ ꢃ
a
colourless oil (flash column chromatography: PE/ AcOEt 93:7).
Deprotection performed following the general procedure 2: pro-
tected 17: 0.046 g, 0.1 mmol, TFA: 0.4 ml. Pure 17 (0.021 g,
0.063 mmol, 63% yield) was obtained as a white foam after flash
column chromatography on silica gel (CH2Cl2/MeOH 9:1, 1% NH3
577
25
435
½
aꢃ
½
a 2D5
(c = 0.1460, MeOH) IR (pure): 3293, 3202, 2918, 2850,
ꢃ
1094, 1079, 981, 827, 720. 1H NMR (400 MHz, CDCl3, lin = linoleyl):
d 5.39–5.30 (m, 4H, H–C(9), H–C(10), H–C(12) and H–C(13) lin) 4.28
(m, 3H, 2ꢂ H–O and H-N) 4.18 (m, 1H, H–C(2)) 3.96 (m, 1H, H–C(3))
3.61 (dd, 2J = 9.7, 3J = 4.1, 1H, HHC(5)) 3.55 (dd, 2J = 9.7, 3J = 5.1, 1H,
HHC(5)) 3.45 (m, 2H, H2C(1) lin) 3.29–3.25 (m, 2H, H–C(4) and
HHC(1)) 3.08 (bd, dd, 2J = 10.2, 1H, HHC(1)) 2.77 (m, 2H, H2C(11)
lin) 2.05 (m, 4H, H2C(8) and H2C(14) lin) 1.54 ((m, 2H, H2C(2) lin)
1.30 (m, 16H, C(2)-(CH2)5-C(8) and C(14)-(CH2)3-C(18) lin) 0.89 (t,
3J = 6.8, 3H, H3C(18) lin). 13C NMR (101 MHz, CDCl3, lin = linoleyl):
d 130.2 (d, 1J = 161, C(9) lin) 130.1 (d, 1J = 161, C(10) lin) 128.0 (d,
1J = 149, C(12) lin) 127.9 (d, 1J = 149, C(13) lin) 74.0 (d, 1J = 143,
C(3)) 71.7 (t, 1J = 142, C(1) lin) 71.2 (d, 1J = 149, C(2)) 70.2 (t,
1J = 143, C(5)) 62.0 (d, 1J = 126, C(4)) 51.5 (t, 1J = 146, C(1)) 31.5 (t,
H2C lin) 29.7 (t, H2C lin) 29.5 (t, 1J = 125, H2C lin) 29.5 (t, 1J = 125,
H2C lin) 29.5 (t, 1J = 125, H2C lin) 29.3 (t, 1J = 120, H2C lin) 29.3 t,
1J = 120, H2C lin) 27.2 (t, H2C lin) 27.2 t, H2C lin) 26.1 (t, 1J = 124,
H2C lin) 25.6 (t, 1J = 125, H2C lin) 22.5 (t, 1J = 117, H2C lin) 14.0 (q,
1J = 123, C(18) lin). HR-ESI-TOF-MS: calcd for C23H43NO3:
382.3316, found 382.3544 ([M+H]+).
25
577
25
435
25
405
aq). ½a 2D5
ꢃ
+27; ½
aꢃ
+25; ½
aꢃ
+50; ½
aꢃ
+65 (c = 0.052, MeOH).
IR (neat): 3417, 3280, 3239, 2917, 2849, 1470, 1376, 1341, 1125,
1094, 832, 718. 1H NMR (400 MHz, CDCl3, td = tetradecyl): d
4.12 (m, 1H, H–C(4)) 3.86 (m, 1H, H–C(3)) 3.49–3.56 (m, 2H,
H2C-C(2)) 3.44 (t, 3J = 6.7, 2H, H–C(1) td) 3.22 (dd, 2J = 12.0,
3J = 5.1, 2H, HHC(5) and H–O) 3.10–3.14 (m, 2H, H–C(2) and H–
O) 2.91 (dd, 2J = 12.0, 3J = 5.1, 1H, HHC(5)) 1.55 (m, 2H, H2C(2)
td) 1.26 (m, 22H, (CH2)11 td) 0.88 (t, 3J = 6.5, 3H, H3C(14) td). 13C
NMR (100 MHz, CDCl3, td = tetradecyl): d 74.4 (d, 1J = 145, C(3))
71.7 (t, 1J = 141, C(1) td) 71.6 (d, 1J = 150, C(4)) 71.3 (t, 1J = 141,
H2C-C(2)) 62.2 (d, 1J = 137, C(2)) 52.0 (t, 1J = 138, C(5)) 31.9 (t,
1J = 124, CH2 td) 29.7 (t, 1J = 124, (CH2)2 td) 29.6 (t, 1J = 124,
(CH2)2 td) 29.6 (t, 1J = 124, CH2 td) 29.6 (t, 1J = 124, CH2 td) 29.6
(t, 1J = 124, CH2 td) 29.3 (t, 1J = 124, CH2 td) 26.1 (t, 1J = 125, CH2
td) 22.7 (t, 1J = 127, CH2 td) 14.1 (q, 1J = 124, C(14) td). HR-ESI-
MS: calcd for C19H39NO3: 330.3003, found: 330.2991 ([M+H]+).

7726
C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
4.3.8. 1,4-Dideoxy-5-O-[(9Z,12Z,15Z)-octadec-9,12,15-trien-1-
yl]-1,4-imino- -ribitol 20
1,4-dideoxy-2,3-O-isopropylidene-5-O-(p-phenylbenzyl)-1,4-imi-
no- -ribitol was obtained (0.080 g, 0.18 mmol, 71% yield) as a col-
D
D
General procedure 1. TBI: 0.136 g, 0.37 mmol, 1.2 equiv, R-X: lin-
olenyl methanesulfonate (0.5 ml, excess), 12: 0.082 g, 0.30 mmol,
NaOH: 0.12 ml. Pure N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-
isopropylidene-5-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl]-
ourless oil after flash column chromatography on silica gel
(petroleum ether/ ethyl acetate 5:1). Deprotection performed fol-
lowing the general procedure 2: protected 22: 0.080 g, 0.18 mmol,
TFA: 0.5 ml. Pure 22 (0.029 g, 0.1 mmol, 56% yield) was obtained
as a white foam after flash column chromatography on silica gel
1,4-imino-D-ribitol was obtained (0.103 g, 0.2 mmol, 67% yield)
25
25
as a colourless oil (flash column chromatography: PE/AcOEt
7:1). Deprotection performed following the general procedure
2: protected 20: 0.103 g, 0.2 mmol TFA: 0.34 ml. Pure 20
(0.048 g, 0.13 mmol, 65% yield) was obtained as a white foam
after flash column chromatography on silica gel (CH2Cl2/MeOH
CH2Cl2/MeOH 9:1, 1% NH3 aq). ½a D25
ꢃ
+31; ½
a
ꢃ
+12; ½
a
ꢃ
+27;
577
435
25
405
½
aꢃ
+33 (c = 0.0515, MeOH). IR (pure): 3437, 3309, 2920, 2881,
2693, 1488, 1452, 1092. 1H NMR (400 MHz, CDCl3, bPh = biphe-
nyl): d 7.58–7.30 (m, 9H, H–C bPh) 4.57 (m, 2H, H2C bPh) 4.12
(m, 1H, H–C(2)) 3.91 (m, 1H, H–C(3)) 3.63 (m, 2H, H2-C(5))
3.25–3.18 (m, 4H, H–C(4), HH–C(1) and 2ꢂ H–O)) 2.92 (bd, 2J
=10.0, 1H, HH–C(1)). 13C NMR (101 MHz, CDCl3, bPh = biphenzyl):
d 140.7 (s, C(4) bPh) 140.7 (s, C(1’) bPh) 136.9 (s, C(1) bPh) 128.8
(d, 1J = 160, CH bPh) 128.2 (d, 1J = 162, CH bPh) 127.3 (d, 1J = 162,
C(4’) bPh) 127.2 (d, 1J = 162, CH bPh) 127.1 (d, 1J = 162, CH bPh)
74.3 (d, 1J = 148, C(2)) 73.1 (t, 1J = 141, H2C-bPh) 71.5 (d,
1J = 152, C(3)) 70.9 (t, 1J = 143, C(5)) 62.3 (d, 1J = 137, C(4)) 52.0
(t, 1J = 138, C(1)). HR-ESI-TOF-MS: calcd for C18H21NO3:
300.1600 found 300.1600 ([M+H]+).
25
577
25
435
25
405
9:1, 1% NH3 aq). ½a D25
ꢃ
+30; ½
aꢃ
+54; ½
aꢃ
+73; ½
a
ꢃ
+84
(c = 0.103, MeOH). IR (neat): 3502, 3277, 3007, 2919, 2851,
1081, 981, 872, 721. 1H NMR (400 MHz, CDCl3, lin = linolenyl):
d
5.42–5.28 (m, 6H, H–C(9), H–C(10) H–C(12), H–C(13), H–
C(15) and H–C(16) lin) 4.14 (m, 1H, H–C(2)) 3.89 (m, 1H, H–
C(3)) 3.73 (br s 3H, 2 xH–O and H-N) 3.57 (dd, 2J = 9.7, 3J = 4.3,
1H, HHC(5)) 3.52 (dd, 2J = 9.7, 3J = 5.3, 1H, HHC(5)) 3.45 (t,
3J = 13.2, 2H, H2C(1) lin) 3.23 (dd, 2J = 12.0, 3J = 4.9, 1H,
HHC(1)) 3.18 (m, 1H, H–C(4)) 2.97 (bd, 2J = 9.7, 1H, HHC(1))
2.80 (m, 4H, H2C(11) and H2C(14) lin) 2.11–2.02 (m, 4H,
H2C(8) and H2C(17) lin) 1.55 (m, 2H, H2C(2) lin) 1.28 (br s
10H, C(2)-(CH2)5-C(8) lin) 0.97 (t, 3J = 7.5, 3H, H3C(18) lin). 13C
NMR (101 MHz, CDCl3, lin = linolenyl): d 131.9 (d, 1J = 152,
C(9) lin) 130.3 (d, 1J = 152, C(10) lin) 128.2 (d, 1J = 154, C(12)
lin) 128.2 (d, 1J = 154, C(13) lin) 127.7 (d, 1J = 154, C(15) lin)
127.1 (d, 1J = 155, C(16) lin) 74.1 (d, 1J = 140, C(3)) 71.6 (t,
1J = 139, C(1) lin) 71.4 (d, 1J = 144, C(2)) 70.7 (t, 1J = 134, C(5))
62.1 (d, 1J = 138, C(4)) 51.7 (t, 1J = 140, C(1)) 29.6 (t, 1J = 123,
H2C lin) 29.5 (t, 1J = 123, H2C lin) 29.5 (t, 1J = 123, H2C lin) 29.4
(t, 1J = 123, H2C lin) 29.2 (t, 1J = 123, H2C lin) 27.2 (t, 1J = 124,
H2C lin) 26.1 (t, 1J = 118, H2C lin) 25.6 (t, 1J = 117, H2C lin) 25.5
(t, 1J = 117, H2C lin) 20.5 (t, 1J = 121, H2C lin) 14.2 (q, 1J = 129,
C(18) lin). HR-ESI-TOF-MS: calcd for C23H41NO3: 380.3159,
found 380.2331 ([M+H]+).
4.3.11. 1,4-Dideoxy-5-O-pentyl-1,4-imino-
N-tert-Butoxycarbonyl-1,4-dideoxy-2,3-O-isopropylidene-5-O-
(pent-5en-1-yl)-1,4-imino- -ribitol (0.051 g, 0.15 mmol) (see
D-ribitol 23
D
preparation of 14) was dissolved in methanol (1.5 ml) and the solu-
tion was degassed with argon. Palladium hydroxide on activated
charcoal (0.08 g, 10% mol) was added and the solution was stirred
under hydrogen atmosphere at 25 °C for 1 h (reaction monitored
by 1H NMR). The suspension was filtered on a Celite pad (eluent:
ethyl acetate) in order to eliminate the catalyst, and the solvent
was evaporated in vacuo, giving N-tert-butoxycarbonyl-1,4-dide-
oxy-2,3-O-isopropylidene-5-O-pentyl-1,4-imino-D-ribitol quanti-
tatively. This product was dissolved in cold trifluoroacetic acid
(0.5 ml) and the solution was stirred at 0 °C for 10 min. Water
(0.07 ml) was added and the solution was stirred at 0 °C for
30 min, then at 25 °C for 1 h (reaction monitored by TLC, petroleum
ether/ethyl acetate 3:1 and CH2Cl2/CH3OH 9:1 (1% aqueous ammo-
nia)). The solvent was evaporated in vacuo and the product was dis-
solved in CH2Cl2 and neutralized with ammonia. After solvent
evaporation in vacuo, the product was purified by flash chromatog-
raphy on silica gel (CH2Cl2/CH3OH 88.5:11.5 (1% aqueous ammo-
nia)). Pure 23 was obtained (0.024 g, 0.13 mmol, 82% yield) as
white foam.
4.3.9. 1,4-Dideoxy-5-O-benzyl-1,4-imino-D-ribitol 21
General procedure 1. TBI: 0.124 g, 0.37 mmol, 1.4 equiv, R-X:
benzyl bromide (0.5 ml, excess), 12: 0.074 g, 0.27 mmol, NaOH:
0.17 ml. Pure N-tert-butoxycarbonyl-1,4-dideoxy-2,3-O-isopropyl-
idene-5-O-benzyl-1,4-imino-D-ribitol was obtained (0.066 g,
0.18 mmol, 68% yield) as a colourless oil after flash column chro-
matography on silica gel (petroleum ether/ ethyl acetate 9:1).
Deprotection performed following the general procedure 2: pro-
tected 21: 0.037 g, 0.10 mmol, TFA: 0.4 ml. Pure 21 (0.014 g,
0.065 mmol, 65% yield) was obtained as a white foam after flash
column chromatography on silica gel CH2Cl2/MeOH 9:1, 1% NH3
25
577
25
435
25
405
½
a 2D5
ꢃ
+40; ½
aꢃ
+43; ½
a
ꢃ
+75; ½
aꢃ
+90 (c = 0.067, MeOH). IR
(pure): 3503, 3269, 2931, 2870, 2812, 1080, 990, 873, 658. 1H
NMR (400 MHz, MeOH-d4, pen = pentyl): d 4.05 (m, 1H, H–C(2))
3.80 (dd, 3J = 7.0, 3J = 5.4, 1H, H–C(3)) 3.59 (dd, 2J = 9.8, 3J = 3.6,
1H, HH–C(5)) 3.46–3.52 (m, 3H, H2-C(1)pen and HH–C(5)) 3.17
(dd, 2J = 12.0, 3J = 5.4, 1H, HH-C(1)) 3.09 (m, 1H, H-C(4)) 2.81 (dd,
2J = 12.0, 3J = 3.9, 1H, HH-C(1)) 1.59 (m, 2H, H2-C(2) pen) 1.30–
1.38 (m, 4H, H2-C(3) and H2-C(4) pen) 0.93 (t, 3J = 7.0, 3H, H3-
C(5) pen). 13C NMR (101 MHz, MeOH-d4, pen = pentyl): d 74.6 (d,
1J = 144, C(2)) 72.5 (d, 1J = 148, C(3)) 72.5 (t, 1J = 148, C(5)) 71.7
(t, 1J = 149, C(1)pen) 63.3 (d, 1J = 139, C(4)) 52.4 (t, 1J = 138, C(1))
30.4 (t, 1J = 123, C(2)pen) 29.5 (t, 1J = 125, C(3) pen) 23.6 (t,
1J = 126, C(14) pen) 14.4 (q, 1J = 124, C(5) pen). HR-ESI-MS: calcd
for C10H21NO3: 204.1594, found 204.1601 ([M+H]+).
25
577
25
435
25
405
aq). ½a 2D5
ꢃ
+57; ½
aꢃ
+52; ½
aꢃ
+94; ½
aꢃ
+109 (c = 0.0635, MeOH)
IR (neat): 3465, 3279, 2948, 2919, 2900, 2871, 2564, 1088, 1020,
728. 1H NMR (400 MHz, MeOH-d4, bn = benzyl): d 7.38–7.28 (m,
5H, H–C bn) 4.56 (m, 2H, H2-C bn) 4.05 (m, 1H, H–C(2)) 3.84 (dd,
3J = 7.0, 3J = 5.4, 1H, H–C(3)) 3.66 (dd, 2J = 9.7, 3J = 3.6, 1H, HH–
C(5)) 3.56 (dd, 2J = 9.7, 3J = 5.5, 1H, HH–C(5)) 3.20–3.12 (m, 2H,
H–C(4) and HH–C(1)) 2.81 (dd, 2J = 12.0, 3J = 3.7, 1H, HHC(1)). 13C
NMR (101 MHz, MeOH-d4, bn = benzyl): d 139.6 (s, C bn) 129.4
(d, 1J = 159, C bn) 128.9 (d, 1J = 158, C bn) 128.7 (d, 1J = 158, C bn)
74.6 (d, 1J = 143, C(2)) 74.3 (t, 1J = 141, H2C bn) 72.5 (d, 1J = 148,
C(3)) 71.3 (t, 1J = 139, H2C(5)) 63.3 (d, 1J = 138, C(4)) 52.3 (t,
1J = 139, C(1)). HR-ESI-TOF-MS: calcd for C12H17NO3: 224.1287,
found 224.1280 ([M+H]+).
4.3.12. 1,4-Dideoxy-5-O-octadecyl-1,4-imino-
N-tert-Butoxycarbonyl-1,4-dideoxy-2,3-O-isopropylidene-5-O-
[(9Z)-octadec-9-en-1-yl]-1,4-imino- -ribitol (0.043 g, 0.08 mmol)
D-ribitol 24
D
4.3.10. 1,4-Dideoxy-5-O-(p-phenylbenzyl)-1,4-imino-
D
-ribitol 22
(see preparation of 13) was dissolved in methanol (1 ml) and the
solution was degassed with argon. Palladium hydroxide on acti-
vated charcoal (0.007 g, 12% mol) was added and the solution
was stirred under hydrogen at 25 °C for 1 h (reaction monitored
General procedure 1. TBI: 0.126 g, 0.34 mmol, 1.3 equiv, R-X: p-
phenylbenzyl bromide (0.5 ml, excess), CH3CN (0.4 ml), 12:
0.070 g, 0.26 mmol, NaOH: 0.35 ml. Pure N-tert-butoxycarbonyl-

C. Bello et al. / Bioorg. Med. Chem. 19 (2011) 7720–7727
7727
by 1H NMR). The suspension was filtered on a Celite pad (eluent:
ethyl acetate) in order to eliminate the catalyst, and the solvent
was evaporated in vacuo, obtaining N-tert-butoxycarbonyl-1,
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4-dideoxy-2,3-O-isopropylidene-5-O-octadecyl-1,4-imino-D-ribitol
quantitatively. This product was dissolved in cold trifluoroacetic
acid (0.32 ml) and the solution was stirred at 0 °C for 10 min.
Water (0.08 ml) was added and the solution was stirred at 0 °C
for 30 min, then at 25 °C for 1 h (reaction monitored by TLC, petro-
leum ether/ethyl acetate 7:1 and CH2Cl2/CH3OH 9:1 (1% aqueous
ammonia)). The solvent was evaporated in vacuo and the product
was dissolved in CH2Cl2 and neutralized with ammonia. After
solvent evaporation in vacuo, the product was purified by flash
chromatography on silica gel (CH2Cl2/CH3OH 9:1 (1% aqueous
ammonia)). Pure 1,4-dideoxy-5-O-octadecyl-1,4-imino-D-ribitol
24 (0.022 mg, 0.06 mmol, 75% yield) was obtained as white foam.
25
577
25
435
25
405
½
a 2D5
ꢃ
+20; ½
aꢃ
+56; ½
a
ꢃ
+70; ½
aꢃ
+80 (c = 0.087, MeOH). IR
(pure): 3294, 2917, 2848, 1470, 1118, 1103, 717. 1H NMR
(400 MHz, Me OH-d4, od = octadecyl): d 4.06 (m, 1H, H-C(2))
3.84 (dd, 2J = 7.2, 3J = 5.0, 1H, H–C(3)) 3.56 (dd, 2J = 10.1, 3J = 3.5,
1H, HH–C(5)) 3.52–3.41 (m, 3H, HHC(1) od, H–C(4) and HH–C(5))
3.20–3.14 (m, 2H, HHC(1) and HHC(1) od) 2.88 (dd, 2J = 12.1,
3J = 3.4, 1H, HHC(1)) 1.55 (m, 2H, H2C(2) od) 1.26 (br s 30 H,
C(2)-(CH2)15-C(18) od) 0.87 (t, 3J = 6.7, 3H, H3C(18) od). 13C NMR
(101 MHz, MeOH-d4, od = octadecyl): d 74.3 (C(3)) 72.6 (C(1) od)
72.2 (C(2)) 70.9 (H2C(5)) 63.1 (C(4)) 52.1 (C(1)) 33.1 (CH2 od)
30.8 ((CH2)10 od) 30.7 (CH2 od) 30.6 (CH2 od) 30.5 (CH2 od) 27.3
(CH2 od) 23.7 (CH2 od) 14.4 (C(14) od). HR-ESI-TOF-MS: calcd for
C
23H47NO3: 386.3629, found: 386.3492 ([M+H]+).
Acknowledgments
This work has been generously supported by the Associazione
Italiana per la ricerca sul cancro (AIRC) by the Swiss National
Science Foundation and the European FP7 – HEALTH-2010-2-4-1-6
(PANACREAS) programme. CB was supported by an EPFL Innogrant;
AN was supported by FBM Interdisciplinary Research Grant and Fon-
dation Dr H Dubois-Ferrière Dinu Lipattic
A. Supplementary data
Supplementary data (viability assays experimental protocols)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2011.07.053.
20. Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, J. J. Med. Chem. 2008, 51, 3661–
3680.
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