Improving the Antitumor Activity of Squalenoyl-Paclitaxel Conjugate Nanoassemblies by Manipulating...

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Improving the Antitumor Activity of Squalenoyl-Paclitaxel Conjugate Nanoassemblies by Manipulating the Linker between Paclitaxel and Squalene

Joachim Caron , Andrei Maksimenko , Séverine Wack , Elise Lepeltier , Claudie Bourgaux , Estelle Morvan , Karine Leblanc , Patrick Couvreur , and Didier Desmaële *

A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to over-come the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2 ′ -OH of paclitaxel through a 1,4- cis , cis -dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an effi cient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl–paclitaxel nano-particles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis -squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2 ′ -squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor effi cacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis -deca-5,8-dienoyl linker shows comparable antitumor effi cacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

1. Introduction

Paclitaxel (PTX, 1a ) is a naturally occurring diterpene alkaloid originally isolated from the bark of the Pacifi c Yew tree ( Taxus brevifolia ). It is now one of the most important chemotherapeutic agents for clinical treatment of lung, ovarian, and breast cancers, and Kaposi’s sarcoma. [ 1 ] Due to its low aqueous solubility, the clinical material is formulated in a 50:50 mixture of ethanol and

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DOI: 10.1002/adhm.201200099

J. Caron, Dr. A. Maksimenko, Dr. S. Wack, Dr, E. Lepeltier, C. Bourgaux, Dr. P. Couvreur, Prof. D. DesmaëleUniversité Paris-SudUMR CNRS 8612, Faculté de Pharmacie, 5, Rue Jean-Baptiste ClémentChâtenay-Malabry, F-92296, France E-mail: [email protected]

E. Morvan, K. LeblancUniversité Paris-SudUMR CNRS 8076 BIORue Jean-Baptiste ClémF-92296, France

Cremophor EL (polyoxyethylene triricini-noleate 35) and is diluted with buffer prior to administration. This excipient has been implicated in clinically important adverse effects, including acute hypersensitivity reactions and peripheral neuropathy. [ 2 , 3 ] Thus, there are many reports in the lit-erature describing attempts to improve the formulation of PTX using micelles, lipo-somes, microspheres or emulsions. [ 4 ] These efforts culminated to the elaboration of a newer formulation, in which PTX is bound to albumin, which is sold under the trade-mark Abraxane for the treatment of breast cancer after failure of combination chemo-therapy. [ 5 ] Alternative strategies to improve the effi ciency of PTX rely on the design of prodrugs. Many attempts have been made to produce water soluble prodrugs by cova-lently coupling small solubilizing moiety at the C-2 or C-7 position of PTX such as car-boxylic acid, [ 6–8 ] amino-acid, [ 9 ] polyamine, [ 10 ] sugar derivatives, [ 11 , 12 ] sulfonate, [ 13 ] or phosphate. [ 14 , 15 ] Several other promising prodrug approaches have been developed in which PTX is bound to polymer, [ 16 ] pep-tide, [ 17–23 ] polysaccharide, [ 24–26 ] and lipid. [ 27 ] Additionally these prodrugs have been dec-orated with tumor-targeting molecules. [ 28 , 29 ] Extensive research has been done to fi nd a

way to mitigate the side effects of PTX, by covalently linking fatty acid to PTX since such lipid prodrugs were supposed to benefi t of the enhanced permeability and retention (EPR) effect when incorporated into lipid vehicles such as liposomes, oil emulsions or micelles. These prodrugs included conjugates with phospholi-pids, [ 30 , 31 ] cholesterol, [ 32 ] α -bromo fatty acids, [ 33 , 34 ] fullerenes, [ 35 ] oleic acid, [ 36 , 37 ] linoleic acid [ 38 ] and docosahexaenoic-acid. [ 39 ] Notably, docosahexaenoic-acid-PTX (DHA-PTX ( 1b ), Taxoprexin),

im

CIS, Faculté de Pharmacie, 5ent, Châtenay-Malabry

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Figure 1 . Structure of previously reported polyenic and squalenoyl PTX prodrugs and structure of the hybrid prodrugs of the present study.

OOAc

OHOAcO

HOBz

O

O

OR

BzNH

HO

1b: R =

O

1c: R =

NH

O

O

1a: R = H, Paclitaxel (PTX)

1d: R =

OO

O

OO1e: R =

72'

3'

O

OPTX

1,4-cis,cis-pentadiene unit squalene moiety

O

O

PhO

BzHN

O

O

O

PhO

BzHN

O

unfavourable attackEasy attack

DHA-PTX (1b) SQ-PTX (1c)

Structure of docosahexaenoic acid-squalene hybrid PTX prodrugs

(DHA)

(SQ)

a very promising slow-release conjugate, is undergoing continued testing and reached the phase III clinical trial for the treatment of metastatic malignant melanoma. [ 40 ]

In this context and to address the problems associated with the formulation of PTX we recently reported its bioconjugation to squalene, a natural triterpene, which has previously dem-onstrated to provide conjugates with self-assembling proper-ties. [ 41–44 ] A series of prodrugs were thus obtained by covalent coupling of the PTX-2 ′ -OH as direct ester ( 1c ), as well as with succinate ( 1d ), diglycolate ( 1e ) or small (polyethylene) glycol chain linkers. [ 45 ] Remarkably, although such a strategy was ini-tially developed with hydrophilic molecules such as nucleoside analogues, the highly lipophilic squalene-paclitaxel conjugates

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self-aggregated in water as nanoassemblies (NA). Unfortunately, 2 ′ -squalenoyl paclitaxel (SQ-PTX, 1c ) NA exhibited a low in vitro anti-cancer activity on M109 Murine lung carci-noma cell line (IC 50 16 500 n M , vs 9 n M for free PTX). This lack of activity has been attrib-uted to resistance of the prodrug to hydro-lysis. Indeed, compound 1c demonstrated a very high stability with no more than 40% of the conjugate being hydrolyzed after 96 h in human serum at 37 ° C. Since it was demon-strated that a polar spacer could improve the relative partitioning rate out of the particle of the lipid anchor and therefore the effi cacy of the lipidic PTX-prodrugs, [ 27 ] polar spacers were introduced. The prodrugs bearing a succinate or a short PEG spacer showed no evidence of improved activity, whereas the glycolate linker partially restored the biological activity but this conjugate remained one order of magni-tude less active than the parent drug. The dif-ference of in vitro anticancer activity between DHA-PTX and SQ-PTX is striking in respect of the very similar structural environment of the two compounds around the C-2 ′ hydroxyl group. Indeed both compound displayed the same ester linkage bound to two methylene groups. The fi rst difference, namely the methyl group attached to the double bond, appeared three-bonds away of the crucial ester function. In the instance, since no major dif-ference could be expected in partitioning rate for these closely related prodrugs, we hypothe-sized that polyunsaturared lipid carriers might be released by an alternative mechanism involving either a specifi c hydrolase (such as fatty acid amide hydrolase [ 46 , 47 ] ) or an oxida-tive cleavage via lipoxygenases. [ 48 ] In this con-text, we set out to investigate a new series of squalene PTX prodrugs in which a 1,4- cis,cis- pentadiene unit was intercalated between the PTX and the squalene chain. Such new con-jugates are expected to benefi t of the effi cient release of the parent drug observed with DHA-PTX while keeping the ability to form NAs inherent to squalene derivatives ( Figure 1 ).

We report herein the synthesis of six new DHA-Squalene hybrid prodrugs of PTX, their formulation into nanoparticles, the characterization of the nanoassemblies, and the in vitro bio-logical activity of the different prodrugs on several cancer cell lines. Finally, the preliminary evaluation of the anticancer effi cacy of these DHA-squalene hybrid prodrugs nanoassemblies in the A549 human lung carcinoma subcutaneous model is reported.

2. Chemistry

The prodrugs used in this study were based on the general struc-ture 1f-k in which the lipid anchors are fi xed by acylation of the

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Figure 2 . Synthesis of DHA-squalene hybrid prodrugs of PTX 1f-k.

XY

f: X = CH2, Y = CH2g: X = CH2CH2, Y = CH2h: X = C(CH3)2, Y = CH2i: X = CH2, Y =

j: X = C(CH3)2, Y =

k

CH2OCCH2CH2

OCH2OCCH2CH2

O

O

OAc

OHOAcO

HOBz

O

O

OR

BzNH

HO

1aEDCI, DMAP

1f-k

2f-k

72'

ROH

R =

R =

O

O

2 ′ -hydroxyl group of PTX. As previously reported the selectivity can be easily achieved exploiting the difference in reaction rates between the PTX 2 ′ - and 7-OH groups ( Figure 2 ). [ 49 ] The lipid anchors used were derived from 1,1 ′ ,2-trisnorsqualenic acid, which was easily available from squalene in four steps. [ 50 , 51 ] This polyisoprenyl moiety was combined with a set of 1,4- cis,cis- pentadiene units as illustrated in Figure 1 . To provide an insight into the infl uence of the number of bonds between the ester linkage and the diene we have designed acids 2f and 2g with two or three methylene groups respectively. To delineate the exact role of the diene, acid 2k which possessed only one cis -double bond motive was designed as control product. To inves-tigate the effect of the steric hindrance around the crucial C-2 ′ carbon, we designed acids 2h , j bearing a gem-dimethyl group. Finally an additional ester linkage was introduced to adjust the hydrophobicity of the lipid anchor (acids 2i , j ).

The syntheses of acids 2f-h are depicted in Figure 3 . Thus, 4-pentenoic acid tert- butyl ester ( 3 ) was ozonolyzed to give the tert- butyl ester of succinic semialdehyde 5 . [ 52 ] Addition of the ylide derived from phosphonium salt 7 [ 53 ] afforded the ( Z )-olefi n 8a (61%, Z / E > 95:5). Cleavage of the THP protecting group of 8a with TsOH provided the unmasked alcohol 8b which was next converted to the phosphonium salt 8e through the iodide 8d . The fi nal connection of 8e with the 1,1 ′ ,2-trisnorsqualenal-dehyde ( 10 ) was achieved using NaHMDS as base followed by hydrolysis in refl uxing 1 N NaOH to provide the desired acid 2f in 68% yield. The gem-dimethyl substituted acid 2h was similarly obtained from the 3,3-dimethylpent-4-enoic acid ( 4 ) , which was in turn, easily available through Claisen–Johnson rearrangement of 3-methyl-2-buten-1-ol. [ 54 ] Ozonolysis of 4, fol-lowed by Wittig condensation with the ylide derived from phos-phonium salt 7 , delivered the expected ester 9a . Manipulation

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of the protected alcohol group of 9a as previ-ously described gave the phosphonium salt 9e in 58% overall yield. Wittig condensation with aldehyde 10 and hydrolysis of the ethyl ester group afforded the target acid 2h in 33% overall yield. The synthesis of the acid 2g , exhibiting three methylene groups between the dienic system and the carboxyl function was achieved using Arndt-Eistert homo-logation from the acid 2f . [ 55 ] Accordingly, the mixed anhydride formed upon reaction of acid 2f with ethyl chloroformate was reacted with ethereal diazomethane. The intermediate diazoketone was next rearranged in methanol in the presence of silver trifl uoroacetate salt to afford the methyl ester of 2g in 83% yield. Finally, saponifi cation of the ester group deliv-ered the corresponding acid 2g (Figure 3 ).

For the synthesis of acids 2i,j bearing an additional ester group, a suitable carboxyl protecting group had to be incorporated at an appropriate stage to allow the Wittig condensa-tion and to ensure the removal of the terminal carboxyl group while preserving the intrac-hain ester linkage. We anticipated being able to fulfi ll this task using the 9-fl uorenylmethyl ester group. [ 56 ] Thus, Wittig condensation of

the phosphonium salt 13 , which was prepared in a similar way as its tert -butyl ester counterpart 8e , with 3-tetrahydropyranyloxy-propionaldehyde ( 14 ) [ 57 ] gave ester 15a in 22% yield. Saponifi -cation of the methyl ester group followed by esterifi cation with 9-fl uorenemethanol ( 18 ) provided ester 15c . TsOH deprotection of the hydroxyl group and EDCI-catalyzed re-esterifi cation with 1,1 ′ ,2-trisnor-squalenic acid ( 17 ) [ 50 , 51 ] afforded diester 21 . The desired acid 2i was fi nally obtained upon treatment with an excess of triethylamine in 32% overall yield from 15a . The same reaction sequence starting from the gem-dimethyl-substituted phosphonium salt 9e delivered the corresponding acid 2j in 8.2% overall yield over the 6 steps. Finally, synthesis of acid 2k was achieved in three steps from bromoester 23 , through Wittig condensation of the phosphonium salt 24 [ 58 ] with 1,1 ′ ,2-trisnor-squalenaldehyde ( 10 ) followed by sodium hydroxide hydrolysis of the ethyl ester group ( Figure 4 ).

The 2 ′ -acyl-PTX derivatives 1f-k were selectively prepared by condensing acids 2f-k with PTX using EDCI/DMAP as cou-pling agents in 70–86% yield ( Table 1 ). Under these conditions only a minute amount of 2 ′ ,7-diacyl products was observed. The products were easily purifi ed by column chromatography and their purity assessed by HPLC.

3. Preparation of and Characterization of Supramolecular Assemblies of 1f–k

The prodrugs based nanoassemblies suspensions (1 mg mL − 1 ) suitable for intravenous infusion were prepared in a single step by nanoprecipitation of an ethanolic solution (2 mg mL − 1 ) in isotonic 5% aqueous dextrose solution. Formation of NAs occurred immediately without the use of any surfactant. Ethanol

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Figure 3 . Synthetic approaches to acids 2f - h . Reagents and conditions: a) i. O 3 /O 2 , CH 2 Cl 2 , −78 ° C, ii. PPh 3 , 16 h, RT ( 5 : 76%, 6 : 27%); b) i. NaHMDS, THF, −78 ° C, ii. 5 or 6 , THF, 16 h, RT ( 8a : 61%, 9a : 66%); c) TsOH, MeOH, 2 h, RT ( 8b : 90%, 9b : 72%); d) MsCl, Et 3 N, −20 ° C, 45 min ( 8-9c : quant); e) NaI, refl uxing acetone, 4 h ( 8d : 90%, 9d : 98%); f) PPh 3 , CH 3 CN, refl ux, 16 h, ( 8e : 73%, 9e : 98%); g) i. NaHMDS, THF, −40 ° C, ii. SqCHO ( 10 ), 15 h, RT; h) 2 N NaOH, EtOH, refl ux 2h ( 2f : 68% overall from 8e , 2h : 87% overall from 9e ); i) i. EtOCOCl, THF, Et 3 N, 0 ° C, 30 min, ii. CH 2 N 2 , Et 2 O, 2 h, RT, iii. AgOCOCF 3 , Et 3 N, THF, MeOH, 59%.

THPO PPh3

Br

R1O2C

3: R1 = t-Bu, R2 = H4 :R1 = Et, R2 = Me

7

8a: X = OTHP, R1 = t-Bu, R2 = H

8b: X = OH, R1 = t-Bu, R2 = H

8c: X = OMs, R1 = t-Bu, R2 = H

8d: X = I, R1 = t-Bu, R2 = H

8e: X = PPh3.I, R1 = t-Bu, R2 = H

9a: X = OTHP, R1 = Et, R2 = Me

9b: X = OH, R1 = Et, R2 = Me

9c: X = OMs, R1 = Et, R2 = Me

9d: X = I, R1 = Et, R2 = Me

9e: X = PPh3.I, R1 = Et, R2 = Me

c

d

e

R2R2

CHOR1O2C

R2R2

5: R1 = t-Bu, R2 = H6 : R1 = Et, R2 = Me

R1O2CR2

R2

c

ed

f

f

a

b

Sq

8e, 9e

SqCHO 10

HO2CR2R2

Sq

g

h

11: R1 = t-Bu, R2 = H12 :R1 = Et, R2 = Me

2f: R2 = H2h R2 = Me

2gi

X

R1O2CR2R2

Sq

Figure 4 . Synthetic approaches to acids 2i-k. Reagents and conditions: a) i. NaHMDS, THF, −78 ° C, 10 min. ii. 14 , THF, 16 h ( 15a: 45%; 16a: 51%); b) 1 N NaOH, EtOH, 3 h, RT ( 15b : 88%, 16b : 95%); c) FmOH, EDCI, DMAP, CH 2 Cl 2 , RT 16 h (1 5c : 80%, 16c 72%); d) TsOH, MeOH, 4 h, RT ( 19 : 94%, 20 , 95%); e) SqCO 2 H ( 17 ), EDCI, DMAP, CH 2 Cl 2 , RT 16 h ( 21 : 66%, 22 : 79%); f) Et 3 N, CH 3 CN, 20 h, RT ( 2i : 81%, 2j : 65%); g) PPh 3 , CH 3 CN, refl ux, 16 h, 98%; h i. NaHMDS, THF, −40 ° C, ii. SqCHO ( 10 ), 15 h, RT, THF; i) 2 N NaOH, EtOH, RT (25% overall from 24 ).

I

R1O2C

13: R1 = Me, R2 = H9e :R1 = Et, R2 = Me

bc

R2R2

R1O2CR2R2

SqCO2H 17

15a: R1 = Me, R2 = H

15b: R1 = H, R2 = H

15c: R1 = Fm, R2 = H

16a: R1 = Et, R2 = Me

16b: R1 = H, R2 = Me

16c: R1 = Fm, R2 = Me

OHCOTHP

14PPh3

a

OTHP

cb

FmO2C

R2R2OH15c, 16c

FmO2C

R2R2O Sq

O

19: R2 = H20 :R2 = Me

d

e21: R2 = H22 :R2 = Me

2i,j

EtO2C

f

gEtO2C

Br-

SqCHO (10)

EtO2C Sq

23 24

25

2k

Br PPh3

h

i

HO

FmOH:18

15a,16a

was then evaporated at 37 ° C under vacuum using a Rotavapor and the organic solvent-free colloidal dispersions were stored at 4 ° C. The mean diameters of the obtained NAs are depicted in Table 1 (Figures S3–S8 of the Supporting Information (SI)). These particles that exhibited the characteristic Tyndall effect of colloidal systems had a polydispersity index lower than 0.20 as measured by quasi-elastic light scattering. No signifi cant change in the size of the NAs was detected over a 30-day storage period at 20 ° C (Figure S1 of the SI). Such colloidal stability could be correlated with the fairly negative Zeta potentials observed

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( ζ -potential = − 19.8 mV and − 26.4 mV for 1f and 1g respec-tively at 5 mg mL − 1 ). No major size difference was observed between the various prodrugs that possessed a dienic linker ( 1f-j ) whereas conjugate 1k , which displayed only one double bond, gave slightly bigger nanoparticles.

Since higher concentration of active drug is required for in vivo experiment, attempts were made to prepare NAs sus-pensions up to 5 mg mL − 1 , using prodrug 1g as a model com-pound. However, particle sizes tended to increase roughly linearly with increasing concentration, leading to an average

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Table 1. Chemical yields of the paclitaxel coupling with acids 2f-k and col-loidal characteristics of the DHA-squalene hybrid prodrugs of PTX 1f-k .

Compound Chemical yield (%)

Average nanoparticle size [nm] a)

Polydispersity Index (PdI)

1f 71 101.3 0.200

1g 83 93.6 0.050

1h 80 122.8 0.156

1i 76 97,9 0.079

1j 85 118.3 0.173

1k 86 152.7 0.181

a) Size distribution by number at 1 mg/mL .

size up to 250 nm at 5 mg mL − 1 . Noteworthy, it is known that such large colloidal particles should be rapidly taken up by the reticulo-endothelial system when administrated intra-venously. [ 59 ] Thus, it was ultimately found that the initial

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Figure 5 . Transmission electron micrographs of nanoparticles of prodrugs of nanoassemblies of prodrug 1g obtained by DLS and CryoTEM (D). The nanoprecipitation of an ethanolic solution (1 mg mL − 1 ) for DLS measureme

concentration of the squalenoyl prodrug in the organic phase was a crucial parameter to control the fi nal size of the nano-particles and that, higher the concentration in ethanol, smaller the size of the NAs. Thus, NAs of prodrug 1g at 5 mg mL − 1 , with a mean diameter of 142 nm (PdI 0.073) were success-fully obtained using an initial ethanol concentration of 1g of 10 mg mL − 1 (instead of 2 mg mL − 1 initially). Thus, tuning of the initial organic phase concentration provided the ability to modulate the nanoparticle size distribution. When high initial concentration in ethanol was used, a complete precipitation occurred upon mixing in the aqueous phase since these com-pounds were almost insoluble in water. On the other hand, when a larger amount of organic solvent was used, a large fraction of the conjugate remained soluble in the mixture. In this case, the nanoprecipitation occurred essentially during the evaporation of the organic solvent inducing the growth of the small particles already formed.

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1f (A) and 1g (B); CryoTEM image (C). Comparison of the size repartition nanoassemblies were prepared at a fi nal concentration of (1 mg mL − 1 ) by nt and at 2 mg mL − 1 (10 mg mL − 1 in ethanol) for cryoTEM imaging.



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NAs of conjugates 1f,g (5 mg mL − 1 ) were imaged by trans-mission electron microscopy (TEM) using phosphotungstic acid as contrast agent, revealing a population of nearly spher-ical nanoparticles with diameter ranging from 50 to 200 nm ( Figure 5 A,B). Under cryo-transmission electron microscopy, NA of conjugate 1g appeared as round-shaped structures with an external corona probably due to a more hydrated area (Figure 5 C). Figure 5 D shows the corresponding particle size distribution histogram, based on the observation of more than 200 particles in comparison with the distribution arising from DLS experiments. Although, some larger nanoparticles could be visualized in the cryoTEM images, probably because a fi nal concentration of 2 mg mL − 1 was used instead of 1 mg mL − 1 for DLS, both methods provided a roughly similar size distribution.

Small angle X-ray scattering (SAXS) was also carried out on a suspension of NAs of conjugate 1g . The scattering curve displayed only a broad bump, indicative of a distribution of repeat distances between moieties having different elec-tronic densities (Figure S2 of the SI). The peak maximum at q = 0.14 Å − 1 corresponded to a mean distance of 45 Å, arising probably from the average distance between PTX moieties in the nano particles. However, no well - defi ned internal structure could be evidenced in these assemblies of highly hydrophobic bioconjugates, unlike in assemblies of amphiphilic squalene-based nucleolipids. [ 60 ] SAXS results were consistent with trans-mission electron microscopy after freeze-fracture observations (Figure S3 of the SI).

4. In Vitro Biological Evaluation

Nanoparticles of PTX prodrugs 1f-k were evaluated for cytotoxic activity in vitro against six human cancer lines (Lung cell line A549, colon cell line HT-29, hormone-dependent breast cell lines MCF-7, Nasopharyngeal epidermoid cell line KB 3.1, pan-creatic carcinoma cell line Mia PaCa-2, hepatocellular liver car-cinoma cell line Hep G2) using free PTX and SQ-PTX ( 1c ) as control. The cell viability was checked by a proliferation assay utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Results showing the concentrations required to inhibit cell growth by 50% (IC 50 values) are presented in Table 2 . In all cases, owing to their prodrug nature, compounds

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Table 2. In vitro cytotoxic activities of nanoassemblies of DHA-Squalene hybrid prodrugs of PTX against various human cancer cell lines.

Compound IC 50 [ μ M ]

A549 HT-29 MCF-7 KB 3.1 Mia PaCa-2 Hep G2

PTX ( 1a ) 0.012 0.044 0.022 0.004 0.047 0.12

SQ-PTX ( 1b ) 2.7 0.72 1.75 0.71 0.45 3

1f 11.6 6.4 3 8.5 2.35 27

1g 0.62 0.53 1.75 0.36 0.62 –

1h 1.9 1.7 – 0.76 0.77 43

1i 0.8 0.78 0.76 0.56 0.63 6.2

1j 4.7 4.4 6.7 1.9 28 –

1k 38 8.6 10.1 8 31 –

Figure 6 . Survival rate of female nude mice treated with PTX-Cremophor EL (A), prodrug 1f NAs (B), and prodrug 1g NAs (C). The mice were injected for fi ve consecutive days. ∗ PTX equivavelent dose.


1f-k NAs showed lower activity than paclitaxel. Prodrugs 1g and 1i displayed improved activity on most cancer cell lines as com-pared to the control to SQ-PTX ( 1c ) devoid of the diene linker. Unexpectedly, the optimal cytotoxic activity was observed with 1g that possessed three methylene groups between the diene system and the acyl group, whereas 1f , whose the dienic linker

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Table 3. Dosing information for MTD studies in nude mice.

Dose [mg/kg/injection] a)

Total Dose [mg/kg] a)

No. of mice

Death

Cremophor-PTX

10 50 5 0

20 100 5 5

32 160 5 5

prodrug 1f NAs

10 50 5 0

20 100 5 0

32 160 5 0

prodrug 1g NAs

10 50 5 0

20 100 5 0

32 160 5 0

SqCO 2 H NAs

100 500 5 0

a) Equivalent to PTX .

exactly mimicked the DHA-PTX, exhibited a reduced activity. On the other hand, the gem-dimethyl group in 2h and 2j reduced the activity presumably due to a steric hindrance in the hydrol-ysis step. Conjugate 1k which possessed only one double bond exhibited a marked lower cytotoxicity with IC 50 between 10 to 40 μ M . The latter result suggested that our working hypothesis of a positive effect of the 1,4-diene moiety on the rate of hydro-lysis at the C-2 ′ hydroxyl group was correct although the overall effect remained modest. Current hypothesis is the release by a specifi c hydrolase which remains to be identifi ed. Interest-ingly, prodrugs 1g and 1i self-assembled into slightly smaller nanoparticles than the other conjugates, revealing that, beside structural chemical factors, the size of the nanoparticle may

Figure 7 . Body weight variations observed for 20 days after intravenous administration of PTX-Cremophor EL, prodrug 1f NAs, and prodrug 1g NAs into female nude mice. The mice were injected for fi ve consecutive days.

-5

0

5

10

0 10 20 30Va

ria

tio

n in

Bo

dy W

eig

ht (%

)

PTX Dose (mg/kg/injection)

1f 1g PTX-Cremophor EL

be a key issue for optimal activity. Assuming that these prodrugs did not enter the cells as NAs through endocytosis, but rather as indi-vidual molecules through a protein-enhanced passive diffusion as demonstrated with other squalenoyl prodrugs, [ 61 ] we may expect that smaller nanoparticles with high surface expo-sure, allowed more effi cient release of the individual molecules through protein-driven dissociation of the NAs.

5. In Vivo Toxicity

The systemic toxicity of DHA-squalene hybrid prodrug NAs was fi rstly investigated, compar-atively to PTX-Cremophor EL, by determining the maximum tolerated dose (MTD) after fi ve consecutive i.v. injections into female nude mice ( Figure 6 ). Dosing and survival details are summarized in Table 3 . All mice treated with control SQCO 2 H NAs tolerated well injected doses. In the PTX-Cremophor

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EL group, mice receiving doses up to 10 mg/kg/injection sur-vived, but all mice in the 20- and 32-mg/kg/injection groups died. No toxicity of squalenoyl-paclitaxel nanoassemblies 1g or 1f was observed at dose levels tested. To further defi ne MTD, the systemic toxicity was investigated by monitoring the body weight loss over 20 days ( Figure 7 ). Using a 10% weight loss as a threshold value for animal health status, [ 62 ] we therefore con-cluded that the MTD for PTX-Cremophor EL was 10 mg/kg/injection and that of squalenoyl-paclitaxel nanoassemblies 1g or 1f was more than 32 mg/kg/injection ( Table 4 ). Noteworthy, the injected dose of squalenoyl-paclitaxel nanoassemblies 1g or 1f was limited by the maximum concentration of nanopar-ticles possible in suspension and the maximum volume able to be injected, both corresponding to a PTX equivalent dose of 32 mg/kg. Whatever the dosing protocol, SQ-PTX nanoassem-blies appeared dramatically less toxic (more than three times) than PTX-Cremophor (Table 4 ).

6. Antitumor Evaluation in Mouse of DHA-Squalene Hybrid Prodrug Nanoassemblies in the A549 Human Lung Carcinoma Subcutaneous Model

The antitumor effi cacy of DHA-squalene hybrid prodrug NAs has been investigated on the human lung (A549) carcinoma. According to the measured anticancer activity of prodrugs NA in vitro , prodrugs 1f,g were selected for in vivo evaluation on human lung carcinoma xenograft model developed by injection of A549 cells in the fl ank of athymic nude mice. After tumors had grown to 80-100 mm 3 , the animals were divided into fi ve groups (n = 8) in such a manner as to minimize weight and tumor size differences among the groups. Based on the MTD previously determined (Table 4 ), the following treatments and doses were administered by intravenous injections in the lateral tail vein for fi ve consecutive days with either (i) saline 0.9%,

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Figure 8 . Tumor growth inhibition by squalenoyl-paclitaxel nanoassemblies of mice bearing human lung (A549) tumor. Tumor volume (A) and body weight (B) were regularly measured during the experimental period. The mice were treated for fi ve consecutive days with PTX-Cre-mophor EL (10 mg/kg/injection i.v. injected, MTD) ( ), prodrug 1g NAs (32 mg/kg/injection PTX equivalent) ( ), and prodrug 1f NAs (32 mg/kg/injection PTX equivalent) ( ), saline (NaCl 0.9%) ( ), or squalenic acid NAs (SqCO 2 H NAs) (100 mg/kg/injection) ( ) (P < 0.01; n = 8).

0

3

6

9

12

0 5 10 15 20 25 30

Vol

um

e R

atio

, V

(dn)/

V(d

0)

Time (days)

1g

1f

PTX

SqCO2H NA

Saline

-15,00

-10,00

-5,00

0,00

5,00

10,00

15,00

20,00

25,00

0 5 10 15 20 25 30

Rel

ativ

e bo

dy w

eigh

t ch

ange

(%

)

Time (days)

1g

1f

PTX

SqCO2H NA

Saline

A

B

Table 4. Maximum tolerated dose (MTD) of free PTX and DHA-squalene hybrid prodrugs NAs.

Drugs MTD [mg/kg/injection]

Cremophor-PTX 10 a) ; [50] a,b)

Prodrug 1f NAs > 32 a) ; [ > 160] a,b,c)

Prodrug 1g NAs > 32 a) ; [ > 160] a,b,c)

SqCO 2 H NAs > 100; [ > 500] b,c)

a) Equivalent to PTX; b) Total dose of treatment after 5 administrations; c) MTD is not defi ned because the given dose was limited by maximum possible concentration of nanoassemblies suspension and injection volume.

(ii) SqCO 2 H NAs 100 mg/kg, (iii) PTX-Cremophor EL at a PTX dose of 10 mg/kg (MTD), (iv) DHA-squalene hybrid prodrugs 1f NAs at a PTX equivalent dose of 32 mg/kg or (v) prodrug 1g NAs at a PTX equivalent dose of 32 mg/kg. The mice were moni-tored regularly for changes in tumor size and weight. As indicated in Figure 8 A, the growth of A549 tumors were not affected by the treatment with the control squalenic acid nanoparticles (SqCO 2 H NAs), when com-pared with saline treated tumors. On the contrary, the treatment with prodrugs 1g and 1f NA reduced the tumor volume of by 45% and 35% at day 30, respectively (p < 0.01). At the same time, mice treated with PTX-Cre-mophor showed slightly more inhibition of tumor growth (ie. 53%) comparatively to 1g NAs (p < 0.1). Conjugate 1f which possessed two methylene groups between the diene system and the acyl group exhibited a lower activity than prodrug 1g with three methylene groups. These results correlated well with the in vitro anticancer activity on A549 cell line. Noteworthy, the absolute weight loss differ-ences in the PTX- and in the DHA-squalene hybrid prodrugs NA-treated groups were more drastic (Figure 8 B) at the doses used for the treatment of A549 subcutaneously grafted tumor model (Figure 8 A). Indeed, a signifi -cant weight loss was observed only in PTX-Cremophor treated mice (5–10%) which was the expression of drug’s toxicity. Thus, in a nutshell, the squalenoylation of PTX allowed to deliver larger amounts of drug without observable toxicity, thus improving the thera-peutic index of PTX.

7. The Morphological and Immunohistochemical Analysis of Tumor Biopsies

Immunohistochemical analysis of biopsies demonstrated enlarged cells with necrotic

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changes only for PTX-treated tumor tissues ( Figure 9 A). The TUNEL staining of tumor sections (5 μ m) from mice was used to assess the induction of apoptosis by the treatments in vivo (Figure 9 ). TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identifi ed by terminal deoxynucleotidyl transferase (TdT), an enzyme that will catalyze the addition of dUTPs that are sec-ondarily labelled with a marker. The mean value of TUNEL pos-itive cells (red colour, Figure 9 A)/fi eld for PTX-Cremophor EL, prodrug 1f NAs, and prodrug 1g NAs treatment groups were 18%, 2.9%, and 3.1%, whereas tumors from saline or SQCO 2 H

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Figure 9 . Immunohistochemical staining of tumor tissues derived from injected human lung (A549) cancer cells. The tumors were treated for fi ve consecutive days by squalenoyl–paclitaxel nanoassemblies 1f or 1g (160 mg/kg equivalent to PTX), PTX-Cremophor EL (50 mg/kg), by saline (0.9%) and squalenic acid nanoparticles (500 mg/kg) and excised at day 8. (A) Paraffi n sections from tumor biopsies were submitted to hematoxylin-eosin-safranin staining (HES, left panels) for morphology study, TUNEL staining (middle-left panels) for visualization of apoptotic cells, caspase-3 staining (middle-right panels) for indication of caspase-3 positive cells, and Ki-67 (right panels) staining for proliferated cells. (B) Quantifi cation of the apoptotic, caspase-3 and Ki - 67-positive cells in the tumor tissue sections. (C) Inhibition of the cell proliferation in the tumor tissues.

NAs control groups had a mean value of 0.5% of TUNEL posi-tive cells/fi eld. Among the different signalling pathways of programmed cell death, caspase-3 plays a central role in the execution-phase of cell apoptosis. Immunostaining of the active form of caspase-3 protease revealed the increased caspase-3 acti-vation (10%) in free PTX-treated mice only (Figure 9 B). Tumors from mice receiving the prodrug 1f NAs or prodrug 1g NAs had a mean value of 2.9% and 3.1% positive cells/fi eld respectively, whereas tumors from saline or SQCO 2 H NAs control groups had a mean value of 0.5% of TUNEL positive cells/fi eld. The low activation of apoptosis and caspase-3 protease by prodrugs 1g and 1f in the tumor may be explained by slow liberation inside cells of the parent active molecule from nanoassemblies which led to more late antitumor effect.

The mean number of proliferating cells determined by Ki-67 staining was assessed in tumor sections (Figure 9 ). The Ki-67 protein (also known as MKI67 ) is a cellular marker for cell pro-liferation. [ 63 ] During interphase, the Ki-67 antigen can be exclu-sively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. Tumors from mice receiving PTX-Cremophor, prodrug 1f NAs, and prodrug 1g NAs had a mean value of 24%, 59%, and 30% Ki-67 positive cells/fi eld respectively, whereas tumors receiving saline or SQCO 2 H NAs had a mean value of 67% or 65% Ki-67 positive cells/fi eld respectively (Figure 9 B). In spite of a low induction of apoptosis by activation of caspase-3, DHA-squalene hybrid prodrug 1g generated a comparable decrease of the tumor proliferative activity (55%) that the parent drug (63%), as

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indicated by the reduced number of Ki67-positive tumor cells, showing a signifi cant decrease in proliferation in comparison to control (Figure 9 B). At the same time, prodrug 1f did not reduce the number of proliferating tumor cells. These fi ndings show that prodrug 1g NAs can effectively inhibit the tumor cell growth in vivo .

8. Conclusion

In the present study, we have synthesized new prodrugs of PTX, by covalent linkage of a squalenyl chain through cis,cis -1,4-dienic linkers at the C-2 ′ position of PTX. Interestingly the introduction of a hydrophobic non-polyisoprenic linker did not disrupt the self-organizing properties of the squalene conju-gates. Indeed, all these prodrugs self-assembled into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy. Zeta potential measurements revealed that all squalenoyl–paclitaxel nanoassemblies held a global negative charge and appeared stable in water for several days. In vitro biological assessment showed that these squalenoyl-paclitaxel nanoparticles displayed notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis -squalenyl-deca-5,8-dienoate prodrug showed improved activity over simple 2 ′ -squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. Our results indicate that the enhanced cytotoxicity


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depends not only on structure of the prodrug, but also on the size of the nanoparticles. The antitumor effi cacy of the two more active prodrug NAs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis -deca-5,8-dienoyl linker showed comparable antitumor effi cacy to the parent drug, but revealed much lower subacute toxicity as seen in body weight loss. Thus, these nanoassem-blies with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

9. Experimental Section General methods : IR spectra were obtained as solid or neat liquid

on a Fourier Transform Bruker Vector 22 spectrometer. Only signifi cant absorptions are listed. Optical rotations were measured on a Perkin-Elmer 241 Polarimeter at 589 nm. The 1 H and 13 C NMR spectra were recorded on Bruker Avance 300 (300 MHz and 75 MHz, for 1 H and 13 C, respectively) or Bruker Avance 400 (400 MHz and 100 MHz, for 1 H and 13 C, respectively) spectrometers. The 31 P NMR spectra were recorded on Bruker AC 200 P (81 MHz) or Bruker Avance 400 (162 MHz). Recognition of methyl, methylene, methine, and quaternary carbon nuclei in 13 C NMR spectra rests on the J -modulated spin-echo sequence. Mass spectra were recorded on a Bruker Esquire-LC spectrometer. High-resolution mass spectra (ESI) were recorded on a ESI/TOF (LCT, Waters) LC-spectrometer. Elemental analyses were performed by the Service de microanalyse, Centre d’Etudes Pharmaceutiques, Châtenay-Malabry, France, with a Perkin Elmer 2400 analyzer. The sizes of the obtained nanoassemblies were measured using a Malvern particle size analyzer (Zetasizer). Purity of tested compounds 1g-j was determined by HPLC analysis to be > 95% pure. Analytical thin-layer chromatography was performed on Merck silica gel 60F 254 glass precoated plates (0.25 mm layer). Column chromatography was performed on Merck silica gel 60 (230–400 mesh ASTM). Tetrahydrofuran (THF) was distilled from sodium/benzophenone ketyl. DMF and CH 2 Cl 2 were distilled from calcium hydride, under a nitrogen atmosphere. All reactions involving air- or water-sensitive compounds were routinely conducted in glassware which was fl ame-dried under a positive pressure of nitrogen. Paclitaxel was purchased from Sequoia Research Product Ltd (UK). Squalene and 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co., France. RPMI 1640 GlutaMAX I and fetal bovine serum were purchased from Dulbecco (Invitrogen, France). Penicillin and streptomycin solution were purchased from Lonza (Verviers, Belgium). Chemicals obtained from commercial suppliers were used without further purifi cation.

HPLC conditions : colonne Waters SUNFIRE C18, 150 × 4,6 mm; solvent A, 100% acetonitrile; solvent B, 100% methanol; fl ow rate of 0.9 mL/min, from 85% B to 100% B in 7 min; and then 100% methanol.

General procedure for the synthesis of compounds 1f-j from acids 2f-j and paclitaxel : To a solution of acid 2 (0.024 mmol) in dry CH 2 Cl 2 (1 mL) were sequentially added paclitaxel (10 mg, 0.012 mmol), EDCI (6.0 mg, 0.031 mmol) and a catalytic amount of 4-DMAP. The reaction mixture was stirred at room temperature for 1h and concentrated under reduced pressure. The residue was purifi ed by column chromatography on silica gel eluting with AcOEt/petroleum ether, 1:3, to give the corresponding conjugates as amorphous white solids.

Compound 1f : (71%), [ α ] D 20 = − 33.5 (c = 2.75, in EtOH); 1 H NMR (400 MHz, CDCl 3, δ ): 8.14 (d, J = 7.1 Hz, 2H, PhCO 2 ), 7.73 (d, J = 7.1 Hz, 2H, PhCONH), 7.61 (t, J = 7.1 Hz, 1H, PhCO 2 ), 7.55–7.46 (m, 3H, C H -ar), 7.45–7.31 (m, 7H, C H -ar), 6.87 (d, J = 9.3 Hz, 1H, N H ), 6.30 (s, 1H, H-10), 6.27 (t, J = 8.4 Hz, 1H, H-13), 5.96 (dd, J = 9.3 Hz, J = 3.2 Hz, 1H, H-3′), 5.69 (d, J = 6.9 Hz, 1H, H-2), 5.52 (d, J = 3.2 Hz, 1H, H-2’), 5.42-5.22 (m, 4H, C H = C H CH 2 C H = C H ), 5.20-5.06 (m, 5H, (CH 3 )C = C H CH 2 ), 4.98 (d, J = 9.9 Hz, 1H, H-5), 4.46 (dd, J = 9.0 Hz, J = 7.9 Hz, 1H, H-7), 4.32 (d, J = 8.4 Hz, 1H, H-20), 4.20 (d, J = 8.4 Hz, 1H, H-20), 3.82 (d, J = 6.9 Hz, 1H, H-3), 2.75 (t, J = 6.8 Hz,


2H, CH = CHC H 2 CH = CH), 2.60-2.35 (m, 7H, 1H-14, 1H-6, OH, = CHC H 2 C H 2 CO 2 Ptx), 2.46 (s, 3H, C H 3 CO 2 ), 2.23 (s, 3H, C H 3 CO 2 ), 2.19–1.96 (m, 21H, CHC H 2 C H 2 (CH 3 )C = , 1H-14), 1.95 (s, 3H, H-18), 1.89 (m, 1H, H-6), 1.75 (br s, 1H, OH), 1.68 (s, 6H, CH = C(C H 3 ) 2 , H-19), 1.60 (s, 15H, CH = C(C H 3 )), 1.24 (s, 3H, H-16), 1.14 (s, 3H, H-17); 13 C NMR (100 MHz, CDCl 3, δ ): 203.9 (C, C9), 172.2 (C, PtxO 2 C CH 2 ), 171.3 (C, CH 3 C O 2 ), 169.9 (C, CH 3 C O 2 ), 168.1 (C, C1’), 167.2 (C, Ph C ONH or Ph C O 2 ), 167.1 (C, C ONH or Ph C O 2 ), 142.9 (C, C12), 137.1 (C, C-ar), 135.2 (C, (CH 3 ) C = CHCH 2 ), 135.1 (C, (CH 3 ) C = CHCH 2 ), 135.0 (C, (CH 3 ) C = CHCH 2 ), 134.4 (C, (CH 3 ) C = CHCH 2 ), 133.8 (C, C-ar), 133.7 (CH, CH-ar), 132.9 (C, C11), 132.1 (CH, CH-ar), 131.3 (C, (CH 3 ) 2 C = CH), 130.3 (2CH, CH-ar), 130.1 (CH, C H = C HCH 2 C H = C H), 129.2 (C, C-ar), 129.1 (3CH, 2CH-ar, C H = C HCH 2 C H = C H), 128.8 (4CH, CH-ar), 128.7 (CH, CH-ar), 127.3 (CH, C H = C HCH 2 C H = C H), 127.1 (2CH, CH-ar), 127.0 (CH, C H = C HCH 2 C H = C H), 126.5 (2CH, CH-ar), 124.8 (CH, (CH 3 )C = C HCH 2 ), 124.5 (CH, (CH 3 )C = C HCH 2 ), 124.4 (CH, (CH 3 )C = C HCH 2 ), 124.3 (2CH, (CH 3 )C = C HCH 2 ), 84.5 (CH, C5), 81.1 (C, C4), 79.3 (C, C1), 76.5 (CH 2 , C20), 75.7 (CH, C10), 75.2 (CH, C2), 74.0 (CH, C2’), 72.2 (CH, C7), 71.8 (CH, C13), 58.6 (C, C8), 52.8 (CH, C3’), 45.6 (CH, C3), 43.2 (C, C15), 39.8 (3CH 2 ), 39.7 (CH 2 ), 35.7 (CH 2 , C6 or C14), 35.6 (CH 2 , C6 or C14), 33.8 (CH 2 , CH 2 C H 2 CO 2 Ptx), 28.3 (2CH 2 ), 26.9 (CH 3 , C16), 26.8 (CH 2 ), 26.7 (2CH 2 ), 25.8 (CH 2 ), 25.7 (CH 3 , ( C H 3 ) 2 C = CH), 25.6 (CH 2 ), 22.7 (CH 3 , C H 3 CO 2 ), 22.5 (CH 2 ), 22.2 (CH 3 , C17), 20.8 (CH 3 , C H 3 CO 2 ), 17.7 (CH 3 , CH = C( C H 3 )), 16.1 (3CH 3 , CH = C( C H 3 )), 16.0 (CH 3 , CH 2 CH = C( C H 3 )), 14.9 (CH 3 , C18), 9.6 (CH 3 , C19); IR (neat): ν = 3100-2800, 1737, 1640, 1445, 1371, 1241, 1069, 708 cm − 1 ; MS ( + ESI) m/z (%): 1353.0 (100) [M + Na + ]; HRMS ( + ESI) m/z : [M + Na + ] calcd for C 81 H 103 NO 15 , 1352.7225; found, 1352.7198. Anal. calcd for C 81 H 103 NO 15 : C 73.11, H 7.80, N 1.05; found: C 73.19, H 7.73, N 1.03. HPLC: 94.0% (t R = 18.7 min).

Compound 1h: (80%); [ α ] D 20 = − 35.5 (c = 0.99, in EtOH); 1 H NMR (400 MHz, CDCl 3, δ ): 8.14 (d, J = 7.1 Hz, 2H, PhCO 2 ), 7.73 (d, J = 7.1 Hz, 2H, PhCONH), 7.61 (t, J = 7.1 Hz, 1H, PhCO 2 ), 7.55-7.46 (m, 3H, C H -ar), 7.45-7.31 (m, 7H, C H -ar), 6.85 (d, J = 9.2 Hz, 1H, N H ), 6.30 (s, 1H, H-10), 6.27 (t, J = 8.3 Hz, 1H, H-13), 5.96 (dd, J = 9.2 Hz, J = 3.3 Hz, 1H, H-3’), 5.69 (d, J = 6.9 Hz, 1H, H-2), 5.49 (d, J = 3.3 Hz, 1H, H-2′), 5.40-5.22 (m, 3H, C H = C H CH 2 C H = C H ), 5.20-5.07 (m, 6H, (CH 3 )C = C H CH 2 , C H = C H CH 2 C H = C H ), 4.98 (d, J = 9.8 Hz, 1H, H-5), 4.46 (dd, J = 9.0 Hz, J = 7.9 Hz, 1H, H-7), 4.31 (d, J = 8.4 Hz, 1H, H-20), 4.20 (d, J = 8.4 Hz, 1H, H-20), 3.82 (d, J = 6.9 Hz, 1H, H-3), 2.86 (t, J = 7.1 Hz, 2H, CH = CHC H 2 CH = CH), 2.46 (s, 3H, C H 3 CO 2 ), 2.60–2.35 (m, 4H, O 2 CC H 2 C(CH 3 ) 2, 1H-14, 1H-6), 2.23 (s, 3H, C H 3 CO 2 ), 2.26–1.95 (m, 23H, = CH CH 2 CH 2 C(CH 3 ), 1H-14), 1.96 (s, 3H, H-18), 1.89 (m, 1H, H-6), 1.68 (s, 6H, ( C H 3 ) 2 C = CH, H-19), 1.60 (s, 15H, CH = C(C H 3 )CH 2 ), 1.23 (s, 3H, H-16), 1.21 (s, 3H, CH 2 (C H 3 ) 2 CCH = ), 1.17 (s, 3H, CH 2 ( C H 3 ) 2 CCH = CH), 1.13 (s, 3H, H-17); 13 C NMR (100 MHz, CDCl 3, δ ): 204.0 (C, C9), 171.4 (C, CH 3 C O 2 ), 170.8 (C, PtxO 2 C CH 2 ), 169.9 (C, CH 3 C O 2 ), 168.2 (C, C1’), 167.2 (2C, Ph C ONH, Ph C O 2 ), 143.1 (C, C12), 137.2 (C, C-ar), 137.1 (CH, (CH 3 ) 2 C C H = CHCH 2 CH = CH), 135.3 (C, CH 2 (CH 3 ) C = CH), 135.2 (C, CH 2 (CH 3 ) C = CH), 135.0 (C, CH 2 (CH 3 ) C = CH), 134.4 (C, CH 2 (CH 3 ) C = CH), 133.9 (C, C-ar), 133.8 (CH, CH-ar), 132.9 (C, C11), 132.1 (CH, CH-ar), 131.4 (C, (CH 3 ) 2 C = CHCH 2 ), 130.4 (2CH, CH-ar), 130.3 (CH, (CH 3 ) 2 CCH = C HCH 2 C H = C H), 129.3 (C, C-ar), 129.2 (2CH, CH-ar), 128.9 (4CH, CH-ar), 128.8 (CH, (CH 3 ) 2 CCH = C HCH 2 C H = C H), 128.5 (CH, CH-ar), 127.7 (CH, (CH 3 ) 2 CCH = C HCH 2 C H = C H), 127.2 (2CH, CH-ar), 126.5 (2CH, CH-ar), 124.9 (CH, (CH 3 )C = C HCH 2 ), 124.6 (CH, (CH 3 )C = C HCH 2 ), 124.5 (CH, (CH 3 )C = C HCH 2 ), 124.4 (2CH, (CH 3 )C = C HCH 2 ), 84.6 (CH, C5), 81.2 (C, C4), 79.3 (C, C1), 76.6 (CH 2 , C20), 75.8 (CH, C10), 75.3 (CH, C2), 74.1 (CH, C2’), 72.3 (CH, C7), 71.8 (CH, C13), 58.7 (C, C8), 52.8 (CH, C3’), 47.5 (CH 2 , O 2 C C H 2 C(CH 3 ) 2 CH = ), 45.7 (CH, C3), 43.3 (C, C15), 39.9 (3CH 2 ), 39.6 (CH 2 ), 35.7 (CH 2 and C, C6 or C14, CH 2 (CH 3 ) 2 C CH = ), 35.6 (CH 2 , C6 or C14), 29.0 (CH 3 , CH 2 (C H 3 ) 2 CCH = ), 28.8 (CH 3 , CH 2 (C H 3 ) 2 CCH = ), 28.4 (2CH 2 ), 27.0 (CH 3 , C16), 26.9 (2CH 2 ), 26.8 (2CH 2 ), 25.9 (CH 2 ), 25.8 (CH 3 , ( C H 3 ) 2 C = CH), 22.9 (CH 3 , C H 3 CO 2 ), 22.3 (CH 3 , C17), 20.9 (CH 3 , C H 3 CO 2 ), 17.8 (CH 3 , CH = C( C H 3 )), 16.2 (2CH 3 , CH = C( C H 3 )), 16.1 (2CH 3 , CH = C( C H 3 )), 15.0 (CH 3 , C18), 9.7 (CH 3 , C19); IR (neat): ν = 3500-3100, 3050-2850, 1734, 1665, 1645, 1451, 1371, 1260, 1238,



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182

1177, 1108, 1068, 1025, 982, 908, 732 cm − 1 ; MS ( + ESI) m/z (%): 1381.0 (100) [M + Na + ]; HRMS (ESI) m/z : [M + Na + ] calcd for C 83 H 107 NO 15 , 1380.7538; found, 1380.7567; HPLC: 93.0% (t R = 19.9 min).

Compound 1g: (83%); [ α ] D 20 = − 35.6 (c = 1.83, in EtOH); 1 H NMR (400 MHz, CDCl 3, δ ): 8.14 (d, J = 7.1 Hz, 2H, Ph C O 2 ), 7.73 (d, J = 7.1 Hz, 2H, Ph C ONH), 7.60 (t, J = 7.1 Hz, 1H, Ph C O 2 ), 7.55–7.46 (m, 3H, C H -ar), 7.45-7.31 (m, 7H, C H -ar), 6.87 (d, J = 9.2 Hz, 1H, N H ), 6.30 (s, 1H, H-10), 6.26 (t, J = 8.3 Hz, 1H, H-13), 5.96 (dd, J = 9.2 Hz, J = 3.3 Hz, 1H, H-3’), 5.68 (d, J = 6.9 Hz, 1H, H-2), 5.51 (d, J = 3.3 Hz, 1H, H-2′), 5.43-5.23 (m, 4H, C H = C H CH 2 C H = C H ), 5.19-5.05 (m, 5H, (CH 3 )C = C H CH 2 ), 4.98 (d, J = 9.8 Hz, 1H, H-5), 4.45 (m, 1H, H-7), 4.31 (d, J = 8.4 Hz, 1H, H-20), 4.21 (d, J = 8.4 Hz, 1H, H-20), 3.82 (d, J = 6.9 Hz, 1H, H-3), 2.73 (t, J = 10.7 Hz, 2H, CH = CHC H 2 CH = CH), 2.63-2.33 (m, 5H, 1H-14, 1H-6, OH, CH 2 C H 2 CO 2 ), 2.45 (s, 3H, C H 3 CO 2 ), 2.22 (s, 3H, C H 3 CO 2 ), 2.25-1.84 (m, 28H, = CHC H 2 C H 2 C(CH 3 ), = CHC H 2 C H 2 C(CH 3 ), CHC H 2 CH 2 CH 2 CO 2 , 1H-14, 3H-18, 1H-6, OH), 1.72–1.64 (m, 8H, H-19, CHCH 2 C H 2 CH 2 CO 2 , ( C H 3 ) 2 C = CH), 1.60 (s, 15H, CH 2 CH = C(C H 3 )), 1.23 (s, 3H, H-16), 1.13 (s, 3H, H-17); 13 C NMR (100 MHz, CDCl 3, δ ): 204.0 (C, C9), 172.7 (C, PtxO 2 C CH 2 ), 171.4 (C, CH 3 C O 2 ), 169.9 (C, CH 3 C O 2 ), 168.3 (C, C1’), 167.3 (C, Ph C ONH or Ph C O 2 ), 167.2 (C, Ph C ONH or Ph C O 2 ), 143.0 (C, C12), 137.1 (C, C-ar), 135.3 (C, (CH 3 ) C = CHCH 2 ), 135.2 (C, (CH 3 ) C = CHCH 2 ), 135.0 (C, (CH 3 ) C = CHCH 2 ), 134.5 (C, (CH 3 ) C = CHCH 2 ), 133.8 (C, C-ar), 133.7 (CH, CH-ar), 132.9 (C, C11), 132.2 (CH, CH-ar), 131.4 (C, (CH 3 ) 2 C = CHCH 2 ), 130.4 (2CH, CH-ar), 130.1 (CH, C H = C HCH 2 C H = C H), 129.6 (CH, C H = C HCH 2 C H = C H), 129.4 (C, C-ar), 129.2 (2CH, CH-ar), 128.9 (4CH, CH-ar), 128.6 (CH, CH-ar), 128.4 (CH, C H = C HCH 2 C H = C H), 127.7 (CH, C H = C HCH 2 C H = C H), 127.2 (2CH, CH-ar), 126.6 (2CH, CH-ar), 124.9 (CH, (CH 3 )C = C HCH 2 ), 124.6 (CH, (CH 3 )C = C HCH 2 ), 124.5 (CH, (CH 3 )C = C HCH 2 ), 124.4 (2CH, (CH 3 )C = C HCH 2 ), 84.6 (CH, C5), 81.2 (C, C4), 79.3 (C, C1), 76.6 (CH 2 , C20), 75.8 (CH, C10), 75.3 (CH, C2), 74.0 (CH, C2’), 72.3 (CH, C7), 71.9 (CH, C13), 58.7 (C, C8), 52.9 (CH, C3’), 45.7 (CH, C3), 43.3 (C, C15), 39.9 (3CH 2 ), 39.6 (CH 2 ), 35.8 (CH 2 , C6 or C14), 35.7 (CH 2 , C6 or C14), 33.3 (CH 2 , CH 2 C H 2 CO 2 ), 28.4 (2CH 2 ), 27.0 (CH 3 , C16), 26.9 (CH 2 ), 26.8 (2CH 2 ), 26.4 (CH 2 ), 25.9 (CH 2 ), 25.8 (CH 3 , ( C H 3 ) 2 C = CH), 25.7 (CH 2 ), 24.7 (CH 2 , CH 2 C H 2 CH 2 CO 2 ), 22.8 (CH 3 , C H 3 CO 2 ), 22.3 (CH 3 , C17), 20.9 (CH 3 , C H 3 CO 2 ), 17.8 (CH 3 , CH 2 CH = C( C H 3 )), 16.2 (2CH 3 , CH 2 CH = C( C H 3 )), 16.1 (2CH 3 , CH 2 CH = C( C H 3 ) 2 ), 14.9 (CH 3 , C18), 9.7 (CH 3 , C19); IR (neat): ν = 3600-3200, 3100-2800, 1738, 1640, 1450, 1371, 1280, 1238, 1178, 1143, 1069, 1025, 984, 708 cm − 1 ; MS ( + ESI) m/z (%): 1367.0 (100) [M + Na + ]; HRMS (ESI) m/z : [M + Na + ] calcd for C 82 H 105 NO 15, 1366.7382; found, 1366.7440; HPLC: 95.0% (t R = 19.2 min).

Compound 1i: (78%); [ α ] D 20 = − 24.9 (c = 0.85, in EtOH); 1 H NMR (400 MHz, CDCl 3, δ ): 8.14 (d, J = 7.1 Hz, 2H, Ph C O 2 ), 7.73 (d, J = 7.1 Hz, 2H, Ph C ONH), 7.61 (t, J = 7.1 Hz, 1H, Ph C O 2 ), 7.55-7.46 (m, 3H, C H -ar), 7.42-7.30 (m, 7H, C H -ar), 6.92 (d, J = 9.0 Hz, 1H, N H ), 6.30 (s, 1H, H-10), 6.26 (t, J = 8.3 Hz, 1H, H-13), 5.96 (dd, J = 9.0 Hz, J = 3.2 Hz, 1H, H-3′), 5.68 (d, J = 6.9 Hz, 1H, H-2), 5.51 (d, J = 3.2 Hz, 1H, H-2′), 5.48-5.25 (m, 4H, C H = C H CH 2 C H = C H ), 5.20–5.05 (m, 5H, (CH 3 )C = C H CH 2 ), 4.97 (d, J = 9.8 Hz, 1H, H-5), 4.44 (dd, J = 9.0 Hz, J = 7.9 Hz, 1H, H-7), 4.31 (d, J = 8.3 Hz, 1H, H-20), 4.20 (d, J = 8.3 Hz, 1H, H-20), 4.05 (t, J = 7.3 Hz, 2H, CH 2 C H 2 O 2 CH 2 ), 3.82 (d, J = 7.0 Hz, 1H, H-3), 2.76 (t, J = 6.2 Hz, 2H, CH = CHC H 2 CH = CH), 2.45 (s, 3H, C H 3 CO 2 ), 2.63-2.42 (m, 5H, 1H-14, 1H-6, OH, = CHCH 2 C H 2 CO 2 Ptx), 2.42-2.32 (m, 6H, = CHC H 2 CH 2 CO 2 Ptx, = CHC H 2 CH 2 O 2 CC H 2 CH 2 C(CH 3 )), 2.27 (m, 2H, CH 2 O 2 CCH 2 C H 2 C(CH 3 )), 2.22 (s, 3H, C H 3 CO 2 ), 2.20-1.93 (m, 20 H, CHC H 2 C H 2 C(CH 3 ), 1H-14, 3H-18), 1.89 (m, 1H, H-6), 1.75 (m, 1H, OH), 1.68 (s, 6H, CH = C(C H 3 ) 2 , H-19), 1.60 (s, 15H, CH = C(C H 3 )CH 2 ), 1.23 (s, 3H, H-16), 1.14 (s, 3H, H-17); 13 C NMR (100 MHz, CDCl 3, δ ): 204.0 (C, C9), 173.5 (C, CH 2 C O 2 CH 2 ), 172.2 (C, PtxO 2 C CH 2 ), 171.4 (C, CH 3 C O 2 ), 169.9 (C, CH 3 C O 2 ), 168.2 (C, C1’), 167.2 (2C, Ph C ONH, Ph C O 2 ), 143.0 (C, C12), 137.2 (C, C-ar), 135.3 (C, CH 2 C = CH(CH 3 )), 135.0 (2C, CH 2 (CH 3 ) C = CH), 133.9 (C, C-ar), 133.8 (CH, CH-ar), 133.3 (C, CH 2 C = CH(CH 3 )), 132.9 (C, C11), 132.1 (CH, CH-ar), 131.4 (C, (CH 3 ) 2 C = CH), 130.4 (3CH, 2CH-ar, C H = C HCH 2 C H = C H), 129.6 (CH, C H = C HCH 2 C H = C H), 129.3 (C, C-ar), 129.2 (3CH, 2CH-ar, C H = C HCH 2 C H = C H), 128.9 (4CH, CH-ar), 128.6 (CH, CH-ar), 127.5 (CH,

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C H = C HCH 2 C H = C H), 127.2 (2CH, CH-ar), 126.7 (2CH, CH-ar), 125.3 (CH, (CH 3 )C = C HCH 2 ), 124.6 (2CH, (CH 3 )C = C HCH 2 ), 124.4 (2CH, (CH 3 )C = C HCH 2 ), 84.6 (CH, C5), 81.2 (C, C4), 79.4 (C, C1), 76.6 (CH 2 , C20), 75.7 (CH, C10), 75.3 (CH, C2), 74.1 (CH, C2’), 72.3 (CH, C7), 71.9 (CH, C13), 63.8 (CH 2 , CH 2 C H 2 O 2 CCH 2 ), 58.7 (C, C8), 52.9 (CH, C3’), 45.7 (CH, C3), 43.3 (C, C15), 39.9 (2CH 2 ), 39.7 (CH 2 ), 35.7 (2CH 2 , C6, C14), 34.8 (CH 2 , CH 2 O 2 CCH 2 C H 2 C(CH 3 ) = ), 33.8 (CH 2 , CH 2 C H 2 CO 2 Ptx), 33.4 (CH 2 , CH 2 O 2 C C H 2 CH 2 (CH 3 )C = ), 28.4 (2CH 2 ), 27.0 (CH 2 ), 26.9 (CH 3 , C16), 26.8 (3CH 2 ), 25.8 (CH 3 , ( C H 3 ) 2 C = CH), 25.7 (CH 2 , CH = CH C H 2 CH = CH), 22.8 (CH 3 , C H 3 CO 2 ), 22.6 (CH 2 , CH C H 2 CH 2 CO 2 Ptx), 22.3 (CH 3 , C17), 20.9 (CH 3 , CH 3 CO 2 ), 17.8 (CH 3 , CH = C( C H 3 )CH 2 ), 16.2 (2CH 3 , CH 2 CH = C( C H 3 )), 16.1 (CH 3 , CH 2 CH = C( C H 3 )CH 2 ), 16.0 (CH 3 , CH 2 CH = C( C H 3 )CH 2 ), 15.0 (CH 3 , C18), 9.7 (CH 3 , C19); IR (neat): ν = 2911, 1726, 1639, 1533, 1451, 1371, 1239, 1178, 1147, 1070, 1025, 982, 908, 799, 775, 731 cm − 1 ; MS ( + ESI) m/z (%): 1425.2 (100) [M + Na + ]; HRMS ( + ESI) m/z : [M + Na + ] calcd for C 84 H 107 NO 17 , 1424.7437; found, 1424.7472. HPLC: 96.0% (t R = 17.3 min).

Compound 1j: (85%); [ α ] D 20 = − 25.9 (c = 1.28, in EtOH); 1 H NMR (400 MHz, CDCl 3, δ ): 8.14 (d, J = 7.1 Hz, 2H, Ph C O 2 ), 7.73 (d, J = 7.1 Hz, 2H, Ph C ONH), 7.61 (t, J = 7.1 Hz, 1H, Ph C O 2 ), 7.55-7.46 (m, 3H, C H -ar), 7.43-7.30 (m, 7H, C H -ar), 6.89 (d, J = 8.9 Hz, 1H, N H ), 6.30 (s, 1H, H-10), 6.26 (t, J = 8.3 Hz, 1H, H-13), 5.96 (dd, J = 8.9 Hz, J = 3.2 Hz, 1H, H-3′), 5.69 (d, J = 6.9 Hz, 1H, H-2), 5.49 (d, J = 3.2 Hz, 1H, H-2’), 5.45-5.28 (m, 3H, C H = C H CH 2 C H = C H ), 5.20-5.05 (m, 6H, (CH 3 )C = C H CH 2 , C H = C H CH 2 C H = C H ), 4.98 (d, J = 9.8 Hz, 1H, H-5), 4.45 (m, 1H, H-7), 4.32 (d, J = 8.3 Hz, 1H, H-20), 4.21 (d, J = 8.3 Hz, 1H, H-20), 4.04 (t, J = 7.2 Hz, 2H, = CHCH 2 C H 2 O 2 CCH 2 ), 3.82 (d, J = 7.0 Hz, 1H, H-3), 2.87 (m, 2H, CH = CHC H 2 CH = CH), 2.49 (s, 2H, O 2 C CH 2 C(CH 3 ) 2 CH = ), 2.46 (s, 3H, C H 3 CO 2 ), 2.63-2.31 (m, 6H, 1H-14. 1H-6. = CHC H 2 CH 2 O 2 CC H 2 CH 2 C(CH 3 )), 2.23 (s, 3H, C H 3 CO 2 ), 2.20-1.93 (m, 20 H, 1H-14, 3H-18, CHC H 2 C H 2 C(CH 3 )), 1.89 (m, 1H, H-6), 1.74 (br s, 1H, OH), 1.68 (s, 6H, ( C H 3 ) 2 C = CH, 3H-19), 1.65 (br s, 1H, OH), 1.60 (s, 15H, CH 2 CH = C(C H 3 )), 1.23 (s, 3H, H-16), 1.21 (s, 3H, CH 2 ( C H 3 ) 2 CCH = CH), 1.17 (s, 3H, CH 2 ( C H 3 ) 2 CCH = CH), 1.14 (s, 3H, H-17); 13 C NMR (100 MHz, CDCl 3, δ ): 204.0 (C, C9), 173.6 (C, CH 2 C O 2 CH 2 ), 171.4 (C, CH 3 C O 2 ), 170.8 (C, PtxO 2 C CH 2 ), 169.9 (C, CH 3 C O 2 ), 168.2 (C, C1’), 167.2 (2C, Ph C ONH, Ph C O 2 ), 143.1 (C, C12), 137.4 (CH, (CH 3 ) 2 C C H = CHCH 2 CH = CH), 137.2 (C, C-ar), 135.3 (C, (CH 3 ) C = CHCH 2 ), 135.1 (2C, (CH 3 ) C = CHCH 2 ), 133.9 (C, C-ar), 133.8 (CH, CH-ar), 133.3 (C, (CH 3 ) C = CHCH 2 ), 132.9 (C, C11), 132.1 (CH, CH-ar), 131.4 (C, (CH 3 ) 2 C = CHCH 2 ), 130.7 (CH, (CH 3 ) 2 CCH = C HCH 2 C H = C H), 130.4 (2CH, CH-ar), 129.4 (C, C-ar), 129.2 (2CH, CH-ar), 128.9 (4CH, CH-ar), 128.5 (CH, CH-ar), 128.2 (CH, (CH 3 ) 2 CCH = C HCH 2 C H = C H), 127.2 (2CH, CH-ar), 126.5 (2CH, CH-ar), 125.3 (2CH, (CH 3 )C = C HCH 2 , (CH 3 ) 2 CCH = C HCH 2 C H = C H 2 ), 124.6 (2CH, (CH 3 )C = C HCH 2 ), 124.4 (2CH, (CH 3 )C = C HCH 2 ), 84.6 (CH, C5), 81.2 (C, C4), 79.4 (C, C1), 76.6 (CH 2. C20), 75.8 (CH, C10), 75.3 (CH, C2), 74.1 (CH, C2’), 72.3 (CH, C7), 71.9 (CH, C13), 63.7 (CH 2 , CH = CHCH 2 C H 2 O 2 CCH 2 ), 58.7 (C, C8), 52.8 (CH, C3’), 45.7 (CH, C3), 47.4 (CH 2. O 2 C C H 2 C(CH 3 ) 2 CH = ), 43.3 (C, C15), 39.9 (2CH 2 ), 39.8 (CH 2 ), 35.8 (CH 2 , C6 or C14), 35.7 (CH 2 and C, C6 or C14, O 2 CCH 2 C (CH 3 ) 2 CH = ), 34.8 (CH 2 , CH 2 O 2 CCH 2 C H 2 C(CH 3 ), 33.4 (CH 2 , CH 2 O 2 C C H 2 CH 2 C(CH 3 ), 29.0 (CH 3. ( C H 3 ) 2 CCH = CH), 28.8 (CH 3 , ( C H 3 ) 2 CCH = CH), 28.4 (2CH 2 ), 27.1 (CH 2 ), 27.0 (CH 3 , C16), 26.9 (2CH 2 ), 26.8 (2CH 2 ), 25.8 (CH 3 , ( C H 3 ) 2 C = CH), 22.9 (CH 3 , C H 3 CO 2 ), 22.3 (CH 3 , C17), 21.0 (CH 3 , C H 3 CO 2 ), 17.8 (CH 3 , CH 2 CH = C( C H 3 )), 16.2 (3CH 3 , CH 2 CH = C( C H 3 )), 16.1 (CH 3 , CH 2 CH = C( C H 3 )), 15.0 (CH 3 , C18), 9.8 (CH 3 , C19); IR (neat): ν = 2926, 1728, 1662, 1582, 1525, 1487, 1450, 1371, 1239, 1177, 1108, 1069, 1025, 984, 908, 855, 802, 775, 731 cm − 1 . MS ( + ESI) m/z (%) = 1453.1 (100) [M + Na + ]; HRMS (ESI) m/z : [M + Na + ] calcd for C 86 H 111 NO 17 , 1452.7750; found, 1452.7791; HPLC: 94.0% (t R = 18.2 min).

Compound 1k: (86%); [ α ] D 20 = − 35.0 (c = 0.80, in EtOH); 1 H NMR (400 MHz, CDCl 3, δ ): 8.12 (d, J = 7.1 Hz, 2H, Ph C O 2 ), 7.73 (d, J = 7.1 Hz, 2H, Ph C ONH), 7.61 (t, J = 7.1 Hz, 1H, Ph C O 2 ), 7.56-7.48 (m, 3H, C H -ar), 7.45–7.31 (m, 7H, C H -ar), 6.88 (d, J = 9.2 Hz, 1H, NH), 6.30 (s, 1H, H-10), 6.27 (t, J = 8.4 Hz, 1H, H-13), 5.96 (dd, J = 9.2 Hz, J = 3.3 Hz, 1H, H-3′), 5.68 (d, J = 6.9 Hz, 1H, H-2), 5.51 (d, J = 3.3 Hz, 1H, H-2’), 5.40-5.26 (m, 2H, CH 2 C H = C H CH 2 ), 5.20-5.06 (m, 5H,



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(CH 3 )C = C H CH 2 ), 4.97 (d, J = 9.8 Hz, 1H, H-5), 4.45 (m, 1H, H-7), 4.31 (d, J = 8.4 Hz, 1H, H-20), 4.20 (d, J = 8.4 Hz, 1H, H-20), 3.82 (d, J = 6.9 Hz, 1H, H-3), 2.46 (s, 3H, C H 3 CO 2 ), 2.64-2.33 (m, 5H, 1H-14, 1H-6, OH, CH 2. CH 2 CH 2 C H 2 CO 2 ), 2.22 (s, 3H, C H 3 CO 2 ), 2.20-1.94 (m, 28H, CH 2 CH 2 C H 2 C H 2 CH = CHCH 2 , = CHC H 2 C H 2 C(CH 3 ) = CH, 1H-14, 3H-18), 1.88 (m, 1H, H-6), 1.81 (s, 1H, OH), 1.68 (s, 6H, ( C H 3 ) 2 C = CH, H-19), 1.60 (s, 17H, CH 2 CH = C(C H 3 ), CH 2 CH 2 C H 2 CH 2 CO 2 ), 1.29 (m, 2H, CH 2 C H 2 CH 2 CH 2 CO), 1.23 (s, 3H, H-16), 1.13 (s, 3H, H-17); 13 C NMR (100 MHz, CDCl 3, δ ): 203.9 (C, C9), 172.8 (C, CH 2 C O 2 Ptx), 171.4 (C, CH 3 C O 2 ), 169.9 (C, CH 3 C O 2 ), 168.2 (C, C1′), 167.2 (2C, Ph C ONH, Ph C O 2 ), 143.0 (C, C12), 137.2 (C, C-ar), 135.2 (C, CH 2 (CH 3 ) C = CH), 135.1 (C, CH 2 (CH 3 ) C = CH), 135.0 (C, CH 2 (CH 3 ) C = CH), 134.6 (C, CH 2 (CH 3 ) C = CH), 133.8 (C, C-ar), 133.7 (CH, CH-ar), 132.9 (C, C11), 132.1 (CH, CH-ar), 131.4 (C, (CH 3 ) 2 C = CHCH 2 ), 130.4 (2CH, CH-ar), 129.9 (CH, CH 2 C H = C HCH 2 ), 129.5 (CH, CH 2 C H = C HCH 2 ), 129.3 (C, C-ar), 129.2 (2CH, CH-ar), 128.9 (4CH, CH-ar), 128.6 (CH, CH-ar), 127.2 (2CH, CH-ar), 126.6 (2CH, CH-ar), 124.7 (CH, (CH 3 )C = C HCH 2 ), 124.5 (2CH, (CH 3 )C = C HCH 2 ), 124.4 (2CH, (CH 3 )C = CH CH 2 ), 84.6 (CH, C5), 81.2 (C, C4), 79.3 (C, C1), 76.6 (CH 2 , C20), 75.7 (CH, C10), 75.3 (CH, C2), 73.9 (CH, C2’), 72.3 (CH, C7), 71.9 (CH, C13), 58.7 (C, C8), 52.9 (CH, C3’), 45.7 (CH, C3), 43.3 (C, C15), 39.9 (3CH 2 ), 39.7 (CH 2 ), 35.7 (CH 2 , C6 or C14), 35.6 (CH 2 , C6 or C14), 33.9 (CH 2 , CH 2 C H 2 CO 2 ), 29.4 (CH 2 ), 28.7 (CH 2 , CH 2 C H 2 CH 2 CH 2 CO 2 ), 28.4 (2CH 2 ), 27.1 (CH 2 , CH 2 C H 2 CH 2 CH = CH), 27.0 (CH 3 , C16), 26.9 (CH 2 ), 26.8 (2CH 2 ), 26.0 (CH 2 , CH 2 CH 2 C H 2 CH = CH), 25.8 (CH 3 , ( C H 3 ) 2 C = CH), 24.8 (CH 2 , CH 2 C H 2 CH 2 CO 2 Ptx), 22.8 (CH 3 , C H 3 CO 2 ), 22.3 (CH 3 , C17), 20.9 (CH 3 , C H 3 CO 2 ), 17.8 (CH 3 , CH 2 CH = C( C H 3 )), 16.2 (2CH 3 , CH 2 CH = C( C H 3 )), 16.1 (2CH 3 , CH 2 CH = C( C H 3 )), 14.9 (CH 3 , C18), 9.7 (CH 3 , C19); IR (neat): ν = 2930, 1738, 1638, 1529, 1488, 1450, 1371, 1239, 1178, 1069, 1025, 983, 907, 855, 801, 777, 733, 708 cm − 1 ; MS ( + ESI) m/z (%): 1355.5 (100) [M + Na + ]; HRMS ( + ESI) m/z : [M + Na + ] calcd for C 81 H 105 NO 15 : 1354.7382, found, 1354.7419. Anal. calcd C 81 H 105 NO 15 .1/2(H 2 O): C 72.33, H 8.10, N 1.05, found: C 72.51, H 7.89, N 1.04.

Preparation of squalenoyl-paclitaxel conjugates nanoassemblies (1c, 1f, 1g, 1h, 1i, 1j, 1k) : A solution of the above mentioned corresponding drugs (1 mg) in 0.5 mL of ethanol was added dropwise, under stirring (500 rpm) into 1 mL of 5% aqueous dextrose solution. Precipitation of the nanoassemblies occurred spontaneously. Stirring was continued for 2 or 3 min. The nanoassemblies suspension was then transferred into a weighted round bottom fl ask and ethanol was evaporated at ambient temperature using a Rotavapor setting the vacuum to about 50–100 mbar for about 10 min. Then, the fl ask was dipped into water bath (water temperature 37 ° C) for about 3–5 min. Evaporation was continued till the weight of the contents decreased to 0.8–0.9 g. Then, the volume of the suspension in the fl ask was made-up to 1.0 g using either 5% dextrose solution or sterile water. The mean diameter of the nanoassemblies was determined at 20 ° C by quasi-elastic light scattering at a selected angle of 90 ° . A Malvern Zetasizer Nano ZS instrument (Malvern Instruments SA, Orsay, France) with back scattering detector was used for measuring the hydrodynamic size (diameter) in batch mode at 25 ° C in a low volume quartz cuvette (path length 10 mm). Samples were prepared by diluting stock solutions 10-fold in water. Hydrodynamic size is reported as the number-weighted average over all size populations (Z-avg). Zeta potential was determined in 1 mM aqueous NaCl at pH 6.5. Measurements were performed in triplicate on at least three independent experiments.

Transmission electron microscopy (TEM). experiments were performed on nanoassemblies samples of 1f and 1g using a Jeol 2000EX (staining with phosphotungstic acid 1% water, Ni carbon-coated grids), at the “Centre Commun de Microscopie Electronique” (UMR 8080 CNRS, Orsay).

Cryogenic transmission electron microscopy : The cryo-TEM investigations were performed with a Cryo-TEM (JEOL 2100) electron microscope, at the “Service de Microscopie Electronique de l’Institut de Biologie Intégrative” (IFR 83 CNRS, Paris). A 4 μ L drop of the suspension of 1f or 1g nanoassemblies (2mg/mL) was deposited on a carbon coated copper grid. Excess liquid was removed with a blotting fi lter paper and the sample was quickly vitrifi ed by plunging it into liquid ethane using a guillotine-like frame. The sample was then transferred to a cryo-sample

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holder. Observations were made at an accelerating voltage of 200 kV under low electron dose. Analysis was made using the ImageJ software.

Transmission electron microscopy (TEM) after freeze-fracture. The procedure consisted of three steps: (a) high-pressure freezing (HPF) including sandwich preparation and freezing of the specimen; (b) freeze-fracture (FF) which included fracturing, replication and cleaning of the replicas, and (c) transmission electron microscopic investigation of the replicas. For the preparation of the sandwich, a small amount of the sample was placed on a 100 μ m deep symmetrical cup, made of copper and able to conduct heat rapidly away from the specimen. Enclosing the nanoassemblies into a sandwich made of two cups permitted the bulk properties to be preserved as long as possible. A high pressure cooling device HPM 010 (Bal-Tec) was used. The sample was subjected to a pressure of about 2100 bars by the injection of a little warm alcohol as a pressurizing medium, a few milliseconds before the sample was cooled by two jets of liquid nitrogen. After cryo-fi xation, it was essential to keep the sample below the devitrifi cation temperature in liquid nitrogen in order to avoid any modifi cation. For fracturing, the sandwiches were mounted on a cold table, which was inserted inside the vacuum chamber of a Bal-Tec Model BAF 400T apparatus on a nitrogen-cooled support kept at 103 K. Once the vacuum was below 10-7 Torr, fracturing was achieved by displacing the single-edge scalpel blade precooled to 83 K. The replication of the surface involved two steps. First, a thin layer (2 nm) of platinum was evaporated onto the specimen at a shadowing angle of 45 ° to provide contrast enhancement of the topographic features of the fracture surface. The second step consisted in deposition of a thicker (20 nm) layer of carbon, which did not add much electron opacity, but did strengthen to the shadow cast, particularly in regions where only one layer of metal had been deposited. All fi lm thickness determinations were monitored by a quartz crystal thickness gauge (4.96 MHz, quartz crystal holder QSK 301 and monitor QSG 060, Bal-Tec). After complete deposition, the vacuum chamber was vented and specimens removed. The replicas of the surfaces were then fl oated off the specimen by submerging in acetone and collected onto naked 400 mesh grids, which were subsequently mounted in a transmission electron microscope (TEM) for observation. TEM measurements were performed on a LEO 912 Omega high-resolution microscope operated at 120 kV.

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 using a position- sensitive linear gaz detector fi lled with an argon-ethane mixture. The scattered intensity was reported as a function of the scattering vector q = 4 π sin θ / λ , where 2 θ is the scattering angle and λ the wavelength. The calibration of the q-range was carried out using pure tristearine (2L β form) and silver behenate. Sample-to-detector distance and beam energy were chosen to cover the required q-range.

In vitro anticancer activity : A549 (lung carcinoma cell line) cells were cultured in F-12K medium supplemented with 10% foetal calf serum, 50 U/mL penicillin and 50 μ g/mL streptomycin. Hep G2 (hepatocellular liver carcinoma cell line), HT-29 (colorectal adenocarcinoma cell line), MCF-7 (breast adenocarcinoma cell line), KB 3.1 (nasopharyngeal epidermoid cell line) and Mia PaCa-2 (pancreatic carcinoma cell line) cells were cultured in a mixture of DMEM/Ham’s F12 (1:1) media supplemented with 10% fetal calf serum, 50 U/mL penicillin, 50 μ g/mL streptomycin and L-glutamine (2 mM). The cytotoxic activity op paclitaxel ( 1a ) and squalenoyl-paclitaxel conjugates 1b , 1f , 1g , 1h , 1i , 1j and 1k towards these cell lines was determined using the 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide (MTT) test, measuring mitochondrial dehydrogenase activity. The cells in exponential growth phase were seeded into 96-well plates and were pre-incubated for 24 h at 37 ° C in a humidifi ed atmosphere of 5% CO 2 in air. Different dilutions of 1a , 1b , 1f , 1g , 1h , 1i , 1j and 1k were added to the cells in the culture medium. Each dilution was tested in triplicate. After 72 h at 37 ° C, culture medium was removed and then 100 μ L of MTT solution in cell culture medium (0.5 mg/mL) were added to each well. After incubation for 2.5 h at 37 ° C, 100 μ L of SDS solution (10% in water, 10 mM HCl) were added and the plates were incubated for 24 hours at 37 ° C. The absorbance

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of converted dye, which is proportional to the number of viable cells, was measured at 570 nm using a microplate reader (Metertech Σ 960, Fisher Bioblock, Illkirch, France). The percentage of surviving cells was calculated as the absorbance ratio of treated to untreated cells. The inhibitory concentration 50% (IC50) of the treatments was determined from the dose-response curve. To avoid the differences in the data due to difference in cell passage number and other conditions of each individual experiment done at different times, the data plotted in the in vitro graphs are an average of three different experiments performed simultaneously on the same day using different well plates.

In Vivo Tumor Study. Six-eight-week old female athymic nude mice (weighing 18–20g) were purchased from Harlan Laboratory. All animals were housed in appropriate animal care facilities during the experimental period, and were handled according to the principles of laboratory animal care and legislation in force in France.

The systemic toxicity of squalenoyl-paclitaxel nanoassemblies was fi rstly investigated, comparatively to PTX, by determining the maximum tolerated dose (MTD) after repeated intravenous injection into female nude mice. Practically, all drugs were administrated on fi ve consecutive days by intravenous injection in the lateral tail vein (10 μ L/g of body weight). All groups (n = 5) received PTX-Cremophor EL or squalenoyl-paclitaxel nanoassemblies 1g or 1f at a PTX equivalent dose of 10, 20, and 32 mg/kg. Three groups of nude mice (n = 5) received PTX-Cremophor at a PTX equivalent dose of 10, 20 and 30 mg/kg and two groups of nude mice received squalenoyl-paclitaxel nanoassemblies 1g or 1f at a PTX equivalent dose of 32 mg/kg. The given dose of squalenoyl-paclitaxel nanoassemblies was limited by maximum possible concentration of nanoparticles suspension (5 mg mL − 1 ) and injection volume (10 μ L/g of body weight). The body weight change and physical states of mice were monitored for a period of 20 days. The MTD was estimated based on the threshold at which all animals survived with a body weight loss less than 10%. Any toxicity of squalenoyl-paclitaxel nanoassemblies 1g or 1f was observed at dose levels tested. Whatever the dosing protocol, SQ-PTX nanoassemblies appeared dramatically (more than three times) less toxic than PTX-Cremophor (Table 4 ).

The antitumor effi cacy of squalenoyl-paclitaxel nanoassemblies was further evaluated, comparatively to free PTX, on A549-bearing mice. 200 μ L of the A549 cell suspension, equivalent to 1 × 10 6 , were injected subcutaneously into nude mice toward the upper portion of the right fl ank, to develop a solid tumor model. Tumors were allowed to grow until a volume ∼ 90 mm 3 before initiating the treatment. Tumor length and width were measured with calipers, and the tumor volume was calculated using the following equation: Tumor volume (V) = length × width 2 /2. Tumor-bearing nude mice were randomly divided into 4 groups of 8 each and injected for fi ve consecutive days with either (i) saline 0.9%, (ii) SqCO 2 H NA 100 mg/kg, (iii) PTX-Cremophor 10 mg/kg (MTD), (iv) squalenoyl-paclitaxel nanoassemblies 1f at a PTX equivalent dose of 32 mg/kg or (v) squalenoyl-paclitaxel nanoassemblies 1g at a PTX equivalent dose of 32 mg/kg by intravenous injection in the lateral tail vein. The injected volume was 10 μ L/g of body weight. The mice were monitored regularly for changes in tumor size and weight.

Immunohistochemical analysis of xenografts. Tumors were excised at day 8, fi xed in Finefi x (Milestone, Italy), paraffi n-embedded, and cut into 4- μ m-thick sections. Hematoxylin-eosin-safranin (HES) staining was performed on all of the xenografts for analysis of morphology. Apoptosis study was performed by caspase-3 staining and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labelling) System (Roche Diagnostics GmbH, Mannheim, Germany). Proliferating cells were visualised by staining with Ki-67 antibody. Ki-67, TUNEL and caspase-3 positive cells were analysed by microscopy (Leica) and counted per view at 40-times magnifi cation. Five representative fi elds were chosen for counting. Necrotic fi elds were excluded.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author. This includes full experimental details for the synthesis

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of all new compounds, spectral data and characterization, size evolution of nanoassemblies made from prodrug 1g, SAXS pattern of the DHA-SQ Paclitaxel prodrug 1g nanoassemblies, image after freeze-fracture of conjugate 1g nanoassemblies, and DLS profi les of nanoassemblies 1f-1k .

Acknowledgements The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Program FP7/2007-2013 Grant Agreement no.249835. The fi nancial support of the Ministère de la Recherche et de la Technologie (fellowship to J.C.) and from CNRS (Grant Ingénieur de valorisation to S.W.) are acknowledged. We thank Olivia BAWA for technical assistance and Service commun d’expérimentation animale from the IFR141 (Châtenay–Malabry). This work has benefi ted from the facilities and expertise of the IMAGIF Cell Biology Unit of the Gif campus which is supported by the Conseil Général de l’Essonne. The authors thank Ghislaine Frébourg and Dr. Jean-Pierre Lechaire from the Service de Microscopie Electronique of IFR de Biologie Intégrative (IFR 83) for the Cryo-TEM experiments. Ludivine Houel-Renault is acknowledged for recording transmission electron micrographs.

Received: March 30, 2012 Revised: July 24, 2012

Published online: December 4, 2012

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