Synthesis of a novel tri-antennary galactoside

with high hepatocyte targeting

Lei Tang, Yong Wu, Jiao Lu, Zhi Rong Zhang, Jin Cheng Yang, Li Hai *

Key Laboratory of Drug Targeting Education Ministry, West China School of Pharmacy,

Sichuan University, Chengdu 610041, PR China

Received 4 December 2006

Abstract

A novel bifunctional compound carrying cluster thiogalactoside as the cell targeting ligands was synthesized for gene delivery to

hepatocytes. Tetra-antennary dendr-OMs4 5 was used as a scaffold for the attachment of three galactosides, while the other mesylate

end was linked with cholesterol through poly(ethylene glycol) chain. This design provided an effective entry for the synthesis of the

bifunctional compound.

# 2007 Li Hai. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Cluster; Cholesterylated galactoside; Targeting ligand; Hepatocyte-specific delivery; Gene delivery

Receptor-mediated drug delivery is a promising approach to site-selective drug delivery. Mammalian parenchymal

liver cells express a unique receptor protein, the galactose-recoganized asialoglycoprotein receptor (ASGPr) [1]. In

view of its exclusive and abundant presence on parenchymal cells, and of its high affinity and a rapid internalization

rate, the ASGPr was considered as one of the most promising candidate targets in many drug carriers [2]. To date,

various synthetic galactosylated derivatives have been developed as liposome ligands for ASGPr [3]. Compared with

polymer–drug conjugates, liposomes can offer various advantages, such as encapsulation of the drugs without any

chemical modification, prevention of early degradation of drugs and the absence of covalent linkages with polymer

facilitating the intracellular release of the drugs from the liposomes [4].

Recently, we have reported a series of mono-antennary galactosides M1–M6 (Fig. 1) coupled with cholesterol by

ether linkage [5]. Liposomes–DNA complexes containing these galactosylated derivatives M1–M4 exhibited higher

transfection activity than non-galactosylated liposome in hepatoma cells HepG2 and SMMC-7721 [6]. Structure–

activity studies of galactosides with the ASGPr have showed that the receptor–ligand interaction exist significant

‘‘cluster effect’’ in which a multivalent interaction results in extremely strong binding of ligand to the receptor [7].

Taking these factors into considerations, therefore we focused on the potential ligands with higher affinity than

mono-antennary galactosides, but with the compatible distance between the galactosyl moieties and lipophilic

moieties, and synthesized a novel tri-antennary thiogalactoside L (Scheme 1) with tri-antennary galactose residues,

spacer length coupled by ether linkage containing several ethylene glycol units. The derivative possesses bi-functional

properties, i.e. lipophilic cholesterol, one of the lipid components used to form liposomes, as the lipophilic anchor

www.elsevier.com/locate/cclet

Chinese Chemical Letters 18 (2007) 513–515

* Corresponding author.

E-mail address: smile.hl@163.com (L. Hai).

1001-8417/$ – see front matter # 2007 Li Hai. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2007.03.018

moiety for stably introducing the galactosyl moiety onto liposomes surface [8], and three hydrophilic galactose

residues outside the liposomes for recognizing the ASGPr on hepatocytes.

Commercially available diethylene glycol was chosen as a hydrophobic linker, its two hydroxyl functionalities

could bridge cholesterol, making stable incorporation into liposomes and tetra-antennary core. Treatment of the

known mesylate 1 [9] with the reported alcohol 2 [10] afforded the corresponding conjugate 3, remaining three mesyl

groups were substituted by NaI in refluxing butanone, then directly converted into iodide 4. The compound 4 was used

as a potential scaffold to conjugate with the known 2,3,4,6-tetra-O-acetyl-1-thio-b-D-galactopyranose [11] activated

with diisopropylethylamine (DIPEA) in butanone to furnish the clustered trisaccharide derivative 5 [12].

Deacetylation of compound 5 was achieved under mild condition to afford the desired products L [13].

Preliminary application of these compounds as ligands of cation liposomes for delivering reporter gene

b-galactosidase plasmid into hepatoma cells HepG2 in vitro showed that liposomes–polycation–DNA (LPD)

complex, containing galactoside L exhibited higher transfection activities compared with non-galactosylated LPD

complex and LPD complex containing galactoside M1–M6.

In summary, we described an efficient route for the synthesis of cluster galactosides derivative L which was applied

to develop targeting hepatocyte liposomal carrier. In hepatoma cells HepG2, the liposome exhibited higher

transfection activities. The results will be reported elsewhere.

L. Tang et al. / Chinese Chemical Letters 18 (2007) 513–515514

Fig. 1. The structure of mono-antennary galactosides.

Scheme 1. The synthetic route of tri-antennary thiogalactoside L. Reagents and conditions: (a) NaH, THF, DMSO, 60 8C, 50%; (b) NaI, butanone,

reflux, 90%; (c) 2,3,4,6-tetra-O-acetyl-1-thio-b-D-galactopyranose, DIPEA, butanone, r.t., 85%; (d) 0.1 mol/L NaOMe, MeOH, r.t., 84%.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 30672537) and Ministry of

Education of P.R. China (No. 20050610085).

References

[1] G. Ashwell, J. Harford, Annu. Rev. Biochem. 51 (1982) 531.

[2] L.A. Sliedregt, P.C. Rensen, E.T. Rump, et al. J. Med. Chem. 42 (4) (1999) 609 (and references therein).

[3] (a) M. Hashida, M. Nishikawa, F. Yamashita, et al. Adv. Drug Deliv. Res. 52 (3) (2001) 187;

(b) S.N. Wang, Y.H. Deng, H.B. Wu, et al. Eur. J. Pharm. Biopharm. 62 (1) (2006) 32.

[4] M. Hashida, K. Akamatsu, M. Nishikava, et al. J. Control. Rel. 62 (1–2) (1999) 253.

[5] L. Hai, X. Sun, Z.R. Zhang, Y. Wu, Chin. Chem. Lett. 16 (8) (2005) 1021.

[6] X. Sun, L. Hai, Y. Wu, et al. J. Drug Targeting 13 (2) (2005) 1021.

[7] E.A. Biessen, D.M. Beuting, H.C. Roelen, et al. J. Med. Chem. 38 (9) (1995) 1538.

[8] S. Kawakami, J. Wong, A. Sato, et al. Biochim. Biophys. Acta 1524 (2–3) (2000) 258.

[9] (a) J. Hukkamaki, P.T. Pakkanen, J. Mol. Catal. A: Chem. 174 (1/2) (2001) 205;

(b) Y. Wu, T. Ji, H. Zheng, J. West China Univ. Med. Sci. 30 (1) (1999) 37.

[10] L. Hai, J. Fan, Z.Y. Zhang, G.Y. Zhang, Y. Wu, Synth. Commun. 36 (18) (2006) 2633.

[11] H.C. Roelen, M.K. Bijsterbosch, H.F. Bakkeren, et al. J. Med. Chem. 34 (3) (1991) 1036.

[12] Selected data for compound 9: IR (KBr): 2932, 2868, 1751, 1225, 1086, 1056 cm�1; 1H NMR (400 MHz, CDCl3, d ppm): 5.43 (d, 3H,

J = 3.2 Hz, gal. H-40), 5.34 (d, 1H, J = 4.4 Hz, chol H-6), 5.22 (t, 3H, J = 10 Hz, gal. H-20), 5.05 (dd, 3H, J = 3.2, 10 Hz, gal. H-30), 4.51 (d, 3H,

J = 10 Hz, gal. H10), 4.13 (m, 6H, gal. H60, H60 0), 3.94 (t, 3H, J = 6.8 Hz, gal. H50), 3.66–3.43 (m, 18H, 4 � OCH2, 2 � OCH2CH2O, CCCH2O),

3.34 (s, 6H, 3 � CCH2O), 3.33 (s, 2H, CCH2O), 3.18 (m, 1H, chol H-3), 2.79–2.69 (m, 2H, SCH-a, SCH-b), 2.15, 2.07, 2.05 and 1.98 (4 � s,

48H, 16 � CH3 acetyl), 2.39–0.68 (remaining chol protons and 4 � CCH2C) with 0.99 (s, 3H, CH3-19), 0.91 (d, 3H, J = 6.8 Hz, CH3-21), 0.86

(d, 6H, J = 6.8 Hz, CH3-26 and CH3-27), 0.68 (s, 3H, CH3-18); MS (m/z): 1885.8 ([M+Na]+); anal. calcd. for C90H142O34S3: C, 57.98; H, 7.68;

S, 5.16. Found: C, 57.73; H, 7.44; S, 5.26.

[13] Selected data for compound L: IR (n, KBr): 3385, 2933, 2867. 1374, 1088 cm�1; 1H NMR (400 MHz, CD3OD, d ppm): 5.36 (m, 1H, chol H-6),

4.31 (d, 3H, J = 9.6 Hz, 3 � gal. H1), 3.88 (d, 3H, J = 3.2 Hz, 3 � gal. H4), 3.76–3.45 (m, 33H, 3 � gal. H2, H3, H5, H6, H60 protons and

9 � CH2O), 3.37 (s, 8H, 4 � CCH2O), 3.19 (m, 1H, chol H-3), 2.84 (m, 1H, SCH-a), 2.75 (m, 1H, SCH-b), 2.35–0.72 (remaining chol protons

and 4 � CCH2C) with 1.02 (s, 3H, CH3-19), 0.91 (d, 3H, J = 6.4 Hz, CH3-21), 0.86 (d, 6H, J = 6.4 Hz, CH3-26 and CH3-27), 0.72 (s, 3H, CH3-

18), 13C NMR (100 MHz, CD3OD, d ppm): 142.0 (chol C-5), 122.7 (chol C-6), 87.9 (chol C-1), 80.9 (chol C-3), 80.5 (gal C-5), 76.3 (gal C-3),

1.9, 71.6, 71.3, 70.9, 70.6, 69.4, 68.4 (C0s from ethylene glycol, S-CH2-, O-CH2), 71.5 (gal C-2), 70.4 (gal C-4), 62.6 (gal C-6), 58.2 (chol C-

17), 57.6 (chol C-14), 51.7 (chol C-9), 43.5 (chol C-13), 42.1 [C(CH2O)4–], 41.1, 40.7, 40.2 (chol C-4, C-12, C-24), 38.5, 38.0, 37.4 (chol C-1,

C-10, C-12), 37.1 (chol C-20), 33.3 (chol C-8), 33.1 (chol C-7), 31.4 (SCH2), 31.0 (chol C-2, C-16), 29.1 (chol C-25), 28.0 (SCH2CH2CH2O),

27.8 (OCH2CH2CH2O), 25.3 (chol C-15), 25.0 (chol C-23), 23.2, 23.0 (chol C-26, C-27), 22.2 (chol C-11), 19.9 (chol C-19), 19.3 (chol C-21),

12.4 (chol C-18); MS (m/z): 1381.6 ([M+Na]+); Anal. Calcd for C66H118O22S3: C, 58.29; H, 8.75; S, 7.07. Found: C, 58.43; H, 8.92; S, 6.84.

L. Tang et al. / Chinese Chemical Letters 18 (2007) 513–515 515