Synthesis of new covalently bound κ-carrageenan-AZT conjugates with improved anti-HIV activities

Article abstract of DOI:10.1021/jm010969d

This paper describes the first covalent synthesis of κ-carrageenan-3′-azido-3′-deoxythymidine (AZT) conjugates. A succinate diester spacer was used to covalently couple AZT onto κ-carrageenan, resulting in a tripartite prodrug. Two methods (UV and radioac

Full text of DOI:10.1021/jm010969d

J . Med. Chem. 2002, 45, 1275-1283
1275
Syn th esis of New Cova len tly Bou n d K-Ca r r a geen a n -AZT Con ju ga tes w ith
Im p r oved An ti-HIV Activities
Patrick Vlieghe,†,‡ Thierry Clerc,‡ Christophe Pannecouque,§ Myriam Witvrouw,§ Erik De Clercq,§
J ean-Pierre Salles,‡ and J ean-Louis Kraus*,†
Laboratoire de Chimie Biomole´culaire, Faculte´ des Sciences de Luminy, 163 avenue de Luminy, case 901,
13288 Marseille Cedex 9, France, Laboratoires LAPHAL, Avenue de Provence, BP 7, 13718 Allauch Cedex, France,
and Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received J une 27, 2001
This paper describes the first covalent synthesis of κ-carrageenan-3′-azido-3′-deoxythymidine
(AZT) conjugates. A succinate diester spacer was used to covalently couple AZT onto
κ-carrageenan, resulting in a tripartite prodrug. Two methods (UV and radioactive counting)
are described and validated to determine the AZT loading onto the κ-carrageenan carrier. This
polymeric carrier, through its own intrinsic anti-HIV activity, is expected to act not only as a
drug delivery agent but also as an anti-HIV agent. Synergism between the two drugs
(κ-carrageenan and AZT) was demonstrated when MT-4 cells were preincubated with the
κ-carrageenan-AZT conjugate prior to HIV-1-infection. A threshold of AZT loaded onto the
κ-carrageenan was required to achieve this synergistic effect. Such κ-carrageenan-AZT
conjugates could be of great therapeutic interest because these conjugates, which contain a
low AZT concentration, present improved anti-HIV activities relative to free AZT. Moreover,
κ-carrageenan is a well-tolerated biopolymer, already used in the food industry.
In tr od u ction
Intensive efforts are underway worldwide to develop
chemotherapeutic agents effective against the human
immunodeficiency virus (HIV),1,2,3 the etiological agent
of the acquired immunodeficiency syndrome (AIDS).4
The search for an effective chemotherapeutic treatment
against HIV infection has led to the development of
agents that target specific and critical events in the HIV
replicative cycle.5,6 Among the current diversity of
compounds active against HIV, the 2′,3′-dideoxynucleo-
sides (ddNs) remain by far the most potent.7-9 The most
extensively studied of these agents is 3′-azido-3′-deoxy-
thymidine (AZT, zidovudine, Retrovir; Figure 1).10 This
ddN was the first that was approved by the Food and
Drug Administration11 for the treatment of AIDS.12,13
AZT, after conversion into its 5′-O-triphosphate ana-
F igu r e 1. Structures of AZT, the first FDA-approved drug
for HIV-treatment, and κ-carrageenan, one of the first sulfated
polysaccharides found to be active against HIV in vitro.
logue (AZT-TP) by cellular enzymes (kinases),14-16
inhibits HIV reverse transcriptase (HIV-RT) by com-
petitive inhibition of the viral reverse transcriptase (RT)
and/or incorporation and subsequent chain termination
of the growing viral DNA strand.17 The major limita-
tions of AZT are due to clinical toxicities18-23 and to the
development of AZT resistance by HIV.9,24-31 In at-
tempts to overcome these problems, numerous chemical
strategies have been developed by medicinal chemists
for designing AZT prodrugs (generally 5′-O-carboxylic
esters).10 The mechanism of action of these ester con-
jugates is based on hydrolysis and/or enzymatic cleavage
of their 5′-O-bonds, between the drug (AZT) and its
spacer group, to AZT within the cells. The expected
advantages of these 5′-O-substitued AZT prodrugs can
be multiple: improvement in anti-HIV activity,10 syn-
ergistic drug interactions,32-34 enhancement of AZT
intracellular uptake,35-37 and decrease of toxicity.10
Moreover, free AZT is known to act synergistically
with numerous compounds, including sulfated poly-
saccharides.38-43 Taking into account these findings, we
considered a strategy to synthesize new covalent con-
jugates between κ-carrageenan and AZT.
Carrageenans are natural sulfated polysaccharides
extracted from different species of red seaweed that
have a common structural backbone of D-galactose
residues. The three families of carrageenans are the λ
family (λ-, π-, and ꢀ-), the â family (â- and γ-), and the
κ family (µ-, ν-, κ-, and ι-). The κ-form is characterized
by a repeating unit of 4-sulfate-â-D-galactopyranose
linked 1f3 and 3,6-anhydro-R-D-galactopyranose linked
* To whom correspondence should be addressed. E-mail: kraus@
luminy.univ-mrs.fr. Phone: (33) 491 82 91 41. Fax: (33) 491 82 94 16.
† Faculte´ des Sciences de Luminy.
‡ Laboratoires LAPHAL.
§ Katholieke Universiteit Leuven.
10.1021/jm010969d CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/16/2002

1276 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Vlieghe et al.
Sch em e 1a
a
Reagents: (a) succinic anhydride, DMAP, DMF; (b) 3,4,5-trimethoxybenzoyl chloride, Et3N, EtOAc; (c) κ-carrageenan, DMF, 60 °C,
72 h.
1f4. The degree of sulfation for the κ-form is 25%
The combination of different antiviral drugs (e.g.,
mutual tripartite prodrugs)45 that target different events
in the HIV replicative cycle (e.g., virus attachment and
RT) has a triple aim: (i) it may lead to increased activity
(ideally synergism); (ii) it may allow reduction of the
individual doses and thus decrease drug toxicity; (iii) it
may prevent or delay the emergence of drug-resistant
virus strains.46
This paper describes, to our knowledge, the first
synthesis of κ-carrageenan-AZT conjugates,55 the meth-
ods used to determine the AZT loading onto the carrier,
and their in vitro anti-HIV activities correlating with
some of their biophysical properties.
(Figure 1).44
An ideal drug carrier (ideally a biomacromolecule)
must (i) protect the drug until it is delivered to the site
of action, (ii) localize the drug at or near the site of
action, (iii) allow the drug release through a chemical
and/or an enzymatic pathway, (iv) minimize host toxic-
ity, (v) be biodegradable, biochemically inert, and non-
immunogenic, (vi) be easily prepared inexpensively, and
(vii) be chemically and biochemically stable in its
formulation.45
In our concept, κ-carrageenan is expected to act not
only as a drug delivery carrier (for AZT release within
or near the infected cells) but also as an anti-HIV agent
by itself, which could act synergistically with AZT.
Indeed, carrageenans such as other sulfated polysac-
charides were found to be active against a wide variety
of enveloped virus.44,46 Carrageenans were among the
first sulfated polysaccharides found to possess anti-HIV
activity.47-49 They inhibit the binding of the virions to
the cell (by shielding the positively charged sites of
amino acids in the V3 loop of the viral envelope
glycoprotein (gp120))46,50 and the cell-to-cell fusion.51
Moreover, carrageenans seem to possess a combination
of good anti-HIV-1 activities and low anticoagulant
properties (in contrast with heparin) that may justify
clinical trials for their therapeutic potential (oral
absorption).44,52-54
Ch em istr y
We describe herein the synthesis of κ-carrageenan-
AZT conjugates via a succinate diester linkage (Scheme
1).
Prior to the coupling of AZT onto the κ-carrageenan
moiety, 5′-O-(succinate)-AZT 1 was first synthesized by
acylation of the 5′-hydroxyl group of AZT with succinic
anhydride in DMF in the presence of 4-(dimethylamino)-
pyridine (DMAP) in 85% yield.37
Since carrageenans are highly soluble in aqueous
solutions, we first tried to couple 1 onto the κ-carrag-
eenan carrier in water using N-ethyl-N′-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDCI) as an

Synthesis of κ-Carrageenan-AZT Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1277
activating agent, which is well-known to activate car-
boxylic acids in aqueous medium. Unfortunately, the
AZT loading onto the κ-carrageenan could not be
determined by a UV spectrophotometric technique
(measuring absorption at 266 nm) because of the strong
absorption (interference) of EDCI probably complexed
with the negatively charged sulfate groups of κ-carra-
geenan. The same problems of spectroscopic interference
were encountered when we attempted to link covalently
1 onto the κ-carrageenan in DMF (in which carrageen-
ans swell but do not dissolve, like some polymers used
in solid-phase synthesis) using various activating
agents: dicyclohexylcarbodiimide (DCC)/1-hydroxyben-
zotriazole (HOBt), DCC/pentachlorophenol (Pcp)/HOBt,
DCC/N-hydroxysuccinimide (HOSu), benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophos-
phonate (BOP), or 1,1′-carbonyldiimidazole (CDI). Un-
fortunately, each assay led to abnormal UV absorbancies
(interference), which impeded the determination of the
amount of AZT covalently bound to the κ-carrageenan
carrier. Finally, we utilized a chemical strategy involv-
ing the formation of a mixed anhydride using the 3,4,5-
trimethoxybenzoyl group as the leaving part of the
anhydride without another activating agent.56 This kind
of strategy has never been described for the coupling of
drugs onto a carrageenan carrier.
no free AZT derivatives were detected from the UV
reading of the blank assays.
To confirm the AZT loading onto the κ-carrageenan
moiety, the synthesis of the corresponding κ-carrag-
eenan-[2-14C]-AZT conjugate 3′ was achieved. For this
synthesis, a total of 808 nmol of [2-14C]-AZT was added
to 4.5 mmol of AZT (50 mCi/mmol), which was coupled
onto the κ-carrageenan via the synthesis of the 5′-O-
(succinate)-[2-14C]-AZT precursor.
Resu lts a n d Discu ssion
The succinate diester linkage for conjugation between
κ-carrageenan and AZT was initially selected with the
thought that release of AZT would be facilitated through
the action of esterases in human serum (normal human
serum, NHS). Indeed, from literature data, it has
already been demonstrated that tripartite prodrugs
containing the succinate diester linkage are hydrolyzed
more rapidly than their corresponding succinate mo-
noesters (Figure 2).45 The determination of the AZT
loading onto the κ-carrageenan carrier was required in
order to study the sensitivity to enzymatic hydrolysis
and the anti-HIV activities of conjugates 3 and 3′. Two
different techniques (UV and radioactive counting) were
used for the quantification of the AZT loading onto the
κ-carrageenan carrier.
AZT Loa d in g on to th e K-Ca r r a geen a n . (i) UV
Deter m in a tion . AZT content in the κ-carrageenan-
succinate diester-AZT conjugates 3 and 3′ was deter-
mined by measuring the absorption at 266 nm, which
is the absorption maximum for AZT. κ-Carrageenan
does not absorb at this wavelength. We first established
a standard curve at 266 nm, plotting absorbancies (OD)
versus variable AZT concentrations in water in the
presence of a constant amount of κ-carrageenan (1 mg/
mL). This standard curve was linear (r ) 1.000) with
respect to AZT concentration up to 90 µM (data not
shown). The AZT loading on each conjugate sample was
determined by interpolation of the standard curve at
266 nm with a conjugate concentration of 1 mg/mL.
This method (performed in triplicate assays) allowed
us to determine that conjugates 3 and 3′ contained (2.0
( 0.5) × 10-5 and (6.8 ( 0.5) × 10-5 mmol of AZT
covalently bound to 1 mg of κ-carrageenan, respectively.
As reported previously for curdlan sulfate,58 the esteri-
fication occurred predominately at the primary hydroxyl
function in this polysaccharidic family (hydroxyl func-
tion in the 6-C position of the 4-sulfate-â-D-galactopy-
ranose part of the κ-carrageenan disaccharidic unit). In
fact, a repeating disaccharidic unit of κ-carrageenan
possesses only one primary hydroxyl function likely to
be esterified. So the number of primary hydroxyl func-
tions per milligram of κ-carrageenan is equal to the
number of mmoles represented by 1 mg of κ-carrageenan
(2.45 × 10-3). For conjugates 3 and 3′, the degree of AZT
substitution calculated on a repeating disaccharidic unit
is only 0.8% and 2.8%, respectively.
In fact, conjugation of carboxylic acid 1 onto κ-carra-
geenan was achieved through the formation of a mixed
anhydride 2 using 3,4,5-trimethoxybenzoyl chloride in
the presence of triethylamine (Et3N) in EtOAc in 94%
yield (Scheme 1).56 The resulting anhydride 2 was
conjugated onto the κ-carrageenan in DMF at 60 °C over
72 h to give the κ-carrageenan-AZT conjugates 3 and
3′ via a succinate diester linkage (Scheme 1). Conjugates
3 and 3′ only differed by the percentage of AZT coupled
onto the κ-carrageenan. These new coupling conditions
make it possible to determine the AZT loading onto the
κ-carrageenan through a simple UV spectrophotometric
technique (at 266 nm) without any interference due to
the remaining coupling reagent, to byproducts of the
reaction (removed by filtration after the precipitation
of the covalent conjugate), or to κ-carrageenan. Then,
we investigated what was the optimum ratio of the
mixed anhydride 2 to the κ-carrageenan (equivalent
amount of mixed anhydride 2 versus equivalent amount
of κ-carrageenan calculated on its repeating disaccha-
ridic unit) to obtain the best procedure for AZT loading
onto the κ-carrageenan moiety.57 We found that conju-
gate 3 synthesized using a ratio of 1.25 equiv of AZT to
1 equiv of κ-carrageenan led to an AZT loading of 2.0 (
0.5 × 10-5 mmol of AZT covalently bound to 1 mg of
κ-carrageenan. However, the best procedure for AZT
loading onto the κ-carrageenan (conjugate 3′) was
obtained using a ratio of 10 to 1, respectively. This
synthesis led to an AZT loading of (6.8 ( 0.5) × 10-5
mmol of AZT covalently bound to 1 mg of κ-carrageenan
(as described below).
From these results, it can be deduced that an 8-fold
excess of anhydride 2 results in a three-and-one-half-
fold increase of the AZT loading onto the κ-carrageenan
carrier (conjugate 3′ versus conjugate 3). This apparent
weak AZT loading onto the κ-carrageenan carrier could
be explained in part by the gelling properties of κ-car-
rageenan, which forms double helices in solution, and
Blanks consisting of mixtures of 5′-O-(succinate)-AZT
and κ-carrageenan (treated under the same conditions
used for the corresponding covalent coupling reactions
but in absence of activating agents) were always per-
formed for UV determination of the AZT covalent
loading onto the κ-carrageenan. Moreover, as expected,

1278 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Vlieghe et al.
F igu r e 2. Concept of tripartite prodrugs applied to AZT and κ-carrageenan.
Ta ble 1. Chemical and Enzymatic Stability of Prodrug 1 and Conjugate 3′ in Acid, Neutral, and Basic Buffers and in Human Serum
(NHS)
% of nucleoside derivatives released (AZT in the case of compound 1;
AZT and compound 1 in the case of conjugate 3′) after 48 h of hydrolysis
citric acid,
pH 1.5, 0.1 M
acetate buffer,
pH 5.5, 0.1 M
PBS buffer,
pH 7.4, 0.1 M
PBS buffer,
pH 8.5, 0.1 M
NaOH,
pH 13.0, 0.5 M
human serum
(NHS), pH 7.2
compd
1
3′
16 ( 0.5
25 ( 0.5
4 ( 0.5
6 ( 0.5
5 ( 0.5
8 ( 0.5
21 ( 0.5
33 ( 0.5
100
100
100a
39 ( 0.5
a
t1/2 (half-life), the time required for 50% hydrolysis of prodrug 1 to AZT at 37 °C upon incubation in NHS, was 25 h.
by the steric hindrance between the two polymeric units
of the helix.59,60
1, release of AZT was less than 4% and that for
conjugate 3′, for which two hydrolysis products can be
expected (AZT and compound 1), the total amount of
hydrolysis products was less than 6%. (iii) Under
neutral conditions (PBS buffer, pH 7.0, 0.1 M), prodrug
1 and conjugate 3′ appeared to be very stable. No
residual-free AZT or prodrug 1 was detected by HPLC
after conjugate 3′ was incubated over 48 h under these
conditions (data not shown). (iv) In weakly basic condi-
tions, at pH 7.4 (PBS buffer, 0.1 M), simulating extra-
cellular fluids, prodrug 1 and conjugate 3′ appeared to
be quite stable. HPLC analytic profiles showed that for
prodrug 1, release of AZT was less than 5% and that
for conjugate 3′, for which two hydrolysis products can
be expected (AZT and compound 1), the total amount
of hydrolysis products was less than 8%. The whole
results clearly indicate that the ester bonds between
AZT and κ-carrageenan were stable in neutral and
weakly acid or basic aqueous solutions (Table 1). (v) In
contrast, under strong basic conditions (pH 13.0), both
compounds (prodrug 1 and conjugate 3′) were totally
hydrolyzed in a base-dependent manner to give 100%
of free AZT. This alkaline hydrolysis permits us to
confirm by HPLC analysis the total amount of AZT
linked onto κ-carrageenan previously determined by UV
and radioactivity. At pH 8.5 (PBS buffer, 0.1 M),
conjugate 3′ was slightly more sensitive to hydrolysis
(33% of hydrolysis products released) than prodrug 1
(21% of AZT released) (Table 1). These results are in
accordance with the concept of tripartite prodrugs via
(ii) Ra d ioa ctive Cou n tin g Deter m in a tion . To
confirm the above results, we performed the radiola-
beled chemical synthesis of conjugate 3′ with [2-14C]-
AZT using the same conditions as previously described.
The radioactive counting (triplicate assays) indicated
that (5.7 ( 0.5) × 10-5 mmol of AZT was covalently
bound to 1 mg of κ-carrageenan. Moreover, UV deter-
mination (triplicate assays) of this radiolabeled conju-
gate showed that (5.9 ( 0.5) × 10-5 mmol of AZT was
covalently bound to 1 mg of κ-carrageenan. In conclu-
sion, radiolabeled chemical synthesis of conjugate 3′
with [2-14C]-AZT confirmed the results obtained by UV
spectrophotometry, in agreement with our previous
findings.
Sta bility Stu d ies in Acid a n d Ba sic Bu ffer s a n d
in Hu m a n Ser u m (NHS).61,62 In Table 1, the stabilities
of prodrug 1 and conjugate 3′, incubated for 48 h at 37
°C, in acid and basic buffered solutions and in human
serum are summarized. The results obtained indicate
the following observations. (i) Under strong acid condi-
tions, at pH 1.5 (citric acid, 0.1 M), simulating gastric
juice, conjugate 3′ was slightly more sensitive to hy-
drolysis (25% of hydrolysis products released: AZT and
compound 1) than prodrug 1 (16% of AZT released)
(Table 1). (ii) Under weakly acid conditions, at pH 5.5
(acetate buffer, 0.1 M), mimicking endosomal compart-
ments, prodrug 1 and conjugate 3′ appeared to be quite
stable. HPLC analytic profiles showed that for prodrug

Synthesis of κ-Carrageenan-AZT Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1279
Ta ble 2. Anti-HIV Evaluation of Prodrug 1 and Conjugates 3 and 3′ in MT-4 Cellsa Infected with HIV-1 (Strain BRU)
mmol of AZT/
b
compd
EC50 (µg/mL)
CC50c (µg/mL)
mg of κ-carrageenand
EC50e (nM)
CC50f (µM)
SIg
FICh
1
3
3′
15
80
6.8
>50
>3333
75
3000
30
4
0.1
10
300
300
300
2.0 × 10-5
6.8 × 10-5
i
i
3.60
0.28
κ-carrageenan
AZT
25
>100
>4000
a
MT-4 cells were pretreated with antiviral compounds before HIV-1-infection (see Anti-HIV Activity Assays, (i), in Experimental Section).
b
EC50: concentration in µg/mL (expressed in κ-carrageenan equivalent) required to inhibit the cytopathicity of HIV-1 by 50% in MT-4
cells. c CC50: concentration in µg/mL (expressed in κ-carrageenan equivalent) required to cause 50% death of uninfected MT-4 cells.
d
Amount of AZT covalently bound onto κ-carrageenan. e EC50: concentration in nM (expressed in AZT equivalent) required to inhibit the
cytopathicity of HIV-1 by 50% in MT-4 cells. f CC50: concentration in µM (expressed in AZT equivalent) required to cause 50% death of
uninfected MT-4 cells. g SI: selectivity index ) CC50/EC50
.
FIC: fractional inhibitory concentration. i Not calculable because the cytotoxicity
h
of these conjugates are only due to their κ-carrageenan moieties at the concentration of 300 µg/mL.
Ta ble 3. Anti-HIV Evaluation of Prodrug 1 and Conjugate 3′ in MT-4 Cellsa Infected with HIV-1 (Strain IIIB)
mmol of AZT/
compd
EC50b (µg/mL)
CC50c (µg/mL)
mg of κ-carrageenand
EC50e (nM)
CC50f (µM)
SIg
FICh
9.22
1
3′
65
109
130
2000
188
25
1.6
12
300
300
6.8 × 10-5
i
κ-carrageenan
AZT
12
42
3500
a
MT-4 cells were infected by HIV-1 and then treated with antiviral compounds (see Anti-HIV Activity Assays, (ii), in Experimental
Section).70 EC50: concentration in µg/mL (expressed in κ-carrageenan equivalent) required to inhibit the cytopathicity of HIV-1 by 50%
b
in MT-4 cells. c CC50: concentration in µg/mL (expressed in κ-carrageenan equivalent) required to cause 50% death of uninfected MT-4
cells. Amount of AZT covalently bound onto κ-carrageenan. e EC50: concentration in nM (expressed in AZT equivalent) required to inhibit
d
the cytopathicity of HIV-1 by 50% in MT-4 cells. f CC50: concentration in µM (expressed in AZT equivalent) required to cause 50% death
g
h
i
of uninfected MT-4 cells. SI: selectivity index ) CC50/EC50
cytotoxicity of this conjugate is only due to its κ-carrageenan moiety at the concentration of 300 µg/mL.
.
FIC: fractional inhibitory concentration. Not calculable because the
a succinate diester linkage as mentioned above.45 (vi)
Interestingly, in NHS (pH 7.2), conjugate 3′ appeared
to be more stable (only 39% of AZT and prodrug 1 are
released from the macromolecular carrier) than prodrug
1 (100% of hydrolysis) (Table 1). These results (in
contradiction with the concept of tripartite prodrugs via
a succinate diester linkage, as demonstrated in Figure
2) suggest that the presence of the κ-carrageenan moiety
protects the ester linkages from hydrolysis by the serum
esterases. As has already been reported for curdlan
sulfate,62 polymeric drug delivery agents, because of
their steric hindrance, can protect the ester bonds from
hydrolysis within the active center of esterase. The total
release of AZT (100%) in NHS from a free mixture of
prodrug 1 and κ-carrageenan (data not shown) con-
firmed the effect of the steric hindrance observed when
the two drugs are covalently bound. Another very
interesting point, demonstrated through HPLC analysis
during the hydrolysis of conjugate 3′ in NHS, is that
the ester bond between the κ-carrageenan and the
succinate spacer is 6-fold more stable than the ester
bond between AZT and the succinate spacer.
In conclusion, the AZT release rate is prolonged in
the case of conjugate 3′ in NHS compared with a
prodrug without steric hindrance. This latter conjugate
is stable at a neutral pH range in aqueous solutions and
is hydrolyzed slowly through esterase catalysis in NHS
to release free AZT and prodrug 1, which is then
hydrolyzed to AZT. These results suggest that κ-carra-
geenan may be expected to act as a suitable drug
delivery agent (with intrinsic anti-HIV activity) giving
a prolonged release of AZT.
conjugates 3 and 3′, and the parent drugs (AZT and
κ-carrageenan). In the first series, MT-4 cells were
pretreated for 1 h with the drugs before HIV-1-infection
(Table 2). In the second series, the drugs were added to
the MT-4 cell cultures that had already been infected
by HIV-1 (Table 3). The 50% effective concentration
(EC50) represents the concentration required to inhibit
the virus-induced cytopathic effect by 50%.
(i) Effect of P r etr ea tm en t of MT-4 Cells w ith
An tivir a l Com p ou n d s on An ti-HIV P oten cy (Ta ble
2). To estimate the contribution to anti-HIV activity
(inhibition of virus attachment) from the κ-carrageenan
moiety, we first administered the antiviral compounds
before HIV-1-infection. As shown in Table 2, a three-
and-one-half higher loading of AZT covalently bound
onto κ-carrageenan resulted in a 40-fold (expressed in
κ-carrageenan equivalent) or in an 11-fold (expressed
in AZT equivalent) higher anti-HIV activity for conju-
gate 3′ (0.1 µg/mL or 6.8 nM, respectively) compared to
conjugate 3 (4 µg/mL or 80 nM, respectively). Probably
there is a critical ratio of the amount of the AZT
covalently bound onto the carrier to the amount of
κ-carrageenan to obtain optimal anti-HIV activity. In
the present experiment, conjugate 3′ was found to be
100-fold more active (0.1 µg/mL) than κ-carrageenan (10
µg/mL) and 4 times more potent (6.8 nM) than AZT itself
(25 nM).
(ii) An ti-HIV P oten cy of An tivir a l Com p ou n d s
on MT-4 Cells Alr ea d y In fected by HIV-1 (Ta ble
3). As shown in Table 3, conjugate 3′ was found to be
seven-and-one-half-fold more active (1.6 µg/mL) than
κ-carrageenan (12 µg/mL), while in contrast, AZT was
8 times more active than conjugate 3′ (expressed in AZT
equivalent) in this experiment.
When the outcomes of both experiments i and ii are
compared, pretreatment of MT-4 cells with conjugate
3′ before HIV-1-infection resulted in an increase of anti-
An tivir a l Activity Resu lts. The different drugs
were evaluated for their inhibitory effect on HIV-1
replication and their cytotoxicity in MT-4 cells (Tables
2 and 3). Two series of experiments were performed in
order to compare the anti-HIV properties of prodrug 1,

1280 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Vlieghe et al.
HIV potency (0.1 µg/mL with pretreatment compared
to 1.6 µg/mL without pretreatment). These results
suggest that preincubation of MT-4 cells with conjugate
3′ reduced virus entry into the cells. The κ-carrag-
eenan-succinate diester-AZT conjugate 3′ inhibited the
binding of the virions to the MT-4 cells and concomi-
tantly delivered AZT to these cells (giving a prolonged
release of AZT) to further inhibit the reverse transcrip-
tion step during the HIV replicative cycle. In the case
of conjugate 3′, a synergistic anti-HIV activity occurred
between the κ-carrageenan and AZT compounds. In fact,
this synergistic effect is only observed when the cells
are pretreated with conjugate 3′ before HIV-1-infection,
as demonstrated below.
centration that causes 50% toxicity in MT-4 cells) of
conjugates 3 and 3′ were considered, it appeared that
these conjugates did not prove to be more toxic than
κ-carrageenan itself. As shown by the results in Tables
2 and 3, the cytotoxicity of a conjugate is only due to its
κ-carrageenan moiety. In fact, the cytotoxicity of the
AZT moiety is masked by that of the κ-carrageenan
moiety. The AZT loading onto the κ-carrageenan carrier
is too weak for observation of the cytotoxicity due to AZT
itself. Thus, a decreased EC50 (expressed in κ-carrag-
eenan equivalent) reflected a gain in the selectivity
index (SI) of the conjugates (Tables 2 and 3).
Con clu sion
(iii) Syn er gistic Activity of K-Ca r r a geen a n a n d
AZT in MT-4 Cells.63 EC50 values were used for
calculation of the fractional inhibitory concentration
(FIC), which allows one to demonstrate a synergistic
effect between the two covalently linked drugs. The
following formula allows one to calculate this value:
Here, we report, to our knowledge for the first time,
the synthesis and anti-HIV properties of new covalently
bound κ-carrageenan-AZT conjugates (tripartite pro-
drugs). Synergistic activity of the two drugs (being part
of the covalent conjugate) was found when MT-4 cells
were pretreated with conjugate 3′ before HIV-1 infec-
tion. However, a critical ratio of AZT bound onto
κ-carrageenan is required to obtain a synergistic anti-
HIV activity (Table 2). At the present time, investiga-
tions are underway in our laboratories in order to
optimize the covalent coupling of AZT (or other nucleo-
side analogues) onto the κ-carrageenan carrier55,57 and
to select the best spacer between AZT and κ-carrag-
eenan.55,56,62 κ-Carrageenan-AZT conjugates may re-
duce the toxicity of AZT. At the present time, conjugate
3′ represents the hit compound of a new family of
covalent κ-carrageenan-AZT conjugates that will re-
quire further studies for safety and anti-HIV efficacy.
FIC ) FICκ-carrageenan + FICAZT
in which
FICκ-carrageenan
)
(EC50 of κ-carrageenan-AZT conjugate
(expressed in κ-carrageenan equivalent))/
(EC50 of κ-carrageenan alone)
and
FICAZT ) (EC50 of κ-carrageenan-AZT conjugate
Exp er im en ta l Section
(expressed in AZT equivalent))/(EC50 of AZT alone)
Nuclear magnetic resonance spectra (1H and 13C NMR) were
recorded with a Bru¨ker AC-250 spectrometer; chemical shifts
are expressed as δ units (part per million) downfield from TMS
(tetramethylsilane). Results from fast atom bombardment
(FAB+) mass spectral analysis were obtained by Dr. Astier
(Laboratoire de Mesures Physiques-RMN, USTL, Montpellier,
France) on a J EOL DX-100 using a cesium ion source and
glycerol/thioglycerol (1:1) or m-nitrobenzyl alcohol (NOBA) as
the matrix. Mass calibration was performed using cesium
iodide. IR spectra were recorded on a Perkin-Elmer FTIR 1605
spectrophotometer. UV spectra were obtained with an Uvikon
930 spectrophotometer (Kontron Instrument). Microanalyses
were carried out by Service Central d’Analyses du CNRS
(Venaison, France) and were within 0.4% of the theoretical
values. Analytical thin layer chromatography (TLC) experi-
ments were performed using silica gel plates 0.2 mm thick
(60F254 Merck). Preparative flash column chromatography
experiments were carried out on silica gel (230-240 mesh, G60
Merck). Analytical HPLC was performed on a Waters 600E
instrument with a M991 detector using the following condi-
tions: 4.6 mm × 150 mm column (Waters Spherisorb S5 ODS2,
5 µM); mobile phases A ) 0.1% TFA in H2O, B ) CH3OH; flow
rate of 1 mL/min. All reagents were of commercial quality
(Aldrich Company, Saint Quentin Fallavier, France) from
One should recall that depending on the FIC values
calculated from the two participating drug moieties, the
following conclusions can be drawn: when FIC < 0.5,
there is a synergistic biological effect between the two
drugs; when 0.5 < FIC < 1.0, there is a subsynergistic
biological effect between the two drugs; when FIC ) 1.0,
there is an additive biological effect between the two
drugs; when 1.0 < FIC < 2.0, there is subantagonistic
biological effect between the two drugs; and when FIC
> 2.0, there is an antagonist biological effect between
the two drugs.63,64
As shown by the calculated FIC indexes in Tables 2
and 3, a synergistic anti-HIV activity was observed
between κ-carrageenan and AZT only when MT-4 cells
were pretreated with conjugate 3′ before HIV-1-infection
(FIC ) 0.28, Table 2). Under the same experimental
conditions, mixtures of free κ-carrageenan and AZT in
the same ratio as for the κ-carrageenan-AZT conjugates
showed only additive effects (data not shown). Interest-
ingly, conjugate 3, which possessed a lower AZT loading
than conjugate 3′, did not show a synergistic anti-HIV
activity (FIC ) 3.60, in Table 2) after pretreatment of
the MT-4 cells with this conjugate before HIV-1-infec-
tion. Probably there is a critical ratio between the AZT
covalently bound onto the carrier and the κ-carrageenan
amount that is needed to achieve an optimal anti-HIV
response resulting in a synergistic activity.
freshly opened containers. κ-Carrageenan (MW ) 600 000,
DPw ) 1470, I ) 1.35; determined by size exclusion chroma-
tography/multiangle laser light scattering (SEC-MALLS)) was
obtained from SKW Biomaterials (l’Isle-sur-la-Sorgue, France).
[2-14C]-AZT (50 mCi/mmol) was purchased from Isotopchim
(Peyruis, France). Radioactive counting in Emulsifier-Safe
Packard was performed with a Packard 1600 TR 103656 liquid
scintillation analyzer with internal chemiluminescence cor-
rection.
(iv) Cytotoxicity a n d Selectivity In d ex. When the
cytotoxicity (CC50, 50% cytotoxic concentration, or con-
Cells a n d Vir u ses. (i) MT-4 Cells a n d HIV-1 (Str a in
BRU) (Ta ble 2). The CEM cell line and the T-leukemia virus

Synthesis of κ-Carrageenan-AZT Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1281
type I (HTLV-I) CD4+ T-cell line, MT-4, were cultured in
RPMI/10% fetal calf serum (FCS), and the medium was
replaced twice a week. The laboratory-adapted strain HIV
clade B (strain BRU) stock was prepared from the supernatant
of the infected CEM cell line, and aliquots were kept frozen
at -80 °C until use.1
(ii) MT-4 Cells a n d HIV-1 (Str a in IIIB) (Ta ble 3). MT-4
cells65 were grown and maintained in RPMI 1640 medium
supplemented with 10% heat-inactivated FCS, 2 mM L-
glutamine, 0.1% sodium bicarbonate, and gentamicin (20 µg/
mL). The origin of the HIV-1 (strain IIIB) stock has been
described elsewhere.66
NaOH, or normal human serum (NHS) was added a total of 5
mg of the conjugate 3′, and the mixture was incubated at 37
°C in a water bath. At various time intervals, the samples (150
µL) were withdrawn and added immediately to ice-cold
methanol (350 µL). The resulting samples were centrifuged
(7 min, 3000 rpm). A total of 300 µL of the supernatants was
withdrawn, evaporated under reduced pressure, and diluted
in 50 µL of methanol in order to obtain 6-fold concentrated
samples. The supernatants were filtered through nylon filters
(0.45 µm) and then analyzed by HPLC using the following
method: 20% of solvent B in solvent A to 100% of solvent B in
20 min. The injection volume was 20 µL. The absorption
maximum for the studied conjugate 3′ was at 267 nm;
therefore, this wavelength was used for the HPLC detection.
Peak retention times (tr) were 10.0 min for AZT and 14.1 min
for compound 1. The percentages of AZT and compound 1
release calculated from peak areas for conjugate 3′ hydrolysis
in buffers and in NHS are summarized in Table 1.
An ti-HIV Activity Assa ys. (i) Anti-HIV activity was
monitored by the efficiency of the compounds to inhibit the
cytopathicity of HIV-1 (strain BRU) in MT-4 cells (Table 2) as
already described.67,68 Briefly, 3 × 105 MT-4 cells were first
preincubated with 100 µL of various concentrations of com-
pounds dissolved in DMSO or in H2O and then were diluted
in phosphate-buffered saline (PBS) solution for 1 h at 37 °C.
Then 100 µL of an appropriate virus dilution was added, and
the cells were incubated for 1 h at 37 °C. After three washes,
cells were resuspended in a culture medium in the presence
or absence of the compounds. Cultures were then continued
for 7 days at 37 °C under 5% CO2 atmosphere, and the medium
was replaced on day 3 postinfection with culture medium
supplemented or not with drug compounds. Each culture
condition was carried out in duplicate. Virus-induced cyto-
pathicity was followed each day with an inverted optical
microscope. Typically, the virus dilution used in this assay
(multiplicity of infection of 0.1 CCID/cell) led to cytopathicity
on day 5 postinfection. The inhibitory concentration of drug
compounds was expressed as the concentration that caused
50% inhibition of viral cytopathicity (EC50) without direct
toxicity for the cells. The cytotoxic concentration (CC50) of drug
compounds was monitored on the basis of the growth of
noninfected cells by the trypan blue exclusion method and
corresponded to the concentration required to cause 50% cell
death. It should be emphasized that when compounds required
the addition of DMSO to be solubilized in water, the concen-
tration in the volume of DMSO used was always less than 10%
with respect to water (the final concentration of DMSO in
MT-4 cells incubation medium being less than 2%). As far as
DMSO could affect the antiviral activity of the tested com-
pounds,69 antiviral assays in which solutions containing equal
concentrations of DMSO in water were performed and used
as standard assays for each tested drug. EC50 and CC50
reported values were then calculated from these standard
assays. Moreover, final DMSO dilutions (1/1000) were much
lower than those enhancing in vitro HIV-1-infection of T-cells.69
Ch em ica l Syn th eses. Mon o(3′-a zid o-3′-d eoxyth ym id in -
5′-yl) Ester 1,4-Bu ta n ed ioic Acid or 3′-Azid o-3′-d eoxy-5′-
O-(su ccin a te)th ym id in e 1. To a solution of AZT (0.802 g,
3.00 mmol, 1 equiv) in anhydrous DMF (15 mL), under N2,
containing DMAP (0.367 g, 3.00 mmol, 1 equiv), was added
succinic anhydride (0.315 g, 3.15 mmol, 1.05 equiv). The
reaction mixture was stirred at room temperature for 14 h.
Then, the solvent was evaporated under reduced pressure. The
residual oil was dissolved in CH2Cl2 (20 mL) and successively
washed with 1 N HCl (2 × 15 mL) and water (2 × 15 mL).
The combined aqueous solutions were extracted twice with
CH2Cl2. The combined organic layers were dried over anhy-
drous Na2SO4, and the solvent was removed under vacuum.
The residue was purified by flash chromatography on silica
gel, using CH3OH/CH2Cl2 8:92 as eluent, to give the title
compound 1 as a white foam (0.937 g, 85%): Rf ) 0.22 (CH3-
OH/CH2Cl2 10:90); IR (CH2Cl2, cm-1) 3482 (CO2H), 3051 (NH),
2110 (N3), 1720 and 1704 (broad CdO, acid and ester), 1690
(CdO, CONH); 1H NMR (CD3OD) δ 1.82 (s, 3H, CH3Thy), 2.3-
2.4 (m, 2H, H-2′,2′′), 2.5-2.6 (m, 4H, AZT-O2C-CH2-CH2-
CO2H), 3.9-4.0 (m, 1H, H-4′), 4.1-4.4 (m, 3H, H-3′, H-5′,5′′),
6.08 (t, 1H, J ) 6.5 Hz, H-1′), 7.44 (s, 1H, H-6); 13C NMR (CD3-
OD) δ 10.76 (CH3Thy), 28.44 (AZT-O2C-CH2-CH2-CO2H),
28.52 (AZT-O2C-CH2-CH2-CO2H), 35.98 (C-2′), 60.03 (C-
3′), 62.70 (C-5′), 81.33 (C-4′), 84.50 (C-1′), 110.13 (C-5), 135.97
(C-6), 150.34 (C-2), 164.46 (C-4), 172.27 (AZT-O2C-(CH2)2-
CO2H), 175.41 (AZT-O2C-(CH2)2-CO2H); MS (FAB+) 368 (M
+ H)+; HPLC tr ) 14.1 min. Anal. (C14H17N5O7) C, H, N.
Mixed An h yd r id e of 3,4,5-Tr im eth oxyben zoic Acid
a n d Mon o-(3′-a zid o-3′-d eoxyth ym id in -5′-yl) Ester 1,4-
Bu ta n ed ioic Acid 2. To a solution of the ω-carboxylic acid 1
(1.025 g, 2.79 mmol, 1 equiv) in anhydrous EtOAc (15 mL),
under N2, containing Et3N (1.17 mL, 8.37 mmol, 3 equiv) was
added dropwise at 0 °C 3,4,5-trimethoxybenzoyl chloride (0.708
g, 3.07 mmol, 1.1 equiv) in anhydrous EtOAc (10 mL). The
reaction mixture was then stirred for 4 h at room temperature,
diluted with EtOAc (15 mL), and washed twice with 0.5 N HCl
(17 mL). After extraction, the organic layer was washed twice
with water (2 × 20 mL) and dried over anhydrous Na2SO4.
EtOAc was removed under reduced pressure, and the product
slowly crystallized under vacuum to give the title compound
2 as a white powder (0.937 g, 85%). The resulting mixed
anhydride was used in the next step without further purifica-
tion: IR (CH2Cl2, cm-1) 3020 (NH), 2111 (N3), 1790 (CdO,
anhydride), 1734 (CdO, ester), 1694 (CdO, CONH), 1590
(trimethoxybenzoyl), 1333 (OCH3); 1H NMR (CDCl3) δ 1.84 (s,
3H, CH3Thy), 2.3-2.4 (m, 2H, H-2′,2′′), 2.64 (pt, 2H, AZT-
O2C-CH2-CH2-CO2CO-Ph(OCH3)3), 2.73 (pt, 2H, AZT-
O2C-CH2-CH2-CO2CO-Ph(OCH3)3), 3.82 (s, 6H, OCH3 meta),
3.87 (s, 3H, OCH3 para), 3.9-4.0 (m, 1H, H-4′), 4.1-4.4 (m,
3H, H-3′, H-5′,5′′), 6.02 (t, 1H, J ) 6.2 Hz, H-1′), 7.25 (s, 1H,
H-6), 7.1-7.3 (pd, 2H, 2H ortho), 9.76 (brs, 1H, NH-3); 13C
NMR (CDCl3) δ 12.73 (CH3Thy), 28.64 (AZT-O2C-CH2-CH2-
CO2CO-Ph(OCH3)3), 30.70 (AZT-O2C-CH2-CH2-CO2CO-
Ph(OCH3)3), 37.60 (C-2′), 56.63 (2H3CO meta), 60.53 (C-3′),
61.27 (H3CO para), 63.77 (C-5′), 81.96 (C-4′), 85.89 (C-1′),
(ii) The inhibitory effects of AZT, κ-carrageenan, and their
prodrugs on HIV-1 (strain IIIB) replication (Table 3) were
monitored by the inhibition of the virus-induced cytopathicity
in MT-4 cells 5 days after infection, as described elsewhere.70
Cytotoxicity of the compounds was determined by measuring
the viability of mock-infected cells on day 5.
Hyd r olysis of P r od r u g 1 in Bu ffer s a n d in Hu m a n
Ser u m (NHS).56 To 990 µL of acetate buffer, water, PBS
buffers, NaOH, or NHS was added a total of 10 µL of a solution
of the desired prodrug 1 (10 mg/mL in DMSO), and the mixture
was incubated at 37 °C in a water bath. At various time
intervals, the samples (100 µL) were withdrawn and added
immediately to ice-cold methanol (400 µL). The resulting
samples were centrifuged (7 min, 3000 rpm). The supernatants
were filtered through nylon filters (0.45 µm), and then
analyzed by HPLC using the following method: 20% of solvent
B in solvent A to 100% of solvent B in 20 min. The injection
volume was 20 µL. The absorption maximum for the studied
prodrug 1 was at 267 nm; therefore, this wavelength was used
for the HPLC detection. Peak retention times (tr) were 10.0
min for AZT and 14.1 min for compound 1. The percentages
of AZT release calculated from peak areas for compound 1 for
hydrolysis in buffers and in NHS are summarized in Table 1.
Hyd r olysis of Con ju ga te 3′ in Bu ffer s a n d in Hu m a n
Ser u m (NHS). To 1 mL of acetate buffer, water, PBS buffers,

1282 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
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108.21 (2C ortho), 111.56 (C-5), 123.76 (C ipso), 136.14
(C-6), 144.08 (C para), 150.50 (C-2), 153.51 (2C meta), 162.48
(AZT-O2C-(CH2)2-CO2CO-Ph(OCH3)3), 164.47 (C-4), 168.94
(AZT-O2C-(CH2)2-CO2CO-Ph(OCH3)3), 171.72 (AZT-O2C-
(CH2)2-CO2CO-Ph(OCH3)3); MS (FAB+) 562 (M + H)+. Anal.
(C24H27N5O11) C, H, N.
K-Ca r r a geen a n -Su ccin a te Diester -AZT Con ju ga tes 3
a n d 3′. To a solution of κ-carrageenan (0.100 g, 0.245 mmol,
1 equiv for 3; 0.113 g, 0.276 mmol, 1 equiv for 3′; calculated
on the repeating disaccharidic unit) in anhydrous DMF (15 or
18 mL for 3 or 3′, respectively), under N2, was added dropwise
mixed anhydride 2 (0.172 g, 0.306 mmol, 1.25 equiv for 3; 1.550
g, 2.760 mmol, 10 equiv for 3′) in anhydrous DMF (10 mL).
The reaction mixture was stirred at 60 °C for 72 h and then
precipitated with 1-propanol (30 or 40 mL for 3 or 3′,
respectively), as is done for some polymers in solid-phase
synthesis. The precipitate was filtered on a Bu¨chner funnel
and washed with acetone, methanol, dichloromethane, and
acetone again to remove all the unbound products and the
byproducts. The conjugates were dried under vacuum to give
82 mg of conjugate 3 and 92 mg of conjugate 3′.
For conjugate 3, OD266 ) 0.1952, corresponding to (2.0 (
0.5) × 10-5 mmol of AZT covalently bound to 1 mg of
κ-carrageenan.
For conjugate 3′, OD266 ) 0.6608, corresponding to (6.8 (
0.5) × 10-5 mmol of AZT covalently bound to 1 mg of
κ-carrageenan.
Ack n ow led gm en t. This research was supported by
grants from LAPHAL Laboratories and PACA Regional
Council (P.V.). INSERM U-322 is acknowledged for
financial support and for antiviral testing of drugs
against HIV-1 (strain BRU). We thank Kristien Erven
for excellent technical assistance.
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