2060 New J. Chem., 2012, 36, 2060–2069 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

Cite this: New J. Chem., 2012, 36, 2060–2069

New potential prodrugs of aciclovir using calix[4]arene as a lipophilic

carrier: synthesis and drug-release studies at the air–water interfacew

Guillaume Sautrey, Igor Clarot, Ewa Rogalska and

Jean-Bernard Regnouf-de-Vains*

Received (in Montpellier, France) 30th April 2012, Accepted 20th July 2012

DOI: 10.1039/c2nj40338b

Two tetra-p-tert-butyl-calix[4]arene species bearing one or two anti-HSV aciclovir units tethered

via carbodiester linkages at the lower rim were synthesized as possible antiviral prodrugs.

The amphiphilic properties of these derivatives were studied using Langmuir balance at the

air–water and air–carbonate buffer interfaces; the monolayers formed were stable on both

subphases. Monolayers formed with these molecules on a carbonate buffer subphase at pH 10

and 37 1C were then used for monitoring hydrolysis of the diester linkage. The release of free

aciclovir of around 30% in 3 days was observed with both derivatives, as shown with HPLC.

Introduction

Since the pioneering work of Hitchings,1 nucleoside analogues

became a major therapeutical class of molecules involved

notably in virology and cancerology.2,3 After intracellular

conversion to phosphorylated forms, they are able to mimic

the structural and functional features of natural nucleosides,

and interact with viral or cellular enzymatic systems involved

in nucleic acid biosynthesis. However, their low solubility in

water,4,5 and in vivo enzymatic degradation6 result in a poor

bioavailability.

Consequently, in order to bypass these problematics, intense

research programmes have been dedicated to the development

of prodrugs of nucleoside or nucleotide analogues.7–12

Our group is involved in the design of new potential

therapeutic molecules build on calixarene scaffolds thought

to play as drug-organizers or dispensers. Indeed, as recently

reviewed by de Fatima et al.,13 Kalchenko et al.,14 and Coleman

et al. or Pourabdollah et al.,15 a few reports describe therapeutic

activities of calixarenes and their derivatives. Some of them

have been conceived as potentially orally administrable, hydro-

phobic but amphiphilic prodrugs of model antibiotics, e.g.

penicillins or ‘‘quinolones’’, in which the latter are attached to

the calixarene platform via an amide16 or a labile ester17

junction. The amphiphilic properties of the latter have been

confirmed by studies of their behavior at the air–water interface

using Langmuir balance techniques.18,19 Nevertheless, the lack

of solubility of organosoluble derivatives in aqueous media

made them non-compliant for in vitro standard evaluation as

antimicrobial agents, as well as for chemical hydrolysis studies.

For these reasons, a water-soluble analogue bearing a nalidixic

acid arm was designed and demonstrated the effective drug

release in biological medium (rat serum), and a pronounced

antibacterial activity.20 Very recently, antibacterial activities of

some organosoluble calixarene-drug conjugates have been

studied by disk diffusion assays using pure DMSO as solvent.21

On the other hand, we developed an extensible methodology

enabling hydrolytic studies of this kind of derivatives, more

specifically shaped as ‘‘quinolone’’ prodrugs, by means of

Langmuir and HPLC techniques.22 The results thus obtained

led us to extend this concept to other classes of therapeutical

agents, more precisely in the field of virology. For this purpose,

with the aim of developing amphiphilic derivatives prone to

interact with cell membranes, and to improve the bioavailability

of the attached drug, we focused on antiviral nucleoside analo-

gues, using the 9-(2-hydroxyethoxy)-methylguanine (aciclovir),

the well-known cutaneous, mucosal, ophthalmic and systemic

anti-HSV or anti-VZV precursor, as a model compound.

In the literature, very few reports are dedicated to the synthesis

and study of calixarene-nucleoside conjugates. Davis et al. devel-

oped tetra-guanosine– and tetra-adenosine–calix[4]arene conju-

gates in the 1,3-alternate conformation with the aim of producing

ion-channels and ion pair receptors by self-assembling.23,24

Kim et al. have studied self-assembling properties of calix[4]arene

derivatives involving 20-deoxy-thymidine analogues grafted on the

upper rim via amide linkers.25 More recently, Consoli et al.

reported the synthesis of several calixarene-nucleoside conjugates,

comprising 20-deoxy-thymidine, -adenosine, -cytosine and

-guanosine nucleosides, grafted via phosphodiester linkers on

the lower rim of the tetra-p-tert-butylcalix[4]arene.26 The authors

described the self-assembling behavior of these derivatives in

chloroform27 and water,28 as well as a preliminary investigation

Universite de Lorraine, SRSMC, UMR 7565 CNRS; equipe GEVSM,Faculte de Pharmacie, 5, rue Albert Lebrun, 54001 Nancy cedex,France. E-mail: jean-bernard.regnouf@pharma.uhp-nancy.fr;Fax: +33 3 83 68 23 45; Tel: +33 3 83 68 23 15w Electronic supplementary information (ESI) available: See DOI:10.1039/c2nj40338b

NJC Dynamic Article Links

www.rsc.org/njc PAPER

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online / Journal Homepage / Table of Contents for this issue

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 2060–2069 2061

dedicated to their capability to inhibit in vitro DNA replication

during PCR amplification.29

Most of these contributions deal with natural nucleosides,

with the aim of generating self-assembled nanostructures or a

biological activity by means of pre-organization of nucleoside

subunits. Nevertheless, and as far as we know, no specific

studies devoted to a prodrug behavior of such structures have

been described.

Indeed, the tetra-p-tert-butylcalix[4]arene could be considered as

a hydrophobic drug carrier. In addition, the combination of this

hydrophobic core with polar nucleoside moieties leads to an

amphiphilic structure. It could be thus expected an affinity of

calixarene–aciclovir adducts, shaped as prodrug, for physiological

interfaces, with an efficient transport of bioactive molecules to

their target. In this sense, we report here the synthesis of two new

potential prodrugs of aciclovir, chosen as a model compound of

nucleoside analogue drugs, using the tetra-p-tert-butylcalix[4]arene

as a carrier via formation of carbonate linkage. The interfacial

hydrolysis of these amphiphilic substances was studied using the

air–water interface by formation of Langmuir monolayers on the

surface of an alkaline buffer.

Experimental

Synthesis

Melting points (1C, uncorrected) were determined using an

Electrothermal 9200 Capillary apparatus. 1H- and 13C-NMR

spectra were recorded on a Bruker DRX 400 MHz (chemical

shifts in ppm, J in Hz), in CDCl3 (7.26 and 77.00 ppm) or

DMSO-d6 (2.50 and 39.50 ppm). 19F-NMR spectra were recorded

on a Bruker DRX 250MHz (chemical shifts in ppm), inDMSO-d6,

without calibration. Mass spectra (electrospray-ESI positive mode)

were recorded on aMicromass Platform II apparatus, at the Service

Commun de Spectrometrie de Masse Organique, Nancy. Infrared

analysis was performed using a Bruker Vector 22 apparatus

(KBr, n in cm�1) and UV spectra were recorded on a SAFAS

UVmc2 apparatus, lmax in nm, e in mol�1 dm3 cm�1. Elemental

analyses were performed at the Service de Microanalyse,

Nancy. Merck TLC plates were used for chromatography

analysis (SiO2, ref. 1.05554; Al2O3, ref. 1.05581). All commer-

cially available products were used without further purification

unless otherwise specified. For NMR data of compounds 2, 5, 6

and 8, letter A refers to free phenols and B to substituted ones;

for 1, 7 and 9, C refers to the central free phenol ring.

5,11,17,23-Tetra-(tert-butyl)-25-(3-hydroxypropoxy)-

26,27,28-tris-hydroxy-calix[4]arene (1). A suspension of tetra-

p-tert-butylcalix[4]arene (1.22 g, 1.87 � 10�3 mol), K2CO3

(0.14 g, 1.03 � 10�3 mol) and KI (1.00 g, 6.02 � 10�3 mol) in

freshly distilled MeCN (110 mL) was refluxed under Ar for

1 h. 3-Bromopropanol (0.18 mL, 2.06 � 10�3 mol) was then

added, and the reflux was maintained for 24 h (TLCmonitoring

SiO2, CH2Cl2 (95) :MeOH (5) v/v). The solvent was evaporated

to dryness, the solid residue was triturated in CH2Cl2 (20 mL),

filtered over kieselguhr, and rinsed with CH2Cl2 (2 � 20 mL).

The solvent was evaporated and the crude product was

purified by chromatography (SiO2, CH2Cl2 (100) to CH2Cl2(99.5) :MeOH (0.5) v/v) to give pure 1 as an amorphous white

solid (0.523 g, 40%). M.p.: 4230 1C. IR (KBr): 3566, 3294,

3160 (n(O-H)); 2960 (nas(CH3)); 2870 (ns(CH2)); 1485, 1462

(d(CH3)); 1203 (n(CQC)); 1047 (n(C-O)). 1H NMR (CDCl3):

1.19 (s, 9H, C(CH3)3 B); 1.22 (s, 18H, C(CH3)3 A); 1.23 (s, 9H,

C(CH3)3 C); 2.27 (m, 2H, OCH2CH2CH2OH); 3.46–4.26 (AB,

J= 13.6, 4H, Ar(A)CH2Ar(C)); 3.46–4.31 (AB, J= 13.6, 4H,

Ar(A)CH2Ar(B)); 3.57 (t, J = 6.2, 1H, OCH2CH2CH2OH);

4.17 (t, J = 5.9, 2H, OCH2CH2CH2OH); 4.27 (m, 2H,

OCH2CH2CH2OH); 7.00 (d, J = 2.4, 2H, ArHm A); 7.06

(s, 2H, ArHm C); 7.09 (d, J = 2.4, 2H, ArHm A); 7.10 (s, 2H,

ArHm B); 9.78 (s, 2H, ArOH A); 10.26 (s, 1H, ArOH C). 13C

NMR (CDCl3): 29.0 (OCH2CH2CH2OH); 31.2 (C(CH3)3 B);

31.4 (C(CH3)3 C); 31.5 (C(CH3)3 A); 31.8 (Ar(A)CH2Ar(B));

31.9 (Ar(A)CH2Ar(C)); 33.9 (C(CH3)3 A); 34.0 (C(CH3)3 C);

34.2 (C(CH3)3 B); 58.8 (OCH2CH2CH2OH); 73.7 (OCH2CH2-

CH2OH); 125.7; 125.8 (Cm A); 125.9 (Cm C); 126.6 (Cm B);

127.4 ((Co A)CH2Ar(B)); 128.0 (Co C); 128.2 ((Co A)

CH2Ar(C)); 133.3 (Co B); 143.5 (Cp A); 143.8 (Cp B); 147.5

(Ci C); 148.1 (Ci A); 148.3 (Cp C); 148.9 (Ci B). Anal. calcd for

C47H62O5 (706.99): C 80.01, H 9.22%; found: C 80.21, H

9.17%. ESI-MS (pos. mode): 707.47 [M + H]+, 729.45 [M +

Na]+. labs (CH2Cl2): 280 (7889); 287 (sh, 7082).

5,11,17,23-Tetra-(tert-butyl)-25,27-bis-(3-hydroxypropoxy)-

26,28-bis-hydroxy-calix[4]arene (2).A suspension of tetra-p-tert-

butylcalix[4]arene (2.00 g, 3.09 � 10�3 mol), K2CO3 (1.02 g,

7.36 � 10�3 mol) and KI (2.50 g, 15.06 � 10�3 mol) in freshly

distilled MeCN (100 mL) was refluxed under Ar for 1 h.

3-Bromopropanol (0.8 mL, 9.15 � 10�3 mol) was then added,

and the reflux was maintained for 16 h (TLC monitoring SiO2,

CH2Cl2 (95) :MeOH (5) v/v). The solvent was evaporated to

dryness, the solid residue was triturated in CH2Cl2 (50 mL),

filtered over kieselguhr, and rinsed with CH2Cl2 (3 � 50 mL).

The solvent was evaporated and the crude product was

purified by chromatography (SiO2, CH2Cl2 (100) to CH2Cl2(98) :MeOH (2) v/v) to give pure 2 as an amorphous white

solid (1.28 g, 54%). M.p.: 4230 1C. IR (KBr): 3406 (n(O-H));

2960 (nas(CH3)); 2869 (ns(CH2)); 1486 (d(CH3)); 1204

(n(CQC)); 1053 (n(C-O)). 1H NMR (CDCl3): 0.99 (s, 18H,

C(CH3)3 A); 1.28 (s, 18H, C(CH3)3 B); 2.20 (m, 4H,

OCH2CH2CH2OH); 3.36–4.20 (AB, J= 13.1, 8H, ArCH2Ar);

4.13 (m, 8H, OCH2CH2CH2OH and OCH2CH2CH2OH); 4.37

(t, J = 5.4, 2H, OCH2CH2CH2OH); 6.85 (s, 4H, ArHm A);

7.06 (s, 4H, ArHm B); 7.74 (s, 2H, ArOH). 13C NMR (CDCl3):

31.0 (C(CH3)3 A); 31.7 (C(CH3)3 B); 31.9 (ArCH2Ar); 33.1

(OCH2CH2CH2OH); 33.8 (C(CH3)3 B); 34.0 (C(CH3)3 A);

61.3 (OCH2CH2CH2OH); 75.5 (OCH2CH2CH2OH); 125.1

(Cm B); 125.7 (Cm A); 127.3 (Co B); 132.5 (Co A); 141.8 (Cp

B); 147.2 (Cp A); 149.5 (Ci A); 150.3 (Ci B). Anal. calcd for

C50H68O6 (765.07): C 78.49, H 8.96%; found: C 78.66, H

8.91%. ESI-MS (pos. mode): 765.51 [M + H]+, 787.49 [M +

Na]+. labs (CH2Cl2): 284 (7118).

2-N-Monomethoxytrityl-aciclovir (3). Preparation in three

steps, according to Martin et al.30 First, the hydroxyl group of

native aciclovir was selectively acetylated using Ac2O in the

presence of catalytic DMAP. Secondly, the amino group of

guanine was tritylated using monomethoxytrityl chloride in

DMF, in the presence of NEt3 and catalytic DMAP. Finally,

the acetyl protective group was removed using NH4OH 25%

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

2062 New J. Chem., 2012, 36, 2060–2069 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

in MeOH. The crude tritylated aciclovir derivative was purified

by chromatography (SiO2, CH2Cl2 (100) to CH2Cl2 (90) :MeOH

(10) v/v) to give pure 3 as an amorphous white solid with a global

yield of 84%. 1H NMR (CDCl3): 3.13 (m, 2H, C(40)H2); 3.45

(m, 2H, C(50)H2); 3.70 (s, 3H, NHC(Ph)2(PhOCH3)); 4.94 (s, 2H,

C(10)H2); 6.72 (d, J = 9.0, 2H, NHC(Ph)2(PhOCH3 CoH)); 7.11

to 7.34 (m, 13H, aromatics NHMMT + C(8)H); 7.80 (br s, 1H,

C(2)NHMMT); 11.41 (br s, 1H, N(1)H). Analysis in agreement

with ref. 30.

2-N-Monomethoxytrityl-50-O-imidazolylcarbonyl-aciclovir (4). A

mixture of solid aciclovir derivative 3 (0.151 g, 0.30 � 10�3 mol)

and 1,10-carbonyldiimidazole (0.122 g, 0.75� 10�3 mol) was dried

under high vacuum for 45 min at room temperature. Under Ar

atmosphere, freshly distilled MeCN (7 mL) was added and the

mixture was stirred at room temperature for 3 h (TLCmonitoring,

SiO2, CH2Cl2 (85) :MeOH (15) v/v). The solvent was evaporated

and the solid residue was dissolved in CH2Cl2 (20 mL) and

washed with water (20 mL). The organic phase was dried over

Na2SO4 and evaporated to dryness, to give 4 as a white solid

(0.173 g, 97%). A pure sample was obtained by a rapid

chromatography (SiO2, CH2Cl2 (90) :MeOH (10) v/v).1H NMR (CDCl3): 3.20 (m, 2H, C(40)H2); 3.70 (s, 3H,

NHC(Ph)2(PhOCH3)); 4.18 (m, 2H, C(50)H2); 4.85 (s, 2H,

C(10)H2); 6.71 (d, J = 9.3, 2H, NHC(Ph)2(PhOCH3 CoH));

7.01 (br s, 1H, C(40 0)H); 7.05 (br s, 1H, C(50 0)H); 7.11 to 7.38

(m, 13H, aromatics NHMMT + C(8)H); 8.05 (br s, 1H,

C(2)NHMMT); 8.09 (s, 1H, C(20 0)H); 11.80 (br s, 1H,

N(1)H). 13C NMR (CDCl3): 55.1 (C(Ph)2(PhOCH3)); 66.3

(C(50)H2); 66.8 (C(40)H2); 70.4 (C(Ph)2(PhOCH3)); 71.6

(C(10)H2); 112.9; 126.6; 127.6; 128.9; 130.2; 137.1; 144.8;

158.1 (MMT); 116.9 (C(5)); 117.1 (C(40 0)); 130.7 (C(50 0));

136.9 (C(20 0)); 137.2 (C(8)); 148.5 (N(CO)O); 150.9 (C(4));

151.7 (C(2)); 159.1 (C(6)O).

5,11,17,23-Tetra-(tert-butyl)-25,27-bis-(3-imidazolylcarbony-

loxypropoxy)-26,28-bis-hydroxy-calix[4]arene (5). A mixture

of calixarene derivative 2 (0.200 g, 0.26 � 10�3 mol) and

1,10-carbonyldiimidazole (0.137 g, 0.84 � 10�3 mol) was dried

under high vacuum for 45 min. Freshly distilledMeCN (5 mL) was

added under Ar, and the mixture was stirred at room temperature

for 1 h (TLC monitoring SiO2, CH2Cl2 (96) :MeOH (4) v/v). The

solvent was evaporated and the oily residue was dissolved in

CH2Cl2 (7.5 mL) and washed with water (10 mL). The organic

phase was dried over Na2SO4 and evaporated to dryness. The solid

residue was purified by chromatography (SiO2, CH2Cl2 (100) to

CH2Cl2 (98) :MeOH (2) v/v) to give pure 5 as a vitreous solid

(0.245 g, 98%). 1H NMR (CDCl3): 0.99 (s, 18H, C(CH3)3 A); 1.28

(s, 18H, C(CH3)3 B); 2.49 (quint, J = 6.0, 4H, OCH2CH2CH2O-

(CO)); 3.35–4.23 (AB, J = 12.9, 8H, ArCH2Ar); 4.14 (t, J =

6.0, 4H, OCH2CH2CH2O(CO)); 4.99 (t, J = 6.0, 4H, OCH2CH2-

CH2O(CO)); 6.85 (s, 4H, ArHm A); 7.05 (br s, 6H, C(4)H+ArHm

B); 7.44 (br s, 4H, C(2)H+C(5)H); 8.16 (s, 2H, ArOH). 13CNMR

(CDCl3): 29.3 (OCH2CH2CH2O(CO)); 31.0 (C(CH3)3 A); 31.6

(C(CH3)3 B); 31.7 (ArCH2Ar); 33.8 (C(CH3)3 B); 34.0 (C(CH3)3A); 65.0 (OCH2CH2CH2O(CO)); 71.6 (OCH2CH2CH2O(CO));

117.1 (C(4)); 125.2 (Cm B); 125.7 (Cm A); 127.4 (Co B); 130.7

(C(5)); 132.5 (Co A); 137.0 (C(2)); 141.9 (Cp B); 147.5 (Cp A); 148. 6

(O(CO)N); 149.1 (Ci A); 150.4 (Ci B).

5,11,17,23-Tetra-(tert-butyl)-25,27-bis-[3-(2-N-monomethoxy-

trityl-aciclovir)-carbonyloxypropoxy]-26,28-bis-hydroxy-calix[4]-

arene (6). A mixture of calixarene derivative 2 (0.200 g, 0.26 �10�3 mol) and 1,10-carbonyldiimidazole (0.137 g, 0.84 �10�3 mol) was dried under high vacuum for 45 min. Freshly

distilled MeCN (5 mL) was added under Ar, and the mixture

was stirred at room temperature for 1 h (TLC monitoring SiO2,

CH2Cl2 (96) :MeOH (4) v/v). The solvent was evaporated and

the oily residue was dissolved in CH2Cl2 (10 mL) and washed

with water (10 mL). The organic phase was dried over Na2SO4

and evaporated to dryness. The resulting carbamate 5 was

mixed with aciclovir derivative 3 (0.285 g, 0.57 � 10�3 mol)

and was dried under high vacuum for 30 min. Freshly distilled

MeCN (5 mL) then 1,1,3,3-tetramethylguanidine (0.25 mL,

1.99 � 10�3 mol) was added under Ar, and the mixture was

stirred at room temperature for 20 h (TLC monitoring SiO2,

CH2Cl2/MeOH 90 : 10 v/v). The solvent was evaporated, and

the oily residue was dissolved in CH2Cl2 (25 mL) and washed

with 5% citric acid aqueous solution (25 mL). The aqueous

phase was extracted with CH2Cl2 (10 mL) and the combined

organic phases were dried over Na2SO4 and evaporated to

dryness. The crude solid residue was purified by chromato-

graphy (SiO2, CH2Cl2 (100) to CH2Cl2 (95) :MeOH (5) v/v) to

give pure 6 as an amorphous white solid (0.238 g, 50%). M.p.:

207 1C. IR (KBr): 3402 (n(O-H) + n(N-H)); 2958 (nas(CH3));

2870 (ns(CH2)); 1748 (n(CQO)); 1688, 1571 (Amide I and

amide II); 1464 (d(CH3)); 1261 (n(CQC)); 1033 (n(C-O)).1H NMR (DMSO-d6): 1.12 (s, 18H, C(CH3)3 B); 1.15 (s, 18H,

C(CH3)3 A); 2.33 (br s, 4H, OCH2CH2CH2O(CO)); 3.06 (br s, 4H,

C(40)H2); 3.42–4.13 (AB, J = 12.4, 8H, ArCH2Ar); 3.67 (s, 6H,

NHC(Ph)2(PhOCH3)); 3.82 (br s, 4H, C(50)H2); 4.02 (br s, 4H,

OCH2CH2CH2O(CO)); 4.63 (br s, 4H, OCH2CH2CH2O(CO));

4.82 (s, 4H, C(10)H2); 6.84 (d, 4H, J = 8.9, C(Ph CoH)2-

(PhOCH3)); 7.13 to 7.28 (m, 20H, MMT + ArHm A + ArHm

B); 7.63 (s, 2H, C(8)H); 7.71 (s, 2H, C(2)NHMMT); 8.50 (s, 2H,

ArOH); 10.63 (s, 2H, N(1)H). 13C NMR (DMSO-d6): 28.8

(OCH2CH2CH2O(CO)); 30.9 (C(CH3)3 B); 31.2 (ArCH2Ar);

31.4 (C(CH3)3 A); 33.6 (C(CH3)3 A); 34.1 (C(CH3)3 B); 54.9

(C(Ph)2(PhOCH3)); 64.3 (OCH2CH2CH2O(CO)); 66.3 (C(50)H2);

66.9 (C(40)H2); 69.5 (C(Ph)2(PhOCH3)); 71.4 (C(10)H2); 72.3

(OCH2CH2CH2O(CO)); 112.9 (C(Ph Co)2(PhOCH3)); 126.5,

127.6, 128.5, 129.9, 144.8, 157.7 (MMT); 116.5 (C(5)); 125.3 (Cm

A); 125.7 (Cm B); 127.3 (Co A); 133.2 (Co B); 137.5 (C(8)); 141.4

(Cp A); 147.3 (Cp B); 149.3 (Ci B); 150.0 (Ci A); 150.1 (C(4)); 151.1

(C(2)); 154.5 (O(CO)O); 156.5 (C(6)O). Anal. calcd for

C108H118N10O16, 1.5 H2O (1839.17): C 70.53, H 6.63, N 7.62, O

15.22%; found: C 70.78, H 6.24, N 7.79, O 15.89%. ESI-MS (pos.

mode): 906.94 [M + 2H]2+/2, 1812.89 [2M + 2H]2+/2. labs(CH2Cl2): 233 (52626); 265 (sh, 35690); 280 (36292).

5,11,17,23-Tetra-(tert-butyl)-25-[3-(2-N-monomethoxytrityl-

aciclovir)-carbonyloxypropoxy]-26,27,28-tris-hydroxy-calix[4]-

arene (7). A mixture of calixarene derivative 1 (0.520 g, 0.73 �10�3 mol) and 1,10-carbonyldiimidazole (0.125 g, 0.77� 10�3 mol)

was dried under high vacuum for 45 min. Freshly distilled MeCN

(10 mL) was added under Ar, and the mixture was stirred at room

temperature for 1 h (TLC monitoring SiO2, CH2Cl2 (96) :MeOH

(4) v/v). The aciclovir derivative 3 (0.440 g, 0.88 � 10�3 mol)

then 1,1,3,3-tetramethylguanidine (0.64 mL, 5.15 � 10�3 mol)

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 2060–2069 2063

was then added and the mixture was stirred under Ar at room

temperature for 15 h (TLC monitoring). The solvent was

evaporated, and the oily residue was dissolved in CH2Cl2(50 mL) and washed with 5% citric acid aqueous solution

(50 mL). The organic phase was dried over Na2SO4 and

evaporated to dryness. The crude solid residue was purified

by chromatography (SiO2, CH2Cl2 (100) to CH2Cl2 (96) :MeOH

(4) v/v) to give pure 7 as an amorphous white solid (0.151 g,

17%). M.p.: 180 1C. IR (KBr): 3346 (n(O-H) + n(N-H)); 2958

(nas(CH3)); 2870 (ns(CH2)); 1750 (n(CQO)); 1691, 1568 (Amide I

and amide II); 1484 (d(CH3)); 1257 (n(CQC)); 1034 (n(C-O)).1H NMR (DMSO-d6): 1.15 (s, 9H, C(CH3)3 B); 1.16 (s, 27H,

C(CH3)3 A, C); 2.41 (m, 2H, OCH2CH2CH2O(CO)); 3.11 (m,

2H, C(40)H2); 3.47–4.06 (AB, J = 13.3, 4H, Ar(A)CH2Ar(C));

3.51–4.22 (AB, J = 12.6, 4H, Ar(A)CH2Ar(B)); 3.86 (m, 2H,

C(50)H2); 4.15 (t, J = 6.1, 2H, OCH2CH2CH2O(CO)); 4.58 (t,

J = 6.3, 2H, OCH2CH2CH2O(CO)); 4.86 (s, 2H, C(10)H2); 6.85

(d, J = 9.0, 2H, MMT); 7.10 (d, J = 2.2, 2H, ArHm(A)-

CH2Ar(C)); 7.12 (s, 2H, ArHm C); 7.18 to 7.29 (16H, MMT,

ArHm(A)CH2Ar(B), ArHm B); 7.65 (s, 1H, C(8)H); 7.70 (s, 1H,

C(2)NHMMT); 9.26 (s, 2H, ArOH A); 9.71 (s, 1H, ArOH C);

10.62 (s, 1H, N(1)H). 13C NMR (DMSO-d6): 28.8 (OCH2CH2-

CH2O(CO)); 30.9 (C(CH3)3 B); 31.1 (C(CH3)3 C); 31.2 (C(CH3)3A); 31.5 (ArCH2Ar); 33.6 (C(CH3)3 A); 33.7 (C(CH3)3 C); 34.0

(C(CH3)3 B); 54.9 (C(Ph)2(PhOCH3)); 64.5 (OCH2CH2CH2O-

(CO)); 66.4 (C(50)H2); 66.8 (C(40)H2); 69.5 (C(Ph)2(PhOCH3));

71.3 (C(10)H2); 73.0 (OCH2CH2CH2O(CO)); 112.9, 125.5, 126.0,

126.5, 127.6, 127.8, 128.0, 128.4, 129.3, 129.8, 133.5, 136.6, 142.7,

144.8, 157.7 (MMT, (Co A)CH2Ar(C), (Co A)CH2Ar(B), Co B,

Co C, (Cm A)CH2Ar(B), Cm B, Cp A, Cp B, Cp C); 116.5 (C(5));

125.3 (Cm C); 127.4 ((Cm A)CH2Ar(C)); 137.7 (C(8)); 147.9

(Ci C); 148.0 (Ci A); 149.0 (Ci B); 150.0 (C(4)); 151.0 (C(2));

154.4 (O(CO)O); 156.5 (C(6)O). Anal. calcd for C76H87 N5O10

(1031.12): C 74.18, H 7.13, N 5.69%; found: C 73.99, H 7.04,

N 5.60%. ESI-MS (pos. mode): 1230.65 [M + H]+. labs(CH2Cl2): 232 (35211); 266 (sh, 20878); 279 (23295).

5,11,17,23-Tetra-(tert-butyl)-25,27-bis-[3-(50-O-aciclovir)-

carbonyloxypropoxy]-26,28-bis-hydroxy-calix[4]arene (8). To a

solution of 6 (0.102 g, 0.056� 10�3 mol) and triisopropylsilane

(1.0 mL, 4.881 � 10�3 mol) in distilled CH2Cl2 (5 mL) was

added trifluoroacetic acid (0.3 mL). The resulting intense

orange coloration disappeared after 15 min at room tempera-

ture (TLC monitoring SiO2, CH2Cl2 (90) :MeOH (10) v/v).

The solvent was evaporated, and the residual trifluoroacetic

acid was removed by successive dissolution/evaporation cycles

using CH2Cl2 until formation of a solid material. Residual

triisopropylsilane and apolar by-products were removed by

trituration and sonication in pentane (5 mL) to give pure 8 as a

crystalline white solid (0.078 g, 93%). M.p.: 175 1C. IR (KBr):

3366, shoulder 3134 (n(O-H) + n(N-H)); 2960 (nas(CH3));

2871 (ns(CH2)); 1747 to 1603 (n(CQO) + Amide I and amide

II); 1483 (d(CH3)); 1267 (n(CQC)); 1041 (n(C-O)). 1H NMR

(DMSO-d6): 1.12 (s, 18H, C(CH3)3 B); 1.16 (s, 18H, C(CH3)3 A);

2.30 (quint, J = 6.0, 4H, OCH2CH2CH2O(CO)); 3.43–4.11 (AB,

J = 12.6, 8H, ArCH2Ar); 3.66 (m, 4H, C(40)H2); 3.95 (broad,

H+); 3.99 (m, 4H, C(50)H2); 4.15 (t, J = 6.0, 4H, OCH2CH2-

CH2O(CO)); 4.61 (t, J = 6.0, 4H, OCH2CH2CH2O(CO)); 5.33

(s, 4H, C(10)H2); 6.60 (br s, 4H, C(2)NH2); 7.13 (s, 4H, ArHm A);

7.15 (s, 4H, ArHm B); 8.00 (s, 2H, C(8)H); 8.47 (s, 2H, ArOH);

10.78 (s, 2H, N(1)H). 13C NMR (DMSO-d6): 28.8 (OCH2CH2-

CH2O(CO)); 30.9 (C(CH3)3 B); 31.1 (ArCH2Ar); 31.4 (C(CH3)3A); 33.6 (C(CH3)3 A); 34.1 (C(CH3)3 B); 64.4 (OCH2CH2CH2O-

(CO)); 66.2 (OCH2CH2CH2O(CO)); 66.5 (C(40)); 72.075 (C(10));

72.3 (C(50)); 115.1 (C(5)); 125.3 (Cm A); 125.7 (Cm B); 127.3

(Co A); 133.1 (Co B); 137.4 (C(8)); 141.5 (Cp A); 147.3 (Cp B);

149.3 (Ci B); 150.1 (Ci A); 151.2 (C(4)); 154.2 (C(2)); 154.5

(O(CO)O); 156.5 (C(6)O). 19F NMR (DMSO-d6): �74.771(s, CF3CO2

�). Anal. calcd for C72H88F6N10O18, 0.25 C5H12

(1513.55): C 58.13, H 6.06, N 9.25%; found: C 58.25, H 6.09, N

9.41%. ESI-MS (pos. mode): 634.32 [M�2CF3CO2�]2+/2. labs

(CH2Cl2): 233 (28464); 259 (28657); shoulder 280 (23630).

5,11,17,23-Tetra-(tert-butyl)-25-[3-(50-O-aciclovir)-carbony-

loxypropoxy]-26,27,28-tris-hydroxy-calix[4]arene (9). To a

solution of 7 (0.121 g, 0.098� 10�3 mol) and triisopropylsilane

(0.4 mL, 1.960 � 10�3 mol) in distilled CH2Cl2 (8 mL) was

added trifluoroacetic acid (0.4 mL). The resulting intense

orange coloration disappeared after 15 min at room tempera-

ture (TLC monitoring SiO2, CH2Cl2/MeOH 90 : 10 v/v). The

solvent was evaporated, and residual trifluoroacetic acid was

removed by successive dissolution/evaporation cycles using

CH2Cl2 until formation of a solid material. The crude product

was purified by chromatography (SiO2, CH2Cl2 (100) to

CH2Cl2 (95) :MeOH (5) v/v) to give pure 9 as a vitreous solid

(81 mg, 82%). 9 was dissolved in MeCN (2 mL) and stored at

4 1C for 24 h. The resulting crystalline white powder was

recovered by filtration (0.033 g, 40% cryst.).

M.p.: 170 1C. IR (KBr): 3449 (n(O-H) + n(N-H)); 2960

(nas(CH3)); 2872 (ns(CH2)); 1747 (n(CQO)); 1634 (Amide I and

amide II); 1484 (d(CH3)); 1265 (n(CQC)); 1037 (n(C-O)).1H NMR (DMSO-d6): 1.152, 1.155 (s + s, 18H, C(CH3)3 B, C);

1.162 (s, 18H, C(CH3)3 A); 2.39 (m, 2H, OCH2CH2CH2O(CO));

3.49–4.08 (AB, J = 13.3, 4H, Ar(A)CH2Ar(C)); 3.52–4.21 (AB,

J = 13.2, 4H, Ar(A)CH2Ar(B)); 3.70 (m, 2H, C(40)H2); 3.92

(br s, H+); 4.13 (t, J = 6.1, 2H, OCH2CH2CH2O(CO)); 4.20

(m, 2H, C(50)H2); 4.56 (t, J= 6.3, 2H, OCH2CH2CH2O(CO));

5.35 (s, 2H, C(10)H2); 6.60 (br s, 2H, C(2)NH2); 7.10 (d, J =

2.3, 2H, (ArHm A)CH2Ar(C)); 7.12 (s, 2H, ArHm C); 7.22 (d,

J = 2.3, 2H, (ArHm A)CH2Ar(B)); 7.26 (s, 2H, ArHm B); 8.01

(s, 1H, C(8)H); 9.21 (s, 2H, ArOH A); 9.72 (s, 1H, ArOH C);

10.76 (s, 1H, N(1)H). 13C NMR (DMSO-d6): 28.8 (OCH2CH2-

CH2O(CO)); 30.9 (C(CH3)3 B) + (Ar(A)CH2Ar(B)); 31.1

(C(CH3)3 C); 31.2 (C(CH3)3 A); 31.5 (Ar(A)CH2Ar(C)); 33.6

(C(CH3)3 A); 33.7 (C(CH3)3 C); 34.0 (C(CH3)3 B); 64.5

(OCH2CH2CH2O(CO)); 66.3 (C(50)); 66.5 (C(40)); 72.0

(C(10)); 73.0 (OCH2CH2CH2O(CO)); 115.2 (C(5)); 125.3

((Cm A)CH2Ar(B)); 125.5, 125.6 ((Cm A)CH2Ar(C) and Cm

C); 126.1 (Cm B); 127.4 ((Co A)CH2Ar(B)); 127.9, 128.0

((Co A)CH2Ar(C) + Co C); 133.5 (Co B); 137.6 (C(8)); 142.8

(Cp A); 143.5 (Cp C); 146.6 (Cp B); 147.9 (Ci C); 147.9 (Ci A);

149.0 (Ci B); 151.1 (C(4)); 154.1 (C(2)); 154.5 (O(CO)O); 156.2

(C(6)O). 19F NMR (DMSO-d6): �74.755 (s, CF3CO2�). Anal.

calcd for C56H71N5O9, 0.5 CF3CO2H (1015.20): C 67.44, H

7.10, N 6.90%; found: C 67.71, H 7.05, N 7.05%. ESI-MS

(pos. mode): 958.53 [M�CF3CO2�]+. labs (CH2Cl2): 232

(25748); shoulder 256 (17266); 277 (17665).

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

2064 New J. Chem., 2012, 36, 2060–2069 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

Monomolecular films

Chloroform used for preparing calixarene derivative solutions

was from Sigma-Aldrich (ref. 288306; 100 mL, 99.9% pure;

0.5–1.0% EtOH), and subphases were prepared with Milli-Q

water which had a surface tension of 72.8 mN m�1 at 20 1C

(pH 5.6). Buffers were prepared with analytical grade NaHCO3

and NaOH. All solvents used were of analytical grade.

Compression isotherms. The surface pressure (P) measure-

ment was carried out with a computer-interfaced KSV 2000

Langmuir balance (KSV Instruments Ltd, Helsinki, Finland).

A Teflons trough (6.5 cm � 58 cm � 1 cm) with two moving

hydrophilic Delrins barriers (symmetric compression) was

used in monomolecular film. The system was equipped with

an electrobalance and a platinum Wilhelmy plate (perimeter

3.94 cm) as a surface pressure sensor. Before each run, the

trough and the barriers were washed using cotton soaked in

chloroform and ethanol and finally rinsed with Milli-Q water.

The platinum Wilhelmy plate was cleaned by rinsing with

Milli-Q water, ethanol, and chloroform, and then heating to a

red glow. All impurities were removed from the subphase

surface by sweeping and suction. When the surface pressure

fluctuation was lower than 0.2 mN m�1 during a compression

stage, monolayers were spread at the air–water interface from

chloroform solutions (concentration around 0.75 mg mL�1)

using a microsyringe (Hamilton Co., USA). After the equili-

bration time of 15 min, the films were compressed at the rate of

5 mm min�1 per barrier (3.25 cm2 min�1 per barrier). Each

compression isotherm was performed at least three times. The

P–A compression isotherms allowed calculating the compressibility

modulus Cs�1 (Cs

�1 = �A(@P/@A)T).31,32

Hydrolysis study. The hydrolysis studies were performed in

borosilicate glass Petri dishes (diameter of 18.5 cm; 268.8 cm2

or 268.8 � 1016 A2, 3 cm depth). The monolayers were spread

on carbonate buffer NaHCO3/Na2CO3 50 mM pH 10, using a

microsyringe (Hamilton Co., USA). After the equilibration

time of 15 minutes, the Petri dishes were covered and kept in

an incubator at 37 1C. All experiments were performed in

triplicate. The treatment of samples was carried out according

to the procedure described below.

Chromatographic procedure

The isocratic HPLC system consisted of the following Merck

Hitachi modules: a model 7100 pump, a model 7200 auto-

sampler set to inject 20 mL and a model 7450 diode array

detector (200–350 nm). The analytical column was a Nucleosil

C18 (125 mm � 4.0 mm id, 3 mm) reversed phase column from

Macherey Nagel. The column temperature was controlled with

a Merck Hitachi model T6300 external oven.

Chromatographic conditions. The chromatographic study

was carried out using a H2O–MeOH 90/10 (v/v) binary

mixture as mobile phase, with a flow rate of 1.0 mL min�1

at 35 1C. For a wavelength of 254 nm (lmax) the limit of

quantification (LOQ), corresponding to a signal-to-noise ratio

of 10, was determined at 5.0 � 10�7 M. The higher theoretical

concentration of aciclovir which could be found in the sub-

phase (100% hydrolysis) was evaluated around 5 � 10�7 M,

not consistent with a chromatographic drug-release kinetic

survey. It was thus necessary to concentrate the subphase by

an adequate sample treatment procedure.

Sample treatment. The Standard Operating Procedure (SOP)

for sample treatment is described as follow: the hydrolysis reaction

kinetic was evaluated from 0 to 3 days. For each time point, the

monolayer spread on the buffer surface was rapidly and carefully

removed by suction, and then 100.0 mL of the subphase were

collected and lyophilized. The residue was dissolved in a minimum

amount of water (5 mL) and the carbonate salts were precipitated

by addition of MeCN in a large excess (45 mL). Carbonate salts

were removed by filtration and the filtrate was evaporated

under vacuum to dryness. Finally, the residue (5 mg approx.)

was dissolved in 1000 mL of pure water. This procedure leads

to a 100-order increase in the aciclovir concentration.

Method validation. The analytical procedure (ST followed

by HPLC analysis) was validated using three calibrated solutions

(500.0 mL) of aciclovir in carbonate buffer, around the theoretical

concentrations in the hydrolysis range 10–100%, i.e. 5 � 10�8 M

(10%), 2� 10�7 M (40%) and 5� 10�7 M (100%) corresponding

to 5 � 10�6 M, 2 � 10�5 M and 5 � 10�5 M after ST (100-order

increase in the aciclovir concentration). Each solution was divided

into three 100.0 mL portions using volumetric flasks and the

ST was performed on these 3 � 3 solutions. The SST samples

were analyzed by HPLC (n = 3), in order to determine

precision and accuracy. The linearity was determined by five

treated aciclovir samples (n = 3 for each) in the concentration

range from 5 � 10�7 M to 5 � 10�5 M. The specificity of the

method was checked by treatment and analysis of a 100.0 mL

carbonate buffer solution (blank without aciclovir) in order to

confirm that there were no interferences in the buffer media.

Results and discussion

Synthesis

In order to develop a versatile, simple, and rapid synthetic way

towards calixarene-based nucleoside analogue prodrugs, several

strategies were examined. The primary alcohol of the sugar

moiety in aciclovir, present in a large number of biologically

active nucleoside analogues (pseudo 50-hydroxyl group), was

considered as the most suitable binding site to the calixarene

platform, via an ester linkage. This implied to prepare an

aciclovir derivative in which the 2-amino group of the guanine

moiety is immobilized by an acido-labile protective group. As

previously reported, the propyl alkyl chain, quickly identifiable

by 1H-NMR, was chosen as a spacer.

According to this approach, three types of linkers, potentially

labile in vivo and in an alkaline environment, were envisaged,

namely phosphodiester, carboxylic ester and carbodiester. The

phosphodiester linkage could lead to an efficient prodrug

behavior, as recently reported in the literature,33 and appears

thus as an interesting option, mimicking natural nucleotides.

Indeed, this group was successfully employed in the conjugation

of calixarene and nucleoside derivatives.26 However, the phos-

phoramidite chemistry required in the coupling stage is delicate

and involves numerous steps compared to other strategies

envisaged (see below), that is not consistent with our goal.

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 2060–2069 2065

Furthermore, the hydrolytic reactivity of nucleoside-based

phosphodiesters could be less pronounced in alkaline medium

than for carboxylic or carbonate esters.34,35 For these reasons,

the phosphodiester linker was not selected for the preliminary

study presented in this work.

The carboxylic ester linker strategy involves the preparation

of calixarene O-(2-carboxyethyl) derivatives which are readily

obtained in two steps by regioselective O-alkylation with an

alkyl-3-bromopropionate, followed by saponification. Never-

theless, attempts to esterify aciclovir derivatives were not

found satisfactory.

The carbodiester linker seemed to us an interesting option.

Indeed, dissymmetric carbonate derivatives can be formed by

‘‘one-pot’’ reaction involving two alcohols and 1,10-carbonyl-

diimidazole (CDI), undermoderate conditions and good yields.36–42

In our case, this convergent strategy involved the preparation of a

2-N-protected aciclovir derivative, and of calixarene-O-(o-hydro-xyalkyl) derivatives. The spacer was chosen identical to previous

works, i.e. the propyl one (Fig. 1).17–19

The mono-hydroxypropyl derivative 1 was prepared with a

yield of 40% by reaction of tetra-p-tert-butylcalix[4]arene with

1.1 equiv. of 3-bromopropanol in dry MeCN at reflux, using

0.6 equiv. of K2CO3 as base and in the presence of an excess of

KI. The di-substituted derivative 2 was prepared by adapta-

tion of the procedure of Armaroli and co-workers,43 under

concentrated conditions and using all reactants in excess, with

a yield of 54% after chromatography. 1H and 13C NMR

experiments showed that cone conformation was preserved

in 1 and 2, as indicated by Ar–CH2–Ar 13C-resonance signals

located at 31–32 ppm and their AB profile for 1H.44,45

The aciclovir derivative 3 bearing the monomethoxytrityl

(MMT) protective group at the 2-amino position of the

guanine was prepared as reported by Martin et al.30

The MMT protective group, labile under anhydrous acidic

conditions, is suitable for our strategy as it can be eliminated

easily in the final step of the synthesis. The 1,10-carbonyldiimi-

dazole (CDI) was used in order to prepare calixarene–aciclovir

conjugates from alcohols 1 and 3 or 2 and 3, via carbodiester

linkages. This synthetic way to dissymmetric carbonate passes

first by the condensation of an alcohol with CDI to afford the

corresponding O-(1-imidazolyl)carbamate. Then, the condensa-

tion of the second alcohol in the presence of an organic base

leads to the desired carbonate derivative.

According to Chmielewski et al. who reported the synthesis

of deoxyribonucleoside 50-O-carbonates, shaped as thermo-

lytic pro-nucleoside for potential use in solid-phase synthesis

of oligonucleotides,36 MeCN was chosen as solvent of reac-

tion, and 1,1,3,3-tetramethylguanidine (TMG) as base for the

second condensation.

The first strategy involved the formation of the O-(1-imida-

zolyl)carbamate 4 from the 2-N-protected aciclovir derivative

3 and CDI, followed by the substitution of the residual

imidazole by the calixarene alcohol derivatives 1 or 2,

in MeCN and in the presence of TMG as base, at room

temperature. Nevertheless, in both cases, complex mixtures

were obtained.

Fig. 1 Synthesis of calix[4]arene–aciclovir derivatives 8 and 9. (i) CDI (2.5 equiv.), MeCN, rt, 3 h, 97%. (ii) 1 or 2 (1.2 equiv. or 0.5 equiv.,

respectively), TMG (7.0 equiv.), MeCN, rt, 24 h, 0%. (iii) CDI (3.2 equiv.), MeCN, rt, 1 h, 98%. (iv) 3 (2.2 equiv.), TMG (7.6 equiv.), MeCN, rt,

20 h, 50%. (v) CDI (1.0 equiv.), MeCN, rt, 1 h. (vi) 3 (1.2 equiv.), TMG (7.1 equiv.), MeCN, rt, 15 h, 17%. (vii) TFA and TIS in large excess,

CH2Cl2, rt, 15 min, 93% (8) and 82% (9).

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

2066 New J. Chem., 2012, 36, 2060–2069 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

The second strategy (Fig. 1) consisted of the direct carba-

moylation of the calixarene alcohol derivatives 1 and 2,

followed by the substitution of residual imidazoles by aciclovir

derivative 3, in the presence of TMG.

Calixarene derivatives 1 or 2 and CDI were dissolved in

anhydrous MeCN under Ar atmosphere at room temperature.

After 1 h reaction time, the disappearance of 1 or 2 was

confirmed by TLC.

In the case of the di-substituted derivative 2, only one new

product was observed that was stable enough for purification on

silica gel. 1H NMR analysis of this compound was consistent

with the structure of the desired bis-imidazocarbamoyl calixarene

derivative 5. Crude 5 was simply obtained by evaporation of

MeCN, dissolution in CH2Cl2 and washing with water to remove

excess of CDI. It was then reacted with 3 in MeCN, in the presence

of TMG in excess, to give, after chromatography, the bis-(2-N-

MMT-aciclovir)–calixarene derivative 6 with a yield of 50%.

The reaction of the mono hydroxypropyl derivative 1 with

1.0 equiv. of CDI gave a main compound, nevertheless too

unstable to be purified by chromatography. On the assump-

tion that it was the good imidazocarbamoyl derivative, the

mono-(2-N-MMT-aciclovir) calixarene derivative 7 was thus

prepared ‘‘one-pot’’, by reaction of 1 with 1.0 equiv. of CDI in

MeCN, followed by addition of both 3 and TMG in excess.

Under these conditions, 7 was isolated in a yield of 17% after

chromatography. In order to understand this low yield, and

with respect to the literature that reports that TMG can be

used as a nucleophilic agent,46,47 we attempted to react TMG

alone with imidazolylcarbamate of 1. This reaction afforded

effectively the TMG carbamate derivative, not described here.

The cleavage of the MMTr protective groups of 6 and 7 was

performed with TFA in CH2Cl2, in the presence of an excess of

trialkylsilane (here triisopropylsilane; TIS) as a carbocation

scavenger.48,49 Detritylation was immediately accomplished

after addition of TFA (TLC monitoring) to give the calixarene

bis- and mono-aciclovir conjugates 8 and 9, respectively. The

MMT+ carbocation was consumed in 15 min reaction time, as

indicated by the disappearance of the orange coloration. The

removal of residual TFA necessitated successive co-evaporations

with CH2Cl2. Pure 8 was obtained as a crystalline solid by a

simple trituration with pentane. 9 was purified by chromato-

graphy on silica gel, then crystallised from aMeCN solution at

4 1C. Both final compounds 8 and 9 were fully characterized;

NMR experiments showed notably that they were in the cone

conformation, as assessed by their 13C Ar–CH2–Ar resonance

signals at ca. 31 ppm. Calixarene bis-(8) and mono-aciclovir

(9) conjugates were obtained as trifluoroacetate salts, as

indicated by the 19F NMR signal located in both cases at

around �74.77 ppm. Nevertheless, combustional analyses

showed that the bis-aciclovir derivative 8 was obtained as

bis-trifluoroacetate salt, while the mono-aciclovir derivative 9

was obtained as hemi-trifluoroacetate salt. Electrospray mass

spectrometry analyses (positive mode) were found to be con-

sistent with the proposed structures, showing the presence of

the corresponding protonated derivative molecular peaks.

Monolayer studies. The interfacial properties of amphiphilic

calixarene derivatives 8 and 9 were studied using Langmuir

monolayers.22 Surface pressure–mean molecular area (P–A)

and surface potential–mean molecular area (DV–A) compres-

sion isotherms of monolayers formed with 8 or 9 spread on

pure water (20 1C) are shown in Fig. 2.

Both compounds formed stable monolayers at the air–water

interface. The stability of Langmuir films is not significantly

affected by the number of aciclovir residues present on the

lower rim of the calixarene, as indicated by the values of P at

the collapse of the films (B21–23 mN m�1) with both deriva-

tives. The values of A at the collapse point are around 126 and

113 A2 with 8 and 9, respectively. However, the maximal

values of the compressibility modulus (Cs�1),31,32 which are

216 and 89 mN m�1 for 8 and 9, respectively, show that 8

forms more rigid films compared to the mono-aciclovir con-

jugate 9. Interestingly, the profile of the P–A isotherm of 8

shows one plateau between B280 and 260 A2 and the second

between B210 and 175 A2. We propose that these plateaus

indicate phase transitions between phases of increasing

condensation; the different phases may correspond to mole-

cules 8 adopting different conformations. While in the case of

9 no phase transitions can be seen in the P–A isotherm, the

jumps observed in the DV–A isotherm between 250 and 200 A2

indicate a stepwise reorientation of the molecules within the

film upon compression.

The results obtained show that the degree of substitution of

the lower rim of the calixarene crown has no significant impact

on the interfacial behavior of 8 and 9, which is contrary to

the effects observed with calixarene derivatives described

earlier.19,22 This effect may be related to the length (10 atoms)

and the polar nature (three oxygen atoms) of the flexible linker

situated between the calixarene and guanine moieties.

Drug-release at the air–water interface

Study of the hydrolytic reactivity on the air–aqueous interface

of amphiphilic calixarene-based prodrugs was recently

described by our group.22 The methodology developed for

this purpose consisted of spreading monolayers at an arbitra-

rily chosen surface pressure on alkaline buffer, and HPLC

quantification of the time-dependent drug release into the

subphase. Due to the low amounts of drug to be released,

Fig. 2 Bottom: surface pressure–mean molecular area (P–A) and

top: surface potential–mean molecular area (DV–A) compression

isotherms of monolayers of 8 (solid lines) and 9 (dotted lines) spread

on pure water (pH 5.6) at 20 1C.

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 2060–2069 2067

the quantification was made possible by increase in its concen-

tration using a sample treatment (ST).

Langmuir monolayers of 8 and 9 were formed at the surface

of an alkaline buffer (NaHCO3/Na2CO3 50 mM, pH 10.0),

and characterized by P–A compression isotherms at 37 1C

(Fig. 3). The mean molecular areas A8 = 140 A2 and A9 =

120 A2 corresponding to P = 25 mN m�1, that is to

condensed phases of 8 or 9 situated below the collapse point

(Pcoll B 30 mN m�1) were chosen for the hydrolysis study,

i.e. for calculating the amount of calixarene solutions to be

spread over buffer in the Petri dishes.

The analytical procedure, consisting of the concentration

of the subphase by ST, followed by HPLC analysis, was

validated in regards to the Q2(R1) guideline of the Inter-

national Conference on Harmonization (ICH).50 Details on

the ST modus operandi and HPLC conditions are depicted in

Experimental Part.

A blank sample, 100.0 mL of carbonate buffer without

aciclovir and treated by ST, was analyzed in order to

check the specificity. It was observed no interferences in the

buffer media.

Several treated standard samples of aciclovir were analyzed

in order to determine sensitivity and linearity of the HPLC-

UV method. The sensitivity was determined by means of the

limit of detection (LOD) and quantification (LOQ), which

were found at 1.5 � 10�7 and 5.0 � 10�7 M, respectively. The

linearity (peak area against aciclovir concentration) was

determined by five treated aciclovir samples (n = 3 for each)

in the concentration range from 5� 10�7 M to 5� 10�5 M, and

fitted using least squares linear regression. A good linearity was

observed with a correlation coefficient higher than 0.999 (data

not shown). The ST was evaluated using three carbonate buffer

solutions of aciclovir, in a concentration range corresponding to

monolayer hydrolysis of 10% (5 � 10�8 M, i.e. 5 � 10�6 M

after ST), 40% (2 � 10�7 M, i.e. 2 � 10�5 M after ST) and

100% (5 � 10�7 M, i.e. 5 � 10�5 M after ST).

In order to determine the precision of the method (repeat-

ability and intermediate precision), each solution was frac-

tioned in three portions of 100.0 mL, representatives

of subphase sampling. All of nine solutions were treated

according to the ST, and the corresponding standard-ST

samples, that are SST(10), SST(40) and SST(100), were analyzed

by HPLC.

The precision of the method was expressed as the relative

standard deviation (RSD). The intra-day variation (repeat-

ability, n = 3) was determined on a single day whereas the

inter-day variation (intermediate precision, n = 9) was

assayed for three consecutive days. Satisfactory results

were obtained (RSD values lower than 4.0% whatever the

concentration in aciclovir under study). The accuracy was

determined by comparing the concentration found for SST

samples (10, 40 and 100%) covering the linearity range, to the

theoretical ones. Results are shown in Table T1 (ESIw) and are

satisfactory.

The drug-release kinetic of 8 and 9 monolayers spread on

alkaline buffer (pH 10.0) at 37 1C was determined from 0 to

3 days by the validated method described in the previous part.

The yield of aciclovir (drug release percentage) was calculated

versus the maximal theoretical quantity that could be obtained

after total hydrolysis from 8 (bearing two aciclovir moieties) or

9 (bearing one aciclovir moiety). Typical chromatograms

and the kinetic survey of aciclovir release are given in Fig. 4.

Fig. 3 Surface pressure–mean molecular area (P–A) compression

isotherms of 8 (solid line) and 9 (dotted line) monolayers spread on

carbonate buffer (pH 10.0) at 37 1C. Compression rate: 5 mm min�1

(3.25 cm2 min�1).

Fig. 4 Interfacial hydrolysis survey of calixarene–aciclovir conjugates. (left) Typical chromatograms (compound 8, l= 254 nm) of, from bottom

to top, subphase samples after ST at t=0, 24, 48, 72 h, and standard-ST sample. (right) Kinetic of the aciclovir release frommonolayers of 8 (solid

line) or 9 (dotted line) spread on carbonate buffer (pH 10.0) at 37 1C.

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

2068 New J. Chem., 2012, 36, 2060–2069 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

It appears that the release of aciclovir into the subphase is effective

and regular. After 3 days at 37 1C, we observe that around 30% of

aciclovir was released from monolayers of 8 or 9, higher than the

previously described nalidixic ester analogues.22

In parallel to these studies, other hydrolysis assays were

conducted with compound 8. Microdispersion of compound 8

in pH 10.0 carbonate buffer was prepared. For this, a solution

of 8 in THF (0.82 mg mL�1, 100 mL) was injected into the

vortex of highly stirred carbonate buffer (10 mL). The resulting

mixture was stirred at room temperature for several days, and

analyzed by HPLC each day. In the time scale of one week, no

release of aciclovir was detected. This observation suggests how

Langmuir monolayers, orienting the polar aciclovir moieties in

the subphase, favor hydrolysis.

Conclusions

In order to evaluate the potency of the calix[4]arene platform

to act as a prodrug scaffold, two new amphiphilic derivatives

integrating the tetra-p-tert-butyl calix[4]arene and one or two

anti-HSV aciclovir subunits tethered at the lower rim via

carbodiester linkages were synthesized. The evaluation of their

amphiphilic behavior performed using Langmuir balance at

the air–water or air–pH 10 buffer interface and 20 or 37 1C showed

that they form stable monolayers suitable for hydrolysis studies.

Monolayers of these two compounds were prepared on pH 10

carbonate buffer subphase, and aciclovir release at 37 1C was

followed by HPLC according to sample treatment developed for

this purpose. A 30% release of aciclovir was observed after 3 days

with both derivatives, which was significantly higher compared

to liquid–liquid interfaces. These results confirm the interest of

engineering the interfacial processes, and show that Langmuir

monolayers spread at the air–water interface are a relevant

model for studying water insoluble prodrugs. This approach

could become an alternative method for pre-biological screen-

ing of such compounds, and, in addition to pre-evaluation of

cellular toxicities on eukaryotic cell membrane models using

similar technology, should allow us to by-pass unadapted

in vitro evaluations, directly to animal models. These two last

points are now under consideration.

Acknowledgements

The authors thank the French Ministere de la Recherche et de

l’Enseignement Superieur, particularly G. Sautrey for a PhD

grant, the CNRS and the Region Lorraine for financial

support. We also thank Mr Eric Dubs for synthesis of starting

materials, Mrs B. Fernette for NMR experiments, as well as

Mrs S. Adach for elemental analyses and Mr F. Dupire for

mass measurements.

Notes and references

1 For an history: G. H. Hitchings, Annu. Rev. Pharmacol., 1992,32, 1.

2 C. Perigaud, G. Gosselin and J.-L. Imbach,Nucleosides Nucleotides,1992, 11, 903.

3 (a) H. J. Field and E. De Clercq, J. Clin. Virol., 2004, 30, 115;(b) A. Matsuda and T. Sasaki, Cancer Sci., 2004, 95, 105.

4 D. C. Baker, T. H. Haskell and S. R. Putt, J. Med. Chem., 1978,21, 1218.

5 W. J. Wechter, D. T. Gish, M. E. Greig, G. D. Gray, T. E. Moxley,S. L. Kuentzel, L. G. Gray, A. J. Gibbons, R. L. Griffin andG. L. Neil, J. Med. Chem., 1976, 19, 1013.

6 E. K. Hamamura, M. Prystasz, J. P. H. Verheyden, J. G. Moffatt,K. Yamaguchi, N. Uchida, K. Sato, A. Nomura and O. Shiratori,J. Med. Chem., 1976, 19, 667.

7 (a) L. W. Peterson and C. E. McKenna, Expert Opin. DrugDelivery, 2009, 6, 405; (b) P. Poijarvi-Virta and H. Lonnberg,Curr. Med. Chem., 2006, 13, 3441.

8 E. De Clercq and H. J. Field, Br. J. Pharmacol., 2006, 147, 1.9 F. Li, H. Maag and T. Alfredson, J. Pharm. Sci., 2008, 97, 1109.10 M. J. Sofia, Antiviral Chem. Chemother., 2011, 22, 23.11 D. R. Bobeck, R. F. Schinazi and S. J. Coats, Antiviral Ther., 2010,

15, 935.12 E. de Clercq, Rev. Med. Virol., 2009, 19, 287.13 A. de Fatima, S. A. Fernandes and A. A. Sabino, Curr. Drug

Discovery Technol., 2009, 6, 151.14 R. V. Rodik, V. I. Boyko and V. I. Kalchenko, Curr. Med. Chem.,

2009, 16, 1630.15 (a) F. Perret and A. W. Coleman, Chem. Commun., 2011, 47, 7303;

(b) B. Mokhtari and K. Pourabdollah, J. Coord. Chem., 2011,64, 3189.

16 A. Ben Salem and J. B. Regnouf-de-Vains, Tetrahedron Lett., 2001,42, 7033.

17 A. Ben Salem and J. B. Regnouf-de-Vains, Tetrahedron Lett., 2003,44, 6769.

18 B. Korchowiec, A. Ben Salem, Y. Corvis, J.-B. Regnouf de Vains,J. Korchowiec and E. Rogalska, J. Phys. Chem. B, 2007,111, 13231.

19 B. Korchowiec, M. Orlof, G. Sautrey, A. Ben Salem,J. Korchowiec, J.-B. Regnouf-de- Vains and E. Rogalska,J. Phys. Chem. B, 2010, 114, 10427.

20 H. Massimba Dibama, I. Clarot, S. Fontanay, A. B. Salem,M. Mourer, C. Finance, R. E. Duval and J.-B. Regnouf-de-Vains,Bioorg. Med. Chem. Lett., 2009, 19, 2679.

21 A. Ben Salem, G. Sautrey, S. Fontanay, R. E. Duval andJ.-B. Regnouf de Vains, Bioorg. Med. Chem., 2011, 19, 7534.

22 G. Sautrey, I. Clarot, A. Ben Salem, E. Rogalska andJ.-B. Regnouf de Vains, New J. Chem., 2012, 36, 78.

23 V. Sidorov, F. W. Kotch, M. El-Kouedi and J. F. Davis, Chem.Commun., 2000, 2369.

24 F. W. Kotch, V. Sidorov, Y.-F. Lam, K. J. Kayser, H. Li,M. S. Kaucher and J. F. Davis, J. Am. Chem. Soc., 2003,125, 15140.

25 (a) S. J. Kim and B. H. Kim, Tetrahedron Lett., 2002, 43, 6367;(b) S. J. Kim and B. H. Kim, Nucleic Acids Res., 2003, 31, 2725.

26 G. M. L. Consoli, G. Granata, E. Galante, F. Cunsolo andC. Geraci, Tetrahedron Lett., 2006, 47, 3245.

27 G. M. L. Consoli, G. Granata, D. Garozzo, T. Mecca andC. Geraci, Tetrahedron Lett., 2007, 48, 7974.

28 G. M. L. Consoli, G. Granata, R. Lo Nigro, G. Malandrino andC. Geraci, Langmuir, 2008, 24, 6194.

29 G. M. L. Consoli, G. Granata, E. Galante, I. Di Silvestro,L. Salafia and C. Geraci, Tetrahedron, 2007, 63, 10758.

30 J. C. Martin, D. P. C. McGee, G. A. Jeffrey, D. W. Hobbs,D. F. Smee, T. R. Matthews and J. P. H. Verheyden, J. Med.Chem., 1986, 29, 1384.

31 J. T. Davies and E. K. Rideal, Interfacial Phenomena, AcademicPress, New York, USA, 2nd edn, 1963.

32 G. L. Gaines, Insoluble monolayers at liquid–gas interfaces, WileyInterscience, New York, USA, 1966.

33 (a) S. Dong and M. C. Schroeder, Int. Patent, 2009, WO 2009/085267 A1; (b) T. Rodriguez-Perez, S. Fernandez, Y. S. Sanghvi,M. Detorio, R. F. Schinazi, V. Gotor and M. Ferrero, BioconjugateChem., 2010, 21, 2239.

34 N. S. Pujar, N. F. Huang, C. L. Daniels, L. Dieter, M. G. Gaytonand A. L. Lee, Biopolymers, 2004, 75, 71.

35 A. M. Ramadan, J. M. Calatayud Sala and T. N. Parac-Vogt,Dalton Trans., 2011, 40, 1230.

36 M. K. Chmielewski, V. Marchan, J. Cieslak, A. Grajkowski,V. Livengood, U. Muench, A. Wilk and S. L. Beaucage, J. Org.Chem., 2003, 68, 10003.

37 J. Ho, F. M. N. Al-Deen, A. Al-Abboodi, C. Selomulya,S. D. Xiang, M. Plebanski and G. M. Forde, Colloids Surf., B,2011, 83, 83.

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 2060–2069 2069

38 A. P. Goodwin, S. S. Lam and J. M. J. Frechet, J. Am. Chem. Soc.,2007, 129, 6994.

39 L. Zhang, L. Song, Z. Liu, H. Li, Y. Lu, Z. Li and S. Ma, Eur. J.Med. Chem., 2010, 45, 915.

40 E. Utagawa, M. Sekine and K. Seio, J. Org. Chem., 2006, 71, 7668.41 D.-K. Kim, N. Lee, Y.-W. Kim, K. Chang, J.-S. Kim, G.-J. Im,

W.-S. Choi, I. Jung, T.-S. Kim, Y.-Y. Hwang, D.-S. Min,K. A. Um, Y.-B. Cho and K. H. Kim, J. Med. Chem., 1998,41, 3435.

42 M. A. Raviolo, P. A. M. Williams, S. B. Etcheverry, O. E. Piro,E. E. Castellano, M. S. Gualdesi andM. C. Brinon, J. Mol. Struct.,2010, 970, 59.

43 A. Hosseini, S. Taylor, G. Accorsi, N. Armaroli, C. A. Reed andP. D. W. Boyd, J. Am. Chem. Soc., 2006, 128, 15903.

44 C. Jaime, J. De Mendoza, P. Prados, P. M. Nieto and C. Sanchez,J. Org. Chem., 1991, 56, 3372.

45 L. C. Groenen, J. D. Van Loon, W. Verboom, S. Harkema,A. Casnati, R. Ungaro, A. Pochini, F. Ugozzoli andD. N. Reinhoudt, J. Am. Chem. Soc., 1991, 113, 2385.

46 S. Li, Y. Lin, J. Cao and S. Zhang, J. Org. Chem., 2007, 72, 4067.47 F. S. Pereira, E. Ribeiro de Azevedo, E. F. da Silva,

T. J. Bonagamba, D. L. da Silva Agostini, A. Magalhaes,A. E. Job and E. R. Perez Gonzalez, Tetrahedron, 2008, 64, 10097.

48 D. A. Pearson, M. Blanchette, M. L. Baker and C. A. Guindon,Tetrahedron Lett., 1989, 30, 2739.

49 P. B. Garland and P. J. Serafinowski,Org. Biomol. Chem., 2009, 7, 451.50 ICHGuidelines Q2(R1): Note for Guidance on Validation of Analytical

Procedures: Text and Methodology Ref. CPMP/ICH/381/95.

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ite d

e L

orra

ine

on 2

8/11

/201

4 10

:30:

46.

View Article Online