Synthesis and antitumour activity of a new series of nitrosoureido sugars

Article abstract of DOI:10.1016/S0223-5234(00)00114-8

New nitrosoureido derivatives of di- or tri-deoxy-sugars have been synthesized. Very potent antitumour activity against L1210 leukaemia was exhibited by the compounds derived from methyl 3-amino-3,4-dideoxy-β- and α- and 4-amino-2,4-dideoxy-β- and α-D-arabino-hexopyranosides, 24, 26, 28 and 29, respectively. In further evaluation against B16 melanocarcinoma bearing mice, only compounds 24 and 26 displayed significant activity. Owing to its lower acute toxicity, methyl 3-[3-(2-chloroethyl)-3-nitrosoureido]- 3,4-dideoxy-β-D-arabino-hexopyranoside 24 appeared as the best candidate for preclinical studies. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00114-8

Eur. J. Med. Chem. 35 (2000) 137−146
© 2000 Editions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001148/FLA
137
´
Original article
Synthesis and antitumour activity of a new series of nitrosoureido sugars
Claude Monnereta*, Simone Risséa, Patrick Ardouinb, Alain Gouyetteb
aLaboratoire de pharmacochimie, unité mixte 176 CNRS/ Institut Curie, 26, rue d’Ulm, F-75248 Paris cedex 05, France
bLaboratoire de pharmacologie clinique, pavillon de recherche 2, Institut Gustave-Roussy, 39, rue Camille Desmoulins,
F-94805 Villejuif cedex, France
Received 8 November 1999; accepted 8 November 1999
Abstract – New nitrosoureido derivatives of di- or tri-deoxy-sugars have been synthesized. Very potent antitumour activity against L1210
leukaemia was exhibited by the compounds derived from methyl 3-amino-3,4-dideoxy-b- and a- and 4-amino-2,4-dideoxy-b- and
a-D-arabino-hexopyranosides, 24, 26, 28 and 29, respectively. In further evaluation against B16 melanocarcinoma bearing mice, only
compounds 24 and 26 displayed significant activity. Owing to its lower acute toxicity, methyl 3-[3-(2-chloroethyl)-3-nitrosoureido]-3,4-
´
dideoxy-â-D-arabino-hexopyranoside 24 appeared as the best candidate for preclinical studies. © 2000 Editions scientifiques et médicales
Elsevier SAS
amino-deoxy-sugars / nitrosourea / antitumour
1. Introduction
ticular, structural variations of C-1, C-2, C-3, C-5 and C-6
nitrosoureas, as well as structural variations at two
monosaccharide positions (bis-N-nitrosourea analogues)
and disaccharide N-nitrosourea analogues were dis-
cussed.
N-(2-haloethyl)-N-nitrosoureas CCNU, MeCCNU and
BCNU are representatives of one of the principle classes
of anticancer agents, displaying both a wide range of
activity in human cancers and being widely used to treat
brain tumours, melanomas and various leukaemias [1].
However, as these drugs produce delayed and cumulative
bone marrow toxicity, their clinical application was
somewhat limited. On the other hand, streptozocin (SZT),
a natural N-nitroso-N-methyl urea of 2-deoxy-D-glucose
retains antitumour activity with less bone marrow toxicity
than the non-carbohydrate nitrosourea [2]. SZT has been
used clinically to treat islet cell carcinoma of the pancreas
and carcinoid carcinoma, but its clinical use was limited
because of the damage to the pancreas and kidneys, this
being the consequence of its diabetogenic activity.
Since the discovery of SZT, many other chloroethyl-
nitrosoureido derivatives of amino-sugars have been
prepared and evaluated. All these compounds were re-
cently reviewed [3] in terms of their synthetic and mecha-
nistic aspects. Antitumour data and structure–activity
relationships were also included in this review. In par-
For our part, several years ago, we described a series of
new nitrosoureido derivatives of di- and trideoxy-
sugars [4]. All these compounds were deoxygenated at
the C-2 position and derived from 3-amino-2,3-dideoxy,
3-amino-2,3,6-trideoxy, 6-amino-2,6-dideoxy and 3,6-
diamino-2,3,6-trideoxy hexopyranoses of α- or â-D or
L-configurations. The influence of the hydroxyl substitu-
tion pattern, as well as the configuration at the anomeric
centre, and of the absolute configuration of the sugar
moiety on the antitumour activity of this series were
studied. All these compounds displayed significant activ-
ity in vivo against L1210, B16 melanocarcinoma, and
Lewis lung carcinoma.
Among the compounds which were endowed with a
moderate lipophilicity, i.e. those situated between the
lipophilic nitrosoureas of the first generation and the
hydrophilic sugar derivatives, methyl 3-[3-(2-chloro-
ethyl)-3-nitrosoureido]-2,3-dideoxy-α-D-arabino-hexopy-
ranoside (ecomustine, CY 233, NCS-609224) was found
to be very potent against L1210 leukaemia [5], as well as
*Correspondence and reprints: Claude.Monneret@curie.fr

138
against solid tumours such as the established B16 mela-
noma and colon 38 adenocarcinoma [6]. It was also
highly active via the i.v. route against xenograft human
tumours [7]. Moreover it had limited and reversible
haematological toxicity, failing to cross the blood-brain
barrier or enter the bone marrow [8].
In the present paper we report the synthesis and the
preliminary antitumour evaluation of two new and
closely related series of nitrosoureido compounds derived
either from 3-amino-3,4-dideoxy- or 4-amino-2,4-dide-
oxy-sugars of the D-series. In addition, a third series,
including 4-nitrosoureido sugars dideoxygenated simul-
taneously at the C-2 and C-3 positions, was also synthe-
sized and evaluated. As for the previous derivatives, their
preparation was undertaken with the aim of attaining a
better therapeutic index, and a lack of cross-resistance.
Furthermore, an increased stability was expected for the
same reason mentioned above.
Figure 1. Preparation of 3-amino-3,4-dideoxysugars 1–4.
it was expected that removal of this atom prior to this
cleavage would lead to the α-anomer. Therefore, hydro-
genolysis of compound 6a was undertaken (H2, Pd/C,
Et3N, EtOAc, 95%) to prepare the 2-deoxy-derivative 6b,
which was subsequently treated by sodium methoxide in
MeOH, leading to 7b (85% yield). Indeed, the target
amino-sugar 12 was successfully obtained in ≈ 38%
overall yield in three steps (7b → 8b → 11 → 12) which
included azidolysis (NaN3, NH4Cl, EtOH, 90 °C), acidic
hydrolysis (Amberlystt 15 ion-exchange resin, MeOH,
80 °C), and catalytic hydrogenation (Pd/C 10%, Et3N,
EtOAc, 80%). On the other hand, 4-amino-1,6-anhydro-
2,4-dideoxy-â-D-arabino-hexopyranose 13 was obtained
by catalytic hydrogenation of 8b.
2. Chemistry
2.1. Synthesis of amino-deoxy-sugars
The preparation of the 3-amino-3,4-dideoxysugars 1–4
(figure 1) has already been reported [9, 10]. The regiose-
lective synthesis of the 4-amino-2,4-dideoxy-â- and α-D-
arabino-hexopyranosides 10 and 12 was achieved from
D-glucal (figure 2). Oxidative cleavage of D-glucal, after
activation of hydroxy groups by O-tributyl-stannylation
as reported by Veyrières et al. [11], afforded 5 in 80%
yield. As expected, regioselective p-toluenesulfonylation
of 5 in pyridine led to 6a in high yield (90%) and 6a was
subsequently treated with sodium methoxide in methanol
to give 1,6:3,4-dianhydro-2-iodo-2-deoxy-â-D-galacto-
pyranose 7a in 94% yield. Cleavage of the 3,4-oxirane
ring of 2-substituted dianhydrohexopyranoses with a
wide range of nucleophiles is known to give the
4-substituted derivatives [12]. Indeed, the 4-azido com-
ponent 8a was the only isolable product, in 80% yield, by
addition of sodium azide to a solution of 7a in EtOH in
the presence of NH4Cl. It remained therefore to cleave
the 1,6-anhydro bond. This was achieved by addition of
Amberlystt ion-exchange resin to a hot methanolic solu-
tion of 8a. This led mainly to the â-methyl glycoside 9
which was easily separated (84%) and characterized. In
contrast, the corresponding α-anomer, also present in
small amount (25/75 ratio), could not be isolated as pure
compound. Amino-sugar 10 was finally obtained by
radical reduction (Bu3SnH, AlBN, 65%) of 9.
In order to ascertain for both series of derivatives
whether optimum antitumour activity is, as for 3-amino-
2-3-dideoxy nitrosoureido derivatives [4], connected or
not to the presence of two free OH groups in the sugar
moiety, the synthesis of ethyl 4-amino-2,3,4-trideoxy-α-
D-threo and α-D-erythro-hexopyranosides 17 and 22 was
also investigated. They were prepared from a common
intermediate, ethyl 6-O-benzyl-2,3-dideoxy-4-O-metha-
nesulfonyl-α-D-erythro-hexopyranoside 14 (figure 3).
Azidolysis of 14 was achieved as previously de-
scribed [13] and the azido-derivative 15 was converted to
16. Under more drastic hydrogenation conditions (Pd/C,
10%, Et3N, EtOAc), 16 was subsequently debenzylated
to afford the amino-sugar of threo configuration 17. The
corresponding erythro amino-sugar 22 was obtained from
14 in 5 steps in 51% overall yield. This involved
inversion of configuration at C-4 leading to 18
(CsCOOEt, 80%), quantitative transesterification of 18
(NaOMe, MeOH) methanesulfonylation of 19 (MsCl,
CH2Cl2, Et3N, 85%), and azidolysis of 20 to give 21.
Finally, catalytic hydrogenation of 21 afforded the target
amino-sugar 22 in 60% yield.
As we postulated that the presence of the bulky iodine
at C-2 may be responsible for the selective formation of
the â-anomer 9 during the cleavage of the anhydro-bond,

139
Figure 2. Regioselective synthesis of the 4-amino-2,4-dideoxy-â- and α-D-arabino-hexopyranosides 10 and 12.
2.2. Synthesis of the 3-(2-chloroethyl)-4-nitrosoureidos
23–26 and of the 4-(2-chloroethyl)-3-nitrosoureido
derivatives 27–32
between 40 and 50 mg/kg (i.p.), except for compounds 25
and 26 which had LD50 values of 25–30 mg/kg, close to
the LD50 values of BCNU (25 mg/kg).
Antineoplastic activity of compounds 23–32 was first
evaluated using an oily suspension at 5 mg/kg/day on
days 1, 5 and 9, after intraperitoneal inoculation of mice
with 105 L1210 leukemia cells. Higher activity was
observed (table II) with compounds 24, 26, 28 and 29.
The median survival time of mice treated with these
compounds was greatly prolonged (T/C > 600) and 8 of
the 10 animals treated survived for more than 60 days
with compounds 24, 28 and 29.
Amino-sugars 1–4 and 10, 12, 13, 16, 17 and 22 were
treated with 2,4,5-trichlorophenyl-N-(2-chloroethyl)-N-
nitrosocarbamate [14] to afford, in a one-step reaction,
the corresponding nitrosourea derivatives 23–32 (fig-
ure 4, table I).
3. Pharmacology
The acute toxicity of compounds 23–32 administered
intraperitoneally was determined in mice (DBA2) before
the evaluation of the antineoplastic activity of those
compounds. The LD50 values for all compounds were
The most active compounds 24, 26, 28 and 29 were
next evaluated against B16 melanocarcinoma (table III).
Compounds were administered intraperitoneally at
10 mg/kg/day at days 1, 5 and 9 after intraperitoneal

140
Figure 3. Synthesis of ethyl 4-amino-2,3,4-trideoxy-α-D-threo and α-D-erythro-hexopyranosides 17 and 22.
inoculation of mice with 2 × 106 B16 melanocarcinoma.
Nitrosourea derivatives 28 and 29 did not display any
significant antiproliferative activity and none of the 10
animals treated survived. In contrast, compounds 24 and
26 displayed high antitumour activity (T/C 257) as 7–8 of
the 10 animals treated survived for more than 60 days
with those derivatives.
Further experiments were conducted with the ni-
trosoureido derivative 24. Indeed, this compound dis-
plays similar in vivo activity against L1210 leukaemia
cells and B16 melanocarcinoma to that of the correspond-
ing α anomer 26, but was less toxic as indicated (vide
supra) by the LD50. Thus, the effect of the i.v. adminis-
tration of 24 on s.c. established B16 melanoma was
Table I. Analytical data of nitrosoureido sugars 23–32.
20b
Compound
M.p. (°C)a
[α]D
MS (DCI/NH3)b
m/z = (M + NH4)+
Analytical data
M.W.
23
24
25
26
27
28
29
30
31
32
108
+7.5 (c 0.3, MeOH)
+11 (c 0.75, CHCl3)
+139 (c 0.5, MeOH)
+121 (c 0.5, MeOH)
–78 (c 0.4, CHCl3)
–47 (c 0.3, CHCl3)
+49 (c 0.5, CHCl3)
+4 (c 0.8, CHCl3)
+62 (c 0.8, CHCl3)
+81.5 (c 0.8, CHCl3)
433
329
364
329
297
329
329
417
327
327
C17H22ClN3O7
C10H18ClN3O6
C10H17Cl2N3O6
C10H18ClN3O6
C9H14ClN3O5
C10H18ClN3O6
C10H18ClN3O6
C18H26ClN3O5
C11H20ClN3O5
C11H20ClN3O5
415.83
311.72
346.17
311.72
279.5
311.72
311.72
399.87
309.75
309.75
109–111
129–130
109–110
syrup
140
145
syrup
syrup
syrup
b
aSolvent: isopropylether. See experimental protocols.

141
Figure 4. 3-(2-chloroethyl)-4-nitrosoureidos 23–26 and the 4-(2-chloroethyl)-3-nitrosoureido derivatives 27–32.
investigated. Compound 24 was injected on days 3, 7 and
11 post implantation at daily doses of 10 and 20 mg/kg.
For comparison, BCNU was injected at 20 mg/kg by the
same i.v. route and schedule. After randomization of the
mice, the median tumour weight in treated and control
groups was almost the same at day 3 (36 mg for 24 versus
40 mg for BCNU and untreated mice). However, as
indicated in table IV, a large difference was observed at
day 30 between treated mice with 24 at 20 mg/kg and
Table II. Antitumour activity of nitrosoureido sugars 23–32 ver-
sus L1210 leukaemia in vivoa.
Compoundb
T/C (%)c
LTSd
(on day 60/total)
23
24
25
26
27
28
29
30
31
32
180
> 600
188
> 600
53
> 600
> 600
100
0
8/10
0/10
6/10
toxic
8/10
8/10
0
Table III. Antitumour activity of nitrosoureido sugars 24, 26, 28
and 29 against B16 melanocarcinomaa.
Compoundb
T/C (%)c
LTSd
(on day 60/total)
24
26
28
29
258
257
143
130
8/10
7/10
0/10
0/10
128
137
0
0
aNumber of control mice: 20. Number of treated mice for each
aNumber of control mice: 20. Number of treated mice for each
b
b
compound: 10. Weight of the mice 20 ± 1 g. Administration of
compound: 10. Weight of the mice 20 ± 1 g. Administration of
c
c
5 mg/kg i.p. at days 1, 5 and 9. T: median survival time in the
10 mg/kg i.p. at days 1, 5 and 9. T: median survival time in the
treated mice/C: median survival time in the control group × 100.
treated mice/C: median survival time in the control group × 100.
dLong time survival.
dLong time survival.

142
Table IV. Antitumour activity of 24 against established B16 me-
analytical values are within ± 0.3% of the calculated
compositions. Reactions were monitored by analytical
thin layer chromatography performed on Merck 60F254
precoated plates. Silica gel Merck (230–400 Mesh
ASTM) was used for column chromatography (table V).
lanocarcinomaa.
Compounda Doseb
Median tumour weight T/I %c
(mg/kg i.v.) (mg) on day
(on day 30)
3
30
24
20
10
20
0
36
32
40
40
688
91.2
75.0
67.0
0
4.1. 1,6-Anhydro-2-deoxy-2-iodo-4-O-
(p-toluenesulfonyl)- â-D-glucopyranose 6a
2 025
2 581
7 812
BCNU
Controls
To a solution of 1,6-anhydro-2-deoxy-2-iodo-â-D-
glucopyranose 5 [11] (14.7 g, 54 mmol) in pyridine
(150 mL), p-toluenesulfonyl chloride (11.59 g, 61 mmol)
was added and the mixture was stirred for 48 h at 20 °C.
The reaction mixture was then diluted with water
(200 mL) and extracted with ethyl acetate (3 × 250 mL).
The combined extracts were washed with a 5% aqueous
solution of hydrochloric acid (v/v), with water and with a
saturated aqueous solution of sodium hydrogencarbonate.
The organic fraction was dried over anhydrous magne-
sium sulfate, filtered and the solvent was removed in
vacuo to give a syrup (17.85 g). This was purified by flash
chromatography using dichloromethane and a mixture of
dichloromethane/methanol (99:1, v/v) as eluent. This
afforded a crystalline residue (14.7 g, 62%) of 9a and a
sample was recrystallized from dichloromethane: m.p.
aThe tumour was implanted s.c. on day 0 in female B6C3F1 mice
(weight ≈ 20 g; bdrugs were given i.v. on days 3, 7 and 11. cT/I was
calculated as 100 – [(median tumour weight in the treated mice/
median tumour weight in the control) × 100].
untreated mice, since the values of the median tumour
weight were 688 mg and 7 812, respectively. For com-
parison, the value observed for animals treated with
BCNU was 2 581. For the tumour-growth inhibition,
antitumour activity was assessed on the basis of the
percentage of tumour inhibition (% TI), calculated from
median tumour weight in treated and control mice on the
day of evaluation, as indicated in table IV. The % TI was
calculated as 100 – [(median tumour weight in treated
mice/median tumour weight in control) × 100. At 20 mg/
kg, the best inhibition was found (TI = 91%) for 24,
versus 67% for BCNU. Even at 10 mg/kg compound 24
exhibited a better activity than BCNU with a TI = 75%.
From these preliminary experiments, it appears that the
nitrosoureido sugar derivative 24 is a serious candidate
for further development.
20
141 °C; [α]D + 20° (c 1, CHCl3); IR (CHCl3): 3 602
(OH) and 1 600 (Ar) cm–1; Anal. Calcd. for C13H15O6SI:
C, 36.63; H, 3.55; I, 29.77; S, 7.52. Found: C, 36.83; H,
3.60; I, 29.38; S, 7.70.
4.2. 1,6:3,4-Di-anhydro-2-deoxy-2-iodo-
â-D-galactopyranose 7a
A 2 M solution of sodium methoxide in methanol
(100 mL) was added to a solution of 6a (19.63 g,
46 mmol) in dry chloroform (800 mL). The mixture was
stirred for 2.5 h at 20 °C and diluted with water
(100 mL). The organic layer was separated, washed with
water (50 mL), dried over anhydrous magnesium sulfate
and evaporated in vacuo to give 12.86 g of a crude syrup.
Flash chromatography with dichloromethane as eluent
4. Experimental protocols
The melting points were measured on a Köfler hot
stage apparatus and are uncorrected. Mass spectra were
obtained with a Nermag-Ribermag R10-10C spectrom-
eter using either the electron impact method (EI) at 70 eV
or applying a desorption chemical ionization technique
(CI) using ammonia as the reagent gas. Infrared spectra
were registered with a Perkin-Elmer 1710 spectropho-
tometer, either as chloroform solutions or KBr discs. The
1H-NMR spectra (250 or 300 MHz) were recorded on a
Bruker AC 250 or a Bruker AC 300 spectrometer.
Chemical shifts are expressed as parts per million down-
field from tetramethylsilane. Splitting patterns have been
designated as follows: s (singlet), d (doublet), t (triplet),
q (quadruplet), dd (doublet of doublet), m (multiplet), bs
(broad singlet). Coupling constants (J values) are listed in
hertz (Hz). Microanalyses were carried out by the ‘Ser-
vice Central d’Analyse du C.N.R.S., Vernaison’ and
20
gave 11.10 g (94%) of pure 7a: syrup; [α]D – 68° (c 1,
CHCl3); MS (DCI/NH3): m/z 272 (M + NH4)+, 255 (M +
H)+; Anal. Calcd. for C6H7O3I: C, 28.37; H, 2.78; I,
49.96. Found: C, 28.53; H, 2.56; I, 50.02.
4.3. 1,6-Anhydro-4-azido-2,4-dideoxy-2-
iodo-â-D-arabino-hexopyranose 8a
A solution of 7a (11.1 g, 43.7 mmol) in ethanol
(130 mL) was heated at 90 °C for 24 h in the presence of
NaN3 (11.6 g, 178.4 mmol) and NH4Cl (46.5 g), dis-
solved in water (26 mL). After cooling to room tempera-

143
Table V. 1H-NMR data of compounds 6–13 and 17–22 in CDCl3, unless otherwise indicated.
Compound H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
3.72
Others
6a
7a
8a
6b
7b
8b
9
5.73
bs
5.72
bs
5.76
bs
5.68
bs
5.46
d (3.5)
5.73
s
4.47
d (8, 5)
3.88
bd (3.1)
4.17
t (1.5, 1.5)
3.95
bd (2.5, < 1)
1.87 bd (15, < 1)
2.19 dd (15.5)
1.96 m
2.10 d (15)
1.98 bd (15, < 1)
2.27 m
4.19
bs
3.70
dd (4.1)
4.21
bs
3.99
m
3.19
m
4.48
4.65
4.18
2.46 (CH3), 7.37 and
t (2.5, 2.5) dd (2.5, 5) dd (8.2)
3.76 4.96 4.17
t (4.5, 4.5) t (4.5, 4.5) d (6)
3.60
≈ t (2.2)
3.54
s
dd (8.5, 5.5.) 7.86 (2d, CH2Ph)
3.61
dd (6.5)
3.81
dd (7.5, 5) (OH)
3.85
dd (7.5, 5)
3.57
dd (6.5, 5)
3.85
dd (65.5)
4.62
dd (5.2)
4.70
d (5)
4.87
4.23
d (6.5)
4.36
d (6.5)
4.08
d (6.5)
4.36
2.74
3.62
dd (5, 4.5) t (5.5)
3.54
s
3.99
m
4.70
d (5)
3.60
d (6.5)
3.94
←
3.85–3.82
→ 3.54
t (9)
3.85
3.54 s (OCH3)
m
m (9, 4, 2) m (12, 5.5, m
2)
2.95
m (9.5, 5, 3) dd (12, 5) dd (12, 3) s (OCH3)
3.50
m
3.22
m
4.27
d (5.5)
←
10*
11
4.31
1.93 (m, 12, 5, 2)
3.23
2.23
3.63
3.51
3.30
dd (10, 2) 1.24 (m, 12, 10, 10) (m)
t (9.9)
3.36
m (11, 9, 5) t
4.82
d (3)
4.70
d (3)
5.40
s
4.74
bs (1)
4.99
bs (1)
2.15 m (13, 5, 1)
1.70 m (13, 10, 3)
1.86 m (12, 5, 1)
1.43 m (12, 10, 10)
1.93 m (14, 8, 2)
1.53 d (14)
4.01
3.86
dd (12, 2.5) dd (12, 4) s (OCH3)
3.60 3.48 3.34
dd (12, 3) dd (12, 5) s (OCH3)
4.20
d (5.5)
3.79
3.30
12*
13*
17*
18
3.32
m
3.52
bs
2.32
t (9.5, 9.5)
2.64
3.52
s
bs
←
1.80–1.65
→
3.72–3.45
→
1.15 t (7, CH3)
3.25 q (CH2)
m
m
←
2.09–1.78
m
→ 5.00
4.12
m
3.79–3.71 3.54–3.44 4.52 dd (9, 4, CH2Ph)
m
s
m
2.30 and 2.28 2q (CH2)
1.13 and 1.22 2t (7, CH3)
19
20
21
4.90
d (3)
←
←
←
2.12–1.19
m
→
← 3.93–3.78 →
378–3.68
m
3.54–3.49 1.25 t (7, CH3)
m
m
m
3.48 q (CH2)
4.60 dd (9, 4, CH2Ph)
→ 1.28 t (7, CH3)
3.45 q (CH2)
4.92
d (3)
2.25–1.25
m
→ 3.70
←
←
←
3.85–3.75
m
m
4.55 dd (9, 4, CH2Ph)
→ 1.25 t (7, CH3)
3.48 q (CH2)
4.60 dd (9, 4, CH2Ph)
→ 1.14 t (7, CH3)
3.20 q (CH2)
4.86
dd (2, 1)
1.95–1.83
m
→
3.73–3.64
m
m
22*
4.72
bs (1)
←
1.63–1.57
m
→
3.68–3.33
m
m
*
in DMSO.
ture, the reaction mixture was filtered and the filtrate was
diluted with dichloromethane (500 mL). The organic
layer was separated, washed with water (2 × 25 mL) and
dried over anhydrous magnesium sulfate. Evaporation in
vacuo gave a crude product which was purified by flash
chromatography with pentane/acetone (5:1, v/v). This
afforded 8a (10.47 g, 80%) as a crystalline compound:
4.4. Methyl 4-azido-2,4-dideoxy-2-iodo-
â-D-arabino-hexopyranoside 9
A methanolic solution of 8a (5.1g in 500 mL) was
heated under reflux for 48 h in the presence of Amberlite
R15 (H)+. The solution was then filtered and the filtrate
evaporated under reduced pressure. The residue (7.1 g)
was chromatographed using pentane/acetone (5/1, v/v) as
eluent. This afforded successively unreacted starting
material 8a (0.69 g, 13%) and 9 (4.79 g, 84%) as a
20
m.p. 67 °C; [α]D + 24.5° (c 0.8, CHCl3); IR (CHCl3):
2 107 cm–1 (N3); MS (DCI/NH3): m/z 315 (M + NH4)+.
Anal. Calcd. for C6H8N3O3I: C, 24.26; H, 2.71; N, 14.15.
Found: C, 24.53; H, 2.56; N, 14.66.
20
crystalline residue: m.p. 118 °C; [α]D + 187° (c 1.06,

144
CHCl3); IR (CHCl3) 3 600 (OH), 2 116 (N3) cm–1; MS
(DCI/NH3): m/z 347 (M + NH4)+. Anal. Calcd. for
C7H12N3O4I: C, 25.55; H, 3.68; N, 12.77. Found: C,
25.96; H, 3.77; N, 12.45.
(8 mL), by sodium azide (0.72 g, 11 mmol) and NH4Cl
(2.88 g, 53.8 mmol) dissolved in water (1.6 mL). After
refluxing for 24 h, the reaction mixture was concentrated
in vacuo and the residue was taken up in dichloro-
methane. Usual work-up of the organic solution followed
by chromatography on silica gel with a mixture of
cyclohexane/EtOAc as eluent (2:1, v/v) afforded succes-
sively the 3-azido (0.118 g, 25%) and the 4-azido 8b
(0.276 g, 55%) sugar derivatives. Compound 8b: m.p.
85 °C; [α]D20 – 154° (c 1, CHCl3); IR (CHCl3): 3 563
(OH), 2 104 (N3) cm–1; MS (DCI/NH3): m/z 189 (M +
NH4)+; 171 (M + NH4 – H2O)+. Anal. Calcd. for
C6H9N3O3: C, 42.11; H, 5.30; N, 24.55. Found: C, 42.37;
H, 5.10; N, 24.50.
4.5. Methyl 4-amino-2,4-dideoxy-
â-D-arabino-hexopyranoside 10
To a solution of 9 (1.2 g, 3.8 mmol) in toluene (60 mL)
kept under argon atmosphere, tributyltin hydride (4 mL,
14.9 mmol) and AIBN (0.3 g) were added. The reaction
mixture was stirred under reflux for 3 h, by which time
the reaction has gone to completion. The toluene was
subsequently removed under vacuum and flash chroma-
tography of the residue (1.18 g) with dichloromethane
and dichloromethane/saturated NH3 methanol (98:2, v/v)
afforded 10 (441 mg, 65%) as crystals: m.p. 96 °C; [α]D20
– 65° (c 1, MeOH); IR (CHCl3): 3 336 cm–1 (OH, NH2);
MS (DCI/NH3): m/z 178 (M + H)+. Anal. Calcd. for
C7H15NO4: C, 47.45; H, 8.53; N,: 7.90. Found: C: 47.18;
H, 8.31; N, 7.82.
4.9. Methyl 4-azido-2,4-dideoxy-
α-D-arabino-hexopyranoside 11
The title compound was prepared from 8b following
the procedure already described for 9. Purification was
carried out by flash chromatography with a mixture of
cyclohexane/EtOAc (1:1, v/v) as eluent, giving 86% of
pure 11 as a crystalline residue: m.p. 85 °C; [α]D20 + 142°
(c 0.8, CHCl3); IR (CHCl3) 3 600 (OH) 2 109 cm–1 (N3);
MS (DCI/NH3): m/z 221 (M + NH4)+, 204 (M + H)+.
Anal. Calcd. for C7H13N3O4: C, 41.38; H, 6.45; N, 20.68.
Found C, 41.48; H, 6.61; N, 20.74.
4.6. 1,6-Anhydro-2,4-dideoxy-4-O-
(p-toluenesulfonyl)-â-D-glucopyranose 6b
A solution of 6a (10.24 g, 24 mmol) in ethyl acetate
(250 mL) was stirred under hydrogen atmosphere (1
atm.) for 18 h in the presence of 10% palladium-on-
charcoal (2 g) and Et3N (5 mL). The catalyst was re-
moved by filtration and the filtrate was evaporated under
reduced pressure to afford 6.87 g (95%) of 6b as a
crystalline residue pure enough for the next step. A
sample was recrystallized from ethyl acetate for analysis:
m.p. 101 °C; [α]D20– 69° (c 0.8, CHCl3); IR 3 558 cm–1
(OH); MS (DCI/NH3): m/z 318 (M + NH4)+. Anal. Calcd.
for C13H16O6S: C, 51.99; H, 5.37; S, 10.68. Found: C,
51.90; H, 5.50; S, 10.69.
4.10. Methyl 4-amino-2,4-dideoxy-
α-D-arabino-hexopyranoside 12
A solution of 11 (0.9 g, 4.43 mmol) in ethyl acetate
(50 mL) was stirred for 24 h under H2 atmosphere (1
atm.) in the presence of 10% Pd-on-charcoal (0.5g) and
Et3N (0.9 mL). The catalyst was then removed by filtra-
tion and the filtrate concentrated in vacuo. This afforded
the amino-sugar 12 (0.62g, 80%) as amorphous crystals:
[α]D20 + 101° (c 0.8, MeOH); IR (CHCl3) 3 347 (OH)
cm–1; MS (DCI/NH3): m/z 178 (M + H)+. Anal. Calcd. for
C7H15NO4: C, 47.45; H, 8.53; N, 7.90. Found: C, 46.88;
H, 8.44; N, 7.72.
4.7. 1,6:3,4-Dianhydro-2-deoxy-â-D-galactopyranose 7b
Following the procedure for the preparation of 7a, the
title compound was prepared from the tosyl derivative 6b
and isolated as a syrup (0.38 g, 85%): [α]D20 – 108° (c 1,
CHCl3); MS (DCI/NH3): m/z 146 (M + NH4)+. Anal.
Calcd. for C6H8O3: C, 56.25; H, 6.29. Found: C, 56.29;
H, 6.20.
4.11. 4-Amino-1,6-anhydro-2,4-dideoxy-
â-D-arabino-hexopyranoside 13
Following the procedure already described for the
preparation of 12, reduction of 8b readily afforded 13 in
81% yield: crystal; m.p. 139 °C; [α]D20 – 106° (c 0.8,
MeOH); IR (CHCl3): 3 360 cm–1 (OH); MS (DCI/NH3):
m/z 146 (M + H)+. Anal. Calcd. for C6H8O3: C, 49.65; H,
6.29; N, 9.65. Found: C, 49.29; H, 6.64; N, 9.45.
4.8. 1,6-Anhydro-4-azido-2,4-dideoxy-
â-D-arabino-hexopyranoside 8b
Following the procedure for the preparation of 8a, the
title compound was prepared by treatment of the anhydro
derivative 7b (0.35 g, 2.7 mmol) in solution, in methanol

145
4.12. Ethyl 4-amino-6-O-benzyl-2,3,4-
trideoxy-α-D-threo-hexopyranoside 16
(DCI/NH3): m/z 284 (M + NH4)+, 267 (M + H)+. Anal.
Calcd. for C15H22O4: C, 67.65; H, 8.33. Found: C, 67.84;
H, 8.22.
To an ethanolic solution of 15 (1.62g, 5.6 mmol in
50 mL) were added Et3N (900 mL) and 5% Pd-on-
charcoal (140 mg). The suspension was stirred for 6 h
under hydrogen atmosphere (1 atm.), filtered and the
filtrate was concentrated in vacuo to give 16 (1.5g, 95%),
pure enough for the next step: syrup; MS (DCI/NH3): m/z
266 (M + H)+.
4.16. Ethyl 6-O-benzyl-2,3-dideoxy-4-O-
methanesulfonyl-α-D-threo-hexopyranoside 20
A solution of 19 (1.55 g, 5.82 mmol) in anhydrous
dichloromethane (60 mL) containing triethylamine
(1.23 mL) was cooled to 0 °C and then methanesulfonyl
chloride (0.65 mL, 8.43 mmol) was added. The solution
was stirred at 0 °C for 5 h and then poured into 30 mL of
ice-water. Extraction with dichloromethane (2 × 150 mL)
and usual work-up afforded 2.49 g of residue. This was
purified by flash chromatography with cyclohexane-
EtOAc (4:1, v/v), giving 2.14 g (85%) of pure 20 as a
syrup: [α]D20 + 31° (c 1, CHCl3); MS (DCI/NH3): m/z 362
(M + NH4)+. Anal. Calcd. for C16H24O6S: C, 67.65; H,
8.33. Found: C, 67.84; H, 8.22.
4.13. Ethyl 4-amino-2,3,4-trideoxy-α-
D-threo-hexopyranoside 17
Following the previous procedure used for 16 but with
10% Pd-on-charcoal, compound 16 was converted into
17: syrup; MS (DCI/NH3): m/z 176 (M + H)+. Anal.
Calcd. for C8H17NO3: C, 54.84; H, 9.78; N, 7.99. Found:
C, 55.01; H, 9.82; N, 7.62.
4.14. Ethyl 6-O-benzyl-2,3-dideoxy-4-O-
propionyl-α-D-threo-hexopyranoside 18
4.17. Ethyl 4-azido-6-O-benzyl-2,3,4-
trideoxy-α-D-erythro-hexopyranoside 21
Caesium propionate (6.55 g) was added to a solution of
ethyl 6-O-benzyl-2,3-dideoxy-4-O-methanesulfonyl-α-
D-erythro-hexopyranoside [13] 14 (5.8 g, 16.86 mmol) in
DMF (80mL) and the reaction mixture was heated with
stirring for 30 h at 120 °C. After cooling to room tem-
perature and addition of water (80 mL), the mixture was
extracted with ether (1 L, then 0.5 L). The combined
organic layers were washed several times with water,
dried over MgSO4 and concentrated in vacuo. The crude
residue (4.85 g) was purified by flash chromatography
using cyclohexane, then a mixture of cyclohexane/EtOAc
(90:10, v/v) as eluent. This led to isolation of 4.33 g
(80%) of 18. A sample was recrystallized from cyclohex-
ane; m.p. ≈ 50 °C: [α]D20 + 30° (c 0.8, CHCl3); IR
(CHCl3): 1 731 cm–1 (CO); MS (DCI/NH3): m/z 340 (M
+ NH4)+, 321 (M + H)+, 294 (M + NH4 – 46)+, 277 (M
+ H – 46)+. Anal. Calcd. for C18H24O5: C, 67.48; H, 7.55.
Found: C, 67.83; H, 7.38.
Sodium azide (0.46 g, 7.07 mmol) was added to a
solution of 20 (1.6 g, 4.65 mmol) in DMF (25 mL) and
the mixture was stirred at 100 °C for 24 h. After cooling
to room temperature, extraction was carried out with
ether (85 mL) after addition of H2O (15 mL). Usual
work-up afforded a crude residue (1.24 g, 91%), pure
enough for the next step; [α]D20 + 134.5° (c 1, CHCl3); IR
(CHCl3): 2 103 cm–1 (N3); MS (DCI/NH3): m/z 309 (M +
NH4)+. Anal. Calcd. for C15H21N3O3: C, 61.84; H, 7.27;
N, 14.42. Found: C, 61.92; H, 7.42; N, 14.12.
4.18. Ethyl 4-amino-2,3,4-trideoxy-
α-D-erythro-hexopyranoside 22
Following the procedure already described for 12
azido-sugar 21 was reduced in ethanol solution to afford
compound 22 which was isolated as a pure compound,
after flash chromatography with dichloromethane-NH3-
saturated methanol (95:5, v/v), in 60% yield as a syrup:
[α]D20 + 157° (c 0.8, MeOH); MS (DCI/NH3): m/z 176 (M
+ H)+, 130 (M + H – C2H5OH)+. Anal. Calcd. for
C8H17NO3. C, 54.84; H, 9.78; N, 7.99. Found: C, 54.76;
H, 9.77; N, 8.04.
4.15. Ethyl 6-O-benzyl-2,3-dideoxy-
α-D-threo-hexopyranoside 19
To a solution of 18 (2 g, 6.2 mmol) in methanol
(40 mL), 1 M sodium methoxide in methanol (4 mL) was
added. After stirring for 5 h at room temperature and
neutralization by IRC 50S ion-exchange resin, the solu-
tion was concentrated in vacuo. Flash chromatography
using a mixture of cyclohexane/EtOAc (90:10, v/v) as
4.19. Typical procedure for the synthesis of
nitrosoureido derivatives 23–32
To a cooled solution (≈ 0 °C) of the amino-sugar
(1 mmol) in anhydrous DMF (5 mL), N-(2-chloroethyl)-
N-nitrosocarbamate of p-nitrophenyl [14] (1.2 mmol)
eluent afforded 19 (1.71 g, ≈ 100%) as a syrup: [α]D20
+
82° (c 1, CHCl3); IR (CHCl3): 3 497 cm–1 (OH); MS

146
was added and the mixture was stirred at the same
temperature for 3–5 h. The DMF was removed under
reduced pressure and the residue was dissolved in EtOAc
(100 mL). The organic solvent was washed several times
with water, dried over MgSO4 and evaporated in vacuo.
The residue was purified by flash chromatography
(cyclohexane-EtOAc) and the pure product crystallized
from isopropyl ether.
10 mg/kg/day in 0.5 mL. Ten mice for each treated group
and 20 mice for control groups were used.
5.1.3. Activity against established B16
melanocarcinoma (table IV)
B16 melanoma was maintained in syngenetic adult
C57BL/6 mice. For evaluation, the B16 tumour was
subcutaneously (s.c.) implanted on day 0 in B6CFF1
male mice. After 1 g of tumour was mixed with 9 mL of
cold balanced salt, the preparation was homogenized and
each mouse was inoculated with 0.5 mL tumour homo-
genate. Compounds 24 and BCNU were given i.v. on
days 3, 7 and 11 at the doses indicated. BCNU was kindly
donated by the Drug Research and Development (DRD),
Division of Cancer Treatment (DCI, NCI, Bethesda, Md,
USA).
5. Antineoplastic evaluation
5.1. In vivo tests
The experimental procedures, the type of mice used
(B6D2F1 or C57BL/6), and the calculation of the median
survival times of the groups of control and treated
animals were in accordance with the protocols of the
National Cancer Institute (Instr. 271F, November 1983).
References
5.1.1. Test against L1210 leukaemia in
oily suspension (table II)
[1] Schabel F.M., Cancer Treat. Rep. 60 (1976) 665–697.
[2] Weiss R.B., Cancer Treat. Rep. 66 (1982) 427–438.
[3] Gnewuch C.T., Sosnovsky G., Chem. Rev. 97 (1997) 829–1013.
On day 0, mice (B6D2F1) were inoculated intraperi-
toneally with 105 L1210 leukaemia cells. On days 1, 5
and 9, the mice (10 for each group treated) received
intraperitoneally the dose of 5 mg/kg of compounds
23–32 in 0.2 mL of neutralized and sterilized olive oil
suspension. The control group (20 mice) received the
same volume of solvent only. The mortality of the mice
was monitored daily and autopsies were performed to
find out whether deaths were due to leukaemia or to a
toxic action of the drug. The observation period lasted at
least 60 days.
[4] Roger P., Monneret C., Fournier J.P., Choay P., Gagnet R., Gosse C.
et al., J. Med. Chem. 32 (1989) 16–23.
[5] Gosse C., Atassi G., Letourneux Y., Ardouin P., Gouyette A.,
Fournier J.P., Roger P., Anticancer Res. 8 (1988) 1419–1422.
[6] Atassi G., Dumont P., Gosse C., Fournier J.P., Gouyette A., Roger P.,
Cancer Chemother. Pharmacol. 25 (1989) 205–209.
[7] Dumont P., Atassi G., Roger P., In Vivo 4 (1990) 61–64.
[8] El Abed I., thesis, Université Paris V, France.
[9] Martin A., Monneret C., Gautier C., Fournier J.P., Roger P., J.
Carbohydr. Chem. 9 (1990) 853–861.
[10] Rissé S., Roger P., Monneret C., J. Carbohydr. Chem. 12 (1993)
1105–1115.
[11] Czernecki S., Leteux C., Veyrières C., Tetrahedron Lett. 33 (1992)
221–224.
5.1.2. Test against B16 melanocarcinoma
in oily suspension (table III)
On day 0, mice (C57BL/6) were inoculated intraperi-
toneally with 2 × 106 B16 melanocarcinoma cells. The
treatment protocol and the observation period are similar
to the above procedure, except the dose which was
[12] Cerny M., Stanek Jr J., Adv. Carbohydr. Chem. Biochem. 34 (1977)
23–177.
[13] Pontikis R., Monneret C., Nucleosides Nucleotides 12 (1993)
547–558.
[14] Roger P., Leroy R., (1988) Eur. Patent 88. 400 999.4.