Synthesis, spectroscopic characterization and biological properties of new natural aldehydes thiosemicarbazones

Article abstract of DOI:10.1016/S0968-0896(99)00260-6

As part of a research programme aimed at the synthesis of compounds with antiviral, antibacterial and antitumor properties and their spectroscopic characterization, new thiosemicarbazones deriving from natural aldehydes have been investigated. These substances contain in the same molecule both a chain with nucleophilic centres N, S with tubercolostatic activity, and a glycosidic or alkyl moiety (modified glycosides and nucleosides have recently received a great deal of attention in the fields of neoplastic diseases and viral infections). In this paper the synthesis and the characterization of these compounds by means of ¹H NMR, IR, and MS techniques is reported. Biological studies have involved both inhibition of cell proliferation and apoptosis tests on human leukemia cell line U937. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00260-6

Bioorganic & Medicinal Chemistry 8 (2000) 157±162
Synthesis, Spectroscopic Characterization and Biological
Properties of New Natural Aldehydes Thiosemicarbazones
Pieralberto Tarasconi, a,* Silvia Capacchi, a Giorgio Pelosi, a Mara Cornia, b
Roberto Albertini, c,d Antonio Bonati, c,d Pier Paolo Dall'Aglio, c,d Paolo Lunghi c,d
and Silvana Pinelli c,d
a
Á
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Universita di Parma, Viale delle Scienze,
I-43100 Parma, Italy
b
Á
Dipartimento di Chimica Organica e Industriale, Universita di Parma, Viale delle Scienze, I-43100 Parma, Italy
c
Á
Istituto di Patologia Speciale Medica, Universita di Parma, I-43100 Parma, Italy
Á
Centro di Ricerca Interuniversitario per Diagnosi, Terapia e Prognosi di Tumori Umani, Universita di Milano, I-20122, Milan, Italy
d
Received 27 July 1999; accepted 8 September 1999
AbstractÐAs part of a research programme aimed at the synthesis of compounds with antiviral, antibacterial and antitumor
properties and their spectroscopic characterization, new thiosemicarbazones deriving from natural aldehydes have been investi-
gated. These substances contain in the same molecule both a chain with nucleophilic centres N, S with tubercolostatic activity, and
a glycosidic or alkyl moiety (modi®ed glycosides and nucleosides have recently received a great deal of attention in the ®elds of
1
neoplastic diseases and viral infections). In this paper the synthesis and the characterization of these compounds by means of H
NMR, IR, and MS techniques is reported. Biological studies have involved both inhibition of cell proliferation and apoptosis tests
on human leukemia cell line U937. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Then, as part of a research programme aimed at the
synthesis of compounds with antiviral, antibacterial and
antitumor properties,9±11 new thiosemicarbazones
deriving from natural aldehydes, such as citronellal,
citral, xylopentadialdofuranose, glyceraldehyde, and
from a nucleoside as uridine were synthesized, and
subsequently characterized by IR and NMR spectro-
scopies. Moreover, trying to correlate experimental data
and biological activity of these new compounds, assays
on inhibition of proliferation and on apoptosis were
made on cellular hystiocytic lines U937, in collaboration
with the Institute of Medical Special Pathology of the
University of Parma. All compounds have demon-
strated to inhibit cell growth and one of them, the (3R)-
(+)-citronellal thiosemicarbazone (1; Scheme 1), to
induce apoptosis.
Compounds potentially useful for their pharmacological
properties have been often obtained by combining, in
the same organic molecule, chemical species biologically
active that already possess, by themselves, di€erent
reaction selectivity or di€erent action mechanism.1±4
A
proper choice of these interacting molecules5 should
make easier the creation of non-covalent interactions
with DNA, so that the resulting compounds could play
an important role for the biological activity.
Recently, the antiviral and antineoplastic activity of
glycosides and modi®ed nucleosides has been dis-
covered.6±8 Since the biological activity carried out in
vitro by thiosemicarbazones is widely known in the
pharmacological ®eld,1±4 we have synthesized com-
pounds that bear in the same molecule di€erent chemi-
cal species, possessing biological properties, with the
aim to examine their biological cooperative e€ect.
Results and Discussion
Synthesis of ligands 1±5
All compounds were obtained by an improved con-
densation reaction of the aldehyde with thiosemicarb-
azide, under ultrasonic irradiation to increase the
Keywords: natural products; antitumor compounds; leukemia; DNA.
*Corresponding author. Tel.: +39-0521-905420; fax: +39-0521-
905557; e-mail: chimic8@ipruniv.cce.unipr.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00260-6

158
P. Tarasconi et al. / Bioorg. Med. Chem. 8 (2000) 157±162
Scheme 1.
reagents solubility, to a€ord the corresponding thio-
semicarbazones in a quantitative yield: the case of (3R)-
(+)-citronellal (95% yield) is reported in Scheme 1.
4, derived from glyceraldehyde, synthesis needed an
intermediate step, in fact 2,3-O-isopropylidene-d-gly-
ceraldehyde was obtained from 1,2;5,6-di-O-isopropyl-
idene-d-mannitol according to literature.13
By following the same procedure, also citral reacted
with thiosemicarbazide to give thiosemicarbazone 2
(92% yield) (Fig. 1). We chose the natural aldehydes
(3R)-(+)-citronellal and citral to be transformed into
the corresponding thiosemicarbazones because, in the
course of our studies about porphyrins bearing natural
aldehydes residues in the meso positions, we found that
lipophylic compounds were more e€ective than the
hydrophylic ones as far as the biological activity was
concerned (apoptosis tests on human leukemia cell line
U937).12
We used the following strategy to obtain the uridine
thiosemicarbazone (5; Scheme 2): a few more steps were
needed before the condensation with thiosemicarbazide.
The uridine thiosemicarbazone (5) was a€orded starting
from uridine (6), after protecting the C-20 and C-30 car-
bons (7), to enhance its solubility in organic apolar
aprotic solvents in which the subsequent synthetic steps
took place, and contemporaneously avoid the oxidation
of the hydroxylic secondary functions. The reaction of
protection was practically quantitative if carried out in
dry acetone in the presence of sulphuric acid.14 Com-
pound 7 was oxidized to aldehyde (8), with a mixture of
CrO3 and pyridine in CH2Cl2:DMF 4:1, according to
our precedent optimization studies.15 Then the aldehyde
function was condensed with thiosemicarbazide in
MeOH, under ultrasonic irradiation for 2 h, to get after
TLC puri®cation with ethylacetate:MeOH (80:20) the
product 5 (90% yield).
Compounds 3 and 4 (Fig. 1) were synthesized in the
same way as the previous products (90% yield), but for
The choice of uridinil-50-carboxyaldehyde was made in
agreement with the recent results reported in the litera-
ture6±8 any modi®cations in the nucleosides are of great
interest in the biomedical ®eld because they can lead to
compounds with antitumor and/or antiviral properties.
Moreover, we believe that the isopropylidene protected
hydroxyls could favour the passage of the molecule
through the cell membrane. In fact compound 5 showed
a good cell proliferation inhibition (see also the Biolo-
gical tests paragraph, Fig. 2, lane 3), but did not result
active in the apoptosis induction. At this point it could
be important to understand whether the whole nucleo-
side molecule or just the sugar moiety was responsible
for the biological activity, thus we synthesized and tes-
ted a few thiosemicarbazones derived from sugars, such
as the protected cyclic carbohydrate xylopentadialdo-
furanose (3) and the less hindered 2,3-O-isopropylidene-
d-glyceraldehyde (4). Both these compounds did not
show remarkable activities, and this feature made us
consider that the whole nucleobase would act as to
determine the biological activity.
Characterization of compounds 1±5
The 1H NMR spectrum of compound 1 (300 MHz,
CDCl3) shows, at room temperature, a singlet at
9.76 ppm relative to the NH next to CˆS, very deshiel-
ded as expected, while the signal of the proton on the
CˆN double bond appears at 7.29 ppm. It is interesting
Figure 1.

P. Tarasconi et al. / Bioorg. Med. Chem. 8 (2000) 157±162
159
Scheme 2.
Figure 2. E€ects of compounds 1±5 on U937 proliferation on the 4th day.
to notice the presence of two broad singlets for the two
NH2 protons, respectively at 7.05 and 6.38 ppm: it
means that the free rotation around the C±N bond is
blocked because of its partial double bond character.16
In the IR spectrum of 1 the bands in the region 3407±
3261 cm 1 are due to the stretching frequencies n (NH2),
while n (NH) is present at 3162 cm 1. The typical bands
of the aliphatic CH appear in the frequency range
3026±2915 cm 1, while n (CN) and n (CˆC) give just one
in agreement with the thionic form of ligand 1 and with
1
the presence of bands n (CS) at 928 and 820 cm
.
The 1H NMR spectrum of compound 2 (300 MHz,
CDCl3) shows at 10.48 ppm the singlet relative to the
NH next to CˆS, at 7.94 ppm the signal of the proton
on the CˆN double bond, and, in the same way as
compound 1, two broad singlets for the two NH2 pro-
tons, respectively at 7.03 and 6.77 ppm.16 In the IR
1
band, by superimposition, at 1595cm 1. No band due to
spectrum of 2 the bands at 3381 and 3264 cm are
due to the stretching frequencies n (NH2), while n (NH)
1
the SH group is observed between 2600 and 2500cm
,

160
P. Tarasconi et al. / Bioorg. Med. Chem. 8 (2000) 157±162
is present at 3161 cm 1. The spectrum is very similar to
the previous one: in the frequency range 3030±2945
cm 1 appear the typical bands of the aliphatic CH, the n
1
(CN) is present at 1641 cm 1, the n (CˆC) at 1607 cm
,
1
and the n (CS) at 835 cm
.
For compound 3 (300 MHz, CD3COCD3) the singlet of
the NHCS appears at 11.39 ppm, very deshielded; the
two broad singlets of the NH2 protons are observed at
8.12 and 7.74 ppm, and the signal of the proton on the
CˆN double bond at 7.28 ppm.
Thiosemicarbazones 4 and 5 (300 MHz, DMSO) behave
in a similar way, giving rise to signals in the same ranges
as found for 1±3. For compound 4 the singlet of the
NHCS appears at 9.86 ppm, the two broad singlets of
NH2 at 7.95 and 7.48 ppm, and the CHˆN signal at
Figure 3. Agarose gel electrophoresis of cellular DNA. The ethidium
bromide-stained agarose gel shows DNA from U937 cells treated as
follows: lane L ladder; lane + positive control; lane untreated con-
trol cells; lane 3 compound 3; lane 4 compound 4; lane 5 compound 5;
lane 1 compound 1; lane 2 compound 2.
7.06 ppm. In the IR spectrum of 4 the bands in the
1
region 3396±3280 cm
are due to the stretching
frequencies n (NH2), while n (NH) is observed at
3197 cm 1. In the frequency range 3064±2926 cm
1
appear the typical bands of the aliphatic CH, while the n
(CO) and n (CN) produce two bands, respectively, at
1605 and 1562 cm 1. Finally, in analogy with the
Experimental
General
behaviour shown by products 1 and 2, no band due to
1
the SH group is observed between 2600 and 2500 cm
in agreement with the thionic form of ligand 4 and with
,
the presence of bands n (CS) at 935 and 828 cm 1. For
compound 5 the H NMR spectrum is quite compli-
All the materials and solvents were obtained from com-
mercial suppliers and used without further puri®cation.
Thiosemicarbazide was purchased from Janssen, citral
from Sigma, while (3R)-(+)-citronellal, 1,2-O-iso-
propylidene-3-O-methyl- a-d-xylopentodialdofuranose,
uridine and 1,2;5,6-di-O-isopropylidene-d-mannitol from
Fluka. 2,3-O-Isopropylidene-d-glyceraldehyde was
obtained from 1,2;5,6-di-O-isopropylidene-d-mannitol
according to literature.13 TLC were performed on E.
Merck plates precoated with SiO2 F254. Sonication was
performed by a Branson Type 2200 instrument (120 W,
220±240 V, 50±60 Hz) operating at a 30±50 kHz fre-
1
cated. A broad singlet relative to the two NH, the one
next to CˆS and the heterocyclic NH-3, appear at
11.3 ppm; the NH2 gives rise to two broad singlets at
8.15 and 7.96 ppm, while the doublet of the CHˆN
proton is observed at 7.45 ppm.
Biological tests: e€ects on cell proliferation and
apoptosis induction
Compounds 1±5 have been tested in vitro on human
leukemic cell lines U937, focusing our attention on their
activity with respect to cell proliferation inhibition and
apoptosis induction. It has been reported that a number
of anticancer drugs may exert their activity by inducing
apoptosis.17 The mechanism underlying the e€ects varies
depending on the initiating stimulus but a common feature
is the activation of the endonuclease leading to DNA
fragmentation. Thiosemicarbazones 1, 2 and 5 inhibited
remarkably cell proliferation when used at 10 mg/mL,
while 4 (100 mg/mL) was less e€ective, and ligand 3,
even at larger concentrations (100 mg/mL), did not show
any e€ect (Fig. 2).
1
quency. H NMR spectra were obtained at room tem-
perature on a Brucker AMX-300 spectrometer, and
chemical shifts are given in units of d relative to TMS as
an internal reference. For compounds 4 and 5, dissolved
in DMSO-d6, the two signals of DMSO and water arose
respectively at 2.5 and 3.4 ppm. Optical rotations were
measured on a Rudolph Autopol III polarimeter. FT±
IR spectra were recorded on a Nicolet 5PCFT±IR
infrared spectrophotometer as KBr pellets, in the fre-
quency range 4000±400 cm 1. CI±MS (m/z, 70 eV) were
obtained on a Finnegan 1020 6c mass spectrometer.
Elemental analyses were performed by the Micro-
analytical Laboratory of the University of Parma
(Dipartimento di Chimica Generale ed Inorganica).
Using these products at the minimum concentration
values at which the maximum inhibition was observed,
only compound 1, derived from (3R)-(+)-citronellal,
was able to cause the typical DNA fragmentation at
10 g/mL, inducing apoptosis (Fig. 3). A striking feature
is that, notwithstanding the similarity between com-
pounds 1 and 2 and their analogous behaviour with
respect to cell inhibition, only compound 1 showed
apoptotic activity. Further studies are now in progress
in our laboratories to ascertain a possible di€erent
mechanism of action between the two.
Synthesis of the natural aldehydes thiosemicarbazones
(1±5)
(3R)-(+)-Citronellal thiosemicarbazone (1): [(3R)-3,7-di-
methyl-6-octenyl]-1-carboxyaldehydethiosemicarbazone
(1). To a solution of thiosemicarbazide (178 mg,
1.95 mmol) in EtOH 95% (10 mL) was added, with
stirring, a solution of (3R)-(+)-citronellal (0.35 mL,
1.95 mmol) in EtOH 95% (10 mL). After 60 min under

P. Tarasconi et al. / Bioorg. Med. Chem. 8 (2000) 157±162
161
ultrasonic irradiation (40 ꢀC) the mixture was cooled,
and compound 1 was isolated as a white powder (95%
yield). ‰aŠ ‡ 30:9; ‰aŠ ‡ 46:5 (c=0.082, CHCl3). H
2.16 mmol) and CH2Cl2 (10 mL) were poured in the
reaction vessel.13 After 10 min the solution was ®ltered
on Buchner and dried over MgSO4 (5 g) to give, after
®ltering and concentrating, 2,3-O-isopropylidene-d-gly-
ceraldehyde (325 mg, 2.5 mmol) (70% yield), that was
subsequently added, in EtOH 95% (5 mL), to a solution
of thiosemicarbazide (228 mg, 2.5 mmol) in EtOH 95%
(20 mL), under stirring and sonication (40 ꢀC). After 3 h
the mixture was cooled, and compound 4 was obtained
20
546
20
589
1
NMR (300 MHz, CDCl3) d 9.76 (1H, s, NHCS), 7.29
(1H, t, J=6.0 Hz, CHˆN), 7.05 and 6.38 (2H, 2 br s,
1H each, NH2), 5.05 (1H, m, CHˆC(CH3)2), 2.24 (1H,
m, CH2 in C-2, H-a), 1.94 (2H, m, CH2 in C-5), 1.73
(1H, m, CH2 in C-2, H-b), 1.66 and 1.57 (6H, 2s, 3H
each, CH3CˆC), 1.35 (1H, m, CHCH3), 1.32 and 1.23
(2H, 2m, 1H each, CH2 in C-4, H-a+H-b), 0.92 (3H, d,
J=7.0, CHCH3). FT IR (KBr, cm 1) n (NH2) 3407 (s),
3261 (s, br); n (NH) 3162 (s); n (CH3) 3026 (m); n (CH2)
2962 (s), 2915 (s); n (CN) + n (CˆC) 1595 (s); n (CS)
928 (w), 820 (m). MS (CI, CH4) 228 (M+1). Anal. calcd
for C11H21N3S: C 58.11, H 9.32, N 18.49, S 14.08;
found: C 58.02, H 9.22, N 18.61, S 14.15.
20
20
as a yellow powder (90% yield). ‰aŠ ‡ 56:4; ‰aŠ
‡
546
589
87:6 (c=0.093, DMSO). 1H NMR (300 MHz, DMSO) d
9.86 (1H, s, NHCS), 7.95 and 7.48 (2H, 2 br s, 1H each,
NH2), 7.06 (1H, br s, CHˆN), 4.05 (1H, m, H-20), 3.95
(1H, m, CH2 in C-30, H-a), 3.80 (1H, m, CH2 in C-30, H-
b), 1.29 and 1.25 (6H, 2s, 3H each, CH3). FT IR (KBr,
cm 1) n (NH2) 3396 (s), 3280 (s, br); n (NH) 3197 (s); n
(CH) 3064 (m), 3013 (m), 2926 (m); n (CO) + n (CN)
1605 (s), 1562 (s); n (CS) 935 (w), 828 (m). MS (CI,
CH4) 204 (M+1). Anal. calcd for C7H13 N3O2S: C
41.36, H 6.45, N 20.69, S 15.74; found: C 41.25, H 6.38,
N 20.77, S 15.89.
Citral thiosemicarbazone (2): [3,7-dimethyl-2,6-octadi-
enyl]-1-carboxyaldehydethiosemicarbazone (2). To a solu-
tion of thiosemicarbazide (178 mg, 1.95 mmol) in EtOH
95% (10 mL), was added, with stirring, a solution of
citral (0.33 mL, 1.95 mmol) in EtOH 95% (10 mL).
After 60 min under ultrasonic irradiation (40 ꢀC) the
mixture was cooled, and compound 2 was isolated as a
yellow powder (92% yield). 1H NMR (300 MHz,
CDCl3) d 10.48 (1H, s, NHCS), 7.94 (1H, d, J=12.1 Hz,
CHˆN), 7.03 and 6.77 (2H, 2 br s, 1H each, NH2), 5.80
(1H, dm, CHˆC in C-2), 4.98 (1H, m, CH=C(CH3)2 in
C-6), 2.15 (2H, m, CH2 in C-4), 1.82 (2H, m, CH2 in C-
5), 1.73 (3H, d, J=3.0, CH3 in C-3), 1.58 and 1.49 (6H,
2s, 3H each, CH3CˆC). FT IR (KBr, cm 1) n (NH2)
3381 (s, br), 3264 (s); n (NH) 3161 (s); n (CH3) 3030 (m);
n (CH2) 2971 (s), 2945 (m); n (CN) 1641 (m); n (CˆC)
1607 (s); n (CS) 835 (m). MS (CI, CH4) 226 (M+1).
Anal. calcd for C11H19N3S: C 58.63, H 8.51, N 18.66, S
14.2; found: C 58.51, H 8.41, N 18.78, S 14.3.
Uridine thiosemicarbazone (5): 20,30-O-isopropylidene-40-
dehydroxymethyluridinil-50 -carboxyaldehydethiosemicar-
bazone (5). To a solution of uridine (6, 601 mg,
2.46 mmol) in distilled acetone (60 mL) was added
dropwise H2SO4 96% (12 drops, pH 3) under nitrogen
atmosphere and stirring. After 3 h the reaction mixture
was neutralised with solid BaCO3 (5 g, pH 6) and ®l-
tered on a Buchner apparatus. The resulting light yellow
oil was evaporated under vacuum and puri®ed by ¯ash
chromatography (ethyl acetate:MeOH 20%) to give the
protected uridine (20,30-O-isopropylidene-uridine)
7
(83% yield) as a yellow oil. Then a mixture consisting of
CrO3 (704 mg, 7.04 mmol) and pyridine (1.14 mL,
14.08 mmol) in CH2Cl2:DMF 4:1 (10 mL), was stirred
under nitrogen atmosphere, at room temperature: after
30 min compound 7 (500 mg, 1.76 mmol) in CH2Cl2:
DMF 4:1 (10 mL) was added, together with acetic
anhydride (0.66 mL, 7.04 mmol), and oxidized. The
reaction mixture was stirred for another 15 min, and
then quenched with EtOH 95% (1 mL), poured in ethyl
acetate (100 mL), ®ltered on silica recovered with dry
Na2SO4, and evaporated under vacuum a€ording 8
(20,30-O-isopropylidene-uridine-50-carboxyaldehyde, 80%
yield) as a colourless oil. 8 (401 mg, 1.42 mmol) in
MeOH (10 mL) was subsequently condensed with the
thiosemicarbazide (129 mg, 1.42 mmol) previously dis-
solved in MeOH (10 mL), under nitrogen atmosphere
and stirring. After 2 h under ultrasonic irradiation
(40 ꢀC) the reaction mixture was ®ltered and puri®ed by
TLC puri®cation with ethylacetate:MeOH (80:20) to
obtain 5 (uridine thiosemicarbazone) as a yellow
Xylopentadialdofuranose thiosemicarbazone (3): 10,20-O-
isopropylidene-30-O-methyl-50-deoxy-ꢀ-D-xylo-pentafur-
anosil-50-carboxyaldehydethiosemicarbazone (3). To a
solution of thiosemicarbazide (50 mg, 0.55 mmol) in
EtOH 95% (15 mL), under stirring, was added 1,2-O-
isopropylidene-3-O-methyl-a-d-xylopentodialdofuranose
(111 mg, 0.55 mmol) in EtOH 95% (5 mL). After 2 h
under ultrasonic irradiation (40 ꢀC) the mixture was
cooled, and compound 3 was obtained as a colourless
20
20
oil (90% yield). ‰aŠ ‡ 57:2; ‰aŠ ‡ 112:5 (c= 0.070,
546
589
CH3COCH3). 1H NMR (300 MHz, CD3COCD3) d
11.39 (1H, s, NHCS), 8.12 and 7.74 (2H, 2 br s, 1H
each, NH2), 7.28 (1H, d, J=7.0 Hz, CHˆN), 5.89 (1H,
d, J=4.0 Hz, H-10), 4.77 (1H, dd, J=7.0, 3.0 Hz, H-40),
4.62 (2H, m, H-20+H-30), 3.32 (3H, s, OCH3), 1.39 and
1.26 (6H, 2s, 3H each, CH3). MS (CI, CH4) 276 (M+1).
Anal. calcd for C10H17N3O4S: C 43.62, H 6.23, N 15.27,
S 11.62; found: C 43.5, H 6.14, N 15.38, S 11.77.
20
20
oil (361 mg, 90% yield). ‰aŠ ‡ 88:3; ‰aŠ ‡ 128:4
546
589
(c=0.055, DMSO). 1H NMR (300 MHz, DMSO) d
11.30 (2H, br s, NHCS+NH-3), 8.15 and 7.96 (2H, 2s,
1H each, NH2), 7.78 (1H, d, J=8.1 Hz, H-6), 7.45 (1H,
d, J=4.5 Hz, CHˆN), 5.85 (1H, d, J=8.1 Hz, H-5),
5.62 (1H, m, H-40), 5.18 (1H, d, J=0.7 Hz, H-10), 4.60
(1H, m, H-20), 4.05 (1H, m, H-30), 2.90 and 2.70 (6H, 2s,
3H each, CH3). MS (CI, CH4) 356 (M+1). Anal. calcd
for C13H17N5O5S: C 43.93, H 4.82, N 19.72, S 9.00;
found: C 43.81, H 4.73, N 19.81, S 9.16.
20,30 -O-Isopropylidene-D-glyceraldehyde thiosemicarb-
azone (4): 20,30-O-isopropylidene-20,30-dihydroxyethyl-1-
carboxyaldehydethiosemicarbazone (4). An aqueous
solution (10 mL) of NaIO4 (462 mg, 2.16 mmol) was
added to SiO2 (4282 mg, 71.28 mmol) and CH2Cl2
(80 mL), under stirring and at room temperature.
Then 1,2;5,6-di-O-isopropylidene-d-mannitol (569 mg,

162
P. Tarasconi et al. / Bioorg. Med. Chem. 8 (2000) 157±162
Biological data: materials and methods
References
The cells U937 were seeded at 2Â105/mL in the presence
of RPMI 1640 supplemented with l-glutamine 200 mM,
10% fetal bovine serum (FBS) and with antibiotics
(penicillin 100 U/mL and streptomycin 100 mg/mL), and
then treated with compounds 1±5. All compounds were
previously stored dry at room temperature and dis-
solved in dimethyl sulphoxide (DMSO) just before their
use. Cell mortality, evaluated on the fourth day by the
trypan blue exclusion method and determined by using
a hemocytometer, never exceeds 5%. For the apoptosis
assay the cells U937 were seeded at 5Â105/mL in the
presence of the above mentioned products at the same
concentrations as used for the proliferation inhibition.
After 18 h the cells (106) were washed twice with PBS at
2000 rpm for 10 min at 4 ꢀC, and then 20mL of a lysis
bu€er solution (EDTA 10 mM, Tris HCl 50 mM pH 8.0
and 0.5% (w/v) sodium laurylsarkosinate) were added.
The pellet was subsequently redissolved and 2.5 ml of
Proteinase K was added (4 mg/mL). After 1 h at 50 ꢀC,
2.5 mg/mL of RNAse A (2 mg/mL) was added and
incubated for 1 h at 40 ꢀC and then for 10 min at 70 ꢀC.
10mL of loading bu€er (EDTA 10mM pH 8, 1% agarose,
0.25% bromophenol blue, 40% sucrose) were subse-
quently added to the extracted DNA, which was loaded
on the agarose gel (2%) with 0.1 mg/mL ethidium bro-
mide to evaluate the characteristic e€ects of apoptosis
by means of electrophoresis.
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This work was supported by Italian MURST (60%) and
by AIRC (Associazione Italiana per la Ricerca sul Can-
cro), Italy. The authors acknowledge the Centro Inter-
facolta di Misure ``Giuseppe Casnati'' dell'Universita di
Parma for NMR and MS instrumental facilities.