Preparation and evaluation of the in vitro erythroid differentiation induction properties of some esters of methyl 3,4-O-isopropylidene-β-D-galactopyranoside and 2,3-O-isopropylidene-D-mannofuranose

Article abstract of DOI:10.1016/S0968-0896(01)00285-1

Two series of glycide esters of short fatty acids, designed for avoiding intramolecular transesterification, were prepared and tested for in vitro erythroid differentiation induction activities using the K562 cell line as experimental system. The 6-O-isob

Full text of DOI:10.1016/S0968-0896(01)00285-1

Bioorganic & Medicinal Chemistry 10 (2002) 347–353
Preparation and Evaluation of the In Vitro Erythroid
Differentiation Induction Properties of Some Esters of Methyl
3,4-O-Isopropylidene-ꢀ-D-galactopyranoside and
2,3-O-Isopropylidene-D-mannofuranose
Giorgio Catelani,a,* Felicia D’Andrea,a Ettore Mastrorilli,a Nicoletta Bianchi,b
Cristiano Chiarabelli,b Monica Borgatti,b Dino Martellob and Roberto Gambarib,*
a
`
Dipartimento di Chimica Bioorganica e Biofarmacia, Universita di Pisa, Via Bonanno, 33-56126, Pisa, Italy
`
Dipartimento di Biochimica e Biologia Molecolare, Universita di Ferrara, Via L. Borsari, 46-44100, Ferrara, Italy
b
Received 9 May 2001; accepted 6 August 2001
Abstract—Two series of glycide esters of short fatty acids, designed for avoiding intramolecular transesterification, were prepared
and tested for in vitro erythroid differentiation induction activities using the K562cell line as experimental system. The 6- O-iso-
butiryl and pivaloyl derivatives of methyl 3,4-O-isopropylidene-b-d-galactopyranosides as well the same 1-O-esters of 2,3-O-iso-
propylidene-a- and b-d-mannofuranose exhibit biological activities much higher that the corresponding acids and could be
proposed as possible agents to modulate production of embryo-fetal hemoglobins by human erythroid cells. # 2001 Elsevier
Science Ltd. All rights reserved.
Introduction
mine the therapeutic potential of new differentiating
compounds.10 K562cells exhibit a low proportion of
hemoglobin-synthetizing cells under standard cell
growth conditions, but are capable to undergo erythroid
differentiation when treated with a variety of com-
pounds, including hemin, cytosine arabinoside (ara-C),
butyric acid, 5-azacytidine, mithramycin, and chromo-
Pharmacologically-mediated regulation of the expres-
sion of human g-globin genes has been suggested in the
search of potential therapeutic agents in hematological
disorders, since increases in fetal Hb levels can be clini-
cally beneficial in sickle cell disease1 and b-thalassemia.2
Therefore, a number of studies have been performed to
identify compounds able to stimulate g-globin gene
expression.3 Among possible biological response modi-
fiers, compounds of great interest are short fatty
acids.4,5 Accordingly, butyrates have been used in ther-
apeutic trials to stimulate fetal hemoglobin gene
expression in patients with hemoglobinopathies.6,7
mycin,
cisplatin
and
cisplatin
analogues,
tallimustine.9ꢀ15 Following erythroid induction of K562
cells, a sharp increase of cytoplasmic accumulation of
Hb Portland (z2g2) and Hb Gower 1 (z2e2) is observed,
accompanied by an increase in the expression of human
e and g globin genes.11ꢀ15
Interestingly, erythroid inducers of K562cells, for
instance hydroxyurea, butyric acid, hemin and talli-
mustine, were found to be able to stimulate the increase
HbF production by normal erythroid precursor
cells.16ꢀ20 Therefore, K562cells represent an experi-
mental system for a first screening of compounds of
possible interest in the pharmacological treatment of
hemoglobinathies, including b-thalassemia.15
The K562cell line, isolated and characterized by Lozzio
and Lozzio8 from a patient with chronic myelogenous
leukemia in blast crisis, has been proposed as a very
useful in vitro model system (a) to study the molecular
mechanism(s) regulating the expression of embryonic
and fetal human globin genes,9 as well as (b) to deter-
We have recently reported21 the synthesis of 123- O-
acyl-1,2-O-isopropylidene-d-glucofuranose derivatives,
*Corresponding authors. Tel.: +39-050-44074; fax: +39-050-43321;
e-mail: glocate@farm.unipi.it
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00201-2

348
G. Catelani et al. / Bioorg. Med. Chem. 10 (2002) 347–353
and tested their effects in augmenting the proportion of
benzidine-positive (hemoglobin-containing) cells in
treated K562cell populations. The results obtained
demonstrated that two of the newly synthesised com-
pounds were potent inducers of erythroid differentiation
of K562cells. In addition, these same compounds
potentiate erythroid induction of K562cells treated
with sub-optimal concentrations of cytosine arabinoside
(ara-C), retinoic acid and mithramycin.21 Table 1 shows
a summary of the results obtained with the compounds
1a, 1b and 1c. Compounds 1b and 1c were considered of
interest for further experiments aimed at the induction
of g-globin gene expression.21
Unfortunately, a rapid intramolecular acyl shift from
O-3 to O-6 was observed both under chemical22 and
biological23 catalysis for 1b. A similar behaviour was
previously reported by Villa et al.24 for 1a. In order to
circumvent this unwanted modification of our a-bran-
ched glycide esters, we have now prepared some other
derivatives that, owing to the unfavourable relative
orientation of the acyl and the free hydroxyl groups, are
expected to be not subjected to intramolecular transes-
terification. We present in this paper the preparation of
some esters of the d-galactopyranoside and the d-man-
nofuranose series, and their biological activity as ery-
throid differentiation agents on human K562cells.
Chemistry
The preparation of methyl 2-O-acyl-3,4-O-iso-
propylidene-b-d-galactopyranosides (4) was achieved
from the mixed diacetal 225 employing a one pot proce-
dure involving the esterification of 2 with the appro-
priate acyl chloride in toluene and trietylamine,
followed by removal of the acyclic acetal function with
pyridinium tosylate in methanol. Analytically pure
compounds 4a–c were thus obtained with satisfactory
yield (53–65%) after flash chromatography of the crude
reaction mixtures followed by crystallization. The 6-O-
acyl isomers 5a–c were obtained from the benzyl ether
3,26 that was routinely 6-O-acylated and debenzylated
by hydrogenolysis with 10% Pd(OH)2 on charcoal in
ethyl acetate. Compounds 5a–c were finally obtained in
excellent overall yields (94–96%) by flash chromato-
graphy.
Table 1. Effects of 3-O-acyl-1,2-O-isopropylidene-d-glucofuranose
derivatives on in vitro growth and erythroid differentiation of human
leukemic K562cells
Compound
Cell proliferation
IC50 (mM)
Erythroid inductiona
% of benzidine-positive
cells (mM)
1a
1b
1c
0.8
2.5
1.5
25.4ꢁ3.8 (2mM)
54.2ꢁ8.2(4 mM)
52.8ꢁ2.9 (3 mM)
The preparation of the 1-O-acyl-2,3-O-isopropylidene-
a- and-b-d-mannofuranose derivatives (8 and 9) was
performed, with minor modifications, following the
procedure described by Baldwin et al.24a for 8a. The
composition of the anomeric ester mixtures obtained by
aResults are presented as maximum level of % of benzidine-positive
(hemoglobin-containing) cells ꢁSD, reached within a 9 days induction
period at the indicated concentrations of tested compounds.

G. Catelani et al. / Bioorg. Med. Chem. 10 (2002) 347–353
349
treatment of commercial 6 (Sigma) with the appropriate
1
mL streptomycin.11 Cell growth was studied by deter-
mining the cell number/mL after different days of in
vitro cell cultures.11 In order to study the ability of the
compounds to induce erythroid differentiation, K562
cells were cultured in the absence or in the presence of
the indicated concentrations of the studied esters of
acyl chloride, was esthablished by H NMR analysis on
the basis of the integration of the signals of the anome-
ric protons (see Experimental). The reaction of 6 with
butanoyl chloride led, in agreement with the results by
Baldwin et al.,24a to a larger amount of a anomer (a/b
ratio >9:1), while with 2-methylpropanoyl chloride the
proportion of the a anomer decreased (a/b ratio 3:1),
and, finally, with 2,2-dimethylpropanoyl chloride an
inverted prevalence of the b anomer was found (a/b
ratio 4:6). The selective hydrolysis of the 5,6-O-iso-
propylidene group of derivatives 7 was performed with
CuCl2 in ethanol, as previously proposed by Iwata and
Ohrui.27 In the case of the a anomers, a-7a–c, the rate of
the hydrolysis was rather slow, requiring about 16 h for
the complete disappearance of the starting products,
and the selectivity of the process was good giving the
partially deprotected products 8a–c in high yields
(about 80%). Conversely, the b anomers reacted faster
(about 4 h) but the yields of the desired 2,3-mono-
isopropylidene derivatives 9b,c were rather low (about
40%), some lower moving products, probably the com-
pletely deisopropylidenated derivatives, being formed.
methyl
3,4-O-isopropylidene-b-d-galactopyranoside
and 2,3-O-isopropylidene-d-mannofuranose, and most
of the corresponding fatty acids, ara-C, tallimustine and
mithramycin. The percent of benzidine-positive K562
cells during the induction was determined daily in order
to quantify the percent of hemoglobin-containing
cells.15 As clearly evident, from the results presented in
Table 2, most of the compounds (for instance 5b, 5c, 8b,
8c, 9c) showed capacity to induce erythroid differentia-
tion (percent of benzidine positive cells higher than
40%) of K562cells, although to a lower level when
compared to ara-C,12 tallimustine15 and mithramycin13
(Table 2) as well as other inducing compounds, includ-
ing cisplatin and cisplatin analogues14 (data not shown).
By contrast, other tested compounds (for instance, 4a–c,
5a, 8a, 9b) showed lower activity. Interestingly, in
agreement with our previous observations21 compounds
with R=CH(CH3)2 and R=C(CH3)3 exhibited high
activity in inducing erythroid differentiation of K562
cells. In addition, Table 2shows also that most of the
newly synthetized esters of methyl 3,4-O-iso-
Biological Studies
Human myeloid leukemia K562(S) cells10 were main-
tained in RPMI 1640 (Gibco, BRL, Milan, Italy) in
10% fetal bovine serum (Gibco, BRL, Milan, Italy), 5%
CO2 supplemented with 50 units/mL penicillin, 50 mg/
propylidene-b-d-galactopyranoside
and
2,3-O-iso-
propylidene-d-mannofuranose cause a decrease in the
proliferation efficiency of K562cells. 50% inhibition of
cell growth (IC50) occurs when K562cells are cultured
for 4 days in the presence of drugs concentrations ran-
ging from 5 mM (5b) to 1.5 mM (9c). This phenomenon
is expected, since it is known that induction of erythroid
differentiation of K562cells is associated with a
decrease of cell growth rates of differentiated cells.11
The induction ability of compounds 5b, 5c, 8b, 8c, 9c is
significantly higher than that exhibited by isobutyric
and pivalic acid.
Table 2. Effects of esters of methyl 3,4-O-isopropylidene-b-d-
galactopyranoside and 2,3-O-isopropylidene-d-mannofuranose on in
vitro growth and erythroid differentiation of human leukemic K562
cells
Compound
Cell proliferation
IC50
Erythroid
inductiona % of
benzidine-positive
cells (mM, nM)
Table 3. Synergism between ara-C and esters of methyl 3,4-O-iso-
propylidene-b-d-galactopyranoside and 2,3-O-isopropylidene-d-man-
nofuranose on in vitro growth and erythroid differentiation of human
leukemic K562cells
4a
4b
4c
4 mM
4 mM
4 mM
24.4ꢁ2.1 (6 mM)
20.3ꢁ1.4 (3 mM)
14.6ꢁ1.8 (4 mM)
5a
5b
5c
3 mM
5 mM
2.5 mM
32.6ꢁ2.3 (6 mM)
43.5ꢁ5.7 (6 mM)
42.5ꢁ3.5 (4 mM)
Compound
Concentration
Erythroid
inductiona % of
benzidine-positive cells
8a
8b
8c
2mM
4 mM
2mM
30.6 ꢁ1.7 (1 mM)
57.3ꢁ4.9 (6 mM)
48 ꢁ8.6 (4 mM)
4a
4b
4c
5a
5b
5c
8a
8b
8c
9b
9c
2mM
3 mM
2mM
34.2 ꢁ3.5
30.3ꢁ4.1
24.4 ꢁ4.5
35.1 ꢁ3.3
42.6ꢁ4.8
36ꢁ1.4
2mM
9b
9c
3 mM
1.5 mM
29.4ꢁ9.1 (3 mM)
49.33ꢁ7.9 (2mM)
6 mM
4 mM
1 mM
5.5 mM
3.5 mM
3 mM
1.5 mM
200 nM
83.7ꢁ3.2
69.5ꢁ6.6
60.2ꢁ3.1
82.2ꢁ5.2
56ꢁ4.6
Butyric acid
Isobutyric acid
Pivalic acid
2.4 mM
4.8 mM
4.5 mM
35.3ꢁ3.7 (3 mM)
14ꢁ2.7 (5 mM)
12ꢁ1.7 (5 mM)
Mithramycin
ara-C
Tallimustine
10 nM
40 nM
15 mM
91.5ꢁ5.1 (20 nM)
78.3ꢁ4.1 (500 nM)
85.7ꢁ3.4 (50 nM)
ara-C
28.2ꢁ2.1
aResults are presented as maximum level of % of benzidine-positive
(hemoglobin-containing) cells ꢁSD reached within a 9 days induction
period at the indicated concentrations of tested compounds. Sub-
optimal concentrations of ara-C (200 nM) were used in combination
with the tested compounds.
aResults are presented as maximum level of % of benzidine-positive
(hemoglobin-containing) cells ꢁSD reached within a 9 days induc-
tion period at the indicated concentrations of tested compounds.

350
G. Catelani et al. / Bioorg. Med. Chem. 10 (2002) 347–353
A second general feature of erythroid inducing agents is
related to the possibility to act in synergism with other
compounds. This could be of interest for the design of
agents useful for efficient combined therapy. Table 3
shows that when sub-optimal concentrations of ara-C
are administered to K562cells together with the tested
compounds, high induction of K562erythroid differ-
entiation was in some cases obtained. This was particu-
larly evident for compounds 8a and 9b. These data
suggest that these compounds could potentiate the
effects of other known inducers of K562cells erythroid
differentiation.
methoxy-1-methylethyl)-b-d-galactopyranoside
(2)25
and methyl 2-O-benzyl-3,4-O-isopropylidene-b-d-galac-
topyranoside (3).26
Methyl 2-O-acyl-3,4-O-isopropylidene-ꢀ-D-galactopyra-
nosides (4a–c). To a solution of 225 (919 mg, 3.0 mmol)
in anhydrous toluene (12mL), anhydrous Et 3N (1.0
mL, 7.2mmol) and the required acyl chloride (6.6
mmol) were added at room temperature. The reaction
mixture was stirred at room temperature until TLC
analysis showed the complete disappearance of the
starting material, then it was filtered and concentrated
to dryness in vacuo. The crude product was dissolved in
MeOH (18 mL) containing pyridinium tosylate (18 mg,
0.11 mmol), stirred at room temperature for 30 min,
neutralized by addition of Na2CO3, stirred for 15 min at
room temperature, filtered and concentrated under
reduced pressure. The crude reaction product, purified
by flash chromatography, gave the desired 2-O-acyl
derivatives 4a–c.
Conclusions
The data presented in this paper demonstrate that some
of the studied esters of methyl 3,4-O-isopropylidene-b-
d-galactopyranoside and 2,3-O-isopropylidene-d-man-
nofuranose (5b, 5c, 8b, 8c, and 9c; see Table 2) could be
considered as inducers of K562cell erythroid differ-
entiation exhibiting a biological activity much higher
than that of the corresponding isobutyric and pivalic
acid. The studied compounds are expected to be not
subjected to intramolecular transesterification, unlike 3-
Methyl 2-O-butanoyl-3,4-O-isopropylidene-ꢀ-D-galacto-
pyranoside (4a). White solid, 53% yield, Rf 0.29 (4:6
hexane–EtOAc); mp 79–81 ꢂC (from EtOAc–hexane);
1
[a]d +20.1 (c 5.0, CHCl3). H NMR (CDCl3), d: 0.96 (t,
O-acyl-1,2-O-isopropylidene-d-glucofuranose
deriva-
3H, J=7.4 Hz, CH3CH2); 1.34, and 1.56 (2s, each 3H,
C(CH3)2); 1.68 (m, 2H, CH3CH2); 2.35 (t, 2H, J=7.3
Hz, CH2CO); 2.58 (bs, 1H, OH); 3.49 (s, 3H, OCH3);
3.84–4.01 (m, 3H, H-5, H-6, and H-60); 4.20 (m, 2H, H-
3, and H-4); 4.28 (d, 1H, J1,2=8.2Hz, H-1), 4.98 (m,
1H, H-2). Anal. calcd for C14H24O7: C, 55.25%; H,
7.95%. Found C, 55.11%; H, 8.02%.
tives.21 In addition, we like to underline that some of the
compounds tested (8a and 9b; see Table 3) exhibit cap-
ability to act in synergism with suboptimal concentrations
of ara-C in inducing erythroid differentiation.
Our results do not explain the mechanism of action of
the tested compounds; in order to study this specific, but
very important, point further experiments should be
undertaken, including analysis of the uptake of the
compounds,28 gene expression profile of treated cells,29
alteration of erythroid-specific markers during induc-
tion.30 We hypothesise, however, that the set of biolo-
gical functions altered by compound 8b and 9a could be
different to those regulated following treatment with
ara-C. We found indeed that a combined treatment (8b
plus ara-C or 9a plus ara-C) cause a synergistic effect on
erythroid induction.
Methyl 2-O-(2-methylpropanoyl)-3,4-O-isopropylidene-
ꢀ-D-galactopyranoside (4b). White solid, 65% yield, Rf
0.25 (1:1 petroleum ether–EtOAc); mp 74–75 ꢂC (from
1
hexane); [a]D+17.4 (c 1.1, CHCl3). H NMR (CDCl3),
d: 1.19 (d, 6H, J=7.0 Hz, (CH3)2CH); 1.34, and 1.56 (2
s, each 3H, C(CH3)2); 2.31 (s, 1H, OH); 2.59 (e, 1H,
CHCO); 3.49 (s, 3H, OCH3), 3.83–4.02(m, 3H, H-5, H-
6, and H-60); 4.20 (m, 2H, H-3, and H-4); 4.29 (d, 1H,
J1,2=8.1 Hz, H-1), 4.96 (m, 1H, H-2). Anal. calcd for
C14H24O7: C, 55.25%; H, 7.95%. Found C, 55.43%; H,
7.84%.
In any case, the results presented in this paper suggest
that some of the esters of methyl 3,4-O-isopropylidene-
b-d-galactopyranoside and 2,3-O-isopropylidene-d-
mannofuranose we have studied in the present paper
could be proposed as possible agents to modulate pro-
duction of embryo-fetal hemoglobins by human ery-
throid cells. In this respect, these compounds are
candidates to determine whether they stimulate HbF
production in normal erythroid precursors, as already
observed for many erythroid inducers of K562cells. 15ꢀ20
Methyl 2-O-(2,2-dimethylpropanoyl)-3,4-O-isopropyli-
dene-ꢀ-D-galactopyranoside (4c). White solid, 58%
yield, Rf 0.25 (1:1 hexane–EtOAc); mp 85–87 ꢂC (from
1
petroleum ether); [a]d +19.3 (c 1.1, CHCl3). H NMR
(CDCl3), d 1.22 (s, 9H, (CH3)3C); 1.34 and 1.56 (2s,
each 3H, C(CH3)2); 2.33 (s, 1H, OH); 3.49 (s, 3H,
0
OCH3); 3.83–4.02(m, 3H, H-5, H-6 and H-6 ); 4.20 (m,
2H, H-3, and H-4); 4.28 (d, 1H, J1,2=8.1 Hz, H-1), 4.95
(m, 1H, H-2). Anal. calcd for C15H26O7: C, 56.59%; H,
8.23%. Found C, 56.69%; H, 8.38%.
Methyl 6-O-acyl-3,4-O-isopropylidene-ꢀ-D-galactopyra-
nosides (5a–c). To a solution of 326 (648 mg, 2.0 mmol)
in anhydrous toluene (12mL), anhydrous Et N (0.68
mL, 4.8 mmol) and the required acyl chloride (4.4
mmol) were added at room temperature. The reaction
mixture was stirred at room temperature until TLC
analysis (3:7 hexane–EtOAc) showed the complete dis-
Experimental
General chemical methods were previously reported by
some of us26. 2,3:5,6-Di-O-isopropyꢂlidene-a-d-manno-
furanose (6) (Sigma) was dried at 50 C and 0.1 torr for
5 h. Previously described procedures were employed for
the preparation of methyl 3,4-O-isopropylidene-6-O-(1-
3

G. Catelani et al. / Bioorg. Med. Chem. 10 (2002) 347–353
351
appearance of the starting material, then it was filtered
and concentrated to dryness in vacuo. The crude pro-
duct was dissolved in EtOAc (16 mL) containing 10%
Pd(OH)2 on charcoal (160 mg), stirred at room tem-
perature under H2 for 2–5 h until the starting material
disappeared (TLC analysis). The suspension was filtered
over Celite, concentrated under reduced pressure and
the residue purified by flash chromatography to give the
desired 6-O-acyl derivatives 5a–c.
EtOAc); [a]d +40.8 (c 1.2, CHCl3), lit:24a [a]d +43.5 (c
1.1, CHCl3).
1-O-(2-Methylpropanoyl)-2,3:5,6-di-O-isopropylidene-ꢁ-
D-mannofuranose (ꢁ-7b). Syrup, 72% yield, Rf 0.46 (1:1
1
hexane–EtOAc); [a]d +40.8 (c 1.7, CHCl3). H NMR
(CDCl3), d: 1.17 (d, 6H, J=6.9 Hz, (CH3)2CH); 1.34,
1.37, 1.45, and 1.48 (4 s, each 3H, 2ꢃC(CH3)2), 2.54 (m,
0
0
1H, CHCO); 4.00 (dd, 1H, J6,6 =8.9 Hz, J5,6 =4.5 Hz,
H-60); 4.00 (dd, 1H, J4,5=7.9 Hz, H-4); 4.09 (dd, 1H,
J5,6=6.1 Hz, H-6); 4.39 (ddd, 1H, H-5); 4.70 (d, 1H,
J2,3=5.9 Hz, H-2); 4.87 (dd, 1H, J3,4=3.7 Hz, H-3);
6.12(s, 1H, H-1). Anal. calcd for C 16H26O7: C, 58.17%;
H, 7.93%. Found C, 58.32%; H, 8.02%.
Methyl 6-O-butanoyl-3,4-O-isopropylidene-ꢀ-D-galacto-
pyranoside (5a). White solid, 93% yield, Rf 0.32(1:1
hexane–EtOAc); mp 76–78 ꢂC (from EtOAc–hexane);
1
[a]d +18.8 (c 1.1, CHCl3). H NMR (CDCl3), d: 0.96 (t,
3H, J=7.4 Hz, CH3–CH2); 1.34 and 1.52(2s, each 3H,
C(CH3)2); 1.67 (m, 2H, CH3CH2); 2.34 (t, 2H, J=7.3
Hz, CH2CO); 2.76 (d, 1H, J2,OH=2.4 Hz, OH); 3.54 (m,
1H, H-2); 3.55 (s, 3H, OCH3); 4.03 (m, 1H, J5,6=6.2
Hz, H-5); 4.09 (d, 1H, J1,2=8.4 Hz, H-1); 4.12(m, 2H,
H-3, and H-4); 4.38 (m, 2H, H-6, and H-60). Anal. calcd
for C14H24O7: C, 55.25%; H, 7.95%. Found C, 55.19%;
H, 8.09%.
1-O-(2-Methylpropanoyl)-2,3:5,6-di-O-isopropylidene-ꢀ-
D-mannofuranose (ꢀ-7b). Syrup, 24% yield, Rf 0.37 (1:1
hexane–EtOAc); [a]d +27.2 (c 1.0, CHCl3). H NMR
1
(C6D6), d: 1.03 (d, 3H, J=5.4 Hz, CH3CH); 1.07 (d, 3H,
J=5.4 Hz, CH3CH); 1.13, 1.27, 1.42, and 1.45 (4 s, each
3H, 2ꢃC(CH3)2), 2.40 (m, 1H, CHCO); 3.45 (dd, 1H,
0
0
J4,5=7.4 Hz, H-4); 4.02(dd, 1H, J6,6 =8.7 Hz, J5,6 =6.4
Hz, H-60); 4.12(dd, 1H, J5,6=5.3 Hz, H-6); 4.26 (dd,
1H, J3,4=3.4 Hz, H-3); 4.31 (dd, 1H, J2,3=6.0 Hz, H-2);
4.53 (ddd, 1H, H-5); 5.78 (d, 1H, J1,2=3.8 Hz, H-1).
Anal. calcd for C16H26O7: C, 58.17%; H, 7.93%. Found
C, 58.02%; H, 7.88%.
Methyl 6-O-(2-methylpropanoyl)-3,4-O-isopropylidene-
ꢀ-D-galactopyranoside (5b). White solid, 95% yield, Rf
0.20 (4:6 hexane–EtOAc); mp 104–107 ꢂC (from petro-
leum ether); [a]d +21.7 (c 1.1, CHCl3). 1H NMR
(CDCl3), d: 1.19 (d, 6H, J=6.9 Hz, (CH3)2CH); 1.34,
and 1.52(2s, each 3H, C( CH3)2), 2.64 (e, 1H, CHCO);
2.75 (bs, 1H, OH); 3.54 (s, 3H, OCH3); 3.54 (dd, 1H,
J2,3=6.9 Hz, H-2); 4.01 (ddd, 1H, J4,5 =2.0 Hz,
J5,6=6.3 Hz, H-5); 4.09 (d, 1H, J1,2=8.3 Hz, H-1); 4.11
(m, 2H, H-3, and H-4); 4.37 (m, 2H, H-6 and H-60).
Anal. calcd for C14H24O7: C, 55.25%; H, 7.95%. Found
C, 55.32%; H, 8.05%.
1-O-(2,2-Dimethylpropanoyl)-2,3:5,6-di-O-isopropylidene
-ꢁ-D-mannofuranose (ꢁ-7c). White solid, 37% yield, Rf
0.61 (1:1 hexane–EtOAc); mp 69–71 ꢂC (directly from
1
flash chromatography); [a]d +49.0 (c 1.0, CHCl3). H
NMR (CDCl3), d: 1.20 (s, 9H, (CH3)3C); 1.35, 1.38,
1.46, and 1.50 (4 s, each 3H, 2ꢃC(CH3)2); 3.97 (dd, 1H,
0
0
J4,5=8.0 Hz, H-4); 4.00 (dd, 1H, J6,6 =8.8 Hz, J5,6 =4.4
Hz, H-60); 4.11 (dd, 1H, J5,6=6.1 Hz, H-6); 4.40 (ddd,
1H, H-5); 4.69 (d, 1H, J2,3=5.9 Hz, H-2); 4.87 (dd, 1H,
J3,4=3.6 Hz, H-3); 6.12(s, 1H, H-1). Anal. calcd for
C17H28O7: C, 59.28%; H, 8.19%. Found C, 59.26%; H,
8.31%.
Methyl 6-O-(2,2-dimethylpropanoyl)-3,4-O-isopropyli-
dene-ꢀ-D-galactopyranoside (5c). White solid, 96%
yield, Rf 0.29 (4:6 hexane–EtOAc); mp 125–129 ꢂC
1
(from petroleum ether); [a]d +21.4 (c 1.0, CHCl3). H
NMR (CDCl3), d: 1.22 (s, 9H, (CH3)3C); 1.34, and 1.52
(2s, each 3H, C( CH3)2); 2.65 (bs, 1H, OH); 3.55 (s, 3H,
OCH3); 3.55 (m, 1H, H-2); 3.97 (m, 1H, J4,5=2.0 Hz,
J5,6=6.4 Hz, H-5); 4.09 (d, 1H, J1,2=8.4 Hz, H-1); 4.10
(m, 2H, H-3, and H-4); 4.36 (d, 2H, H-6 and H-60).
Anal. calcd for C15H26O7: C, 56.59%; H, 8.23%. Found
C, 56.69%; H, 8.28%.
1-O-(2,2-Dimethylpropanoyl)-2,3:5,6-di-O-isopropylidene-
ꢀ-D-mannofuranose (ꢀ-7c). White solid, 52% yield, Rf
0.51 (1:1 hexane–EtOAc); mp 67–68 ꢂC (directly by
1
flash-chromatography); [a]d +25.0 (c 1.4, CHCl3). H
NMR (C6D6), d: 1.14, 1.26, 1.42, and 1.45 (4 s, each 3H,
2ꢃC(CH3)2); 1.19 (s, 9H, (CH3)3C); 3.35 (dd, 1H,
0
0
J4,5=7.6 Hz, H-4); 4.00 (dd, 1H, J6,6 =8.6 Hz, J5,6 =6.3
Hz, H-60); 4.09 (dd, 1H, J5,6=5.3 Hz, H-6); 4.27 (dd,
1H, J3,4=3.3 Hz, H-3); 4.33 (dd, 1H, J2,3=6.0 Hz, H-2);
4.53 (ddd, 1H, H-5); 5.68 (d, 1H, J1,2=3.8 Hz, H-1).
Anal. calcd for C17H28O7: C, 59.28%; H, 8.19%. Found
C, 59.33%; H, 8.26%.
1-O-Acyl-2,3:5,6-di-O-isopropylidene-ꢁ- and ꢀ-D-man-
nofuranose (7a–c). To a stirred solution of dry 2,3:5,6-
di-O-isopropylidene-a-d-mannofuranose (6) (1.30 g, 1.0
mmol) in anhydrous toluene (15 mL), anhydrous Et3N
(1.75 mL, 12mmol) and the required acyl chloride (11.0
mmol) were added. The reaction mixture was stirred
under reflux until TLC analysis (1:1 hexane–EtOAc)
revealed the complete disappearance of the starting
material, filtered, and concentrated under reduced pres-
sure. A flash chromatography on silica gel of these
crude reaction products gave the desired 1-O-acyl deri-
vatives 7.
1-O-Acyl-2,3-O-isopropylidene-ꢁ- and ꢀ-D-mannofura-
noses (8–9). To a solution of di-O-isopropylidene deri-
vatives 7 (1.0 mmol) in EtOH (9 mL), copper(II)
chloride dihydrate (850 mg, 4.9 mmol) was added at
room temperature, and the reaction mixture was stirred
at room temperature until TLC analysis (3:7 hexane–
EtOAc) revealed the complete disappearance of the
starting material (about 4 h for b anomers and about 16
h for a anomers). The reaction was stopped by addition
1-O-Butanoyl-2,3:5,6-di-O-isopropylidene-ꢁ-D-mannofur-
anose (ꢁ-7a). Syrup, 95% yield, Rf 0.60 (1:1 hexane–

352
G. Catelani et al. / Bioorg. Med. Chem. 10 (2002) 347–353
of solid NaHCO3 (900 mg) and the mixture was stirred
for 30 min until no more CO2 was formed. Then, 1.0
mL of H2O was added, a further amount of CO2 being
produced, and the mixture was diluted with 4.0 mL of
H2O to promote precipitation. The pale blue precipitate
was filtered on Celite and the filtrate was extracted with
CHCl3 (3ꢃ10 mL). The organic extracts were combined,
dried, concentrated under reduced pressure, and the
resulting residue was directly subjected to flash chro-
matography on silica gel.
4.95 (dd, 1H, J3,4=4.8 Hz, H-3); 5.90 (d, 1H, J1,2=4.3
Hz, H-1). Anal. calcd for C14H24O7: C, 55.26%; H,
7.95%. Found C, 55.35%; H, 7.89%.
Acknowledgements
R.G. is supported by C.N.R. P.F. Biotechnology. C.C.
and D.M. were supported by fellowships from ‘‘Asso-
ciazione Veneta per la lotta alla talassemia — Rovigo,
Italy’’ and from ‘‘Associazione per la lotta alla talasse-
mia di Ferrara — Ferrara, Italy’’. Partial support from
Universities of Pisa and Ferrara is also acknowledged.
1-O-Butanoyl-2,3-O-isopropylidene-ꢁ-D-mannofuranose
(8a). White solid, 80% yield, mp 76–77 ꢂC (directly by
flash chromatography) Rf 0.18 (95:5 CH2Cl2–MeOH);
[a]d +57.3 (c 1.5, CHCl3); lit24a: [a]d +54.6 (c 1.2,
CHCl3), mp 79 ꢂC.
1-O-(2-Methylpropanoyl)-2,3-O-isopropylidene-ꢁ-D-man-
nofuranose (8b). White solid, 80% yield, Rf 0.45
(EtOAc); mp 98–99 ꢂC (directly by flash-chromato-
graphy); [a]d +58.3 (c 1.2, CHCl3). H NMR (CDCl3),
d: 1.16 (d, 6H, J=7.0 Hz, (CH3)2CH); 1.36, and 1.50 (2
s, each 3H, C(CH3)2), 2.05 (bs, 1H, OH); 2.52 (m, 1H,
References and Notes
1. Platt, O. S.; Thorington, B. D.; Brambilla, D. J.; Milner,
P. F.; Rosse, W. F.; Vichinsky, E.; Kinney, T. R. New Engl. J.
Med. 1991, 325, 11.
2. Rochette, J.; Craig, J. E.; Thein, S. L. Blood Rev. 1994, 8,
213.
3. Lowrey, C. H.; Nienhuis, A. W. New Engl. J. Med. 1993,
329, 845.
4. Dover, G. J.; Brusilow, S.; Samid, D. New Engl. J. Med.
1992, 327, 569.
1
0
CHCO); 2.73 (bs, 1H,0 OH); 3.71 (dd, 1H, J6,6 =11.42
0
Hz, J5,6 =5.0 Hz, H-6 ); 3.87 (dd, 1H, J5,6=2.9 Hz, H-
6); 4.04 (ddd, 1H, H-5); 4.05 (dd, 1H, J4,5=8.1 Hz, H-
4); 4.70 (d, 1H, J2,3=5.9 Hz, H-2); 4.93 (dd, 1H,
J3,4=3.5 Hz, H-3); 6.17 (s, 1H, H-1). Anal. calcd for
C13H22O7: C, 53.78%; H, 7.64%. Found C, 53.86%; H,
7.76%.
5. Ikuta, T.; Atweh, G.; Boosalis, V.; White, G. L.; Da Fon-
seca, S.; Boosalis, M.; Faller, D. V.; Perrine, S. P. Ann. New
York Acad. Sci. 1998, 850, 87.
6. Perrine, S. P.; Ginder, G. D.; Faller, D. V.; Dover, G. H.;
Ikuta, T.; Witkowska, H. E.; Cai, S. P.; Vichinsky, E. P.; Oli-
vieri, N. F. New Engl. J. Med. 1993, 328, 81.
7. Atweh, G. F.; Sutton, M.; Nassif, I.; Boosalis, V.; Dover,
G. J.; Wallenstein, S.; Wright, E.; McMahon, L.; Stama-
toyannopoulos, G.; Faller, D. V.; Perrine, S. P. Blood 1999,
93, 1790.
8. Lozzio, C. B.; Lozzio, B. B. Blood 1975, 45, 321.
9. Rutherford, T. R.; Clegg, J. B.; Weatherall, D. J. Nature
1979, 280, 164.
10. Cioe, L.; McNab, A.; Hubbell, H. R.; Meo, P.; Curtis, P.;
Rovera, G. Cancer Res. 1981, 41, 237.
11. Gambari, R.; Barbieri, R.; Buzzoni, D.; Bernardi, F.;
Marchetti, G.; Amelotti, F.; Piva, R.; Viola, L.; Del Senno, L.
Biochem. Biophysic. Acta 1986, 886, 203.
1-O-(2,2-Dimethylpropanoyl)-2,3-O-isopropylidene-ꢁ-D-
mannofuranose (8c). White solid, 80% yield, Rf 0.45
(EtOAc); mp 149 ꢂC (directly by flash-chromatography);
[a]d +60.4 (c 1.1, CHCl3). H NMR (CD3CN–D2O), d:
1.15 (s, 9H, (CH3)3C); 1.31, and 1.42(2s, each 3H,
1
0
0
C(CH3)2); 3.45 (dd, 1H, J6,6 =11.5 Hz, J5,6 =5.6 Hz, H-
60); 3.63 (dd, 1H, J5,6=2.9 Hz, H-6); 3.81 (ddd, 1H, H-
5); 3.96 (dd, 1H, J4,5=8.7 Hz, H-4); 4.67 (d, 1H,
J2,3=5.9 Hz, H-2); 4.87 (dd, 1H, J3,4=3.4 Hz, H-3);
5.94 (s, 1H, H-1). Anal. calcd for C14H24O7: C, 55.26%;
H, 7.95%. Found C, 55.07%; H, 8.03%.
1-O-(2-Methylpropanoyl)-2,3-O-isopropylidene-ꢀ-D-man-
nofuranose (9b). Syrup, 50% yield, Rf 0.20 (EtOAc);
[a]d +11.4 (c 2.5, CHCl3). 1H NMR (CDCl3), d: 1.21 (d,
6H, J=7.0 Hz, (CH3)2CH); 1.39, and 1.56 (2s, each 3H,
12. Bianchi Scarra, G. L.; Romani, M.; Coviello, D. A. Can-
cer Res. 1986, 46, 6327.
13. Bianchi, N.; Osti, F.; Rutigliano, C.; Ginanni Corradini,
F.; Borsetti, E.; Tomassetti, M.; Mischiati, C.; Feriotto, G.;
Gambari, R. Br. J. Haematol. 1999, 104, 258.
14. Bianchi, N.; Ongaro, F.; Chiarabelli, C.; Gualandi, L.;
Mischiati, C.; Bergamini, P.; Gambari, R. Biochem. Pharma-
col. 2000, 60, 31.
15. Bianchi, N.; Chiarabelli, C.; Borgatti, M.; Mischiati, C.;
Fibach, E.; Gambari, R. Br. J. Haematol. 2001, 113, 951.
16. Fibach, E.; Burke, K. P.; Schechter, A. N.; Noguchi, C. T.;
Rodgers, G. P. Blood 1993, 81, 1630.
C(CH3)2), 2.10 (bs, 2H, OH); 2.62 (m, 1H, CHCO); 3.73
0
0
0
(dd, 1H, J6,6 =11.5 Hz, J5,6 =5.3 Hz, H-6 ); 3.87 (dd,
1H, J5,6=3.2Hz, H-6); 3.97 (dd, 1H, J4,5=8.9 Hz, H-4);
4.10 (ddd, 1H, H-5); 4.85 (dd, 1H, J2,3=6.6 Hz, H-2);
4.94 (dd, 1H, J3,4=4.6 Hz, H-3); 5.93 (d, 1H, J1,2=4.2
Hz, H-1). Anal. calcd for C13H22O7: C, 53.78%; H,
7.64%. Found C, 53.77%; H, 7.64%.
17. Dalyot, N.; Fibach, E.; Rachmilewitz, E. A.; Oppenheim,
A. Exp. Hematol. 1992, 20, 1141.
18. Fibach, E.; Kollia, P.; Schechter, A. N.; Noguchi, C. T.;
Rodgers, G. P. Blood 1995, 85, 2967.
19. Fibach, E.; Prasanna, P.; Rodgers, G. P.; Samid, D. Blood
1993, 82, 2203.
20. Kollia, P.; Fibach, E.; Politou, M.; Noguchi, C. T.;
Schechter, A. N.; Loukopoulos, D. Ann. New York. Acad. Sci.
1998, 850, 449.
1-O-(2,2-Dimethylpropanoyl)-2,3-O-isopropylidene-ꢀ-D-
mannofuranose (9c). Syrup, 40% yield, Rf 0.35
1
(EtOAc); [a]d +16.4 (c 1.5, CHCl3). H NMR (CDCl3),
d: 1.25 (s, 9H, (CH3)3C); 1.39, and 1.56 (2s, each 3H,
C(CH3)2); 1.99 (bs, 1H, OH); 2.90 (bs, 1H, OH); 3.73
0
0
0
(dd, 1H, J6,6 =11.5 Hz, J5,6 =5.3 Hz, H-6 ); 3.88 (dd,
1H, J5,6=3.1 Hz, H-6); 3.95 (dd, 1H, J4,5=8.8 Hz, H-4);
4.08 (ddd, 1H, H-5); 4.86 (dd, 1H, J2,3=6.9 Hz, H-2);

G. Catelani et al. / Bioorg. Med. Chem. 10 (2002) 347–353
353
21. (a) Catelani, G.; Osti, F.; Bianchi, N.; Bergonzi, M. C.;
D’Andrea, F.; Gambari, R. Bioorg. Med. Chem. Lett. 1999, 9,
3153. (b) Bergonzi, M. C.; Bianchi, N.; Catelani, G.; D’An-
drea, F.; Gambari, R.; Lipucci di Paola, M.; Osti, F. Eur.
Patent 1004590 A2, 2000. Chem. Abstr. 2000, 133, 4898.
22. Catelani, G.; D’Andrea, F. Unpublished results.
23. Chiesi Farmaceutici. Personal communication.
24. (a) Balwin, P.-J.; Douillet, O. C.; Pouillart, P. R.; Ronco,
G.; Villa, PTC Int. Appl. WO 9603411, 1996, Chem. Abstr.
1996, 125, 34034 (b) Goueth, P. Y.; Gogalis, P.; Bikanga, R.;
Gode, P.; Postel, D.; Ronco, G.; Villa, P. J. Carbohydr. Chem.
1994, 13, 249.
25. Barili, P. L.; Berti, G.; Catelani, G.; Colonna, F.; Marra,
A. Tetrahedron Lett. 1986, 27, 2307.
26. Barili, P. L.; Berti, G.; D’Andrea, F.; Di Bussolo, V.;
Granucci, I. Tetrahedron 1992, 48, 6273.
27. Iwata, M.; Ohrui, H. Bull. Chem. Soc. Jpn. 1981, 54, 2837.
28. Meyron-Holtz, E. G.; Vaisman, B.; Cabantchik, Z. I.;
Fibach, E.; Rouault, T. A.; Hershko, C.; Konijn, A. M. Blood
1999, 94, 3205.
29. Mitchell, T.; Plonczynski, M.; McCollum, A.; Hardy, C. L.;
Safaya, S.; Steinberg, M. H. Blood Cells Mol. Dis. 2001, 27, 309.
30. Furukawa, Y.; Kikuchi, J.; Nakamura, M.; Iwase, S.;
Yamada, H.; Matsuda, M. Br. J. Haematol. 2000, 110, 663.