Synthesis of chloramphenicol and mandelonitrile galactose-containing prodrugs

This article was downloaded by: [Australian Catholic University]

On: 01 October 2014, At: 00:32

Publisher: Taylor & Francis

Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,

37-41 Mortimer Street, London W1T 3JH, UK

Journal of Carbohydrate Chemistry

Publication details, including instructions for authors and subscription information:

http://ww w .tandfonline.com/loi/lcar20

SYNTHESIS OF CHLORAMPHENICOL AND

MANDELONITRILE GALACTOSE-CONTAINING PRODRUGS

Yves L. Janin ^a, Grégory Zoltobroda ^a, Christiane Huel ^b& Claude Monneret ^a

^aUMR 176 , CNRS-Institut Curie , 26 rue d'Ulm, Paris, Cedex 05, 75248, France

^bU 350 , INSERM-Institut Curie , Bat. 110 Campus Universitaire, Orsay, F-91405, France

Published online: 16 Aug 2006.

To cite this article: Yves L. Janin , Grégory Zoltobroda , Christiane Huel & Claude Monneret (2002) SYNTHESIS OF

CHLORAMPHENICOL AND MANDELONITRILE GALACTOSE-CONTAINING PRODRUGS, Journal of Carbohydrate Chemistry, 21:4,

275-286, DOI: 10.1081/CAR-120013493

To link to this article: http://dx.doi.org/10.1081/CAR-120013493

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained

in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no

representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the

Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and

are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and

should be independently verified with primary sources of information. Taylor and Francis shall not be liable for

any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever

or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of

the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematic

reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any

form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://

www.tandfonline.com/page/terms-and-conditions

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

JOURNAL OF CARBOHYDRATE CHEMISTRY

Vol. 21, No. 4, pp. 275–286, 2002

SYNTHESIS OF CHLORAMPHENICOL

AND MANDELONITRILE

GALACTOSE-CONTAINING PRODRUGS

1

2

Yves L. Janin,^1,Gre´gory Zoltobroda, Christiane Huel,

*

and Claude Monneret¹

¹UMR 176, CNRS-Institut Curie, 26 rue d’Ulm, 75248 Paris,

Cedex 05, France

²U 350, INSERM-Institut Curie, Bat. 110 Campus Universitaire,

F-91405 Orsay, France

ABSTRACT

The synthesis of O¹-b-D-galactopyranosylchloramphenicol and O¹-b-D-

galactopyranosylmandelonitrile as prodrugs potentially substrates of b-

galactosidase, are reported. Preparation of O¹-(2,3,4,6-tetra-O-acetyl-b-D-

galactopyranosyl) chloramphenicol from unprotected chloramphenicol was

successful using b-^D-galactopyranose pentaacetate and boron trifluoride di-

ethyl etherate in acetonitrile. However, the b-galactosylated diastereoisomers

of racemic mandelonitrile had to be made via O¹-(2,3,4,6-tetra-O-acetyl-b-D-

galactopyranosyl)mandelamide in dichloromethane prior to dehydration to

obtain the nitrile moiety. Indeed, galactosylation trials starting directly from

mandelonitrile in acetonitrile led to the O¹-(2,3,4,6-tetra-O-acetyl-a-D-galac-

topyranosyl)mandelonitrile diastereoisomers. From a methodological point of

view, this work extends the use of the galactosylation method to new hy-

droxyl bearing compounds. It also points out that the solvent used

(acetonitrile or dichloromethane) and the purity of boron trifluoride diethyl

etherate can be crucial factors in the use of this method as an eventual

*

Corresponding author. E-mail: yves.janin@curie.u-psud.fr

275

DOI: 10.1081/CAR-120013493

Copyright D 2002 by Marcel Dekker, Inc.

0732-8303 (Print); 1532-2327 (Online)

www.dekker.com

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

276

JANIN ET AL.

alternative to heavy metal-based Lewis acids usually employed in glyco-

sylation reactions.

Key Words: Glycosylation; Galactosepentacetate; Prodrug; Mandelonitrile;

Chloramphenicol

INTRODUCTION

The antibiotic chloramphenicol and the cyanide-generating mandelonitrile are

both compounds displaying biological activity that can be (temporarily) blocked by a

chemical transformation. Indeed, choramphenicol palmitate (1) has been developed^[1]

as an inert prodrug which is selectively hydrolysed by lipases thus releasing the

antibacterial drug. Naturally occurring glycosylated derivatives of mandelonitrile, such

as the gentobioside derivative amygdalin (2), are selectively hydrolysed by b-glu-

cosidase, thus leading to the release of toxic cyanide.^[2–4]Our current interest in drug

targeting led us to elaborate prodrugs which would be specifically released by the

action of b-galactosidase. Many successful examples of such prodrugs^[5–9]have been

reported for their potential application in selective chemotherapy, such as ADEPT^[10,11]

or GDEPT^[12](Antibody or Gene Directed Enzyme Prodrug Therapy). Thus,

chloramphenicol or mandelonitrile b-galactosides 3 and 4 seemed good candidates

for inactivation via galactosylation, since compounds 1 and 2 have proved to be pro-

drugs specifically activated by lipases or b-glucosidase, respectively (Figure 1).

RESULTS AND DISCUSSION

The synthesis of prodrug 3 was achieved via the selective galactosylation of

chloramphenicol 5, using b-galactopyranose pentaacetate in the presence of boron

trifluoride diethyl etherate in dry acetonitrile, to form the b-anomer 6 in 56% yield.

The a-galactopyranose pentaacetate which is unreactive^[13,14]toward these glycosyla-

tion conditions was also isolated. The regioselectivity of this galactosylation reaction

1

was ascertained by NMR spectroscopy notably from the chloramphenicol methylene H

signals shift from 3.40 and 3.61 ppm to 3.54 and 4.01 ppm, respectively. Removal of

NO₂

HO

NC

HO

NC

Cl

NH

O

NH Cl

O

Galactose

β−

β−Gentobiose

O O

Palmitate O O

Cl

Galactose

β−

1

2

3

4

Figure 1.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

GALACTOSE-CONTAINING PRODRUGS

277

NO₂

HO

O

ii

HO

i

NH Cl

O

O O

OAc

Cl

O O

OH

Cl

AcO

HO

O

Cl

HO

AcO

HO

5

OAc

OH

6

3

Scheme 1. i: b-D-Galactopyranose pentaacetate, BF₃ÁOEt₂, MeCN. ii: NH₃/MeOH.

the acetyl groups was then achieved using methanolic ammonia to give the galactoside

3 (Scheme 1).

Synthesis of compound 4 was far less straightforward. The reported preparation

of mandelonitrile b-glucoside or b-glucuronide requires glycosylation of mandelamide

7 using tetraacetylglycopyranoside bromide and a mercuric salt.^[15]In order to avoid

the use of mercury, we focused on the galactosylation of mandelamide (7) using b-

galactopyranose pentaacetate and boron trifluoride diethyl etherate complex.

Our first unsuccessful attempts led us to try the known b-galactosylation of

benzyl alcohol. At first, using boron trifluoride diethyl etherate complex in dichlo-

romethane, we were unable to reproduce the 72% yield previously reported.^[16]As

judged by TLC, the only observed result was an extensive decomposition. However, the

b-galactosylated benzyl ether was prepared by simply changing the reaction solvent

from dichloromethane to acetonitrile. Eventually, we found out that the purity of the

boron trifluoride etherate was essential for this reaction. It was only by boiling boron

trifluoride diethyl etherate and a small amount of diethyl ether over calcium hydride

overnight, prior to its distillation, that we were able to obtain a Lewis acid pure enough

for the reaction to proceed in dichloromethane (distilled over calcium hydride). On the

other hand, if acetonitrile was used as solvent, the use of a boron trifluoride diethyl

etherate of any reasonable purity or age led to an acceptable yield (55%) of b-

galactosylated benzyl alcohol.

Dichloromethane is the solvent commonly used in this very simple galactosyla-

tion method which was first reported to give, from trichloroethyl alcohol, either the b-

anomer^[17]or, with a longer reaction time, the a-anomer primarily.^[18]A later study,^[13]

with b-glucopyranose pentaacetate, illustrates some fascinating kinetic features of this

method which seem highly dependent on the nature of the alcohol studied. Good b-

glycosylation results have been obtained with different kinds of alcohols such as serine

or threonine derivatives,^[19–23]and even hydroxyproline.^[24]Many other b-functiona-

lised aliphatic alcohols can react^[25–31]as well as benzyl,^[16]allyl,^[32–34]propargyl^[35,36]

or some other lipophilic alcohols.^[37–39]The use of acetonitrile as solvent has been

reported.^[40]In some instances it was beneficial to the reaction outcome,^[19]in another

instance it was not beneficial.^[22]Operating procedures usually favour the use of an excess

or of an equivalent of boron trifluoride diethyl etherate. However, a remarkable 2-

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

278

JANIN ET AL.

a

CONH₂

HO

H₂NOC

O

H₂NOC

O

i

O

AcO

or ii

OAc

iii

Mandelamide (7)

Ca = R : 9a

Ca = S : 9b

8a,b

a

NC

O

NC

O

CN

HO

O

i

AcO

OAc

Ca = S : 12a

Ca = R : 12b

OAc

Mandelonitrile (11)

Ca = R : 10a

Ca = S : 10b

Scheme 2. i: b-D-Galactopyranose pentaacetate, BF₃ÁOEt₂, MeCN. ii: b-D-Galactopyranose

pentaacetate, pure BF₃ÁOEt₂, CH₂Cl₂. iii: (CF₃CO)₂O, pyridine, CH₂Cl₂.

deacetylation effect was reported^[41]when an excess of allyl alcohol was present. In that

case, the reaction led to the allyl 3,4,6-tri-O-acetyl-b-^D-galactopyranoside in 62% yield.

In our cases, the reaction was carried out with an excess of the boron trifluoride

diethyl etherate complex and we cannot over-emphasize the need for thorough

purification of the Lewis acid, if dichloromethane is used. Acetonitrile and boron

trifluoride may form a complex which allows the galactosylation reaction to proceed.

However, at least one example of a chemical reaction dependent on the impurities

content of the boron trifluoride diethyl etherate complex used has been reported.^[42]

Thus such impurities, which seem in our case to be detrimental to the galactosylation

reaction in dichloromethane, are actually ‘‘neutralized’’ in acetonitrile.

In this context, we proceeded to try the galactosylation of mandelamide^[43]7. In

acetonitrile, using commercial boron trifluoride diethyl etherate, the reaction (i on

Scheme 2) led to a complex mixture. From the ¹H NMR analysis of the partially

purified material, the presence of the four possible diastereoisomers 8a, 8b, 9a and 9b

was deduced, with predominance of the a-galactosides 8a and 8b. On the other hand,

the galactosylation reaction (ii on Scheme 2) led mainly to the two b-diastereoisomers

9a and 9b in dichloromethane only if properly purified boron trifluoride diethyl

etherate (as described above) was used. Diastereoisomer 9a could be isolated via

chromatography followed by recrystallization from 2-propanol in 33% yield whereas,

despite repeated purification attempts, the oily diastereoisomer 9b remained conta-

minated by small amounts of a-diastereoisomers 8a and 8b.

1

Each H NMR spectra of the two b-anomers 9a and 9b displays a characteristic

anomeric proton as doublets at 4.28 ppm or 4.62 ppm, with coupling constants of 7.9

Hz or 7.8 Hz, respectively. Traces of the a-anomers were also detectable in the reaction

mixture mainly by their acetyl signals. The S-enriched mandelamide was prepared from

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

GALACTOSE-CONTAINING PRODRUGS

279

optically pure (S)-(+)-mandelic acid using a classical procedure^[43]followed by re-

crystallization from toluene (ee^[44]=69%). Starting from this mandelamide, a large

excess of compound 9b was observed in the ¹H NMR spectra of the unchromato-

graphed reaction mixture. This allowed us to assign the absolute configuration for all

the b-configured derivatives, i.e. for 9a and 9b and, as described below, for compounds

10a and 10b.

A dehydration reaction^[15]using pyridine and trifluoroacetic anhydride (iii on

Scheme 2) was conducted on amide 9a and on the fraction containing mostly 9b. This

enabled us to obtain the pure b-anomers 10a and, from the fraction containing 9b, the

other b-diasteroisomer 10b along with a small amount of the a-anomer 12b.

Attempts to galactosylate mandelonitrile (11) with b-^D-galactopyranose penta-

acetate in acetonitrile (i on Scheme 2) led almost exclusively to the two a-di-

astereoisomers 12a and 12b. Diastereoisomer 12a was isolated in 26% yield by re-

crystallization whereas the oily 12b could only be partially purified by chromatography

and still contained a small amount of 10a and 10b. Starting from (R)-( +)-enriched

mandelonitrile (Aldrich, ee (in our hands) = 44%) the reaction led to a 2:3 mixture of

1

compound 12a:12b (as measured by their respective H NMR signals integration ratio),

thus allowing assignment of absolute configurations of compounds 12a and 12b.

Direct galactosylation of mandelonitrile in dichloromethane was unsuccesful,

possibly because of nitrile chelation by boron trifluoride rendering its hydroxyl function

unreactive. This seems reasonable as a similar lack of glycosylation was reported in

trials using acetobromoglucose, mandelonitrile and mercuric salts.^[15]

Deacetylation was conducted on b-anomer 10a. Surprisingly, the use of meth-

anolic ammonia led to partial amminolysis of the nitrile moiety along with racemization

of the cyanohydrin asymmetric centre. The use of sodium methoxide in methanol was

more efficient, but also led to the racemized target compound 4 as a hygroscopic syrup.

CONCLUSION

This work illustrates some additional results on the known use of boron

trifluoride etherate as an alternative to heavy metal-based Lewis acids employed in

glycosylation reactions. Preparation of the b-galactosyl-containing prodrug 3 via direct

galactosylation of the primary hydroxyl function of chloramphenicol turned out to be

very easy. On the other hand, our studies toward the preparation of compound 4 led to

quite a lot more work. Indeed the galactosylation of mandelonitrile trials in dichloro-

methane and acetonitrile illustrates the subtlety of this reaction. In this work we

observed the importance of employing an appropriate solvent (dichloromethane versus

acetonitrile) and a pure catalyst (boron trifluoride), factors that one should be aware of

in the course of trials carried out with this method. The potential use of prodrugs 3 and

4 in biology will be reported elsewhere.

EXPERIMENTAL

General methods. ¹H and ¹³C NMR spectra were recorded on a Bruker AC-

200 or Varian multi-400 or 500 spectrometers. Shifts are given in ppm with respect to

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

280

JANIN ET AL.

the TMS signal and coupling constants (J) are given in Hertz. Signals assignment was

often confirmed by two dimensional NMR experiments (COSY, NOESY, HMQC,

HMBCR). Low and high resolution mass spectra were obtained by Mrs Nicole Morin

(ENS, 24 rue Lhomond F-75231 Paris) on a MS 700 Jeol and ammonia-based chemical

ionization was used. Column chromatography was performed on Merck silica gel 60

˚

(0.035–0.070 mm). Solvents were usually dried using activated 3 A or 4 A molecular

sieves. Activation of the molecular sieves was done by using a plastic-free domestic

microwave oven (irradiation in a quartz beaker of 100–200 g of new molecular sieves

until partial melting, i.e. from 1 to 8 min by periods of 1 min alternated with cooling).

CAUTION: due to residual traces of solvents, microwave irradiation of molecular

sieves previously used can result in a serious explosion.

O¹-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)chloramphenicol (6). b-D-

galactopyranose pentaacetate (2.5 g, 6.4 mmol) and chloramphenicol (12.4 g, 38.3

mmol) were dissolved in dry acetonitrile (300 mL). Boron trifluoride diethyl etherate

(8 mL, 63.1 mmol) was added, and the solution was stirred under an inert atmosphere

for 15 min. The solution was poured into water (500 mL), the organic materials were

extracted with dichloromethane and the organic phase was dried over magnesium

sulfate and concentrated to dryness. The residue was chromatographed over silica

eluting with a mixture of heptane-ethyl acetate 4:5 yielding compound 6 as a hard

1

foam (2.34 g, 56%). mp 85–89°C (dec.). H NMR ((CD₃)₂SO, 400 MHz): 1.96, 2.04,

2.05, 2.16 (4s, 12 H, CH₃); 3.54 (dd, 1 H, J =7.3 and 10, CH-1); 4.01 (dd, 1 H, J =6

and 10, CH-1); 4.1 (m, 2 H, GalH-6); 4.11 (m, 1 H, CH-2); 4.25 (t, 1 H, J =6.5,

GalH-5); 4.82 (d, 1 H, J= 8.0, GalH-1); 4.92-5.05 (m, 2 H, GalH-2 and CH-3); 5.19

(dd, 1 H, J= 3.2 and 10.4, GalH-3); 5.31 (d, 1 H, J=3.6, GalH-4); 6.21 (d, 1 H,

J=4.5 OH-3); 6.44 (s, 1 H, CHCl₂); 7.61 (d, 2 H, J=5.8, CHAr-2); 8.22 (d, 2 H,

J=5.8, CHAr-3); 8.54 (d, 1 H, J= 8.8, NH). ¹³C NMR ((CD₃)₂SO, 100 MHz): 20.0

(CH₃); 54.6 (CH-2); 61.3 (GalC-6); 66.4 (CHCl₂); 67.4 (GalC-4); 67.7 (CH₂-1); 68.6

(GalC-2); 69.5 (CH-3); 70.0 (GalC-5); 70.2 (GalC-3); 100 (GalC-1); 123.0 (Ar-2);

127.4 (Ar-3); 146.6 (Ar-4); 150.4 (Ar-1); 163.4 (CONH); 169.2, 169.5, 169.9 (COCH₃).

m/z (MH + NH₃)=670. HRMS: Calcd for C₂₅H₃₄N₃O₁₄³⁵Cl₂: [M ⁺+NH₄], 670.1417.

Found: m/z, 670.1422.

O¹-b-D-Galactopyranosylchloramphenicol (3). Compound 6 was treated with

methanol saturated with ammonia (50 mL) in a sealed flask for 48 h at room tem-

1

perature. The solution was concentrated to dryness to give compound 3 as a syrup. H

NMR ((CD₃)₂SO, 400 MHz): 3.37 (m, 1 H, CH-S3); 3.40 (m, 1 H, GalH-2); 3.44 (m,

1 H, GalH-5); 3.45 (m, 1 H, CH-1); 3.56 (m, 2 H, GalH-6); 3.70 (s (br), 1 H, GalH-4);

3.93 (t (br), 1 H, J= 9.0, CH-1); 4.13 (s(br), 1 H, CH-2); 4.22 (d, 1 H, J= 7.2, GalH-1);

5.22 (s, 1 H, CH-3); 6.51 (s, 1 H, CHCl₂); 6.21, 7.33 (2s (br), 2 H, 2 OH); 7.69 (d, 2 H,

J=8.7, CHAr-2); 8.19 (d, 2 H, J= 8.7, CHAr-3); 8.50 (m, 1 H, NH). ¹³C NMR

((CD₃)₂SO, 100 MHz): 51.5 (CH-2); 57.5 (GalC-6); 63.6 (CHCl₂); 64.2 (CH₂-1); 65.3

(GalC-4); 66.1 (CH-3); 67.5 (GalC-5); 70.5 (GalC-3); 72.5 (GalC-2); 100.9 (GalC-1);

120.0 (Ar-2); 124 (Ar-3); 143.7 (Ar-4); 148.2 (Ar-1); 160.6 (CONH). m/z (MH +

NH₃)=502. HRMS: Calcd for C₁₇H₂₆N₃O₁₀³⁵Cl₂: [M ⁺+ NH₄], 502.0995. Found: m/z,

502.1007.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

GALACTOSE-CONTAINING PRODRUGS

281

Preparation of compounds 9a and 9b. Mandelamide (6 g, 35.2 mmol) and b-D-

galactopyranose pentaacetate (5.2 g, 13.3 mmol) were dispersed in dry dichloromethane

(150 mL, distilled over calcium hydride) and the suspension was cooled to 0°C under an

inert atmosphere. Very pure boron trifluoride diethyl etherate (11.8 mL, 93.1 mmol,

refluxed with a small amount of ether over calcium hydride for 24 h prior to a dis-

tillation) was added and the solution was stirred overnight, allowing the temperature to

rise back to 25°C. The solution was then poured into water and extracted with di-

chloromethane. The organic layer was cautiously washed with water, saturated sodium

hydrogenocarbonate, water, then dried over magnesium sulfate and concentrated to dry-

ness. The residue was chromatographed over silica gel eluting with a mixture of heptane-

ethyl acetate with proportions gradually varying from 1:2 to 1:4. The fractions containing

compounds 9a and 9b were recrystallized from 2-propanol yielding 9a (2.17 g, 33%).

The filtrate was concentrated to dryness and chromatographed over silica gel (eluting

with heptane-ethyl acetate 1:4) to yield 9b as a syrup (1.1 g, still containing small

amounts of a-diastereoisomers).

O¹-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-(R)-mandelamide (9a). mp

1

158°C. H NMR (CDCl₃, 400 MHz): 1.95, 2.00, 2.05, 2.15 (4s, 12 H, CH₃); 3.77 (t, 1

H, J= 7.0, CH-5); 4.14 (m, 2 H, GalH-6); 4.28 (d, J= 7.9, GalH-1); 4.91 (dd, 1 H,

J= 3.4 and 10.5, GalH-3); 5.15 (s, 1 H, HCCONH₂); 5.25 (dd, 1 H, J= 7.8 and 10.5,

GalH-2); 5.33 (m, 1 H, GalH-4); 5.53 and 6.81 (dependent on sample’s concentration)

(2s (br), 2 H, NH₂); 7.28–7.38 (m, 5 H, Ar). ¹³C NMR (CDCl₃, 100 MHz): 20.5, 20.6,

20.8 (CH₃); 61.2 (GalC-6); 66.9, 69.0, 70.2, 71.0 (GalC-3, GalC-2, GalC-4, GalC-5);

79.3 (CHCONH₂); 97.9 (GalC-1); 127.7 (CH-Ar); 128.8 (CH-Ar); 129.2 (CH-Ar);

135.1 (C-Ar); 170.0, 172.1 (CO and CONH₂).

Anal. Calcd for C₂₂H₂₇NO₁₁(481.46): C, 54.88; H, 5.65; N, 2.91; O, 36.55.

Found: C, 54.75; H, 5.63; N, 2.84; O, 36.56.

O¹-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-(S)-mandelamide (9b). ¹H

NMR (CDCl₃, 400 MHz): 1.95, 1.97, 2.06, 2.16 (4s, 12 H, CH₃); 3.81 (m, 1 H,

GalH-5); 3.94 (m, 2 H, GalH-6); 4.62 (d, J =7.8, GalH-1); 5.00 (dd, 1 H, J=3.5 and 10.5,

GalH-3); 5.08 (s, 1 H, HCCONH₂); 5.30 (m, 2 H, GalH-2 and GalH-4); 5.46 and 6.50

(dependent on sample’s concentration), (2s (br), 2 H, NH₂) ; 7.30–7.42 (m, 5 H, Ar). ¹³

C

NMR (CDCl₃, 100 MHz): 20.6 (CH₃); 61.1 (GalC-6); 66.9, 69.3, 70.2, 70.9 (GalC-3,

GalC-2, GalC-4, GalC-5); 81.1 (CHCONH₂); 100.8 (GalC-1); 127.1 (CH-Ar); 128.4

(CH-Ar); 128.8 (CH-Ar); 136.2 (C-Ar); 170.2, 172.6 (CO and CONH₂). m/z

(MH +NH₃)= 499. HRMS: Calcd for C₂₂H₃₁N₂O₁₁: [M ⁺+NH₄], 499.1927. Found:

m/z, 499.1924.

Preparation of compound 10a and 10b. The crystalline amide 9a (1.33 g, 8.8

mmol) was dissolved in dry dichloromethane (200 mL, distilled over calcium hydride).

˚

Pyridine (1.46 mL, 18.0 mmol, dried over 4 A molecular sieves) and trifluoroacetic

anhydride (1.27 mL, 9.0 mmol) were added. The solution was stirred under an inert

atmosphere for 20 min. If TLC monitoring showed that the conversion was not com-

pleted, another portion of pyridine and trifluoroacetic anhydride were added. The

organic solution was then poured into water, extracted with dichloromethane, washed

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

282

JANIN ET AL.

with water, dried over magnesium sulfate and concentrated to dryness. The residue was

chromatographed over silica gel eluting with a 2:1 mixture of heptane and ethyl acetate,

to provide compound 10a as a hard foam (0.81 g, 63%). Using the same procedure,

starting from 0.4 g of the fraction containing amide 9b (still containing some a-

diastereoisomers), 0.3 g of compound 10b was obtained as an oil, along with small

amount of 12b.

O¹-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-(R)-mandelonitrile (10a).

mp 130°C. ¹H NMR (CDCl₃, 400 MHz): 1.95, 1.97, 2.05, 2.10 (4s, 12 H, CH₃);

3.85 (t, 1 H, J=6.7, GalH-5); 4.13 (m, 2 H, GalH-6); 4.47 (d, J= 8.0, GalH-1); 4.93

(dd, 1 H, J =3.4 and 10.0, GalH-3); 5.26 (dd, 1 H, J= 8.0 and 10.0, GalH-2); 5.35 (d,

1 H, J=3.4, GalH-4); 5.49 (s, 1 H, CHCN); 7.43 (m, 5 H, Ar). ¹³C NMR (CDCl₃,

100 MHz): 20.4 (CH₃); 61.0, 66.6, 68.2, 68.6, 70.5, 71.1 (GalC-6, GalC-4, GalC-3,

GalC-2, GalC-5, CHCN); 99.0 (GalC-1); 116.6 (CN); 127.5 (CH-Ar); 129.1 (CH-Ar);

130.2 (CH-Ar); 132.1 (C-Ar); 169.8, 169.9, 170.0, 170.2 (CO). Anal. Calcd for

C₂₂H₂₅NO₁₀(463.44): C, 57.02; H, 5.44; N, 3.02; O, 34.52. Found: C, 57.05; H, 5.41;

N, 2.89; O, 34.41.

O¹-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-(S)-mandelonitrile (10b).

¹H NMR (CDCl₃, 400 MHz): 1.97, 2.05, 2.07, 2.12 (4s, 12 H, CH₃); 4.01 (t, 1 H,

J=6.5, GalH-5); 4.17 (m, 2 H, GalH-6); 4.87 (d, J =7.4, GalH-1); 5.07 (dd, 1 H, J =3.4

and 10.2, GalH-3); 5.26 (dd, 1 H, J =7.7 and 10.2, GalH-2); 5.42 (d, 1 H, J= 3.4,

GalH-4); 5.66 (s, 1 H, CHCN); 7.45 (m, 5 H, Ar). ¹³C NMR (CDCl₃, 100 MHz): 20.45

(CH₃); 61.1, 66.7, 67.9, 68.1, 70.5, 71.3 (GalC-6, GalC-4, GalC-3, GalC-2, GalC-5,

CHCN); 98.7 (GalC-1); 116.3 (CN); 127.0 (CH-Ar); 128.9 (CH-Ar); 129.9 (CH-Ar);

132.0 (C-Ar); 169.2, 169.8, 169.9, 170.2 (CO). m/z (MH +NH₃) =481. HRMS: Calcd

for C₂₂H₂₉N₂O₁₀: [M ⁺+NH₄], 481.1822. Found: m/z, 481.1826.

Preparation of compound 12a and 12b. Mandelonitrile 85–90% (3.8 mL,

27.1 mmol) and b-^D-galactopyranose pentaacetate (3 g, 7.6 mmol) were dispersed in

dry acetonitrile (200 mL). Commercial boron trifluoride diethyl etherate (9.8 mL, 77.3

mmol) was added and the solution was stirred at 60°C for 30 min. The solution was

then cautiously poured into a solution of sodium hydrogenocarbonate (25 g, 0.3 mol).

The aqueous phase was extracted with dichloromethane, the organic layers were

washed with water, dried over magnesium sulfate, then concentrated to dryness. The

residue was chromatographed over silica gel, eluting with a mixture of heptane-ethyl

acetate 3:2. The fraction containing compounds 12a and 12b (and to a much smaller

extent isomer 10b) were recrystallized from 2-propanol, yielding pure 12a (0.92 g,

26%). The resulting filtrate was concentrated to dryness and chromatographed again

over silica gel (eluting with heptane ethyl acetate 2/1) to give 12b as a syrup (0.8 g,

still containing a small proportion of 12a).

O¹-(2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl)-(S)-mandelonitrile

1

(12a). mp 193°C. H NMR (CDCl₃, 400 MHz): 1.95, 1.97, 2.06, 2.12 (4s, 12 H,

CH₃); 4.08 (dd, 1 H, J=6.5 and 11.8, GalH-6); 4.20 (dd, 1 H, J=6.5 and 11.8, GalH-

6); 4.43 (t, 1 H, J=6.5, GalH-5); 5.09 (dd, 1 H, J= 3.8 and 11.0, GalH-2); 5.29 (d, 1

H, J =3.8, GalH-1); 5.33 (s, 1 H, CHCN); 5.37 (dd, 1 H, J=3.3 and 11, GalH-3);

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

GALACTOSE-CONTAINING PRODRUGS

283

5.51 (d, 1 H, J=3.3, GalH-4); 7.43 (m, 5 H, Ar). ¹³C NMR (DMSOd6, 100 MHz):

20.39, 20.4, 20.46, 20.57 (CH₃); 61.1 (GalC-6); 67.0, 67.57, 67.61, 67.7 (GalC-3,

GalC-2, GalC-4, GalC-5); 68.5 (CHCN); 96.5 (GalC-1); 116.8 (CN); 127.0 (CH-Ar);

129.2 (CH-Ar); 130.3 (CH-Ar); 132.0 (C-Ar); 169.8, 169.9, 173.1, 173.3 (CO).

Anal. Calcd for C₂₂H₂₅NO₁₀(463.44): C, 57.02; H, 5.44; N, 3.02. Found: C,

56.81; H, 5.39; N, 3.15.

O¹-(2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl)-(R)-mandelonitrile (12b).

¹H NMR (CDCl₃, 400 MHz): 1.96, 2.04, 2.11, 2.13 (4s, 12 H, CH₃); 4.08 (m, 2 H,

GalH-6); 4.13 (m, 1 H, GalH-5); 5.23 (m, 2 H, GalH-3 and GalH-2); 5.42 (m, 1 H,

GalH-4); 5.45 (d, J=3.0, GalH-1); 5.50 (s, 1 H, CHCN); 7.46 (m, 5 H, Ar). ¹³C NMR

(CDCl₃, 100 MHz): 20.5 (CH₃); 61.4 (GalC-6); 66.9; 67.0; 67.5 (GalC-2, GalC-3,

GalC-5 and GalC-4); 67.9 (CHCN); 95.1 (GalC-1); 116.4 (CN); 127.6 (CH-Ar); 129.2

(CH-Ar); 130.3 (CH-Ar); 132.0 (C-Ar); 169.8; 169.9; 170.1 (CO). m/z (MH +NH₃) =

481. HRMS: Calcd for C₂₂H₂₉N₂O₁₀: [M ⁺+NH₄], 481.1822. Found: m/z, 481.1819.

O¹-b-D-Galactopyranosylmandelonitrile (4). Compound 10a (0.15 g, 0.3

mmol.) and sodium methanolate (0.04 g, 0.7 mmol) were stirred in dry methanol

˚

(30 mL, dried over 3 A molecular sieves) under an inert atmosphere for 25 min. The

solution was neutralized with Dowex 50, filtered, then concentrated to dryness to

afford compound 4 as a hygroscopic mixture of the two b-diastereoisomers in a 60–

40% proportion. H NMR (CD₃OD, 500 MHz): 3.30 to 3.74 (m, 6 H, GalH-6, GalH-5,

1

GalH-4, GalH-3, GalH-2); 4.26 (d, 2/5 H, J =7.0, GalH-1 minor); 4.52 (d, 3/5 H,

J= 7.0, GalH-1 major); 6.03 (s, 2/5 H, CHCN minor); 6.10 (s, 3/5 H, CHCN major);

7.52 (m, 3 H, CHAr major); 7.60 (m, 2 H, CHAr minor). ¹³C NMR (CD₃OD, 125

MHz): Only small differences of signals were observed between the two diasteromers,

thus hampering full assignment on their spectra: 60.2, 60.5, 66.7, 66.9, 67.8, 68, 70.3,

70.5, 73.0, 73.3, 75.8, 76 (GalC-2, GalC-3, GalC-4, GalC-5, GalC-6 and CHCN); 101

and 102 (GalC-1), 118 and 119 (CN), 127, 129, 129.6 (CHAr), 134 (CAr). m/z

(MH +NH₃)= 313. HRMS: Calcd for C₁₄H₂₁N₂O₆: [M ⁺+ NH₄], 313.1399. Found: m/z,

313.1407.

REFERENCES

1. Edgerton, W.H.; Maddox, V.H.; Controulis, J. The structure of chloramphenicol

palmitate. J. Am. Chem. Soc. 1955, 77, 27–29.

2. Haisman, D.R.; Knight, D.J. The enzyme hydrolysis of amygdalin. Biochem. J.

1967, 103, 528–534.

3. Fenselau, C.; Pallante, S.; Batzinger, R.P.; Benson, W.R.; Barron, W.R.; Sheinin,

E.B.; Maienthal, M. Mandelonitrile b-glucuronide: Synthesis and characterisation.

Science 1977, 198, 625–627.

4. Syrigos, K.N.; Rowlinson-Busza, G.; Epenetos, A.A. In vitro cytotoxicity following

specific activation of amygdalin by b-glucosidase conjugated to a bladder cancer-

associated monoclonal antibody. Int. J. Cancer 1998, 78, 712–719.

5. Abraham, R.; Aman, N.; von Borstel, R.; Darsley, M.; Kamireddy, B.; Kenten, J.;

Morris, G.; Titmas, R. Conjugates of COL-1 monoclonal antibody and b-^D-galac-

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

284

JANIN ET AL.

tosidase can specifically kill tumor cells by generation of 5-fluorouridine from the

prodrug b-^D-galactosyl-5-fluorouridine. Cell Biophys. 1994, 24/25, 127–133.

6. Leenders, R.G.G.; Damen, E.W.P.; Bijsterveld, E.J.A.; Scheeren, H.W.; Houba,

P.H.J.; van der Meulen-Muileman, I.H.; Boven, E.; Haisma, H.J. Novel anthra-

cycline-spacer-b-glucuronide, -b-glucoside, and -b-galactoside prodrugs for applica-

tion in selective chemotherapy. Bioorg. Med. Chem. 1999, 7 (8), 1597–1610.

7. Bakina, E.; Farquhar, D. Intensely cytotoxic anthracycline prodrugs: Galactosides.

Anti-Cancer Drug Des. 1999, 14, 507–515.

8. Bru¨sselbach, S.; Korn, T.; Wo¨lkel, T.; Mu¨ller, R.; Kontermann, R.E. Enzyme and

recruitment and tumor cell killing in vitro by secreted bispecific single-chain

diabody. Tumor Targeting 1999, 4, 115–123.

9. Ghosh, A.K.; Khan, S.; Marini, F.; Nelson, J.A.; Farquhar, D. A daunorubicin b-

galactoside prodrug for use in conjunction with gene-directed enzyme prodrug

therapy. Tetrahedron Lett. 2000, 41 (25), 4871–4874.

10. Bagshawe, K.D. Towards generating cytotoxic agents at cancer sites. Br. J. Cancer

1989, 60, 275–281.

11. Syrigos, K.N.; Epenetos, A.A. Antibody directed enzyme prodrug therapy

(ADEPT): A review of the experimental and clinical considerations. Anticancer

Res. 1999, 19, 605–613.

12. Melton, R.; Connors, T.; Knox, R.J. The use of prodrugs in targeted anticancer

therapies. S.T.P. Pharma Sci. 1999, 9, 13–33.

13. Ellervik, U.; Jansson, K.; Magnusson, G.J. Gas chromatographic investigation of

the boron trifluoride etherate-induced formation and anomerization of glucopyrano-

sides. J. Carbohydr. Chem. 1998, 17 (4–5), 777–784.

14. Veerneman, G.H. Chemical Synthesis of O-Glycosides. In Carbohydrate Chem-

istry; Boons, G.-J., Ed.; Chapman and Hall: New York, 1998; 98–174.

15. Junghans, B. Darstellung des Cyanogen Glycosides Mandelglucuronid [2-(b-^D-

glucopyranuronosyloxy)-2-phenylacetonitrile]. Pharmazie 1982, 37, 172–175.

16. Kobayashi, Y.; Shiozaki, M.; Ando, O. Syntheses of trehazolin derivatives and

evaluation as glycosidase inhibitors. J. Org. Chem. 1995, 60 (8), 2570–2580.

17. Magnusson, G.; Noori, G.; Dahme´n, J.; Frejd, T.; Lave, T. BF₃-etherate induced

formation of 2,2,2-trichloroethyl glycopyranoside. Selective visualization of carbo-

hydrate derivatives on TLC plates. Acta Chem. Scand., Ser. B 1981, 35, 213–216.

18. Risbood, P.A.; Reed, L.A., III; Goodman, L. The preparation and study of some

novel glycosides of ^D-galactose. Carbohydr. Res. 1981, 88 (2), 245–251.

19. Elofsson, M.; Walse, B.; Kihlberg, J. Building blocks for glycopeptide synthesis:

Glycosylation of 3-mercaptopropionic acid and Fmoc amino acids with unprotected

carboxyl groups. Tetrahedron Lett. 1991, 32 (51), 7613–7616.

20. de la Torre, B.G.; Torres, J.L.; Bardaj´ı, E.; Clape´s, P.; Xaus, N.; Jorba, X.; Clavet,

S.; Albericio, F.; Valencia, G. Improved method for the synthesis of O-glycosylated

Fmoc amino acids to be used in solid-phase glycopeptide synthesis. J. Chem. Soc.,

Chem. Commun. 1990, 965–967.

˚

21. Kihlberg, J.; Ahman, J.; Walse, B.; Drakenberg, T.; Nilsson, A.; So¨deberg-Ahlm,

C.; Bengtsson, B.; Olsson, H. Glycosylated peptide hormones: Pharmacological

properties and conformational studies of analogues of [1-desamino, 8-^D-arginin]-

vasopressin. J. Med. Chem. 1995, 38 (1), 161–169.

22. Salvador, L.A.; Elofsson, M.; Kihlberg, J. Preparation of building blocks for

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

GALACTOSE-CONTAINING PRODRUGS

285

glycopeptide synthesis by glycosylation of Fmoc amino acids having unprotected

carboxyl groups. Tetrahedron 1995, 51 (19), 5643–5656.

23. Sjo¨lin, P.; Kihlberg, J. Deacetylation of N-a-methylated glycopeptides reveals that

Aza-enolates provide protection against b-elimination of carbohydrates O-linked to

serine. Tetrahedron Lett. 2000, 41 (22), 4435–4439.

24. Arsequell, G.; Sa`rries, N.; Valencia, G. Synthesis of glycosylated hydroxyproline

building blocks. Tetrahedron Lett. 1995, 36 (40), 7323–7326.

25. Dahme´n, J.; Frejd, T.; Gro¨nberg, G.; Lave, T.; Magnusson, G.; Noori, G. 2-

bromoethyl glycosides: Synthesis and characterisation. Carbohydr. Res. 1983, 116

(2), 303–307.

26. Tang, P.W.; Williams, J.M. The deamination of 1-aminohexan-2-ol and 1-(amino-

methyl)pentyl b-^D-galactopyranoside; a model study of the selective cleavage of the

hydroxylysine-bound glycosyl residues of collagen. J. Chem. Soc., Perkin Trans. 1

1984, 1199–1203.

27. Sasaki, A.; Murahashi, N.; Yamada, H.; Morikaw, A. Syntheses of novel galactosyl

ligands for liposomes and the influence of the spacer on accumulation in the rat

liver. Biol. Pharm. Bull. 1995, 18 (5), 740–746.

28. Chernyak, A.Y.; Sharma, G.V.M.; Kononov, L.O.; Khrisna, P.R.; Levinsky, A.B.;

Kochetkov, N.K.; Roa, A.V.R. 2-azidoethyl glycosides: Glycosides potentially

useful for the preparation of neoglycoconjugates. Carbohydr. Res. 1992, 223,

303–309.

29. Davis, B.G.; Lloyd, R.C.; Jones, J.B. Controlled site-selective glycosylation of

proteins by a combinatorial site-directed mutagenesis and chemical modification

approach. J. Org. Chem. 1998, 63 (26), 9614–9615.

30. Davis, B.G.; Maughan, M.A.T.; Green, M.P.; Ullman, A.; Jones, J.B. Glycometha-

nethiosulfonates: Powerful reagents for protein glycosylation. Tetrahedron: Asym-

metry 2000, 11 (1), 245–262.

31. Suhr, R.; Scheel, O.; Thiem, J. Synthesis of glycosyl glycerols and related

glycolipids. J. Carbohydr. Chem. 1998, 17 (6), 937–968.

32. Lyubeshkin, A.B.; Anikin, M.V.; Gurine, A.V.; Sebyakin, Y.S. An alternative syn-

thesis of 1,2-trans-glycosyl diglycerides. Russ. J. Org. Chem. 1994, 30 (4), 604–609.

33. Mereyela, H.B.; Lingannagaru, S.R. A study of Pd(II)Cl2/CuCl catalysed wacker

reaction for the deprotection of prop-2-enyl and prop-1-enyl ethers. Tetrahedron

1997, 53 (51), 17501–17512.

34. Mandai, T.; Okumoto, H.; Oshitari, T.; Nakanishi, K.; Mikuni, K.; Hara, K.; Hara,

K. A practical synthetic method for a- and b-glycosyloxyacetic acids. Heterocycles

2000, 52 (1), 129–132.

35. Mereyala, H.B.; Gurrala, S.R. A highly diastereoselective, practical synthesis of

allyl, propargyl 2,3,4,6-tetra-O-acetyl-b-^D-gluco-b-D-galactopyranosides and allyl,

propargyl heptaacetyl-b-^D-lactosides. Carbohydr. Res. 1998, 307 (3–4), 351–

354.

36. Mereyela, H.B.; Gurrala, S.R. Design, development and utility of glycosyl donors

bearing and acetoxymethoxy leaving group. Chem. Lett. 1998, 863–864.

37. Ansari, A.A.; Frejd, T.; Magnusson, G. 3-bromo-2-bromomethylpropyl glycosides

in the preparation of double-chain bis-sulfide neo-glycolipids. Carbohydr. Res.

1987, 161 (2), 225–233.

38. Susaki, H.; Suzuki, K.; Ikeda, M.; Yamada, H.; Watanabe, H.K. Synthesis of

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

286

JANIN ET AL.

artificial glycoconjugates of arginine-vasopressin and their antidiuretic activities.

Chem. Pharm. Bull. 1994, 42 (10), 2090–2096.

39. Ye, X.-S.; Wong, C.-H. Anomeric reactivity-based one-pot oligosaccharides syn-

thesis: A rapid route to oligosaccharide libraries. J. Org. Chem. 2000, 65 (8), 2410–

2431.

40. Sivanandaiah, K.M.; Babu, V.V.S.; Shankaramma, S.C. Solid phase synthesis of

O-glycoopioid peptides related to dermorphin. Indian J. Chem. 1998, 37B, 760–

767.

41. Mei-Zheng, L.; Hong-Ni, F.; Zhong-Wu, G.; Yong-Zheng, H. One-step glycosyla-

tion and selective deprotection of peracetylated monosaccharides for facile

syntheses of allyl glycosides with a free C-2 hydroxyl group. Carbohydr. Res.

1996, 290 (2), 233–237.

42. Davies, J.A.; Petersohn, C.; Kukushkin, V.Y. Unprecedented formation of isoxazole

in the reaction between nitromethane and BF3ÁOEt2 in the presence of ethylene.

J. Chem. Soc., Perkin Trans. 1 1998, 3139–3140.

43. Audreith, L.F.; Sveda, M. Mandelamide. In Organic Syntheses; Horning, E.C., Ed.;

John Wiley & Sons: New York, 1955; Vol. III, 536–538.

44. Myers, A.I.; Slade, J. Asymmetric addititon of organometallics to chiral ketooxa-

zolines. Preparation of enantiomerically enriched a-hydroxy acids. J. Org. Chem.

1980, 45 (14), 2785–2791.

Received August 9, 2001

Accepted April 21, 2002

Article Doi

Synthesis of chloramphenicol and mandelonitrile galactose-containing prodrugs

DOI: 10.1081/CAR-120013493

Source and publish data:

Authors:

Article abstract of DOI:10.1081/CAR-120013493

Full text of DOI:10.1081/CAR-120013493

Products guided by the article

R&D Labs maybe for 488817-84-1

Relevant to this article

Hot Product