Synthesis and induction of apoptosis in B cell chronic leukemia by diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside hydrochloride and its derivatives

Article abstract of DOI:10.1016/S0008-6215(02)00407-X

2-Acetamido-2-deoxy-D-glucose hydrochloride (D-glucosamine hydrochloride) has been used for the preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-β- (4) and 2-tetrachlorophthalimido-α,β-D-glucopyranose (6), which have been transformed into the appropriate bromides and the chloride. Both bromo and chloro sugars were used as a glycosyl donors for the glycosylation of diosgenin [(25R)-spirost-5-en-3β-ol]. These condensations were conducted under mild conditions, using silver triflate as a promoter, and gave diosgenyl glycosides 9 and 12. Each of them was converted into diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside hydrochloride (11) and N-acylamido derivatives. The structures of all new glycosides were established by ¹H and ¹³C NMR spectroscopy. These diosgenyl glycosides are the first saponins containing the D-glucosamine residue that have been synthesized. These compounds show promising antitumor activities. The synthetic saponins increase the number of apoptotic B cells, in combination with cladribine (2-CdA), that are isolated from chronic lymphotic leukemia (B-CLL) patients.

Full text of DOI:10.1016/S0008-6215(02)00407-X

Carbohydrate Research 338 (2003) 133–141
www.elsevier.com/locate/carres
Synthesis and induction of apoptosis in B cell chronic leukemia by
-glucopyranoside hydrochloride and
diosgenyl 2-amino-2-deoxy-b-
D
its derivatives
Henryk Myszka,a,* Dorota Bednarczyk,a Maria Najder,b Wiesław Kacac,d
aFaculty of Chemistry, Uni6ersity of Gdan´sk, Sobieskiego 18, 80-952 Gdan´sk, Poland
bDepartment of Tumor Pathology, Medical Uni6ersity of Ło´dz´, Paderewskiego 4, 93-513 Ło´dz´, Poland
cMicrobiology and Virology Center of the Polish Academy of Sciences, Lodowa 106, 93-232 Ło´dz´, Poland
d
´
´
Swie˛tokrzyska Academy, Department of Microbiology, Swie˛tokrzyska 15, 25-405 Kielce, Poland
Received 31 July 2002; accepted 16 October 2002
Abstract
2-Acetamido-2-deoxy-
D
-glucose hydrochloride (
D
-glucosamine hydrochloride) has been used for the preparation of 1,3,4,6-
-glucopyranose (6), which have been trans-
tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-b- (4) and 2-tetrachlorophthalimido-a,b-
formed into the appropriate bromides and the chloride. Both bromo and chloro sugars were used as a glycosyl donors for the
D
glycosylation of diosgenin [(25R)-spirost-5-en-3b-ol]. These condensations were conducted under mild conditions, using silver
triflate as a promoter, and gave diosgenyl glycosides 9 and 12. Each of them was converted into diosgenyl 2-amino-2-deoxy-b-D-
1
glucopyranoside hydrochloride (11) and N-acylamido derivatives. The structures of all new glycosides were established by H and
13C NMR spectroscopy. These diosgenyl glycosides are the first saponins containing the
D
-glucosamine residue that have been
synthesized. These compounds show promising antitumor activities. The synthetic saponins increase the number of apoptotic B
cells, in combination with cladribine (2-CdA), that are isolated from chronic lymphotic leukemia (B-CLL) patients. © 2002
Published by Elsevier Science Ltd.
Keywords: 2-Acetamido-2-deoxy-D-glucose; D-Glucosamine; Diosgenin; (25R)-Spirost-5-en-3b-ol; Diosgenyl glycosides; Trifluoroacetamido deriva-
tives; Tetrachlorophthalimido derivatives; Glycosylation
1. Introduction
substituted at the 2-position and another sugar or sugar
chain at the 3- or 4-position.1–9
We have synthesized diosgenyl glycosides containing
Saponins are steroid or triterpenoid glycosides that are
widely distributed in plants and in some marine organ-
isms.1,2 The carbohydrate residue, usually a mono-, di-,
tri- or tetrasaccharide, is covalently attached to the
sapogenin backbone. Examples include diosgenin, ti-
gogenin or sarsapogenin. The carbohydrate chain con-
stitutes a hydrophilic part, while the appropriate
sapogenin is a hydrophobic fragment in this kind of
D
-glucosamine derivatives as the carbohydrate residue.
These glycosides have not been found thus far in natu-
ral sources. The presence of the -glucosamine residue,
D
particularly the NH2 group, creates an opportunity to
synthesize new derivatives of glycosides. We have re-
ported general methods for the synthesis of this kind of
diosgenyl glycosides. The most widely glycosylation
methods are based on the participation of a group at
C-2 of glycosyl donor, which favor formation of a
1,2-trans glycosidic bond. Also the leaving groups of
the glycosyl donors have been recognized as one of the
most important parameters responsible for the glycosy-
lation reaction. Among the many methods of glycosyla-
tion now available, we decided to use the appropriate
bromo and chloro sugars as glycosyl donors and silver
triflate as the promoter.10–12
glycoside. Usually, in natural diosgenyl glycosides b-
glucopyranose is the first sugar attached to diogenin,
which in turn has very often an a- -rhamnopyranose
D-
L
* Corresponding author. Tel.: +48-58-345-0344; fax: +
48-58-341-0357
E-mail address: myszka@chemik.chem.univ.gda.pl (H.
Myszka).
0008-6215/03/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0008-6215(02)00407-X

134
H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
Some of saponins exhibit a wide spectrum of biolog-
glycosyl donors for glycosylation of diosgenin. Among
many methods the most widely used way involved a
glycosyl donor containing a participating group as the
amino-protecting function.10 The Koenigs–Knorr reac-
tion is one of the oldest method for the preparation of
1,2-trans-glycosides requiring 3,4,6-tri-O-acetyl-2-2-ac-
ical activities, which can be ascribed to the diosgenin
moiety and/or to the carbohydrate residue. Diosgenyl
glycosides are often found as the major components in
traditional Oriental medicines and have been used there
as pharmaceutical agents to treat malaria, helminth
infections and snake bites.13–16 They are also good
antifungal and antibacterial agents.5,17 In the last two
decades many pharmacological studies have shown that
diosgenin and its glycosides exhibit cytotoxicity toward
human cancer cells.7,18–27 Dioscin,4,6,28 polyphyllin D,4,5
gracillin,3 trillin3 and saponin named Pb or PF315,20,29
are the most known diosgenyl saponins with these
properties.
In a set of experiments we have investigated cells
used from B cell chronic lymphocytic leukemia (B-
CLL) patients, the most common form of leukemia in
the Western world.30 In this article we examined and
compared the effects of the newly synthesized diosgenyl
glycosides and 2-chlorodeoxyadenosine (2-CdA) on
apoptosis B-CLL. Cladribine (2-CdA) is a purine
analog recently introduced for the treatment of
lymphoid malignancies.31,32 The aims of the biological
studies were to find out if the two diosgenyl glycosides
and 2-CdA increase apoptosis and necrosis of B cells
isolated from blood of B-CLL patients.
ylamido-2-deoxy-D-glucopyranosyl bromides and chlor-
ides as donors and silver salts as promoters. In our
route the synthetic strategy is based on preparation
NTFAc- and NTCP-protected per-O-acetyl bromides
and chlorides and subsequent coupling with diosgenin.
The yield of the coupling reaction was usually moder-
ate, probably due to the low reactivity of the glycosyl
donors and the possibility of oxazoline derivative for-
mation, as well as the reactivity of the acceptor.
Compound 3 was obtained from commercially avail-
able
D-glucosamine hydrochloride in a procedure that
used p-anisaldehyde for protection of NH2 (“1),
acetylation (“2) and removal of the p-methoxybenzyli-
dene group with HCl in acetone (Scheme 1, route a–c).
This product was acylated again, but now with tri-
fluoroacetic anhydride (“4), and finally 4 was treated
with excess of TiBr4 to give glycosyl donor 5 in good
yield (80%). Application of trifluoroacetyl as a protect-
ing group for the NH2 function gave the possibility of
selective deprotection of the hydroxyl and amino
groups in the final glycoside. Subsequently, NTCP-
derivative 6, as a mixture of anomers, was obtained
2. Results and discussion
2.1. Synthesis
from
D-GlcNH2·HCl in a one-pot reaction sequence.
First, the 3,4,5,6-tetrachlorophthalimido (TCP) group
was installed by adding one equivalent of sodium
methoxide in MeOH, followed by 3,4,5,6-tetra-
chlorophthalic anhydride. The NTCP-intermediate was
As demonstrated in the preceding paper,33 the appro-
priate derivatives of
D-glucosamine proved to be useful
Scheme 1. Reagents and conditions: (a) aq NaOH, CH3O–C6H4–CHO; (b) Py, Ac2O; (c) acetone, 5 M HCl; (d) Py, (CF3CO)2O;
(e) CH2Cl2−EtOAc (10:1), TiBr4; (f) NaOMe in MeOH, TCPO, Py, Ac2O; (g) HBr–CH3COOH; (h) CHCl3, Cl2HCOCH3.

H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
135
Scheme 2. Reagents and conditions: (a) CH2Cl2, AgOTf, −15 °C; (b) CH2Cl2, AgOTf, rt; (c) NaOMe in MeOH; (d) acetone, 0.1
M NaOH, 1.3% HCl in MeOH; (e) EtOH, H2NCH2CH2NH2 or EtOH, NH2NH2·H2O; (f) MeOH, Ac2O; (g) C6H5NCO.
then O-acetylated with Ac2O in pyridine which resulted
in 6. This procedure was adapted from Schmidt, who
applied NTCP derivatives for the synthesis of series
O-glycosides.34,35 Treatment of 6 with excess of HBr in
HOAc or with TiBr4 resulted in the formation of
glucosyl bromides 7 (white foam), also as an anomeric
mixture.36 Compound 6 was also transformed into the
chloride 8 via the modified method (dichloromethyl+
methyl ether and BF3·Et2O) that has been used in the
synthesis of the phthalimido derivatives in the galacto
series.37 Under these conditions only the crystalline
anomer b was obtained (Scheme 1, route f–h).
neighboring participation group in the coupling
reaction.
All three reactions were realized in the presence of an
activator (AgOTf), which is called the reaction pro-
moter, and in all of them only the b-glycosides have
been obtained. The methods with use of glycosylation
donors containing a participating group as the amino
protective function in Koenigs–Knorr reactions are the
so-called ‘oxazoline’ and ‘phthalimido’ procedures.10
The role of AgOTf is to assist the departure of the
anomeric leaving group (here Br or Cl) in a way giving
intermediates that have a pronounced tendency to form
the 1,2-trans-glycosides 9 and 12.
The glycoside 9 was O-deacetylated by the Zemple´n
procedure38 to give product 10 in high yield (98%).
Finally, removal of all of the acyl groups on 9 with
sodium hydroxide provided hydrochloride 11 (82%),
(Scheme 2, entries c and d), which will be used as
substrates for the synthesis of a new series of diosgenyl
glycosides (e.g., N-ureido derivatives).
A TCP group in 12 could be rapidly and cleanly
cleaved by use of a slight excess of ethylenediamine in
ethanol.34,35,39 Thus treatment of 12 with six equivalents
of ethylenediamine in EtOH during 6 h at 60 °C gave
fully deprotected glycoside 13 in 70% yield and N-ace-
tyl-glycoside 14 in 29% yield, (Scheme 2, entries e and
f). Alternatively, treatment of 12 with hydrazine hy-
drate results in only 13 (almost quantitatively). For
biological purposes 13 was converted into hydrochlo-
ride 11.
Glycosylation of diosgenin with 3,4,6-tri-O-acetyl-2-
deoxy-2-trifluoroacetamido-a-
D
-glucopyranosyl
bro-
mide (5) in the presence of AgOTf used in conjunction
,
with activated and finally powdered 4A molecular
sieves in CH2Cl2 afforded exclusively b-glycoside 9
(Scheme 2, entry a, yield 69%). The 1,2-trans-linkage
was ensured by the N-trifluoroacetyl neighboring-group
participation.
Bromide 7 was also used as a glycosyl donor in the
coupling reaction with diosgenin as an acceptor.36 This
reaction was carried out under almost the same condi-
tions as those above and also gave the b glycoside 12 in
73% yield. The structures of these crystalline com-
pounds have previously been determined in our labora-
1
tory by H and 13C NMR spectroscopy, together with
X-ray analysis.36
In the next step, the coupling reaction of 3,4,6-tri-O-
acetyl-2-deoxy-2-tetrachlorophthalimido-b-D-glucopy-
ranosyl chloride (8) and diosgenin as an acceptor was
carried out in the same manner as that for bromide 7
(Scheme 2, entry b). Here also the 1,2-trans-linkage was
formed (“12, 65%), because of the bulky NTCP group
at carbon C-2, which is simultaneously serving as the
To confirm the structure of 14, glycoside 13 was
treated with acetic anhydride in methanol, which after
workup in the usual manner gave the N-acetyl deriva-
tive 14.40

136
H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
Saponin 10 was also obtained in a good yield by
B cells were cultivated 24 h alone or in the presence of
11 and 15 and 2-CdA. The B-cell apoptosis was de-
tected by staining with FITC-labeled monoclonal anti-
bodies against annexin V, which recognized
phosphatidylserine on the surface apoptotic cells. Dur-
ing apoptosis the B-cell plasma membrane change in-
cluded redistribution of phosphatidylserine from the
cytoplasmatic surface to the outer leaflet making it
available for annexin V binding. During later stages of
apoptosis the plasma membranes become increasingly
permeable and propidium iodide (PI) could move
across the membrane and bind to DNA. Flow cytome-
try with annexin V/PJ double staining was used to
distinguish necrotic and apoptotic B cells.32 The effects
of 24 h cells in incubation alone, with saponins 11, 15,
and 2-CdA, and the mixture of 11 or 15 with 2-CdA on
the amount of apoptotic (A) and necrotic (PI) cells are
shown in Table 1. The spontaneous apoptosis and
necrosis of leucemic B-cells (control) are indicated on
very heterogeneous patient cell populations. The
amount of apoptotic cells varies from a few percentage
points (samples 7, 12) up to more than 50% (samples
22, 25). The incubation of leucemic B-cells with 2-CdA,
N-acylation of the 2-amino sugar derivative 13 with
trifluoroacetic anhydride in pyridine, followed by
methanolysis.41 Hydrochloride 11 (as a free base 13)
has been applied for the preparation of its N-aryl-ure-
ido derivative. The most convenient method for the
synthesis of ureido derivatives of amino sugars is based
on the reaction of the appropriate isocyanate with the
amino group.42–44 Phenyl isocyanate was added to the
solution of 13 (free base) in 1:1 chloroform–methanol,
and after 30 minutes product 15 was isolated as a
crystalline precipitate (Scheme 2, entry g). The struc-
1
tures of 9–15 were established on the basis of the H
and 13C NMR spectroscopy.
2.2. Biological activity
A total of 27 patients with progressive or symptomatic
B-CLL were enrolled in studies.45 All patients were
CD5+, CD19+ and CD23+ as were tested with
monoclonal antibodies (DAKO, Denmark) with the use
of a flow cytometry method (data not shown). Patients’
Table 1
The percentage of apoptotic (A) and necrotic (PI) leukemic B cell in the presence of Cladribine (2-CdA), 11 and 15
n
Control
A
+2-CdA
+ 11
+2-CdA+11
+15
+2-CdA+15
PI
A
PI
9.2
A
PI
8.5
A
PI
A
PI
7.2
8.9
13.4
7
18.6
1.1
6
6.3
0.2
15.8
2.6
0.8
5.4
33.5
0.2
0.4
0.9
2.2
24
6.1
5.1
10
1.4
3.6
24.2
3.3
6.3
A
PI
1
2
3
4
5
6
7
8
42.6
29.9
12.7
31.4
24.3
5.2
23
30.8
34
27.1
21.5
1.4
3.7
6.1
1.3
20
8.8
1
11.7
46.4
0.5
1.5
4.3
15.3
34.9
9.2
23.4
49.4
12.1
29.7
20.5
4.4
10.2
9.8
32.9
55.8
21
3.8
29.4
47
2.5
6.3
24.4
48
12.9
23.3
21.5
5.3
10.7
10.9
15.3
54.1
13.2
4.2
28.7
47.2
2.9
19.1
46.7
11.7
30
7.7
3.1
36
20.1
45.5
14.7
25.1
21
16.5
46.9
13.1
41.9
19
5.7
4.1
23.2
10.8
19.1
1.1
7.9
6
10.5
37.6
7.8
15.3
0.8
6.3
4.3
2.2
12.7
3.2
0.9
4.9
33.3
0.3
0.4
1
2.9
23
9.2
8.1
14.8
17
6.4
19.7
14.9
5.5
9
38.8
7.2
18.9
1.4
7.3
5.3
0.9
14.3
3
0.9
5.1
32.9
0.3
0.8
1.3
3.4
23.4
13.6
10
7
21.1
7.2
10
16.2
0.8
6.5
6
5.4
6.3
7.2
11.9
10.6
10
53.3
12.3
3.6
28.3
43.6
2.1
10.9
10.2
35.5
–
12.3
18.3
44.3
18.1
4
37.5
35.7
2.3
12.2
12.3
14
30.2
21.3
15.7
52.5
37
9.4
9
37.5
56.7
17.8
9
24.1
45.4
3.3
1.4
14.1
3.5
5.8
2.2
27.9
0.4
0.6
0.7
23.6
27.8
8.3
7
10.4
4.6
7.4
28
4
2.2
2
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
15.2
5.7
17.1
52
3
6.2
2.6
2.4
1.1
21.4
0.2
0.4
0.4
3.8
20.8
8.8
10
15.2
6
6.6
13.4
6.2
2.9
7
9
7.7
5.4
5.3
6.6
7.5
11.4
8.1
10.1
11.4
20.9
16.1
24.4
54.9
19.2
27.9
50.7
15.1
17.8
10.7
6
26.2
22.4
19.6
62.5
33.7
27.2
59.4
24.5
27
25.9
18.6
16.4
61.3
40.9
14.9
60.5
12.9
27.8
26.9
18.1
17.2
59.8
24.5
13.2
53.2
13.8
27
22.7
15.3
22.9
59.5
28.7
31
57.5
17.1
15.6
11
21.4
15.9
14.5
31.7
7.7
12.2
8.8
3.2
23.6
2.7
6.1
23.1
51.1
17.1
28.8
12.9

H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
137
Table 2
Evaluation of apoptosis (A) and necrosis (PI) of B cell isolated from B cell chronic lymphocytic leukemia patients
+2-CdA
+11
+2-CdA+11
+15
+2-CdA+15
A
PI
A
PI
A
PI
A
PI
A
PI
no effect
increase
decrease
11 (41) a
11 (41)
5 (18)
18 (67)
4 (15)
5 (18)
14 (52)
6 (22)
7 (26)
21 (78)
2 (7)
4 (15)
13 (48)
3 (11)
11 (40)
13 (48)
9 (33)
5 (18)
16 (59)
6 (22)
5 (18)
22 (81)
0
5 (18)
12 (44)
6 (22)
9 (33)
13 (48)
5 (18)
9 (33)
a In brackets the percentage of total samples tested; 27=100%.
11 and 15 alone or with mixtures of the two shows no
trend, as increases or decreases in apoptosis and necro-
sis depends on the samples tested. The results of Table
1 are summarized in Table 2. The effects of 11, 15 and
2-CdA were estimated as significant if the amount of
apoptotic cells decrease or increase for more then 5%,
in comparison to each samples of untreated B cells
(spontaneous A and PI). 2-CdA increases the amount
of apoptotic cells in 11 (41%) and necrosis in 4 (15%) of
the samples. The 11 and 15 were less effective than
2-CdA and increases apoptosis in 6 (22%) of the sam-
ples. Both saponins were also less effective on inducing
the PI penetration, and only 11 induced it on 2 samples
(7%). However, 11 and 15 enhanced the PI penetration
in the presence of 2-CdA to 33% and 18%, respectively.
It is worth stressing that 11 and 15 reduced the amount
of annexin V binding samples, in comparison to 2-CdA
alone. That may suggest changes on the outer surface
membranes of tumor cells by both diosgenin deriva-
tives. In summary, 11 and 15 are able to induce apopto-
sis and necrosis of some leukemic B-cells. The most
important effect of 11 and 15 seems to be related with
the increase of B-cell membrane permeability in some
samples in the presence of 2-CdA. The testing of bio-
logical activities of 10 and 14 are planned. The com-
parison of all four diosgenyl derivatives may allow one
to choose the most effective compound for consider-
ation in antitumor therapy.
Column chromatography was performed on Kieselgel
60 (B0.08 mm; Macherey–Nagel). The organic ex-
tracts were dried over anhydrous Na2SO4, unless other-
wise noted. For NMR measurements the samples were
dissolved in the appropriate solvent, and Me4Si was
used as the internal reference. NMR spectra were
recorded with a Varian Mercury spectrometer (400
1
MHz for H and 100 MHz for 13C). Elemental analyses
were performed on a Carlo Erba EA1108 analyzer.
3.2. Preparation of 1,3,4,6-tetra-O-acetyl-b-D-glu-
cosamine hydrochloride (3)
To a solution of
D-glucosamine hydrochloride (25 g,
0.12 mol) in a freshly prepared aqueous solution of 1 M
NaOH (120 mL) under stirring was added p-anisalde-
hyde (17 mL, 0.14 mol). After a short time crystalliza-
tion began. Then the mixture was refrigerated and after
2 h the precipitated product was filtered off and washed
with cold water, and followed by a mixture of 1:1
EtOH–Et2O to give 2-deoxy-2-[p-methoxybenzylide-
ne(amino)]- -glucopyranose (1) (25.7 g, 72%): mp 165–
D
166 °C, lit.46 163–164 °C. This intermediate product (25
g, 0.084 mol) was added successively to a cooled (ice-
water) mixture of pyridine (135 mL) and Ac2O (75 mL).
The mixture was stirred in ice-bath ꢀ1 h and then at
room temperature overnight. The yellow solution was
poured into 500 mL of ice-water. The precipitated
white product was filtered, washed with cold water and
dried. This product was identified as 1,3,4,6-tetra-O-
3. Experimental
acetyl-2-deoxy - 2 - [p - methoxybenzylidene(amino)]-b-D-
glucopyranose (2) (34.3 g, 88%): mp 180–182 °C, lit.46
188 °C; [h]2D0+95° (c 1.0, CHCl3), lit.46+98.6°
(CHCl3). Subsequently this compound (34 g, 0.073 mol)
was dissolved in warm acetone (300 mL), and then HCl
(5 M, 15 mL) was added with immediate formation of
a precipitate. The mixture was cooled, and after addi-
tion of Et2O (300 mL), it was stirred for 2 h and
refrigerated overnight. The precipitated product was
filtered, washed with Et2O and dried to give 3 (24.9 g,
89%): at 235 °C this compound decomposed; [h]2D0+32°
(c 1.0, MeOH), lit.46+29.7° (H2O).
3.1. General methods
Melting points were determined on a standard appara-
tus and were uncorrected. Optical rotations were mea-
sured on a Perkin–Elmer 343 polarimeter in a 1-dm cell
at 20 °C. Thin-layer chromatography (TLC) was per-
formed on aluminum sheets coated with Kieselgel 60
F254 (E. Merck, Darmstadt). The following solvent
systems were used: A, 4:1 CCl4–acetone; B, 5:1 tolu-
ene–ethyl acetate; C, 6:1 CCl4–acetone; D, 2:1 tolu-
ene–ethyl acetate; E, 5:1 chloroform–methanol.

138
H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
3.3. Preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-
trifluoroacetamido-b- -glucopyranose (4)
J4,5 10 Hz, H-4b), 5.68 (dd, 1 H, J2,3 10 Hz, J3,4 9.2 Hz,
H-3b), 6.26 (d, 1 H, J1,2 3.6 Hz, H-1a), 6.46 (d, 1 H, J1,2
8.8 Hz, H-1b), 6.48 (dd, 1 H, J2,3 11.6 Hz, J3,4 9.2 Hz,
H-3a), 5.16 (dd, 1 H, J3,4 9.2 Hz, J4,5 10.4 Hz, H-4a).
Anal. Calcd for C22H19Cl4NO11: C, 42.95; H, 3.11; N,
2.28. Found: C, 43.02; H, 3.10; N, 2.28.
D
To a suspension of 3 (11.5 g, 30 mmol) in CH2Cl2 (100
mL) and pyridine (12 mL), under cooling (ice-water)
and stirring, trifluoroacetic anhydride (12 mL) was
added. After stirring for 0.5 h the solution was poured
into ice-water. The organic phase was washed with
water (100 mL), dried and concentrated. Addition of
Et2O yielded crystals of 4 (12.1 g, 91%): mp 164–
165 °C, lit.47: mp 167 °C; [h]D20 −10° (c 1.0, CHCl3),
lit.47 −13° (c 2.4, CHCl3).
3.6. 3,4,6-Tri-O-acetyl-2-deoxy-2-(3,4,5,6-tetra-
chlorophthalimido)-b-D-glucopyranosyl chloride (8)
To a solution of 6 (0.3 g, 0.5 mmol) in dry CHCl3 (2
mL) Cl2HCOCH3 (0.55 mL, 4.4 mmol) and BF3·Et2O
(0.3 mL) were added. After being stirred for 10 min
under N2, the reaction mixture was heated for 48 h at
60 °C. Then it was diluted with CHCl3 (20 mL), washed
with H2O and satd aq NaHCO3, dried over anhydrous
MgSO4, and concentrated to give a foam, which was
crystallized from Et2O–petroleum ether to afford 8 (0.2
g, 67%): mp 154–156 °C; [h]2D0 + 70° (c 0.5, CHCl3); Rf
0.57 (system C); 1H NMR (CDCl3): l 1.92 (s, 3 H,
OAc), 2.05 (s, 3 H, OAc), 2.14 (s, 3 H, OAc), 3.94 (m,
1 H, J4,5 10.26 Hz, H-5), 4.21 (dd, 1 H, J5,6% 2.2 Hz,
H-6%), 4.34 (dd, 1 H, J5,6 4.8 Hz, H-6), 4.51 (dd, 1 H,
J2,3 10.26 Hz, J3,4 9.15 Hz, H-2), 5.27 (dd, 1 H, J2,3
10.26 Hz, J3,4 9.15 Hz, H-3), 6.7 (d, 1 H, J1,2 9.2 Hz,
H-1). Anal. Calcd for C20H16Cl5NO9: C, 40.60; H, 2.73;
N, 2.37. Found: C, 40.79; H, 2.66; N, 2.31.
3.4. Preparation of 3,4,6-tri-O-acetyl-2-deoxy-2-tri-
fluoroacetamido-a-D-glucopyranosyl bromide (5)
To a solution of 4 (6.65 g, 15 mmol) in dry CH2Cl2 (150
mL) and dry EtOAc (15 mL) was added TiBr4 (6 g, 16
mmol). The mixture was stirred at room temperature
and monitored by TLC (solvent A). After 0.5 h a
second portion of TiBr4 (6 g, 16 mmol) was added, and
after stirring for 4 h the red solution was diluted with
CHCl3 (350 mL) and washed with water (2×150 mL).
The organic layer was dried and filtered, and the sol-
vents were evaporated. The residue was crystallized
from Et2O–hexane to yield bromide 5 (5.5 g, 80%): mp
92–94 °C, lit.47 mp 96 °C; [h]D20 +122° (c 1.0, CHCl3),
lit.47+126° (c 2.92, CHCl3).
3.7. Diosgenyl 3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroac-
etamido-b-D-glucopyranoside (9)
3.5. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(3,4,5,6-tetra-
chlorophthalimido)-a,b-
D-glucopyranose (6)
To a cooled (ca. −15 °C) solution of diosgenin (0.42 g,
1 mmol) in anhyd CH2Cl2 (100 mL) with stirring under
D-Glucosamine hydrochloride (2.16 g, 0.01 mol) was
,
N2 type 4 A crushed molecular sieves (6 g) and bromide
added to a NaOMe solution prepared from Na (0.23 g,
0.01 mol) and MeOH (10 mL). After 15 min the
mixture was treated with 3,4,5,6-tetrachlorophthalic an-
hydride (1.43 g, 5 mmol) and shaken for 20 min.
Afterwards Et3N (1.4 mL, 0.01 mol) was added, and
the reaction mixture was treated again with the anhy-
dride (1.43 g, 5 mmol). The mixture was then warmed
at 50 °C and stirred for 1 h. After concentration, the
residue was treated with pyridine (20 mL) and Ac2O (20
mL) at room temperature for 24 h. The solution was
poured into ice-water, and the aqueous mixture was
extracted with CHCl3 (3×100 mL). The organic ex-
tracts were combined, successively washed with H2O,
HCl (3%), satd aq NaHCO3 and H2O. The organic
layer was dried and filtered, and the solvent was evapo-
rated. Addition of MeOH caused precipitation of 6 as a
white solid (2.7 g, 44%). An additional portion of 6 (1.1
g, 18%) was obtained after column chromatography
5 (0.93 g, 2 mmol) were successively added. AgOTf
(0.77 g, 3 mmol) dissolved in anhyd Et2O (25 mL) was
then added dropwise during 50 min. This mixture was
stirred under N2 at ca. −15 °C for 30 min. After this
time TLC (solvent A and D) showed the conversion of
substrate into products. The mixture was filtered and
evaporated to dryness. The syrup was chro-
matographed (solvent B) to remove unreacted dios-
genin and to purify the coupling product. It afforded
pure 9 (0.55 g, powder, 69%): mp \220 °C (dec); [h]D20
−74° (c 0.6, MeOH); 1H NMR data see ref.33; 13C
NMR (100 MHz, CDCl3): l 14.73 (C-21), 16.48 (C-18),
17.34 (C-27), 19.51 (C-19), 20.68 (COCH3), 20.81
(COCH3), 20.97 (COCH3), 21.03 (C-11), 28.98, 29.58,
30.49, 31.57 (C-8, C-24, C-25, C-23, C-2), 32.03 (C-15),
32.26 (C-7), 37.03 (C-10), 37.31 (C-1), 38.78 (C-4),
39.93 (C-12), 40.46 (C-13), 41.80 (C-20), 50.26 (C-9),
55.50 (C-2%), 56.68 (C-14), 62.28 (C-6%, C-17), 67.05
(C-26), 68.70 (C-4%), 71.83 (C-3%), 72.00 (C-5%), 80.06
(C-3), 80.99 (C-16), 99.01 (C-1%), 109.51 (C-22), 122.23
(C-6), 140.32 (C-5), 169.55, 170.93, 171.27 (COCH3×
3, COCF3). Anal. Calcd for C41H58F3NO11: C, 61.72;
H, 7.33; N, 1.76. Found: C, 61.72; H, 7.40; N, 1.65.
1
(system B). H NMR (CDCl3) for 6: l 1.9–2.21 (4s, 12
H, 4 OAc), 4.25 (dd, 1 H, J5,6 4.8 Hz, H-6b), 4.35 (dd,
1 H, H-6%b), 4.28–4.4 (m, 2 H, H-5a, H-6a), 4.45 (dd,
1 H, J1,2 8.8 Hz, J2,3 10 Hz, H-2b), 4.71 (dd, 1 H, J1,2
3.6 Hz, J2,3 11.6 Hz, H-2a), 5.23 (dd, 1 H, J3,4 9.2 Hz,

H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
139
1
3.8. Diosgenyl 2-deoxy-2-trifluoroacetamido-b-
glucopyranoside (10)
D
-
[h]2D0 −56° (c 0.4, 1:1 CHCl3–MeOH); H NMR data
see ref.33; 13C NMR (100 MHz, CDCl3–CD3OD): l
15.00 (C-21), 16.94 (C-18), 17.62 (C-27), 19.99 (C-19),
21.90 (C-11), 29.18, 30.64, 31.25, 32.31, 32.59, 32.65,
33.07 (C-8, C-24, C-25, C-23, C-2, C-7, C-15), 37.86
(C-10), 38.29 (C-1), 39.71 (C-4), 40.76 (C-12), 41.29
(C-13), 42.72 (C-20), 51.33 (C-9), 57.56 (C-2%), 57.95
(C-14), 62.61 (C-6%), 63.29 (C-17), 67.74 (C-26), 71.60
(C-4%), 77.10, 77.62 (C-3%, C-5%), 79.58 (C-3), 81.96
(C-16), 102.11 (C-1%), 110.38 (C-22), 122.47 (C-6),
141.25 (C-5). Anal. Calcd for C33H53NO7·HCl·1.5H2O:
C, 62.00; H, 8.99; N, 2.19. Found: C, 61.94; H, 8.67; N,
1.94.
To a solution of 9 (0.32 g, 0.4 mmol) in MeOH (100
mL) a 1 M solution of NaOMe in MeOH (5 mL) was
added. The mixture was stirred at room temperature
for 0.5 h and was then neutralized with Dowex-50W
(H+) ion-exchange resin (pH was adjusted to 7; pH
meter) and filtered, and the solvent was evaporated.
The semisolid residue was dissolved 1:1 in CHCl3–
MeOH and filtered and again concentrated to dryness
to give 10 (0.26 g, powder, 98%): mp \217 °C (dec.);
1
[h]2D0 −53° (c 0.8, MeOH); H NMR ((CD3)2SO): l 3.1
(m, 2 H, H-3, H-5), 3.4 (m, 3 H, H-2, H-4, H-6%), 3.75
(dd, 1 H, H-6), 4.45–4.5 (m, 1 H, 6-OH), 4.5 (d, 1 H,
J1,2 9.2 Hz, H-1), 5.05 (m, 1 H, 3-OH), 5.15 (m, 1 H,
4-OH), 9.15 (d, 1 H, J 8.4 Hz, NH); diosgenyl protons:
0.72 (d, CH3), 0.76 (s, CH3), 0.9 (d, CH3), 0.92 (s, CH3),
1.9 (m, C(15)-H), 2.28 (m, C(15)-H%), 4.28 (dd, C(3)ꢀH),
5.3 (m, C(6)ꢀH). 13C NMR (100 MHz, (CD3)2SO): l
14.56 (C-21), 15.91 (C-18), 16.99 (C-27), 18.96 (C-19),
20.30 (C-11), 28.42, 29.10, 29.74, 30.90, 31.40, 31.48
(C-8, C-24, C-25, C-23, C-2, C-7, C-15), 36.28 (C-10),
36.64 (C-1), 38.25 (C-4), 38.88 (C-12), 40.13 (C-13),
41.03 (C-20), 49.47 (C-9), 55.67 (C-14), 56.36 (C-2%),
60.87 (C-6%), 61.75 (C-17), 65.86 (C-26), 70.44 (C-5%),
73.10 (C-4%), 77.02 (C-3%), 77.53 (C-3), 80.13 (C-16),
98.53 (C-1%), 108.35 (C-22), 121.14 (C-6), 140.23 (C-5),
158.00 (COCF3). Anal. Calcd for C35H52F3NO8·
1.5H2O: C, 60.15; H, 7.93; N, 2.00. Found: C, 60.24; H,
7.63; N, 1.97.
3.10. Diosgenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(3,4,5,6-
tetrachlorophthalimido)-b-D-glucopyranoside (12)
To a mixture of 8 (0.87 g, 1.4 mmol), diosgenin (0.4 g,
,
1 mmol) and 4 A molecular sieves (4 g) in anhyd
CH2Cl2 (68 mL) at room temperature under N2, AgOTf
(0.75 g, 2.92 mmol) in dry Et2O (45 mL) was added.
After stirring 1 h, the mixture was neutralized by Et3N
(2 mL), diluted with CHCl3, filtered and concentrated.
Addition of MeOH caused precipitation of 12 as a
white solid (0.62 g, 65%); mp 257–258 °C, [h]2D0 +12°
1
(c 0.45, CHCl3); for H, 13C NMR and X-ray crystal-
lography data see ref.36.
3.11. Diosgenyl 2-amino-2-deoxy-b-D-glucopyranoside
(13) and diosgenyl 2-acetamido-2-deoxy-b-D-glucopyran-
oside (14)
Compound 10 was also obtained from 13 in the
following procedure: to 0.1 g of 13 dissolved in dry
pyridine (2 mL) and cooled in ice, 0.3 mL trifl-
uoroacetic anhydride in dry Et2O (2 mL) was added
dropwise. After 24 h, the solution was diluted with
EtOAc, washed with H2O, dried (MgSO4) and after
concentration purified by silica gel-column chromatog-
raphy (10:1 CHCl3–MeOH) to afford 10 as a white
solid (0.085 g, 60%).
Compound 12 (0.2 g, 0.21 mmol) was dissolved in
EtOH (4 mL), and then ethylenediamine (0.074 g, 1.23
mmol) was added. The reaction mixture was heated at
60 °C for 6 h and then concentrated. The residue was
applied to a silica gel column (5:1 CHCl3–MeOH,
containing 0.2% Et3N) to give 13 as a white solid (0.083
g, 70%) and 14 (0.037 g, 29%). For 13: mp 230 °C
(dec.); [h]2D0 −102° (c 0.5, 1:1 CHCl3–CH3OH); 1H
NMR (CD3OD): l 2.62 (dd, 1 H, J1,2 8.1 Hz, J2,3 9.3
Hz, H-2), 3.26 (m, 1 H, J4,5 8.3 Hz, J5,6 5 Hz, H-5), 3.30
(m, 1 H, J2,3 9.3 Hz, J3,4 8.3 Hz, H-3), 3.33 (dd, 1 H,
J3,4 8.3 Hz, J4,5 8.3 Hz, H-4), 3.71 (dd, 1 H, J5,6 9 Hz,
H-6), 3.84 (dd, 1 H, J5,6% 2.3 Hz, H-6%), 4.37 (d, 1 H, J1,2
8.1 Hz, H-1); diosgenyl protons: 0.72 (s, CH3), 0.76 (s,
CH3), 0.9 (d, CH3), 0.92 (s, CH3), 1.9 (m, C(15)ꢀH), 2.28
(m, C(15)ꢀH%), 3.32 (m, C(26)ꢀH(a)), 3.45 (m, C(26)ꢀH(e)),
3.58 (m, C(16)ꢀH), 4.4 (dd, C(3)ꢀH), 5.35 (m, C(6)ꢀH);
13C NMR (100 MHz, CDCl3–CD3OD): l 14.97 (C-21),
16.90 (C-18), 17.59 (C-27), 19.96 (C-19), 21.81 (C-11),
29.61, 30.56, 31.16, 32.22, 32.47, 32.57, 32.98 (C-8,
C-24, C-25, C-23, C-2, C-7, C-15), 37.77 (C-10), 38.19
(C-1), 39.65 (C-4), 40.66 (C-12), 41.21 (C-13), 42.62
(C-20), 51.20 (C-9), 57.46 (C-2%), 57.83 (C-14), 62.57
(C-6%), 63.13 (C-17), 67.67 (C-26), 71.51 (C-4%), 77.07,
3.9. Diosgenyl 2-amino-2-deoxy-b-D-glucopyranoside
hydrochloride (11)
The coupled product 9 (0.33 g, 0.4 mmol) was dissolved
in acetone (65 mL) and then a 0.1 M solution of NaOH
(32 mL) was added. After stirring for 2 h at room
temperature, the pH of the mixture was adjusted to 7
(pH meter) by adding Dowex-50W ion-exchange resin,
and then the mixture was filtered. Concentration of the
filtrate yielded a syrup, which was dissolved in EtOH
and concentrated again. The semisolid residue was dis-
solved in 1:1 CHCl3–MeOH and filtered, and a stoi-
chiometric amount of 1.3% HCl in MeOH was added.
The hydrochloride was precipitated with petroleum
ether to give 11 as an amorphous powder (0.21 g, 82%):

140
H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
77.43 (C-3%, C-5%), 79.52 (C-3), 81.88 (C-16), 102.14
(C-1%), 110.33 (C-22), 122.41 (C-6), 141.13 (C-5). For
14: mp \238 °C (dec.); [h]2D0−75° (c 0.5, 1:1 CHCl3–
trifuged again to give 15 as an amorphous powder (0.14
1
g, 80%): [h]2D0 −11° (c 0.4, 1:1 CHCl3–CH3OH); H
NMR (CD3OD): l 3.26–3.34 (m, 4 H, H-2, H-3, H-4,
H-5), 3.68 (dd, 1 H, J5,6 5.2 Hz, H-6), 3.88 (dd, 1 H,
J5,6% 2.0 Hz, H-6%), 4.60 (d, 1 H, J1,2 8.4 Hz, H-1), 6.98
(t, 1 H, Ph), 7.25 (t, 2 H, Ph), 7.35 (d, 2 H, Ph), 7.41 (d,
1 H, J 8.8 Hz, NH); diosgenyl protons: 0.78 (s, CH3),
0.80 (s, CH3), 0.96 (d, CH3), 0.99 (s, CH3), 2.20 (m,
C(15)ꢀH), 2.39 (m, C(15)ꢀH%), 3.39 (m, C(26)ꢀH(a)), 3.44
(m, C(26)ꢀH(e)), 3.54 (m, C(16)ꢀH), 4.39 (dd, C(3)ꢀH), 5.32
(m, C(6)ꢀH). Anal. Calcd for C40H58N2O8·1.5H2O: C,
66.55; H, 8.52; N, 3.88. Found: C, 66.21; H, 8.19; N,
4.01.
1
CH3OH); H NMR ((CD3)2SO): l 3.05 (m, 2 H, H-3,
H-5), 3.3–3.4 (m, 3 H, H-2, H-4, H-6%), 3.66 (dd, 1 H,
J5,6 5.6 Hz, H-6), 4.4 (d, 1 H, J1,2 9.2 Hz, H-1),
4.35–4.45 (m, 1 H, 6-OH), 4.82 (m, 1 H, 4-OH), 4.89
(m, 1 H, 3-OH), 7.61 (d, 1 H, J 8.4 Hz, NH); diosgenyl
protons: 0.73 (d, CH3), 0.74 (s, CH3), 0.9 (d, CH3), 0.95
(s, CH3), 1.9 (m, C(15)ꢀH), 2.3 (m, C(15)ꢀH%), 4.28 (dd,
C(3)ꢀH), 5.31 (m, C(6)ꢀH); 13C NMR (100 MHz,
(CD3)2SO): l 14.57 (C-21), 15.92 (C-18), 17.00 (C-27),
19.06 (C-19), 20.32 (C-11), 23.03 (COCH3), 28.42,
29.17, 29.74, 30.88, 30.93, 31.41, 31.49 (C-8, C-24,
C-25, C-23, C-2, C-7, C-15), 36.00 (C-10), 36.74 (C-1),
38.43 (C-4), 38.88 (C-12), 40.13 (C-13), 41.04 (C-20),
49.51 (C-9), 55.69 (C-14), 55.83 (C-2%), 61.06 (C-6%),
61.76 (C-17), 65.87 (C-26), 70.67 (C-5%), 74.19 (C-4%),
76.88 (C-3%), 77.41 (C-3), 80.13 (C-16), 99.33 (C-1%),
108.35 (C-22), 120.99 (C-6), 140.46 (C-5), 168.90
(COCH3).
3.14. Biological methods
3.14.1. Cell separation. Periferial blood mononuclear
cells (PBMC) were collected into sterile, versanian-con-
taining tubes. The PMBC were separated by differential
centrifugation on
a
Ficoll-Hypaque centrifuge
(Lymphoprep; Nyegard, Oslo, Norway).
When 13 (0.025 g, 0.0434 mmol) in MeOH (6 mL)
was treated with Ac2O (1.2 mL) and Et3N (0.12 mL), it
gave 0.02 g of 14, identical with the sample prepared
during deprotection of 12.
3.14.2. Culture conditions. B-CLL separated cells were
cultured (2×106 cells/mL) in complete medium (RPMI
1640, Bio-Whittaker, Walkersville, MD, USA), supple-
mented with 10% heat-inactivated FCS with or without
2-chlorodeoxyadenosidne (Leustain; Ortho Biotech,
Raritan, NJ, USA), 11 and 15 for 24 h, at final concen-
tration 1.75×10−7 M (50 ng/mL) in a fully humidified
atmosphere of 5% CO2 at 37 °C. In some experiments
11 or 15 were added simultaneously with 2-CdA, each
at a concentration 1.75×10−7 M (50 ng/mL).
3.12. Diosgenyl 2-amino-2-deoxy-b-D-glucopyranoside
(13) from the reaction of 12 with hydrazine hydrate
Glycoside 12 (0.2 g, 0.206 mmol) in EtOH (4 mL) was
heated with hydrazine hydrate (0.35 mL of an 85%
solution) for 24 h. After this time the solvent was
evaporated, and the residue was dried under high vac-
uum to remove traces of hydrazine. Silica gel column
chromatography (solvent E) of this residue afforded 13
(0.12 g, 97%), which was isolated as the hydrochloride
11, after adding to the solution stoichiometric amount
of 1.3% HCl in MeOH.
3.14.3. Analysis of apoptosis. The cells were centrifuged,
washed in PBS and stained with Annexin V and propid-
ium iodide according the manufacturer’s protocol (Bio-
Source International, Inc., Camarillo, CA, USA).
The distribution of cellular DNA content and apop-
totic cells were determined after staining with propid-
ium iodide and Annexin V by flow cytometry using a
Epics XL Coulter flow cytometer with argon laser
excitation at 488 nm.
3.13. Diosgenyl 2-deoxy-2-phenylureido-b-D-glucopyran-
oside (15)
To a solution of 11 (0.15 g, 0.25 mmol) in 1:1 CHCl3–
MeOH (30 mL) under stirring was added Dowex 2X-8
ion-exchange resin (HCO−3 ) to obtain 13 (the free base
of 11). After a short time (ca. 10 min), the mixture was
filtered, and phenyl isocyanate (86 mL, 0.8 mmol) was
added. It was stirred for 1 h at room temperature until
TLC (solvent E) showed the complete conversion of 13
into the product. Then the solvents were evaporated to
dryness. The semisolid residue was dissolved in 1:1
chloroform–methanol, and the mixture was filtered and
concentrated to a low volume. Addition of hexane
afforded the product as a white solid, which was sepa-
rated on a centrifuge, washed with hexane and cen-
Acknowledgements
The authors are very gratefully dr Tadeusz Robak
(Medical University, Ło´dz´, Poland) for their valuable
suggestion and dr Katarzyna Bednarska (MVC PAS,
Ło´dz´, Poland) for her help with data evaluations. This
research was partly supported by The Polish State
Committee for Scientific Research under grants: 6PO4C
05620, 7 T09A 129 21, BW/8000-5-0260-1 and BW/
8000-5-0021-1.

H. Myszka et al. / Carbohydrate Research 338 (2003) 133–141
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