Novel anthracycline oligosaccharides: Influence of chemical modifications of the carbohydrate moiety on biological activity

Bioorganic & Medicinal Chemistry 10 (2002) 1459–1470

Novel Anthracycline Oligosaccharides: Inﬂuence of Chemical

Modiﬁcations of the Carbohydrate Moiety on Biological Activity

A. Cipollone,^a,* M. Berettoni,^aM. Bigioni,^aM. Binaschi,^aC. Cermele,^aE. Monteagudo,^a

L. Olivieri,^aD. Palomba,^aF. Animati,^aC. Goso^aand C. A. Maggi^b

^aMenarini Ricerche, via Tito Speri 10, 00040 Pomezia, Italy

^bMenarini Ricerche, via Sette Santi 3, 50131 Firenze, Italy

Received 8 October 2001; accepted 19 November 2001

Abstract—Several observations highlight the importance of the carbohydrate moiety for the biological activity of antitumoural

anthracyclines. Here is reported the synthesis, cytotoxicity and topoisomerase II-mediated DNA cleavage intensity of the new oli-

gosaccharide anthracyclines 1–4 modiﬁed in the sugar residue. Evaluation of cytotoxic potency on diﬀerent cell lines, resulted in

quite similar values among the diﬀerent analogues. On the other hand, topoisomerase II-mediated DNA breaks level was diﬀerent

for the various compounds, and was not related to cytotoxicity, thus supporting previous observations reported for some mono-

Introduction

experimental results demonstrate that chemical mod-

iﬁcations in the carbohydrate moiety of the molecule,

Anthracycline antibiotics are still among the most useful

drugs in cancer therapy because of their clinical eﬃcacy

against a broad spectrum of solid tumours and leuke-

mias. The importance of this class of antitumour agents

has stimulated a number of studies aimed to achieve a

deeper knowledge of the mechanism of action of these

drugs and to identify the critical structural motives,

which might improve their pharmacological properties

and reduce their toxicity. DNA intercalation was

proved to be a necessary but not suﬃcient condition for

exhibiting antitumoural activity. Interference with the

catalytic cycle of the enzyme topoisomerase II is con-

sidered as the main biological event responsible for the

cytotoxic eﬀect of anthracyclines.¹Experimental data

show that antracyclines stabilize a DNA–enzyme com-

plex, wherein DNA strands are cut and covalently

linked to the protein, hindering DNA strand religation

by topoisomerase II and thus enhancing DNA breaks

level.^2ꢀ5From the model of DNA–drug complex, the

non-intercalating portions, that is the a-hydroxy car-

bonyl side chain and the carbohydrate moiety, appear

positioned in the DNA minor groove.^6,7Moreover,

can determine changes in the drug preferred base

sequence at the cleavage site.⁸From these observations

it was supposed that, in the DNA–enzyme–drug com-

plex, the anthracycline molecule is placed at the pro-

tein–DNA interface and that the non-intercalating

chemical moieties could play an important role in the

formation and stabilisation of the ternary complex.^9,10

We started a research program aimed at the synthesis of

new anthracyclines modiﬁed in the carbohydrate moiety

in order to obtain derivatives with improved pharma-

cological proﬁle. In particular, on the base of the cur-

rently accepted model of the ternary complex, an

elongation of the sugar portion could increase mole-

cular interactions between drug and DNA and/or

topoisomerase II and provide more information on the

structural requirements of this part of the molecule to

obtain an improved biological activity.

We have already described new disaccharide anthracy-

clines belonging both to the daunomycin series and the

doxorubicin series in which the amino sugar, diﬀerent

from the natural and synthetic disaccharide anthracy-

clines till then reported, was not directly linked to the

aglycone moiety.¹¹Evaluation of topoisomerase II-

mediated DNA breaks and of antitumour activity

*Corresponding author. Fax:+39-06-9106137; e-mail: acipollone@

menarini-ricerche.it

PII: S0968-0896(01)00411-4

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A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

Scheme 1. (i) AllOCOCl, py, CH₂Cl₂, rt, 4 h; (ii) PhSSiMe₃, CF₃SO₃SiMe₃, CH₂Cl₂, rt, 2 h; (iii) (p-MeO) C₆H₄CH₂OH (p-MBnOH), Ph₃PHBr,

CH₂Cl₂, rt, 1 h; (iv) 1 N MeONa, MeOH, rt, 2 h; (v) (a) Bu₂SnO, toluene, reﬂux, 12 h; (b) AllOCOCl, Et₃N, CH₂Cl₂, ꢀ40 ^ꢁC, 2 h; (vi) IDCP,

(CH₂Cl)₂–Et₂O, M.S. 4 A, 10 ^ꢁC, 2 h; (vii) Ph₃P, 1 h; (viii) Ce(NH₄)₂(NO₃)₆, CH₃CN–H₂O, rt, 2 h; (ix) (p-NO₂)C₆H₄COCl (p-NBzCl), py, CH₂Cl₂,

0 ^ꢁC, 1 h; (x) PhSSiMe₃, CF₃SO₃SiMe₃, CH₂Cl₂, 0 C, 1 h; (xi) p-NBzCl, py, CH₂Cl₂, rt, 1 h.

conﬁrmed the critical role of the sugar moiety for the

pharmacological properties of this new class of anthra-

cyclines.¹²In particular, 1,4-a-linkage between the C-4

position of the ﬁrst sugar and the anomeric carbon of

the second carbohydrate unit of the disaccharide,

turned out to be the optimal conﬁguration for the bio-

logical activity. One of these new anthracyclines, MEN

10755, was identiﬁed as the best compound in this ser-

ies. This new derivative showed remarkable eﬃcacy in a

number of xenografted (gynaecological, lung, prostatic)

human tumour in nude mouse, both sensitive and resis-

tant to doxorubucin.¹³Besides, in animal models MEN

10755 evidenced lower cardiotoxic eﬀects with respect to

doxorubicin.¹⁴

deoxy derivative of MEN 10755 (3), and the compound

bearing a combination of the two modiﬁcations, that is

epimerization in 4⁰⁰and removal of the hydroxy group

in 3⁰(4) (Fig. 1).

A convergent synthetic strategy was adopted for the

synthesis of these compounds, implying the separate

construction of the aglycone and of the oligosaccharide

moiety and their successive coupling to give the desired

glycoside.

Oligosaccharides were assembled starting from glycosyl

donors activated at the anomeric position with thio-

phenyl group. Thioglycosides have been recently intro-

duced as potential glycosyl donors as they have the

advantage to be stable to normal protecting group

manipulation encountered in oligosaccharide synthesis,

although they may be selectively activated to become

glycosyl donors.^15,16Condensations between the thio-

glycosides and the glycosyl acceptors were performed in

the presence of a preformed soluble complex of iodo-

nium ion.¹⁷This protocol allowed to overcome the

known low reactivity of the C-4 axial hydroxyl group of

the glycosyl acceptor,¹⁸to obtain the glycosidic bond in

the desired a conﬁguration and to avoid the formation

of transglycosylation products. All the glycosyl accep-

tors were protected at the anomeric position with p-

methoxybenzyl group. This group, in contrast to the

common anomeric protecting groups, which require

acid or reductive conditions for their deprotection, was

removable under neutral condition without aﬀecting the

glycosidic bond.¹⁹Allyloxycarbonyl group, used for

In order to establish more precise structure–activity

relationships and to achieve a deeper knowledge of the

inﬂuence of the carbohydrate portion on the biological

activity for this new class of oligosaccharide anthracy-

clines, we designed novel oligosaccharide analogues

containing chemical and conﬁgurational modiﬁcations

in the carbohydrate moiety.

Results

Synthesis

Here we report the synthesis of new oligosaccharide

anthracyclines 1—4. They were respectively the 4⁰⁰-epi-

derivative of MEN 10755 (1), the trisaccharide (2), in

which the aminosugar daunosamine is separated from

the aglycone by two 2-deoxy-l-fucose units, the 3⁰-

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

1461

protecting the other hydroxyl or amino functions pre-

sent in the carbohydrate molecule, was equally cleavable

under neutral condition, allowing the base sensitive

hydroxy function in the side chain of the anthracycline

molecule, not to be aﬀected in the ﬁnal deprotection.²⁰

The trisaccharide was obtained through a stepwise syn-

thetic sequence ﬁrst obtaining the disaccharide moiety,

then coupling it with another molecule of glycosyl

acceptor, with both the glycosylations carried out in the

presence of iodonium ion as promoter.

The two 3⁰-deoxy disaccharides 33 and 37 were obtained

condensing, respectively, the thiophenyl daunosaminide

18 and thiophenyl acosaminide 7, with p-methoxybenzyl

2, 3, 6-trideoxy-l-lyxo-hexopyranoside 32 (Scheme 2).

Starting materials for the disaccharide 12 were phenyl 3-

N,4-O-di-allyloxycarbonyl-1-thio- acosaminide 7 and p-

methoxybenzyl 3 - O - allyloxycarbonyl-2-deoxy-l-fuco-

side 11 (Scheme 1).

Compound 7 was obtained from methyl-l-acosaminide

5,^21,22after protection of the free hydroxyl and amino

functions with allyloxycarbonyl group, and activation at

the anomeric position by treatment with phenylthiotri-

methylsilane and trimethylsilyl triﬂuoromethanesulfonate

(trimethylsilyltriﬂate).²³Addition of p-methoxybenzyl

alcohol²⁴to the easily available 3, 4-di-O-acetyl-l-fucal

8,²⁵de-O-acetylation and successive regioselective pro-

tection in C-3 position, through formation of a cyclic

stannyl ether, of the resulting p-methoxybenzyl fucoside

10^26,27gave the glycosyl acceptor 11. Condensation of

this latter with a slight excess of glycosyl donor 7 in the

presence of iodonium dicollidine perchlorate (IDCP),²⁸

resulted in the formation of the desired disaccharide 12,

exclusively in a conﬁguration, in addition to the corre-

sponding sulfenamide derivative 13 (30%).²⁹This latter

was easily converted into the useful disaccharide by

adding triphenylphosphine to the reaction mixture.

Quantitative removal of the anomeric p-methoxybenzyl

group in 12 with ammonium cerium (IV) nitrate, fol-

lowed by activation with 4-nitrobenzoyl chloride furn-

ished the glycosyl donor 15 in 80% of yield.

Scheme 2. (i) p-MBnOH, 0.22 M SnCl₄, CH₂Cl₂, rt, 2 h; (ii) H₂,

[(Ph)₃P]₃RhCl, benzene, rt, dark, 2 h; (iii) 1 M NaOMe, MeOH, rt, 2

h; (iv) DEAD, Ph₃P, PhCO₂H, CH₂Cl₂, rt, 2 h; (v) K₂CO₃, MeOH, rt,

2 h; (vi) IDCP, (CH₂Cl)₂–Et₂O, M.S. 4 A, 5–10 ^ꢁC, 3 h; (vii) Ph₃P, 1 h;

(viii) Ce(NH₄)₂(NO₃)₆, CH₃CN–H₂O, rt, 2 h; (ix) p-NBzCl, py,

CH₂Cl₂, 0 ^ꢁC, 2 h.

The carbohydrate fragments forming the trisaccharide

moiety were p-methoxybenzyl 2-deoxy fucoside 11 and

thiophenyldaunosaminide 18 (Scheme 1). This latter

was obtained from 3-N-allyloxycarbonyl-l-daunosamine

16³⁰after treatment with p-nitrobenzoyl chloride to give

17 and successive reaction with (phenylthio)-

trimethylsilane and trimethylsilyl triﬂate. The trisacchar-

ide 24 was easily accessible through a stepwise sequence

ﬁrst condensing the thiophenyldaunosaminide 18 with

the p-methoxybenzyl 2-deoxy fucoside 11 in the pre-

sence of iodonium dicollidine perchlorate to obtain the

disaccharide 19 with the desired a 1,4 linkage. Activation

of the anomeric position of this disaccharide through

direct substitution of the p-methoxybenzyl group with the

thiophenyl group, failed. Therefore, this conversion was

performed through deprotection with ammonium cer-

ium(IV) nitrate, reaction with p-nitrobenzoyl chloride to

give 22 and than with (phenylthio)trimethylsilane and

trimethylsilyltriﬂate to obtain the glycosyl donor 23.

This latter was coupled, following the same procedure

of the previous glycosylation, with another molecule of

2-deoxyfucoside 11 yielding to the trisaccharide 24. The

usual sequence of deprotection and activation at the

anomeric position, gave the activated trisaccharide 26.

Figure 1. Molecular structures of MEN 10755 and oligosaccharide

modiﬁed anthracyclines.

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A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

Scheme 3. (i) TMSOTf, CH₂Cl₂–Et₂O, M.S. 4 A, ꢀ20 ^ꢁC, 2 h; (ii) 0.5 M K₂CO₃, MeOH–CH₂Cl₂, ꢀ10 ^ꢁC, 1 h; (iii) (a) [(Ph₃P)₄]Pd, Me₂NTMS,

TMSOAc, CH₂Cl₂, rt, dark, 0.5 h; (b) 0.001 M aq HCl.

Table 1. Cytotoxic activity of the disaccharide anthracyclines on

human carcinoma cell lines^a

The fully protected glycosides 42–45 were obtained by

coupling the 14-acetoxyidarubicinone 41 with the acti-

vated carbohydrate residues 15, 26, 36 and 40, using

trimethylsilyltriﬂate as promoter, according to a known

procedure.³³The new glycosides were produced in good

yields and with the glycosidic bond in the desired a con-

ﬁguration. Mild deprotection of the ester groups with

potassium carbonate aﬀorded the partially deprotected

glycosides 46–49. Successive removal of the allyloxy-

carbonyl groups in neutral conditions with tetrakis(tri-

phenylphosphine)palladium led respectively to the ﬁnal

glycosides 1–4 isolated as hydrochloride salt (Scheme 3).

Cell line MEN 10755

1

2

3

4

A2780

GCL-4

H460

33Æ11

36Æ22

76Æ12

1250Æ778

239Æ9

80Æ20

23Æ1

11Æ5

11Æ3

109Æ42

353Æ127

>1000

7Æ1

16Æ4

9Æ1

24Æ13

29Æ19

17.5Æ18

890Æ610

58Æ13

12Æ2

7.8Æ5.2

PC-3

350Æ212 215Æ65

CALU-3

DU-145

SW-684

123Æ40

18Æ0.5

106Æ23

13Æ2

14Æ7

19Æ2

41Æ13

^aIC₅₀(nM) values were determined from dose–response curves from at

least three indipendent experiments. Human tumor cell lines are

A2780 (ovarian), GCL-4 (SCLC), H460 (lung), PC-3 (prostate),

CALU-3 (lung), DU-145 (prostate), SW-684 (ﬁbrosarcoma).

Cytotoxic activity

Cytotoxicity of the anthracycline analogues 1–4 was

evaluated on a panel of diﬀerent human tumour cell

lines and compared with the cytotoxic eﬀect of MEN

10755 (Table 1). The disaccharide analogues showed a

cytotoxic potency comparable to that of the reference

compound, except in the PC-3 tumour line which was

resistant to all the drugs. Obtained results pointed out

that neither epimerization in C-4⁰⁰(1) nor removal of the

OH group in C-3⁰(3 and 4) notably aﬀected the cellular

response of the tested compounds. Introduction of a

further carbohydrate residue in the trisaccharide deri-

vative 2, almost abolishes cytotoxic activity.

This latter was obtained in a multistep sequence from

3,4-di-O-acetyl-l-rhamnal 27²⁵through a SnCl₄cata-

lyzed addition of p-methoxybenzyl alcohol with allylic

rearrangement,³¹reduction of the double bond in non-

hydrogenolytic conditions to 29 and de-O-acetylation to

30. Inversion of the conﬁguration of the hydroxy group

in C-4 position was performed following Mitsunobu

procedure³²to give 31, later deprotected according to

the standard procedure to 32.

IDCP promoted coupling of 32 with the two glycosyl

donors 18 and 7, followed the same chemical and ste-

reochemical course as above described, giving dis-

accharides 33 and 37 with an a interglycosidic linkage.

Conversion of 33 and 37 to the glycosylating dis-

accharides 36 and 40 was performed according to the

previously described sequence of deprotection with

ammonium cerium(IV) nitrate and successive activation

with p-nitrobenzoyl chloride at the anomeric position.

Topoisomerase II-mediated DNA cleavage

The DNA cleavage activity of the new compounds 1–4

in the presence of the enzyme topoisomerase II a was

investigated in order to determine the inﬂuence of the

chemical modiﬁcations at the 3⁰and 4⁰⁰positions of

these anthracycline analogues on the DNA cleavage

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

1463

as intensity of topoisomerase II mediated DNA clea-

vage appeared to be slightly reduced.

These results seem to indicate that no direct correlation

exist between the extent of enzyme-induced DNA clea-

vage and the cytotoxicity in this class of oligosaccharide

anthracyclines. The same observations were reported

for anthracycline monosaccharides modiﬁed in C-3⁰posi-

tion. It was found that these derivatives had an altered site

speciﬁcity with respect to the parent compounds and that

quantitative relations between levels of DNA cleavage

and cytotoxicity were diﬀerent for compounds that had

diﬀerent sequence speciﬁcity.^8,10Thus, in addition to the

level and persistence, the site selectivity of drug-stimu-

lated DNA breaks may also contribute to the cytotoxic

activities of anthracyclines. Hence, the need of further

experiments to determine the sequence speciﬁcity of the

oligosaccharide anthracyclines here reported.

Experimental

General remarks

NMR experiments were recorded on a Bruker Avance

400 MHz spectrometer equipped with a 5 mm inverse

probe, and processed using Xwin-NMR version 2.1.

The ESI-MS experiments were performed on a model

QUATTRO triple Quadrupole mass spectrometer

(Micromass, UK) equipped with an ESI interface. Sol-

vents and reagents were dried before use according to

standard procedures.³⁴All reactions requiring anhyd-

rous conditions were performed under a nitrogen

atmosphere and starting materials were dried by

coevaporations with anhydrous toluene. Molecular

sieves 4 A were activated by ﬂaming under reduced pres-

sure. 60 PF254 silica gel-coated aluminum sheets (Merck)

were used for TLC analysis. Column chromatography

was performed on silica gel 60, 230–400 mesh (Merck).

Figure 2. (a) Stimulation of topoisomerase II mediated DNA cleavage

by the anthracyclines analogues 1–4 and the reference compound

MEN 10755. Lane C, control DNA; lane T, topoisomerase II alone.

(b) Quantiﬁcation of topoisomerase II dependent DNA cleavage sti-

mulated by anthracycline analogues in SV40 DNA. DNA cleavage

levels were determined at diﬀerent sites with an analysis performed on

a Macintosh computer using the public domain NIH Image program

(developed at the US National Institute of Health and available on the

internet at http://RSB.info.nih.gov/nih-image). To normalize among

diﬀerent experiments, the stimulation factor of MEN 10755 at 1 M,

which was included in all the gels, was used as an internal standard.

Methyl 3-N,4-O-di-allyloxycarbonyl-2, 3, 6-trideoxy-ꢀ-

L-arabino-hexopyranoside (6). To a mixture of methyl

3-amino-2, 3, 6-trideoxy-a-l-arabino-hexopyranoside 5

(3 g, 19 mmol) in anhydrous dichloromethane (140 mL),

anhydrous pyridine (2.44 mL, 30.4 mmol) and allyl-

chloroformate (3.22 mL, 30.4 mmol) were added at

ꢀ10 ^ꢁC under nitrogen atmosphere. After 4 h at room

temperature, water (50 mL) was added at 0 ^ꢁC and after

stirring 10 min, the organic layer was washed with

saturated aq NaHCO₃(100 mL), water (100 mLÂ2) and

dried with Na₂SO₄. Evaporation of the solvent gave a

residue, chromatographed on silica gel using petroleum

intensity. The 4⁰⁰-epimer of MEN 10755, 1, and the tri-

saccharide derivative 2, showed an enzyme-mediated DNA

cleavage level similar to MEN 10755. The two 3⁰-deoxy

derivatives 3 and 4 were less active in mediating topoi-

somerase II a DNA breaks than the parent drug (Fig. 2).

Conclusions

With the exception of the trisaccharide analogue 2, for

which other biological events, such as cellular uptake,

must be involved in order to explain its lack of cyto-

toxicity, the disaccharide derivatives 1, 3, 4 showed a

cytotoxic potency similar to that of the reference com-

pound. On the other hand, ability to poison topoi-

somerase II was diﬀerent for these compounds.

Epimerization of the C-4⁰⁰position (1) or introduction

of a further carbohydrate residue (2), did not aﬀect the

capacity to induce DNA breaks, while removal of the

hydroxyl group in C-3⁰position of compounds 3 and 4,

seemed to inﬂuence the stability of the ternary complex,

1

ether/ethyl acetate (7:3) to aﬀord 4.3 g (70%) of 6. H

NMR (CDCl3) d 6.0–5.8 (2H, m, 2ÂCH₂¼CH), 5.4–5.2

(4H, m, 2ÂCH₂¼CH), 4.8 (1H, br s, NH), 4.75 (1H, d,

H-1), 4.65–4.55 (4H, m, 2ÂOCH₂–CH), 4.0–3.75 (1H,

m, H-3), 3.7–3.5 (1H, m, H-5), 3.3 (3H, s, OCH₃), 3.15–

3.0 (1H, m, H-4), 2.15–2.0 (1H, dd, H-2eq), 1.75–1.55

(1H, td, H-2ax), 1.3 (3H, d, H-6).

Phenyl 3-N,4-O-di-allyloxycarbonyl-1-thio-2, 3, 6-tri-

deoxy-ꢀ-^L-arabino-hexopyranoside (7). To a solution of

methyl 3-N,4-O-di-allyloxycarbonyl-acosaminide 6 (5.7

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A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

g, 17.3 mmol) in anhydrous dichloromethane (60 mL),

phenylthiotrimethyl silane (4.8 mL, 26 mmol), and,

dropwise, trimethylsilyl triﬂate (5.0 mL, 26 mmol) were

added at 0 ^ꢁC. After stirring at room temperature for 2

h, the mixture was diluted with dichloromethane (60

mL), washed with saturated aq NaHCO₃(60 mL),

water (2Â30 mL), dried with Na₂SO₄and concentrated

in vacuo. The residue was chromatographed on silica

gel using petroleum ether/ethyl acetate (8:2) to give 7

temperature. After ﬁltration of the precipitate oxalates,

the ﬁltrate was concentrated in vacuo and chromato-

graphed on silica gel using petroleum ether/diethyl ether

(9:1) to aﬀord 11 (0.64 g, 62% considering the recovered

1

starting material). H NMR (CDCl₃) d 7.3–7.2 (2H, m,

Ph), 6.9–6.8 (2H, m, Ph), 6.0–5.8 (1H, m, CH₂¼CH),

5.4–5.2 (2H, m, CH₂¼CH), 5.1–5.0 (1H, m, H-3), 5.0

(1H, d, H-1), 4.7–4.55 (3H, m, OCH₂–CH, OCH₂–Ph),

4.4 (1H, d, OCH₂–Ph), 4.0 (1H, q, H-5), 3.8 (1H, d, H-

4), 3.7 (3H, s, OCH₃), 2.1 (1H, td, H-2ax), 2.0 (1H, dd,

H-2eq), 1.3 (3H, d, H-6).

1

(6.6 g, 94%). H NMR (CDCl₃) d 7.5–7.4 (2H, m, Ph),

7.3–7.2 (2H, m, Ph), 6.0–5.8 (2H, m, 2ÂCH₂¼CH), 5.5

(1H, d, H-1), 5.4–5.2 (4H, m, 2ÂCH₂¼CH), 4.9 (1H, br

s, NH), 4.7–4.5 (4H, m, 2ÂOCH₂–CH), 4.5–4.3 (2H, m,

H-4, H-5), 4.2–4.1 (1H, m, H-3), 2.5 (1H, dd, H-2eq),

2.1 (1H, td, H-2ax), 1.2 (3H, d, H-6).

3-N-allyloxycarbonyl-1,4-di-O-p-nitrobenzoyl-2, 3, 6-tri-

deoxy-ꢀ-^L-lyxo-hexopyranose (17). To a solution of 2.7

g (11.7 mmol) of 3-N-allyloxycarbonyldaunosamine 16

in anhydrous pyridine (50 mL), p-nitrobenzoylchloride

(6.5 g, 35 mmol), was added at 0 ^ꢁC under nitrogen

atmosphere. After 1 h at room temperature, the mixture

was diluted with dichloromethane (70 mL) and stirred

for 5 min with ice-cold water (100 mL). The organic

layer was washed with ice-cold 2 M H₂SO₄(3Â50 mL),

saturated aq NaHCO₃(3Â50 mL), water (50 mL), dried

over Na₂SO₄. After evaporation of the solvent, the

residue was puriﬁed by crystallization (acetone/chloro-

form/hexane=1:1:1) to aﬀord 2.2 g of a anomer of 17.

The mother liquor were chromatographed using petro-

leum ether/ethyl acetate (7:3) to give 2.7 g of the

anomeric mixture with a 79% total yield. ¹H NMR

(CDCl₃) a anomer d 8.45–8.15 (8H, m, Ph), 6.6 (1H, s,

H-1), 6.0–5.8 (1H, m, CH₂¼CH), 5.55 (1H, s, H-4),

5.35–5.15 (2H, m, CH₂¼CH), 4.8 (1H, d, NH), 4.6–4.5

(3H, m, OCH₂–CH, H-3), 4.35 (1H, q, H-5), 2.25–2.15

(2H, m, H-2), 1.2 (3H, d, H-6).

p-Methoxybenzyl 3,4-di-O-acetyl-2, 6-dideoxy-ꢀ-^L-lyxo-

hexopyranoside (9). To a solution of crude 3,4-di-O-

acetyl-l-fucal 8 (4.7 g, 21.9 mmol) and p-methoxybenzyl

alcohol (3.9 g, 28.0 mmol) in anhydrous dichloro-

methane (70 mL), triphenylphosphine hydrobromide

(0.35 g, 1 mmol) was added at 0 ^ꢁC under nitrogen

atmosphere. After 1 h at room temperature, the reaction

mixture was diluted with dichloromethane (230 mL),

the organic phase was washed with saturated aq

NaHCO₃(30 mL), water (2Â30 mL) and dried over

Na₂SO₄. Evaporation of the solvent furnished a residue

(9 g) used without puriﬁcation in the successive reac-

1

tion. H NMR (CDCl₃) d 7.3–7.2 (2H, m, Ph), 6.9–6.8

(2H, m, Ph), 5.35–5.25 (1H, m, H-3), 5.15 (1H, s, H-4),

5.0 (1H, s, H-1), 4.7–4.6 (1H, d, OCH₂-Ph), 4.55–4.45

(1H, d, OCH₂–Ph), 4.1 (1H, q, H-5), 2.15 (3H, s,

OCOCH₃), 2.1–2.0 (1H, td, H-2ax), 1.95 (3H, s,

OCOCH₃), 1.95–1.85 (1H, dd, H-2eq), 1.1 (3H, d, H-6).

Phenyl 3-N-allyloxycarbonyl-4-O-p-nitrobenzoyl-1-thio-

2, 3, 6-trideoxy-ꢀ-^L-lyxo-hexopyranoside (18). To a

solution of 3-N-allyloxycarbonyl-1, 4-di-O-p-nitro-

benzoyl-l-daunosamine 17 (1.14 g, 2.15 mmol) in

anhydrous dichloromethane (11.4 mL) phenylthio-

trimethyl silane (0.59 mL, 3.22 mmol), and, dropwise,

trimethylsilyl triﬂate (0.62 mL, 3.22 mmol) were added

at 0 ^ꢁC and under nitrogen atmosphere. After stirring at

room temperature for 2 h, the mixture was diluted with

dichloromethane (10 mL), washed with saturated aq

NaHCO₃(10 mL), water (2Â10 mL), dried with Na₂SO₄

and concentrated in vacuo. The residue was chromato-

graphed on silica gel using petroleum ether: ethyl acetate

p-Methoxybenzyl 2,6-dideoxy-ꢀ-^L-lyxo-hexopyranoside

(10). To a solution of the crude p-methoxybenzyl fuco-

side 9 in methanol (70 mL), 1 N methanolic MeONa (4

mL) was added at 0 ^ꢁC under nitrogen atmosphere.

After 2 h at room temperature, the solution was neu-

tralized with 0.75% HCl in ethanol. Removal of the

solvent gave a residue, puriﬁed by ﬂash chromato-

graphy using petroleum ether/ethyl acetate (1:1), and

after the elution of p-methoxybenzyl alcohol, petroleum

1

ether/acetone (1:2) to give 3.5 g of 10 (60%). H NMR

(CDCl₃) d 7.3–7.2 (2H, m, Ph), 6.9–6.8 (2H, m, Ph),

4.95 (1H, s, H-1), 4.6–4.5 (1H, d, OCH₂–Ph), 4.4–4.3

(1H, d, OCH₂–Ph), 4.05–3.95 (1H, m, H-3), 3.95 (1H, q,

H-5), 3.75 (3H, s, OCH₃), 3.6 (1H, s, H-4), 1.95–1.85 (1H,

dd, H-2eq), 1.8–1.7 (1H, td, H-2ax), 1.35 (3H, d, H-6).

1

(8:2) to give 18 (0.96 g, 95%). H NMR (CDCl₃) d 8.4–

8.1 (5H, m, Ph), 7.5–7.2 (4H, m, Ph), 6.0–5.8 (1H, m,

CH₂¼CH), 5.75 (1H, d, H-1), 5.5 (1H, s, H-4), 5.35–

5.15 (2H, m, CH₂¼CH), 4.8–4.65 (2H, m, H-5, NH),

4.6–4.5 (2H, m, OCH₂–CH), 4.4 (1H, m, H-3), 2.35 (1H,

td, H-2ax), 2.2 (1H, dd, H-2eq), 1.2 (3H, d, H-6).

p-Methoxybenzyl 3-O-allyloxycarbonyl-2,6-dideoxy-ꢀ-^L-

lyxo-hexopyranoside (11). A mixture of p-methoxy-

benzylfucoside 10 (1 g, 3.73 mmol) and dibutyltin oxide

(1.1 g, 4.46 mmol) in anhydrous toluene (100 mL) was

reﬂuxed for 12 h with continuous azeotropic removal of

water till concentration to half the volume. After cool-

ing at ꢀ78 ^ꢁC, anhydrous triethylamine (0.623 mL, 4.48

mmol) and allylchloroformate (0.474 mL, 4.48 mmol),

were added. After 2 h at ꢀ45 ^ꢁC, a precooled solution of

oxalic acid (0.402 g, 4.46 mmol) in anhydrous acetoni-

trile (20 mL) was added at ꢀ78 ^ꢁC. The reaction mixture

was stirred for about 1 h and allowed to warm to room

General procedure A for the synthesis of oligosaccharides

12, 19, 24, 33, 37

To a solution of the glycosyl acceptor (1 mmol) and the

glycosyl donor (1.4 mmol) in anhydrous diethyl ether

(16 mL) and 1,2 dichloroethane (4 mL), activated

molecular sieves 4 A (2.2 g) were added. The mixture

was stirred for 20 min at room temperature and then, at

10 ^ꢁC, iodonium dicollidine perchlorate (IDCP) (1.95

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

1465

mmol) was added. After 1 h at this temperature, TLC

showed the formation of the disaccharide with the

desired a glycosidic bond, and of the N-sulphenamide

derivative of the same disaccharide (ratio 6:4). After

adding triphenylphosphine (1.9 mmol), the reaction

mixture was stirred till complete conversion of the sul-

phenamide derivative to the desired disaccharide (1 h).

The mixture was diluted with diethyl ether (20 mL) and

ﬁltered, washed with 10% aq Na₂S₂O₃(20 mL), satu-

rated aq NaHCO₃(20 mL), water (2Â20 mL) and dried

over Na₂SO₄. Evaporation of the solvent and ﬂash

chromatography of the residue on silica gel using pet-

roleum ether/ethyl acetate (8:2), gave the desired dis-

accharides.

(15). Compound 12 was deprotected and activated as

described in the general procedure B to give 15 (80%).

¹H NMR (CDCl₃) d 8.4–8.1 (4H, m, Ph), 6.5 (1H, d, H-

1), 6.0–5.8 (3H, m, 3ÂCH₂¼CH), 5.4–5.1 (8H, m,

3ÂCH₂¼CH, NH, H-3⁰), 4.9–4.5 (9H, m, 3ÂOCH₂–

CH, H-1⁰, H-3, H-4⁰), 4.4–4.1 (2H, m, H-5, H-5⁰), 3.95

(1H, s, H-4), 2.5–1.7 (4H, m, H-2, H-2⁰), 1.3–1.1 (6H, m,

H-6, H-6⁰). ESMS m/z calcd for C₃₁H₃₈N₂O₁₅

(M+H)+ 679.6, found 679.8.

p-Methoxybenzyl 4- O-(3-N-allyloxycarbonyl-2,3,6-tri-

deoxy-4-O-p-nitrobenzoyl-ꢀ-^L-lyxo-hexopyranosyl)-3-O-

allyloxycarbonyl-2,6-dideoxy-ꢀ-L-lyxo-hexopyranoside

(19). Compounds 11 and 18 were reacted according to

1

the general procedure A to aﬀord 19 in 75% yield. H

NMR (CDCl₃) d 8.4–8.2 (4H, m, Ph), 7.3–7.2 (2H, m,

Ph), 7.0–6.8 (2H, m, Ph), 6.0–5.8 (2H, m, 2ÂCH₂¼CH),

5.5–5.0 (8H, m, 2ÂCH₂¼CH, H-1, H-1⁰, H-3, H-4⁰),

4.7–4.4 (9H, m, 2ÂOCH₂–CH, OCH₂–Ph, NH, H-3⁰, H-

5⁰), 4.0 (1H, q, H-5), 3.9 (1H, s, H-4), 3.8 (3H, s, OCH₃),

2.3–1.8 (4H, m, H-2, H-2⁰), 1.3 (3H, d, H-6), 1.1 (3H, d,

H-6⁰). ESMS m/z calcd for C₃₅H₄₂N₂O₁₄(M+H)+

715.7, found 715.9.

General procedure B for the synthesis of the activated

oligosaccharides 15, 22, 26, 36, 40

To a solution of the protected disaccharides 12, 19, 24,

33 and 37 (1 mmol) in acetonitrile/water (9:1, 10 mL),

ammonium cerium (IV) nitrate (2 mmol) was added.

After 2 h at room temperature, the reaction mixture was

diluted with ethyl acetate (10 mL). The separated aqu-

eous layer was extracted with ethyl acetate (2Â10 mL)

and the combined organic extracts were treated with

10% aq Na₂S₂O₅(20 mL). The obtained precipitate was

ﬁltered and the organic phase washed with saturated aq

NaHCO₃(20 mL), water (2Â20 mL) and dried over

Na₂SO₄. Evaporation of the solvent under reduced

pressure gave a residue (quantitative reaction) used in

the next reaction without puriﬁcation. To a solution of

crude deprotected disaccharide 14, 21, 25, 35 and 39 (1

mmol), in anhydrous pyridine (6 mL), p-nitro-

benzoylchloride (3 mmol) was added at 0 ^ꢁC. After stir-

ring for 2 h, at the same temperature, water (3 mL) was

added to the reaction mixture and after 10 min, it was

diluted with dichloromethane (10 mL). The organic

layer was washed with cold 2 M H₂SO₄(4Â10 mL) and

ice, cold saturated aq NaHCO₃(4Â10 mL), water (2Â10

mL), and dried over Na₂SO₄. Concentration in vacuo

and ﬂash chromatography on silica gel using petroleum

ether/ethyl acetate (8:2) gave the activated dis-

accharides.

4-O-(3-N-allyloxycarbonyl-2,3,6-trideoxy-4- O-p-nitro-

benzoyl-ꢀ-^L-lyxo-hexopyranosyl)-3-O-allyloxycarbonyl-

2,6-dideoxy-1-O-p-nitrobenzoyl-ꢀ-L-lyxo-hexopyranoside

(22). Following the general procedure B, 19 gave the

activated disaccharide 22 in 83% yield. ¹H NMR

(CDCl₃) d 8.4–8.1 (8H, m, Ph), 6.5 (1H, s, H-1), 6.0–5.8

(2H, m, 2ÂCH₂¼CH), 5.5 (1H, s, H-4⁰), 5.4–5.2 (5H, m,

2ÂCH₂¼CH, H-3), 5.1 (1H, s, H-1⁰), 4.7–4.4 (7H, m,

2ÂOCH₂–CH, NH, H-3⁰, H-5⁰), 4.2 (1H, q, H-5), 4.0

(1H, s, H-4), 2.5 (1H, td, H-2eq), 2.2–2.1 (2H, m, H-2⁰),

2.0 (1H, td, H-2ax), 1.3 (3H, d, H-6), 1.1 (3H, d, H-6⁰).

ESMS m/z calcd for C₃₄H₃₇N₃O₁₆(M+H)+ 744.7,

found 744.9.

Phenyl 4-O-(3-N-allyloxycarbonyl-2,3,6-trideoxy-4-O-p-

nitrobenzoyl-ꢀ-^L-lyxo-hexopyranosyl)-2,6-dideoxy-3-O-

allyloxycarbonyl-1-thio-ꢀ-L-lyxo-hexopyranoside (23).

To a solution of the disaccharide 22 (0.5 g, 0.67 mmol)

in anhydrous dichloromethane (25 mL), phenylthio-

trimethyl silane (0.31 mL, 1.68 mmol), and, dropwise,

trimethylsilyl triﬂate (0.26 mL, 1.34 mmol) were added

at 0 ^ꢁC. After stirring at 0 ^ꢁC for 1 h, the mixture was

diluted with dichloromethane (10 mL), washed with

saturated aq NaHCO₃(10 mL), water (2Â10 mL), dried

with Na₂SO₄and concentrated in vacuo. The residue

was chromatographed on silica gel using petroleum

p-Methoxybenzyl 4- O-(3-N,4- O-di-allyloxycarbonyl-

2,3,6 - trideoxy - ^L- arabino - hexopyranosyl) - 3 - O -

allyloxycarbonyl-2,6-dideoxy-L-lyxohexopyranoside (12)

The thiophenyl acosaminide 7 and the p-methoxy-

benzylfucoside 11 were reacted according to the general

procedure A to give 12 (74%). ¹H NMR (CDCl₃) d 7.2–

6.9 (4H, m, Ph), 6.0–5.8 (3H, m, 3ÂCH₂¼CH), 5.4–5.0

(8H, m, 3ÂCH₂¼CH, NH, H-1 ), 4.85 (1H, s, H-1⁰),

4.7–4.1 (11H, m, 3ÂOCH₂–CH, OCH₂–Ph, H-3, H-4,

H-5⁰), 3.9 (1H, q, H-5), 3.85 (1H, s, H-4), 3.8 (3H, s,

OCH₃), 2.4 (1H, dd, H-2⁰eq), 2.2 (1H, td, H-2⁰ax), 1.9

(1H, dd, H-2eq), 1.8 (1H, td, H-2ax), 1.3–1.1 (6H, m, H-

6, H6⁰). ESMS m/z calcd for C₃₂H₄₃NO₁₃(M+H)+

650.7, found 650.5.

1

ether/ethyl acetate (8:2) to give 23 (0.25 mg, 54%). H

NMR (CDCl₃) d 8.4–8.2 (4H, m, Ph), 7.6–7.2 (5H, m,

Ph), 6.0–5.8 (2H, m, 2ÂCH₂¼CH), 5.7 (1H, d, H-1), 5.5

(1H, s, H-4⁰), 5.4–5.0 (6H, m, 2ÂCH₂¼CH, H-1⁰, H-3),

4.7—4.4 (8H, m, 2ÂOCH₂–CH, NH, H-3⁰, H-5, H-5⁰),

4.0 (1H, s, H-4), 2.5 (1H, td, H-2eq), 2.2–1.8 (3H, m, H-

2⁰, H-2ax), 1.3 (3H, d, H-6), 1.1 (3H, d, H-6⁰).

p-Methoxybenzyl 4-O-4-O-(3-N-allyloxycarbonyl-4-O-p-

nitrobenzoyl-2, 3, 6-trideoxy-ꢀ-^L-lyxo-hexopyranosyl)-3-

O-allyloxycarbonyl-2,6-dideoxy-ꢀ-L-lyxo-hexopyranosyl

-3-O-allyloxycarbonyl-2,6-dideoxy-ꢀ-^L-lyxo-hexopyrano-

side (24). To a solution of the disaccharide 23 (220

4-O-(3-N,4-O-di-allyloxycarbonyl - 2,3,6 - trideoxy - ꢀ - ^L-

arabino-hexopyranosyl)-3-O-allyloxycarbonyl-2, 6-di-

deoxy-1-O-p-nitrobenzoyl-ꢀ -L -lyxo-hexopyranosyde

1466

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

mg, 0.32 mmol) and the p-methoxybenzyl fucoside 11

(113 mg, 0.32 mmol) in anhydrous diethyl ether (6 mL)

and 1,2 dichloroethane (1.5 mL), activated molecular

sieves 4 A (750 mg) were added. The mixture was stirred

for 20 min at room temperature and then, at 10 ^ꢁC,

iodonium dicollidine perchlorate (220 mg, 0.48 mmol)

was added. After 1.5 h at this temperature, the mixture

was diluted with diethyl ether (10 mL) and ﬁltered,

washed with 2% aq Na₂S₂O₃(10 mL), water (2Â10

mL) and dried over Na₂SO₄. Evaporation of the sol-

vent and ﬂash chromatography of the residue on silica

gel using petroleum ether/ethyl acetate (7.5:1.5), aﬀor-

chromatography on silica gel using dichloromethane to

1

obtain 29 (450 mg, 90%). H NMR (CDCl₃) d 7.3–6.9

(4H, m, Ph), 4.8 (1H, s, H-1), 4.6–4.4 (2H, m, OCH₂–

Ph), 4.5 (1H, m, H-4), 3.9–3.8 (1H, m, H-5), 3.7 (3H, s,

OCH₃), 2.1 (3H, s, COCH₃), 1.9–1.7 (4H, m, H-2, H-3),

1.2 (3H, d, H-6).

p-Methoxybenzyl 4-O-benzoyl-2,3,6-trideoxy-ꢀ-^L-lyxo-

hexopyranoside (31). Removal of the acetyl group was

performed according to the known procedures adding,

at 0 ^ꢁC, 1 N methanolic NaOMe (400 mL) to a solution

of compound 29 (90 mg, 0.3 mmol) in methanol (700

mL). After 2 h at room temperature, the mixture was

neutralized with 0.75% HCl in ethanol. After con-

centration of the solvent the residue was chromato-

graphed on silica gel, using petroleum ether/ethyl

acetate (1:1) to give the deprotected p-methoxybenzyl

2,3,6-trideoxy-a-l-lyxo hexopyranoside 30 (60%). 252

mg (1 mmol) of this compound, triphenylphosphine

(393 mg, 1.5 mmol) and benzoic acid (183 mg, 1.5

mmol) were dissolved in anhydrous tetrahydrofuran (10

mL). A solution of diethyl azodicarboxylate (0.236 mL,

1.5 mmol) in tetrahydrofuran (2 mL) was added drop-

wise while stirring at room temperature. After 2 h the

reaction was stopped. Prolonged reaction times did not

increase the yield of the reaction. After removal of the

solvent the residue was redissolved in dichloromethane

(20 mL), washed with saturated aq NaHCO₃(10 mL),

water (10 mL), dried over Na₂SO₄and evaporated in

vacuo. Flash chromatography of the residue on silica

gel using dichloromethane, gave 31 (178 mg, 50% yield,

80% considering the recovered starting material). ¹H

NMR (CDCl₃) d 7.7–7.4 (5H, m, Ph), 7.3–6.9 (4H, m,

Ph), 5.1 (1H, s, H-1), 5.0 (1H, s, H-4), 4.7–4.5 (4H, m,

OCH₂–Ph), 4.2 (1H, q, H-5), 3.8 (3H, s, OCH₃), 2.3–1.9

(4H, m, H-2, H-3), 1.3 (3H, d, H-6).

1

ded the desired trisaccharide in 41% yield. H NMR

(CDCl₃) d 8.4–8.2 (4H, m, Ph), 7.3 (2H, m, Ph), 6.9

(2H, m, Ph), 6.0–5.8 (3H, m, 3ÂCH₂¼CH), 5.5 (1H, s,

H-4⁰⁰), 5.4–5.0 (11H, m, 3ÂCH₂¼CH, H-1, H-1⁰, H-1⁰⁰,

H-3, H-3⁰), 4.7–4.4 (11H, m, 3ÂOCH₂–CH, OCH₂–Ph,

NH, H-3⁰⁰, H-5⁰⁰), 4.3 (1H, q, H-5), 4.0–3.8 (3H, m, H-

4, H-4⁰, H-5), 3.8 (3H, s, OCH₃), 2.4–1.8 (6H, m, H-2,

H-2⁰, H-2⁰⁰), 1.4–1.1 (9H, m, 3 Â H-6, H-6⁰, H-6⁰⁰).

ESMS m/z calcd for C₄₅H₅₆N₂O₁₉(M+H)+ 929.9,

found 929.7.

4-O-[4-O-(3-N-allyloxycarbonyl-2,3,6-trideoxy-4-O-p-ni-

trobenzoyl-ꢀ-^L-lyxo-hexopyranosyl)-3-O-allyloxycarbo-

nyl - 2,6 - dideoxy - ꢀ - L - lyxo - hexopyranosyl] - 3 - O-

allyloxycarbonyl-2,6-dideoxy-1-O-p-nitrobenzoyl-ꢀ-^L-

lyxo-hexopyranoside (26). The trisaccharide 24 was

treated according to the general procedure B to give 26

(60%). ¹H NMR (CDCl₃) d 8.4–8.2 (8H, m, Ph), 6.0–5.8

(4H, m, 3ÂCH₂=CH, H-1), 5.5 (1H, s, H-4⁰⁰), 5.4–5.3

(6H, m, 3ÂCH₂¼CH), 5.2 (1H, m, H-3), 5.1 (1H, s, H-

1), 5.0 (1H, s, H-1⁰), 4.8 (1H, dt, H-3), 4.7–4.5 (8H, m,

3ÂOCH₂–CH, NH, H-5⁰⁰), 4.4 (1H, m, H-3⁰⁰), 4.3 (1H,

q, H-5⁰⁰), 4.0 (1H, s, H-4⁰), 3.9 (1H, s, H-4), 3.8 (1H, q,

H-5), 2.4–1.8 (6H, m, H-2, H-2⁰, H-2⁰⁰), 1.3 (3H, d, H-6),

1.2 (3H, d, H-6⁰), 1.1 (3H, d, H-6⁰⁰). ESMS m/z calcd for

C₄₄H₅₁N₃O₂₁(M+H)+ 958.9, found 958.7.

p-Methoxybenzyl 2,3,6-trideoxy-ꢀ-^L-lyxo-hexopyrano-

side (32). K₂CO₃(15 mg, 0.11 mmol) was added at

room temperature to a solution of p-methoxybenzyl 4-

O-benzoyl-2,3,6-trideoxy fucose 31 (392 mg, 1.1 mmol)

in methanol (50 mL). After 2 h the solvent was evapo-

rated to dryness and the residue ﬂash chromatographed

on silica gel using dichloromethane/methanol (98:2) to

p-Methoxybenzyl 4-O-acetyl-2,3,6-trideoxy-ꢀ-^L-arabino-

hex-2-enopyranoside (28). To a solution of 27 (2 g, 9.3

mmol) and p-methoxybenzyl alcohol (1.9 g, 14 mmol) in

anhydrous dichloromethane (60 mL), 0.22 M SnCl₄in

dichloromethane (4.0 mL, 0.88 mmol) was added at

0 ^ꢁC. After 2 h at room temperature the reaction mix-

ture was diluted with dichloromethane (20 mL). The

organic phase was washed with saturated aq NaHCO₃

(30 mL) and water (2Â20 mL), then dried over Na₂SO₄.

Evaporation of the solvent in vacuo aﬀorded a residue

chromatographed on silica gel using petroleum ether/

1

give 32 (260 mg, 94%). H NMR (CDCl₃) d 7.3–6.9

(4H, m, Ph), 4.8 (1H, s, H-1), 4.6–4.4 (2H, m, OCH₂–

Ph), 4.0 (1H, q, H-5), 3.8 (3H, s, OCH₃), 3.5. (1H, d, H-

4), 2.2–1.5 (4H, m, H-2, H-3), 1.3 (3H, d, H-6).

p-Methoxybenzyl 4-O-(3-N-allyloxycarbonyl-4- O-p-ni-

trobenzoyl-2,3,6-trideoxy-ꢀ-^L-lyxo-hexopyranosyl)-2,3,6

- trideoxy - ꢀ - L - lyxo - hexopyranoside (33). The p-

methoxybenzyl 2,3,6-trideoxy fucoside 32 was reacted

with the thiophenyl daunosaminide 18, following the

general procedure A to give 33 (84%). ¹H NMR

(CDCl₃) d 8.4–8.2 (4H, m, Ph), 7.3–6.9 (4H, m, Ph),

6.0–5.8 (1H, m, CH₂¼CH), 5.5 (1H, s, H-5⁰), 5.3–5.1

(2H, m, CH₂¼CH), 5.1 (1H, s, H-1⁰), 4.9 (1H, s, H-1),

4.7–4.4 (6H, m, OCH₂–CH, OCH₂–Ph, NH, H-3⁰), 4.3

(1H, q, H-5), 3.9 (1H, m, H-5⁰), 3.8 (3H, s, OCH₃), 3.5.

(1H, s, H-4), 2.2–1.5 (6H, m, H-2, H-2⁰, H-3), 1.3 (3H,

d, H-6), 1.2 (3H, d, H-6⁰). ESMS m/z calcd for

C₃₀H₃₆N₂O₁₁(M+H)+ 601.2, found 601.4.

1

diethyl ether (8:2) to aﬀord 28 (1.9 g, 70% yield). H

NMR (CDCl₃) d 7.3–6.9 (4H, m, Ph), 6.2 (1H, q, H-3),

5.8 (1H, dd, H-2), 4.8 (1H, s, H-1), 4.6–4.4 (2H, m,

OCH₂–Ph), 4.2 (1H, m, H-5), 3.7 (3H, s, OCH₃), 3.6

(1H, m, H-4), 2.1 (3H, s, COCH3), 1.2 (3H, d, H-6).

p-Methoxybenzyl 4-O-acetyl-2, 3, 6-trideoxy-ꢀ-^L-arabino-

hexopyranoside (29). To a solution of compound 28

(500 mg, 1.7 mmol) in anhydrous benzene (25 mL)

chlorotris(triphenylphosphine)rhodium(I) (78.6 mg,

0.085 mmol) was slowly added. After 2 h in the dark,

under hydrogen atmosphere (1 atm), the solvent was

evaporated to dryness. The residue was puriﬁed by ﬂash

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

1467

4-O-(3-N-allyloxycarbonyl-4-O-p-nitrobenzoyl-2,3,6-tri-

deoxy-ꢀ-^L-lyxo-hexopyranosyl)-2,3,6-trideoxy-1-O-p-ni-

trobenzoyl - ꢀ - L - lyxo - hexopyranoside (36). The

disaccharide 33 was treated according to the general

procedure B to give 36 (80%). ¹H NMR (CDCl₃) d 8.4–

8.2 (8H, m, Ph), 6.5 (1H, br s, H-1), 6.0–5.8 (1H, m,

CH₂¼CH), 5.5 (1H, s, H-4⁰), 5.4–5.2 (2H, m,

CH₂¼CH), 5.1 (1H, br s, H-1⁰), 4.7–4.4 (4H, m, OCH₂–

CH, H-3⁰, NH), 4.3 (1H, q, H-5⁰), 4.2 (1H, q, H-5), 3.7

(1H, s, H-4), 2.4–1.8 (6H, m, H-2, H-2⁰, H-3), 1.3 (3H,

d, H-6), 1.2 (3H, d, H-6⁰). ESMS m/z calcd for

C₃₀H₃₃N₃O₁₃(M+H)⁺644.2, found 644.5.

m, 3ÂCH₂¼CH), 5.6 (1H, s, H-1⁰), 5.0–5.4 (11H, m,

3ÂCH₂¼CH, H-1⁰⁰, H-3⁰, H-7, H-14), 4.9 (1H, d, H-4⁰⁰),

4.7–4.5 (6H, m, 3ÂOCH₂–CH), 4.4 (1H, q, H-5⁰⁰), 4.3

(1H, m, H-3⁰⁰), 4.1 (1H, q, H-5⁰), 3.9 (1H, s, H-4⁰), 3.2

(2H, q, H-10), 2.5–1.7 (6H, m, H-2⁰, H-2⁰⁰, H-8), 2.2

(3H, s, COCH₃), 1.4–1.2 (6H, m, H-6⁰, H-6⁰⁰). ESMS m/

z calcd for C₄₆H₅₁NO₂₀(M+H)+ 938.9, found 938.7.

7-{3-O-allyloxycarbonyl-4-O-[3-O-allyloxycarbonyl-2,6-

dideoxy-4-O-(3-N-allyloxycarbonyl-2,3,6-trideoxy-4-O-p

-nitrobenzoyl-ꢀ-^L-lyxo-hexopyranosyl)-ꢀ-L-lyxo-hexo-

pyranosyl]-2,6-dideoxy-ꢀ-^L-lyxo-hexopyranosyl-14-acet-

oxy-4-demethoxyadriamycinone (43). The aglycone 41

and the trisaccharide 26 were coupled according to the

general procedure C to aﬀord the glycoside 43 (60%).

¹H NMR (CDCl₃) d 13.6 (1H, s, OH-6), 13.2 (1H, s,

OH-11), 8.4 (2H, m H-1, H-4), 8.3–8.2 (4H, m, Ph), 7.8

(2H, m, H-2, H-3), 6.0–5.7 (3H, m, 3ÂCH₂¼CH), 5.55

(1H, br s, H-1⁰), 5.45 (1H, br s, H-4⁰⁰⁰), 5.4–5.2 (9H, m,

3ÂCH₂¼CH, H-7, H-14), 5.1 (1H, m, H-3⁰⁰), 5.0 (1H, br

s, H-1⁰⁰⁰), 4.9 (1H, br s, H-1⁰⁰), 4.8 (1H, m, H-3⁰), 4.7–4.5

(7H, m, 3ÂOCH₂–CH, NH), 4.35 (1H, q, H-5⁰⁰⁰), 4.2

(1H, q, H-5⁰⁰), 4.1 (1H, m, H-3⁰⁰⁰), 4.05 (1H, q, H-5⁰),

3.95 (1H, br s, H-4⁰⁰), 3.9 (1H, br s, H-4⁰), 3.15 (2H, q,

H-10), 2.4–2.2 (2H, m, H-8), 2.2 (3H, s, COCH₃), 2.1–

1.6 (6H, m, H-2⁰, H-2⁰⁰, H-2⁰⁰⁰), 1.3 (3H, d, H-6⁰) 1.2 (3H,

d, H-6⁰⁰), 1.1 (3H, d, H-6⁰⁰⁰). ESMS m/z calcd for

C₅₉H₆₄N₂O₂₆(M+H)⁺1218.2, found 1218.4.

p-Methoxybenzyl 4- O-(3-N, 4 -O-di-allyloxycarbonyl-

2,3,6 - trideoxy - ꢀ - ^L- arabino - hexopyranosyl) - 2,3,6-tri-

deoxy-ꢀ-L-lyxo-hexopyranoside (37). The p-methoxy-

benzyl 2,3,6-trideoxy fucoside 32 was reacted with the

thiophenyl acosaminide 7, following the general proce-

1

dure A to give 37 (75%). H NMR (CDCl₃) d 7.3–6.8

(4H, m, Ph), 6.0–5.8 (2H, m, 2ÂCH₂¼CH), 5.4–5.2 (4H,

m, 2ÂCH₂¼CH), 5.0 (1H, d, NH), 4.9 (1H, s, H-1⁰), 4.8

(1H, dt, H-3), 4.7–4.5 (5H, m, 2ÂOCH₂–CH, H-1), 4.5–

4.3 (2H, m, OCH₂–Ph), 4.0 (1H, q, H-5), 3.9 (1H, m, H-

3⁰), 3.8 (3H, s, OCH₃), 3.7. (1H, br s, H-4), 3.5 (1H, m,

H-5⁰), 2.2–1.5 (6H, m, H-2, H-2⁰, H-3), 1.3 (3H, d, H-6),

1.2 (3H, d, H-6⁰). ESMS m/z calcd for C₂₆H₃₅NO₁₀

(M+H)+ 522.2, found 522.3.

4-O-(3-N,4-O-di-allyloxycarbonyl-2,3,6-trideoxy-ꢀ-^L-

arabino - hexopyranosyl) - 2,3,6 - trideoxy-1-O-p-nitroben-

zoyl-ꢀ-L-lyxo-hexopyranoside (40). Application of the

general procedure B to the disaccharide 37 gave 40 in

80% yield. ¹H NMR (CDCl₃) d 8.4–8.2 (4H, m, Ph), 6.5

(1H, bs, H-1), 6.0–5.8 (2H, m, CH₂¼CH), 5.4–5.2 (4H,

m, CH₂¼CH), 5.0 (1H, d, NH), 4.9–4.8 (2H, m, H-3⁰,

H-1⁰), 4.7–4.5 (5H, m, OCH₂–CH, H-4⁰), 4.3 (1H, m, H-

5⁰), 4.1 (1H, q, H-5), 4.0 (1H, s, H-4), 2.2–1.5 (6H, m, H-2,

H-2⁰, H-3), 1.3 (3H, d, H-6), 1.2 (3H, d, H-6⁰). ESMS m/z

calcd for C₂₆H₃₂N₂O₁₂(M+H)+ 565.2, found 565.4.

7-[4-O-(2, 3, 6-Trideoxy-3-N-allyloxycarbonyl-4-O-p-ni-

trobenzoyl-ꢀ-^L-lyxo-hexopyranosyl)-2,3,6-trideoxy-ꢀ-L-

lyxo-hexopyranosyl]-14-acetoxy-4-demethoxyadriamyci-

none (44). The aglycone 41 and the disaccharide 36

were reacted following the general procedure C obtain-

1

ing the glycoside 44 in 45% yield. H NMR (CDCl₃) d

13.6 (1H, s, OH-6), 13.3 (1H, s, OH-11), 8.4 (2H, m, H-

1, H-4), 7.8 (2H, m, H-2, H-3), 6.0–5.8 (1H, m,

CH₂¼CH), 5.5 (1H, s, H-1⁰), 5.4–5.2 (3H, m, CH₂¼CH,

H-7), 4.5 (3H, m, H-14, NH), 4.9 (1H, s, H-1⁰), 4.8 (2H,

d, OCH₂–CH), 4.6 (1H, d, OH-9), 4.2–3.9 (3H, m, H-3⁰,

H-5⁰, H-5⁰⁰), 3.7 (1H, d, H-4⁰⁰), 3.6 (1H, s, H-4⁰), 3.4–3.0

(2H, q, H-10), 3.0 (1H, t, OH-14), 2.4–1.8 (11H, m,

COCH₃, H-2⁰, H-2⁰⁰, H-3⁰, H-8), 1.3–1.2 (6H, m, H-6⁰,

H-6⁰⁰). ESMS m/z calcd for C₄₅H₄₆N₂O₁₈(M+H)+

903.9, found 903.7.

General procedure C for the synthesis of fully protected

glycosides 42–45

To a mixture of 14-acetoxy-4-demethoxyadriamycinone

(1 mmol) 41, disaccharide (1 mmol) and activated

molecular sieves (5 g) in anhydrous dichloromethane/die-

thyl ether (3:1, 110 mL), trimethylsylil triﬂate (1.5 mmol)

was added dropwise at ꢀ30 ^ꢁC. After 1.5 h, 2% aq

NaHCO₃(60 mL), at 0 ^ꢁC, and then dichloromethane

(125 mL) were added to the reaction mixture. The organic

layer was washed with water (100 mL), dried over Na₂SO₄

and evaporated in vacuo. The crude residue was ﬂash

chromatographed on silica gel using dichloromethane/

acetone (19:1) to aﬀord the protected glycosides.

7-[4-O-(3-N,4-O-di-allyloxycarbonyl-2,3,6-trideoxy-ꢀ-^L-

arabino-hexopyranosyl)-2,3,6-trideoxy-ꢀ-L-lyxo-hexo-

pyranosyl]-14-acetoxy-4-demethoxyadriamycinone (45).

The aglycone 41 and the disaccharide 40 were coupled

according to the general procedure C. The obtained

residue was puriﬁed by preparative HPLC (Merck col-

umn, LiChrosphere RP-18,

7

mm, 250Â25 mm,

CH₃CN+0.1% TFA/H₂O + 0.1% TFA, 80:20, ﬂow 8

mL/min) giving the glycoside 45 (55%). ¹H NMR

(CDCl₃) d 13.6 (1H, s, OH-6), 13.3 (1H, s, OH-11), 8.4

(2H, m, H-1, H-4), 7.8 (2H, m, H-2, H-3), 6.0–5.8 (2H,

m, 2ÂCH₂¼CH), 5.5 (1H, s, H-1⁰), 5.4–5.2 (5H, m,

2ÂCH₂¼CH, H-1⁰⁰), 4.9 (1H, s, H-4⁰⁰), 4.8 (1H, d, NH),

4.7–4.5 (4H, m, 2ÂOCH₂–CH), 4.4 (2H, m, H-14), 4.2

(1H, m, H-3⁰⁰), 4.1–3.9 (2H, m, H-5⁰, H-5⁰⁰), 3.6 (1H, s,

H-4⁰), 3.4–3.0 (2H, q, H-10), 2.4–2.0 (8H, m, H-2⁰, H-2⁰⁰,

H-3⁰, H-8), 2.1 (3H, s, COCH₃), 1.4–1.2 (6H, m, H-6⁰,

7-[4-O-(2,3,6-trideoxy-3-N,4-O-di-allyloxycarbonyl-ꢀ-^L-

arabino-hexopyranosyl)-2,6-dideoxy-3-O-allyloxycarbo-

nyl-ꢀ-L-lyxo-hexopyranosyl]-14-acetoxy-4-demethoxy-

adriamycinone (42). The aglycone 41 and the

disaccharide 15 were reacted following the general pro-

1

cedure C to obtain the glycoside 42 (73%). H NMR

(CDCl₃) d 13.6 (1H, s, OH-6), 13.3 (1H, s, OH-11), 8.4

(2H, m, H-1, H-4), 7.8 (2H, m, H-2, H-3), 6.0–5.8 (3H,

1468

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

H-6⁰⁰). ESMS m/z calcd for C₄₂H₄₇NO₁₇(M+H)⁺

837.8, found 837.6.

(3H, m, H-3⁰, H-5⁰, H-5⁰⁰), 3.6 (2H, m, H-4⁰, H-4⁰⁰), 3.3

(1H, d, H-10), 3.2–3.0 (2H, m, H-10, OH-14), 2.4–1.9

(8H, m, H-2⁰, H-2⁰⁰, H-3⁰, H-8), 1.3–1.2 (6H, m, H-6⁰, H-

6⁰⁰). ESMS m/z calcd for C₃₆H₄₁NO₁₄(M+H)⁺712.7,

found 712.9.

General procedure D for the synthesis of partially

protected glycosides 46–49

To a solution of fully protected glycoside (1 mmol) in

methanol/dichloromethane (2:1, 106 mL), 0.5 M aq

K₂CO₃(3 mmol) was added at ꢀ10 ^ꢁC. After stirring for

1 h at ꢀ10/ꢀ5 ^ꢁC the reaction mixture was quenched

with 0.1 M HCl. The aq layer was washed with chloro-

form until a colourless extract was obtained. The com-

bined organic extracts were washed with water (2Â50

mL), dried over Na₂SO₄and concentrated in vacuo.

Flash chromatography on silica gel using dichloro-

methane/ethanol (96:4) gave the partially deprotected

glycoside.

7-[4-O-(3-N,4-O-di-allyloxycarbonyl-2,3,6-trideoxy-ꢀ-^L-

arabino-hexopyranosyl)-2,3,6-trideoxy-ꢀ-L-lyxo-hexo-

pyranosyl]-4-demethoxyadriamycinone (49). The glyco-

side 45 was reacted following the general procedure D

to aﬀord the glycoside 49 (71%). ¹H NMR (CDCl₃)

13.55 d (1H, s, OH-6), 13.3 (1H, s, OH-11), 8.4 (2H, m,

H-1, H-4), 7.9 (2H, m, H-2, H-3), 6.0–5.8 (2H, m,

2ÂCH₂¼CH), 5.5–5.1 (6H, m, 2ÂCH₂¼CH, H-1⁰, H-7),

5.0 (1H, s, H-1⁰⁰), 4.9 (1H, s, H-4⁰⁰), 4.8 (1H, m, NH),

4.75 (2H, d, H-14), 4.7–4.5 (4H, m, 2ÂOCH₂–CH), 4.4

(1H, q, H-5⁰), 4.2 (1H, m, H-3⁰⁰), 4.0 (1H, m, H-5⁰⁰), 3.6

(1H, s, H-4⁰), 3.3 (1H, d, H-10), 3.1–3.0 (2H, m, H-10,

OH-14), 2.25–1.6 (8H, m, H-2⁰, H-2⁰⁰, H-3⁰, H-8), 1.2

(6H, m, H-6⁰, H-6⁰⁰). ESMS m/z calcd for C₅₀H₅₄N₂O₂₁

(M+H)⁺852.3, found 852.6.

7-[4-O-(2,3,6-trideoxy-3-N,4-O-di-allyloxycarbonyl-ꢀ-^L-

arabino-hexopyranosyl)-2,6-dideoxy-3-O-allyloxycarbo-

nyl-ꢀ-L-lyxo-hexopyranosyl]-4-demethoxyadriamycinone

(46). The fully protected glycoside 42 was partially

deprotected according to the general procedure D to

General procedure E for the synthesis of glycosides 1–4

1

aﬀord 46 (70%). H NMR (CDCl₃) d 13.6 (1H, s, OH-

6), 13.3 (1H, s, OH-11), 8.4 (2H, m H-1, H-4), 7.9 (2H,

m, H-2, H-3), 6.0–5.8 (3H, m, 3ÂCH₂¼CH), 5.6 (1H, s,

H-1⁰), 5.4–5.1 (8H, m, 3ÂCH₂¼CH, H-7, OH-9), 4.9–

4.5 (12H, m, 3ÂOCH₂–CH, NH, H-14, H-1⁰⁰, H-3⁰, H-

4⁰⁰), 4.4–4.0 (3H, m, H-3⁰⁰, H-5, H-5⁰), 3.9 (1H, s, H-4⁰),

3.2 (2H, q, H-10), 2.5–1.8 (6H, m, H-2⁰, H-2⁰⁰, H-8), 1.4–

1.2 (6H, m, H-6⁰, H-6⁰⁰). ESMS m/z calcd for

C₄₄H₄₉NO₁₉(M+H)+ 896.9, found 896.7.

To a solution of the partially deprotected glycoside (1

mmol) in dichloromethane (125 mL), N,N-dimethyl-

trimethylsilylamine (5 mmol), trimethylsilylacetate (5

mmol) and tetrakis-(triphenylphosphine)palladium

(0.03 mmol) were added at room temperature. After

stirring for 30 min in the dark, dichloromethane (500

mL) was added and the organic layer was washed with

5% aq NaCl (2Â370 mL) and percolated through a

column of Na₂SO₄(125 g). The organic phase was con-

centrated to a small volume and evaporated several

times with ethyl acetate, until complete removal of di-

chloromethane. After concentration of the solution to a

small volume (125 mL), 0.001 M HCl (75 mL) was

added and the separated organic layer was extracted

with 0.001 M HCl (4Â12 mL) until a colourless extract

was obtained. The combined aqueous extracts were

washed with ethyl acetate (3Â250 mL), concentrated to

a volume of about 55 mL (bath temperature <30 ^ꢁC),

and after adjusting the pH to a value of 4.0, added with

acetonitrile (6 mL). This solution was ﬁltered on a 10

cm reverse phase bad (Merck, LiChropep RP-18, 40–63

mm, substance/reverse phase 1/100, water (pH 4)/ace-

tonitrile 2:1). After removal of the residual acetonitrile

and adjusting the pH to 4.5, the eluate was lyophilized

to give the ﬁnal glycoside as hydrochloride.

7-{3-O-allyloxycarbonyl-4-O-[3-O-allyloxycarbonyl-2,6-

dideoxy-4-O-(3-N-allyloxycarbonyl-2,3,6-trideoxy-ꢀ-^L-

lyxo-hexopyranosyl)-ꢀ-L-lyxo-hexopyranosyl]-2, 6-di-

deoxy-ꢀ-^L-lyxo-hexopyranosyl}-4-demethoxyadriamyci-

none (47). Compound 43 was reacted following the

general procedure D to obtain the glycoside 47 (73%).

¹H NMR (CDCl₃) d 13.55 (1H, s, OH-6), 13.3 (1H, s,

OH-11), 8.35 (2H, m H-1, H-4), 7.85 (2H, m, H-2, H-3),

6.0–5.8 (3H, m, 3ÂCH₂¼CH), 5.57 (1H, s, H-1⁰), 5.4–

5.2 (7H, m, 3ÂCH₂¼CH, H-7), 5.15–5.0 (2H, m, OH-9,

H-3⁰⁰), 4.95 (1H, s, H-1⁰⁰⁰), 4.87 (1H, s, H-1⁰⁰), 4.85–4.7

(3H, m, H-3⁰, H-14), 4.65–4.5 (7H, m, 3ÂOCH₂–CH,

NH), 4.4–4.3 (1H, m, H-5⁰⁰⁰), 4.3–4.2 (1H, m, H-5⁰⁰),

4.2–4.0 (2H, m, H-3⁰⁰⁰, H-5⁰), 3.95 (1H, br s, H-4⁰⁰), 3.9

(1H, s, H-4⁰), 3.6 (1H, s, H-4⁰⁰⁰), 3.3 (1H, d, H-10), 3.05

(1H, d, H-10), 3.0 (1H, br s, OH-14), 2.4–1.6 (8H, m, H-

2⁰, H-2⁰⁰, H-2⁰⁰⁰, H-8), 1.3 (3H, d, H-6⁰) 1.2 (3H, d, H-6⁰⁰),

1.1 (3H, d, H-6⁰⁰⁰). ESMS m/z calcd for C₅₀H₅₉NO₂₂

(M+H)⁺1027.0, found 1027.1.

7-[4-O-(2,3,6-trideoxy-3-amino-ꢀ-^L-arabino-hexopyrano-

syl)-2,6-dideoxy-ꢀ-L-lyxo-hexopyranosyl]-4-demethoxya-

driamycinone (1). The partially protected glycoside 46

was treated as described in the general procedure E to

7-[4-O-(2,3,6-trideoxy-3-N-allyloxycarbonyl - ꢀ - ^L- lyxo -

hexopyranosyl)-2,3,6-trideoxy-ꢀ-L-lyxo-hexopyranosyl]-

4- demethoxyadriamycinone (48). The fully protected

glycoside 44 was partially deprotected according to the

general procedure D to aﬀord 48 (70%). ¹H NMR

(CDCl₃) d 13.55 (1H, s, OH-6), 13.25 (1H, s, OH-11),

8.4 (2H, m, H-1, H-4), 7.8 (2H, m, H-2, H-3), 5.9 (1H,

m, CH₂¼CH), 5.45 (1H, s, H-1⁰), 5.4–5.2 (3H, m,

CH₂¼CH, H-7), 5.1 (3H, m, H-14, NH), 4.9 (1H, s, H-

1⁰), 4.8 (2H, d, OCH₂–CH), 4.6 (1H, d, OH-9), 4.2–3.9

1

give 1 (40%). H NMR (DMSO) d 8.4 (2H, m H-1, H-

4), 8.1 (2H, m H-2, H-3), 5.6 (1H, d, OH-4⁰⁰), 5.5 (1H, s,

OH-9), 5.3 (1H, s, H-1⁰), 5.0 (1H, m, H-7), 4.9 (1H, s, H-

1⁰⁰), 4.8 (1H, t, OH-14), 4.7–4.5 (3H, m, OH-3⁰, H-14),

4.3–4.1 (2H, m, H-5⁰⁰, H-5⁰), 3.8 (1H, m, H-3⁰), 3.6 (1H,

s, H-4⁰), 3.3 (1H, m, H-3⁰⁰), 3.1–3.0, (3H, m, H-4⁰⁰, H-

10), 2.2 (3H, m, H-2⁰⁰eq, H-8), 1.9–1.6 (3H, m, H-2⁰⁰ax,

H-2⁰), 1.2 (3H, d, H-6⁰), 1.1 (3H, d, H-6⁰⁰). ESMS m/z

calcd for C32H37NO13 (M+H)⁺644.6, found 644.7.

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

1469

7-{4-O-[4-O-(2,3,6-trideoxy-3-amino-ꢀ-L-lyxo-hexopyra-

Topoisomerase II mediated DNA cleavage

nosyl)-2,6-dideoxy-ꢀ-L-lyxo-hexopyranosyl]-2,6-dideoxy-

ꢀ - ^L- lyxo - hexopyranosyl}-4-demethoxyadriamycinone

(2). Following the general procedure E the glycoside 47

SV 40 DNA fragments were 5⁰³²P-labelled with T4

kinase and g-³²P ATP. DNA cleavage reactions were

performed at 37 ^ꢁC for 20 min in 10 mM TRIS–HCl pH

6, 10 mM MgCl₂, 100 mM KCl, 1 mM ATP with or

without drugs. Cleavage reactions were stopped with

1% SDS and 0.1 mg/ mL proteinase K and incubated at

42 ^ꢁC for 45 min and then analyzed on 1% agarose gel.

Autoradiograms of dried gels were carried out using

Amersham Hyperﬁlms.

1

gave 2 in 60% yield. H NMR (DMSO) d 13.6 (1H, s,

OH-6), 13.2 (1H, s, OH-11), 8.35 (2H, m, H-1, H-4), 8.0

(2H, m, H-2, H-3), 5.50 (1H, s, OH-9), 5.30 (1H, d, OH-

4⁰⁰⁰), 5.25 (1H, br s, H-1⁰), 5.95 (1H, t, OH-14), 4.90–4.8

(3H, m, H-1⁰⁰, H-1⁰⁰⁰, H-7), 4.65 (1H, d, OH-3⁰), 4.60

(2H, d, H-14), 4.55 (1H, d, OH-3⁰⁰), 4.40 (1H, q, H-5⁰⁰),

4.30 (1H, q, H-5⁰⁰), 4.15 (1H, q, H-5⁰), 3.90 (1H, m, H-

3⁰⁰), 3.75 (1H, m, H-3⁰), 3.50 (3H, m, H-4⁰, H-4⁰⁰, H-4⁰⁰⁰),

3.40 (1H, m, H-3⁰⁰⁰), 3.00 (2H, s, H-10), 2.25–2.05 (2H,

m, H-8), 1.95–1.50 (6H, m, H-2⁰, H-2⁰⁰, H-2⁰⁰⁰), 1.15 (3H,

d, H-6⁰) 1.10 (3H, d, H-6⁰⁰), 1.05 (3H, d, H-6⁰⁰⁰). ESMS m/

z calcd for C₃₈H₄₇NO₁₆(M+H)+774.8, found 774.6.

Acknowledgements

The work was supported by A. Menarini-Industrie

Farmaceutiche Riunite and Istituto Mobiliare Italiano

(Grant no. 63216).

7-[4-O-(2,3,6-trideoxy-3-amino-ꢀ-^L-lyxo-hexopyranosyl)

-2,3,6-trideoxy-ꢀ-L-lyxo-hexopyranosyl]-4-demethoxy-

adriamycinone (3). According to the general procedure

1

E, 48 aﬀorded the ﬁnal glycoside 3 (70%). H NMR

References and Notes

(DMSO) d 8.4 (2H, m, H-1, H-4), 7.8 (2H, m, H-2, H-

3), 5.5 (1H, s, OH-9), 5.4 (1H, d, OH-4⁰⁰), 5.2 (1H, s, H-

1⁰), 5.0 (1H, br s, H-7), 5.0 (1H, s, H-1⁰⁰), 4.9 (1H, s, H-

1⁰⁰), 4.8 (1H, t, OH-14), 4.6 (2H, d, H-14), 4.2 (1H, q, H-

5⁰⁰), 3.8 (1H, m, H-5⁰), 3.6 (1H, s, H-3⁰), 3.5 (2H, d, H-4⁰,

H-4⁰⁰), 3.0 (2H, m, H-10), 2.2 (2H, m, H-8), 1.8–1.4 (6H,

m, H-2⁰, H-2⁰⁰, H-3⁰), 1.3–1.2 (6H, m, H-6⁰, H-6⁰⁰).

ESMS m/z calcd for C₃₂H₃₇NO₁₂(M+H)+ 628.6,

found 628.7.

1. Wang, J. C. Annu. Rev. Biochem. 1996, 65, 635.

2. Capranico, G.; Binaschi, M.; Borgnetto, M. E.; Zunino, F.;

Palumbo, M. Trends Pharmacol. Sci. 1997, 18, 323.

3. Gatto, B.; Capranico, G.; Palumbo, M. Curr. Pharm. Des

1999, 5, 195.

4. Capranico, G.; Zunino, F. Eur. J. Cancer 1992, 28A, 2055.

5. Pommier, Y.; Schwartz, R. E.; Kohn, K. W.; Zwelling,

L. A. Biochemistry 1984, 23, 3194.

6. Wang, A. H. J.; Ughetto, G.; Quigley, G. J.; Rich, A. Bio-

chemistry 1987, 26, 1152.

7. Moore, M. H.; Hunter, W. N.; Langlois d’Estanoit, B.;

Kennard, O. J. Mol. Biol. 1989, 206, 693.

8. Capranico, G.; Supino, R.; Binaschi, M.; Capolongo, L.;

Grandi, M.; Suarato, A.; Zunino, F. Mol. Pharmacol. 1994,

45, 908.

7-[4-O-(2,3,6-trideoxy-3-amino-ꢀ-^L-arabino-hexopyrano-

syl) - 2,3,6 - trideoxy - ꢀ - L - lyxo-hexopyranosyl]-4-

demethoxyadriamicinone (4). From the protected glyco-

side 49 following the general procedure E the glycoside

1

4 was obtained in 65% yield. H NMR (DMSO) d 8.4

(2H, m, H-1, H-4), 7.8 (2H, m, H-2, H-3), 5.9 (1H, d,

OH-4⁰⁰), 5.6 (1H, s, OH-9), 5.3 (1H, s, H-1⁰), 5.0 (1H, s,

H-7), 5.0 (1H, s, H-1⁰⁰), 4.9 (1H, s, H-1⁰⁰), 4.8 (1H, t,

OH-14), 4.5 (2H, d, H-14), 4.1 (1H, q, H-5⁰), 3.7 (1H, m,

H-5⁰⁰), 3.5 (1H, s, H-4⁰), 3.2 (1H, m, H-3⁰⁰), 3.1–3.0 (2H,

m, H-10), 2.2–2.0 (3H, m, H-2⁰ax, H-8), 1.8–1.5 (5H, m,

H-2⁰eq, H-2⁰⁰, H-3⁰), 1.3–1.2 (6H, m, H-6⁰, H-6⁰⁰). ESMS

m/z calcd for C₃₂H₃₇NO₁₂(M+H)+ 628.2, found

628.1.

9. Capranico, G.; Butelli, E.; Zunino, F. Cancer Res. 1995, 55,

312.

10. Binaschi, M.; Capranico, G.; Dal Bo, L.; Zunino, F. Mol.

Pharmacol. 1997, 51, 1053.

11. Animati, F.; Arcamone, F.; Berettoni, M.; Cipollone, A.;

Franciotti, M.; Lombardi, P. J. Chem. Soc., Perkin Trans.

1996, 1, 1327.

12. Arcamone, F.; Animati, F.; Bigioni, M.; Capranico, G.;

Caserini, C.; Cipollone, A.; De Cesare, M.; Ettorre, A.;

Guano, F.; Manzini, S.; Monteagudo, E.; Pratesi, G.; Salva-

tore, C.; Supino, R.; Zunino, F. Biochem. Pharmacol. 1999, 57,

1133.

Cytotoxicity assays

13. Pratesi, G.; De Cesare, M.; Caserini, C.; Perego, P.; Dal

Bo, L.; Polizzi, D.; Supino, R.; Bigioni, M.; Manzini, S.; Iaf-

rate, E.; Salvatore, C.; Casazza, A. M.; Arcamone, F.; Zunino,

F. Clin. Cancer Res. 1998, 4, 2833.

14. Arcamone, F.; Animati, F.; Berettoni, M.; Bigioni, M.;

Capranico, G.; Casazza, A. M.; Caserini, C.; Cipollone, A.;

De Cesare, M.; Franciottti, M.; Lombardi, P.; Madami, A.;

Manzini, S.; Monteagudo, E.; Polizzi, D.; Pratesi, G.; Righ-

etti, S. C.; Salvatore, C.; Supino, R.; Zunino, F. J. Natl. Can-

cer Inst. 1997, 89, 1217.

15. Nicolaou, K. C.; Seitz, S. P.; Papahatjis, D. P. J. Am.

Chem. Soc. 1983, 105, 2430.

16. Veeneman, G. H.; van Boom, J. H. Tetrahedron Lett.

1990, 31, 275.

17. Smid, P.; de Ruiter, G. A.; van der Marel, G. A.; Romb-

outs, F. M.; van Boom, J. H. J. Carbohydr. Chem. 1991, 10,

833.

All cell lines were maintained in RPMI 1640 (Gibco/

BRL, Gaithersburg, MD) supplemented with 10% fetal

calf serum, 2 mM glutamine, 100 units penicillin and

100 mg streptomycin at 37 ^ꢁC in a 5% CO₂, 95% air

humidiﬁed incubator. After 24 h of incubation in the

medium, cells were exposed to drugs. After 24 h, cells

were washed with saline and incubated in drug-free med-

ium for about three doubling times (72 h). Cellular viabi-

lity was then measured by Sulforhodamine B (SRB)

assay.³⁵The IC₅₀(the concentration achieving 50% cel-

lular mortality compared to untreated control) was

evaluated by a curve in which the surviving percentage

of cells was reported as a function of the drug con-

centration. The numbers in Table 1 are the means Æ SE

of three independent determinations.

1470

A. Cipollone et al. /Bioorg. Med. Chem. 10 (2002) 1459–1470

18. Thiem, J.; Klaﬀke, W. J. Org. Chem. 1989, 54, 2006.

19. Classon, B.; Garegg, P. J.; Samuelsson, B. Acta Chem.

Scand. B38 1984, 419.

20. Merzouk, A.; Guibe, F.; Loﬀet, A. Tetrahedron Lett.

1992, 33, 477.

27. Reginato, G.; Ricci, A.; Roelens, S.; Scapecchi, S. J. Org.

Chem. 1990, 55, 5132.

28. Lemieux, R. U.; Morgan, A. R. Can. J. Chem. 1965, 43,

2190.

29. Craine, L.; Raban, M. Chem. Rev. 1989, 89, 689.

30. Obtained from hydrolysis of 3-N-allyloxycarbonyl dauno-

mycin

21. Pelyvas, I.; Sztarickai, F.; Bognar, R. Carbohydr. Res.

1979, 76, 257.

22. Pelyvas, I.; Hasegawa, A.; Wisthler, R. L. Carbohydr. Res.

1986, 146, 193.

31. Bhate, P.; Horton, D.; Priebe, W. Carbohydr. Res. 1985,

´

144, 331.

23. Nambiar, S.; Daeuble, J. F.; Doyle, R. J.; Taylor, K. G.

Tetrahedron Lett. 1989, 30, 2179.

24. Bolitt, V.; Mioskowski, C.; Lee, S.-G.; Falck, J. R. J. Org.

Chem. 1990, 55, 5812.

25. Roth, W.; Pigman, W. Methods Carbohydr 1963, 2,

405.

26. Monneret, C.; Gagnet, R.; Florent, J.-C. J. Carbohydr.

Chem. 1987, 6, 221.

32. Mitsunobu, O. Synthesis 1981, 1.

33. Kimura, Y.; Suzuki, M.; Matsumoto, T.; Abe, R.; Tera-

shima, S. Bull. Chem. Soc. Jpn. 1986, 59, 423.

34. Perrin, D. D.; Armarego, W. L. F. Puriﬁcation of

Laboratory Chemicals, 3rd ed.; Pergamon: Oxford, 1988.

35. Skehan, P.; Storeng, R.; Scudiero, D. A.; Monks, A.;

McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Ken-

ney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107.

Article Doi

Novel anthracycline oligosaccharides: Influence of chemical modifications of the carbohydrate moiety on biological activity

DOI: 10.1016/S0968-0896(01)00411-4

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