Squalenoyl gemcitabine monophosphate: Synthesis, characterisation of nanoassemblies and biological evaluation

FULL PAPER

DOI: 10.1002/ejoc.201100036

Squalenoyl Gemcitabine Monophosphate: Synthesis, Characterisation of

Nanoassemblies and Biological Evaluation

Joachim Caron,^[a]Elise Lepeltier,^[b]L. Harivardhan Reddy,^[b][‡]Sinda Lepêtre-Mouelhi,^[b]

Séverine Wack,^[b]Claudie Bourgaux,^[b]Patrick Couvreur,^[b]and Didier Desmaële*^[b]

Keywords: Nucleosides / Nucleotides / Prodrugs / Antitumor agents / Medicinal chemistry / Nanostructures

Trialkylammonium 4-(N)-1,1Ј,2-trisnor-squalenoylgemcitab- thickness of about 70 Å. Such liposomal trialkylammonium

ine monophosphate (SQdFdC-MP) salts were prepared from

1,1Ј,2-trisnor-squalenoylgemcitabine by phosphoramidite-

type chemistry. This amphiphilic molecule self-assembled

into nanoassemblies of about 100 nm in size in aqueous solu-

tions. Cryo-TEM and small-angle X-ray scattering (SAXS)

investigations revealed that SQdFdC-MP molecules self-

organized into unilamellar liposomes with a membrane

SQdFdC-MP formulations displayed significant anticancer

activity on leukaemia cells. These results suggested that

squalene conjugates of negatively charged nucleotide ana-

logues efficiently penetrated into tumour cells. Furthermore,

triethylammonium SQdFdC-MP nanoassemblies were found

to be more active than the parent nucleoside towards the

L1210 10 K resistant cell line.

form by deoxycytidine kinase (dCK). The primary phos-

phorylation end product (dFdC-MP, 2) is subsequently

phosphorylated by pyrimidine kinases to the active 5Ј-di-

phosphate (dFdC-DP) and then to the active triphosphate

(dFdC-TP) derivative. This primary phosphorylation by

dCK is the rate-limiting step for the activation of gemcitab-

ine.^[6]Poor phosphorylation due to low dCK expression in

the cells represents another important resistance mecha-

nism limiting the activity of gemcitabine.^[7]Furthermore,

gemcitabine suffers a short drug half-life, due to rapid de-

Introduction

Gemcitabine (2Ј,2Ј-difluorodeoxycytidine, dFdC, 1, Fig-

ure 1) is a synthetic nucleoside analogue of deoxycytidine

with geminal fluorine atoms at the C-2Ј carbon. This anti-

neoplastic drug is currently marketed under the name

Gemzar® and approved as a first-line agent in the treat-

ment of pancreatic, non-small cell lung and breast can-

cers.^[1]In preclinical studies, besides efficient antitumour ac-

tivity against pancreatic cancer, it has also shown efficacy

against a wide range of human solid tumours including co-

lon, bladder, ovarian, head and neck, cervical and hepato-

cellular cancers.^[2]Gemcitabine acts as an antimetabolite,

inhibiting ribonucleotide reductase and DNA synthesis.^[3]

This drug has a complex metabolism pathway, and there are

many mechanisms that contribute to reduce its efficiency.

Because of its hydrophilic character, this compound can be

transported intracellularly only through an active mecha-

nism driven by membrane nucleoside transporters including

the hENT1, hENT2, hCNT1 and hCNT3 proteins.^[4]De-

ficiency in membrane nucleoside transporter activity in-

duces resistance to gemcitabine,^[5]which is an important

cause of treatment failure. After entry into the cells, gemcit-

abine is first phosphorylated to its monophosphorylated

[a] UMR 8076 BIOCIS Université Paris Sud,

5 rue J.-B. Clément, 92296 Châtenay-Malabry, France

[b] UMR 8612, Université Paris Sud,

5 rue J.-B. Clément, 92296 Châtenay-Malabry, France

Fax: +33-1-46835752

E-mail: didier.desmaele@u-psud.fr

[‡] Present address: sanofi-aventis,

13 Quai Jules Guesdes, 94403 Vitry-sur-Seine, France

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201100036.

Figure 1. Structures of gemcitabine, gemcitabine monophosphate

and their squalenoyl derivatives.

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D. Desmaële et al.

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amination to its inactive uracil analogue by endogenous de-

Therefore, in order to reduce the phosphate negative

oxycytidine deaminase.^[8]As a consequence, the clinical im- charge and to enable the modified nucleotide to enter the

pact of gemcitabine on pancreatic adenocarcinomas re- cells, many pronucleotides modified by masking groups on

mains modest, owing to a narrow therapeutic index and the phosphate moiety have been designed.^[18]Although

a high degree of inherent and acquired chemoresistance.^[9]many imaginative and clever approaches have been tried,

Methods that have been employed to improve the half-lives no single platform method has proved to be successfully

and to reduce the toxicities of gemcitabine and other anti- applicable to all the NAs. In particular, the delivery of 2Ј,3Ј-

cancer nucleoside-based drugs have included encapsulation disubstituted nucleotides remains troublesome.^[19]In in-

in colloidal drug delivery systems^[10]and the development vestigations directed towards overcoming these drawbacks,

of lipidic prodrugs.^[11]These prodrugs are designed to pen- we recently reported that squalenoyl monophosphate nu-

etrate cells through passive diffusion, bypassing the nucleos- cleosides with antiviral or anticancer activities were also

idic transporter system.

able to assemble into nanoparticles that displayed promis-

To enhance cellular uptake of nucleoside analogues, we ing biological activities.^[20]In this paper we give a full ac-

have recently developed a new strategy known as “squa- count of the synthesis of conjugate SQdFdC-MP (5, Fig-

lenoylation”, involving conjugation to squalene (3, Fig- ure 1), we provide key features about the supramolecular

ure 1), a natural precursor in cholesterol biosynthesis. The organisation of the nano-objects and finally we disclose new

resulting squalenoyl prodrugs possessed amphiphilic char- biological results relating to the efficacy of SQdFdC-MP

acter and spontaneously self-organized into nanoassemblies towards gemcitabine-resistant cancer cells.

(NAs) of 100–300 nm in water. Similarly, coupling of gemci-

tabine to squalene resulted in the formation of squalenoyl

gemcitabine (SQdFdC, 4), which when dispersed in water

Results and Discussion

formed nanoassemblies of 130 nm mean diameter.^[12]

SQdFdC NAs are unique and possess important character-

Although there are certainly ample instances of nucleo-

istic properties in relation to other gemcitabine conjugates, tides modified by lipid appendages fixed on their phospho-

such as self-association into NAs in water without the aid rus moieties, lipidic conjugates possessing unmodified phos-

of surfactants unlike other fatty acid/gemcitabine conju- phate groups are much scarcer. The synthesis of such deriv-

gates described in the literature [i.e., 4-(N)-valeroyl-, 4-(N)- atives is usually achieved by acylation of the exocyclic

lauroyl- and 4-(N)-stearoylgemcitabine, which were highly amino function of a cytosine or adenine nucleoside prior to

lipophilic and formed large aggregates in water].^[13]After implementation of the 5Ј-phosphorylation. Nevertheless, at

intravenous administration, this SQdFdC nanomedicine the outset of this study we decided first to explore the direct

displayed impressively enhanced anticancer activity (relative acylation of the exocyclic amino function of the cytosine

to free gemcitabine) against experimental leukaemia, devel- moiety of the unprotected 5Ј-phosphorylated cytidine deriv-

oped either by intravenous injection of leukaemia cells lead- ative 2 through the use of deoxycytidine monophosphate

ing to metastasis^[14]or by subcutaneous grafting to induce (8, Scheme 1) as a simplified, commercially available, model

solid tumours.^[15]In mice with L1210 leukaemia, the intra- compound of dFdC-MP (2).^[21]

venously injected SQdFdC nanomedicine led to 75% long-

However, condensation of deoxycytidine monophosphate

term survivors, which was not observed after treatment with (8) with the mixed anhydride 7 derived from 1,1Ј,2-trisnor-

free gemcitabine at equitoxic doses.^[16]It has been shown squalenic acid (6), readily available in few steps from squa-

that this improved anticancer activity is probably due to the lene,^[12,22]provided only a minute amount of an inseparable

ability of SQdFdC to diffuse into tumour cells passively, mixture of the monosqualenoyl derivatives 9 and 10 (4%)

leading to further accumulation within cellular membranes, together with the bis-squalenoyl material 11 (6%)

including those of organelles such as the endoplasmic retic- (Scheme 1). The failure of this direct procedure prompted

ulum. SQdFdC is then released from this transient reservoir us to develop a new synthetic route involving the phosphor-

in the cell cytoplasm and cleaved into dFdC, which leads ylation of SQdFdC (4). Many very efficient procedures for

either to the build-up of its biologically active triphosphate the phosphorylation of nucleosides are available,^[23]but we

metabolite, or to the efflux of dFdC through equilibrative quickly discovered that the presence of the isoprenylated

membrane transporters.^[17]

Beyond these results, we were keen to circumvent the troublesome than we had originally thought.

side chain bound to the cytosine nucleus was much more

cellular kinases by taking advantage of the squalenoyl car-

Several attempts to phosphorylate the 5Ј-hydroxy group

rier to allow passive diffusion of charged nucleotide of squalenoyl-dideoxycytidine (12, Scheme 2),^[12]used as

through cell membranes. Indeed, the direct delivery of the a simplified surrogate of SQdFdC, with various phos-

nucleoside monophosphate (NMP) into the tumour cells phorus(V) reagents were unsuccessful. Condensation of 12

has been considered as a potential strategy for overcoming with POCl₃/triethyl phosphate,^[24]for example, induced the

the rate-limiting primary phosphorylation step. Unfortu- competitive cleavage of the squalene chain. On the other

nately, NMPs were found to be inefficient because they are hand, diethyl phosphorochloridate^[25]and dibenzyl phos-

not transported by nucleoside transporters and they do not phorochloridate^[26]reacted smoothly with 12 in the presence

penetrate freely through the cellular membranes because of of N-methylimidazole as catalyst to provide the expected

their high polarities.

triester phosphates 13a and 13b in 90% and 48% yields,

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Squalenoyl Gemcitabine Monophosphate

Scheme 2. Synthetic approach to the triethylammonium phosphate

salt 9a. Reagents and conditions: a) i. 14 (1.2 equiv.), iPr₂NEt,

THF, 0 °C, 1 h, ii. 1H-tetrazole, NCCH₂CH₂OH, CH₃CN, THF,

20 °C, 1 h iii. H₂O₂(30 wt.-%), 0 °C, 30 min, 23%; b) DBU,

CH₃CN, 24 h, 20 °C; c) i. 16 (2.5 equiv.), 1H-tetrazole, CH₃CN,

CH₂Cl₂(1:1), 20 °C, 3 d; ii. H₂O₂(30 wt.-%), 0 °C, 4 h, 52%;

d) Et₃N, CH₃CN, 20 °C, 24 h, 98%.

Scheme 1. Attempted synthesis of squalenoyl-deoxycytidine mono-

phosphate from deoxycytidine monophosphate.

respectively. Unfortunately, attempts to remove the ethyl or

benzyl groups of 13a and 13b by use of TMSBr^[27]met with

failure once again, because of participation by the double

bonds of the squalene chain.

The use of phosphoramidite reagents for the introduc- under anhydrous conditions.^[32]The phosphoramidite 16

tion of the desired phosphate group was therefore investi- was prepared in a one-pot process from PCl₃via N,N-di-

gated.^[28]Because of the potentially easy deprotection of the isopropylphosphoramidous dichloride by a slight modifica-

resulting phosphate through a retro-Michael process, (2- tion of the reported procedure.^[33]Condensation of 12 with

cyanoethyl) N,N-diisopropylchlorophosphoramidite^[29](14) 16 in the presence of 1H-tetrazole, followed by oxidative

was chosen. In the event, sequential treatment of 12 with workup, gave the expected phosphotriester 13d in 52%

14 in the presence of N,N-diisopropylethylamine, followed yield. Compound 13d, on treatment with a large excess of

by 3-hydroxypropionitrile and 1H-tetrazole, and oxidative triethylamine in acetonitrile, followed by removal of the 9-

workup with iodine gave the expected phosphotriester 13c methylene-9H-fluorene by-product by ether washing, gave

in a very low yield, due to partial addition of iodine to the rise to the desired triethylammonium phosphate salt 9a in

squalene double bonds. However, when hydrogen peroxide 98% yield as an amorphous white solid.

(30%) was used, 13c was obtained in a modest 23% yield.

With a viable synthetic route to hand, we turned our at-

Attempts to improve this yield through the use of the corre- tention to the more challenging case of gemcitabine. The

sponding bis(2-cyanoethyl) N,N-diisopropylphosphoramid- synthesis of SQdFdC (4) has been described by us pre-

ite turned out to be fruitless.^[30]The final deprotection of viously.^[12]Briefly, 1,1Ј,2-trisnor-squalenic acid (6,

13c through β-elimination was attempted with DBU. Un- Scheme 1) was first activated with ethyl chloroformate be-

fortunately, even in the presence of TMSCl as catalyst, only fore coupling to gemcitabine to provide 4 routinely in 55%

one cyanoethyl group could be removed.^[31]

isolated yield. Although we were aware that both hydroxy

A more rewarding route was found on changing to N,N- groups of 4 could react, we first checked the direct conden-

diisopropyl-phosphoramidous acid bis(9H-fluoren-9-ylmeth- sation of the phosphoramidite 16 with SQdFdC (4), as-

yl) ester (16, Scheme 2). The 9-fluorenylmethyl group was suming that we could expect such a bulky reagent to phos-

demonstrated by Watanabe et al. to be an efficient base- phorylate the less hindered primary alcohol selectively. Un-

labile protecting group allowing for complete deprotection fortunately, though, treatment of SQdFdC (4) with the

of phosphotriesters by use of an excess of a tertiary amine phosphoramidite 16, followed by oxidative workup, gave

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the desired phosphate 17a (Scheme 3) in only 10% yield, 5Ј-DMT group by treatment with acetic acid was unsatis-

together with the 3Ј-isomer 17b and the 3Ј,5Ј-diphosphoryl- factory. In contrast, however, upon brief treatment with for-

ated compound 18.

mic acid^[35]the expected 5Ј-free alcohol 24 was obtained in

77% yield. Condensation of 24 with the phosphoramidite

16 in the presence of 1H-tetrazole, followed by hydrogen

peroxide workup, gave the phosphotriester 25 in 80% yield.

At this stage the 3Ј-hydroxy group was unmasked by treat-

ment with HF·pyridine^[36]to afford 26 in 69% yield. The

final deprotection of the phosphate group was effected by

treatment with a large excess of triethylamine in acetonitrile

to give the triethylammonium phosphate salt 5a in 98%

yield as an amorphous white solid.

Scheme 3. Attempted phosphorylation of SQdFdC (4) and the 1-

ethoxyethyl-ether-protected compound 21. Reagents and condi-

tions: a) i. 16 (1.1 equiv.), 1H-tetrazole, CH₃CN, CH₂Cl₂(1:1),

20 °C, 3 d; ii. H₂O₂(30 wt.-%), 0 °C, 4 h; b) TBDMSCl, Im, DMF,

20 °C, 36 h, 89%; c) ethyl vinyl ether, PPTS cat. CH₂Cl₂, 20 °C,

16 h, quantitative; d) nBu₄NF, THF, 20 °C, 2 h, 86%; e) i. 16, 1H-

tetrazole, CH₃CN, CH₂Cl₂(1:1), 20 °C, 3 d; ii. H₂O₂(30 wt.-%),

0 °C, 4 h.

We therefore decided to protect the 3Ј-hydroxy group be-

fore addressing the phosphorylation of the 5Ј-hydroxy

group. In contrast with the phosphorylation reaction, the

silylation of the 5Ј-hydroxy group with tBuMe₂SiCl was un-

eventful, leading chemoselectively to the 5Ј-silylated deriva-

tive 19 (Scheme 3). Treatment of the alcohol 19 with ethyl

vinyl ether and selective removal of the silyl group at C-5Ј

with TBAF afforded the key intermediate 21 in 86% overall

yield. Unfortunately, though, condensation of 21 with the

phosphoramidite 16 as described above gave a complex

mixture of phosphorylated materials. Examination of the

crude mixture revealed that the 1-ethoxyethyl ether protect-

ing group at C-3Ј did not survive the slightly acidic reaction

conditions.

Scheme 4. Synthetic approach to the tertiary ammonium phos-

phate salts 5a and 5b. Reagents and conditions: a) DMTrCl,

DMAP cat., Py, 20 °C, 3 d, 83%; b) TBDMSCl, DBU, CH₂Cl₂,

room temp., 36 h, 90%; c) HCO₂H/MeOH/CH₂Cl₂(2:1:1), 0 °C,

45 min, 77%; d) i. 16, 1H-tetrazole, CH₃CN, CH₂Cl₂(1:1), 20 °C,

3 d; ii. H₂O₂(30%), 0 °C, 4 h, 80%; e) HF·py, py, 20 °C, 4 h, 68%;

f) R¹R²R³N, CH₃CN, 24 h, 20 °C (5a: 98%; 5b, 85%).

We therefore decided to protect the 3Ј-hydroxy moiety

Because potential toxicity is associated with triethyl-

with a silyl group, assuming that it should be resistant to amine, we looked for a pharmacological more suitable salt.

exposure to tetrazole. Transient protection of the 5Ј-hy- Unfortunately, treatment with currently used amines such

droxy group was achieved through formation of the highly as diethylethanolamine, 4-(2-hydroxyethyl)morpholine or

acid-labile dimethoxytrityl ether (Scheme 4).^[34]Treatment procaine turned out to be inefficient. We finally found that

of 4 with dimethoxytrityl chloride in pyridine, followed by treatment of 26 with dimethylethanolamine smoothly gave

C-3Ј protection with a tert-butyldimethylsilyl group, thus the corresponding dimethylethanolammonium phosphate

provided the fully protected derivative 23. Removal of the salt 5b in 85% yield.

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Squalenoyl Gemcitabine Monophosphate

Preparation and Characterization of Nanoassemblies of 5a

and 5b

oscillation, arising from the bilayer thickness. From the po-

sition of the curve minimum a thickness of about 70 Å

could be estimated for the membrane.

Triethylammonium SQdFdC-MP (5a) NAs were pre-

pared in a single step by nanoprecipitation of an ethanolic

solution in isotonic aqueous dextrose (5%) solution. Nano-

particle formation occurred immediately. Ethanol was then

evaporated at 37 °C under vacuum in a Rotavapor^®and

the organic solvent-free colloidal dispersion was stored at

4 °C.^[37]The mean diameters of the obtained NAs are

shown in Table 1. These particles had a polydispersity index

lower than 0.30 as measured by quasi-elastic light scat-

tering. No significant change in the size of the NA was

detected over a 3 day storage period at 4 °C. Such stability

could be correlated with the high negative Zeta potential (ζ

= –43.1 mV) observed. Nanoprecipitation of an ethanolic

solution of the dimethylethanolammonium phosphate salt

5b also led to the formation of similar small-sized NAs dis-

playing the characteristic Tyndall effect of colloids.

Table 1. Measurement of particle size and polydispersity index of

5a and 5b nanoassemblies.

Figure 3. SAXS pattern of the SQdFdC-MP liposomes shown on

log scale. At low q values the intensity exhibits a q^–2dependence

characteristic of planar membranes.

Concentration [mgmL^–1]

Size [nm]

PDI

5a

5b

1

3

5

2

4

59Ϯ2

104Ϯ2

144

56

153Ϯ15

0.27

0.14

0.18

0.29

0.06

As shown in Table 1, nanoprecipitation of SQdFdC-MP

(5a) solutions with concentration higher than 1 mgmL^–1

yielded larger vesicles, suggesting that increases in nanopar-

ticle size were preferred to increases in nanoparticle num-

Triethylammonium SQdFdC-MP (5a) nanoassemblies bers. With the assumption that the number of vesicles re-

(2 mgmL^–1) were imaged by cryo-TEM, revealing a popula- mained unchanged when the SQdFdC-MP concentration in

tion of unilamellar liposomes with diameters ranging from aqueous solution varied, the ratio between vesicles’ radii

100 to 300 nm (Figure 2). Small-angle X-ray scattering could be estimated from the ratio between the correspond-

(SAXS) was carried out on a suspension of liposomes to ing solution concentrations. If the membrane thickness d is

characterize their organization fully (Figure 3). The ob- small relative to the radius (r) of the vesicle, the volume of

tained scattering curve corresponds to the form factor of a the vesicle shell can be expressed as

locally flat membrane. Indeed, in the range q = 0.02–

Vs_h= 4πr²d

0.06 Å^–1the logI vs. logq plot shows a linear variation with

a slope close to –2 (–1.97), which is the fingerprint of a

planar structure. At higher q values the scattering intensity

was characterized by a broad bump followed by a small

The bilayer thickness is constant. Consequently, the fol-

lowing relation should hold

rЈ/r = √cЈ/c

where r and rЈ are the radii of the vesicles formed by nano-

precipitation of solutions of concentration c and cЈ, respec-

tively. The values for the 5a compound reported in Table 1

are in reasonable agreement with this relationship, suggest-

ing that, in this concentration range, the concentration of

SQdFdC-MP is the most important parameter determining

the sizes of the nanoparticles.

Both cryo-TEM and SAXS provided evidence that tri-

ethylammonium SQdFdC-MP bioconjugates self-organized

as liposomal vesicles. In contrast, it has previously been

shown that SQdFdC NAs displayed an inverse hexagonal

structure formed by close-packed cylinders in which the

aqueous cores were surrounded by hydrophilic gemcitabine

molecules linked to the hydrophobic squalene moieties.^[15]

The difference in structure between SQdFdC and SQdFdC-

MP NAs could be explained by simple geometrical packing

Figure 2. Selected cryo-TEM image showing unilamellar vesicles of

SQdFdC-MP (5a).

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considerations. The molecular shape of the SQdFdC mole- mediating the primary phosphorylation step necessary for

cule can be approximated to a truncated cone, with a small the activation of dFdC. Such down-regulation of deoxycyt-

polar headgroup interfacial area and a bulky hydrophobic idine kinase in the cells leads to poor activation of dFdC,

SQ chain. The addition of the 5Ј-phosphate group should resulting in cellular resistance to dFdC. As expected, on the

result in a bulkier and probably more hydrated headgroup. L1210 10 K cell line, dFdC exhibited a considerably lower

Furthermore, electrostatic interactions between polar activity, displaying an IC₅₀value of 19.4 μm (relative to the

groups are believed to increase their interfacial areas. In a 3.2 nm on the L1210 wt cell line). In contrast, and unlike in

nutshell, this should correspond to a more cylindrical shape the case of the L1210 wt cell line, the SQdFdC (4) NAs

of the SQdFdC-MP molecules, stabilizing bilayers upon displayed an improved activity (IC₅₀value of 9.8 μm) rela-

self-assembly in water. The electrostatic repulsion between tive to free dFdC (19.4 μm). This lower IC₅₀value was at-

bilayers should favour the existence of unilamellar lipo- tributed to the fact that the SQdFdC NAs were slowly re-

somes.

leasing the gemcitabine intracellularly, not overwhelming

the low intracellular dCK enzymatic activity. In contrast,

free gemcitabine is mainly degraded intracellularly by de-

amination, because the dCK activity is too low to convert

a sufficient amount of gemcitabine into its active phosphor-

ylated counterpart.^[14]Interestingly, liposomal formulations

of 5a were still more efficient (IC₅₀value of 3.7 μm) than

those of the parent compound SQdFdC (4) NAs. To rule

out the possibility of interference of the triethylammonium

cation of 5a with its anticancer activity, the cytotoxicity of

aqueous triethylammonium dihydrogen phosphate solution

(as a placebo) on this cell line was studied. This placebo

did not elicit any toxicity toward the L1210 10 K cell line

at least up to 20 μm, although cell survival started decreas-

ing above this concentration, thus pointing to the absence

of any interference of triethylammonium cation in the ob-

served activity of 5a liposomes. The superiority of trieth-

ylammonium SQdFdC-MP liposomes has thus been clearly

demonstrated, because they significantly reduced the resis-

tance displayed by the L1210 10 K cell line in relation to

dFdC (1) and SQdFdC (4). It is hypothesized that the ob-

served improved anticancer activity of 5a liposomes is the

result of bypassing the primary phosphorylation step,

thereby presenting dFdC for the formation of the active tri-

phosphate form of dFdC.

In Vitro Biological Evaluation

The anticancer activities of trialkylammonium salts of

5a and 5b unilamellar liposomes were first tested against

L1210 wt (a dFdC-sensitive murine lymphoid leukaemia

cell line) (Table 2). On this cell line, dFdC exhibited efficient

anticancer activity, displaying a 50% inhibitory concentra-

tion (IC₅₀) value of 3.2 nm. Owing to its prodrug nature,

SQdFdC (4) NA showed a higher IC₅₀(29 nm) and thus a

lower anticancer activity than free dFdC, probably because

of the time lag associated with cellular penetration, dFdC

leakage from SQdFdC and further primary phosphoryl-

ation, before resulting in the formation of the active tri-

phosphate. In contrast, the phosphorylated derivatives of

SQdFdC (i.e., the triethylammonium salt 5a and the di-

methylethanolammonium salt 5b liposomes) displayed con-

siderably lower IC₅₀values (6.1 nm and 6.7 nm, respectively)

and thus more efficient anticancer activity than SQdFdC 4-

NA, whereas interestingly the activities of these phosphory-

lated derivatives were quite similar to that of dFdC. No-

tably, triethylammonium dihydrogen phosphate incubated

as an aqueous solution did not elicit any activity against

this cell line in the dose range tested, thus ruling out any

cytotoxicity of the triethylammonium cation.

Table 3. In vitro anticancer activities of dFdC and nanoassemblies

of 4 and 5a against the L1210 10 K (dFdC-resitant) murine leu-

kaemia cell line.

Table 2. In vitro anticancer activities of dFdC and nanoassemblies

of 4, 5a and 5b against L1210 wt (dFdC-sensitive) murine leu-

kaemia cell line.

IC₅₀on L1210 10 K leukaemia

cell line (μm)

IC₅₀on L1210 wt

leukaemia cell line (nm)

IC₉₀(nm)

dFdC (1)

19.4

9.8

3.7

SQdFdC (4)-NAs^[a]

5a-NAs

dFdC (1)

3.2

38

6.1

6.7

10

73

31

28

SQdFdC (4)-NAs^[a]

5a-NAs

[a] NAs = nanoassemblies.

5b-NAs

[a] NAs = nanoassemblies.

Conclusions

Drug resistance is a serious clinical concern in the treat-

ment of cancer, so the anticancer activity of the SQdFdC-

A new lipidic derivative of gemcitabine monophosphate

MP derivative 5a was also tested against the L1210 10 K with an unmodified phosphate group has been synthesized

murine lymphoid leukaemia cell line (Table 3). This cell line by covalent coupling of a squalenoyl residue at the N-4 po-

is a resistant variant of the L1210 wt cell line developed by sition in the cytidine nucleus by phosphoramidite-type

chronic exposure to incremental concentrations of dFdC.^[38]chemistry. The ammonium salts of this bioconjugate self-

The L1210 10 K cell line thus exhibits ca. 6000-fold resis- organized in water as nanoassemblies of 50–150 nm mean

tance to dFdC measured in terms of the IC₅₀values, due diameters. Cryo-TEM and small-angle X-ray scattering

to the down-regulation of deoxycytidine kinase, the enzyme (SAXS) investigations showed that SdFdC-MP molecules

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Squalenoyl Gemcitabine Monophosphate

(5 mL), dried with MgSO₄, and concentrated in vacuo to leave an

oil. The crude product was purified by flash chromatography on

silica gel (AcOEt/cyclohexane 1:1) to give the phosphotriester 13a

as a colourless oil (70 mg, 90%): R_f= 0.33 (AcOEt/cyclohexane

self-organized in unilamellar liposomes with a membrane

thickness of about 70 Å. The liposomal dFdC-MP trialkyl-

ammonium salts displayed promising activity on L1210 leu-

kaemia cells, demonstrating that the conjugation promotes

passive diffusion through cell membranes, whereas

SQdFdC-MP triethylammonium salts display significant

cytotoxicity on the drug-resistant L1210 10 K cell line. This

platform thus opens exciting prospects for the discovery of

new anticancer nanomedicines with improved pharmaco-

logical activities on resistant cancer cells.

1

1:1). [α]_D= +33.5 (c = 2.9, EtOH). H NMR (300 MHz, CDCl₃):

δ = 9.47 (s, 1 H, NH), 8.22 (d, J = 7.4 Hz, 1 H, 6-H), 7.40 (d, J =

7.4 Hz, 1 H, 5-H), 6.03 (dd, J = 6.4, 2.4 Hz, 1 H, 1Ј-H), 5.16–5.04

[m, 5 H, CH=C(CH₃)], 4.40–4.08 (m, 5 H), 2.56 (t, J = 7.5 Hz, 2

H, NHCOCH₂), overlapped by 2.55–2.45 (m, 1 H, 2Ј-H), 2.30 (t,

J

= 7.5 Hz, 2 H, NHCOCH₂CH₂), 2.16–1.91 [m, 19 H,

=CHCH₂CH₂C(CH₃), 2Ј-H, 3Ј-H], 1.64 [s, 3 H, (CH₃)₂C=], 1.58

(s, 3 H, CH₃), 1.56 (s, 9 H, CH₃), 1.55 (s, 3 H, CH₃), 1.40–1.28 (m,

6 H, POCH₂CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 173.4

(C, NHCO), 162.6 (C, C-4), 155.2 (C, C-2), 144.4 (CH, C-6), 135.2

[C, =C(CH₃)CH₂], 134.9 [2 C, =C(CH₃)CH₂], 132.9 [C, =C(CH₃)-

CH₂], 131.3 [C, =C(CH₃)CH₂], 125.7 [CH, HC=C(CH₃)], 124.4

[2ϫCH, HC=C(CH₃)], 124.3 [2ϫCH, HC=C(CH₃)], 96.0 (CH, C-

5), 88.1 (CH, C-1Ј), 80.3 (d, J_C,P= 7.5 Hz, CH, C-4Ј), 67.2 (d, J_C,P

Experimental Section

General Methods: IR spectra were obtained from solids or neat

liquids with a Fourier Transform Bruker Vector 22 spectrometer.

Only significant absorptions are listed. Optical rotations were

=

4.5 Hz, CH₂, C-5Ј), 64.3 (d, J_C,P= 5.3 Hz, 2ϫCH₂,

1

measured with a Perkin–Elmer 241 polarimeter at 589 nm. The H

CH₃CH₂OP), 39.8 [2ϫCH₂, =C(CH₃)CH₂], 39.6 [CH₂, =C(CH₃)-

CH₂], 36.5 [CH₂, =C(CH₃)CH₂], 34.6 [CH₂, =C(CH₃)CH₂], 33.3

(CH₂, C-2Ј), 28.3 [2ϫCH₂, =C(CH₃)CH₂], 26.8 [2ϫCH₂,

=C(CH₃)CH₂], 26.7 [CH₂, =C(CH₃)CH₂], 25.8 [CH₃, (CH₃)₂C=],

24.6 (CH₂, C-3Ј), 17.8 (CH₃), 16.3–16.0 (6CH₃) ppm. ³¹P NMR

and ¹³C NMR spectra were recorded with Bruker Avance 300

(300 and 75 MHz for ¹H and ¹³C, respectively) or Bruker Av-

ance 400 (400 and 100 MHz for ¹H and ¹³C, respectively) spectrom-

eters. The ¹⁹F and ³¹P NMR spectra were recorded with Bruker

AC 200 P (188 and 81 MHz for ¹⁹F and ³¹P, respectively) or Bruker

Avance 400 (376 MHz and 162 MHz for ¹⁹F and ³¹P, respectively)

spectrometers. Recognition of methyl, methylene, methine, and

quaternary carbon nuclei in ¹³C NMR spectra was based on the J-

modulated spin-echo sequence. Mass spectra were recorded with a

Bruker Esquire-LC instrument. The purities of the tested com-

pounds 5a and 5b were determined by elemental analysis to be at

least 95%. Elemental analyses were performed by the Service de

microanalyse, Centre d’Etudes Pharmaceutiques, Châtenay-Mala-

bry, France, with a Perkin–Elmer 2400 analyzer. The sizes of the

obtained nanoassemblies were measured with a Malvern particle

size analyzer (Zetasizer). Analytical thin-layer chromatography was

performed with Merck silica gel 60F₂₅₄glass precoated plates

(0.25 mm layer). Column chromatography was performed with

Merck silica gel 60 (230–400 mesh ASTM). Diethyl ether and tetra-

hydrofuran (THF) were distilled from sodium/benzophenone ketyl.

Methanol and ethanol were dried with magnesium and distilled.

Benzene, toluene, DMF and CH₂Cl₂were distilled from calcium

hydride under nitrogen. All reactions involving air- or water-sensi-

tive compounds were routinely conducted in glassware that had

been flame-dried under a positive pressure of nitrogen. Gemcitab-

ine and dideoxycytidine were purchased from Sequoia Research

Products Ltd. (UK). Squalene and 3-[4,5-dimethylthiazol-2-yl]-3,5-

diphenyltetrazolium bromide (MTT) were purchased from Sigma–

Aldrich Chemical Co., France. RPMI 1640 GlutaMAX I and foetal

bovine serum were purchased from Dulbecco (Invitrogen, France).

Penicillin and streptomycin solutions were purchased from Lonza

(Verviers, Belgium). Chemicals obtained from commercial suppliers

were used without further purification. L1210 10 K is a deoxycytid-

ine kinase-deficient resistant variant (6080-fold resistant to gemcit-

abine, as measured through the IC₅₀values, obtained from Dr. C.

Dumontet, INSERM, Lyon, France) of the L1210 wt murine cell

line.

(162 MHz, CDCl ): δ = 1.58 (s) ppm. IR (neat): ν = 2984, 2962,

˜

3

2930, 2845, 1715, 1665, 1612, 1560, 1493, 1434, 1395, 1386, 1317,

1266, 1165, 1138, 1098, 1078, 1027, 984, 946, 809, 791, 731 cm^–1.

MS (+APCI): m/z (%) = 730.4 (100) [M + H]⁺.

Compound 13b: N-Methylimidazole (103 mg, 1.25 mmol), Et₃N

(72 mg, 0.72 mmol) and dibenzyl chlorophosphate (300 mg,

1.7 mmol) were added sequentially to a solution of SQddC (12,

100 mg, 0.17 mmol) in CHCl₃(2 mL). The reaction mixture was

stirred at 60 °C for 16 h and was then treated with water (3 mL).

The phases were separated and the aqueous layer was extracted

with AcOEt (3ϫ10 mL). The combined organic layers were

washed with brine (5 mL), dried with MgSO₄and concentrated in

vacuo to afford a yellow oil. The crude product was purified by

flash chromatography on silica gel (AcOEt/cyclohexane 1:1) to give

the phosphotriester 13b as a colourless oil (70 mg, 48%): R_f= 0.33

(AcOEt/cyclohexane 1:1). [α]_D= +32.9 (c = 1.55, EtOH). ¹H NMR

(300 MHz, CDCl₃): δ = 9.34 (s, 1 H, NH), 8.07 (d, J = 7.5 Hz, 1

H, 6-H), 7.41–7.25 (m, 11 H, 5-H, C₆H₅), 5.98 (dd, J = 6.5, 3.2 Hz,

1 H, 1Ј-H), 5.22–4.97 [m, 9 H, CH=C(CH₃)CH_2,OCH₂Ph], 4.33–

3.97 (m, 3 H, 4Ј-H, 5Ј-H), 2.61–2.32 (m, 5 H), 2.12–1.90 (m, 19 H),

1.67 [s, 3 H, (CH₃)₂C=], 1.62 (s, 3 H, CH₃), 1.59 (s, 9 H, CH₃),

1.58 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 173.2

(C, NHCO), 162.5 (C, C-4), 155.1 (C, C-2), 144.4 (CH, C-6), 135.6

[C, (CH)₅CCH₂OP], 135.5 [C, (CH)₅CCH₂OP], 135.2 [C, =C(CH₃)-

CH₂], 135.0 [2 C, =C(CH₃)CH₂], 133.0 [C, =C(CH₃)CH₂], 131.3

[C, =C(CH₃)CH₂], 128.8 (2ϫCH, Ph), 128.7 (4ϫCH, Ph), 128.2

(4ϫCH, Ph), 125.8 [CH, HC=C(CH₃)], 124.5 [2ϫCH,

HC=C(CH₃)], 124.3 [2ϫCH, HC=C(CH₃)], 96.1 (CH, C-5), 88.1

(CH, C_1Ј), 80.1 (d, J_C,P= 8.3 Hz, CH, C-4Ј), 67.6 (d, J_C,P= 5.3 Hz,

2ϫCH₂, PhCH₂OP), 67.6 (d, J_C,P= 5.3 Hz, CH₂, C-5Ј), 39.8

[2ϫCH₂, =C(CH₃)CH₂], 39.7 [CH₂, =C(CH₃)CH₂], 36.6 [CH₂,

=C(CH₃)CH₂], 34.6 [CH₂, =C(CH₃)CH₂], 33.2 (CH₂, C-2Ј), 28.4

Compound 13a: N-Methylimidazole (9 mg, 0.11 mmol), Et₃N [2ϫCH₂, =C(CH₃)CH₂], 26.9 [2ϫCH₂, =C(CH₃)CH₂], 26.8 [CH₂,

(22 mg, 0.22 mmol) and diethyl chlorophosphate (43 mg, =C(CH₃)CH₂], 25.8 [CH₃, (CH₃)₂C=], 24.7 (CH₂, C-3Ј), 17.8

0.24 mmol) were added to a solution of SQddC (12, 63 mg,

0.11 mmol) in CH₂Cl₂(1 mL). The reaction mixture was stirred at

20 °C for 5 h and was then treated with water (3 mL). The phases

were separated and the aqueous layer was extracted with AcOEt

(3ϫ10 mL). The combined organic layers were washed with brine

(CH₃), 16.2 (3ϫCH₃), 16.1 (CH₃), 16.0 (CH₃) ppm. ³¹P NMR

(162 MHz, CDCl ): δ = 0.34 (s) ppm. IR (neat): ν = 2966, 2854,

˜

3

1718, 1665, 1613, 1560, 1493, 1455, 1432, 1383, 1317, 1264, 1215,

1188, 1136, 1100, 1012, 995, 966, 909, 808, 791, 732, 697 cm^–1. MS

(+APCI): m/z (%) = 854.6 (100) [M + H]⁺.

Eur. J. Org. Chem. 2011, 2615–2628

www.eurjoc.org

2621

D. Desmaële et al.

FULL PAPER

Compound 13c: N,N-Diisopropylethylamine (74 mg, 0.57 mmol)

and the chlorophosphoramidite 14 (70 mg, 0.31 mmol) were added

to an ice-cooled solution of SQddC (12, 150 mg, 0.25 mmol) in dry

THF (1 mL). The reaction mixture was stirred at 20 °C for 1 h. 3-

Hydroxypropionitrile (60 mg, 0.88 mmol) and tetrazole (0.45 m in

CH₃CN, 2 mL, 0.9 mmol) were then added and the reaction mix-

ture was stirred for an additional hour at 20 °C. The mixture was

cooled to 0 °C and H₂O₂(30 wt.-%, 0.3 mL, 3 mmol) was added.

Stirring was continued at 0 °C for 30 min, then saturated aqueous

NaHSO₃(10 mL) was added. The mixture was then extracted with

CH₂Cl₂(4ϫ50 mL). The combined organic layers were washed

with brine (20 mL), dried with MgSO₄and concentrated in vacuo.

The crude product was purified by flash chromatography on silica

gel (AcOEt/MeOH 95:5) to provide the phosphotriester 13c as a

colourless viscous oil (45 mg, 23%): R_f= 0.37 (AcOEt/MeOH

(s) ppm. IR (neat): ν = 2964, 2930, 1449, 1396, 1363, 1183, 1153,

˜

1127, 1103, 1009, 975, 887 cm^–1.

Compound 13d: A solution of 1H-tetrazole in acetonitrile (0.45 m,

1.4 mL, 0.64 mmol) and a solution of bis(9H-fluoren-9-ylmethyl)

diisopropylamidophosphite (16, 165 mg, 0.3 mmol) in dry THF

(2 mL) were sequentially added to a solution of SQddC (12, 77 mg,

0.12 mmol) in THF (1 mL). After stirring at 20 °C for 3 days, the

mixture was cooled to 0 °C and H₂O₂(30 wt.-%, 1 mL, 9 mmol)

was added. Stirring was continued at 0 °C for 3 h, then saturated

aqueous NaHSO₃(10 mL) was added. The mixture was then ex-

tracted with CH₂Cl₂(4ϫ50 mL). The combined organic layers

were washed with brine (20 mL), dried with MgSO₄and concen-

trated in vacuo. The residue was purified by flash chromatography

on silica gel (MeOH/CH₂Cl₂5:95) to give the phosphotriester 13d

as a colourless viscous oil (65 mg, 52%): [α]_D= +27.4 (c = 4.05,

1

95:5). H NMR (300 MHz, CDCl₃): δ = 8.29 (d, J = 7.5 Hz, 1 H,

1

CHCl₃). H NMR (300 MHz, CDCl₃): δ = 8.01 (br. s, 1 H, NH),

6-H), 7.50 (d, J = 7.5 Hz, 1 H, 5-H), 6.13–6.01 (m, 1 H, 1Ј-H),

5.29–5.03 [m, 5 H, CH=C(CH₃)], 4.60–4.25 (m, 7 H, CHCH₂OP,

4Ј-H, 5Ј-H), 3.01–2.86 (m, 2 H, NCCH₂CH₂OP), 2.68–2.50 (m, 1

H, 2Ј-H) overlapped by 2.55 (t, J = 6.9 Hz, 2 H, NHCOCH₂-

CH₂)_,2.35 [t, J = 6.9 Hz, 2 H, NHCOCH₂CH₂C(CH₃)], 2.252–

1.78 [m, 21 H, =CHCH₂CH₂C(CH₃), 2Ј-H, 3Ј-H, NCCH₂CH₂OP],

1.67 (s, 6 H, CH₃), 1.61 (s, 12 H, CH₃) ppm. ¹³C NMR (75 MHz,

MeOD): δ = 175.4 (C, NHCO), 164.2 (C, C-4), 157.8 (C, C-2),

145.7 (CH, C-6), 136.0 [C, =C(CH₃)CH₂], 135.8 [2 C, =C(CH₃)-

CH₂], 134.4 [C, =C(CH₃)CH₂], 132.0 [C, =C(CH₃)₂], 126.5 [CH,

HC=C(CH₃)], 125.6 [2ϫCH, HC=C(CH₃)], 125.5 [2ϫCH,

HC=C(CH₃)], 118.4 (2 C, NCCH₂CH₂O), 97.8 (CH, C-5), 89.7

(CH, C-1Ј), 81.8–81.5 (m, CH, C-4Ј), 68.1–67.3 (m, 2ϫCH_2,

NCCH₂CH₂OP), 64.5 (d, J_C,P= 4.5 Hz, CH₂, C-5Ј), 40.9 [2ϫCH₂,

=C(CH₃)CH₂], 40.7 [CH₂, =C(CH₃)CH₂], 37.1 (CH₂,

NHCOCH₂CH₂), 35.7 (CH₂, NHCOCH₂CH₂), 33.8 (CH₂), 29.2

(2ϫCH₂), 27.8 (CH₂), 27.7 (CH₂), 27.6 (CH₂), 25.9 [CH₃, (CH₃)₂-

C=], 25.7 (CH₂), 20.4–20.0 (2ϫCH₂, NCCH₂CH₂OP), 17.8 (CH₃),

16.2 (3ϫCH₃), 16.1 (CH₃) ppm. ³¹P NMR (162 MHz, MeOD): δ

7.92 (d, J = 7.4 Hz, 1 H, 6-H), 7.72 (t, J = 7.2 Hz, 4 H, 4ϫHAr),

7.69 (d, J = 7.4 Hz, 1 H, 5-H), 7.52 (dd, J = 7.4, 3.3 Hz, 2 H, HAr),

7.44–7.19 (m, 10 H, HAr), 5.93 (dd, J = 6.4, 3.3 Hz, 1 H, 1Ј-H),

5.24–4.90 [m, 5 H, CH=C(CH₃)], 4.28–4.17 (m, 4 H, CHCH₂OP),

4.07–3.93 (m, 3 H, CH-CH₂OP 4Ј-H), 3.95 (ddd, J = 11.8, 7.4,

,

3.3 Hz, 1 H, 5Ј-H), 3.84 (ddd, J = 11.8, 6.7, 2.7 Hz, 1 H, 5Ј-H),

2.53–2.30 (m, 5 H, 2Ј-H, NHCOCH₂CH₂), 2.16–1.92 [m, 19 H,

=CHCH₂CH₂C(CH₃), 2Ј-H, 3Ј-H], 1.69 [s, 3 H, (CH₃)₂C=], 1.63

(s, 3 H, CH₃), 1.61 (s, 9 H, CH₃), 1.60 (s, 3 H, CH₃) ppm. ¹³C

NMR (75 MHz, CDCl₃): δ = 172.9 (C, NHCO), 162.3 (C, C-4),

155.0 (C, C-2), 143.7 (CH, C-6), 142.8 (4 C, Ar), 141.3 (2 C, Ar),

135.0 [2 C, =C(CH₃)CH₂], 134.7 [2 C, =C(CH₃)CH₂], 132.8 [C,

=C(CH₃)CH₂], 131.1 [C, =C(CH₃)₂], 127.8 (4ϫCH, Ar), 127.0

(4ϫCH, Ar), 125.5 [CH, HC=C(CH₃)], 124.8 (3ϫCH, Ar), 124.7

(CH, Ar), 124.3 [2ϫCH, HC=C(CH₃)], 124.1 [2ϫCH,

HC=C(CH₃)], 119.9 (CH, Ar), 119.8 (3ϫCH, Ar), 96.0 (CH, C-

5), 87.9 (CH, C-1Ј), 79.6 (d, J_C,P= 7.2 Hz, CH), 69.0 (d, J_C,P

=

5.8 Hz, 2ϫCH₂, FmCH₂OP), 67.5 (d, J_C,P= 5.7 Hz, CH₂, C-5Ј),

47.8 (d, J_C,P= 7.8 Hz, CH), 47.7 (d, J_C,P= 7.9 Hz, 2ϫCH, FmC-

9), 39.6 [2ϫCH₂, =C(CH₃)CH₂], 39.4 [CH₂, =C(CH₃)CH₂], 36.3

(CH₂, NHCOCH₂CH₂), 34.4 (CH₂, NHCOCH₂CH₂), 32.8 (CH₂),

28.1 (2ϫCH₂), 26.6 (2ϫCH₂), 26.5 (CH₂), 25.6 [CH₃, (CH₃)₂C=],

24.5 (CH₂), 17.6 (CH₃), 16.0 (3ϫCH₃), 15.9 (CH₃), 15.8

= –1.77 (s) ppm. IR (neat): ν = 3026, 2930, 2850, 1715, 1659, 1612,

˜

1559, 1493, 1450, 1431, 1383, 1317, 1308, 1266, 1188, 1137, 1096,

1045, 999, 966, 791 cm^–1. MS (+APCI): m/z (%) = 780.5 (30) [M +

H]⁺, 494.3 (100) [M – C₁₁H₁₆N₂O₅P + 2 H]⁺.

(CH ) ppm. IR (neat): ν = 3150–2800, 1715, 1661, 1610, 1652,

˜

3

N,N-Diisopropyl Bis(9-methylfluorenyl)phosphoramidite (16): N,N-

Diisopropylethylamine (1.48 g, 11.5 mmol) was added to an ice-

cooled solution of PCl₃(0.79 g, 5.7 mmol) in dry THF (20 mL).

Diisopropylamine (1.08 g, 10.7 mmol) was then added over 10 min.

After the system had been stirred at 0 °C for an additional period

of one hour, another portion of N,N-diisopropylethylamine (1.48 g,

11.5 mmol) was added at 0 °C, followed by a solution of 9-fluoren-

ylmethanol (2.24 g, 11.4 mmol) in dry THF (5 mL). The reaction

mixture was then stirred at room temperature for 24 h and concen-

trated under reduced pressure. The residue was taken up into a

phosphate buffer solution at pH 7 (20 mL) and was extracted with

1494, 1449, 1384, 1317, 1267, 1191, 1138, 1105, 1017, 989, 759,

740 cm^–1. MS (+APCI): m/z (%) = 1030.3 (100) [M + H]⁺.

Triethylammonium Dideoxycytidine-squalene 5Ј-Monophosphate

(9a): Triethylamine (2.5 mL, 18 mmol) was added to a solution of

dideoxycytidine-squalene

5Ј-O-bis(fluorenylmethyl)phosphate

(13d, 52 mg, 0.05 mmol) in dry CH₃CN (9 mL) and the mixture

was allowed to stand at 20 °C. After 17 h the solution was concen-

trated under reduced pressure. The white powder obtained was

washed with diethyl ether (3ϫ2 mL). The residue was dissolved in

MeOH (5 mL) and the solution was filtered. The clear solution

AcOEt (4ϫ50 mL). The combined organic layers were washed obtained was concentrated under reduced pressure to give the

with the phosphate buffer solution (pH 7, 20 mL), dried with

MgSO₄and concentrated in vacuo to give an oily residue, which

was purified by flash chromatography on silica gel (cyclohexane/

phosphate salt 9a as a white amorphous solid (38.5 mg, 98%):

[α]_D= +18.1 (c = 2.1, EtOH). H NMR (300 MHz, [D₄]MeOH): δ

= 8.57 (d, J = 7.4 Hz, 1 H, 6-H), 7.49 (d, J = 7.4 Hz, 1 H, 5-H),

1

AcOEt/Et₃N 20:1:0.2) to yield the phosphoramidite 16 as a yellow 6.03 (dd, J = 6.6, 2.4 Hz, 1 H, 1Ј-H), 5.20–5.04 [m, 5 H,

1

oil (2.41 g, 81%): H NMR (300 MHz, CDCl₃): δ = 7.75 (dd, J =

7.4, 4.9 Hz, 4 H, H-Ar), 7.65 (t, J = 7.4 Hz, 4 H, H-Ar), 7.39 (t, J

CH=C(CH₃)], 4.41–4.22 (m, 2 H, 4Ј-H, 5Ј-H), 4.00–4.16 (m, 1 H,

5Ј-H), 3.18 [q, J = 7.2 Hz, 6 H, (CH₃CH₂)₃NH⁺], 2.53 (t, J =

= 7.4 Hz, 2 H, H-Ar), 7.37 (t, J = 7.4 Hz, 2 H, H-Ar), 7.30 (dt, J 7.3 Hz, 2 H, NHCOCH₂) overlapped by 2.55 (m, 1 H, 2Ј-H), 2.33

= 7.4, 1.2 Hz, 2 H, H-Ar), 7.28 (dt, J = 7.4, 1.2 Hz, 2 H, H-Ar), (t, J = 7.3 Hz, 2 H, NHCOCH₂CH₂), 2.15–1.90 [m, 19 H,

4.18 (dd, J = 7.2, 6.7 Hz, 2 H, CHCH₂OP), 4.01 (dt, J = 9.9,

6.7 Hz, 2 H, CHCH₂OP), 3.81 (td, J = 9.9, 7.2 Hz, 2 H, (s, 3 H, CH₃), 1.59 (s, 12 H, CH₃), 1.32 [t, J = 7.2 Hz, 9 H,

CHCH₂OP), 3.80–3.58 [m, 2 H, PNCH(CH₃)₂], 1.17 [d, J = 6.8 Hz,

(CH₃CH₂)₃NH⁺] ppm. ¹³C NMR (75 MHz, [D₄]MeOH): δ = 175.2

12 H, P-NCH(CH₃)₂] ppm. ³¹P NMR (81 MHz, CDCl₃): δ = 147.4 (C, NHCO), 164.1 (C, C-4), 157.9 (C, C-2), 146.7 (CH, C-6), 136.0

=CHCH₂CH₂C(CH₃), 2Ј-H, 3Ј-H], 1.66 [s, 3 H, (CH₃)₂C=], 1.64

2622

www.eurjoc.org

Eur. J. Org. Chem. 2011, 2615–2628

Squalenoyl Gemcitabine Monophosphate

[C, =C(CH₃)CH₂], 135.9 [C, =C(CH₃)CH₂], 135.8 [C, =C(CH₃)-

CH₂], 134.5 [C, =C(CH₃)CH₂], 132.0 [C, =C(CH₃)₂], 126.4 [CH,

HC=C(CH₃)], 125.7 [CH, HC=C(CH₃)], 125.6 [CH, HC=C(CH₃)],

125.5 [2ϫCH, HC=C(CH₃)], 97.9 (CH, C-5), 89.5 (CH, C-1Ј), 82.8

(d, J_C,P= 8.6 Hz, CH, C-4Ј), 66.4 (d, J_C,P= 4.5 Hz, CH₂, C-5Ј),

1:1). ¹H NMR (300 MHz, CDCl₃, the presence of two dia-

stereomers induces the splitting of some lines): δ = 8.82 (br. s, 1 H,

NH), 8.09–8.04 (m, 1 H, 6-H), 7.42 (d, J = 7.6 Hz, 1 H, 5-H), 6.39–

6.31 (m, 1 H, 1Ј-H), 5.21–5.05 [m, 5 H, CH=C(CH₃)], 4.92–4.80

[m, 1 H, OCH(CH₃)O], 4.51–4.26 (m, 1 H, 3Ј-H), 4.08–3.98 (m, 2

47.6 [3ϫCH₂, (CH₃CH₂)₃NH⁺], 40.9 [2ϫCH₂, =C(CH₃)CH₂], H), 3.87–3.65 (m, 2 H), 3.60–3.40 (m, 1 H), 2.60–2.50 (m, 2 H,

40.7 [CH₂, =C(CH₃)CH₂], 37.2 (CH₂, NHCOCH₂CH₂), 35.8 (CH₂, NHCOCH₂), 2.34 (t, J = 7.9 Hz, 2 H, NHCOCH₂CH₂), 2.16–1.92

NHCOCH₂CH₂), 34.4 (CH₂), 29.2 (2ϫCH₂), 27.8 (CH₂), 27.7

(CH₂), 27.6 (CH₂), 25.9 [CH₃, (CH₃)₂C=], 25.4 (CH₂), 17.8 (CH₃),

16.3 (2ϫCH₃), 16.2 (2ϫCH₃), 9.2 [3ϫCH₃, (CH₃CH₂)₃-

[m, 16 H, =CHCH₂CH₂C(CH₃)], 1.67 [s, 3 H, (CH₃)₂C=], 1.62 (s,

3 H, CH₃), 1.59 (s, 12 H, CH₃), 1.37–1.33 [m, 3 H, OCH(CH₃)O],

1.21–1.15 (m, 3 H, OCH₂CH₃), 0.95 [s, 9 H, (CH₃)₃CSi], 0.13 [s, 6

NH⁺] ppm. IR (neat): ν = 3300–2750, 1714, 1660, 1610, 1565, 1494, H, (CH₃)₂Si] ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 174.2 (C,

˜

1450, 1384, 1318, 1235, 1143, 1102, 1050, 991, 926, 792 cm^–1. MS

NHCO), 164.5 (C, C-4), 155.1 (C, C-2), 143.7 (CH, C-6), 135.3 [C,

=C(CH₃)CH₂], 135.1 [C, =C(CH₃)CH₂], 134.9 [C, =C(CH₃)CH₂],

134.0 [C, =C(CH₃)CH₂], 131.1 [C, =C(CH₃)₂], 125.4 [CH,

HC=C(CH₃)], 125.0 [CH, HC=C(CH₃)], 124.9 [CH, HC=C(CH₃)],

124.8 [CH, HC=C(CH₃)], 124.7 [CH, HC=C(CH₃)], 123.3 (t, J_C,F

= 261 Hz, CF₂, C-2Ј), 100.5 and 100.2 [CH, OCH(CH₃)O], 97.6

(CH, C-5), 86.0–84.9 (m, CH, C-1Ј), 80.2–80.1 (m, CH, C-4Ј), 71.1–

70.3 (m, CH, C-3Ј), 62.1 and 61.6 (CH₂, OCH₂CH₃), 60.5 and 60.3

(CH₂, C-5Ј), 40.2 [2ϫCH₂, =C(CH₃)CH₂], 40.1 [CH₂, =C(CH₃)-

CH₂], 36.4 (CH₂, NHCOCH₂CH₂), 34.8 (CH₂, NHCOCH₂CH₂),

28.8 (2ϫCH₂), 27.3 (CH₂), 27.2 (CH₂), 27.1 (CH₂), 25.9 [4ϫCH₃,

(CH₃)₃CSi, (CH₃)₂C=], 20.3 and 20.0 [CH₃, OCH(CH₃)O], 18.4 [C,

(CH₃)₃CSi], 17.7 (CH₃), 16.2 (2ϫCH₃), 16.1 (2ϫCH₃), 15.3 and

15.2 (CH₃, OCH₂CH₃), –5.5 [m, 2ϫCH₃, (CH₃)₂Si] ppm. ¹⁹F

NMR (188 MHz, CDCl₃, the presence of two diastereomers in-

duces the splitting of the lines): δ = –111.3 (d, J = 242.5 Hz, 1 F)

and –112.0 (d, J = 240.6 Hz, 1 F), –114.0 (d, J = 240.6 Hz, 1 F)

(+ESI): m/z (%) = 718.3 (100) [M – HOCH₂CH₂NHMe₂

+

2Na]⁺, 696.4 (76) [M – HOCH₂CH₂NHMe₂+ H + Na]⁺, 674.3

(37) [M – HOCH₂CH₂NHMe₂+ 2H]⁺. MS (–ESI): m/z (%) = 672

(100) [M – HOCH₂CH₂NHMe]^–.

Compound 19: Imidazole (58 mg, 0.85 mmol) and tert-butyldimeth-

ylsilyl chloride (120 mg, 0.79 mmol) were added to an ice-cooled

solution of SQdFdC (4, 410 mg, 0.64 mmol) in dry DMF (4 mL).

The reaction mixture was stirred at room temperature for 24 h and

concentrated under reduced pressure. The yellow residue was

treated with saturated aqueous sodium hydrogencarbonate solution

(10 mL) and was extracted with AcOEt (3ϫ30 mL). The combined

organic layers were washed with brine (20 mL), dried with MgSO₄

and concentrated in vacuo to afford a yellow oil. Purification by

flash chromatography on silica gel (AcOEt/cyclohexane 1:1) gave

19 as a white foam (428 mg, 89%): R_f= 0.50 (AcOEt/cyclohexane

1

1:1). H NMR (300 MHz, CDCl₃): δ = 8.46 (br. s, 1 H, NH), 8.10

and 114.7 (d, J = 242.5 Hz, 1 F) ppm. IR (neat): ν = 2956, 2926,

˜

(d, J = 7.6 Hz, 1 H, 6-H), 7.41 (d, J = 7.6 Hz, 1 H, 5-H), 6.41 (dd,

J_H,F= 9.5, J_H,F= 4.9 Hz, 1 H, 1Ј-H), 5.21–5.06 [m, 5 H,

CH=C(CH₃)], 4.37 (td, J_H,F= 11.6, J = 10.6 Hz, 1 H, 3Ј-H), 4.08–

4.00 (m, 2 H, 5Ј-H), 3.90 (dd, J = 12.0, 2.1 Hz, 1 H, 4Ј-H), 2.55–

2.50 (m, 3 H, NHCOCH₂, 3Ј-H), 2.35 (t, J = 7.3 Hz, 2 H,

NHCOCH₂CH₂), 2.12–1.93 [m, 16 H, =CHCH₂CH₂C(CH₃)], 1.68

[s, 3 H, (CH₃)₂C=], 1.62 (s, 3 H, CH₃), 1.60 (s, 12 H, CH₃), 0.94 [s,

9 H, (CH₃)₃CSi], 0.13 [s, 6 H, (CH₃)₂Si] ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 173.7 (C, NHCO), 163.2 (C, C-4), 155.2 (C, C-2),

144.3 (CH, C-6), 134.9 [C, =C(CH₃)CH₂], 134.8 [C, =C(CH₃)CH₂],

134.7 [C, =C(CH₃)CH₂], 132.7 [C, =C(CH₃)CH₂], 131.0 [C,

=C(CH₃)₂], 125.5 [CH, HC=C(CH₃)], 124.3 [CH, HC=C(CH₃)],

124.2 [CH, HC=C(CH₃)], 124.1 [2ϫCH, HC=C(CH₃)], 122.1 (t,

J_C,F= 260 Hz, CF₂, C-2Ј), 97.2 (CH, C-5), 84.4 (dd, J_C,F= 37.8,

J_C,F= 26.3 Hz, CH, C-1Ј), 81.1 (d, J_C,F= 5.5 Hz, CH, C-4Ј), 68.5

(t, J_C,F= 22.0 Hz, CH, C-3Ј), 60.4 (CH₂, C-5Ј), 39.6 [2ϫCH₂,

=C(CH₃)CH₂], 39.4 [CH₂, =C(CH₃)CH₂], 36.2 (CH₂,

NHCOCH₂CH₂), 34.3 (CH₂, NHCOCH₂CH₂), 28.1 (2ϫCH₂),

26.6 (2ϫCH₂), 26.5 (CH₂), 25.7 [3ϫCH₃, (CH₃)₃CSi], 25.5 [CH₃,

(CH₃)₂C=], 18.2 [C, (CH₃)₃CSi], 17.5 (CH₃), 15.9 (3ϫCH₃), 15.7

(CH₃), –5.6 [CH₃, (CH₃)₂Si], –5.7 [CH₃, (CH₃)₂Si] ppm. ¹⁹F NMR

(188 MHz, CDCl₃): δ = –117.5 (d, J_F,F= 232 Hz, 1 F), –116.3 (d,

2859, 1723, 1681, 1628, 1558, 1492, 1441, 1391, 1322, 1271, 1257,

1196, 1129, 1082, 957, 871, 833, 812, 785 cm^–1. MS (+ESI): m/z

(%) = 832.9 (100) [M + H]⁺, 760.6 (65) [M – H₂C=CHOEt]⁺.

Compound 21: The silyl ether 20 (210 mg, 0.25 mmol) was treated

with TBAF (1 m in THF, 0.5 mL, 0.5 mmol). The reaction mixture

was stirred at room temperature for 2 h and water (1 mL) was then

added. The mixture was extracted with AcOEt (3ϫ5 mL). The

combined organic layers were washed with brine (5 mL), dried with

MgSO₄and concentrated in vacuo. The crude product was then

taken into AcOEt and filtered over silica to give pure 21 as a

colourless oil (154 mg, 86%): R_f= 0.45 (AcOEt/cyclohexane 2:1).

¹H NMR (300 MHz, CDCl₃, the presence of two diastereomers

induces the splitting of most lines): δ = 8.44 (br. s, 1 H, NH), 7.98–

7.92 (m, 1 H, 6-H), 7.45 (d, J = 7.6 Hz, 1 H, 5-H), 6.27–6.16 (m,

1 H, 1Ј-H), 5.22–5.06 [m, 5 H, CH=C(CH₃)], 4.95–4.87 [m, 1 H,

OCH(CH₃)O], 4.62–4.45 (m, 1 H, 3Ј-H), 4.12–4.02 (m, 2 H, 5Ј-H,

4Ј-H), 3.92–3.46 (m, 3 H, 5Ј-H, OCH₂CH₃), 2.56–2.48 (m, 3 H,

NHCOCH₂, 2Ј-H), 2.35 (t, J = 7.3 Hz, 2 H, NHCOCH₂CH₂),

2.12–1.92 [m, 16 H, =CHCH₂CH₂C(CH₃)], 1.68 [s, 3 H, (CH₃)₂-

C=], 1.62 (s, 3 H, CH₃), 1.60 (s, 12 H, CH₃), 1.40–1.35 [m, 3 H,

OCH(CH₃)O], 1.24–1.17 (m, 3 H, OCH₂CH₃) ppm. ¹³C NMR

(75 MHz, CDCl₃): δ = 173.0 (C, NHCO), 162.7 (C, C-4), 155.0 (C,

C-2), 144.9 (CH, C-6), 135.1 [C, =C(CH₃)CH₂], 134.9 [C,

=C(CH₃)CH₂], 134.8 [C, =C(CH₃)CH₂], 132.5 [C, =C(CH₃)CH₂],

131.2 [C, =C(CH₃)₂], 126.0 [CH, HC=C(CH₃)], 125.4 [CH,

HC=C(CH₃)], 124.7 [3ϫCH, HC=C(CH₃)], 100.5 and 100.2 [CH,

OCH(CH₃)O], 97.1 (CH, C-5), 86.0–84.9 (m, CH, C-1Ј), 80.1–79.8

(m, CH, C-4Ј), 71.3–70.4 (m, CH, C-3Ј), 62.1 and 61.6 (CH₂,

OCH₂CH₃), 59.8 (CH₂, C-5Ј), 39.7 [2ϫCH₂, =C(CH₃)CH₂], 39.5

[CH₂, =C(CH₃)CH₂], 36.6 (CH₂, NHCOCH₂CH₂), 34.4 (CH₂,

NHCOCH₂CH₂), 28.3 (2ϫCH₂), 26.8 (CH₂), 26.7 (CH₂), 26.6

(CH₂), 25.9 [CH₃, (CH₃)₂C=], 20.0 [m, CH₃, OCH(CH₃)O], 17.7

(CH₃), 16.0 (3ϫCH₃), 15.9 (CH₃), 15.0 and 14.9 (CH₃,

OCH₂CH₃) ppm, the CF₂carbon was not detected. ¹⁹F NMR

J_F,F= 232 Hz, 1 F) ppm. IR (neat): ν = 2928, 2857, 1725, 1660,

˜

1619, 1559, 1491, 1439, 1384, 1319, 1256, 1196, 1134, 1083, 951,

909, 833, 812, 783, 732 cm^–1. MS (+APCI): m/z (%) = 760.7 (100)

[M + H]⁺.

Compound 20: Ethyl vinyl ether (207 mg, 2.9 mmol) and a catalytic

amount of pyridinium para-toluenesulfonate were added to a solu-

tion of 5Ј-O-silylated gemcitabine-squalene 19 (200 mg, 0.26 mmol)

in CH₂Cl₂(1 mL). The reaction mixture was stirred at room tem-

perature for 16 h and then treated with saturated sodium hydrogen-

carbonate solution (5 mL). The mixture was extracted with AcOEt

(3ϫ5 mL). The combined organic layers were washed with brine

(5 mL), dried with MgSO₄and concentrated in vacuo to afford 20

as a colourless oil (214 mg, 99%): R_f= 0.69 (AcOEt/cyclohexane

Eur. J. Org. Chem. 2011, 2615–2628

www.eurjoc.org

2623

D. Desmaële et al.

FULL PAPER

(188 MHz, CDCl₃, the presence of two diastereomers induces the

1

(AcOEt/cyclohexane 1:1). [α]_D= –13.1 (c = 4.0, EtOH). H NMR

splitting of some lines): δ = –111.4 (d, J = 242 Hz, 1 F), –112.2 (s,

(300 MHz, CDCl₃): δ = 8.63 (br. s, 1 H, NH), 8.18 (d, J = 7.7 Hz,

1 H, 6-H), 7.44–7.25 (m, 9 H, H-Ar), 7.17 (d, J = 7.7 Hz, 1 H, 5-

1 F), –113.8 (d, J = 242 Hz, 1 F) ppm. IR (neat): ν = 2959, 2923,

˜

2857, 1723, 1665, 1624, 1556, 1495, 1438, 1394, 1322, 1274, 1195,

1125, 1099, 1081 cm^–1. MS (+ESI): m/z (%) = 718.6 (100) [M +

H]⁺, 740.6 (12) [M + Na]⁺.

H), 6.85 (d, J = 8.9 Hz, 4 H, H-Ar), 6.38 (dd, J_H,F= 9.7, J_H,F=

4.3 Hz, 1 H, 1Ј-H), 5.22–5.06 [m, 5 H, CH=C(CH₃)], 4.48–4.36 (m,

1 H, 3Ј-H), 4.02 (br. d, J = 8.2 Hz, 1 H, 4Ј-H), 3.81 (s, 6 H,

ArOCH₃), 3.68 (br. d, J = 11.3 Hz, 1 H, 5Ј-H), 3.34 (dd, J = 11.3,

2.8 Hz, 1 H, 5Ј-H), 2.56–2.48 (m, 2 H, NHCOCH₂), 2.34 (t, J =

Compound 22: 4,4Ј-Dimethoxytrityl chloride (680 mg, 2.0 mmol)

and a catalytic amount of DMAP were added to a solution of

gemcitabine-squalene (4, 438 mg, 0.68 mmol) in dry pyridine

(4 mL). The reaction mixture was stirred at room temperature for

3 days. Pyridine was then removed under reduced pressure and the

red residue was treated with saturated sodium hydrogencarbonate

solution (10 mL). The mixture was extracted with AcOEt

(3ϫ25 mL). The combined organic layers were washed with H₂O

(20 mL) and brine (20 mL), dried with MgSO₄and concentrated

in vacuo to afford an oil. Purification by flash chromatography on

silica gel (AcOEt/cyclohexane 1:2) gave 22 as a colourless oil

(534 mg, 83%): R_f= 0.37 (AcOEt/cyclohexane 1:1). [α]_D= +4.7 (c

8.0 Hz,

2

H, NHCOCH₂CH₂), 2.10–1.95 [m, 16 H,

=CHCH₂CH₂C(CH₃)=CH], 1.68 [s, 3 H, (CH₃)₂C=], 1.63 (s, 3 H,

CH₃), 1.60 (s, 12 H, CH₃), 0.80 [s, 9 H, (CH₃)₃CSi], 0.07 [s, 3 H,

(CH₃)₂Si], –0.06 [s, 3 H, (CH₃)₂Si] ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 173.1 (C, NHCO), 162.9 (C, C-4), 158.8 [2 C, Me-

OC(CH)₄C], 154.8 (C, C-2), 144.1 (CH, C-6), 143.8 [C, (HC)₅C],

135.1 [2 C, MeOC(CH)₄C], 135.0 [C, =C(CH₃)CH₂], 134.9 [C,

=C(CH₃)CH₂], 134.8 [C, =C(CH₃)CH₂], 132.8 [C, =C(CH₃)CH₂],

131.2 [C, =C(CH₃)₂], 130.1 [4ϫCH, MeOC(CH)₄C], 128.2

[2ϫCH, HC(HC)₄C], 128.0 [2ϫCH, HC(HC)₄C], 127.3 [CH,

HC(HC)₄C], 125.7 [CH, HC=C(CH₃)], 124.4 [CH, HC=C(CH₃)],

124.3 [CH, HC=C(CH₃)], 124.2 [2ϫCH, HC=C(CH₃)], 121.7 (t,

J_C,F= 261 Hz, CF₂, C-2Ј), 113.3 [4ϫCH, MeOC(CH)₄C], 97.2

(CH, C-5), 86.9 (C, Ar₃C), 84.7 (dd, J_C,F= 40.1, J_C,F= 24.4 Hz,

CH, C-1Ј), 80.8 (d, J_C,F= 8.8 Hz, CH, C-4Ј), 70.6 (dd, J_C,F= 28.0,

J_C,F= 17.3 Hz, CH, C-3Ј), 60.4 (CH₂, C-5Ј), 55.2 (2ϫCH₃, OMe),

39.7 [2ϫCH₂, =C(CH₃)CH₂], 39.5 [CH₂, =C(CH₃)CH₂], 36.4

(CH₂, NHCOCH₂CH₂), 34.4 (CH₂, NHCOCH₂CH₂), 28.3

(2ϫCH₂), 26.7 (2ϫCH₂), 26.6 (CH₂), 25.6 [CH₃, (CH₃)₂C=], 25.4

[3ϫCH₃, (CH₃)₃CSi], 17.9 [C, (CH₃)₃CSi], 17.6 (CH₃), 16.0

(2ϫCH₃), 15.9 (CH₃), 15.8 (CH₃), –4.9 [CH₃, (CH₃)₂Si], –5.4[CH₃,

(CH₃)₂Si] ppm. ¹⁹F NMR (188 MHz, CDCl₃): δ = –114.2 (d, J_F,F

1

= 1.5, MeOH). H NMR (300 MHz, CDCl₃): δ = 9.06 (br. s, 1 H,

NH), 8.12 (d, J = 7.6 Hz, 1 H, 6-H), 7.40–7.35 (m, 2 H, H-Ar),

7.30–7.20 (m, 8 H, H-Ar, 5-H), 6.81 (d, J = 8.5 Hz, 4 H, H-Ar),

6.36 (dd, J_H,F= 8.7, J_H,F= 5.4 Hz, 1 H, 1Ј-H), 5.15–5.02 [m, 5 H,

CH=C(CH₃)], 4.47 (td, J_H,F= 11.1, J = 9.0 Hz, 1 H, 3Ј-H), 4.09

(m, 1 H, 4Ј-H), 3.75 (s, 6 H, ArOCH₃), 3.60 (d, J = 10.7 Hz, 1 H,

5Ј-H), 3.51 (dd, J = 10.7, 3.4 Hz, 1 H, 5Ј-H), 2.42–2.35 (m, 2 H,

NHCOCH₂CH₂C), 2.25 (t, J = 7.9 Hz, 2 H, NHCOCH₂CH₂C),

2.08–1.90 [m, 16 H, CH₂CH₂C(CH₃)=], 1.64 [s, 3 H, (CH₃)₂C=],

1.56 (s, 12 H, CH₃), 1.55 (s, 3 H, CH₃) ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 173.0 (C, NHCO), 162.9 (C, C-4), 158.7 (2 C, MeOC),

155.3 (C, C-2), 144.4 (CH, C-6), 144.1 [C, (HC)₅C], 135.4 [C, Me-

OC(CH)₄C], 135.3 [C, MeOC(CH)₄C], 135.1 [C, =C(CH₃)CH₂],

134.9 [C, =C(CH₃)CH₂], 134.8 [C, =C(CH₃)CH₂], 132.8 [C,

=C(CH₃)CH₂], 131.1 [C, =C(CH₃)₂], 130.0 [2ϫCH, MeOC-

(CH)₄C], 129.9 [2ϫCH, MeOC(CH)₄C], 128.1 [2ϫCH, HC(HC)₄-

C], 128.0 [2ϫCH, HC(HC)₄C], 127.1 [CH, HC(HC)₄C], 125.6

[CH, HC=C(CH₃)], 124.4 [2ϫCH, HC=C(CH₃)], 124.2 [2ϫCH,

HC=C(CH₃)], 122.0 (t, J_C,F= 261 Hz, CF₂, C-2Ј), 113.3 [4ϫCH,

= 239 Hz, 1 F), –116.7 (d, J_F,F= 239 Hz, 1 F) ppm. IR (neat): ν =

˜

2960, 2930, 2857, 1722, 1678, 1625, 1608, 1555, 1510, 1492, 1443,

1389, 1342, 1318, 1252, 1211, 1177, 1133, 1091, 1068, 1036, 857,

838, 782 cm^–1. MS (+APCI): m/z (%) = 1063.6 (100) [M + H]⁺,

303.2 (25) [Ph(MeOPh)₂C]⁺. C₆₃H₈₅F₂N₃O₇Si (1062.46): calcd. C

71.22, H 8.06, N 3.96; found C 70.89, H 7.99, N 3.87.

Compound 24: Formic acid (4 mL) was added dropwise to an ice-

cooled solution of compound 23 (520 mg, 0.49 mmol) in MeOH/

dichloromethane (1:1, 4 mL). The reaction mixture instantly be-

came red and was stirred at 0 °C for 45 min. Saturated aqueous

sodium hydrogencarbonate solution (15 mL) was added and the

mixture was extracted with AcOEt (3ϫ20 mL). The combined or-

ganic layers were washed with H₂O (20 mL) and brine (20 mL).

Drying with MgSO₄and concentration under reduced pressure af-

forded the crude product, which was purified by flash chromatog-

raphy on silica gel (AcOEt/cyclohexane 1:2) to provide the alcohol

24 (288 mg, 77%) as a white foam: R_f= 0.47 (AcOEt/cyclohexane

MeOC(CH)₄C], 97.3 (CH, C-5), 86.9 (C, Ar₃C), 84.4 (dd, J_C,F

=

41.2, J_C,F= 21.1 Hz, CH, C-1Ј), 80.6 (d, J_C,F= 6.5 Hz, CH, C-4Ј),

69.9 (dd, J_C,F= 27.4, J_C,F= 19.5 Hz, CH, C-3Ј), 61.0 (CH₂, C-5Ј),

55.2 (2ϫCH₃, OCH₃), 39.7 [2ϫCH₂, =C(CH₃)CH₂], 39.5 [CH₂,

=C(CH₃)CH₂], 36.2 (CH₂, NHCOCH₂CH₂), 34.3 (CH₂,

NHCOCH₂CH₂), 28.2 (2ϫCH₂), 26.7 (2ϫCH₂), 26.6 (CH₂), 25.6

[CH_3,(CH₃)₂C=], 17.6 (CH₃), 16.0 (2ϫCH₃), 15.9 (CH₃), 15.8

(CH₃) ppm. ¹⁹F NMR (188 MHz, CDCl₃): δ = –115.9 (d, J_F,F

=

240 Hz, 1 F), –118.7 (d, J_F,F= 240 Hz, 1 F) ppm. IR (neat): ν =

˜

2924, 2853, 1724, 1660, 1610, 1557, 1508, 1489, 1442, 1382, 1316,

1251, 1196, 1176, 1131, 1083, 1034, 992, 909, 826 cm^–1. MS

(+APCI): m/z (%) = 949.3 (100) [M + H]⁺, 646.7 (12) [M – Ph(Me-

OPh)₂C + H]⁺, 303.2 (73) [Ph(MeOPh)₂C]⁺. C₅₇H₇₁F₂N₃O₇·

0.5H₂O: calcd. C 71.52, H 7.58, N 4.39; found C 71.66, H 7.44, N

4.37.

1

1:1). [α]_D= +36.6 (c = 3.5, EtOH). H NMR (300 MHz, CDCl₃):

δ = 8.94 (br. s, 1 H, NH), 7.97 (d, J = 7.6 Hz, 1 H, 6-H), 7.46 (d,

J = 7.6 Hz, 1 H, 5-H), 6.24 (t, J_H,F= 7.6 Hz, 1 H, 1Ј-H), 5.24–5.04

[m, 5 H, CH=C(CH₃)], 4.43 (td, J_H,F= 11.9, J = 8.0 Hz, 1 H, 3Ј-

H), 4.07 (br. d, J = 11.9 Hz, 1 H, 5Ј-H), 3.96 (td, J = 8.0, J_H,F

=

Compound 23: DBU (182 mg, 1.2 mmol) and TBDMSCl (170 mg, 2.5 Hz, 1 H, 4Ј-H), 3.81 (br. d, J = 11.9 Hz, 1 H, 5Ј-H), 3.10 (br.

1.13 mmol) were added sequentially to a solution of 5Ј-O-(4,4Ј-di- s, 1 H, OH), 2.60–2.53 (m, 2 H, NHCOCH₂), 2.34 (t, J = 7.4 Hz,

methoxytrityl)gemcitabine-squalene (22, 320 mg, 0.337 mmol) in

dry CH₂Cl₂(4 mL). The reaction mixture was stirred at room tem-

perature for 36 h and saturated aqueous NH₄Cl (10 mL) was

added. The phases were separated and the aqueous phase was ex-

tracted with CH₂Cl₂(3ϫ20 mL). The organic layers were washed

with brine (20 mL), dried with MgSO₄and subsequently concen-

trated under reduce pressure. The viscous residue was purified by

flash chromatography on silica gel (AcOEt/cyclohexane 1:2) to

yield 23 as a colourless viscous oil (322 mg, 90%): R_f= 0.69

2 H, NHCOCH₂CH₂), 2.14–1.97 [m, 16 H, =CHCH₂CH₂C(CH₃)],

1.68 [s, 3 H, (CH₃)₂C=], 1.62 (s, 3 H, CH₃), 1.60 (s, 9 H, CH₃),

1.58 (s, 3 H, CH₃), 0.91 [s, 9 H, (CH₃)₃CSi], 0.13 [s, 3 H, (CH₃)₂-

Si], 0.12 [s, 3 H, (CH₃)₂Si] ppm. ¹³C NMR (75 MHz, CDCl₃): δ =

173.8 (C, NHCO), 163.2 (C, C-4), 155.2 (C, C-2), 145.0 (CH, C-6),

135.0 [C, =C(CH₃)CH₂], 134.8 [C, =C(CH₃)CH₂], 134.7 [C,

=C(CH₃)CH₂], 132.8 [C, =C(CH₃)CH₂], 131.1 [C, =C(CH₃)₂],

125.6 [CH, HC=C(CH₃)], 124.3 [CH, HC=C(CH₃)], 124.2 [CH,

HC=C(CH₃)], 124.1 [2ϫCH, HC=C(CH₃)], 122.0 (t, J_C,F

=

2624

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Eur. J. Org. Chem. 2011, 2615–2628

Squalenoyl Gemcitabine Monophosphate

261 Hz, CF₂, C-2Ј), 97.5 (CH, C-5), 85.3 (t, J_C,F= 31.0 Hz, CH, 1197.1 (100) [M + H]⁺. C₇₀H₈₈F₂N₃O₈PSi (1196.54): calcd. C

C-1Ј), 81.6 (CH, C-4Ј), 69.7 (t, J_C,F= 22.8 Hz, CH, C-3Ј), 59.2 70.27, H 7.41, N 3.51; found C 70.10, H 7.44, N 3.69.

(CH₂, C-5Ј), 39.7 [CH₂, =C(CH₃)CH₂], 39.6 [CH₂, =C(CH₃)CH₂],

Compound 26: HF·pyridine (70%, w/w, 0.5 mL, ca. 19.5 mmol) was

39.5 [CH₂, =C(CH₃)CH₂], 36.3 (CH₂, NHCOCH₂CH₂), 34.4 (CH₂,

added at 0 °C to a solution of the phosphotriester 25 (250 mg,

NHCOCH₂CH₂), 28.2 (2ϫCH₂), 26.7 (2ϫCH₂), 26.6 (CH₂), 25.6

0.21 mmol) in dry pyridine (0.5 mL). After having been stirred at

room temperature for 4 h the reaction mixture was cooled to 0 °C,

then saturated aqueous NaHCO₃(5 mL) was added. The mixture

was extracted with AcOEt (3ϫ10 mL). The combined organic lay-

[CH₃, (CH₃)₂C=], 25.4 [3ϫCH₃, (CH₃)₃CSi], 17.9 [C, (CH₃)₃CSi],

17.6 (CH₃), 16.0 (2ϫCH₃), 15.9 (CH₃), 15.7 (CH₃), –5.0 [CH₃,

(CH₃)₂Si], –5.4 [CH₃, (CH₃)₂Si] ppm. ¹⁹F NMR (188 MHz,

CDCl₃): δ = –113.6 (d, J_F,F= 238 Hz, 1 F), –115.1 (d, J_F,F

=

ers were washed with brine (10 mL), dried with MgSO₄and con-

centrated in vacuo. The crude product was purified by flash

chromatography on silica gel (AcOEt/cyclohexane 1:1 then 2:1) to

yield compound 26 as a viscous colourless oil (155 mg, 68%): R_f=

0.15 (AcOEt/cyclohexane 1:1). [α]_D= +19.0 (c = 2.9, EtOH). ¹H

NMR (300 MHz, CDCl₃): δ = 9.03 (br. s, 1 H, NH), 7.69 (t, J =

7.3 Hz, 4 H, H-Ar), 7.57 (d, J = 7.5 Hz, 1 H, 6-H), 7.49 (t, J =

7.6 Hz, 2 H, H-Ar), 7.42 (t, J = 7.6 Hz, 2 H, H-Ar), 7.38–7.18 (m,

9 H, H-Ar, 5-H), 6.28 (t, J_H,F= 7.6 Hz, 1 H, 1Ј-H), 5.19 [t, J

= 6.6 Hz, 1 H, HNCOCH₂CH₂C(CH₃)=CH], 5.20–5.05 [m, 4 H,

CH₂CH=C(CH₃)], 4.82 (br. s, 1 H, OH), 4.40–3.96 (m, 10 H,

CH₂OP, CHCH₂OP, 3Ј-H, 4Ј-H, 2ϫH-5Ј), 2.55 (t, J = 7.3 Hz, 2

H, NHCOCH₂CH₂), 2.40–2.25 (m, 2 H, NHCOCH₂CH₂), 2.17–

1.90 [m, 16 H, =CHCH₂CH₂C(CH₃)], 1.68 [s, 3 H, (CH₃)₂C=], 1.62

(s, 3 H, CH₃), 1.60 (s, 9 H, CH₃), 1.58 (s, 3 H, CH₃) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 173.2 (C, CONH), 162.9 (C, C-4),

155.0 (C, C-2), 144.3 (CH, C-6), 142.8 (2 C, Ar), 142.7 (2 C, Ar),

141.4 (3 C, Ar), 141.3 (C, Ar), 135.1 [C, =C(CH₃)CH₂], 134.9 [2 C,

=C(CH₃)CH₂], 132.8 [C, =C(CH₃)CH₂], 131.2 [C, =C(CH₃)₂],

127.9 (4ϫCH, Ar), 127.1 (4ϫCH, Ar), 125.7 [CH, HC=C(CH₃)],

124.9 (CH, Ar), 124.8 (3ϫCH, Ar), 124.4 [CH, HC=C(CH₃)],

124.2 [2ϫCH, HC=C(CH₃)], 121.5 (t, J_C,F= 261 Hz, CF₂, C-2Ј),

120.1 (3ϫCH, Ar), 120.0 (CH, Ar), 97.2 (CH, C-5), 84.6 (m, CH,

C-1Ј), 79.6 (m, CH, C-4Ј), 70.2 (m, CH, C-3Ј), 69.4 (m, 2ϫCH₂,

238 Hz, 1 F) ppm. IR (neat): ν = 3600–3000, 2969, 2931, 2857,

˜

1727, 1678, 1659, 1617, 1558, 1495, 1435, 1393, 1322, 1255, 1211,

1136, 1094 cm^–1. MS (–ESI): m/z (%) = 758.9 (100) [M – H]^–.

C₄₂H₆₇F₂N₃O₅Si (760.09): calcd. C 66.37, H 8.88, N 5.53; found C

66.33, H 9.09, N 4.87.

Compound 25: A solution of 1H-tetrazole in acetonitrile (2 mL,

0.45 m, 0.9 mmol) and a solution of bis(9H-fluoren-9-ylmethyl) di-

isopropylamidophosphite (16, 450 mg, 0.86 mmol) in dry dichloro-

methane (2 mL) were added sequentially to a solution of 24

(170 mg, 0.224 mmol) in acetonitrile (0.2 mL). After having been

stirred at room temperature for 3 days, the mixture was cooled to

0 °C and H₂O₂(30 wt.-%, 0.6 mL, 5.5 mmol) was slowly added.

Stirring was continued at 0 °C for 4 h, then saturated aqueous

NaHCO₃(15 mL) was added. The mixture was then extracted with

AcOEt (4ϫ25 mL). The combined organic layers were washed

with saturated aqueous NaHSO₃(20 mL) and brine (20 mL), dried

with MgSO₄and concentrated in vacuo. The viscous residue was

purified by flash chromatography on silica gel (AcOEt/cyclohexane

1:1) to give phosphotriester 25 as a colourless viscous oil (214 mg,

80%): R_f= 0.57 (AcOEt/cyclohexane 1:1). [α]_D= +17.1 (c = 1.4,

EtOH). ¹H NMR (400 MHz, CDCl₃): δ = 9.03 (br. s, 1 H, NH),

7.75–7.68 (m, 4 H, H-Ar), 7.57 (d, J = 7.6 Hz, 1 H, 6-H), 7.55–

7.20 (m, 13 H, H-Ar, 5-H), 6.26 (t, J_H,F= 7.9 Hz, 1 H, 1Ј-H), 5.21

[t, J = 6.6 Hz, 1 H, HNCOCH₂CH₂C(CH₃)=CH], 5.16–5.06 [m, 4

H, CH₂CH=C(CH₃)], 4.37–4.24 (m, 4 H, CHCH₂OP), 4.11 (t, J =

6.0 Hz, 2 H, CH–CH₂OP), 4.05 (m, 1 H, 3Ј-H), 3.93 (br. s, 2 H,

5Ј-H), 3.85 (br. s, 1 H, 4Ј-H), 2.65–2.68 [m, 2 H, NHCOCH₂-

CH₂C(CH₃)], 2.37 [t, J = 7.6 Hz, 2 H, NHCOCH₂CH₂C(CH₃)],

2.12–1.93 [m, 16 H, =CHCH₂CH₂C(CH₃)], 1.68 [s, 3 H, (CH₃)₂-

C=], 1.63 (s, 3 H, CH₃), 1.60 (s, 9 H, CH₃), 1.59 (s, 3 H, CH₃),

0.85 [s, 9 H, (CH₃)₃CSi], 0.06 [s, 3 H, (CH₃)₂Si], 0.00 [s, 3 H, (CH₃)₂-

Si] ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 173.0 (C, NHCO),

162.9 (C, C-4), 154.8 (C, C-2), 144.3 (CH, C-6), 143.0 (C, Ar),

142.9 (3 C, Ar), 141.7 (C, Ar), 141.6 (C, Ar), 141.5 (2 C, Ar), 135.2

[C, =C(CH₃)CH₂], 135.0 [2 C, =C(CH₃)CH₂], 132.9 [C, =C(CH₃)-

CH₂], 131.3 [C, =C(CH₃)₂], 128.1 (4ϫCH, Ar), 127.3 (4ϫCH, Ar),

126.0 [CH, HC=C(CH₃)], 125.0 (3ϫCH, Ar), 124.9 (CH, Ar),

FmCHCH₂OP), 65.0 (d, J_C,P= 4.1 Hz, CH₂, C-5Ј), 47.8 (d, J_C,P

=

8.2 Hz, CH, FmC-9), 47.7 (d, J_C,P= 7.8 Hz, CH, FmC-9), 39.7

[2ϫCH₂, =C(CH₃)CH₂], 39.5 [CH₂, =C(CH₃)CH₂], 36.4 (CH₂,

NHCOCH₂CH₂), 34.5 (CH₂, NHCOCH₂CH₂), 28.2 [2ϫCH₂,

=C(CH₃)CH₂], 26.8 [CH₂, =C(CH₃)CH₂], 26.7 [CH₂, =C(CH₃)-

CH₂], 26.6 [CH₂, =C(CH₃)CH₂], 25.7 [CH₃, (CH₃)₂C=], 17.6

(CH₃), 16.0 (3ϫCH₃), 15.9 (CH₃) ppm. ¹⁹F NMR (188 MHz,

CDCl₃): δ = –115.4 (d, J_F,F= 243 Hz, 1 F), –118.5 (d, J_F,F

=

243 Hz, 1 F) ppm. ³¹P NMR (81 MHz, CDCl₃): δ = 0.00 (s) ppm.

IR (neat): ν = 3500–2900, 3293, 2969, 2921, 2853, 1727, 1678, 1661,

˜

1623, 1558, 1491, 1449, 1385, 1320, 1269, 1199, 1133, 1077, 1022,

741 cm^–1. MS (+APCI): m/z (%) = 1082.5 (10) [M + H]⁺, 646.5

(100) [M – (FmO)₂PO + H]⁺. C₆₄H₇₄F₂N₃O₈P·0.5H₂O: calcd. C

70.44, H 6.93, N 3.85; found C 70.35, H 6.77, N 4.03.

124.6 [2ϫCH, HC=C(CH₃)], 124.4 [2ϫCH, HC=C(CH₃)], 121.3 Triethylammonium Gemcitabine-squalene 5Ј-Monophosphate (5a):

(t, J_C,F= 261 Hz, CF₂, C-2Ј), 120.3–120.1 (m, 4ϫCH, Ar), 97.2 Triethylamine (5 mL, 36 mmol) was added to a solution of gemcita-

(CH, C-5), 84.6 (m, CH, C-1Ј), 80.0 (t, J_C,P= 7.0 Hz, CH, C-4Ј), bine-squalene 5Ј-O-bis(fluorenylmethyl)phosphate (26, 140 mg,

71.2 (dd, J_C,F= 29.2, J_C,F= 18.0 Hz, CH, C-3Ј), 69.5 (m, 2ϫCH_2,

FmCH₂OP), 64.3 (d, J_C,P= 4.3 Hz, CH₂, C-5Ј), 48.0 (d, J_C,P

0.13 mmol) in dry CH₃CN (3 mL). After having been stirred at

room temperature for 24 h, the reaction mixture was concentrated

=

6.7 Hz, CH, FmC-9), 47.9 (d, J_C,P= 6.7 Hz, CH, FmC-9), 39.9 under reduced pressure. The obtained white powder was washed

[2ϫCH₂, =C(CH₃)CH₂], 39.7 [CH₂, =C(CH₃)CH₂], 36.7 (CH₂, with diethyl ether (3ϫ5 mL). The residue was dissolved in MeOH

NHCOCH₂CH₂), 34.6 (CH₂, NHCOCH₂CH₂), 28.4 (2ϫCH₂),

26.9 (2ϫCH₂), 26.8 (CH₂), 25.8 [CH₃, (CH₃)₂C=], 25.6 [3ϫCH₃,

(CH₃)₃CSi], 18.0 [C, (CH₃)₃CSi], 17.8 (CH₃), 16.2 (2ϫCH₃), 16.1

(5 mL) and the solution was filtered. The obtained clear solution

was concentrated under reduced pressure to give the phosphate salt

5a as a white amorphous solid (105 mg, 98%): [α]_D= +25.1 (c =

(CH₃), 16.0 (CH₃), –4.8 [CH₃, (CH₃)₂Si], –5.3 [CH₃, (CH₃)₂- 0.88, EtOH). ¹H NMR (400 MHz, [D₄]MeOH): δ = 8.34 (d, J =

Si] ppm. ¹⁹F NMR (376 MHz, CDCl₃): δ = –115.1 (d, J_F,F

=

7.6 Hz, 1 H, 6-H), 7.54 (d, J = 7.6 Hz, 1 H, 5-H), 6.27 (t, J_H,F=

240 Hz, 1 F), –117.9 (d, J_F,F= 240 Hz, 1 F) ppm. ³¹P NMR

(162 MHz, CDCl₃): δ = –0.86 (hept, J_PH= 6.0 Hz) ppm. IR (neat):

6.9 Hz, 1 H, 1Ј-H), 5.20 [t, J = 6.8 Hz, 1 H, HNCOCH₂-

CH₂C(CH₃)=CH], 5.18–5.05 [m, 4 H, CH₂CH=C(CH₃)CH₂], 4.39

(td, J = 12.7, 8.4 Hz, 1 H, 3Ј-H), 4.28 (br. d, J = 12.7 Hz, 1 H, 5Ј-

ν = 2929, 2855, 1722, 1677, 1625, 1555, 1492, 1449, 1433, 1390,

˜

1319, 1269, 1253, 1212, 1134, 1091, 1014, 989, 839 cm^–1. MS H), 4.15 (ddd, J = 12.7, 6.1, 2.4 Hz, 1 H, 5Ј-H), 4.06 (d, J = 8.4 Hz,

(+ESI): m/z (%) = 1235.8 (15) [M + K]⁺, 1219.8, (40) [M + Na]⁺,

1 H, 4Ј-H), 3.23 [q, J = 7.3 Hz, 6 H, (CH₃CH₂)₃NH⁺], 2.54 (t, J =

Eur. J. Org. Chem. 2011, 2615–2628

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2625

D. Desmaële et al.

FULL PAPER

7.5 Hz,

2

H, NHCOCH₂CH₂), 2.34 (t,

J

=

7.5 Hz,

2

H, (2ϫCH₃) ppm. ¹⁹F NMR (188 MHz, [D₄]MeOH): δ = –117.2 (d,

J = 246 Hz, 1 F), –118.5 (d, J = 246 Hz, 1 F) ppm. ³¹P NMR

NHCOCH₂CH₂), 2.13–1.93 [m, 16 H, =CHCH₂CH₂C(CH₃)], 1.66

[s, 3 H, (CH₃)₂C=], 1.65 (s, 3 H, CH₃), 1.59 (s, 12 H, CH₃), 1.34 (81 MHz, [D ]MeOH): δ = 2.10 (s) ppm. IR (neat): ν = 3500–2500,

˜

4

[t, J = 7.3 Hz, 9 H, (CH₃CH₂)₃NH⁺] ppm. ¹³C NMR (100 MHz,

[D₄]MeOH): δ = 175.2 (C, NHCO), 164.8 (C, C-2), 157.7 (C, C-4),

146.1 (CH, C-6), 136.0 [C, =C(CH₃)CH₂], 135.9 [2 C, =C(CH₃)-

CH₂], 134.8 [C, =C(CH₃)CH₂], 132.1 [C, =C(CH₃)₂], 126.5 [CH,

HNCOCH₂CH₂C(CH₃)=CH], 125.7 [CH, HC=C(CH₃)], 125.6

2991, 2915, 1715, 1667, 1621, 1560, 1490, 1444, 1384, 1315, 1279,

1196, 1136, 1080 cm^–1. MS (+ESI): m/z (%) = 770.6 (37) [M –

HOCH₂CH₂NHMe₂+ 2Na]⁺, 748.6 (50) [M – HOCH₂CH₂-

NHMe₂+ H + Na]⁺, 726.6 (51) [M – HOCH₂CH₂NHMe₂

+

2H]⁺, 646.7 (100) [M – C₅H₁₄NO₄P]⁺. HRMS (–ESI): calcd. for

C₃₆H₅₃N₃O₈F₂P: 724.3538; found 724.3564. C₄₀H₆₅F₂N₄O₉P·H₂O:

calcd. C 57.68, H 8.11, N 6.73; found C 57.52, H 8.11, N 7.11.

[CH, HC=C(CH₃)], 125.5 [2ϫCH, HC=C(CH₃)], 123.9 (t, J_C,F

258 Hz, CF₂, C-2Ј), 98.7 (CH, C-5), 86.5 (dd, J_C,F= 38.1, J_C,F

=

26.8 Hz, CH, C-1Ј), 81.5 (m, CH, C-4Ј), 70.0 (dd, J_C,F= 24.0, J_C,F

= 20.5 Hz, CH, C-3Ј), 63.1 (d, J_C,P= 4.0 Hz, CH₂, C-5Ј), 47.8

[3ϫCH₂, (CH₃CH₂)₃NH⁺], 40.9 [2ϫCH₂, =C(CH₃)CH₂], 40.7

[CH₂, =C(CH₃)CH₂], 37.2 (CH₂, NHCOCH₂CH₂), 35.7 (CH₂,

NHCOCH₂CH₂), 29.2 [2ϫCH₂, =C(CH₃)CH₂], 27.8 [CH₂,

=C(CH₃)CH₂], 27.7 [CH₂, =C(CH₃)CH₂], 27.6 [CH₂, =C(CH₃)-

CH₂], 25.9 [CH₃, (CH₃)₂C=], 17.8 (CH₃), 16.2 (2ϫCH₃), 16.1

(2ϫCH₃), 9.2 [3ϫCH₃, (CH₃CH₂)₃NH⁺] ppm. ¹⁹F NMR

Preparation of Triethylammonium 4-(N)-Trisnor-squalenoylgemcita-

bine Monophosphate Nanoassemblies from 5a and 5b: A solution of

the appropriate drug (3 mg) in ethanol (0.5 mL) was added drop-

wise, with stirring (500 rpm), to aqueous dextrose solution (5%,

1 mL). Precipitation of the nanoassemblies occurred spontane-

ously. Stirring was continued for 2 or 3 min. The nanoassemblies

suspension was then transferred into a weighted round-bottomed

flask and ethanol was evaporated at ambient temperature with a

Rotavapor with vacuum set to about 50–100 mbar for about

10 min. The flask was then dipped into a water bath (water tem-

perature 37 °C) for about 3–5 min. Evaporation was continued un-

til the weight of the contents had decreased to 0.8–0.9 g. The vol-

ume of the suspension in the flask was then made up to 1.0 g either

with dextrose solution (5%) or with sterile water. The mean dia-

meter of the nanoassemblies was determined at 20 °C by quasi-

elastic light scattering with a nanosizer (Coulter N4MD; Beckman

Coulter, Inc., Fullerton, CA).

(188 MHz, [D₄]MeOH): δ = –119.3 (dd, J_F,F= 239.2, J_H,F

=

12.3 Hz, 1 F), –120.2 (br. d, J_F,F= 239.2 Hz, 1 F) ppm. ³¹P NMR

(162 MHz, [D ]MeOH): δ = 1.38 (s) ppm. IR (neat): ν = 3500–

˜

4

2800, 2992, 2933, 2840, 1722, 1677, 1616, 1562, 1494, 1435, 1392,

1320, 1199, 1140, 1091 cm^–1. MS (+ESI): m/z (%) = 827.7 (100) [M

+ H]⁺, 770.6 (86) [M – Et₃NH + 2Na]⁺. HRMS (–ESI): calcd. for

C₃₆H₅₃N₃O₈F₂P: 724.3538; found 724.3552. C₄₂H₆₉F₂N₄O₈P·H₂O:

calcd. C 59.70, H 8.47, N 6.63; found C 59.91, H 8.34, N 6.52.

Dimethylethanolammonium Gemcitabine-squalene 5Ј-Monophos-

phate (5b): Dimethylethanolamine (2.5 mL, 25 mmol) was added to

a solution of gemcitabine-squalene 5Ј-O-bis(fluorenemethyl)phos-

phate (26, 88 mg, 0.081 mmol) in dry CH₃CN (1.5 mL). After having

been stirred at room temperature for 24 h, the reaction mixture was

concentrated under reduced pressure. The obtained white powder

was washed with diethyl ether (3ϫ5 mL). The residue was dis-

solved in MeOH (4 mL) and the solution was filtered. The obtained

clear solution was concentrated under reduced pressure to give the

phosphate salt 5b as a white amorphous solid (56 mg, 85%). [α]_D

= +22.5 (c = 1.7, EtOH). ¹H NMR (400 MHz, [D₄]MeOH): δ =

8.35 (d, J = 7.6 Hz, 1 H, 6-H), 7.55 (d, J = 7.6 Hz, 1 H, 5-H), 6.27

(dd, J_H,F= 6.7, J_H,F= 6.6 Hz, 1 H, 1Ј-H), 5.20 [t, J = 7.5 Hz, 1

Cryogenic Transmission Electron Microscopy: The cryo-TEM in-

vestigations were performed with a Cryo-TEM (JEOL 2100) elec-

tron microscope, at the “Service de Microscopie Electronique de

l’Institut de Biologie Intégrative” (IFR 83 CNRS, Paris). A drop

(4 μL) of the suspension of SQdFdC-MP nanoassemblies

(2 mgmL^–1) was deposited on a carbon-coated copper grid. Excess

liquid was removed with a blotting filter paper and the sample was

quickly vitrified by plunging it into liquid ethane by use of a guillo-

tine-like frame. The sample was then transferred to a cryo-sample

holder. Observations were made at an accelerating voltage of

200 kV under low electron dose. Analysis was performed with the

ImageJ software.

H, HNCOCH₂CH₂C(CH₃)=CH], 5.16–5.06 [m,

4

H,

Small-Angle X-ray Experiments: X-ray scattering experiments were

performed on the Austrian synchrotron beamline at ELETTRA.

Small angles X-ray scattering (SAXS) patterns were recorded with

a position-sensitive linear gas detector filled with an argon/ethane

mixture. The scattered intensity was reported as a function of the

scattering vector q = 4πsinθ/λ, where 2θ is the scattering angle and

λ the wavelength. The calibration of the q range was carried out

with pure tristearine (2 Lβ form) and silver behenate. Sample-to-

detector distance and beam energy were chosen to cover the re-

quired q range. The scattered intensity from a capillary filled with

water was subtracted from the vesicle scattering curve, after proper

normalization.

CH=C(CH₃)], 4.40 (td, J = 12.6, J = 8.6 Hz, 1 H, 3Ј-H), 4.28 (br.

d, J = 12.7 Hz, 1 H, 5Ј-H), 4.14 (ddd, J = 12.7, 6.1, 2.4 Hz, 1 H,

5Ј-H), 4.06 (d, J = 8.6 Hz, 1 H, 4Ј-H), 3.85 [t, J = 6.7 Hz, 2 H,

HOCH₂CH₂NH(CH₃)₂], 3.21 [t,

J

=

6.7 Hz,

2

H,

HOCH₂CH₂NH(CH₃)₂], 2.88 [s, 6 H, HOCH₂CH₂NH(CH₃)₂],

2.54 (t, J = 7.7 Hz, 2 H, NHCOCH₂CH₂), 2.33 (t, J = 7.7 Hz, 2

H, NHCOCH₂CH₂), 2.14–1.92 [m, 16 H, =CHCH₂CH₂C(CH₃)],

1.66 [s, 3 H, (CH₃)₂C=], 1.65 (s, 3 H, CH₃), 1.59 (s, 12 H,

CH₃) ppm. ¹³C NMR (100 MHz, [D₄]MeOH): δ = 175.2 (C,

CONH), 164.8 (C, C-4), 157.7 (C, C-2), 146.1 (CH, C-6), 136.0 [C,

=C(CH₃)CH₂], 135.9 [C, =C(CH₃)CH₂], 135.8 [C, =C(CH₃)CH₂],

134.4 [C, =C(CH₃)CH₂], 132.0 [C, =C(CH₃)₂], 126.5 [CH,

NCOCH₂CH₂C(CH₃)=CH], 125.6 [2ϫCH, HC=C(CH₃)], 125.5

In Vitro Anticancer Activity: L1210 wt (a murine lymphoid leu-

[2ϫCH, HC=C(CH₃)], 123.9 (t, J_C,F= 259 Hz, CF₂, C-2Ј), 98.7 kaemia cell line-wild type) and L1210 10 K (a resistant mutant of

(CH, C-5), 86.4 (dd, J_C,F= 33.0, J_C,F= 29.0 Hz, CH, C-1Ј), 81.5 the L1210 wt cell line) cells were cultured in RPMI 1640 Gluta-

(t, J_C,F= 6.5 Hz, CH, C-4Ј), 69.9 (t, J_C,F= 22.5 Hz, CH, C-3Ј), 63.0 MAX I supplemented with foetal calf serum (10%), penicillin

(d, J_C,P= 4.3 Hz, CH₂, C-5Ј), 60.4 [CH₂, HOCH₂CH₂NH(CH₃)₂], (50 UmL^–1) and streptomycin (50 μgmL^–1) and l-glutamine (2 m).

56.8

(CH₂,

HOCH₂CH₂N),

43.7

[s,

2ϫCH_3,

The anticancer activities of dFdC (1), SQdFdC nanoassemblies (4-

HOCH₂CH₂NH(CH₃)₂], 40.8 [2ϫCH₂, =C(CH₃)CH₂], 40.7 [CH₂, NAs), triethylammonium SQdFdC-MP (5a-NAs) and dimethyleth-

=C(CH₃)CH₂], 37.2 (CH₂, NHCOCH₂CH₂), 35.7 (CH₂, anolammonium SQdFdC-MP (5b-NAs) towards these cell lines

NHCOCH₂CH₂), 29.2 [2ϫCH₂, =C(CH₃)CH₂], 27.8 [CH₂, were determined by the 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl-

=C(CH₃)CH₂], 27.7 [CH₂, =C(CH₃)CH₂], 27.6 [CH₂, =C(CH₃)-

tetrazolium bromide (MTT) test, with measurement of mito-

CH₂], 25.9 [CH₃, (CH₃)₂C=], 17.8 (CH₃), 16.2 (2ϫCH₃), 16.1 chondrial dehydrogenase activity. The cells in exponential growth

2626

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Eur. J. Org. Chem. 2011, 2615–2628

Squalenoyl Gemcitabine Monophosphate

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phase were seeded into 96-well plates and were pre-incubated for

24 h at 37 °C in a humidified atmosphere of CO₂(5%) in air. Dif-

ferent dilutions of dFdC (1), (4-NAs), 5a-NAs and 5b-NAs were

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in triplicate. After 72 h at 37 °C, MTT solution (200 μL) in cell

culture medium (0.5 mgmL^–1) was added to each well. After incu-

bation for 2.5 h at 37 °C, the culture medium was removed and the

resultant formazan crystals were dissolved in DMSO (200 μL). The

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of viable cells, was measured at 570 nm with a microplate reader

(Metertech Σ 960, Fisher Bioblock, Illkirch, France). The percen-

tage of surviving cells was calculated as the absorbance ratio of

treated to untreated cells. The 50% inhibitory concentrations

(IC₅₀s) of the treatments were determined from the dose/response

curves. To avoid the differences in the data due to difference in cell

passage number and other conditions of each individual experi-

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Supporting Information (see footnote on the first page of this arti-

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[11]

Acknowledgments

The research leading to these results received funding from the

European Research Council under the European Community’s

Seventh Framework Program FP7/2007-2013 Grant Agreement

no.249835. The financial support of the Ministère de la Recherche

et de la Technologie (fellowship to J. C.) and the Centre National

de la Recherche Scientifique (CNRS) (grant Ingénieur de valori-

sation to L. H. R) is acknowledged. The authors thank Dr. Estelle

Morvan for 2D NMR spectral analyses, Dr. Heinz Amenitsch from

the ELETTRA synchrotron, Dr. Ghislaine Frébourg and Dr. Jean-

Pierre Lechaire for the cryo-TEM.

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Received: January 11, 2011

Published Online: March 11, 2011

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Article Doi

Squalenoyl gemcitabine monophosphate: Synthesis, characterisation of nanoassemblies and biological evaluation

DOI: 10.1002/ejoc.201100036

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