Synthesis and anti-HIV activity of glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir

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Carbohydrate Research 336 (2001) 161–180

Synthesis and anti-HIV activity of glucose-containing

prodrugs derived from saquinavir, indinavir and

nelﬁnavir

Marielle Rouquayrol,^aBe´range`re Gaucher,^aJacques Greiner,^aAnne-Marie Aubertin,^b

Pierre Vierling,^a,* Roger Guedj^a

^aLaboratoire de Chimie Bio-Organique, UMR 6001 CNRS, Uni6ersite´ de Nice Sophia-Antipolis, Parc Valrose,

F-06108 Nice, France

^bINSERM U74, Institut de Virologie, 3 rue Koeberle´, F-67000 Strasbourg, France

Received 30 July 2001; accepted 21 September 2001

Abstract

With the aim at improving the transport of the current HIV protease inhibitors across the intestinal and blood

brain barriers and their penetration into the central nervous system, the synthesis of various acyl and carbamatoyl

glucose-containing prodrugs derived from saquinavir, indinavir and nelﬁnavir, their in vitro stability with respect to

hydrolysis, and their anti-HIV activity have been investigated.

D-Glucose, which is actively transported across these

barriers, was connected through its 3-hydroxyl to these antiproteases 6ia a linker. The liberation of the active free

drug during the incubation time of the prodrugs with the cells was found to be crucial for HIV inhibition. The labile

ester linking of the glucose-containing moiety to the peptidomimetic hydroxyl of saquinavir or to the indinavir C-8

hydroxyl, which is not part of the transition state isostere, is not an obstacle for anti-HIV activity. This is not the

case for its stable carbamate linking to the peptidomimetic hydroxyl of saquinavir, indinavir and nelﬁnavir. The

chemical stability with respect to hydrolysis of some of the saquinavir and indinavir prodrugs reported here, the

liberation rate of the active free drug and the HIV inhibitory potency are acceptable for an in vivo use of these

prodrugs. These glucose-linked ester and carbamate prodrugs display a promising therapeutic potential provided that

their bioavailability, penetration into the HIV sanctuaries, and/or the liberation of the active free drug from the

carbamate prodrugs are improved. Furthermore, no cytotoxicity was detected for the prodrugs for concentrations as

reserved.

Keywords: HIV; Protease inhibitors;

D-Glucose prodrugs; Indinavir; Saquinavir; Nelﬁnavir

1. Introduction

combinations are the most efﬁcient chemo-

therapies against AIDS known at present. Re-

cent clinical trials have clearly established

their efﬁcacy in drastically reducing the

plasma viral load below its detection threshold

and in substantially increasing the level of T4

lymphocytes [for a recent overview, see Ref.

1]. Despite such polytherapies, HIV replica-

tion continues indicating the existence of

Polytherapies which rely on the use of HIV

reverse transcriptase and protease inhibitor

* Corresponding author. Tel.: +33-4-92076143; fax: +33-

4-92076151.

E-mail address: vierling@unice.fr (P. Vierling).

PII: S0008-6215(01)00260-9

162

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

recent paper from our laboratory which was

dedicated to saquinavir and indinavir pro-

drugs.¹¹These antiproteases were conjugated

to fatty acid chains, amino-acids or hy-

drophilic polyethylene glycol polymers with a

view to facilitate their passive or active diffu-

sion across the physiological barriers, or to

avoid their binding to plasma proteins and

their inactivation and rapid elimination from

the blood circulation, respectively.

reservoirs or sanctuaries for the virus (such as

the lymphatic system and central nervous sys-

tem (CNS)) in which the antiviral agents do

not penetrate at an efﬁcient inhibitory level or

do not penetrate at all.^2–4This is particularly

the case of the protease inhibitors [for a recent

overview, see Ref. 4]. In order to reduce total

body viral replication, an attractive alternative

is to improve the pharmacological properties,

safety and pharmacokinetic proﬁles, and, con-

sequently, the therapeutic potential of the cur-

rent antiproteases (APs) used in clinics, i.e.

indinavir, saquinavir, ritonavir, nelﬁnavir, am-

prenavir, lopinavir. This implies, among oth-

ers, to increase their oral bioavailability and

delivery into the CNS sanctuary by facilitat-

ing, for example, their active permeation

through the intestinal and blood–brain barri-

ers. Aiming at these goals, different strategies

which have been used with some success for

several drugs may be applied. Thus, the diffu-

sion through the intestinal and blood–brain

barriers of pharmacologically signiﬁcant

amounts of drugs can be facilitated via the

2. Results and discussion

Syntheses.—The syntheses of the prodrugs

were performed using a two-step procedure

which consisted of the condensation of an acid

or isocyanate derivative of the isopropylidene

protected sugar and the appropriate an-

tiprotease, followed by TFA-deprotection of

the isopropylidene groups, as shown in

Scheme 2.

Concerning the ester-linked prodrugs, ac-

ylation of the antiproteases was conducted

using conventional EDC–DMAP as coupling

reagent.¹²Actually, EDC was preferred to

DCC due to easier work-up and puriﬁcation

leading to higher yields in the target

compounds.

The unique hydroxyl function of saquinavir

was acylated in the 72–77% yield range. Deac-

etalation was performed using aqueous TFA¹³

and gave Saq-C(O)C4Glc(2TFA) in 80% yield.

Although these deprotection conditions were

previously used to prepare sugar esters with-

out any particular difﬁculty,¹⁴deprotection of

the succinate Saq-C(O)C2C(O)GlcP and car-

boxymethyl Saq-C(O)C1GlcP prodrugs was

most difﬁcult and rapid degradation occurred

during work-up and silica gel puriﬁcation. The

deprotection of Saq-C(O)C2C(O)GlcP was

further poorly reproducible and only possible

on very low amounts giving Saq-C(O)-

C2C(O)Glc(2TFA) in 41% yield. All our at-

tempts using more dilute TFA media, lower

temperature or FeCl₃–6 H₂O¹⁵failed. These

trials produced either hydrolysis of only the

5,6-isopropylidene group that is well-known

as to be the easier group to be cleaved,¹⁶or

several degradation compounds, the major

one being Saq-C(O)C2C(O)OH resulting from

nutrient ( -glucose, aminoacids) carrier-medi-

D

ated transport systems located at these

barriers.^5–9

This paper is devoted to the synthesis of

various -glucose-containing prodrugs derived

D

from saquinavir, indinavir and nelﬁnavir

(Scheme 1), to their chemical stability with

respect to hydrolysis under physiological con-

ditions and to their in vitro anti-HIV evalua-

tion, which are prerequisites for further in

vitro and in vivo investigations. The connec-

tion of

D

-glucose onto the hydroxyl functions

of the APs has been performed, via different

spacer units, through ester or chemically more

stable carbamate bonds which should be hy-

drolysed by cellular enzymes, thus liberating

the active free drug within the cells. This

glucose prodrug strategy is mainly aimed at

improving the penetration of the APs into the

up to now preserved CNS sanctuary.

D

-Glu-

cose, which is actively transported across the

intestinal and blood–brain barriers, was con-

nected through its 3-hydroxyl to the APs via a

linker. This connection should preserve its

recognition and transport capability by the

GLUT-1 transporter system located at these

barriers.^5,6,10This work is in continuation of a

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

163

Scheme 1. Chemical structures and code names of the glucose-containing antiprotease (AP=saquinavir, indinavir, and nelﬁnavir)

prodrugs described in this study and atom numbering used in the description of their NMR spectra.

164

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

the cleavage of the ester bond a to glucose.

Concerning the deacetalation of Saq-C(O)-

C1GlcP, saquinavir was quantitatively regen-

erated, likely as a result of an internal lactoni-

sation involving one of the deprotected sugar

hydroxyl and the ester linkage, by analogy

with previously described intramolecular reac-

tions occurring in ester-linked glucose–pep-

tide adducts.¹⁷

For the acylation of the C-8 ‘external’ hy-

droxyl of indinavir (Scheme 2), a stochastic

reaction was anticipated to be as efﬁcient as a

protection/deprotection strategy of its ‘inter-

nal’ C-14 hydroxyl. Furthermore, the C-8 hy-

droxyl of indinavir was found to be more

easily acylated than its C-14 hydroxyl.¹¹Thus,

reacting indinavir with 1 equiv of acid 1 or 5

resulted mainly in the formation of the mo-

Scheme 2. Synthetic pathway to the glucose-containing saquinavir, indinavir and nelﬁnavir prodrugs: (i) succinic anhydride–

Et₃N–CH₂Cl₂; (ii) EDC–DMAP–CH₂Cl₂; (iii) DCC–DMAP–CH₂Cl₂; (iv) 9:1 TFA–H₂O; (v) NaH–methyl bromoacetate–

THF; (vi) NaOH (1 M); (vii) NaH–ethyl 5-bromopentanoate–THF; (viii) NaOH (1 M)–THF; (ix) Et₃N–ethyl

chloroformate–NaN₃–acetone; (x) toluene–Z; (xi) CuCl–CH₂Cl₂; (xii) CuCl–DMF; (xiii) 7:3 TFA–water.

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

165

a reaction between the isocyanate and the

more accessible, hence more reactive C-8 indi-

navir and C-1 nelﬁnavir hydroxyl, as observed

for their acylation. The surprising issue of the

reaction between the isocyanate group and

indinavir or nelﬁnavir, which occurred only in

the presence of CuCl, indicates likely a com-

plex formation between Cu(I), and indinavir

or nelﬁnavir and, consequently, activation of

the C-14 indinavir and C-18 nelﬁnavir

hydroxyl.

noester Ind(8)-C(O)C2C(O)GlcP (28% yield)

and Ind(8)-C(O)C4GlcP (62% yield), respec-

tively, this latter derivative being accompanied

by a low amount of diester Ind-[C(O)C4-

GlcP]2 (14%). Deacetalation of the C-8 pro-

drugs afforded Ind(8)-C(O)C2C(O)Glc(3TFA)

and Ind(8)-C(O)C4Glc(1TFA) in 48 and 81%

yields, respectively. Deprotection of Ind(8)-

C(O)C2C(O)GlcP was achieved with the same

difﬁculties as for its saquinavir analogue.

The stochastic strategy for the acylation of

nelﬁnavir with acid 1 led mainly to Nelf(1)-

C(O)C2C(O)GlcP which corresponds to the

acylation of the nelﬁnavir aromatic C-1 hy-

droxyl function. Neither the monoester corre-

sponding to the acylation of the C-18 nor the

diester corresponding to the acylation of both

the C-1 and C-18 hydroxyls were detected.

However, Nelf(1)-C(O)C2C(O)GlcP was ob-

tained with poor yield (16%) owing to partial

degradation which occurred during silica gel

chromatography puriﬁcation. Unfortunately,

all our attempts to deprotect Nelf(1)-

C(O)C2C(O)GlcP failed, the expected Nelf(1)-

C(O)C2C(O)Glc compound could even not be

detected in the reaction medium.

Concerning the carbamate-linked Saq-C-

(O)NC4GlcP, Ind(14)-C(O)NC4GlcP, and

Nelf(18)-C(O)NC4GlcP prodrugs, these deri-

vatives were obtained by CuCl-catalysed addi-

tion of the corresponding antiprotease to iso-

cyanate 7 in the 64–77% yield range.¹⁸It is

noticeable that no reaction occurred between

these antiproteases and isocyanate 7 using

more conventional procedures,¹⁹including the

base-catalysed addition with DMAP,²⁰Et₃N,

or pyridine,²¹or the acid-catalysed addition

involving BF₃·Et₂O,²²the antiproteases being

recovered quantitatively. These glucose-pro-

tected carbamates were further converted with

TFA into Saq-C(O)NC4Glc(4TFA), Ind(14)-

C(O)NC4Glc(2TFA) and Nelf(18)-C(O)NC4-

Glc(2TFA), respectively, in high yields (85–

99%).

1

The H and ¹³C NMR spectra of the pro-

tected and deprotected prodrugs are in full

agreement with the proposed structures.^23–25

The prodrugs consist further into mutarotated

mixtures of

D

-glucose anomers in approxi-

mately equal amounts (3:2 to 1:1) as shown by

the anomeric proton integration H-1%a [4.99–

5.16 ppm (J_1%,2%2.7–3.2 Hz)] and H-1%b [4.37–

4.53 ppm (J_1%,2%7.2–8.7 Hz)].

That formation of the esters occurred on

the C-8 indinavir and C-1 nelﬁnavir hydroxyl

and formation of the carbamates on the C-14

indinavir and C-18 nelﬁnavir hydroxyl, was

unambiguously conﬁrmed by ¹³C NMR spec-

troscopy. As expected, the resonances of these

carbon atoms are deshielded (ꢀDlꢀ=2–4.5

ppm) [resp. shielded for Nelf(1)-C(O)C2C-

(O)GlcP (ꢀDlꢀꢀ7 ppm)] in comparison with

those of the free antiprotease (see examples

given in Table 1), while the signals of their

respective vicinal b-carbon atoms are shielded

(ꢀDlꢀ=1.5–3.2 ppm) [resp. deshielded for

Nelf(1)-C(O)C2C(O)GlcP (ꢀDlꢀꢀ7 ppm)].

Furthermore, the resonances of the carbon

atoms bearing the remaining ‘free’ hydroxyl

group and of their vicinal b-carbon atoms are

almost not affected.^24,25

Concerning the synthesis of the glucose

starting materials depicted in Scheme 2, con-

densation of commercial 1,2;5,6-di-O-isopro-

pylidene-a- -glucofuranose with succinic an-

D

hydride, methyl bromoacetate or ethyl 5-bro-

mopentanoate afforded 1, 2 or 4 in 70, 75 and

48% yield, respectively.^26,27Saponiﬁcation of

the two latter esters afforded quantitatively

their corresponding acids 3 and 5. The isocya-

nate derivative 7 was prepared in two steps

from acid 5 (84% overall yield) which con-

sisted ﬁrst into formation of the acyl azide 6,

then into a Curtius rearrangement of this

It is furthermore noteworthy that only one

carbamate derivative was produced with indi-

navir and nelﬁnavir but, in contrast to the

formation of the ester derivatives, the carba-

mate bond was formed surprisingly and quasi

exclusively on the internal C-14 indinavir and

C-18 nelﬁnavir hydroxyl. One expected indeed

166

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

Table 1

¹³C chemical shifts of the indinavir C-14 and C-8, of the nelﬁnavir C-18 and C-1 carbons bearing a hydroxyl, acyl or carbamate

functionality, and of their C-b vicinal carbons

Compound

¹³C chemical shift (in ppm)

C-13(b)

39.2

C-14(ORa)

65.8

C-15(b)

61.5

C-7(b)

38.1

C-8(ORb)

73.0

C-9(b)

57.5

Indinavir

(Ra=Rb=H)

Ind(14)-C(O)NC4GlcP

(Ra=C(O)NC4GlcP; Rb=H)

Ind(8)-C(O)C4GlcP

(Ra=H; Rb=C(O)C4GlcP)

36.4

70.3

59.9

39.1

73.3

57.9

38.2

65.8

61.4

37.6

75.8

54.9

C-10(b)

54.2

C-18(ORa)

70.5

C-19(b)

59.6

C-6(b)

123.4

123.3

129.4

C-1(ORb)

156.8

C-2(b)

116.8

116.3

Nelﬁnavir

(Ra=Rb=H)

Nelf(18)-C(O)NC4GlcP

(Ra=C(O)NC4GlcP; Rb=H)

Nelf(1)-C(O)C2C(O)GlcP

(Ra=H; Rb=C(O)C2C(O)GlcP)

51.0

72.4

56.5

156.0

54.8

70.4

58.8

149.8

123.9

(125.7)

fairly stable azide.²⁸This azide was obtained

by reacting NaN₃with the mixed anhydride

prepared from 5 and ethyl chloroformate.²⁹

Azide 6 was recovered contaminated by isocy-

domimetic hydroxyl was very slow. On the

other hand, no correlation was found between

the hydrolysis rate of the acylated C-8 indi-

navir prodrugs (which is not part of the tran-

sition state isostere) and their anti-HIV

activity.¹¹

The HIV inhibition levels and cytotoxicities

of the new glucose-containing saquinavir, in-

dinavir, and nelﬁnavir prodrugs were evalu-

ated in vitro in CEM-SS and MT4 cells

against HIV-1 according to published proce-

dures.^30–32The data are collected in Table 2

together with those of saquinavir and

nelﬁnavir³³(as their methanesulfonate salt),

and indinavir (as its sulfate salt).

The sensitivity to hydrolysis of some of the

glucose-containing ester and carbamate pro-

drugs and their stability was also checked

using the same hydrolysis protocol as that

described in the previous study.¹¹The hydrol-

ysis experiments were performed in a pH 7.3

buffer at 37 °C and in the absence of serum,

cells and virus using a prodrug concentration

in the 146–239 mM range. A prodrug hydroly-

sis half-life of 180 min was measured for the

representative ester Saq-C(O)C4-Glc(2TFA),

while no hydrolysis was detected for the three

carbamate prodrugs after 7 days of incubation

(Table 2). This stability of the carbamate

function was in accordance with literature

data.^9,34

1

anate 7, as shown by H and ¹³C NMR

[ꢀ2.36 vs. ꢀ3.33 ppm for CH₂aC(O)N₃and

CH₂aNꢀC(O), respectively], and was con-

verted quantitatively into isocyanate 7 by

heating.

Biological acti6ity and chemical stability.—

The saquinavir hydroxyl, the indinavir C-14

hydroxyl—and not the C-8—and the nelﬁ-

navir C-18 hydroxyl—and not the C-1—are

involved in the peptidomimetic noncleavable

transition state isostere responsible for the

protease inhibitory potency of saquinavir, in-

dinavir, and nelﬁnavir, respectively.¹¹There-

fore, it is most important that these hydroxyls

be accessible for antiviral activity.

In the previous study dedicated to the ester

prodrugs of saquinavir and indinavir, a close

correlation between their anti-HIV activity

and the hydrolysis of their acylated ‘pepti-

domimetic’ hydroxyl, hence the liberation of

the active free drug during the time of incuba-

tion, was found: the faster the hydrolysis, the

closer the anti-HIV activity level to that of the

respective parent drug.¹¹Concomitantly, the

level of HIV inhibition was very low for the

prodrugs for which hydrolysis of this pepti-

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

167

the chemically stable carbamate Saq-C(O)-

NC4-Glc(4TFA), Ind(14)-C(O)NC4-Glc-

In the antiviral assays, and based on the

prodrug hydrolysis data measured for the rep-

(2TFA) and Nelf(18)-C(O)NC4-Glc(2TFA)

prodrugs with IC₅₀from 3600 nM to \10

mM are indeed far less active than their respec-

tive parent drug (R values from 400 to ]10⁴).

As far as the two C-8 indinavir ester pro-

drugs, i.e., Ind(8)-C(O)C2C(O)-GlcP and Ind-

(8)-C(O)C4-Glc(1TFA) are concerned, one ex-

pects that the masking of the C-8 hydroxyl,

which is not part of the transition state

isostere, and its liberation rate should not

modify drastically the antiviral activity of in-

dinavir. This is indeed the case as evidenced

by their R-values ranging from 5 to 25. These

results suggest also that these C-8 indinavir

prodrugs themselves may possess an antiviral

activity, which is lower than that of indinavir,

and/or that their hydrolysis rate may however

also contribute to increasing their intrinsic

antiviral potency.

resentative

saquinavir

ester

and

the

saquinavir, indinavir C-14- and nelﬁnavir C-

18 carbamate derivatives, the antiviral activity

levels appear to be correlated to the amount

of the parent drug released during the 4 day

time-span of the antiviral experiments. Indeed,

the glucose-containing ester saquinavir pro-

drugs display high HIV inhibitory levels (IC₅₀

from ꢀ10 to 120 nM on CEM-SS cells, from

40 to 560 nM on MT4 cells). For these ester

prodrugs, e.g., Saq-C(O)C1-GlcP, Saq-C(O)-

C2C(O)-GlcP, Saq-C(O)C2C(O)-Glc(2TFA),

and Saq-C(O)C4-Glc(2TFA), an anti-HIV ac-

tivity (as shown by the R values from 1 to 8

listed in Table 1) close to that of free

saquinavir is detected, likely indicating a rela-

tively fast hydrolysis rate for these saquinavir

derivatives. This is conﬁrmed by the short

half-life (180 min) of Saq-C(O)C4-Glc(2TFA)

for which the highest anti-HIV activity was

measured.

In conclusion, it appears that the labile ester

masking of the peptidomimetic hydroxyl of

the antiproteases is not an obstacle for anti-

HIV activity. This is however not the case for

its stable carbamate masking. The chemical

stability with respect to hydrolysis of some of

the saquinavir and indinavir prodrugs re-

By contrast, the anti-HIV efﬁciency of

saquinavir, indinavir, and nelﬁnavir is consid-

erably reduced when glucose is conjugated to

the peptidomimetic hydroxyl of these an-

tiproteases through a carbamate functionality:

Table 2

Anti-HIV activity (IC₅₀) and cytotoxicity (CC₅₀) data for saquinavir, indinavir and nelﬁnavir prodrugs* in CEM-SS and MT-4 cell

a

cultures infected with HIV-1 LAI and HTLV IIIB, respectively, together with their hydrolysis half life (t_1/2

)

d

Compound

IC₅₀(nM)

CEM-SS

R

IC₅₀(nM)

MT-4

R

CC₅₀(M)

CEM-SS

CC₅₀(M)

MT-4

t_1/2hydrolysis

Saquinavir (MeSO₃H)

Saq-C(O)C2C(O)GlcP

Saq-C(O)C2C(O)Glc(2TFA)

Saq-C(O)C1GlcP

Saq-C(O)C4Glc(2TFA)

Saq-C(O)NC4Glc(4TFA)

Indinavir (H₂SO₄)

Ind(8)-C(O)C2C(O)GlcP

Ind(8)-C(O)C4Glc(1TFA)

Ind(14)-C(O)NC4Glc(2TFA)

Nelﬁnavir (MeSO₃H)

9

18

32

19

B11

3600

]10

120

48

18

\10⁻⁵

\10⁻⁴

\10⁻⁵

\10⁻⁴

\10⁻⁵

8×10⁻⁶

\10⁻⁵

\10⁻⁴

\10⁻⁵

\10⁻⁴

b

ꢀ2

ꢀ3.5

ꢀ2

B1.2

ꢀ400

150

109

41

62

7100

22

560

190

42000

ꢀ8

ꢀ6

ꢀ2

ꢀ3.5

ꢀ395

180 min

c

b

c

]12

]4.8

]5200

ꢀ26

ꢀ9

ꢀ1910

52,000

2 ^e

c

Nelf(18)-C(O)NC4Glc(2TFA)

8200

4100

\10⁴

\10⁻⁵

^at_1/2, which corresponds to the time at which 50% of hydrolysis is observed, has been determined from hydrolysis experiments

performed by incubating the prodrugs in a pH 7.3 DMEM solution at 37 °C.

^bNot tested.

^cNo hydrolysis detected after 7 days of incubation.

^dR is the ratio of the prodrug IC₅₀to that of its parent compound.

^eData from Ref. 33.

168

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

ported here, the liberation rate of the active

free drug and the HIV inhibitory potency are

acceptable for an in vivo use of these prodrugs.

These glucose-linked ester and carbamate pro-

drugs display a promising therapeutic potential

provided that their bioavailability, their pene-

tration into the HIV sanctuaries, and/or the

liberation of the active free drug from the

carbamate prodrugs are improved. Further-

more, no cytotoxicity was detected for the

prodrugs for concentrations as high as 10 or

even 100 mM (Table 2), thus indicating a

promising therapeutic index for these new

derivatives. Studies concerning their pharma-

cological properties (binding to plasma

proteins, permeation across intestinal and

blood–brain barrier models), and their safety

and pharmacokinetic proﬁles are now in

progress.

If not speciﬁed, column chromatography

puriﬁcations were carried out on Silica Gel 60

(E. Merck, 70–230 mesh). The purity of all

new compounds was checked by TLC, NMR,

MS and HPLC. TLC analyses were performed

on precoated Silica Gel F254 plates (E.

Merck) with detection by UV and by charring

with 50% MeOH–H₂SO₄solution, ninhydrin

or KMnO₄. HPLC analyses (ﬂow of 1 mL/

min) were performed using a HP1100 appara-

tus using a Lichrospher 100 RP-18 (5 mm)-

packed column (250×4 mm) (column I) or a

Nucleosil 120-3C8 column (100×4.6 mm)

(column II) or a Lichrospher 100 RP-18 (5

mm) column (250×3.2 mm) (column III) and

water–CH₃CN (v/v) 0.1% TFA gradient as

eluent (solvent A: from 4:1 to 0:1 over 30 min;

solvent B: from 7:3 to 0:1 over 30 min; UV

detection) or isocratic water (15 mM ACS and

15 mM PSA)–CH₃CN (v/v) pH 6.0 buffer

(solvent C: v/v=59:41; solvent: D v/v=

41:59; solvent E: v/v=45:55; UV detection).

With column I and solvent A, retention times

(R_t) of indinavir and saquinavir are 10.7 and

16.6 min, respectively, while with column I

3. Experimental

Syntheses

General.—Unless otherwise indicated, the re-

actions were performed under anhyd nitrogen

using dry solvents and reagents. Anhydrous

solvents were prepared by standard methods.

Acetic acid sodium salt trihydrate (ACS),

1

and solvent E, R_tof nelﬁnavir is 12.9 min. H,

19

¹³C, and F NMR spectra were recorded with

a Brucker AC 200 spectrometer at 200, 50.3,

and 188.3 MHz, respectively. Chemical shifts

(l) are given in ppm relative to the signal (i)

for internal reference Me₄Si or indirectly to

copper(I)

chloride,

dicyclohexylcarbodi-

imide (DCC), 1,2:5,6-di-O-isopropylidene-a-

D

-

glucofuranose, 1-(3-dimethylaminopropyl)-3-

ethyl-carbodiimide hydrochloride (EDC),

4-dimethylaminopyridine (DMAP), 1-pentane

sulfonic acid sodium salt (PSA), anhyd potas-

sium carbonate, potassium hydrogen sulfate,

sodium azide, sodium chloride, sodium hy-

dride, sodium hydrogencarbonate, sodium hy-

droxide, succinic anhydride, triethylamine,

were purchased from Aldrich, methyl bro-

moacetate from Acros, and ethyl chlorofor-

mate, ethyl 5-bromopentanoate, and triﬂuoro-

acetic acid (TFA) from Fluka. All these mate-

rials, except triethylamine, were used without

further puriﬁcation. Saquinavir, indinavir and

nelﬁnavir (as their methanesulfonate salt or

sulfate salt) were a gift from Hoffmann–La

Roche, E. Merck, and Agouron, respectively,

and were deprotonated prior to their use in the

synthetic processes (CHCl₃or EtOAc extrac-

tion of the free base from a NaHCO₃or

Na₂CO₃10% solution of the antiprotease).

1

CHCl₃(l 7.27) for H; (ii) for internal refer-

ence Me₄Si or indirectly to CDCl₃(l 76.9) for

¹³C; (iii) to internal reference CFCl₃for ¹⁹F.

Concerning the description of the prodrug

NMR spectra, the atoms of the antiprotease

part are depicted as C-x and H-y whereas

those of the sugar part are depicted as C-x%

and H-y% according to their standard nomen-

clature numbering (see Scheme 1 for number-

1

ing). COSY H/¹H, H/¹³C NMR correlation,

¹³C DEPT, and/or mass spectrometry data

fully conﬁrm the signal assignments and struc-

ture of the isolated materials. The glucose-de-

protected target compounds consisted of TFA

salts and the TFA anion quantiﬁcation was

assessed by ¹⁹F NMR using 3,3,3-triﬂuoro-

ethanol as internal standard. Electron-spray

ionisation–mass spectra (ESI–MS) were

recorded on a Finnigan MAT TSQ 7000 ap-

paratus equipped with an atmospheric pres-

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

169

0:1 hexane–EtOAc) afforded the white solid 2

(0.96 g, 75%); R_f0.62 (3:2 hexane–EtOAc);

¹H NMR (CDCl₃): l 5.89 (d, J_1,23.7 Hz, 1 H,

H-1), 4.71 (d, 1 H, H-2), 4.37–4.29 (m, 1 H,

H-5), 4.26 (s, 2 H, CH₂), 4.14–4.06 (m, 2 H,

H-4, H-6a), 4.02–3.98 (m, 1 H, H-6b), 3.94 (d,

J_3,42.8 Hz, 1 H, H-3), 3.75 (s, 3 H, OCH₃),

sure ionisation source. This method used in

positive mode gives either M⁺, [M+H]⁺,

[M+Na]⁺, [M−H+Na]⁺and/or [M−H+

K]⁺signals. IR spectra were recorded on a

Brucker FT-IFS 45 spectrometer as Nujol

ﬁlms.

Synthesis of the 1,2:5,6-di-O-isopropylidene-

13

1.48, 1.41, 1.34, 1.30 (4 s, 4×3 H, CH₃C); C

h- -glucofuranose deri6ati6es

D

1,2;5,6 - Di - O - isopropylidene - 3 - O - (3 - car-

boxypropanoyl)-h- -glucofuranose (1).—1,2;5,

6-Di-O-isopropylidene-a- -glucofuranose (5.0

NMR (CDCl₃): l 170.7 [C(O)OCH₃], 112.0,

109.1 (CH₃C), 105.3 (C-1), 83.8 (C-3), 83.4

(C-2), 81.2 (C-4), 72.7 (C-5), 68.4 (CH₂), 67.4

(C-6), 52.0 (CH₃), 26.9, 26.3, 25.4 (CH₃C).

1,2;5,6-Di-O-isopropylidene-3-O-(carboxy-

D

g, 19.2 mmol) and succinic anhydride (5.8 g,

57.6 mmol) were stirred with Et₃N (6.7 mL,

48.0 mmol) in CH₂Cl₂(80 mL) at rt for 30 h.

The reaction mixture was washed with 5%

KHSO₄, then water until neutrality. The or-

ganic layer was dried over Na₂SO₄, ﬁltered

and evaporated to dryness. The brown oil

obtained was chromatographed on silica gel

(EtOAc) and the solid obtained was cen-

trifuged in CH₂Cl₂. After evaporation of the

solvent, 1 (4.8 g, 70%) was obtained as a white

powder; R_f0.85 (9:1 EtOAc–EtOH); ¹H

NMR (CDCl₃): l 10.03 [bs, 1 H, C(O)OH],

5.85 (d, J_1,23.7 Hz, 1 H, H-1), 5.24 (d, J_3,41.9

Hz, 1 H, H-3), 4.48 (d, 1 H, H-2), 4.22–4.15

(m, 2 H, H-4, H-5), 4.11–3.97 (m, 2 H, H-

6a,6b), 2.66 (m, 4 H, 2 CH₂), 1.50, 1.39, 1.30,

methyl)-h- -glucofuranose (3).—Compound 2

D

(960 mg, 2.9 mmol) in 1 M NaOH aq solution

(10 mL) was reﬂuxed for 1.5 h, then stirred at

rt for 12 h. After addition of CHCl₃, the

reaction mixture was made acid up to pH 2–3

with 1 M HCl. The organic layer was then

washed until neutrality, dried over Na₂SO₄,

ﬁltered and evaporated. Colourless oil 3 was

obtained quantitatively; R_f0.56 (9:1 EtOAc–

1

MeOH); H NMR (CDCl₃): l 7.89 [bs, 1 H,

C(O)OH], 5.92 (d, J_1,23.7 Hz, 1 H, H-1), 4.57

(d, 1 H, H-2), 4.33 (m, 1 H, H-5), 4.31 and

4.14 [AB system, J_AB17.3 Hz, 2 H, CH₂C(O)],

4.21–4.12 (m, 2 H, H-4, H-6a), 4.02 (m, 1 H,

H-6b), 3.94 (d, J_3,43.5 Hz, 1 H, H-3), 1.47,

1.44, 1.36, 1.30 (4 s, 4×3 H, CH₃C); ¹³C

NMR (CDCl₃): l 171.7 [C(O)OH], 112.4,

110.0 (CH₃C), 105.8 (C-1), 84.1 (C-3), 82.5

(C-2), 81.0 (C-4), 73.2 (C-5), 67.9 [CH₂], 67.4

(C-6), 26.9, 26.7, 26.3, 25.0 (CH₃C).

13

1.28 (4 s, 4×3 H, CH₃C); C NMR (CDCl₃):

l 177.6 [C(O)OH], 170.9 [OC(O)CH₂], 112.4,

109.5 (CH₃C), 105.1 (C-1), 83.3 (C-2), 79.8

(C-4), 76.6 (C-3), 72.5 (C-5), 67.3 (C-6), 28.9,

28.8 (CH₂), 26.9, 26.8, 26.2, 25.3 (CH₃C).

1,2;5,6-Di-O-isopropylidene-3-O-(methoxy-

1,2:5,6-Di-O-isopropylidene-3-O-(4-ethoxy-

carbonylmethyl)-h- -glucofuranose (2).—So-

D

carbonylbutyl)-h-

D

-glucofuranose

(4).—The

dium hydride (240 mg, 10 mmol) was added

same process described for the preparation of

portionwise to a solution of 1,2;5,6-di-O-iso-

2 was applied to 1,2;5,6-di-O-isopropylidene-

propylidene-a- -glucofuranose (1.0 g, 3.8

D

a- -glucofuranose (3.0 g, 11.5 mmol), NaH

D

mmol) in THF (10 mL) at 0 °C. After the

initial reaction had subsided, the stirred mix-

ture was reﬂuxed for 1.5 h, then cooled down

to 0 °C and methyl bromoacetate (1.8 mL, 19

mmol) was added dropwise. The reaction mix-

ture was stirred at 0 °C for 3 h, then at rt for

24 h. After hydrolysis of excess NaH, the

mixture was evaporated under diminished

pressure. The residual oil was extracted with

CH₂Cl₂(3×30 mL) that was washed with

cold water, dried over Na₂SO₄, and evapo-

rated under diminished pressure. The residue

puriﬁed by chromatography on silica gel (1:0–

(553 mg, 23 mmol) and ethyl 5-bromopen-

tanoate (9.23 mL, 57.6 mmol). After treat-

ment, the residue was chromatographed on

silica gel (3:0–3:1 hexane–EtOAc) to afford 4

(2.15 g, 48%) as a colourless oil; R_f0.7 (3:2

hexane–EtOAc); ¹H NMR (CDCl₃): l 5.85 (d,

J_1,23.7 Hz, 1 H, H-1), 4.50 (d, 1 H, H-2),

4.30–4.15 (m, 1 H, H-5), 4.10–3.90 (m, 5 H,

H-4, H-6a,6b, CH₃CH₂), 3.85 (d, J_3,43.0 Hz, 1

H, H-3), 3.65–3.40 [m, 2 H, OCH₂], 2.30 [t, J

7.0 Hz, 2 H, CH₂C(O)], 1.75–1.55 [m, 4 H,

OCH₂CH₂CH₂], 1.50, 1.40, 1.35, 1.30 (4 s,

4×3 H, CH₃C), 1.25 (t, J 7.1 Hz, 3 H,

170

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

13

4.30–4.19 (m, 1 H, H-5), 4.15–3.90 (m, 3 H,

H-4, H-6a,6b), 3.84 (d, J_3,42.8 Hz, 1 H, H-3),

3.65–3.40 [m, 2 H, OCH₂CH₂], 2.36 [t, J 6.3

Hz, 2 H, CH₂C(O)], 1.75–1.55 [m, 4 H,

OCH₂CH₂CH₂], 1.48, 1.41, 1.33, 1.30 (4 s,

CH₃CH₂); C NMR (CDCl₃): l 173.6 [C(O)],

111.9, 109.1 (CH₃C), 105.5 (C-1), 82.7 (C-3),

82.4 (C-2), 81.4 (C-4), 72.6 (C-5), 70.3

[OCH₂CH₂], 67.5 (C-6), 60.4 (CH₃CH₂), 34.1

[CH₂C(O)], 27.0, 26.9, 26.4, 25.5 (CH₃C), 29.3

[OCH₂CH₂], 21.6 [CH₂CH₂C(O)], 14.4

(CH₃CH₂).

13

4×3 H, CH₃C); C NMR (CDCl₃): l 111.9,

109.1 (CH₃C), 105.4 (C-1), 82.6 (C-3), 82.3

(C-2), 81.3 (C-4), 72.5 (C-5), 69.9 [OCH₂CH₂],

67.4 (C-6), 36.5 [CH₂C(O)], 29.0 [OCH₂CH₂],

26.95, 26.9, 26.3, 25.4 (CH₃C), 21.5

[CH₂CH₂C(O)].

1,2:5,6-Di-O-isopropylidene-3-O-(4-carboxy-

butyl)-h- -glucofuranose (5).—The same pro-

D

cess described for the preparation of 3 was

applied to 4 (1.6 g, 4.1 mmol) in THF (12 mL)

and 1 M NaOH solution (10 mL). After treat-

ment, 5 (1.9 g, 99%) was obtained as a colour-

1,2:5,6-Di-O-isopropylidene-3-O-(4-isocya-

natobutyl)-h- -glucofuranose (7).—A solution

D

1

less oil; R_f0.6 (3:2 CH₂Cl₂–EtOAc); H NMR

of 6 (2.0 g, 5.2 mmol) in toluene (4 mL) was

reﬂuxed until no more nitrogen evolved. Re-

moval of toluene under reduced pressure af-

forded 7 (1.8 g, 99%) as an orange oil; IR (w

cm⁻¹, Nujol): 2262 (NꢀCꢀO); ¹H NMR

(CDCl₃): l 5.85 (d, J_1,23.7 Hz, 1 H, H-1), 4.51

(d, J_2,13.7 Hz, 1 H, H-2), 4.30–4.15 (m, 1 H,

H-5), 4.10–3.90 (m, 3 H, H-4, H-6a,6b), 3.84

(d, J_3,43.0 Hz, 1 H, H-3), 3.53 and 3.64 (AB

part of an ABX₂system, J_AB9.4, J_AX5.4, J_BX

5.7 Hz, 2 H, OCH₂CH₂), 3.32 (t, J 6.3 Hz, 2

H, CH₂N), 1.75–1.55 (m, 4 H, CH₂CH₂-

CH₂N), 1.48, 1.40, 1.33, 1.30 (4 s, 4×3 H,

CH₃C); ¹³C NMR (CDCl₃): l 111.9, 109.1

(CH₃C), 105.4 (C-1), 82.6 (C-3), 82.3 (C-2),

81.3 (C-4), 72.5 (C-5), 69.7 (OCH₂), 67.5 (C-

6), 42.8 (CH₂N), 28.1 (OCH₂CH₂), 26.8

(CH₂CH₂N), 26.95, 26.9, 26.3, 25.4 (CH₃C).

Synthesis of the ester prodrug deri6ati6es

(CDCl₃): l 9.70 [bs, 1 H, C(O)OH], 5.82 (d,

J_1,23.7 Hz, 1 H, H-1), 4.48 (d, 1 H, H-2),

4.30–4.20 (m, 1 H, H-5), 4.08–4.00 (m, 2 H,

H-4, H-6a), 3.93 (dd, J_6%,68.6, J_6%,55.8 Hz, 1 H,

H-6b), 3.80 (d, J_3,43.0 Hz, 1 H, H-3), 3.57 and

3.50 [AB part of an ABX₂system, J_AB9.3, J_AX

5.6, J_BX5.9 Hz, 2 H, OCH₂CH₂], 2.33 [t, J 7.0

Hz,

2

H, CH₂C(O)], 1.62 [m,

4

H,

OCH₂CH₂CH₂], 1.44, 1.37, 1.29, 1.26 (4 s,

4×3 H, CH₃C); ¹³C NMR (CDCl₃): l 179.1

[C(O)OH], 111.8, 109.0 (CH₃C), 105.3 (C-1),

82.6 (C-3), 82.3 (C-2), 81.3 (C-4), 72.5 (C-5),

70.0 [OCH₂CH₂], 67.3 (C-6), 33.6 [CH₂C(O)],

29.0 [OCH₂CH₂], 26.9, 26.8, 26.3, 25.3

(CH₃C), 21.4 [CH₂CH₂C(O)].

1,2:5,6-Di-O-isopropylidene-3-O-(4-azido-

carbonylbutyl)-h- -glucofuranose (6).—Deri-

D

vative 5 (2.4 g, 6.7 mmol) was suspended in

water (1.2 mL) and a sufﬁcient amount of

acetone was added to complete the solution.

The solution was cooled down to 0 °C and

successively Et₃N (1.1 mL, 7.7 mmol) in ace-

tone (13 mL) and ethyl chloroformate (0.83

mL, 8.7 mmol) in acetone (4 mL) were added.

After a stirring period of 30 min at 0 °C,

NaN₃(653 mg, 10 mmol) in water (2.3 mL)

was added dropwise. After another stirring

period of 2 h at 0 °C, the reaction mixture was

poured into ice water. The oil which separated

was extracted with Et₂O (3×30 mL). The

organic layer was washed with cold water,

dried over Na₂SO₄, and evaporated to afford

6 (2.1 g, 85%) as a yellow oil contaminated

with ca. 10% of isocyanate derivative 7 (see

NMR data below); IR (w cm⁻¹, Nujol): 1713

Synthesis of succinic acid 1(R)-[3(S)-tert-

butylcarbamoyloctahydro - 4a(S),8a(S) - iso -

quinolin-2-ylmethyl)]-2(S)-[3-carbamoyl-2(S)-

[(quinoline-2-carbonyl)-amino]-propionylamino]

-3-phenyl-propyl ester 4-[3-O- -glucose]-butyl

D

ester (bis triﬂuoroacetic acid salt) [Saq-C-

(O)C2C(O)Glc(2TFA)]

Succinic acid 1(R)-[3(S)-tert-butylcarba-

moyloctahydro - 4a(S),8a(S) - isoquinolin - 2 - yl-

methyl)]-2(S)-[3-carbamoyl-2(S)-[(quinoline-2-

carbonyl) - amino] - propionylamino] - 3 - phenyl-

propyl ester 4-[3-O-(1,2;5,6-di-O-isopropyli-

dene-h- -glucofuranose)]-butyl ester [Saq-C-

D

(O)C2C(O)GlcP]:—Compound 1 (200 mg, 0.6

mmol) and DMAP (67 mg, 0.6 mmol) were

added to saquinavir (185 mg, 0.3 mmol) in 3

mL CH₂Cl₂. Then, EDC (106 mg, 0.6 mmol)

was added to the mixture at 0 °C and the

solution was stirred for 15 min at 0 °C and for

1

(CꢀO), 2041 (NꢀN); H NMR (CDCl₃): l 5.85

(d, J_1,23.7 Hz, 1 H, H-1), 4.51 (d, 1 H, H-2),

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

171

MeOH); R_t10.7 min (column II, solvent B,

19 h at rt. The reaction mixture was diluted

with CH₂Cl₂and was washed. The organic

layer dried over Na₂SO₄was ﬁltered, and

evaporated under diminished pressure. Puriﬁ-

cation by chromatography on silica gel

(EtOAc) gave the title compound (203 mg,

72%) as a white solid; R_f0.35 (EtOAc); R_t

1

210 nm); H NMR (CD₃OD): l 8.47 (d, J_7,6

8.5 Hz, 1 H, H-7), 8.16 (d, 1 H, H-6), 8.15 (d,

J_1,28.2 Hz, 1 H, H-1), 8.00 (d, J_4,38.1 Hz, 1

H, H-4), 7.83 (dd, J_2,37.2, J_2,18.2 Hz, 1 H,

H-2), 7.69 (dd, J_3,48.1, J_3,27.2 Hz, 1 H, H-3),

7.24 (d, J 7.4 Hz, 2 H, H-21, H-25), 6.99 (t, J

7.4 Hz, 2 H, H-22, H-24), 6.83 (t, J 7.4 Hz, 1

H, H-23), 5.30 (m, 1 H, H-26), 5.15 (d, J_1%,2%

3.5 Hz, 0.5 H, H-1%a), 4.64 (m, 1 H, H-18),

4.45 (d, J_1%,2%9.4 Hz, 0.5 H, H-1%b), 3.90–3.61

(m, 2 H, H6%a,6%b), 3.61–3.30 (m, 4 H, H-2%–

5%), 3.15–2.15 [m, 17 H, H-13, H-19, H-27,

H-29, H-30, H-35–37, C(O)CH₂], 2.15–1.39

(m, 8 H, H-31–34), 1.33 (s, 9 H, H-41); ¹³C

NMR (CD₃OD): l 175.2, 175.1 [Saq-C(O)

a,b], 175.7, 174.0 (C-14, C-16), 174.5, 174.3

[Glc-C(O) a,b], 172.3 (C-38), 166.1, 166.2 (C-

10), 149.0 (C-8), 147.0 (C-9), 139.4 (C-20),

138.9 (C-6), 131.6, 130.8 (C-1, C-2), 130.9

(C-5), 130.3 (C-21, C-25), 129.5 (C-4), 129.2

(C-22, C-24), 129.0 (C-3), 127.1 (C-23), 119.7

(C-7), 98.2 (C-1%b), 94.0 (C-1%a), 79.8 (C-3%b),

77.8, 77.7 (C-5%b, C-3%a), 74.9 (C-26), 74.6

(C-2%b), 72.9 (C-5%a), 72.2 (C-2%a), 70.7 (C-37),

69.9, 69.8 (C-4%a, C-4%b), 62.5, 62.4 (C-6%a,

C-6%b), 59.8 (C-29), 56.6 (C-27), 53.1 (C-12),

52.0 (C-40), 51.6 (C-18), 38.1 (C-13), 37.1

(C-30), 35.2 (C-19), 34.9 (C-35), 31.9 (C-36),

30.5, 30.3 [C(O)CH₂], 29.0 (C-41), 31.5, 27.2,

27.0, 22.0 (C-31–34).

1

19.6 min (column II, solvent B, 210 nm); H

NMR (CDCl₃): l 9.11 (d, J 7.5 Hz, 1 H,

H-11), 8.20 (d, J_7,68.5 Hz, 1 H, H-7), 8.11 (d,

1 H, H-6), 8.08 (d, J_1,28.3 Hz, 1 H, H-1), 7.80

(d, J_4,38.0 Hz, 1 H, H-4), 7.72 (ddd, J_2,37.0,

J_2,18.3, J_2,41.8 Hz, 1 H, H-2), 7.63 (d, J 8.9

Hz, 1 H, H-17), 7.57 (ddd, J_3,48.0, J_3,11.8 Hz,

1 H, H-3), 7.11 (d, J 7.0 Hz, 2 H, H-21, H-25),

7.02 (t, J 7.0 Hz, 2 H, H-22, H-24), 6.90 (t, J

7.0 Hz, 1 H, H-23), 6.57 (bs, 1 H, H-15), 6.37

(bs, 1 H, H-15), 5.90 (bs, 1 H, H-39), 5.84 (d,

J_1%,2%3.7 Hz, 1 H, H-1%), 5.35 (m, 1 H, H-26),

5.23 (d, J_3%,4%2.4 Hz, 1 H, H-3%), 4.80 (m, 1 H,

H-12), 4.48 (d, 1 H, H-2%), 4.38 (m, 1 H,

H-18), 4.23–4.19 (m, 2 H, H-4%, H-5%), 4.14–

3.95 (m, 2 H, H-6%a,6%b), 3.00–2.20 [m, 17 H,

H-13, H-19, H-27, H-29, H-30, H-35–37,

C(O)CH₂], 1.90–1.60 (m, 8 H, H-31–34), 1.31

(s, 9 H, H-41), 1.47, 1.37, 1.29, 1.25 (4 s, 4×3

13

H, CH₃C); C NMR (CDCl₃): l 173.7, 173.5

(C-14, C-16), 171.9, 171.3 [C(O)CH₂], 170.3

(C-38), 164.6 (C-10), 149.1 (C-8), 146.6 (C-9),

137.4 (C-6), 137.2 (C-20), 130.2, 130.1 (C-1,

C-2), 129.4 (C-5), 129.1 (C-21, C-25), 128.5

(C-22, C-24), 128.1 (C-4), 127.7 (C-3), 126.5

(C-23), 118.7 (C-7), 112.3, 109.4 (CH₃C),

105.1 (C-1%), 83.3 (C-2%), 79.7 (C-4%), 76.5 (C-

3%), 73.9 (C-26), 72.5 (C-5%), 70.4 (C-37), 67.2

(C-6%), 59.4 (C-29), 56.5 (C-27), 51.8 (C-12),

51.0 (C-40), 49.7 (C-18), 37.5 (C-13), 35.7

(C-30), 35.1 (C-19), 33.2 (C-35), 30.8 (C-36),

29.3, 29.1 [C(O)CH₂], 28.8 (C-41), 26.9, 26.8,

26.3, 25.4 (CH₃C), 30.7, 26.2, 25.8, 20.7 (C-

31–34); ESI-MS: m/z 1013.5 [M]⁺, 1035.4

[M−H+Na]⁺in agreement with the calcu-

lated mass for [M]=C₅₄H₇₂N₆O₁₃.

Synthesis of [3-O-(1,2;5,6-di-O-isopropyli-

dene-h- -glucofuranose)]acetic acid 1(R)-

D

[3(S)-tert-butylcarbamoyloctahydro-4a(S),8a-

(S)-isoquinolin-2-ylmethyl)]-2(S)-[3-carbamo-

yl - 2(S) - [(quinoline - 2 - carbonyl) - amino] - pro-

pionylamino]-3-phenyl-propyl ester [Saq-C(O)-

C1GlcP].—The same process described for

the preparation of Saq-C(O)C2C(O)GlcP was

applied to 3 (157 mg, 0.5 mmol), DMAP (61

mg, 0.5 mmol), saquinavir (166 mg, 0.25

mmol) and EDC (95 mg, 0.5 mmol) in CH₂Cl₂

(3 mL). After treatment, the residue was chro-

matographed on silica gel (10:0–9:1 CHCl₃–

MeOH) to give Saq-C(O)C1GlcP (180 mg,

74%) as a white solid; R_f0.81 (9:1 CHCl₃–

MeOH); R_t18.6 min (column II, solvent B,

Sa q -C(O)C2C(O)Glc(2TFA):—Saq-C(O)-

C2C(O)GlcP (43 mg, 0.04 mmol) in a 9:1

TFA–water mixture (5 mL) was stirred for 10

min at rt. The residue obtained after evapora-

tion of the solvents was puriﬁed by chro-

matography (10:0 to 4:1 CHCl₃–MeOH) to

give Saq-C(O)C2C(O)Glc(2TFA) (20 mg,

41%) as a white solid; R_f0.24 (9:1 EtOAc–

1

210 nm); H NMR (CDCl₃): l 9.14 (d, J 6.9

Hz, 1 H, H-11), 8.19 (d, J_7,68.5 Hz, 1 H, H-7),

8.11 (d, 1 H, H-6), 8.09 (d, J_1,28.5 Hz, 1 H,

H-1), 7.91 (d, J 8.9 Hz, 1 H, H-17), 7.78 (d,

172

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

1 H, H-7), 8.09 (d, 1 H, H-6), 8.06 (d, J_1,28.4

Hz, 1 H, H-1), 7.78 (d, J_4,38.1 Hz, 1 H, H-4),

7.69 (dd, J_2,37.3, 1 H, H-2), 7.67 (d, J 8.4 Hz,

1 H, H-17), 7.54 (dd, 1 H, H-3), 7.10 (d, J 7.0

Hz, 2 H, H-21, H-25), 7.02 (t, J 7.0 Hz, 2 H,

H-22, H-24), 6.89 (t, J 7.0 Hz, 1 H, H-23),

6.66 (bs, 1 H, H-15), 6.43 (bs, 1 H, H-15), 5.88

(bs, 1 H, H-39), 5.81 (d, J_1%,2%3.6 Hz, 1 H,

H-1%), 5.29 (m, 1 H, H-26), 4.78 (m, 1 H,

H-12), 4.48 (d, 1 H, H-2%), 4.36 (m, 1 H,

H-18), 4.31–4.20 (m, 1 H, H-5%), 4.10–3.99

(m, 2 H, H-4%, H-6%a), 3.97 (dd, J_6%b,6%a8.5,

J_6%b,5%5.8 Hz, 1 H, H-6%b), 3.81 (d, J_3%,4%2.8 Hz,

1 H, H-3%), 3.54 [m, 2 H, CH₂Glc], 2.88–2.15

[m, 19 H, H-13, H-19, H-27, H-29, H-30,

H35–37, C(O)CH₂], 1.98–1.56 [m, 12 H, H-

31–34, CH₂CH₂CH₂Glc], 1.29 (s, 9 H, H-41),

J_4,37.5 Hz, 1 H, H-4), 7.71 (ddd, J_2,18.5, J_2,3

7.0, J_2,41.4 Hz, 1 H, H-2), 7.55 (dd, J_3,47.5,

J_3,27.0 Hz, 1 H, H-3), 7.17–6.99 (m, 5 H,

H-21–25), 6.55 (bs, 1 H, H-15), 6.37 (bs, 1 H,

H-15), 6.06 (bs, 1 H, H-39), 5.88 (d, J_1%,2%3.7

Hz, 1 H, H-1%), 5.48 (m, 1 H, H-26), 4.75 (m,

1 H, H-12), 4.69 (d, J_2%,1%3.7 Hz, 1 H, H-2%),

4.44–3.94 [m, 8 H, H-18, CH₂C(O), H-3%–6%],

3.00–2.23 (m, 13 H, H-13, H-19, H-27, H-29,

H-30, H-35–37), 1.85–1.60 (m, 8 H, H-31–

34), 1.33 (s, 9 H, H-41), 1.46, 1.42, 1.27, 1.23

(4 s, 4×3 H, CH₃C); ¹³C NMR (CDCl₃): l

173.8, 173.6 (C-14, C-16), 170.2, 170.1 [C-38,

CH₂C(O)], 164.4 (C-10), 149.1 (C-8), 146.6

(C-9), 137.4 (C-6), 137.1 (C-20), 130.2, 130.1

(C-1, C-2), 129.4 (C-5), 129.0 (C-21, C-25),

128.6 (C-22, C-24), 128.1 (C-4), 127.6 (C-3),

126.6 (C-23), 118.6 (C-7), 112.0, 109.2

(CCH₃), 105.3 (C-1%), 83.2 (C-3%), 83.1 (C-2%),

81.1 (C-4%), 74.4 (C-26), 72.7 (C-5%), 70.8 (C-

37), 67.9 [CH₂C(O)], 67.2 (C-6%), 59.8 (C-29),

56.6 (C-27), 51.4 (C-12), 51.0 (C-40), 49.3

(C-18), 37.7 (C-13), 35.8 (C-30), 34.8 (C-19),

33.1 (C-35), 30.9 (C-36), 28.8 (C-41), 26.9,

26.4, 25.5 (CH₃C), 30.7, 26.3, 25.9, 20.6 (C-

31–34). ESI-MS: m/z 971.4 [M]⁺, 993.4 [M−

H+Na]⁺, in agreement with the calculated

mass for [M]=C₅₂H₇₀N₆O₁₂.

13

1.44, 1.37, 1.25, 1.21 (4 s, 4×3 H, CH₃C); C

NMR (CDCl₃): l 173.7, 173.5 (C-14, C-16),

173.1 [C(O)CH₂], 170.3 (C-38), 164.6 (C-10),

149.1 (C-8), 146.6 (C-9), 137.4 (C-6), 137.1

(C-20), 130.2, 130.1 (C-1, C-2), 129.3 (C-5),

129.1 (C-21, C-25), 128.5 (C-22, C-24), 128.1

(C-4), 127.6 (C-3), 126.5 (C-23), 118.7 (C-7),

111.8, 109.0 (CCH₃), 105.3 (C-1%), 82.6 (C-3%),

82.3 (C-2%), 81.2 (C-4%), 73.4 (C-26), 72.6 (C-

5%), 70.6 (C-37), 70.2 [CH₂Glc], 67.3 (C-6%),

59.6 (C-29), 56.9 (C-27), 51.8 (C-12), 50.9

(C-40), 49.7 (C-18), 37.4 (C-13), 35.7 (C-30),

35.3 (C-19), 34.1 [C(O)CH₂], 33.2 (C-35), 30.8

(C-36), 29.3 [CH₂CH₂Glc], 28.8 (C-41), 26.9,

26.8, 26.3, 25.5 (CH₃C), 21.5 [C(O)CH₂CH₂],

30.7, 26.1, 25.9, 20.7 (C-31–34).

Synthesis of 5-[3-O-( -glucose)]pentanoic

D

acid 1(R)-[3(S)-tert-butylcarbamoyloctahydro-

4a(S),8a(S)-isoquinolin-2-ylmethyl)]-2(S)-[3-

carbamoyl-2(S)-[(quinoline-2-carbonyl)-amino]

-propionylamino]-3-phenyl-propyl ester (bis tri-

ﬂuoroacetic acid salt) [Saq-C(O)C4Glc(2TFA)]

Saq-C(O)C4Glc(2TFA):—Saq-C(O)C4GlcP

(44 mg, 0.04 mmol) in a 9:1 TFA–water v/v

mixture (2 mL) was stirred for 10 min at rt.

The residue obtained after evaporation of the

solvents was puriﬁed by chromatography

(10:0–4:1 EtOAc–MeOH) to give Saq-

C(O)C4Glc(2TFA) (40 mg, 80%) as a white

solid; R_f0.25 (9:1 EtOAc–MeOH); R_t12.5

5-[3-O-(1,2;5,6-Di-O-isopropylidene-h- -glu-

D

cofuranose)]pentanoic acid 1(R)-[3(S)-tert-

butylcarbamoyloctahydro - 4a(S),8a(S) - iso -

quinolin-2-ylmethyl)]-2(S)-[3-carbamoyl-2(S)-

[(quinoline-2-carbonyl)-amino]-propionylamino]

-3-phenyl-propyl ester [Saq-C(O)C4GlcP]:—

The same process described for the prepara-

tion of Saq-C(O)C2C(O)GlcP was applied to

5 (122 mg, 0.34 mmol), DMAP (63 mg, 0.5

mmol), saquinavir (163 mg, 0.24 mmol) and

EDC (94 mg, 0.5 mmol) in CH₂Cl₂(3 mL).

After treatment, the residue was chro-

matographed on silica gel (EtOAc) to give

Saq-C(O)C4GlcP (188 mg, 77%) as a white

solid; R_f0.55 (EtOAc); R_t21.3 min (column I,

1

min (column I, solvent A, 240 nm); H NMR

(CD₃OD): l 8.37 (d, J_7,68.5 Hz, 1 H, H-7),

8.07 (d, 1 H, H-6), 8.05 (d, J_1,28.4 Hz, 1 H,

H-1), 7.90 (d, J_4,38.0 Hz, 1 H, H-4), 7.74

(ddd, J_2,37.2, J_2,18.4, J_2,41.3 Hz, 1 H, H-2),

7.59 (ddd, J_3,11.3 Hz, 1 H, H-3), 7.14 (d, J 7.3

Hz, 2 H, H-21, H-25), 6.92 (t, J 7.3 Hz, 2 H,

H-22, H-24), 6.77 (t, J 7.3 Hz, 1 H, H-23),

5.20 (m, 1 H, H-26), 4.99 (d, J_1%,2%3.1 Hz, 0.5

H, H-1%a), 4.37 (m, 1.5 H, H-18, H-1%b), 3.75–

1

solvent A, 240 nm); H NMR (CDCl₃): l 9.15

(d, J 7.3 Hz, 1 H, H-11), 8.18 (d, J_7,68.5 Hz,

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

173

9.1 Hz, 1 H, H-9), 5.29 (td, J 5.2, J 1.8 Hz, 1

H, H-8), 5.19 (d, J_3%,4%2.0 Hz, 1 H, H-3%), 4.28

(d, J_2%,1%3.7 Hz, 1 H, H-2%), 4.15–4.08 (m, 2 H,

H-4%, H-5%), 4.04–3.96 (m, 2 H, H-6%a,6%b),

3.81 (m, 1 H, H-14), 3.48 (s, 2 H, H-24),

3.18–1.90 [m, 20 H, H-7, H-12, H-13, H-30,

H-15–19, C(O)CH₂], 1.32 (s, 9 H, H-23), 1.49,

1.37, 1.28, 1.25 (4 s, 4×3 H, CH₃C); ¹³C

NMR (CDCl₃): l 175.1 (C-11), 171.3, 171.9

[C(O)CH₂], 169.4 (C-20), 150.6 (C-29), 149.1

(C-28), 141.0 (C-31), 139.8, 139.3 (C-1, C-6),

136.9 (C-26), 132.5 (C-25), 129.1 (C-32, C-36),

128.4 (C-33, C-35), 128.1 (C-2), 127.1, 126.3

(C-3, C-4), 125.1 (C-34), 123.6, 123.5 (C-5,

C-27), 112.4, 109.5 (CH₃C), 105.1 (C-1%), 83.4

(C-2%), 79.8 (C-4%), 76.5, 76.4 (C-8, C-3%), 72.5

(C-5%), 67.3 (C-6%), 65.9 (C-14), 64.4 (C-19),

61.6 (C-15), 60.3 (C-24), 55.1 (C-9), 54.9, 52.7

(C-16, C-17), 51.2 (C-22), 48.2 (C-18), 46.1

(C-12), 39.4 (C-30), 38.1 (C-13), 37.6 (C-7),

29.1 (C-23), 28.8 [C(O)CH₂], 26.9, 26.8, 26.6,

25.3 (CH₃C).

3.13 (m, 8 H, H-2%–6%, CH₂Glc), 3.10–2.10 [m,

17 H, H-13, H-19, H-27, H-29, H-30, H-35–

37, C(O)CH₂], 2.00–1.40 [m, 12 H, H-31–34,

CH₂CH₂CH₂Glc], 1.25 (s, 9 H, H-41); ¹³C

NMR (CD₃OD): l 175.6, 175.5 [C(O)CH₂,

a,b], 175.2, 175.1 (C-14, C-16), 172.4 (C-38),

166.1 (C-10), 148.6 (C-8), 146.5 (C-9), 139.3

(C-20), 139.0 (C-6), 130.9 (C-5), 131.6, 130.8

(C-1, C-2), 130.3 (C-21, C-25), 129.5 (C-4),

129.2 (C-22, C-24), 129.0 (C-3), 127.2 (C-23),

119.7 (C-7), 98.3 (C-1%b), 94.1 (C-1%a), 86.5

(C-3%b), 83.6 (C-3%a), 78.0 (C-5%b), 76.3 (C-

2%b), 74.5 (C-26), 73.9 (C-5%a), 73.6, 73.5

[CH₂Glc a,b], 73.1 (C-2%a), 71.6, 71.5 (C-4%a,

C-4%b), 70.7 (C-37), 62.9, 62.7 (C-6%a, C-6%b),

59.9 (C-29), 56.8 (C-27), 53.1 (C-12), 52.0

(C-40), 51.5 (C-18), 38.0 (C-13), 37.1 (C-30),

35.5 (C-19), 35.0 [C(O)CH₂], 34.8 (C-35), 31.8

(C-36), 30.7 [CH₂CH₂Glc], 29.0 (C-41), 22.6

[C(O)CH₂CH₂], 31.5, 27.2, 27.1, 22.0 (C-31–

34). ESI-MS: m/z 933.5 [M]⁺, 955.5 [M−

H+Na]⁺, 971.4 [M−H+K]⁺in agreement

with the calculated mass for [M]=C₄₉H₆₈-

N₆O₁₂.

Ind(8)-C(O)C2C(O)Glc(3TFA): — Ind(8)-C-

(O)C2C(O)GlcP (97 mg, 0.1 mmol) in a 9:1

TFA–water v/v mixture (10 mL) was stirred

for 10 min at rt. The residue obtained after

evaporation of the solvents was puriﬁed by

chromatography (10:0 to 4:1 CHCl₃–MeOH)

to give Ind(8)-C(O)C2C(O)Glc(3TFA) (58 mg,

48%) as a white solid; R_f0.45 (7:3 EtOAc–

MeOH); R_t5.4 min (column I, solvent B, 254

Synthesis of succinic acid 1(S)-[2-(R)-benz-

yl-5-(2(S)-tert-butylcarbamoyl-4-pyridin-3-

ylmethyl-piperazin-1-yl)-4(S)-hydroxy-penta-

noylamino]-indan-2(R)-yl ester 4-[3-O- -glu-

D

cose]-butyl ester (tris triﬂuoroacetic acid salt)

[Ind(8)-C(O)C2C(O)Glc(3TFA)]

Succinic acid 1(S)-[2-(R)-benzyl-5-(2(S)-

tert - butylcarbamoyl - 4 - pyridin - 3 - ylmethyl-

piperazin-1-yl)-4(S)-hydroxy-pentanoylamino]-

indan-2(R)-yl ester 4-[3-O-(1,2;5,6-di-O-iso-

1

nm); H NMR (CD₃OD): l 8.50 (m, 2 H,

H-28, H-29), 7.82 (d, J 7.8 Hz, 1 H, H-26),

7.50–7.07 (m, 10 H, H-2–5, H-27, H-32–36),

5.56 (d, J 5.1 Hz, 1 H, H-9), 5.38 (m, 1 H,

H-8), 5.10 (d, J_1%,2%3.5 Hz, 0.5 H, H-1%a), 4.53

(d, J_1%,2%8.7 Hz, 0.5 H, H-1%b), 3.93–1.90 [m,

29 H, H-7, H-12–19, H-24, H-30, C(O)CH₂,

H-2%, H-3%, H-4%, H-5%, H-6%a,6%b], 1.32 (s, 9 H,

H-23); ¹³C NMR (CD₃OD): l 178.0 (C-11),

174.2, 174.0 [Ind-C(O), a,b], 173.6, 173.5

[GlcC(O) a,b], 173.1 (C-20), 150.9 (C-29),

149.1 (C-28), 141.7 (C-31), 140.9, 140.8 (C-1,

C-6), 139.3 (C-26), 135.2 (C-25), 130.2 (C-32,

C-36), 129.5 (C-33, C-35), 129.3 (C-2), 128.1,

127.4 (C-3, C-4), 126.0 (C-34), 125.2, 125.0

(C-5, C-27), 98.1 (C-1%b), 93.9 (C-1%a), 79.6

(C-3%b), 77.7, 77.6 (C-5%b, C-3%a), 77.2 (C-8),

74.5 (C-2%b), 72.9 (C-5%a), 72.1 (C-2%a), 69.9,

69.8 (C-4%a, C-4%b), 68.2 (C-14), 67.8 (C-19),

63.3 (C-15), 62.6, 62.4 (C-6%a, C-6%b), 60.4

propylidene-h-

D

-glucofuranose)]-butyl

ester

[Ind(8)-C(O)C2C(O)GlcP]:—The same pro-

cess described for the preparation of Saq-

C(O)C2C(O)GlcP was applied to 1 (131 mg,

0.36 mmol), DMAP (44 mg, 0.36 mmol), indi-

navir (222 mg, 0.36 mmol) and EDC (75 mg,

0.36 mmol) in CH₂Cl₂(10 mL). After treat-

ment, the residue was chromatographed on

silica gel (10:0–9:1 EtOAc–MeOH) to give

Ind(8)-C(O)C2C(O)GlcP (97 mg, 28%) as a

white solid; R_f0.59 (4:1 EtOAc–MeOH); R_t

1

9.6 min (column I, solvent B, 254 nm); H

NMR (CDCl₃): l 8.55 (m, 2 H, H-28, H-29),

7.67 (bs, 1 H, H-21), 7.59 (dt, J 7.8, J 2.0 Hz,

1 H, H-26), 7.30–7.15 (m, 10 H, H-2–5, H-27,

H-32–36), 6.37 (d, J 9.1 Hz, 1 H, H-10), 5.77

(d, J_1%,2%3.7 Hz, 1 H, H-1%), 5.63 (dd, J 5.2, J

174

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

C(O)], 169.2 (C-20), 150.5 (C-29), 149.1 (C-

28), 141.0 (C-31), 139.7, 139.3 (C-1, C-6),

136.8 (C-26), 132.4 (C-25), 128.9 (C-32, C-36),

128.3 (C-33, C-35), 128.0 (C-2), 127.0, 126.3

(C-3, C-4), 125.0 (C-34), 123.7, 123.4 (C-5,

C-27), 111.7, 108.9 (CCH₃), 105.2 (C-1%), 82.5

(C-3%), 82.2 (C-2%), 81.2 (C-4%), 75.8 (C-8), 72.4

(C-5%), 70.1 [CH₂Glc], 67.3 (C-6%), 65.8 (C-14),

63.9 (C-19), 61.4 (C-15), 60.2 (C-24), 54.9

(C-9), 54.6, 52.7 (C-16, C-17), 51.2 (C-22),

47.8 (C-18), 46.2 (C-12), 39.3 (C-30), 38.2

(C-13), 37.6 (C-7), 33.6 [Ind-C(O)CH₂], 29.6

[CH₂CH₂Glc], 29.0 (C-23), 26.9, 26.8, 26.2,

25.4 (CH₃C), 21.3 [C(O)CH₂CH₂]. ESI-MS:

m/z 956.5 [M]⁺, 978.6 [M−H+Na]⁺, in

agreement with the calculated mass for [M]=

C₅₃H₇₃N₅O₁₁.

(C-24), 56.6 (C-9), 56.4, 53.1 (C-16, C-17),

52.1 (C-18, C-22), 46.2 (C-12), 40.5 (C-30),

38.8 (C-13), 38.1 (C-7), 30.2 [C(O)CH₂], 29.0

(C-23).

Synthesis of 5-[3-O-( -glucose)]pentanoic

D

acid 1(S)-[2-(R)-benzyl-5-(2(S)-tert-butylcar-

bamoyl-4-pyridin-3-ylmethyl-piperazin-1-yl)-

4(S)-hydroxy-pentanoylamino]-indan-2(R)-yl

ester (triﬂuoroacetic acid salt) [Ind(8)-C(O)-

C4Glc(1TFA)]

5-[3-O-(1,2;5,6-Di-O-isopropylidene-h- -glu-

D

cofuranose)]pentanoic acid 1(S)-[2-(R)-benzyl-

5-(2(S)-tert-butylcarbamoyl-4-pyridin-3-yl-

methyl-piperazin-1-yl)-4(S)-hydroxy-pentano-

ylamino]-indan-2(R)-yl ester [Ind(8)-C(O)-

C4GlcP]:—The same process described for

the preparation of Saq-C(O)C2C(O)GlcP was

applied to 5 (120 mg, 0.33 mmol), DMAP (42

mg, 0.35 mmol), indinavir (177 mg, 0.29

mmol) and EDC (66 mg, 0.35 mmol) in

CH₂Cl₂(4 mL). After treatment, the residue

was chromatographed three times on silica gel

(10:0–9:1 EtOAc–MeOH) to give Ind(8)-

C(O)C4GlcP (160 mg, 62%) and 5-[3-O-(1,

Ind-[C(O)C4GlcP]2: R_f0.79 (4:1 EtOAc–

1

MeOH); H NMR (CDCl₃): l 8.50 (dd, J 4.8,

J 1.7 Hz, 1 H, H-28), 8.49 (d, J 1.7 Hz, 1 H,

H-29), 7.62 (dt, J 7.9, J 1.7 Hz, 1 H, H-26),

7.28–7.11 (m, 10 H, H-2–5, H-27, H-32–36),

6.90 (bs, 1 H, H-21), 6.10 (d, J 9.2 Hz, 1 H,

H-10), 5.84 (d, J_1%,2%3.7 Hz, 1 H, H-1%), 5.82 (d,

J_1%,2%3.7 Hz, 1 H, H-1%), 5.63 (dd, J 5.2, J 9.2

Hz, 1 H, H-9), 5.28 (td, J 5.2, J 1.5 Hz, 1 H,

H-8), 4.50 (m, 1 H, H-14), 4.49 (d, 2 H, H-2%),

4.23 (m, 2 H, H-5%), 4.11–3.92 (m, 6 H, H-4_,%

H-6%a,6%b), 3.83 (d, J_3%,4%3.4 Hz, 1 H, H-3%),

3.81 (d, J_3%,4%3.4 Hz, 1 H, H-3%), 3.53 [m, 4 H,

CH₂Glc], 3.46 (s, 2 H, H-24), 3.25–2.00 [m, 20

H, H-7, H-12, H13, H-15–19, H-30,

C(O)CH₂], 1.70–1.50 [m, 8 H, CH₂CH₂CH₂-

Glc], 1.49, 1.40, 1.32, 1.31 (4s, 33 H, H-23,

CH₃C); ¹³C NMR (CDCl₃): l 174.0 (C-11),

173.4, 172.5 [C(O)CH₂], 170.2 (C-20), 150.4

(C-29), 148.9 (C-28), 140.6 (C-31), 139.3,

139.0 (C-1, C-6), 136.7 (C-26), 132.9 (C-25),

128.8 (C-32, C-36), 128.5 (C-33, C-35), 128.2

(C-2), 127.2, 126.6 (C-3, C-4), 125.0 (C-34),

123.8, 123.4 (C-5, C-27), 105.3 (C-1%), 82.6

(C-3%), 82.2, 82.1 (C-2%), 81.2 (C-4%), 75.9 (C-8),

72.4 (C-5%), 70.1, 70.0 [CH₂Glc], 69.8 (C-14),

67.3, 67.2 (C-6%), 67.0 (C-19), 59.9 (C-24), 58.9

(C-15), 55.1 (C-9), 55.8, 52.2 (C-16, C-17),

50.9 (C-22), 49.9 (C-18), 45.7 (C-12), 39.7

(C-30), 37.6 (C-7), 35.5 (C-13), 34.1, 33.6

[C(O)CH₂], 29.7, 29.2 [CH₂CH₂Glc], 28.8 (C-

23), 26.8, 26.3, 25.5, 25.4 (CH₃C), 21.7, 21.3

[C(O)CH₂CH₂]. ESI-MS: m/z 1298.6 [M]⁺,

1320.6 [M−H+Na]⁺, in agreement with the

calculated mass for [M]=C₇₀H₉₉N₅O₁₈.

2;5,6-di-O-isopropylidene-a- -glucofuranose)]

D

pentanoic acid 1(S)-[(2(S)-tert-butylcarba-

moyl - 4 - pyridin - 3 - ylmethyl - piperazin - 1 - yl-

methyl]-3-(R)-[2-(R)-5-[3-O-(1,2;5,6-di-O-iso-

propylidene - a -

D

- glucofuranose)]pentanoyl] -

indan-2(S)-ylcarbamoyl]-4-phenyl-butyl ester

[Ind-[C(O)C4GlcP]2] (46.5 mg, 14%) as white

solids.

Ind(8)-C(O)C4GlcP: R_f0.57 (4:1 EtOAc–

1

MeOH); H NMR (CDCl₃): l 8.52 (dd, J 4.8,

J 1.6 Hz, 1 H, H-28), 8.49 (d, J 1.6 Hz, 1 H,

H-29), 7.75 (bs, 1 H, H-21), 7.59 (dt, J 7.8, J

1.6 Hz, 1 H, H-26), 7.31–7.12 (m, 10 H,

H-2–5, H-27, H-32–36), 6.26 (d, J 9.1 Hz, 1

H, H-10), 5.82 (d, J_1%,2%3.7 Hz, 1 H, H-1%), 5.62

(dd, J 5.2, J 9.1 Hz, 1 H, H-9), 5.23 (td, J 5.2,

J 1.5 Hz, 1 H, H-8), 4.48 (d, 1 H, H-2%), 4.22

(dd, J_5%,6%a5.9, J_5%,6%b6.0 Hz, 1 H, H-5%), 4.08 (d,

J_4%,3%3.0 Hz, 1 H, H-4%), 4.06–3.90 (m, 2 H,

H-6%a,6%b), 3.88–3.73 (m, 1 H, H-14), 3.80 (d,

J_3%,4%3.0 Hz, 1 H, H-3%), 3.48 [m, 4 H, H-24,

CH₂Glc], 3.19–2.43 (m, 14 H, H-7, H-12,

H-15–19, H-30), 2.32 (m, 1 H, H-13b), 2.05–

1.80 [m, 3 H, H-13a, Ind-C(O)CH₂], 1.50 [m, 4

H, CH₂CH₂CH₂Glc], 1.32 (s, 9 H, H-23), 1.47,

1.39, 1.29, 1.24 (4 s, 4×3 H, CH₃C); ¹³C

NMR (CDCl₃): l 174.8 (C-11), 172.4 [Ind-

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

175

mmol), nelﬁnavir (186 mg, 0.33 mmol) and

EDC (68 mg, 0.33 mmol) in DMF (10 mL).

After treatment, the residue was chro-

matographed twice on silica gel (10:0–2:3 hex-

ane–EtOAc, then 10:0–9:1 CHCl₃–MeOH) to

give Nelf(1)-C(O)C2C(O)GlcP (49 mg, 16%)

Ind(8)-C(O)C4Glc(1TFA):—A 9:1 TFA–

water mixture (1 mL) was added dropwise to

a solution of Ind(8)-C(O)C4GlcP (47 mg, 0.05

mmol) in CH₃CN. The solution was stirred

for 3 h at 0 °C and for 1 h at rt. Then, a 9:1

TFA–water mixture (1 mL) was added drop-

wise to the reaction solution at rt and the

mixture stirred for another 30 min. The

residue obtained after evaporation of the sol-

vents was puriﬁed twice by chromatography

(10:0–7:3 EtOAc–MeOH) to give Ind(8)-

C(O)C4Glc(1TFA) (39.5 mg, 81%) as a white

solid; R_f0.63 (7:3 EtOAc–MeOH); R_t8.6 min

1

as a white solid; R_f0.72 (EtOAc); H NMR

(CDCl₃): l 7.45–7.00 (m, 9 H, H-2–4, H13-

17, H-9 or H-30), 5.84 (d, J_1%,2%3.7 Hz, 1 H,

H-1%), 5.53 (bs, 1 H, H-9 or H-30), 5.28 (d,

J_3%,4%2.0 Hz, 1 H, H-3%), 4.51 (d, J 3.7 Hz, 1 H,

H-2%), 4.50–4.40 (m, 1 H, H-10), 4.31–4.01

(m, 4 H, H-4%–6% and H-18), 3.80 and 3.69 (m,

2 H, H-11a,b), 2.90 (dd, J_AB9.7, J_AX4.4 Hz,

1 H, H-20a), 3.00–2.75 (m, 2 H, H-19a, H-

28), 2.70–2.38 (m, 2 H, H-19b, H-20b), 2.28

(s, 3 H, H-7), 2.25–1.30 [m, 16 H, H-21–27,

C(O)CH₂CH₂C(O)], 1.53, 1.42, 1.31, 1.29 (4 s,

4×3 H, CH₃C), 1.17 (s, 9 H, H-32); ¹³C

NMR (CDCl₃): l 174.0 (C-8), 170.8, 170.7

[C(O)CH₂], 170.3 (C-29), 149.8 (C-1), 137.9,

135.8 (C-5, C-12), 130.2 (C-13, C-17), 129.4

(C-6), 129.1 (C-14, C-16), 126.5 (C-3, C-15),

125.7, 123.9 (C-2, C-4), 112.4, 109.5 (CH₃C),

105.2 (C-1%), 83.3 (C-2%), 79.8 (C-4%), 76.7 (C-

3%), 72.5 (C-5%), 70.8 (C-28), 70.4 (C-18), 67.4

(C-6%), 59.8 (C-20), 58.8 (C-19), 54.8 (C10),

51.3 (C-31), 36.2 (C-21), 35.5 (C-11), 33.8

(C-26), 31.1 (C-27), 29.1, 29.0 [C(O)CH₂], 28.5

(C-32), 31.0, 26.4, 26.1, 20.6 (C-22–5), 26.9,

26.8, 26.3, 25.3 (CH₃C), 13.3 (C-7).

1

(column I, solvent A, 254 nm); H NMR

(CD₃OD): l 8.58 (m, 2 H, H-28, H-29), 7.59

(dt, J 7.9 Hz, 1 H, H-26), 7.31–7.17 (m, 10 H,

H-2–5, H-27, H-32–36), 5.58 (d, J 5.0 Hz, 1

H, H-9), 5.37 (td, J 5.0, J 1.5 Hz, 1 H, H-8),

5.08 (d, J_1%,2%3.2 Hz, 0.5 H, H-1%a), 4.47 (d,

J_1%,2%7.2 Hz, 0.5 H, H-1%b), 4.11–3.23 [m, 11

H, H-14, H-24, H-2%-6% CH₂Glc], 3.22–2.62

,

(m, 14 H, H-7, H-12, H-15–19, H-30), 2.30–

1.82 [m, 4 H, H-13, C(O)CH₂], 1.60 [m, 4 H,

CH₂CH₂CH₂Glc], 1.32 (s, 9 H, H-23); ¹³C

NMR (CD₃OD): l 177.6 (C-11), 174.9, 174.8

[C-20, C(O)CH₂a,b], 150.6 (C-29), 149.4 (C-

28), 141.7 (C-31), 141.1, 140.5 (C-1, C-6),

140.4 (C-26), 130.2 (C-32, C-36), 129.6 (C-33,

C-35), 129.3 (C-2), 128.2, 127.7 (C-3, C-4),

126.1 (C-34), 125.8 (C-5), 125.1 (C-27), 98.4

(C-1%b), 94.1 (C-1%a), 86.5 (C-3%b), 83.6 (C-

3%a), 78.0 (C-5%b), 76.9 (C-8), 76.3 (C-2%b), 73.9

(C-5%a), 73.6, 73.5 [CH₂Glc a,b], 73.1 (C-2%a),

71.7, 71.6 (C-4%a, C-4%b), 67.2, 67.1 (C-14,

C-19), 62.9, 62.8 (C-6%a, C-6%b), 62.7 (C-15),

59.3 (C-24), 56.7 (C-9), 54.6 (C-16, C-17), 52.8

(C-22), 51.1 (C-18), 46.1 (C-12), 40.6 (C-30),

38.5 (C-13), 38.3 (C-7), 34.8 [C(O)CH₂], 30.7

[CH₂CH₂Glc], 28.8 (C-23), 22.6 [C(O)CH₂-

CH₂]. ESI-MS: m/z 876.5 [M]⁺, 898.5 [M−

H+Na]⁺, in agreement with the calculated

mass for [M]=C₄₇H₆₅N₅O₁₁.

Synthesis of the carbamate prodrug

deri6ati6es

Synthesis of [4-(3-O- -glucose)-butyl]-car-

D

bamic acid 1(R)-[3(S)-tert-butylcarbamoyloc-

tahydro-4a(S),8a(S)-isoquinolin-2-ylmethyl)]-

2(S)-[3-carbamoyl-2(S)-[(quinoline-2-carbon-

yl) - amino] - propionylamino] - 3 - phenyl - propyl

ester (tetrakis triﬂuoroacetic acid salt) [Saq-

C(O)NC4Glc(4TFA)]

[4-[3-O-(1,2;5,6-Di-O-isopropylidene-h- -

D

glucofuranose)]-butyl]-carbamic acid 1(R)-

[3(S)-tert -butylcarbamoyloctahydro-4a(S),8a-

(S)-isoquinolin-2-ylmethyl]-2(S)-[3-carbamo-

yl - 2(S) - [(quinoline - 2 - carbonyl) - amino] - pro-

pionylamino]-3-phenyl-propyl ester [Saq-C-

(O)NC4GlcP]:—Derivative 7 (214 mg, 0.60

mmol) was added at rt to a stirred solution of

saquinavir (200 mg, 0.30 mmol) in CH₂Cl₂(2

mL), followed by CuCl (59 mg, 0.60 mmol).

After 5 h stirring at rt, the reaction mixture

Synthesis of succinic acid 3-[3-[3(S)-tert-

butylcarbamoyloctahydro - 4a(S),8a(S) - iso -

quinolin-2-yl]-2(R)-hydroxy-1(S)-phenylsulf-

anylmethyl-propylcarbamoyl]-2-methyl-phenyl

ester 4-[3-O-(1,2;5,6-di-O-isopropylidene-h-

D

-

glucofuranose)]-butyl ester [Nelf(1)-C(O)-

C2C(O)GlcP].—The same process as that

used for Saq-C(O)C2C(O)GlcP was applied to

1 (118 mg, 0.33 mmol), DMAP (40 mg, 0.33

176

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

1

solvent D, 240 nm); H NMR (CD₃OD): l

8.42 (d, J_7,68.6 Hz, 1 H, H-7), 8.15 (d, 1 H,

H-6), 8.10 (d, J_1,28.7 Hz, 1 H, H-1), 7.95 (d,

J_4,37.9 Hz, 1 H, H-4), 7.80 (dt, J_2,37.9, J_2,18.7

Hz, 1 H, H-2), 7.65 (t, 1 H, H-3), 7.20 (d, J

7.4 Hz, 2 H, H-21, H-25), 6.98 (t, J 7.4 Hz, 2

H, H-22, H-24), 6.81 (t, J 7.4 Hz, 1 H, H-23),

5.16 (d, J_1%,2%2.7 Hz, 0.5 H, H-1%a), 5.20–5.00

(m, 2 H, H-12, H-26), 4.52 (d, 0.5 H, J_1%,2%7.2

Hz, H-1%b), 4.35 (m, 1 H, H-18), 3.70–3.30

(m, 8 H, H-2%–6%, CH₂Glc), 3.25-2.20 (m, 12

H, H-13, H-19, H-27, H-29, H-30, H-35–37,

CH₂NH), 2.10–1.40 (m, 12 H, H-31–34,

CH₂CH₂Glc), 1.32 (s, 9 H, H-41); ¹³C NMR

(CD₃OD): l 175.6 (C-14, C-16), 172.4 (C-38),

166.5 (C-10), 158.6 [NHC(O)O], 150.1 (C-8),

148.0 (C-9), 139.3 (C-20), 139.1 (C-6), 130.9

(C-5), 131.7, 130.8 (C-1, C-2), 130.2 (C-21,

C-25), 129.7 (C-4), 129.3 (C-22, C-24), 129.1

(C-3), 127.3 (C-23), 119.9 (C-7), 98.3 (C-1%b),

94.1 (C-1%a), 86.3 (C-3%b), 83.5 (C-3%a), 78.1

(C-5%b), 76.2 (C-2%b), 75.3 (C-26), 73.8

(CH₂Glc), 73.7 (C-5%a), 73.2 (C-2%a), 71.5 (C-

4%a,b), 71.1 (C-37), 62.8, 62.6 (C-6%a,b), 60.7

(C-29), 58.0 (C-27), 53.6 (C-12), 52.3 (C-40),

51.5 (C-18), 41.9 (CH₂NH), 38.3 (C-13), 37.2

(C-30), 35.4 (C-19), 34.7 (C-35), 31.9 (C-36),

29.0 (C-41), 28.6 (CH₂CH₂NH), 27.5

(CH₂CH₂Glc), 31.6, 27.2, 26.7, 21.9 (C-31–

34). ESI-MS: m/z 948.5 [M]⁺, 970.4 [M−

H+Na]⁺, in agreement with the calculated

mass for [M]=C₄₉H₆₉N₇O₁₂.

was extracted with CH₂Cl₂(3×20 mL). The

organic layer was washed with water and

brine, dried over Na₂SO₄, and evaporated.

The residue was chromatographed on silica

gel (9:0–9:1 EtOAc–MeOH) to afford Saq-

C(O)NC4GlcP (208 mg, 68%) as a white pow-

1

der; R_f0.30 (EtOAc); H NMR (CDCl₃): l

9.12 (d, J 7.6 Hz, 1 H, H-11), 8.15 (d, J_7,68.6

Hz, 1 H, H-7), 8.06 (d, 1 H, H-6), 8.02 (d, J_1,2

8.0 Hz, 1 H, H-1), 7.74 (d, J_4,38.1 Hz, 1 H,

H-4), 7.65 (dd, J_2,37.8, 1 H, H-2), 7.50 (dd, 1

H, H-3), 7.26–6.92 (m, 4 H, H-21, H-22,

H-24, H-25), 6.84 (m, 1 H, H-23), 6.60 (bs, 1

H, H-17), 6.08 (bs, 2 H, H-15), 5.79 (d, J_1%,2%

3.7 Hz, 1 H, H-1%), 5.39 (m, 1 H, H-39), 5.25

[m, 1 H, NHC(O)O], 5.02 (m, 1 H, H-26),

4.82 (m, 1 H, H-12), 4.45 (d, J_2%,1%3.7 Hz, 1 H,

H-2%), 4.31 (m, 1 H, H-18), 4.20 (m, 1 H,

H-5%), 4.10–3.85 (m, 3 H, H-4%, H-6%a,6%b),

3.77 (d, J_3%,4%2.9 Hz, 1 H, H-3%), 3.48 (m, 2 H,

CH₂Glc), 3.02 (m, 2 H, CH₂NH), 3.00–2.25

(m, 13 H, H-13, H-19, H-27, H-29, H-30,

H-35–37), 1.80–1.45 (m, 12 H, H-31–34,

CH₂CH₂CH₂NH), 1.26 (s, 9 H, H-41), 1.41,

1.34, 1.25, 1.22 (4 s, 4×3 H, CH₃C); ¹³C

NMR (CDCl₃): l 174.0, 173.5 (C-14, C-16),

170.4 (C-38), 164.5 (C-10), 156.3 [NHC(O)O],

149.1 (C-8), 146.5 (C-9), 137.3 (C-6, C-20),

130.1 (C-1, C-2), 129.3 (C-5), 129.2 (C-21,

C-25), 128.3 (C-22, C-24), 128.0 (C-4), 127.6

(C-3), 126.3 (C-23), 118.7 (C-7), 111.7, 108.9

(CH₃C), 105.2 (C-1%), 82.5 (C-3%), 82.2 (C-2%),

81.1 (C-4%), 73.7 (C-26), 72.5 (C-5%), 70.2 (C-

37), 70.1 (CH₂Glc), 67.2 (C-6%), 59.9 (C-29),

57.1 (C-27), 52.1 (C-12), 50.8 (C-40), 49.9

(C-18), 40.8 (CH₂NH), 37.5 (C-13), 35.9 (C-

30), 35.6 (C-19), 33.2 (C-35), 30.9 (C-36), 29.6

(CH₂CH₂Glc), 28.7 (C-41), 27.0 (CH₂CH₂-

NH), 26.8, 26.2, 25.4 (CH₃C), 30.7, 26.5, 25.6,

20.7 (C-31–34).

Synthesis of [3-O-( -glucose)]-carbamic acid

D

1(S)-(2(S)-tert-butylcarbamoyl-4-pyridin-3-yl-

methyl-piperazin-1-ylmethyl)-3(R)-(2(R)-hy-

droxy-indan-1(S)-ylcarbamoyl)-4-phenyl-butyl

ester (bis triﬂuoroacetic acid salt) [Ind(14)-

C(O)NC4Glc(2TFA)]

[4-[3-O-(1,2;5,6-Di-O-isopropylidene-h- -

D

glucofuranose)]-butyl]-carbamic acid 1(S)-

(2(S) - tert - butylcarbamoyl - 4 - pyridin - 3 - yl-

methyl-piperazin-1-ylmethyl)-3(R)-(2(R)-hy-

droxy-indan-1(S)-ylcarbamoyl)-4-phenyl-butyl

ester [Ind(14)-C(O)NC4GlcP]:—The same

process described for the preparation of Saq-

C(O)NC4GlcP was applied to 7 (134 mg, 0.37

mmol), indinavir (230 mg, 0.37 mmol), CuCl

(37 mg, 0.37 mmol) in CH₂Cl₂(2 mL). After

treatment, the residue was chromatographed

on silica gel (9:0–4:1 EtOAc–MeOH) to af-

ford Ind(14)-C(O)NC4GlcP (280 mg, 77%) as

Saq-C(O)NC4Glc(4TFA):—A 7:3 TFA–

water mixture (10 mL) was added at 0 °C to a

solution of Saq-C(O)NC4GlcP (100 mg, 0.097

mmol) in CH₃CN (2 mL). After 2 h at 0 °C

then 24 h at rt, the reaction mixture was

evaporated and chromatographed on silica gel

(10:0–4:1 CH₂Cl₂–MeOH) to afford Saq-

C(O)NC4Glc(4TFA) (135 mg, 99%) as a white

powder; R_f0.25 (9:1 EtOAc–MeOH); R_f0.35

(9:1 CH₂Cl₂–MeOH); R_t3.2 min (column I,

solvent D, 240 nm); 3.8 min (column III,

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

177

11), 170.7 (C-20), 158.9 [NHC(O)O], 151.5

(C-29), 150.5 (C-28), 142.2 (C-31), 140.9 (C-

26), 141.9, 140.5 (C-1, C-6), 130.3 (C-32, C-

36), 129.6 (C-33, C-35), 128.9 (C-2), 127.8,

127.6 (C-3, C-4), 126.2 (C-34), 125.9 (C-5),

98.3 (C-1%b), 94.2 (C-1%a), 86.5 (C-3%b), 83.6

(C-3%a), 78.0 (C-5%b), 76.3 (C-2%b), 74.2 (C-8),

73.9 (C-5%a), 73.8, 73.6 (CH₂CGlc), 73.2 (C-

2%a), 71.7, 71.6 (C-4%a, b), 71.1 (C-14), 62.9,

62.7 (C-6%a, b), 60.0 (C-15), 59.2 (C-24), 58.8

(C-9), 53.0 (C-16, C-17), 52.8 (C-22), 52.2

(C-18), 46.0 (C-12), 41.9 (CH₂NH), 40.9 (C-

30), 40.7 (C-7), 35.6 (C-13), 29.0 (C-23), 28.6

(CH₂CH₂Glc), 27.7 (CH₂CH₂NH). ESI-MS:

m/z 892.0 [M+H]⁺, 914.0 [M+Na]⁺, in

agreement with the calculated mass for [M]=

C₄₇H₆₆N₆O₁₁.

a white powder; R_f0.6 (4:1 EtOAc–MeOH);

¹H NMR (CDCl₃): l 7.65 (d, J 7.8 Hz, 1 H,

H-26), 7.40–7.15 (m, 12 H, H-2–5, H-27–29,

H-32–36), 6.90 (bs, 1 H, H-21), 6.05 (d, J 8.6

Hz, 1 H, H-10), 5.84 (d, J_1%,2%3.7 Hz, 1 H,

H-1%), 5.28 (dd, J 4.7, J 8.6 Hz, 1 H, H-9),

5.10–4.95 [m, 2 H, H-14, NHC(O)O], 4.50 (d,

1 H, H-2%), 4.30–4.20 (m, 2 H, H-8, H-4%),

4.15–3.95 (m, 3 H, H-5%, H-6%), 3.84 (d, J_3%,4%

3.0 Hz, 1 H, H-3%), 3.49 (s, 2 H, H-24), 3.55

(m, 2 H, CH₂CGlc), 3.21 (m, 2 H, CH₂NH),

3.00–2.25 (m, 16 H, H-7, H-12, H-13, H-15–

19, H-30), 1.59 (s, 4 H, CH₂CH₂CH₂NH),

1.36 (s, 9 H, H-23), 1.50, 1.43, 1.34, 1.31 (4 s,

4×3 H, CH₃C); ¹³C NMR (CDCl₃): l 174.1

(C-11), 170.6 (C-20), 156.3 [NHC(O)O], 150.5

(C-29), 149.0 (C-28), 140.3 (C-31), 140.1,

139.7 (C-1, C-6), 136.9 (C-26), 129.1 (C-32,

C-36), 128.7 (C-33, C-35), 127.9 (C-2), 126.9,

126.8 (C-3, C-4, C-34), 125.0, 124.6 (C-5, C-

27), 111.9, 109.0 (CH₃C), 105.3 (C-1%), 82.4

(C-3%), 82.2 (C-2%), 81.1 (C-4%), 73.3 (C-8), 72.5

(C-5%), 70.3 (C-14), 69.9 (CH₂Glc), 67.4 (C-6%),

59.9 (C-15), 59.6 (C-24), 57.9 (C-9), 56.0, 52.2

(C-16, C-17), 50.9 (C-18), 50.8 (C-22), 46.4

(C-12), 40.9 (CH₂NH), 40.1 (C-30), 39.1

(C-7), 36.4 (C-13), 28.8 (C-23), 26.9 (CH₂-

CH₂Glc), 26.8, 26.3, 25.5 (CH₃C), 26.6

(CH₂CH₂NH).

Synthesis of [4-(3-O- -glucose)-butyl]-car-

D

bamic acid 1(R)-[3(S)-tert-butylcarbamoyloc-

tahydro - 4a(S),8a(S) - isoquinolin - 2 - ylmethyl] -

2(S)-(3-hydroxy-2-methyl-benzoylamino)-3-

phenylsulfanyl-propyl ester (bis triﬂuroacetic

acid salt) [Nelf(18)-C(O)NC4Glc(2TFA)]

[4-[3-O-(1,2;5,6-Di-O-isopropylidene-h- -

D

glucofuranose)]-butyl]-carbamic acid 1(R)-

[3(S)-tert-butylcarbamoyloctahydro-4a(S),8a-

(S)-isoquinolin-2-ylmethyl]-2(S)-(3-hydroxy-

2 - methyl - benzoylamino) - 3 - phenylsulfanyl -

propyl ester [Nelf(18)-C(O)NC4GlcP]:—The

same process described for the preparation of

Saq-C(O)NC4Glc was applied to 7 (95 mg,

0.26 mmol), nelﬁnavir (150 mg, 0.26 mmol),

CuCl (26 mg, 0.26 mmol) in 2:0.5 CH₂Cl₂–

DMF (2.5 mL). After treatment, the residue

was chromatographed on silica gel (3:0–3:2

CH₂Cl₂–EtOAc) to afford Nelf(18)-C(O)-

NC4GlcP (155 mg, 63.5%) as a white powder;

R_f0.5 (3:2 CH₂Cl₂–EtOAc); ¹H NMR

(CDCl₃): l 7.88 (bs, 1 H, H-9 or H-30), 7.41

(dd, J 1.5 Hz and J 7.0 Hz, 2 H, H-13, H-17),

7.33–7.14 (m, 5 H, H-14-16), 6.96–6.75 (m, 3

H, H-2–4), 6.11 (bs, 1 H, H-9 or H-30), 5.82

(d, J_1%,2%3.7 Hz, 1 H, H-1%), 5.30–5.12 [m, 2 H,

H-18, NHC(O)O], 4.55 (m, 1 H, H-10), 4.49

(d, 1 H, H-2%), 4.31–4.21 (m, 1 H, H-5%),

3.91–4.14 (m, 3 H, H-4%, H-6%), 3.80 (d, J_3%,4%

3.0 Hz, 1 H, H-3%), 3.60–3.32 (m, 4 H, H-11,

CH₂Glc), 3.10 (m, 2 H, CH₂NH), 2.95 (m, 1

H, H-20a), 2.70–2.40 (m, 2 H, H-19a, H-28),

2.35–2.10 (m, 2 H, H-19b, H-20b), 2.22 (s, 3

H, H-7), 2.00-1.10 (m, 16 H, H-21–27,

Ind(14)-C(O)NC4Glc(2TFA):—A 9:1 TFA–

water mixture (1 mL) was added at 0 °C to a

solution of Ind(14)-C(O)NC4GlcP (160 mg,

0.16 mmol) in CH₃CN (1 mL). After 10 min

stirring at 0 °C, the reaction mixture was

evaporated under diminished pressure and

chromatographed on silica gel (49:1–4:1

CH₂Cl₂–MeOH) to afford Ind(14)-C(O)-

NC4Glc(2TFA) (157 mg, 85%) as a white

powder; R_f0.40 (4:1 CH₂Cl₂–MeOH); R_t4.0

min (column I, solvent C, 210 nm); 5.1 min

1

(column III, solvent C, 210 nm); H NMR

(CD₃OD): l 7.96 (m, 2 H, H-28, H-29), 7.60

(m, 1 H, H-26), 7.35–7.15 (m, 10 H, H-2–5,

H-27, H-32–36), 5.24 (m, 1 H, H-9), 5.05 (d, J

2.7 Hz, 0.5 H, H-1%a), H-14 masked by sol-

vent, 4.45–4.35 (m, 1.5 H, H-1%b, H-8), 4.11

(m, 1 H), 3.85–3.25 (m, 11 H, H-24, H-3%–6%,

CH₂Glc), 3.25–2.65 (m, 16 H, H-7, H-12,

H-15–19, H-30, CH₂NH), 2.02 (m, 2 H, H-

13), 1.55 (m, 4 H, CH₂CH₂CH₂NH), 1.31 (s, 9

H, H-23); ¹³C NMR (CD₃OD): l 177.6 (C-

178

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

with the calculated mass for [M]=C₄₃H₆₄-

N₄O₁₁S.

CH₂CH₂CH₂NH), 1.46, 1.39, 1.31, 1.26 (4 s,

4×3 H, CH₃C), 1.13 (s, 9 H, H-32); ¹³C

NMR (CDCl₃): l 174.1 (C-8), 170.5 (C-29),

156.0 (C-1), 155.2 [NHC(O)O], 138.3, 136.0

(C-5, C-12), 129.8 (C-13, C-17), 129.0 (C-14,

C-16), 126.3 (C-3, C-15), 122.3 (C-6), 118.6

(C-4), 116.3 (C-2), 111.7, 108.9 (CH₃C), 105.2

(C-1%), 82.4 (C-3%), 82.1 (C-2%), 81.1 (C-4%), 72.5

(C-5%), 72.4 (C-18), 70.3 (CH₂CGlc), 70.0 (C-

28), 67.2 (C-6%), 59.4 (C-20), 56.5 (C-19), 51.1

(C-31), 51.0 (C10), 40.8 (CH₂NH), 35.9 (C-

21), 35.6 (C-11), 33.7 (C-26), 31.0 (C-27), 28.5

(C-32), 26.9 (CH₂CH₂Glc), 26.5 (CH₂CH₂-

NH), 30.9, 26.4, 25.8, 20.7 (C-22–25), 26.8,

26.2, 25.4 (CH₃C), 12.7 (C-7).

Hydrolysis kinetics.—Hydrolysis experi-

ments were performed by incubating 22 mL of

a 9:1 DMEM–MeOH solution (pH 7.3) of the

prodrug (156, 146, 239 and 174 mM for Saq-

C(O)C4Glc(2TFA), Saq-C(O)NC4Glc(4TFA),

Ind(14)-C(O)NC4Glc(2TFA), Nelf(18)-C(O)-

NC4Glc(2TFA), respectively) at 37 °C with

stirring. Hydrolysis was followed by HPLC

monitoring of either the disappearance of the

prodrug and appearance of the parent drug,

by injecting 60 mL of the solution onto the

HPLC column. HPLC analysis was performed

using a HP1100 apparatus with a Kromasil

C18 (100–5 mm)-packed column (150×3 mm)

for the saquinavir ester prodrug (column IV),

or with a Lichrospher 100 RP-18 (5 mm)-

packed column (250×3.2 mm) for the carba-

mate prodrugs (column I). The mobile phase

consisted of solvent D for the saquinavir

derivatives, solvent C for the indinavir deriva-

tives, and solvent E for the nelﬁnavir deriva-

tives. The prodrugs and/or drugs were

detected by measuring their UV absorption at

210 or 240 nm and the signals were inter-

preted by the software provided. Under their

respective HPLC conditions, the retention

times measured for the different compounds

were 7.3 min for saquinavir and 4.4 min for

Saq-C(O)C4Glc(2TFA) (column IV), 5.1 min

for saquinavir and 3.2 min for Saq-C(O)-

NC4Glc(4TFA), 11.6 min for indinavir and

4.0 min for Ind(14)-C(O)NC4Glc(2TFA), 12.9

min for nelﬁnavir and 5.2 min for Nelf(18)-

C(O)NC4Glc(2TFA) (column I). The prodrug

and/or drug concentration was determined

from HPLC calibration curves. These curves

were established under the same HPLC condi-

tions, using standard calibrated prodrug and

drug solutions that were prepared in the same

hydrolysis medium as the sample under inves-

tigation. The calibration curves were linear

(correlation coefﬁcient in the 0.9995–1 range)

in a concentration range of 0.00149–0.3737

mM for saquinavir, 0.0022–0.22 mM for Saq-

C(O)C4Glc(2TFA), 0.0007–0.1780 mM for

Saq-C(O)NC4Glc(4TFA), 0.0016–0.4073 for

indinavir, 0.0009–0.2234 mM for Ind(14)-

C(O)NC4Glc(2TFA), 0.0017–0.4403 mM for

nelﬁnavir and 0.0009–0.2330 mM for Nelf-

Nelf(18)-C(O)NC4Glc(2TFA):—TFA de-

protection as described for the preparation of

Ind(14)-C(O)NC4Glc(2TFA) was applied to

Nelf(18)-C(O)NC4GlcP (100 mg, 0.11 mmol)

in CH₃CN (1 mL). After treatment, the

residue was chromatographed on silica gel

(9:1–4:1 CH₂Cl₂–MeOH) to afford Nelf(18)-

C(O)NC4Glc(2TFA) (113 mg, 97%) as a white

powder; R_f0.2 (9:1 CH₂Cl₂–MeOH); R_t5.2

min (column I, solvent E, 210 nm); 5.8 min

1

(column III, solvent E, 210 nm); H NMR

(CD₃OD): l 7.48 (m, 2 H, H-13, H-17), 7.28

(d, J 7.1 Hz, 2 H, H-14, H-16), 7.17 (m, 1 H,

H-15), 7.00 (t, J 7.5 Hz, 1 H, H-3), 6.82 (m, 2

H, H-2, H-4), 5.22 (m, 1 H, H-18), 5.06 (m,

0.6 H, H-1%a), 4.51–4.42 (m, 1.4 H, H-10,

H-1%b), 3.85–3.00 (m, 13 H, H-11, H-20a,

H-2%–6%, CH₂Glc, CH₂NH), 2.75–2.60 (m, 2

H, H-19a, H-28), 2.35–2.15 (m, 2 H, H-19b,

H-20b), 2.22 (s, 3 H, H-7), 2.15–1.10 (m, 16

H, H-21–27, CH₂CH₂CH₂NH), 1.16 (s, 9 H,

H-32); ¹³C NMR (CD₃OD): l 173.6 (C-8,

C-29), 158.3 [NHC(O)O], 157.1 (C-1), 140.0,

137.2 (C-5, C-12), 131.3 (C-13, C-17), 130.1

(C-14, C-16), 127.5, 127.4 (C-3, C-15), 123.4

(C-6), 119.4 (C-4), 117.1 (C-2), 98.3 (C-1%b),

94.1 (C-1%a), 86.4 (C-3%b), 83.6 (C-3%a), 78.1

(C-5%b), 76.3 (C-2%b), 73.8 (CH₂Glc), 73.6 (C-

5%a, C-28), 73.2 (C-2%a), 71.8, 71.6 (C-4%a,b),

71.0 (C-18), 63.0, 62.9 (C-6%a, b), 60.6 (C-20),

57.6 (C-19), 52.2 (C-31), 52.1 (C-10), 41.9

(CH₂NH), 37.5 (C-21), 35.5 (C-11), 35.0 (C-

26), 32.2 (C-27), 29.0 (C-32), 28.6 (CH₂CH₂-

Glc), 27.6 (CH₂CH₂NH), 32.9, 27.5, 27.0, 21.8

(C-22–25), 15.6 (C-7). ESI-MS: m/z 845.9

[M+H]⁺, 867.9 [M+Na]⁺, in agreement

M. Rouquayrol et al. / Carbohydrate Research 336 (2001) 161–180

179

(18)-C(O)NC4Glc(2TFA). Each sample was

analysed in triplicate and each calibration

curve was determined in triplicate and re-

peated on the same day of analysis.

number of living cells. Cell viability of the

prodrugs, as estimated by the CC₅₀values,

was measured after 5 days of incubation

at 37 °C with various concentrations of

the tested product. The prodrug CC₅₀

values were determined from the curves of the

% of living cells against prodrug concen-

tration.

For Saq-C(O)C4Glc(2TFA), ln([prodrug]₀–

[prodrug(t)]) and of ln[drug(t)] against time

was plotted and its half-life of hydrolysis (t_1/2)

was measured from this curve. For the three

carbamate prodrugs investigated, no hydroly-

sis was observed after 7 days of incubation.

Anti6iral assays.—The in vitro antiviral ac-

tivity of the compounds was determined ac-

cording to published procedures.^30–32Brieﬂy,

CEM-SS cells were infected with a dose of

HIV-1 (LAI strain) infecting 50% of the cells.

Four days latter, the growing of HIV-1 was

evaluated by measuring the reverse transcrip-

tase (RT) which expresses the presence of the

virus in the supernatant culture medium. The

tested compounds were added to the cell cul-

tures after viral infection. RT inhibition % was

measured in comparison with the non treated

cells.

Acknowledgements

We thank the Fondation de la Recherche

Me´dicale (Sidaction) and the ANRS for ﬁnan-

cial support, and E. Merck, Hoffmann La

Roche and Agouron for a generous gift of

indinavir,

respectively.

saquinavir

and

nelﬁnavir,

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Synthesis and anti-HIV activity of glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir

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