Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as α-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies

Article abstract of DOI:10.1016/j.bmc.2010.03.009

Novel α-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group = 4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Full text of DOI:10.1016/j.bmc.2010.03.009

Bioorganic & Medicinal Chemistry 18 (2010) 3320–3334
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as
a-mannosidase inhibitors and with antitumor activities against
hematological and solid malignancies
Claudia Bello a, , Michele Cea c, , Giovanna Dal Bello b, , Anna Garuti c, Ilaria Rocco c, Gabriella Cirmena c,
Eva Moran c, Aimable Nahimana e, Michel A. Duchosal e, Floriana Fruscione d, Paolo Pronzato b,
Francesco Grossi b, Franco Patrone c, Alberto Ballestrero c, Marc Dupuis a, Bernard Sordat a,
Alessio Nencioni c, Pierre Vogel a,
*
a Laboratory of Glycochemistry and Asymmetric Synthesis (LGSA), Ecole Polytechnique Fédérale de Lausanne (EPFL), Batochime, CH-1015 Lausanne, Switzerland
b National Cancer Research Institute, 16132 Genoa, Italy
c Department of Internal Medicine, University of Genoa, Italy
d Department of Experimental Medicine, University of Genoa, Italy
e Service of Hematology, University Hospital (CHUV), Lausanne, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel
a-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)
Received 1 February 2010
Revised 1 March 2010
Accepted 4 March 2010
Available online 9 March 2010
pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with
the aryl group = 4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of
different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modifi-
cation of genes involved in cell cycle progression and survival.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Glycosylation
a-Mannosidase inhibitors
Antiproliferative effects
Cell cycle
Haematological malignances
1. Introduction
tion in the endoplasmic reticulum (ER) and Golgi apparatus in an
ordered manner.4,5 Improvement of the metastatic phenotype
Glycosylation is one of the most important post-translational
modifications that proteins undergo. Starting from a basic protein
scaffold, glycosylation leads to proteins endowed with varied bio-
logical properties and thereby allows amplifying the information
contained in a genome.1
Consistently, aberrant glycosylation leads to the abnormalities
observed in numerous hereditary diseases and appears to also have
a role in acquired conditions such as autoimmunity and cancer.2
The development of new therapeutics that target the glycosylation
pathway is therefore a major goal for the treatment of such condi-
tions and it can now take advantage of a fairly well detailed knowl-
edge of the enzymes and substrates involved in this process.3 The
majority of these enzymes are transmembrane proteins that func-
through inhibition of the machinery of complex carbohydrate for-
mation would provide an important weapon in the arsenal of can-
cer treatment.6,7
A key enzyme involved in this process is
a-mannosidase de-
puted to removing mannose residues from the maturing oligosac-
charide by hydrolyzing the mannosyl glycosidic bond.8 Two classes
of processing a-mannosidases, each one with distinct biochemical
properties, catalytic mechanism and amino acid sequences have
been described.9–12 Since the aberrant glycosylation of glycopro-
teins and glycolipids is one of the molecular changes involved in
malignant transformation13,14 the specific inhibition of
a-man-
nosidases has been proposed as an anticancer strategy to prevent
tumor progression and diffusion.15–19 Kino et al. were the first to
report that the subcutaneous administration of swainsonine, a nat-
ural inhibitor of Golgi
a-mannosidase II containing a 4-amino-4-
deoxy-mannofuranoside moiety,20 completely inhibited formation
and growth of lung metastases in a mouse sarcoma model.21
Afterwards clinical trials have demonstrated that swainsonine
* Corresponding author. Tel.: +41 21 693 93 71.
E-mail addresses: claudia.bello@epfl.ch (C. Bello), pierre.vogel@epfl.ch (P. Vogel).
C.B., M.C. and G.D. contributed equally to this study.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.009

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3321
O
O
O
NH2
H
N
O
O
NaHB(OAc)3
ClCH2CH2Cl
OMe
N
OMe
+
Boc
O
O
N
R
Boc
H
1
2 R = Ph
3 R = -CF3Ph
8 R = Ph
R
p
9 R = -CF3Ph
p
4 R = thienyl
5 R = OMe
10 R = thienyl
11 R = OMe
6 R = prop-2-en-1-oxy
12 R = prop-2-en-1-oxy
7 R =
p
-benzyloxy
13 R = p-benzyloxy
Scheme 1. Reductive aminations.
induces clinical responses in patients with solid and haematologi-
cal malignancies.22 However the toxicity observed, possibly
2. Results and discussion
2.1. Chemical synthesis
resulting from the undesired co-inhibition of lysosomal
idase,23,24 prevented further experimentations and resulted in the
search for new and more selective -mannosidases inhibitors.
a-mannos-
a
The synthetic pathway for the preparation of the new inhibitors
was centred on the coupling of two fragments, carbaldehyde 1 and
p-substituted phenylglycine methyl esters 2–7 (Scheme 1), via
reductive amination.
Some analogues of swainsonine as well as simpler derivatives have
shown interesting inhibitory properties.24,25
In order to facilitate the discovery of new specific
a-mannosi-
dase inhibitors, we have developed a new synthesis protocol lead-
ing to the generation of a new family of compounds with selective
The preparation of the starting material, tert-butyl(3aR,4R,6aS)-
4-formyl-2,2-dimethyltetrahydro-5H-[1,3]dioxolo-[4,5-c]pyrrole-
5-carboxylate (1), was performed following a modified procedure
(Scheme 2) based on Fleet’s synthesis.37 The modifications allowed
us to perform the synthesis faster and in better overall yield,
thanks to faster work-up and purification of the intermediates.
and competitive inhibitory activity for
a-mannosidase from jack
bean.26–28 The novel
a-mannosidase inhibitors contain in their
chemical structure a pyrrolidine ring functionalized to allow their
internalization by cells. The introduction of a lipophilic ester
moiety led to derivatives that inhibit the growth of human cancer
cells lines more efficiently than swainsonine and the other
analogues.29,30
The first step was the protection of the two diols present in
D-
gulonic acid -lactone 14 (Scheme 2) as acetonides, in the presence
c
of acetone, dimethoxypropane and p-toluenesulfonic acid as cata-
lyst. In that case, after neutralization with sodium carbonate, the
solvent was evaporated and the product recovered by adding ethyl
acetate and extracting with water, instead of filtering the salts on a
Celite pad and purify by flash chromatography, as reported in the
literature. In the second step lithium aluminium hydride was
substituted with Red-Al as reducing agent: in that way the reaction
was cleaner and the work-up was performed by simple extraction
In the present work, we report on the identification, synthesis
and biological evaluation of novel
a-mannosidase inhibitors with
dihydroxypyrrolidine structure characterized by potent and spe-
cific anticancer activity in cell lines of different histology and in
primary leukemic cells. The choice of biaryl appendices was in-
spired by reports showing that these groups might improve thera-
peutic properties of compounds containing them.31–36
HO
H
O
O
O
O
1. Red-Al, THF, 0°C
2. Rochelle salt
(62%)
acetone, DMP
p-TSA cat, r.t.
(99%)
HO
HO
H
O
O
O
HO
OH
O
N
14
15
O
O
O
O
1. MsCl, NEt3, CH2Cl2, 0°C
2. BnNH2, 65°C
(77% two steps)
AcOH, 55°C
(50% conversion)
H
O
O
HO
O
O
16
17
O
O
O
O
O
O
Pd(OH)2-C/H2
Boc2O, MeOH, r.t.
(73%)
NaIO4
MeOH/H2O
(90%)
OH
OH
O
N
N
N
H
OH
OH
Boc
Boc
18
19
1
Scheme 2. Synthesis of 2,5-dideoxy-2,5-imino-L-arabinose derivative 1.

3322
C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
after stirring with saturated aqueous Rochelle’s salt, instead of fil-
tration on silica gel.
LiAlH4 to convert the esters into the corresponding primary alcohols
26–28. Finally, reaction with trifluoroacetic acid/water 4:1 released
the completely deprotected diamines 29–31 (Scheme 3).
Dimesylate of diol 16 (Scheme 2) was prepared by reaction with
methanesulfonyl chloride in CH2Cl2 using trietylamine as base.
This procedure allowed to isolate the dimesylate by simple extrac-
tion. On the contrary, in the Fleet’s method, pyridine was used as
solvent and its elimination required evaporation in vacuo before
extraction and flash chromatography on silica gel. Formation of
intermediates 17–19 followed the Fleet’s procedure, except for
the purification of 17 where the excess of benzylamine was evap-
orated as its azeotrope with p-xylene. The targeted aldehyde 1 was
obtained then by oxidation of diol 19 using sodium periodate. For
the synthesis of the biarylglycinol moieties, commercially available
For the synthesis of the ether series we used simple William-
son’s etherification.40 Reaction of protected 4-hydroxy-
D-phenyl-
glycine (21) with allyl and benzyl bromide in the presence of
potassium carbonate as base, led, after Boc deprotection with tri-
fluoroacetic acid, to the corresponding ethers 6 and 7 (Scheme 4)
in good yields. Methyl ether 5 (Scheme 4) was prepared starting
from unprotected 4-hydroxy-
tion of both carboxylic and phenol groups with MeI at 0 °C leading
to the desired 4-methoxy- -phenylglycine methyl ester 5 (Scheme
D-phenylglycine (20) through reac-
D
4), after treatment with trifluoroacetic acid.
4-hydroxy-
D
-phenylglycine (20) (Scheme 3) was used as starting
Amines 5–7 were used in the reductive aminations with alde-
hyde 1. Reduction of the ester groups into primary alcohols (LiAlH4
in THF) and subsequent deprotection of the diol and amine of the
pyrrolidines with trifluoroacetic acid led to the final products 32–34.
material. After protection of both amino and carboxylic groups,
the intermediate 21 (Scheme 3) reacted with triflic anhydride to
generate the corresponding triflate 22 (Scheme 3) in good yield.
Suzuki cross-coupling38,39 with phenylboronic acid, 4-(trifluoro-
methyl)phenylboronic acid and thienylboronic acid in toluene
using tetrakis(triphenylphosphine)palladium as catalyst led to the
desired N-tert-butyloxycarbonyl biphenylglycine methyl ester
23, N-tert-butyloxycarbonyl-4-(trifluoromethyl)biphenylglycine
methylester 24 and N-tert-butyloxycarbonyl-4-(thienyl)phenylgly-
cine methyl ester 25 (Scheme 3) in 84%, 70% and 84% yield, respec-
tively. tert-Butylcarbamate was cleaved and the resulting amines
2–4 were submitted to reductive aminations with the pyrrolidine-
carbaldehyde1. The corresponding products 8–10were treated with
HO
OH
H
N
N
H
OH
OR'
32 R' = allyl
33 R' = benzyl
34 R' = Me
NH2
NHBoc
OMe
OH
Tf2O, Py
CH2Cl2, 0°C
(86%)
1. SOCl2,MeOH, reflux
2. Boc2O, NEt3, H2O/dioxane, r.t.
(87%)
O
O
HO
HO
20
21
NHBoc
NHBoc
TFA / H2O
RB(OH)2, K2CO, Pd(PPh3)4
toluene, 90°C
OMe
OMe
O
O
TfO
R
22
23 R = Ph (84% )
24 R = p-F3C-C6H5 (70%)
25 R = thienyl (quant.)
O
O
OMe
NH2
O
H
N
O
LiAlH4
THF, r.t.
+1, NaBH(OAc)3
1,2-DCE, r.t.
OMe
N
Boc
R
R
2 R = Ph (quant.)
3 R = p-F3C-C6H5 (quant.)
4 R = thienyl (75%)
8 R = Ph (48%)
9 R = p-F3C-C6H5 (49%)
10 R = thienyl (46%)
HO
O
O
OH
H
N
H
N
TFA/H2O 4:1
N
H
OH
N
OH
Boc
R
R
26 R = Ph (85%)
27 R = p-F3C-C6H5 (82%)
28 R = thienyl (83%)
29 R = Ph (47%)
30 R = p-F3C-C6H5 (43%)
31 R = thienyl (73%)
Scheme 3. Synthesis of new 2-[(arylmethylamino)methyl]pyrrolidine-3,4-diols.

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3323
NHBoc
OMe
NH2
O
1. RBr, K2CO3, acetone, reflux
2. TFA/ H2O, r.t.
R'O
OMe
O
HO
HO
21
20
6 R' = allyl (76%)
7 R' = benzyl (81%)
NH2
O
NH2
OH
OMe
1. Boc2O, NaOH 1N, dioxane/H2O, r.t.
2. K2CO3, MeI, DMF, 0°C
3. TFA /H2O, r.t. (84%)
O
MeO
5
Scheme 4. Synthesis of ethers of 4-hydroxy-D-phenylglycine.
2.2. Bioassays
leukemia, glioblastoma, prostate cancer, lung cancer, and breast
cancer). Cells were exposed to increasing concentrations of 29–
34 for 72 h and viability was subsequently detected by colorimet-
ric assay. Compound 30 (with the 4-(4-CF3C6H4)-benzylamino
group) showed potent cytotoxic activity in all the cell lines assayed
Compounds 29–34 were assayed for their inhibitory activities
toward 13 commercially available glycosidases41,42 (Table 1). Un-
der enzyme optimal pH and at 1 mM concentration they did not in-
hibit
coffee beans, b-galactosidases from Escherichia coli, bovine liver
and Aspergillus orizae, -glucosidase from rice, amyloglucosidase
a-L-fucosidase from bovine kidney, a-galactosidase from
for concentrations <200
pound 29 and, to a lesser extent, 32 and 34 also exhibited growth
inhibition effects although with IC50s >200 M (data not shown).
lM (Table 2 and Fig. 1 part A and B). Com-
a
l
from Aspergillus niger, b-mannosidase from snail and b-N-acetyl-
Compounds 31 and 33 did not show antiproliferative activity.
glucosaminidases from jack beans and from bovine kidney. Pyrrol-
idines 29–34 showed weak inhibition of a-glucosidase from yeast
Table 2
(24%, <10%, <10%, 41%, 45%, 41%, respectively), of b-glucosidase
from almonds (20%, 50%, 36%, 72%, 40%, 62%, respectively) and of
b-xylosidase from Aspergillus niger (50%, 52%, 50%, 28%, 45%, 28%,
respectively, at 1 mM). However, 29–34 were found to be potent
Evaluation of the ability of compound 30 to inhibit cell growth on nine human tumor
cell lines of different histology
IC50 (lM)
Compound 30
Cell lines
Histology
inhibitors of
malian Golgi
Kinetics measurements gave for 29: Ki = 0.9
a
a
-mannosidase from jack bean, a good model of mam-
-mannosidase II,43 with >98% inhibition at 1 mM.
U266
Jurkat
U87
PC3
A549
Multiple myeloma
T cell Leukemia
Glioblastoma
Prostate adenocarcinoma
Lung cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
50
75
80
132
152
77
66
93
48
l
M, IC50 = 1.4
l
l
M;
M;
for 30: Ki = 2.7
for 32: Ki = 0.5
l
l
M, IC50 = 5
M, IC50 = 0.9
M, IC50 = 0.8 l
l
l
M; for 31: Ki = 0.8
M; for 33: Ki = 0.6
M.
lM, IC50 = 1.1
lM, IC50 = 1.4 lM
MCF7
and for 34: Ki = 0.5
l
MDA-MB-231
BT474
SKBR3
Dihydroxypyrrolidine derivatives 29–34 were then evaluated
for their capacity to inhibit cell growth on nine established human
tumor cell lines of different histology (multiple myeloma, T-cell
Table 1
Calculation of the percentage of inhibition (1 mM) and kinetics measurement (IC50 and Ki, given in
l
M) for products 29, 30, 31, 32, 33 and 34 (optimal pH)
Enzyme (pH)
Compound
Percentage of inhibition
29
30
31
32
33
34
a
-
L
-Fucosidase EC 3.2.1.51
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
1-bovine kidney (6)
a-Galactosidase EC 3.2.1.22
2-coffee beans (6)
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
b-Galactosidase EC 3.2.1.23
5-escherichia coli (7)
8-Aspergillus orizae (4)
n.i.
24
n.i.
n.i.
n.i.
n.i.
n.i.
41
n.i.
45
n.i.
41
a-Glucosidase EC 3.2.1.20
10-yeast (7)
11-Rice (4)
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
Amyloglucosidase EC 3.2.1.3
13-aspergillus niger (5)
b-Glucosidase EC 3.2.1.21
15-almonds (5)
20
50
36
72
40
62
a-Mannosidase EC 3.2.1.24
99
98
97
99
99
98
16-jack beans (5)
IC50 = 1.4
Ki = 0.9
n.i.
IC50 = 5
Ki = 2.7
n.i.
IC50 = 1.1
Ki = 0.8
n.i.
IC50 = 0.9
Ki = 0.5
n.i.
IC50 = 1.4
Ki = 0.6
n.i.
IC50 = 0.8
Ki = 0.5
n.i.
b-Mannosidase EC 3.2.1.25
18-snail (4)
b-Xylosidase EC 3.2.1.37
19-aspergillus niger (5)
b-N-Acetylglucosaminidase EC 3.2.1.30
21-jack beans (5)
50
52
50
28
45
28
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
22-BOVINE kidney (4)
n.i. = no inhibition (inhibition lower than 10% at 1 mM).

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C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
The cytotoxic activity of these drugs is typically detected after 6–
12 h of incubating and is initially characterized by extensive intra-
cellular vacuolization. In line with our previous findings with this
class of compounds26,27 these data confirm that the dihydroxypyr-
rolidine moiety is pivotal for their tumoricidal activity. The side
chains of these small molecules are likely to affect drug potency
by conferring variable membrane-permeability. However, addi-
tional effects of these pharmacophoric moieties cannot, in princi-
ple, be excluded. Experiments conducted in A549 cells showed
that cell death induced by 30 and related analogues is neither af-
fected by the antioxidant N-acetyl-cysteine, nor by the pan-cas-
pase inhibitor zVAD-fmk (data not shown) suggesting that cell
killing by these compounds is unlikely to involve reactive oxygen
species or caspase activity. Since pyrrolidine 30 exhibited the most
potent inhibitory activity on cancer cell growth (Fig. 2) further
experiments were conducted on this compound. Initially, in order
to provide clues to its mechanism of action, we performed cell cy-
cle analysis and monitored the changes in gene expression profile
in breast cancer cell lines treated with this novel compound. Cell
cycle analysis showed that the 30 increases the percentage of cells
in G2/M phase in a time and dose-dependent manner (Fig. 3).
Increasing 30 concentration from 12.5 to 200 lM led to an increase
of the cell fraction with G2/M DNA content from 12.5% to 41.9%
after a 12-h treatment, while S-phase nuclei decrease from 30%
to 16%. These data indicate that the a-mannosidase inhibitor 30 in-
duces cell cycle arrest and accumulation of cells in G2/M phase.
Meanwhile, the effect on gene expression was investigated by
TLDA using as a framework a panel of 90 genes selected for their
relevance in cancer biology and metastasis. As shown in Figure 4,
treatment with this compound consistently downregulated
Figure 1. The novel dihydroxypyrrolidine derivatives provide an antiproliferative activity in 8 human cancer cell lines. (part A–B). 5 ꢀ 104 cells/well were incubated in 96-
well plates, allowed to adhere for 24 h and then incubated with novel compound at concentration ranging between 10ꢁ2 and 400
lM for 72 h. Thereafter, viability was
determined by a commercially available colorimetric assay (CellTiter 96 Aqueous1). Results are shown as means of triplicate wells with SD. IC50s were estimated using
GraphPad prism 4.

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3325
Fig. 1 (continued)
CDC25, ID1, cyclin E1, and hTERT in SKBR3 as well as in MCF7 cells.
Furthermore, it induced upregulation of CDKN1C and CDKN1A,
which inhibit several cyclin/Cdk complexes and act as negative
regulators of cell proliferation. Thus, although restricted to a lim-
ited set of genes, the analysis performed by TLDA detected molec-
ular sequelae of 30 that could possibly underlie the effects of this
compound on cell cycle and cell viability.
These data suggest that this compound is unlikely to act via the
mitochondrial apoptotic pathway and that it could therefore possi-
bly be of use in Bcl2-expressing cancers. Finally, the in vivo cyto-
toxic activity was evaluated on primary tumors cells, collected by
patients with B-cell chronic lymphocytic leukemia (B-CLL) and
multiple myeloma (MM), with increasing concentrations ranging
from 50 to 200 lM for 24 h and subsequently quantifying apopto-
B-cell leukemia/lymphoma-2 (Bcl2) is an antiapoptotic protein
frequently upregulated in hematological and solid malignancies,
where it confers resistance to therapy and a poor prognosis.44–46
Therefore, to investigate the effect of Bcl-2 overexpression on the
cytotoxic activity of the dihydroxypyrrolidine drugs, we used Jur-
kat T cells engineered to overexpress this protein and the respec-
tive control cells.45,47–50 As shown in Figure 5 (A–C), while
almost completely preventing cell death in response to doxorubi-
cin and etoposide, Bcl2 failed to affect the cytotoxic effect of 30.
tic cells by staining with annexin V (AV) and propidium iodide (PI)
and flow cytometric analysis. As expected, the percentage of an-
nexin V-positive cells increased in a time- and dose-dependent
manner (Fig. 6).
3. Conclusion
Despite the existence of numerous therapeutic options for the
treatment of human cancers the results are still disappointing

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C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
Figure 2. Pyrrolidine 30 induces dose-dependent cell death in Jurkat cells. 5 ꢀ 103 Jurkat cells/well were incubated for 24 h and 72 h in the presence of the indicated
concentrations of pyrrolidine 30. Thereafter, cell viability was assessed by flow cytometry using annexin V and 7AAD double staining. 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. Data are derived from at least three
independent experiments.
Figure 3. Effects of pyrrolidine 30 on cell cycle progression. A: 105 SKBR3 cells/well were seeded in 24-well plates. 24 h later cells were treated with increasing concentration
of pyrrolidine 30 from 50 to 200 lM. Cell cycle was analyzed after 48 h by propidium iodide staining of isolated cell nuclei and flow cytometry. Representative graphs show
the percentage of cells in G1, S and G2/M cell cycle phases. B: MCF7, SKBR3, MDA-MB-231 and BT474 cells exposed to novel drug for 72 h were imaged using a 40X
magnification.
and thus mandate the search for new compounds to improve the
clinical outcome. Interfering with the glycosylation pathway has
recently emerged as a promising therapeutic strategy to produce
antitumour effects in numerous types of malignancies.13
Our data show that the new functionalized pyrrolidines affect
the expression of genes involved in cell cycle progression thereby
arresting cells in the G2/M phase of cell cycle and causing the
upregulation of cell cycle inhibitors CDKN1A and CDKN1C, as well
as the downregulation of CDC25A cyclin E1, and of MYBL2 which,
conversely, promote cells cycle progression. These experiments
In a preliminary report, we found that pyrrolidine compounds
with
a-mannosidase inhibitory capacity had cytostatic activity in
melanoma and glioblastoma cells. Here, we extend these observa-
tions using more potent analogues that effectively prevent growth
in cancer cell lines of different histology, including primary leuke-
mic cells. Probably, due to the pharmacophoric properties and the
lipophilic character of the biphenyl moiety compound 30 proved to
be more active toward the cancer cells tested than the simpler ana-
logues 29, 31, 32–34 and those described earlier.28–30
establish the new a-mannosidase inhibitors as effective cell cycle
modulators. Additionally the cell death via dihydroxypyrrolidine
30 happens irrespective of Bcl-2 overexpression, a condition that
normally inhibits apoptosis by preventing mitochondrial pore for-
mation and cytochrome c release into the cytosol.45 The latter
property of this novel compound appears of special importance
as Bcl2 is frequently overexpressed in cancer, mainly in B-CLL, con-

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3327
Figure 4. Effects of pyrrolidine 30 on gene expression profile in Human breast cancer cell lines (SKBR3 and MCF7). 2 ꢀ 105 SKBR3 and MCF7 cells/well were seeded in 6-well
plates and treated with 50 lM for 12 h. Thereafter, RNA was extracted and used for cDNA synthesis and TLDA. Results were calculated using RPLP0 as an housekeeping gene
and changes in gene expression were determined using cells treated with vehicle DMSO alone as a control.
Figure 5. Effect of Bcl-2 overexpression on cell death in response to etoposide, doxorubicin and pyrrolidine 30. A, B, Bcl-2-overexpressing and vector control Jurkat cells were
treated for 72 h with increasing concentrations of pyrrolidine 30. Dead cells were enumerated by flow cytometric analysis of propidium iodide-stained cells. Viability was
calculated using the following formula: 100 ꢁ [(drug ꢁ induced death ꢁ spontaneous death)/(100 ꢁ spontaneous death) ꢀ 100]. Means of triplicate wells with SD are shown.
C, 4 ꢀ 104 Jurkat cells engineered to overexpress Bcl-2 and the respective vector control cells were seeded in 96-well plates and treated for 72 h with 40
lM etoposide, 2 lM
doxorubicin or 50
lM pyrrolidine 30. Thereafter, cells were harvested and dead cells were quantified by flow cytometry after staining with PI. Results are presented as means
of triplicates with SD.

3328
C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
Figure 6. Pyrrolidine 30 efficiently kills primary B-CLL and multiple myeloma cells. A, primary B-CLL cells (>60% CD19+ cells) were isolated from PB samples, while primary
multiple myeloma (MM) cells were obtained from a bone marrow aspirate in a patient at disease diagnosis. 106 cells/well were seeded in 24-well plates and cultured with or
without pyrrolidine 30 at the indicated concentrations. 24 h later cells were harvested, washed, stained with FITC-conjugated Annexin-V and PI, and analyzed by flow-
cytometry.
ferring unfavorable growth conditions and to cancer therapeu-
tics.48 In summary, the novel
-mannosidase inhibitors reported
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. Opti-
cal rotations were recorded at 25 °C on a Jasco P-1020 polarimeter.
Mass spectra: GC-IE/CI/MS experiments were preformed with a
1200 L 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.
(3aR,4R,6aS)-5-Benzyl-4-[(7S)-10,1-dimethyl-9,11-dioxolan-7-yl]-
a
display promising anticancer activity through cell cycle modula-
tion. Additionally and significantly, their cytotoxic potential is
not affected by obstruction of the intrinsic mitochondrial pathway.
More studies are required to define their effectiveness and safety in
in vivo tumor models and to elucidate their own mechanism of
action.51
4. Experimental data
4.1. Synthesis of 3,4-dihydroxypyrrolidine derivatives
2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrole (17). D-Gu-
All commercially available reagents (Fluka, Aldrich, Acros) were
used without further purification. Technical solvents were used for
extraction without any purification. For reactions requiring anhyd
conditions, dry solvents were bought (Fluka) or filtered prior to use
(Innovative Technology). In absence of any particular notification,
experiments were carried out under argon atmosphere. Reactions
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 WATER
(300 ml)] or Pancaldi [(NH4)6Mo4 (21 g), Ce(SO4)2 (1 g), H2SO4
(31 ml) and WATER (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 Bru-
ker ARX-400 and DPX-400 spectrometers at 400 MHz. The signals
of residual solvents were used as reference (MeO-d4: 3.32 ppm,
CDCl3: 7.27 ppm). Coupling constants are given in Hz. When neces-
sary, 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. The signals of residual solvents were used as reference
(MeO-d4: 49 ppm, CDCl3: 77 ppm). When necessary, the 13C-sig-
nals assignments were confirmed by HSQC spectra. IR spectra were
lonolactone (14) (5 g, 28.07 mmol) was dissolved in acetone/DMP
(5/1 vol: vol, 150 ml). p-Toluenesulfonic acid was added until pH
3 and the solution was then stirred at 25 °C until the starting mate-
rial was consumed (control by TLC, AcOEt/petrol ether 4:1). The
solution was neutralized with solid Na2CO3 and, after solvent evap-
oration in vacuo, the residue was poured into water and extracted
with EtOAc (3 ꢀ 50 ml). The combined organic extracts were
washed with brine (50 ml) and dried with MgSO4. After solvent
evaporation in vacuo, pure 2,3:5,6-di-O-isopropylidene-D-gulono-
1,4-lactone (15) was obtained (7.210 g, 27.91 mmol, 99% yield) as
a light yellow solid. This product (7.21 g, 27.9 mmol) was added
portionwise to a Red-Al solution in toluene/THF (16 ml of a 3.5 M
solution in toluene diluted in 29 ml of anhyd THF) at 0 °C. The solu-
tion was stirred at 25 °C for 3 h and then methanol was added until
the excess of Red-Al was consumed. The solution was poured into a
satd aq soln. of sodium potassium tartrate (50 ml) and the mixture
was stirred for 2 h at 25 °C. The organic phase was collected and
the aqueous phase extracted with EtOAc (3 ꢀ 30 ml). The com-
bined organic extracts were washed successively with a satd aq
soln of NaHCO3 (30 ml) and brine (30 ml), then dried (MgSO4).
After solvent evaporation in vacuo, 5,6-di-O-isopropylidene-
D-gul-
itol (16) was obtained as a white solid (4.56 g, 17.4 mmol, 62%

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3329
yield) and used without any further purification. 16 (4.56 g,
17.4 mmol) was dissolved in dry CH2Cl2 (100 ml), Et3N (9 ml,
6.57 g, 64.9 mmol, 3.7 equiv) was added and the solution was
cooled to 0 °C with an ice bath. MeSO2Cl (5 ml, 7.37 g, 51.0 mmol,
3 equiv) was added dropwise and the mixture was stirred at 0 °C
for 2 h (reaction monitored by TLC: EtOAc/petroleum ether 3:1).
The solution was washed with cold water (30 ml), then with 1 M
aq HCl (50 ml) and finally with brine (30 ml), dried with MgSO4
and the solvent was evaporated in vacuo.
The crude mesylate was dissolved in benzylamine (120 ml) and
the solution was stirred at 70 °C for 2 days. CH3Cl was added and
the solution was washed successively with 1 M aq HCl
(2 ꢀ 70 ml) then with water (50 ml) and brine (50 ml). The solvent
was evaporated in vacuo and pure 17 (3.93 g, 13.4 mmol, 77% two
steps) was obtained as white solid after flash column chromatogra-
phy on silica gel (petroleum ether/EtOAc 3:1). 1H NMR (400 MHz,
CDCl3, Ph = phenyl): d 7.36–7.32 (m, 5H Ph) 4.73–4.69 (m, 2H, H-
C(6a) and H-C(3a)) 4.18 (m, 1H, HHC(8)) 4.08–4.01 (m, 2H, N-
HHC-Ph and HHC(8)) 3.84 (d, 2J = 13.2, N-HHC-Ph) 3.69 (dd,
3J = 7.9, 3J = 7.2, H-C(7)) 3.12 (dd, 2J = 11.6, 3J = 4.7, 1H, HHC(6))
3.02 (br d, 3J = 4.0, 1H, H-C(4)) 2.82 (dd, 2J = 11.6, 3J = 2.0, 1H, H-
C(6)) 1.57 (s, 3H, H3C(1’)) 1.42 (s, 3H, H3C(1’’)) 1.34 (s, 6H,
H3C(1’’’) and H3C(1IV)). Analytical and spectroscopic data are in ac-
cord with those reported in the literature.34
tion was diluted with CH2Cl2 (20 ml), water (20 ml) was added and
the two layers were separated. The organic phase was washed with
aqueous NaOH (0.5 N, 2 ꢀ 20 ml), with 10% aq soln of citric acid
(2 ꢀ 20 ml) and finally with water (20 ml), dried over MgSO4 and
the solvent was evaporated in vacuo. Purification by flash chroma-
tography on silica gel (petroleum ether/EtOAc 3:1) led to pure
methyl N-tert-butoxycarbonyl-4-[[(trifluoromethyl)sulfonyl]oxy]-
D-phenylglycinate (22: 1.301 g, 3.1 mmol, 86% yield) as pale yellow
solid. 22 (0.155 g, 0.4 mmol) was dissolved in anhyd toluene
(3.0 ml), phenylboronic acid (0.089 g, 0.7 mmol, 1.9 equiv) and
K2CO3 (0.076 g, 0.6 mmol, 1.5 equiv) were added and the mixture
was degassed under Ar. (Ph3P)4Pd (0.014 g, 3% mol) was dissolved
in degassed toluene (0.5 ml) under nitrogen (glove box) and the
solution was added to the reaction mixture. The suspension was
stirred at 90 °C overnight. EtOAc (4 ml) was added and the mixture
was washed with satd aq soln of NaHCO3 (3 ml), then with water
(3 ml), with 10% aq soln of citric acid (3 ml), again with water
(3 ml) and finally with brine (3 ml). The solution was dried with
MgSO4 and the solvent was evaporated in vacuo. After flash chro-
matography on silica gel (petroleum ether/EtOAc 6:1), pure methyl
N-tert-butoxycarbonyl-4-phenyl-D-phenylglycinate (23: 0.107 g,
0.3 mmol, 84% yield) was recovered as white foam. Spectral data
were the same as those reported for this compound.52 1HNMR
(400 MHz, CDCl3): d 7.60–7.57 (m, 4H, H (bph)) 7.46–7.43 (m,
4H, H (bph)) 7.38–7.34 (m, 1H, H-C(4’) (bph)) 5.60 (bd, 3J = 7.0,
1H, H–N–Boc) 5.38 (d, 3J = 7.0, 1 H, H-C(2)) 3.76 (s, 3H, OCH3)
1.45 (s, 9H, (CH3)3 t-butoxy). CI-MS: 182, 225, 242, 286, 303, 342.
23 (0.107 g, 0.30 mmol) was dissolved in trifluoroacetic acid (80%
aq, 4 ml) at 0 °C. The solution was stirred at 20 °C for 3 h. The sol-
vent was evaporated in vacuo and pure 2 was obtained quantita-
tively after flash chromatography on silica gel (CH2Cl2/MeOH
95:5). Spectral data were the same as those reported for this com-
pound.53 1HNMR (400 MHz, CDCl3, bhp = biphenyl): d 7.59–7.57
(m, 4H, H (bhp)) 7.49–7.44 (m, 4H, H (bhp)) 7.40–7.36 (m, 1H, H-
C(4’) (bhp)) 4.86 (br s, 1H, H-C(2)) 3.76 (s, 3H, OCH3).
N-Benzyl-1,4-dideoxy-5,6-O-isopropylidene-1,4-imino-
(18). N-Benzyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imi-
no- -allitol (17) (3.93 g, 13.4 mmol) was dissolved in AcOH
D-allitol
D
(60 ml of 80% vol/vol in water) and the solution was stirred at
60 °C overnight. The reaction was stopped at about 50% of conver-
sion (monitored by TLC: pure EtOAc), in order to avoid the depro-
tection of the secondary acetate (the starting material was
recovered). After evaporation of the solvent in vacuo, the product
was purified by flash chromatography using pure EtOAc as eluent
yielding 29% of N-benzyl-1,4-dideoxy-5,6-O-isopropylidene-1,4-
imino-D
-allitol (18: 1.005 g, 3.4 mmol) as white solid. 1H NMR
(400 MHz, CDCl3, Ph = phenyl): d 7.33 (m, 5H, H Ph) 4.76 (dd,
3J = 6.7, 3J = 4.0, 1H, H-C(3a)) 4.64 (m, 1H, H-C(7)) 4.15 (m, 1H,
HHC-Ph + AcOEt) 3.95 (m, 1H, H-C(6a)) 3.81 (dd, 2J = 11.5, 2J = 6.3,
1H, HHC(8)) 3.70 (dd, 2J = 11.5, 3J = 5.3, 1H, HHC(8)) 3.62 (d,
2J = 12.9, 1H, HHC-Ph) 3.32 (dd, 2J = 11.2, 2J = 5.9, 1H HHC(6))
3.25 (br s, 2H, 2 ꢀ H–O) 2.92 (m, 1H, H-C(4)) 2.70 (dd, 2J = 11.2,
2J = 3.8, 1H, HHC(4)) 1.56 (s, 3H, H3C(1’)) 1.34 (s, 3H, H3C(1’’)). Ana-
lytical and spectroscopic data are in accord with those reported in
the literature.37
Methyl 4-(4-trifluorophenyl)-
N-tert-butoxycarbonyl-4-[[(trifluoromethyl)sulfonyl]oxy]-
D
-phenylglycinate (3). Methyl
D-phenyl-
glycinate (22: 0.205 g, 0.5 mmol) was dissolved in anh. toluene
(4.0 ml), 4-(trifluoromethyl)phenylboronic acid (0.194 g, 1.0 mmol,
2.0 equiv) and K2CO3 (0.111 g, 0.8 mmol, 1.6 equiv) were added
and the mixture was degassed under Ar. (Ph3P)4Pd (0.060 g, 10%
mol) was dissolved in degassed toluene (1.0 ml) under nitrogen
(gloves box) and the solution was added to the reaction mixture.
The suspension was stirred at 90 °C overnight. EtOAc (4 ml) was
added and the mixture was washed successively with satd aq soln
of NaHCO3 (3 ml), water (3 ml), 10% aq soln of citric acid (3 ml),
water (3 ml) and finally with brine (3 ml). The solution was dried
(MgSO4) and the solvent was evaporated in vacuo. After flash chro-
matography on silica gel (petroleum ether/EtOAc 7:1), pure methyl
Methyl 4-phenyl-
(7.5 ml, 12.23 g, 102.8 mmol, 2.8 equiv) was added dropwise to an-
hyd MeOH (72 ml) at 0 °C. 4-Hydroxy- -phenylglycine (20)
D-phenylglycinate (2). Thionyl chloride
D
(6.054 g, 36.2 mmol) was added slowly and the solution was stir-
red and heated under reflux until consumption of the starting
material (monitored by TLC, CH3CN/NH3 5:1). After solvent evapo-
ration in vacuo the crude was dissolved in dioxane/water (1:1,
130 ml), di-tert-butyldicarbonate (7.81 g, 41.9 mmol, 1.1 equiv)
and then, slowly, Et3N (until pH 8.5) were added and the solution
was stirred at 20 °C overnight. EtOAc (50 ml) was added and the
two phases were separated. The organic layer was washed with
satd aq soln of NH4Cl (30 ml), then with satd aq soln of NaHCO3
(30 ml) and finally with brine, dried over MgSO4 and the solvent
was evaporated in vacuo. Pure methyl N-tert-buthoxycarbonyl 4-
N-tert-butoxycarbonyl-4-(4-trifluorophenyl)-D-phenylglycinate (24:
0.143 g, 0.4 mmol, 70% yield) was recovered as a white foam. 1H
NMR (400 MHz, CDCl3): d 7.66 (m, 4H, arom. H) 7.59 (d, 3J = 8.2,
2H, arom. H) 7.48 (d, 3J = 8.2, 2H, arom. H) 5.66 (bd, 3J = 7.0, 1H,
H–N–Boc) 5.39 (d, 3J = 7.0, 1H, H-C(2)) 3.76 (s, 3H, OCH3) 1.45 (s,
9H, (CH3)3 t-butoxy). N-tert-Butoxycarbonyl-4-(4-trifluorophenyl)-
D
-phenylglycinate (0.1431 g, 0.35 mmol) was dissolved in trifluoro-
acetic acid (80% aq, 3.5 ml) at 0 °C. The solution was stirred at 0 °C
for 1 h and then at 20 °C for 3 h (reaction monitored by TLC, petro-
leum ether/EtOAc 5:1). The solvent was evaporated in vacuo and
hydroxy-D-phenylglycinate (21: 8.815 g, 31.3 mmol, 87% yield)
was obtained after flash column chromatography on silica gel
(petroleum ether/EtOAc 2:1). 21 (1.01 g, 3.6 mmol) was dissolved
in CH2Cl2 (5 ml), pyridine (1.35 ml, 1.26 g, 4.4 equiv) was added
and the solution was cooled to 0 °C. (MeSO2)2O (0.7 ml, 1.179 g,
4.2 mmol, 1.2 equiv) was added dropwise (the temperature should
not be higher than 5 °C) and the solution was stirred at 0 °C for 1 h
(reaction monitored by TLC, petroleum ether/EtOAc 3:1). The solu-
pure 3 was obtained quantitatively after flash chromatography on
25
silica gel (CH2Cl2/MeOH from 95:5 to 90:10).
½
a
ꢂ
¼ ꢁ47;
589
25
25
25
½aꢂ
¼ ꢁ19; ½
a
ꢂ
¼ ꢁ18; ½
a
ꢂ
¼ ꢁ26 (c 0.175, MeOH).
577
435
405
IR(pure): 3377, 3040, 2959, 2919, 2850, 1734 (C@O), 1614,
1324, 1115, 821. 1H NMR (400 MHz, CDCl3): d 7.69 (m, 4H, arom.
H) 7.60 (d, 3J = 8.1, 2H, arom. H) 7.49 (d, 3J = 8.2, 2H, arom. H)
4.71 (br s, 1H, H-C(2)) 3.75 (s, 3H, OCH3). 1H NMR (400 MHz,

3330
C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
MeOH-d4): d 7.80 (d, 3J = 7.7, 2H, arom. H) 7.73 (m, 4H arom. H)
7.56 (d, 3J = 7.6, 2H, arom. H) 3.75 (s, 3H, OCH3). 13C NMR
(101 MHz, MeOH-d4): d 173.05 (s, C(1) (C(O)OCH3)) 145.25 (s,
0.8 mmol, 90% yield) that was used without any further purifica-
tion. NaBH(OAc)3 (0.134 g, 0.6 mmol) was added portionwise to a
stirred solution of aldehyde (1: 0.118 g, 0.4 mmol) and amine 2
(0.102 g, 0.4 mmol) in 1,2-dichloroethane (4.5 ml) at room temper-
ature. After complete disappearance of reagents (reaction moni-
tored by TLC), the solution was poured into a satd aq soln of
NaHCO3 (5 ml per mmol). The organic phase was collected and
the aqueous phase extracted with EtOAc (10 ml per mmol, three
times). The combined organic extracts were washed with brine
(10 ml per mmol, once) and dried (MgSO4). Solvent evaporation
in vacuo and flash chromatography (light petroleum/EtOAc 4:1)
gave pure 8 as light yellow oil (0.094 g, 0.2 mmol, 49% yield). Ester
8 was added portionwise to a cooled suspension (0 °C) of LiAlH4
(0.017 g, 0.4 mmol) in anhyd THF (5 ml). The solution was stirred
at room temperature for 3 h, then WATER was added dropwise
(0.5 ml) and the mixture was filtered on silica gel conditioned with
EtOAc. After solvent evaporation in vacuo, the obtained amino
alcohol 26 (0.077 g, 0.2 mmol, 85% yield) was deprotected without
any further purification. Compound 26 was dissolved in trifluoro-
acetic acid (80% aq, 1.7 ml) at 0 °C. The solution was stirred at
0 °C for 1 h and then at 20 °C for 3 h (reaction monitored by TLC,
petroleum ether/EtOAc 5:1). The solvent was evaporated in vacuo
and pure 29 (0.027 g, 0.1 mmol, 47% yield) was collected as a white
solid after flash chromatography on silica gel (CH3CN/NH4OH 8:1).
Melting point: 151–156 °C (dec).
2
C(1)arom) 141.43 (s, C(4)) 137.77 (s, C(1’)) 130.64 (q, JCF = 32.3,
C(4’)) 129.31 (dd, 1J = 159.9, 2J = 6.0, 2C, arom. C) 128.89 (dd,
1J = 159.9, 2J = 6.6, 2C, arom. C) 128.59 (dd, 1J = 161.2, 2J = 6.3, 2C,
1
3
arom. C) 126.82 (dq, JCH = 109.1, JCF = 3.8, C(3’) and C(5’))
125.74 (q, JCF = 269.5, CF3) 58.30 (d, 1J = 142.3, C(2)) 53.36 (q,
1
1J = 147.1, OCH3). HR-ESI-TOF-MS: calcd for C16H14F3NO2:
310.1049, found 310.1039 ([M+H]+).
Methyl 4-(3-thienyl)-
D-phenylglycinate (4). Methyl N-tert-
butoxycarbonyl-4-[[(trifluoromethyl)sulfonyl]oxy]-
D
-phenylglyci-
nate (22: 0.255 g, 0.6 mmol) was dissolved in anhyd toluene
(6.0 ml), 3-thiopheneboronic acid (0.153 g, 1.2 mmol, 2.0 equiv)
and K2CO3 (0.125 g, 0.9 mmol, 1.5 equiv) were added and the mix-
ture was degassed under Ar. (Ph3P)4Pd (0.140 g, 20% mol) was dis-
solved in degassed toluene (1.0 ml) under nitrogen (gloves box)
and the solution was added to the reaction mixture. The suspen-
sion was stirred at 90 °C for 6 h (reaction monitored by TLC: petro-
leum ether/EtOAc 7:1). EtOAc (4 ml) was added and the mixture
was washed successively with satd aq soln of NaHCO3 (3 ml), water
(3 ml), 10% aq soln of citric acid (3 ml), water (3 ml) and finally
with brine (3 ml). The solution was dried (MgSO4) and the solvent
was evaporated in vacuo. After flash chromatography on silica gel
(petroleum ether/EtOAc 7:1), pure methyl N-tert-butoxycarbonyl-
25
25
25
25
4-(3-thienyl)-
D-phenylglycinate (25) was obtained quantitatively,
½aꢂ
¼ ꢁ12; ½
aꢂ
¼ ꢁ14; ½
aꢂ
¼ ꢁ28; ½
aꢂ
¼ ꢁ360 (c 0.271,
589
577
435
405
white foam. 1H NMR (400 MHz, CDCl3, thi = thienyl): d 7.60 (d,
3J = 8.2, 2H, arom. H) 7.54 (m, 1H thi) 7.41 (m, 4H, arom. H) 5.57
(br s, 1H, H–N) 5.36 (d, 3J = 7.1, 1H, H-C(2)) 3.76 (s, 3H, CH3) 1.46
(s, 9H, (CH3)3 t-butyl). 25 (0.20 g, 0.6 mmol) was dissolved in triflu-
oroacetic acid (80% aq, 4.3 ml) at 0 °C. The solution was stirred at
0 °C for 1 h and then at 20 °C for 3 h (reaction monitored by TLC,
petroleum ether/ EtOAc 5:1). The solvent was evaporated in vacuo
MeOH). UV(CH3CN): 257 (22605) 208 (32249) IR (pure): 3294,
2873, 2445, 1487, 1407, 1109, 1029, 835, 761, 687. 1H NMR
(400 MHz, MeOH-d4, bph = biphenyl): d 7.58 (m, 4H, H (bph))
7.43 (m, 4H, H (bph)) 7.32 (m, 1H, H-C(4’) (bph)) 4.05 (m, 1H, H-
C(4)) 3.82 (dd, 3J = 8.3, 3J = 4.4, 1H, H-C(3)) 3.69 (m, 2H, CHCH2OH)
3.61 (dd, 3J = 10.7, 3J = 8.7, 1H CHCH2OH) 3.18 (dd, 2J = 12.2, 3J = 5.1,
1H, HH-C(5)) 3.12 (m, 1H, H-C(2)) 2.88 (dd, 2J = 12.2, 3J = 3.2, 1H,
HH-C(5)) 2.73 (dd, 2J = 12.1, 3J = 5.0, 1H, HH-C(6)) 2.61 (dd,
2J = 12.1, 3J = 7.4, 1H, HH-C(6)). 13C NMR (101 MHz, MeOH-d4,
bph = biphenyl): d 142.19 (s, C (bph)) 141.88 (s, C (bph)) 141.12
(s, C (bph)) 129.89 (m, 2CH (bph)) 129.29 (m, 2CH (bph)) 128.35
(br s, C(4’) (bph)) 128.20 (d, 1J = 166.0, 2CH (bph)) 127.92 (d,
1J = 160.0, 2CH (bph)) 76.59 (d, 1J = 141.1, C(3)) 72.40 (d,
1J = 150.1, C(4)) 67.77 (t, 1J = 140.7, CHCH2OH) 66.49 (d,
1J = 134.3, CHCH2OH) 62.91 (d, 1J = 140.7, C(2)) 52.07 (t,
1J = 140.7, C(5)) 50.45 (t, 1J = 130.9, C(6)).HR-MALDI-TOF-MS: calcd
for C19H24N2O3 329.1865, found 329.1868 ([M+H]+).
and the pure 4 (0.111 g, 0.5 mmol, 75% yield) was obtained after
25
589
flash chromatography on silica gel (CH2Cl2/MeOH 95:5). ½
aꢂ
¼
25
577
25
435
25
405
ꢁ51; ½aꢂ
¼ ꢁ23; ½
a
ꢂ
¼ ꢁ25; ½
aꢂ
¼ ꢁ36 (c 0.167, MeOH).
IR(pure): 3378, 3039, 2959, 2919, 2853, 1735 (C@O), 1610,
1324, 1111, 821. 1H NMR (400 MHz, MeOH-d4, Ph = phenyl,
thi = thienyl): d 7.62 (d, 3J = 7.8, 2H Ph) 7.46–7.38 (m, 5H, 2H Ph
and 3H thi) 5.31 (s, 1H, H-C(2)) 3.75 (s, 3H, CH3). 13C NMR
(101 MHz, MeOH-d4, Ph = phenyl, thi = thienyl): d 174.15 (s, C@O)
142.68 (s, arom. C) 137.62 (s, arom. C) 137.54 (s, arom. C) 128.89
(d, 1J = 185.6,
C C Ph) 127.51 (d,
thi) 127.77 (d, 1J = 168.1,
1J = 181.7, C thi) 127.03 (d, 1J = 111.3, C Ph) 121.82 (d, 1J = 187.4,
C thi) 58.70 (d, 1J = 142.4, C(2)) 53.10 (q, 1J = 147.8, CH3). HR-ESI-
TOF-MS: calcd for C13H13NO2S: 248.0745, found 248.0746
([M+H]+).
(2R,3R,4S)-2-{{{(1R)-2-Hydroxy-1-[4’-(trifluoromethyl)-[1,1’-
biphenyl]-4-l]ethyl}amino} methyl}pyrrolidine-3,4-diol (30). NaB-
H(OAc)3 (0.112 g, 0.5 mmol) was added portionwise to a stirred
solution of aldehyde
1 (0.124 g, 0.45 mmol) and amine 3
(2R,3R,4S)-2{{[(1R)-1-[1,1’-Biphenyl]-4-yl-2-hydroxyethyl]amino}-
methyl}pyrrolidine-3,4-diol (29). N-Benzyl-1,4-dideoxy-5,6-O-iso-
propylidene-1,4-imino-D-allitol (18: 1.006 g, 3.4 mmol) was dis-
solved 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 Ar. 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 chromatography (diethyl ether/petroleum ether 4:1 to
diethyl ether 100%) giving N-tert-butyloxycarbonyl-1,4-dideoxy-
(0.159 g, 0.4 mmol) in 1,2-dichloroethane (4 ml) at room tempera-
ture. After complete disappearance of reagents (reaction moni-
tored by TLC), the solution was poured into a satd aq soln of
NaHCO3 (5 ml per mmol). The organic phase was collected and
the aqueous phase extracted with EtOAc (10 ml per mmol, three
times). The combined organic extracts were washed with brine
(10 ml per mmol, once) and dried (MgSO4). Solvent evaporation
in vacuo and flash chromatography (light petroleum/EtOAc 4:1 to
2:1) gave pure 9 as light yellow oil (0.094 g, 0.2 mmol, 49% yield).
Ester 9 was added portionwise to a cooled suspension (0 °C) of
LiAlH4 (0.017 g, 0.4 mmol) in anhyd THF (4 ml). The solution was
stirred at room temperature for 3 h, then water was added drop-
wise (0.5 ml) and the mixture was filtered on silica gel conditioned
with EtOAc. After solvent evaporation in vacuo, amino alcohol 27
was obtained (0.077 g, 0.1 mmol, 82% yield). It was deprotected
without any further purification. 27 was dissolved in trifluoroace-
tic acid (80% aq, 1.5 ml) at 0 °C. The solution was stirred at 0 °C for
1 h and then at 20 °C for 3 h (reaction monitored by TLC, petroleum
ether/EtOAc 5:1). The solvent was evaporated in vacuo and pure 30
5,6-O-isopropylidene-1,4-imino-D-allitol (19: 0.760 g, 2.5 mmol,
73% yield) as white sol NaIO4 (0.5289 g, 2.5 mmol, 2.8 equiv) was
added to a solution of 19 (0.261 g, 0.9 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 EtOAc was added. The
two phases were separated and the aqueous phase was extracted
twice with EtOAc. The combined organic phases were washed with
brine, dried with MgSO4 and the solvent was evaporated in vacuo
giving pure tert-butyl(3aR,4R,6aS)-4-formyl-2,2-dimethyltetrahy-
dro-5H-[1,3]dioxolo-[4,5-c]pyr role-5-carboxylate (1: 0.209 g,

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3331
(0.0241 g, 0.06 mmol, 43% yield) was collected as a white foam
after flash chromatography on silica gel (CH3CN/NH4OH 8:1).
0.6 mmol) was dissolved in acetone (5 ml), K2CO3 (0.152,
1.1 mmol, 2 equiv) was added followed by allyl bromide
(0.092 ml, 0.1286 g, 1.1 mmol, 2 equiv) and the solution was stir-
red at 25 °C overnight. The solvent was evaporated in vacuo and
the crude was dissolved in EtOAc (5 ml). Water (5 ml) was added
and the two layers were separated. The aqueous phase was ex-
tracted with EtOAc (3 ꢀ 3 ml), the collected organic phases were
washed with brine (5 ml), dried on MgSO4 and the solvent was
evaporated in vacuo, giving methyl N-tert-butoxycarbonyl-4-
25
25
25
25
½aꢂ
¼ ꢁ4; ½
a
ꢂ
¼ ꢁ3; ½aꢂ
¼ ꢁ8; ½aꢂ
¼ ꢁ11 (c 0.241, MeOH).
589
577
435
405
UV (CH3CN): 260 (15449); 202 (29536). IR (solid): 3043, 1670,
1327, 1120, 824, 720. 1H NMR (400 MHz, MeOH-d4, bph = biphe-
nyl): d 7.77 (m, 2H bph) 7.69 (m, 4H bph) 7.49 (m, 2H bph) 4.20
(m, 1H, H-C(4)) 3.93 (dd, 3J = 8.0, 3J = 4.0, 1H, H-C(3)) 3.88 (dd,
2J = 7.6, 3J = 4.8, 1H CHCH2OH) 3.67 (m, 2H, CHCH2OH and 1H
CHCH2OH) 3.43 (m, 2H, H-C(2) and HH-C(5)) 3.27 (m, 1H, HH-
C(5) + MeOH) 2.94 (bd, 2J = 11.7, 1H, HH-C(6)) 2.81 (dd, 2J = 11.7,
3J = 8.8, 1H, HH-C(6)). 13C NMR (101 MHz, MeOH-d4, bph = biphe-
nyl): d 145.73 (s, C bph) 141.29 (s, C bph) 140.43 (s, C bph)
130.52 (q, 2J = 32, C(4’) bph) 129.44 (d, 1J = 158.9, 2CH bph)
128.63 (dd, 1J = 159, 3J = 64, 2CH bph) 128.50 (dd, 1J = 161, 3J = 63,
2 CH bph) 126.80 (d, 1J = 166.3, 2CH bph) 125.81 (q, 1J = 270, CF3)
74.75 (d, 1J = 143.7, C(3)) 70.92 (d, 1J = 152.4, C(4)) 68.39 (t,
1J = 109, CHCH2OH) 66.22 (d, 1J = 136.3, CHCH2OH) 63.33 (d,
1J = 145.6, C(2)) 50.71 (t, 1J = 148.9, C(5)) 47.29 (t, 1J = 124.0,
C(6)). HR-MALDI-TOF-MS: calcd for C20H23F3N2O3: 397.1739,
found 397.1744 ([M+H]+).
(prop-2-enyloxy)-D-phenylglycinate as a colorless oil (0.136 g,
25
589
25
577
25
435
0.4 mmol, 76% yield). ½
a
ꢂ
¼ ꢁ91; ½
aꢂ
¼ ꢁ99; ½
aꢂ
¼ ꢁ212;
25
405
½aꢂ
¼ ꢁ260 (c 0.1375, MeOH) IR (pure): 3375, 2977, 1745,
1713, 1611, 1586, 1510, 1457, 1437, 1391, 1366, 1342, 1304,
1245, 1166, 1054, 1025, 997, 926, 834, 781, 642, 592, 559, 552,
545, 538. 1H NMR (400 MHz, MeOH-d4): d 7.28 (d, 3J = 8.6, 2H
Ph) 6.90 (d, 3J = 8.6, 2H Ph) 6.05 (m, 1H, H-C(2) allyl) 5.51 (bd,
3J = 6.3, 1H, H–N) 5.41 (dd, 1H, 2J = 17.2, 3J = 1.4, HH-C(3) allyl)
5.31–5.26 (m, 2H, HH-C(3) allyl and H-C(2)) 4.53 (d, 3J = 5.3, 2H,
H2-C(1) allyl) 3.72 (s, 3H, OCH3) 1.44 (s, 9H, (CH3)3 t-butoxy). 13C
NMR (101 MHz, MeOH-d4): d 171.83 (s, C(1) C(O)OCH3) 158.72
(s, C(1) Boc) 154.83 (s, Ph) 133.0 (d, 1J = 155.5, Ph) 129.11 (s, Ph)
128.34 (d, 1J = 157.4, Ph) 117.77 (t, 1J = 156.6, C(3) allyl) 115.06
(d, 1J = 158.8, C(2) allyl) 79.95 (s, C t-butoxy) 68.78 (t, 1J = 142.9,
C(1) allyl) 56.94 (d, 1J = 141.7, C(2)) 52.62 (q, 1J = 147.0, O-CH3)
28.26 (q, 1J = 126.6, 3 ꢀ CH3 t-butoxy). HR-ESI-TOF-MS: calcd for
C12H15NO3: 322.1649, found 322.1638 ([M+H]+). Methyl N-tert-
(2R,3R,4S)-2{{{(1R)-2-Hydroxy-1-[4-(3-thienyl)phenyl]ethyl}-
amino}methyl}pyrrolidine-3,4-diol (31). NaBH(OAc)3 (0.138 g,
0.7 mmol, 1.7 equiv) was added portionwise to a stirred solution
of aldehyde 1 (0.119 g, 0.4 mmol) and amine 3 (0.110 g, 0.4 mmol)
in 1,2-dichloroethane (4.5 ml) at room temperature. After com-
plete disappearance of reagents (reaction monitored by TLC), the
solution was poured into a satd aq soln of NaHCO3 (5 ml). The or-
ganic phase was collected and the aqueous phase extracted with
EtOAc (3 ꢀ 5 ml). The combined organic extracts were washed
with brine (3 ml) and dried (MgSO4). Solvent evaporation in vacuo
and flash chromatography (light petroleum/EtOAc 4:1 to 2:1) gave
pure 10 as light yellow oil (0.089 g, 0.2 mmol, 46% yield). Ester 10
was added portionwise to a cooled suspension (0 °C) of LiAlH4
(0.015 g, 0.4 mmol) in anhyd THF (4.5 ml). The solution was stirred
at room temperature for 3 h, then water was added dropwise
(0.5 ml) and the mixture was filtered on silica gel conditioned with
EtOAc. After solvent evaporation in vacuo, the obtained amino
alcohol 28 (0.071 g, 0.2 mmol, 83% yield) was deprotected without
any further purification. Compound 28 was dissolved in trifluoro-
acetic acid (80% aq, 1.5 ml) at 0 °C. The solution was stirred at
0 °C for 1 h and then at 20 °C for 3 h (reaction monitored by TLC,
petroleum ether/EtOAc 5:1). The solvent was evaporated in vacuo
and pure 31 (0.038 g, 0.1 mmol, 73% yield) was collected as a white
foam after flash chromatography on silica gel (CH3CN/NH4OH 8:1).
butoxycarbonyl-4-(prop-2-enyloxy)-D-phenylglycinate
(0.136 g,
0.4 mmol) was dissolved in trifluoroacetic acid (80% aq, 4.2 ml) at
0 °C and then stirred at 25 °C for 4 h. The solvent was evaporated
in vacuo, the crude was dissolved in CH2Cl2 (2 ml) and neutralized
with 25% aq. NH3 (0.3 ml), the solvent was evaporated in vacuo and
the crude methyl 4-(prop-2-enyloxy)-
used in the subsequent reaction without any further purification.
Methyl 4-(phenylmethoxy)- -phenylglycinate (7). Methyl N-
tert-butoxycarbonyl-4-hydroxy- -phenylglycinate 21 (0.316 g,
D-phenylglycinate 6 was
D
D
1.1 mmol) was dissolved in acetone (5.5 ml), K2CO3 (0.458,
3.3 mmol, 3 equiv) was added followed by benzyl bromide
(0.26 ml, 0.375 g, 2.2 mmol, 2 equiv) and the solution was stirred
at 25 °C for 8 h (reaction monitored by TLC: petroleum ether/EtOAc
4:1). The solvent was evaporated in vacuo and the crude was dis-
solved in EtOAc (5 ml). Water (5 ml) was added and the two layers
were separated. The aqueous phase was extracted with EtOAc
(3 ꢀ 5 ml), the collected organic phases were washed with brine
(5 ml), dried on MgSO4 and the solvent was evaporated in vacuo,
25
589
25
577
25
435
25
405
½aꢂ
¼ þ15;
½aꢂ
¼ þ15;
½aꢂ
¼ þ18;
½aꢂ
¼ þ36 (c 0.062,
giving pure methyl N-tert-butoxycarbonyl-4-(phenylmethoxy)-D-
MeOH). UV (CH3CN): 265 (17169) 228 (17173). IR (pure): 3242,
2916, 2849, 1467, 1237, 1029, 779, 719. 1H NMR (400 MHz,
MeOH-d, Ph = phenyl and thi = thienyl): d 7.57 (d, 3J = 8.1, 2H Ph)
7.54 (m, 1H thi) 7.38 (m, 2H thi) 7.27 (d, 3J = 8.0, 2H Ph) 4.24 (m,
1H, H-C(4)) 3.94 (dd, 3J = 7.7, 3J = 4.1, 1H H-C(3)) 3.81 (dd,
3J = 8.1, 3J = 4.3, 1H, CHCH2OH) 3.71 (dd, 2J = 10.8, 3J = 4.3 1H,
CHCHHOH) 3.64 (dd, 2J = 10.8, 3J = 8.4, 1H, CHCHHOH) 3.47–3.49
(m, 2H, H-C(2) and HH-C(5)) 3.23 (dd, 2J = 12.5, 3J = 2.0, 1H, HH-
C(5)) 2.93 (dd, 2J = 13.3, 3J = 4.3, 1H, HH-C(6)) 2.75 (dd, 2J = 13.3,
3J = 8.8, 1H, HH-C(6)). 13C NMR (101 MHz, MeOH-d, Ph = phenyl
and thi = thienyl): d 141.35 (s, 1 arom. C) 136.77 (s, 1 arom. C)
133.79 (s, 1 arom. C) 130.44 (d, 1J = 132.6, 2CH Ph) 128.36 (d,
1J = 154.3, CH thi) 127.49 (d, 1J = 170.6, 2CH Ph) 126.93 (d,
1J = 194,9 CH thi) 123.06 (d, 1J = 184.1, CH, thi) 74.99 (d,
1J = 148.1, C(3)) 70.23 (d, 1J = 151.8, C(4)) 65.19 (d, 1J = 196.3,
CHCH2OH) 64.21 (t, 1J = 148.1, CHCH2OH) 59.38 (d, 1J = 137.0,
C(2)) 51.36 (t, 1J = 144.4, C(5)) 46.03 (t, 1J = 85.2, C(6)). HR-MAL-
DI-TOF-MS: calcd for C17H22N2O3S: 335.1429, found 335.1434
([M+H]+).
phenylglycinate as a colorless oil (0.325 g, 0.9 mmol, 81% yield).
1H NMR (400 MHz, MeOH-d4): d 7.36 (d, 3J = 8.4, 2H Ph) 7.33 (t,
3J = 7.3, 2H Ph) 7.31 (m, 3H Ph) 7.0 (d, 3J = 8.6, 2H Ph) 5.16 (s, 1H,
H-C(2)) 5.10 (s, 2H, H2C-Ph) 3.71 (s, 3H, CH3). Methyl N-tert-butoxy-
carbonyl-4-(phenylmethoxy)-D-phenylglycinate (0.104 g, 0.3 mmol)
was dissolved in trifluoroacetic acid (80% aq, 2.8 ml) at 0 °C and
then stirred at 25 °C for 3 h. The solvent was evaporated in vacuo,
the crude was dissolved in CH2Cl2 (2 ml) and neutralized with 25%
aq NH3 (0.3 ml), the solvent was evaporated in vacuo and the pure
7 (0.0744 g, 0.3 mmol, quantitative) was obtained as a white foam
after flash column chromatography on silica gel (CH2Cl2/MeOH
9:1). 1H NMR (400 MHz, CDCl3, Ph = phenyl): d 7.42–7.29 (m, 7H
Ph) 6.96 (d, 3J = 8.7, 2H Ph) 5.07 (s, 2H, H2C-Ph) 4.58 (s, 1H, H-
C(2)) 3.71 (s, 3H, CH3). Analytical and spectroscopical data are in
accord with those reported in literature.54
Methyl 4-methoxyphenyl-
D-glycinate (5). 1 N aq NaOH (60 ml)
was added to a solution of 4-hydroxy-D-phenylglycine 20 (9.95 g,
59 mmol) in dioxane/water (1:1, 120 ml). A solution of di-tert-bu-
tyl dicarbonate (14.33 g, 77 mmol, 1.3 equiv) in dioxane (60 ml)
was added and the pale yellow solution was stirred at 20 °C over-
night. The dioxane was evaporated in vacuo and the aqueous phase
Methyl 4-(prop-2-enyloxy)-D-phenylglycinate (6). Methyl N-
tert-butoxycarbonyl 4-hydroxy-
D
-phenylglycinate 21 (0.154 g,

3332
C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
was washed with diethyl ether (25 ml). KHSO4 was added until pH
2.5 and the aqueous layer was extracted with EtOAc (3 ꢀ 30 ml),
dried (MgSO4) and the solvent was evaporated in vacuo. Pure N-
CHCH2OH and CHCH2OH) 3.12 (dd, 2J = 12.3, 3J = 5.2, 1H, HH-C(5))
3.03 (m, 1H, H-C(2)) 2.81 (dd, 2J = 12.3, 3J = 3.4, 1H, HH-C(5)) 2.63
(dd, 2J = 12.0, 3J = 5.2, 1H, HH-C(6)) 2.52 (dd, 2J = 12.0, 3J = 7.6, 1H,
HH-C(6)). 13C NMR (101 MHz, MeOH-d4, Ph = phenyl, all = allyl): d
159.57 (s, C Ph) 134.98 (s, C Ph) 133.99 (d, 1J = 21.3, C(2) all)
129.79 (d, 1J = 156.0, 2 CH Ph) 117.40 (t, 1J = 158.1, C(3) all)
115.83 (d, 1J = 158.0, 2 CH Ph) 76.91 (d, 1J = 141.9, C(3)) 72.58 (d,
1J = 148.0, C(4)) 69.79 (t, 1J = 144.7, C(1) all) 67.81 (t, 1J = 141.2,
CHCH2OH) 66.04 (d, 1J = 132.8, CHCH2OH) 62.81 (d, 1J = 137.9,
C(2)) 52.26 (t, 1J = 138.9, C(5)) 50.88 (t, 1J = 138.3, C(6)). HR-ESI-
TOF-MS: calcd for C16H24N2O4 309.1814, found 309.1818 [M+H]+
and 331.1044 [M+Na]+.
tert-butoxycarbonyl-4-hydroxy-
white solid after recristallization from EtOAc (13.36 g, 50 mmol,
85% yield). N-tert-Butoxycarbonyl-4-hydroxy- -phenylglycine
D-phenylglycine was recovered as
D
(13.36 g, 50 mmol) was dissolved in DMF (450 ml) at 0 °C. K2CO3
(34.51 g, 250 mmol, 5 equiv) and then, dropwise, MeI (12.5 ml,
28.5 g, 201 mmol, 4 equiv) were added and the solution was stirred
at 0 °C for 2 h (monitored by TLC CH2Cl2/MeOH 7:3). Satd aq soln of
NaHCO3 (500 ml) was added and the solution was extracted with
diethyl ether (3 ꢀ 100 ml). The collected organic layers were
washed with brine, dried over MgSO4 and the solvent was evapo-
rated in vacuo. Pure methyl N-tert-butoxycarbonyl-4-methoxy-
(2R,3R,4S)-2{{(1R)-2-Hydroxy-1-{4-[(phenylmethoxy)phenyl]-
hydroxyethyl}amino}methyl} pyrrolidine-3,4-diol (33). NaBH(OAc)3
(0.094 g, 0.4 mmol) was added portionwise to a stirred solution of
aldehyde 1 (0.133 g, 0.5 mmol) and amine 7 (0.075 g, 0.3 mmol) in
1,2-dichloroethane (3.5 ml) at room temperature. After complete
disappearance of reagents (reaction monitored by TLC), the solution
was poured into a satd aq soln of NaHCO3 (5 ml). The organic phase
was collected and the aqueous phase extracted with EtOAc
(3 ꢀ 5 ml). The combined organic extracts were washed with brine
(5 ml) and dried (MgSO4). Solvent evaporation in vacuo and flash
chromatography (light petroleum/EtOAc 4:1 to 2:1) gave pure 13
as colorless oil (0.065 g, 0.1 mmol, 41% yield). This compound was
added portionwise to a cooled suspension (0 °C) of LiAlH4 (0.012,
0.3 mmol) in anhyd THF (3.2 ml). The solution was stirred at room
temperature for 3 h, then water was added dropwise (0.5 ml) and
the mixture was filtered on silica gel conditioned with EtOAc. After
solvent evaporation in vacuo, tert-butyl(3aR,4R,6aS)-2,2-dimethyl-
4{{{(1R)-2-hydroxy-1-[4-(phenylmethoxy)phenyl]ethyl}amino}
methyl}-tetrahydro-3aH-[1, 3]dioxolo[4,5-c]pyrrole-5-carboxylate
was obtained (0.060 g, 0.1 mmol, quant.). It was deprotected with-
out any further purification. tert-Butyl(3aR,4R,6aS)-2,2-dimethyl-
4{{{(1R)-2-hydroxy-1-[4-(phenylmethoxy)phenyl]ethyl}amino}
methyl}-tetrahydro-3aH-[1, 3]dioxolo[4,5-c]pyrrole-5-carboxylate
(0.0598 g, 0.12 mmol) was dissolved in trifluoroacetic acid (80% aq,
1.3 ml) at 0 °C and then stirred at 25 °C for 3 h. The solvent was evap-
orated in vacuo, the crude was dissolved in CH2Cl2 (2 ml) and neu-
tralized with 25% aq NH3 (0.3 ml), the solvent was evaporated in
vacuo and the pure 33 (0.017 g, 0.05 mmol, 42% yield) was obtained
as a pale yellow foam after flash column chromatography on silica
phenyl-D-glycinate was recovered as white foam (7.24 g,
24.4 mmol, 49% yield) after column chromatography on silica gel
(pentane/EtOAc 3:1). 1H NMR (400 MHz, MeOH-d4): d 7.27 (d,
3J = 8.4, 2H, Ph) 6.90 (d, 3J = 8.4, 2H, Ph) 5.13 (s, 1H, H-C(2)) 3.78
(s, 3H, p-OCH3) 3.69 (s, 3H, CH3-OC(1)) 1.44 (s, 9H, (CH3)3 t-but-
oxy). Methyl N-tert-butoxycarbonyl-4-methoxyphenyl-D-glycinate
(0.253 g, 0.9 mmol) was dissolved in trifluoroacetic acid (80% aq,
8.5 ml) at 0 °C and the mixture was stirred for 2 h at 20 °C. The sol-
vent was evaporated in vacuo and pure methyl 4-methoxy-D-phe-
nylglycinate 5 was obtained quantitatively after flash column
chromatography on silica gel (CH2Cl2/MeOH from 95:5 to 9:1).
Analytical and spectroscopic data were in accord to those reported
in the literature.54
(2R,3R,4S)-2{{{(1R)-2-Hydroxy-1-[4-(prop-2-enyloxy)phenyl]-
ethyl}amino}methyl} pyrrolidine-3,4-diol (32). NaBH(OAc)3
(0.141 g, 0.7 mmol) was added portionwise to a stirred solution
of aldehyde 1 (0.129 g, 0.5 mmol) and amine 6 (0.169 g, 0.4 mmol)
in 1,2-dichloroethane (5 ml) at room temperature. After complete
disappearance of reagents (reaction monitored by TLC), the solu-
tion was poured into a satd aq soln of NaHCO3 (3 ml). The organic
phase was collected and the aqueous phase extracted with EtOAc
(3 ꢀ 5 ml). The combined organic extracts were washed with brine
(5 ml) and dried (MgSO4). Solvent evaporation in vacuo and flash
chromatography (light petroleum/EtOAc 4:1 to 2:1) gave pure 12
as light yellow oil (0.085 g, 0.2 mmol, 43% yield). tert-Butyl (3aR,4-
R,6aS)-2,2-dimethyl-4{{{(1R)-2-methoxy-2-oxo-1-[4-(prop-2-eny-
loxy)phenyl]ethyl}amino}methyl}-tetrahydro-3aH-[1,3]dioxolo[4,
5-c]pyrrole-5-carboxylate (12: 0.085 g, 0.2 mmol) was added por-
tionwise to a cooled suspension (0 °C) of LiAlH4 (0.014, 0.4 mmol)
in anhyd THF (4 ml). The solution was stirred at room temperature
for 3 h, then water was added dropwise (0.5 ml) and the mixture
was filtered on silica gel conditioned with EtOAc. After solvent
evaporation in vacuo, tert-butyl(3aR,4R,6aS)-2,2-dimethyl-4{{{(1R)-
2-hydroxy-1-[4-(prop-2-enyloxy)phenyl]ethyl}amino}methyl}-
tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate was ob-
tained (0.081 g, 0.2 mmol, quantitatively). It was deprotected with-
out any further purification. tert-Butyl(3aR,4R,6aS)-2,2-dimethyl-
4{{{(1R)-2-hydroxy-1-[4-(prop-2-enyloxy)phenyl]ethyl}amino}meth-
yl}-tetrahydro-3aH-[1,3]dioxolo[4,5-]pyrrole-5-carboxylate (0.081 g,
0.2 mmol) was dissolved in trifluoroacetic acid (80% aq, 2.8 ml) at
0 °C and then stirred at 25 °C for 3 h. The solvent was evaporated
in vacuo, the crude was dissolved in CH2Cl2 (2 ml) and neutralized
with 25% aq NH3 (0.3 ml), the solvent was evaporated in vacuo and
the pure 32 0.0241 g, 0.08 mmol, 44% yield) was obtained as a
25
589
25
577
25
435
gel (CH3CN/NH4OH 6:1). ½
a
ꢂ
¼ ꢁ6; ½
aꢂ
¼ ꢁ6; ½
a
ꢂ
¼ ꢁ13;
25
405
½aꢂ
¼ ꢁ16 (c 0.342, MeOH). UV (CH3CN): 275 (1754); 226
(23510). IR (solid (KBr), cmꢁ1): 3420, 2923, 1384, 1244, 1014, 839,
744, 697. 1H NMR (400 MHz, MeOH-d): d 7.44 (m, 2Ph) 7.38 (m, 2H
Ph) 7.32 (m, 1H Ph) 7.29 (d, 3J = 8.5, 2H Ph) 7.00 (d, 3J = 8.5, 2H Ph)
5.09 (s, 2H, H2-C-Ph) 4.10 (m, 1H, H-C(4)) 3.73 (m, 2H, CHCH2OH
and H-C(3)) 3.62 (dd, 2J = 10.8, 3J = 4.4, 1H, CHCH2OH) 3.56 (dd,
2J = 10.8, 3J = 8.8, 1H CHCH2OH) 3.24 (dd, 2J = 12.2, 3J = 4.5, 1H, HH-
C(5)) 3.18 (m, 1H, H-C(2)) 2.96 (d, 2J = 12.2, 1H, HH-C(5)) 2.74 (dd,
2J = 12.3, 3J = 4.7, 1H, HH-C(6)) 2.61 (dd, 2J = 12.3, 3J = 7.9, 1H, HH-
C(6)). 13C NMR (100 MHz, MeOH-d): d 158.82 (s, C Ph) 137.75 (s, C
Ph) 132.90 (s, C Ph) 128.71 (d, 1J = 159.9 2CH Ph) 128.51 (dd,
1J = 159.9, 2J = 7.0, 2CH Ph) 127.86 (d, 1J = 86.3, 2CH Ph) 127.49 (d,
1J = 157.1, CH Ph) 115.11 (d, 1J = 158.2, 2CH Ph) 74.40 (d, 1J = 143.3,
C(3)) 70.58 (d, 1J = 150.8, C(4)) 69.96 (t, 1J = 142.0, CHCH2OH) 66.59
(t, 1J = 141.0, H2C-Ph) 65.00 (d, 1J = 132.8, CHCH2OH) 61.81 (d,
1J = 142.5, C(2)) 51.11 (t, 1J = 142.3, C(5)) 48.38 (t, 1J = 134.5, C(6)).
HR-ESI-TOF-MS: calcd for C20H26N2O3: 359.1971, found 359.1969
([M+H]+).
white solid after flash column chromatography on silica gel
25
589
(CH3CN/NH4OH 8:1). Melting point: 160–164° C (dec) ½
aꢂ
¼
25
577
25
435
þ6; ½aꢂ
¼ þ22; ½
aꢂ
¼ þ21 (0.241 g/100 ml, MeOH). IR (pure):
(2R,3R,4S)-2{{[(1R)-2-Hydroxy-1-(4-methoxyphenyl)ethyl]amino}-
methyl}pyrrolidine-3,4-diol (34). NaBH(OAc)3 (0.169 g, 0.8 mmol)
3419, 1384, 1243, 1014, 839,697. 1H NMR (400 MHz, MeOH-d4, al-
l = allyl): d 7.25 (d, 3J = 8.6, 2H, Ar-H) 6.89 (d, 3J = 8.6, 2H, Ar-H) 6.04
(m, 1H, CH2 = CHCH2O) 5.38 (d, 2J = 17.8, 1H, Htrans-C(3) all) 5.22 (d,
2J = 10.0, 1H, Hcis-C(3) all) 4.52 (d, 3J = 5.12, 2H, H2C(1) all) 3.69 (dd,
3J = 8.4, 3J = 4.4, 1H, H-C(3)) 4.01 (m, 1H, H-C(4)) 3.58 (m, 3H
was added portionwise to
a stirred solution of aldehyde 1
(0.168 g, 0.6 mmol) and amine 5 (0.166 g, 0.9 mmol) in 1,2-dichlo-
roethane (6 ml) at room temperature. After complete disappear-
ance of reagents (reaction monitored by TLC), the solution was

C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
3333
poured into a satd aq soln of NaHCO3 (5 ml). The organic phase was
collected and the aqueous phase extracted with EtOAc (3 ꢀ 5 ml).
The combined organic extracts were washed with brine (5 ml)
and dried (MgSO4). Solvent evaporation in vacuo and flash chroma-
tography (light petroleum/EtOAc 4:1 to 2:1) gave pure 11 as as a
yellow oil (0.144 g, 0.3 mmol, 52% yield). This product was added
Ficoll (Biochrom) density gradient centrifugation. The mononu-
clear cell fraction was collected, washed with phosphate-buffered
saline (PBS) and immediately seeded in RPMI 1640-based culture
medium. B-CLL cells, as detected by CD19 and CD23 expression,
were >60% in all of the samples used for this study. Similarly, the
purity of multiple myeloma cells was detected by staining with
an anti-CD138 antibody and flow cytometry.
portionwise to
a cooled suspension (0 °C) of LiAlH4 (0.038,
1.0 mmol) in anhyd THF (8 ml). The solution was stirred at room
temperature for 3 h, then water was added dropwise (0.5 ml) and
the mixture was filtered on silica gel conditioned with EtOAc. After
solvent evaporation in vacuo, tert-butyl (3aR, 4R, 6aS)-2,2-di-
methyl-4{{{(1R)-2-hydroxy-1-[4-(methoxyphenyl)phenyl]-ethyl}
amino}methyl}-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrole-5-car-
boxylate so obtained (0.070 g, 0.2 mmol, 53% yield) was deprotec-
ted without any further purification. Tert-butyl(3aR,4R,6aS)-2,
2-dimethyl-4{{{(1R)-2-hydroxy-1-[4-(methoxyphenyl)phenyl]ethyl}
amino}methyl}-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrole-5-car-
boxylate (0.070 g, 0.2 mmol) was dissolved in trifluoroacetic acid
(80% aq, 1.7 ml) at 0 °C and then stirred at 25 °C for 3 h. The solvent
was evaporated in vacuo, the crude was dissolved in dichlorometh-
ane (2 ml) and neutralized with 25% aq NH3 (0.3 ml), the solvent
was evaporated in vacuo and the pure 34 (0.0370 g, 0.13 mmol,
4.4. Viability assays
5 ꢀ 103 cells/well were plated in 200
plates. 48 h later, the dihydroxypyrrolidine derivative was added
to the wells at concentrations ranging between 1 and 400 M, such
ll medium in 96-well
l
that the vehicle DMSO never exceeded 0.4%. Viability was deter-
mined 72 h later using CellTiter 96 Aqueous1 (Promega Italia, Mi-
lan, Italy) according to the manufacturer’s instructions. Incubation
times with CellTiter96 Aqueous1 ranged between 2 and 4 h. Plates
were read with a spectrophotometer (Labsystems iEMS Reader MF)
at 490 nm wave length. IC50s were estimated using GraphPad
Prism4. For apoptosis detection using Annexin V and propidium io-
dide, 2 ꢀ 106 cells/well were plated in a 24-well plate and stimu-
lated with different concentrations of novel compound. 24 h later,
cells were harvested, washed with PBS and stained with FITC-con-
jugated Annexin V (Becton Dickinson, BD Italia, Milan, Italy) and
76% yield) was obtained as a white foam after flash column chro-
25
589
matography on silica gel (CH3CN/NH4OH 6:1).
½
aꢂ
¼ ꢁ19;
25
25
25
½aꢂ
¼ ꢁ13;
½aꢂ
¼ ꢁ29;
½aꢂ
¼ ꢁ35 (c 0.185, MeOH) UV
propidium iodide (2 lg/ml) according to the manufacturer’s
instructions. FACS analysis was done on a FACS Calibur (Becton
Dickinson) by acquiring 20000 events.
577
435
405
(CH3CN): 277 (1447). IR (pure): 3298, 3270, 2874, 2833, 1512,
1245, 1114, 1025, 807. 1H NMR (400 MHz, MeOH-d4): d 7.29 (d,
3J = 8.8, 2H Ph) 6.93 (d, 3J = 8.8, 2H Ph) 4.05 (m, 1H, H-C(4)) 3.80
(s, 3H, H3-CO) 3.75 (dd, 3J = 9.0, 3J = 4.6, 1H, H-C(3)) 3.62 (m, 3H,
CHCH2OH and 2H CHCH2OH) 3.14 (dd, 2J = 12.0, 3J = 5.2, 1H, HH-
C(5)) 3.07 (m, 1H, H-C(2)) 2.84 (dd, 2J = 12.2, 3J = 3.2, 1H, HH-
C(5)) 2.66 (dd, 2J = 11.8, 3J = 4.6, 1H, HH-C(6)) 2.53 (dd, 2J = 11.8,
3J = 7.8, 1H, HH-C(6)). 13C-NMR (101 MHz, MeOH-d4): d 160.38 (s,
C Ph) 133.52 (s, C Ph) 129.85 (d, 1J = 157.4, 2 CH Ph) 115.01 (d,
1J = 158.1, 2CH Ph) 76.95 (d, 1J = 143.5, C(3)) 72.50 (d, 1J = 149.1,
C(4)) 67.54 (t, 1J = 140.5, CHCH2OH) 65.77 (d, 1J = 133.9, CHCH2OH)
62.40 (d, 1J = 137.3, C(2)) 55.86 (q, 1J = 142.7, CH3OH) 51.95 (t,
1J = 139.7, C(5)) 51.00 (t, 1J = 133.9, C(6)). HR-ESI-TOF-MS: calcd
for C14H22N2O4: 283.1658, found 283.1658 ([M+H]+) and
305.1393 ([M+Na]+).
4.5. Cell cycle analysis
For cell cycle analysis, cells were resuspended in a buffer con-
taining 0.1% sodium citrate, 0.1% TritonX, and 50 lg/ml propidium
iodide. Cell cycle analysis was performed on a FACS Calibur using
ModFit LT software.
4.6. Microscopy
Cells were imaged using the 40X magnification of a Zeiss AXIO-
VERT200 microscope, camera Qlympus C-4040ZOOM. The image
files were downloaded using the software Olympus CAMEDIA
Master 2.5.
4.2. Cells lines
5.6 RNA extraction and Taqman low-density arrays (TLDA).
Total RNA was isolated from cell lysates using QIAGEN
RNeasy Mini anion-exchange spin columns (QIAGEN, Hilden,
Germany) according to the instructions of the manufacturer. To-
tal RNA (1 lg) was subjected to a 20-lL cDNA synthesis reaction
using SuperScript RTII (Invitrogen, Karlsruhe, Germany). Oli-
go(dT) was used as primer. PCR amplification was performed
using 2 lL cDNA TLDA were done for a panel of 90 genes that
were previously selected for their relevance in cancer biology
and metastasis. For these experiments, SKBR3 and MCF7 cells
U266, A549, MCF7, SKBR3, MDA-MB-231, BT474, U87 and PC3
cells were obtained from American Type Culture Collection (ATCC,
Rockville, MD, USA). Jurkat T cells engineered to stably overexpress
Bcl2 and the respective vector control cells were a gift of Dr. Claus
Belka (Department of Radiation Oncology, University of Tuebingen,
Tuebingen, Germany). Adherent cell lines were grown in 10-cm
culture dishes in McCoy’s medium (Sigma Chemical Co., USA) sup-
plemented with 10% (v/v) heat-inactivated fetal bovine serum
(FCS, GIBCO, Grand Island, NY),
L
-glutamine and antibiotics. U266
were plated in 6-well plates and treated for 12 h with 50 lM
and Jurkat cells were maintained in RPMI-1640 medium (Sigma
Chemical Co., USA) supplemented with 10% (v/v) fetal bovine ser-
of novel drug. Thereafter, cells were washed and used for RNA
extraction. cDNA was generated using random hexamers and
used for multiple quantitative PCR (Q-PCR) as described previ-
ously.55 TLDA were performed on a 7900HT Fast Real Time
PCR System using primer collections assembled by Agilent. Re-
sults were plotted as mRNA fold induction in drug-treated ver-
um, -glutamine and antibiotics. Cells were maintained at 37 °C
L
in a humidified 5% CO2 atmosphere. The dihydroxypyrrolidine
derivative was weighted and dissolved in DMSO to prepare a
100 mM stock solution.
DD
sus vehicle-treated cells (calcd as 2ꢁ CT).56 Q-PCRs were
4.3. B-CLL and multiple myeloma cell isolation
performed in duplicate for each gene. RPLP0 was used as an
housekeeping gene to normalized the results.
Peripheral blood (PB) samples from B-chronic lymphocytic leu-
kemia (B-CLL) patients, and bone marrow (BM) samples from mul-
tiple myeloma patients were obtained from routine diagnostic
samples after informed consent of patients and permission by
the local ethics committee. PB and BM samples were subjected to
4.7. Immunoblotting
Bcl2 expression in Jurkat cells was evaluated by immunoblot-
ting as previously described.47

3334
C. Bello et al. / Bioorg. Med. Chem. 18 (2010) 3320–3334
27. Popowycz, F.; Gerber-Lemaire, S.; Demange, R.; Rodriguez-Garcia, E.; Asenjo, A.
Acknowledgments
T.; Robina, I.; Vogel, P. Bioorg. Med. Chem. Lett. 2001, 11, 2489.
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Juillerat-Jeanneret, L. J. Med. Chem. 2005, 48, 4237.
This work was supported by grants from the Associazione Itali-
ana Leucemie (AIL) and University of Genoa, Italy. We thank also
the Swiss National Science Foundation (Bern) for generous finan-
cial support.
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