WO1999010359A2

WO1999010359A2 - Liposomic based sialyl lewis x mimetics

Info

Publication number: WO1999010359A2
Application number: PCT/EP1998/005390
Authority: WO
Inventors: Chi-Huey Wong; Chun-Cheng Lin; Thomas J. Woltering; Gabriele Weitz-Schmidt
Original assignee: Novartis Ag; The Scripps Research Institute
Priority date: 1997-08-26
Filing date: 1998-08-25
Publication date: 1999-03-04
Also published as: WO1999010359A3; AU1144799A

Abstract

Liposomic sLex mimetics of formula MIMETIC-LINKER-LIPID and cross-linked liposomic sLex mimetics of formula (I).

Description

Organic Compounds

The present invention relates to compounds that inhibit cellular adhesion. More particularly, the present invention relates to liposomic based sialyl Lewis X mimetics which mimic the inhibition of selectin-mediated cellular adhesion by sialyl Lewis X. These compounds may be incorporated into lipid vesicles and display E-selectin binding activity.

The early stage of neutrophil migration from the blood stream to the site of inflammation or tissue injury occurs with the rolling adhesion of neutrophils to vascular endotheiium. The adhesion process involves the interaction of sialyl Lewis X (sLe^x), a terminal tetrasaccharide of cell-surface glycoproteins and glycolipids, and the endothelial leukocyte adhesion molecule 1 (E-selectin). Inhibition of the sLe^x/E-selectin interaction has been shown to be effective in animal models of certain inflammatory diseases such as reperfusion injury. Although the synthesis of sLe^x on a large scale has been developed for clinical evaluation, this natural saccharide is relatively weak regarding its activity (IC₅₀ ~ 0.5 mM) against E-selectin and can only be used in its injectable form for acute symptoms as it is orally inactive and unstable in the blood stream. Development of sLe^x mimetics with higher affinity for the receptor and better activity and stability against glycosidase has been of current interest. sLe^x is a cell surface carbohydrate ligand found on neutrophils, anchored onto the outer membrane therof by integral membrane glycoproteins and/or glycolipids. sLe^x mediates binding of neutrophils to vascular endothelial cells by binding to E-selectin.

E-selectin is a cell surface protein inducibly expressed in endothelial cells in response to inflammatory factors such as interleukin Iβ and tumor necrosis factor α, leukotriene B₄, neuro- toxins and bacterial endotoxins, e.g., lipopolysaccharides. These compounds augment poly- morphonuclear leukocyte (neutrophil), and monocyte adhesion. Binding of neutrophils to endothelial cells is observed at an early stage after tissue injury and is associated with various acute and chronic inflammation. Neutrophil-mediated inflammatory diseases may be treated by administration of sLe^x. Administration of sLe inhibits the sLe^x/E-selectin interaction and blocks adhesion of neutrophils to endothelial cells.

In addition to binding to neutrophils, vascular endothelial cells play key roles in a number of biological responses by selectively binding certain cells, for instance phagocytic leukocytes, in the bloodstream. For example, endothelial cells preferentially bind monocytes and granulocytes prior to their migration through the blood vessel wall and into surrounding tissue in an inflammatory response.

Certain inflammation-triggering compounds are known to act directly on the vascular endo- thelium to promote the adhesion of leukocytes to vessel walls. Cells then move through the walls and into areas of injury or infection.

Cellular adhesion to vascular endothelium is also thought to be involved in tumor metastasis. Circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.

Blood platelets are also involved in similar responses. Platelets are known to become activated during the initiation of hemostasis and undergo major morphological, biochemical, and functional changes (e.g., rapid granule exocytosis, or degranulation), in which the platelet alpha granule membrane fuses with the external plasma membrane. As a result, new cell surface proteins become expressed that confer on the activated platelet new functions, such as the ability to bind both other activated platelets and other cells. Activated platelets are recruited into growing thrombi, or are cleared rapidly from the blood circulation. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes that are thought to be mediated by this process include atherosclerosis, blood clotting and inflammation.

Specialized cell surface receptors on endothelial cells and platelets, designated E-selectin (endothelial leukocyte adhesion molecule-1 ; ELAM-1 ) and P-selectin (granule membrane protein-140; GMP-140, also known as PADGEM), respectively, are involved in the recognition of various circulating cells by the endothelium and platelets. For example, E-selectin has been shown to mediate endothelial leukocyte adhesion, which is the first step in many inflammatory responses. Specifically, E-selectin binds human neutrophils, monocytes, eosinophils, certain T-lymphocytes, NK cells, and the promyelocytic cell line HL-60.

P-selectin is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions. Thus, for example, activated platelets that express P-selectin on their surface are known to bind to monocytes and neutrophils, and also to bind monocyte-like cell lines, e.g., HL-60 and U937.

P-selectin is an alpha granule membrane protein of molecular mass 140,000 that is expressed on the surface of activated platelets upon platelet stimulation and granule secretion. It is also found in megakaryocytes within the Weibel-Palade bodies.

A third receptor is the lymphocyte homing receptor, MEL-14 antigen or its human counterpart LAM-1 (L-selectin). In addition to lymphocyte homing, MEL-14 antigen/LAM-1 is believed to function early in neutrophil binding to the endothelium.

The term "selectin" has been suggested for a general class of receptors, which includes E- selectin (ELAM-1 ), P-selectin (GMP-140) and L-selectin (MEL-14), because of their lectin- like domain and the selective nature of their adhesive functions. The structure and function of selectin receptors has been elucidated by cloning and expression of full length cDNA encoding each of the above receptors.

The extracellular portion of selectins can be divided into three segments based on homolo- gies to previously described proteins. The N-terminal region (about 120 amino acids) is related to the C-type mammalian lectin protein family that induces low affinity IgE receptor CD23. A polypeptide segment follows, which has a sequence that is related to proteins containing the epidermal growth factor (EGF) motif. Lastly, after the EGF domain are one or more tandem repetitive motifs of about 60 amino acids each, related to those found in a family of complement regulatory proteins.

WO 91/19501 and WO 91/19502 disclose that oligosaccharides containing the pentameric and hexameric structures shown below inhibited selective cellular binding between cells containing the ligand (below) and those containing a selectin receptor, and that the penta- and hexasaccharides assayed provided better inhibition than did sLe^x. NeuAcα2→3Galβ1 →4(Fucα1 →3)GlcNAcβ1 →3Galβ-; NeuAcα2→3Galβ1 →4(Fucα1→3)GlcNAcβ1→3Galβl→4Glc-; and NeuAcα2→3Galβ1 →4(Fucα1→3)GlcNAc = sLe^x. Mulligan et al. [Nature 364:149-151 (1993)] reported upon the in vivo effects of sLe^x and a pentamer such as that above present as a -O(CH₂)₅CO₂CH3 glycoside in a neutrophil/P-se- lectin-dependent rat model. Intravenous infusion of up to 200 μg of sLe^x or the pentamer dramatically reduced lung injury and diminished tissue accumulation of neutrophils in rats that received an intravenous infusion of cobra venom. Based on the concentrations used, 200 μg, the effective intravenous concentration of sLe^x was calculated to be less than 1 μM.

DeFrees et al. [J.Am.Chem.Soc. 117:66-79 (1995)] reported on the in vitro inhibition of binding between E-selectin and sLe^x-bearing HL-60 cells for a number of sLe^x-related materials including sLe^x itself, an ethyl glycoside of the above pentamer and a number of bivalent sLe^x analogs. Those authors noted that although the affinity of sLe^x for E-selectin is relatively weak in vitro, the IC₅₀ value in vivo for protecting against lung injury in rats was in the 1 μM range.

The search for novel sLe^x mimetics with simpler structure, higher affinity for the receptor, and better stability against glycosidases, especially fucosidase and sialidase, has been of current interest. A sLe^x mimetic is a compound which includes the functional groups of sLe^x and which mimics the active conformation of sLe" in space, but which lacks one or more of the glycosidic bonds of sLe^x and/or one or more of the saccharide subunits or analogs thereof. Of the several active sLe mimetics and sLe^x analogs that have been designed and synthesized two sLe mimetics synthesized by Uchiyama et al. [J.Am.Chem.Soc. 117:5395 (1995)] are of particular note because they exhibit activities similar to sLe in the E-selectin binding assay.

The key structural features of sLe^x required for recognition by E-selectin have been determined by structural and conformational studies and by comparative studies of the blocking activity of sLe^x analog families. It appears that the six functional groups of the sLe^x molecule including the 2-, 3- and 4-OH groups of L-fucose, the 4- and 6-OH groups of Gal and the - CO₂ ^" group of sialic acid are essential for E-selectin recognition.

Although sLe^x and active sLe^x analogs can be employed as anti-inflammatory agents, these tetrasaccharides can only be used in acute symptoms as they are unstable in the blood and orally inactive. In addition, it is generally difficult to synthesize oligosaccharides on a large-

SUBST1TUTE SHEET (RULE 26) scale. The use of sLe^x mimetics can obviate the above problems associated with sLe^x analogs. Unfortunately, sLe^x mimetics generally have low activity.

Recent studies indicate that sLe^x is active in vivo as an anti-inflammatory agent due to its inhibitory activity against E- and P-selectin of endothelial cells, which interact with sLe^x-ex- pressing neutrophils and leukocytes in the rolling adhesion step of inflammatory reactions. Several drawbacks are encountered, however, when considering sLe^x as a drug candidate: the activity is relatively low (IC₅₀ for E- and P-selectin is 0.5 mmol and > 3 m respectively) the rotational barrier is relatively high (5 kcal/mole) for the free sugar binding to E-selectin; sLe^x is difficult to synthesize on large scales; it is relatively unstable and orally inactive.

What are needed are sLe^x mimetics which are easy to synthesize, more stable and more active than sLe^x, and preferably orally active. Moreover, mimetics are needed for stability as compared to sLe^x and sLe^x analogs; which are easier to synthesize than sLe" and sLe^x analogs; and which exhibit greater activity as compared to known sLe^x mimetics.

One aspect of the invention is directed to a liposomic sLe^x mimetic of formula I

MIMETIC-LINKER-LIPID (I) wherein MIMETIC is a sLe mimetic; LIPID is a lipid moiety; and LINKER is a conjugating element which links MIMETIC to LINKER with the proviso that when MIMETIC is a group of formula II

LIPID is not a group of formula III

Preferably MIMETIC is a radical derived from a compound of formulae lla, lib or lie

wherein

X is a direct bond, -NR³- or -O-;

Y is a direct bond, -O- or CrC₃alkylene;

R¹ is hydrogen, methyl, hydroxyl, -O(CH₂)_nCH₂-Ar; -O(CH₂)_nCHCH-Ar;

-O(CH2)nCONHCH(CH₂OCO(CH₂)mCH₃)2 or -O(CH₂)nCONHC(CH2)_mCH₃; R² is hydrogen; -CONH₂; -COOH; -CONH(CH₂)_nCONH(CH₂)_mCH₃; -CONH(CH₂)mCH₃;

-COOEt or -CONH(CH₂CH₂O)pCH₂NH2; R³ is hydrogen, -(CH₂)_nCOOH or -(CH₂)nCONH(CH2)mCH₃; R⁴ is HO₂C(CH₂)_nCONHCH(CH(CH₃)(OH))-; HO₂C(CH₂)_nCONHCH(CH(OH)(CH₂OH))-;

Hθ2C(CH2)nCONHC(CH₂θH)₂-; HO₂CCH( H2)CH2CONHCH(CH2Ph(OH))-; -CH2Pθ3^2';

-OPO₃ ^2" or a radical of formula lla' or lib'

R⁵ is -(CH₂)n[CH(OH)]₂COCH₂θPθ3Na₂; -(CH^_n.CHfOHVjzCOCHaCHzOPOgNaz;

-[CH(OH)]₂CONHCH₂COOH; -CH₂CONHPh-COOH; -[CH(OH)]₂CO(CH₂)nOPO3Na₂;

-CONHCH(COOH)(CH₂)_nCOOH; -NHCO(CH₂)_nCO₂Bn; -NHCO(CH₂)_nCOOH;

-NHCOC[NHCO(CH₂)_nCOOH]CH(OH)(CH₂)_mCH₂OH; -CONHPh-(COOH); hydrogen;

-CH₂CONH(CH₂)i3CH₃; -NHCOC(CH₂OH)₂NHCO(CH₂)_nCOOH; -(CH₂),C(O)-R_x or

-[CH(OH)]₂CONHCHR_yCOOH; R⁶ is -NH₂, -CONH(CH₂CH₂O)pCH₂NH₂; -CONH₂; -COOH, hydrogen, hydroxyl, -O-d-C₆alkyl;

-OBn; -N₃; -OSO₃ ^2'; -OCO[CH₂]₂CONHCH(CH₂CO₂H)COOH or -NHR'; R⁷ is -NH₂₎ -CONH(CH₂CH₂O)pCH₂NH₂; -CONH₂ or -COOH; R⁸ is -O-methyl, -O(CH₂)_nCH₂Ar; -O(CH₂)_nCHCHAr; -O(CH₂)nCONHCH(CH₂OCO(CH₂)mCH3)2 or -O(CH₂)nCONHC(CH2)mCH₃;

R_x is butyl amine, amyl amine, hexyl amine, heptyl amine, octyl amine, nonylamine, decyl- amine, undecylamine, dodecylamine, tridecylamine, 1-tetradecylamine, pentadecyl- amine, hexadecyl amine, octadecyl amine, 1 -amino naphthalene, 2-naphthalene, 2-amino-2-napthol perhydrochloride, 4-pentyl aniline, 4-hexyl aniline, 4-heptyl aniline, 4-octyl aniline, 4-decyl aniline, 4-dodecyl aniline, 4-tetradecyl aniline or 4-hexadecyl- aniline;

R_y is a side chain of an amino acid selected from Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His;

R' is Cι-C₆alkyl, acyl, decanoyi, phenylacetyl, -CO[CH₂]₂COOH, hexanoyi, heptanoyi, octan- oyl, nonanoyl, decanoyi, undecanoyl, laurate, tridecanoyl, myristate, pentadecanoyl, pal- mitate, heptadecanoyl, stearate, nonadecanoyl, eicosanoyl, hexaicosanoyl, docosanoyl, tricosanoyl, tetracosanoyl, hexacosanoyl, heptacosanoyl, octacosanoyl, triacontanoyl, 4-phenylbutyroyl, 5-phenyl valerate, 6-phenyl hexanoyi, oleic acid, 3-trans-7-trans-far- nesoyl, 8-trans-10-trans-dodecandien-1-carboxy, 2-naphthanoyl, 1 -hydroxy-2-naphthan- oyl, 1 ,4-dihydroxy-2-naphthanoyl, -C(O)(CH₂)_uCH₃; -C(O)(CH₂)_wNHCO(CH₂)_qC(O)OH; -C(O)(CH₂)_uC₆H5 θr -C(O)(CH₂)_rCON(CH₂CONH(CH2)CH3)HCONH(CH₂)sCH3;

Ar is phenyl, benzyl, methoxyphenyl, pentafluorophenyl, biphenyl, dibenzocyclopentane or naphthyl; Ph is phenyl; Et is ethyl; Bn is benzyl; and wherein 0 < m < 20; 0 < n < 10; 0 < p < 100; 0 < t < 6; 2 < (w + q) < 22; 1 < u < 22; and 2 < (r + s) < 22.

More preferably, MIMETIC is a group of formula Va, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, Vm, Vn, Vo, Vp, Vq, Vr, Vs, Vt, Vu, Vv, Vw, Vx or Vy

wherein

Bn is benzyl; m_z and n_z individually are 1 or 2;

R is -(CH₂)_nrC₆H₅ or -CH₂CONH(CH₂)_mrCH₃; wherein nr is 2 or 3 and mr is a number of from

1 to 16; and

Ar is hydrogen, phenyl, benzyl, 4-methoxyphenyl, 2,3,4,5,6-pentafluorophenyl, 2-naphthyl,

2-fluorenyl, 4-biphenylyl or dimer.

More preferably, MIMETIC is a group of formula Va, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, Vm, Vn, Vo, Vp, Vq, Vr, Vs, Vt, Vv, Vw, Vx or Vy

Preferably LIPID is a group of formula Ilia or 1Mb

wherein 0 < m_x < 20; 0 < n_x < 10; and R is a radical of formula lllb^a, lllb^b, lllb^c or lllb^d

(lllb^d)

wherein each n_y individually is a number of from 0 to 10.

Preferably LINKER is a group of formula IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh or IVi,

(^IVd)' — NH— PEG-NH- (IVe), — 0— PEG-NH- (IVf),

O

-O-PEG-O- (IVg), →^ ^ (IV ) or __oX^ A₀_ <^IVi>

^ Ό wherein 0 < v < 100.

Another aspect of the invention is directed to a liposomic sLe^x mimetic of formula I wherein MIMETIC is a sLe^x mimetic; LIPID is a lipid moiety; and LINKER is a conjugating element which links MIMETIC to LINKER with the proviso that when MIMETIC is a group derived from a compound of formula II'

wherein Y, R¹, R², R³ and R⁴ are as defined above LIPID is not a group of formula Ilia.

Preferred liposomic sLe^x mimetics are those of formula l^b

wherein MIMETIC is as defined above but not a group of formula II.

The liposomic sLe^x mimetics of formula I may be prepared according to procedures as are e.g. disclosed in WO 98/08854.

The present invention also comprises a process for the preparation of the liposomic sLe^x mimetics of formula I wherein a linker precursor is coupled to the corresponding MIMETIC precursor, followed by coupling to the LIPID precursor. The procedures are generally known to the skilled person or can be deduced from the disclosure of e.g. WO 98/08854.

Another aspect of the invention is directed to a liposomic sLe^x mimetic represented by formula P

(sLe^x)-LINKER-LIPID (P).

Another aspect of the invention is directed to a cross-linked liposomic sLe^x mimetic of formula VI 'LIPID (Bj

^JN

[MIMETIC-LINKER-LIPID (Al (VI)

wherein MIMETIC and LINKER are as defined above and LIPID (A) is a group of formula Ilia or derived therefrom; and

LIPID (B) is a compound selected from compounds of formulae Vila and Vllb or derived therefrom

wherein m_x, n_x and n_y are as defined above; with the proviso that MIMETIC is not a group of formula II.

ba ,bb

Preferred are cross-linked liposomic sLe^x mimetics of formula VI ^a or VI

wherein MIMETIC is as defined above with the proviso that MIMETIC is not a group of formula II.

Another aspect of the invention is directed to a method for preparing a cross-linked liposomic sLe mimetic of formula VI comprising sonicating a lipid conjugate of formula XII

MIMETIC-LINKER-LIPID (A) (XII) wherein MIMETIC and LINKER have the above meanings; and LIPID (A) is a group of formula Ilia, with a compound of formulae Vila or Vllb.

Another aspect of the invention is directed to a method for preparing a cross-linked liposomic sLe^x mimetic of formula VI comprising STEP A sonicating a lipid conjugate of formula XII with a compound of formulae Vila or Vllb; and Step B irradiating the resulting product with UV light. Another aspect of the invention is directed to a method for preparing a cross-linked liposomic sLe mimetic of formula VP

ILIPID (B.

^JN

[ MIMETIC-LINKER-LIPID (Ap (VP)

wherein LINKER, LIPID (A) and LIPID (B) are as defined above; and MIMETIC is a radical derived from a compound of formula lla wherein X, Y, R² and R⁴ are as defined above; and

R¹ is hydrogen, methyl, hydroxyl, -O(CH₂)_nCH₂-Ar; -O(CH₂)_nCHCH-Ar;

-O(CH₂)nCONHCH(CH₂OCO(CH2)mCH3)2, -O(CH₂)nCONHC(CH2)mCH₃; or a radical derived from a compound of formulae Vila or Vllb comprising sonicating a lipid conjugate of formula XIP

MIMETIC-LINKER-LIPID (A) (XIP) wherein MIMETIC, LINKER and LIPID (A) have the above meanings; with a compound of formula Vila or Vllb.

Another aspect of the invention is directed to a method for preparing a cross-linked liposomic sLe^x mimetic of formula VP comprising STEP A sonicating a lipid conjugate of formula XIP with a compound of formula Vila or Vllb; and Step B irradiating the resulting product with UV light.

The procedures are generally known to the skilled person or can be deduced from the Examples, describing further nonlimiting details of the preparation.

The compounds of formulae I, VI and VP exhibit valuable pharmacological properties as indicated in tests and are therefore indicated for therapy. In particular the compounds of formulae I, VI and VP inhibit the binding of E-seiectin to HL-60 cells as disclosed in Example D.

The compounds are particularly indicated for preventing or treating conditions or diseases which are mediated by the binding of selectin in cellular adhesion, e.g. acute or chronic inflammatory or autoimmune diseases such as rheumatoid arthritis, asthma, allergy conditions, psoriasis, contact dermatitis, adult respiratory distress syndrome, inflammatory bowel disease and ophthalmic inflammatory diseases, infection diseases such as septic shock, traumatic shock, thrombosis and inappropriate platelet aggregation conditions, cardiovascular diseases such as heart attacks, reperfusion injury, multiple sclerosis and neoplastic diseases including metastasis conditions, strokes and acute or chronic rejection of organ or tissue transplants.

Acute and chronic rejection play a role in the transplantation of organs or tissues from a donor to a recipient of the same species (allograft) or different species (xenograft). Among such transplanted organs or tissues and given illustratively are heart, lung, combined heart- lung, trachea, liver, kidney, spleen, pancreatic (complete or partial, e.g. Langerhans islets), skin, bowel, or cornea or a combination of any of the foregoing.

For the above uses the required dosage will of course vary depending on the mode of administration, the particular condition to be treated and the effect desired, In general, however, satisfactory results are achieved at dosage rates of from 0.1 to about 100 mg/kg/day, administered in 1 , 2, 3, or 4 doses/day, or in sustained release form. Suitable daily dosages for oral administration to larger mammals, e.g., humans, are generally about 50 to 1500 mg, preferably in the order of from 200 to 800 mg. Unit dosage forms suitably comprise from about 25 mg to 0.750 g of a compound of the invention, together with a pharmaceutical acceptable diluent or carrier therefor.

The compounds of formula I may be administered by any conventional route of administration, e.g. enterally, preferably orally, e.g. in the form of tablets, capsules, solutions or suspensions, or parenterally e.g. in form of injectable solutions or suspensions.

OPO₃H₂, PO₃H₂ and the carboxyl group may be in free acid form or in salt form. Pharmaceutically acceptable salts are to be understood as meaning, in particular, the alkali metal and alkaline earth metal salts, for example sodium, potassium, magnesium and calcium salts. Sodium and potassium ions and their salts are preferred.

In accordance with the foregoing the present invention further provides:

(a) a compound of formula I, VI or VP or a pharmaceutically acceptable salt thereof for use as a pharmaceutical;

(b) a method for preventing or treating conditions or diseases as indicated above in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

(c) a pharmaceutical composition comprising a pharmaceutically effective amount of the compound of formula I, VI or VP or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier;

(d) a compound of formula I, VI or VP or a pharmaceutically acceptable salt thereof for use in the manufacturing of a medicament for use in the method as in (b) above.

The liposomic sLe^x mimetic may be administered alone or in combination with one or more other anti-inflammatory or immunosuppressive agents, for example in combination with cyclosporin A and analogs thereof, FK-506 and analogs thereof, rapamycin and analogs thereof, mycophenolic acid, mycophenolate mofetil, mizoribine, 15-deoxyspergualine, leflunomide, steroids, cyclophosphamide, azathioprene (AZA), or anti-lymphocyte antibodies or immunotoxins such as monoclonal antibodies to leukocyte receptors, e.g. MHC, CD2, CD3, CD4, or CD25; especially in combination with a T-cell suppressant, e.g., cyclosporin A or FK-506. Such combination therapy is further comprised within the scope of the invention, e.g., a method according to 1 above further comprising administration concomitantly or in sequence of a therapeutically or synergistically effective amount of such a second immunosuppressive or anti-inflammatory agent.

The following examples are offered as a way for illustration of this invention and not in a way of limitation.

Examples

Abbreviations used: BnBr: benzyl bromide; CSA: camphorsulfonic acid; DCM: dichloro- methane; DEAD: diethylazodicarboxylate; C-DHAP: 3-keto-4-hydroxy-butanyl-1-phos- phonate; DHAP: dihydroxyacetone phosphate; dppb: 1 ,4-bis(diphenylphosphino)butane; DMAP: 4-dimethylaminopyridine; DMF: dimethylformamide; DMS: dimethylsulfide; EDC: 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI: electrospray ionization; HOBt: 1 -hydroxybenzotriazole; HOSu: N-hydroxy succinimide; NBA: m-nitrobenzylalcohol; NHS: N-hydroxysuccinimide; NMM: 4-methyl morpholine; PPh₃: triphenylphosphine; RT: room temperature; SGCC: silica gel column chromatography; TBAF: tertbutylammonium fluoride; TBAI: tetrabutylammonium iodide; THF: tetrahydrofuran; TMSOTf: trimethylsilyl- triflate.

¹H and ¹³C NMR spectra are recorded either on a Bruker AM-250, a Bruker AMX-400 or a Bruker AMX-500 spectrometer. Residual protic solvent CHCI₃ (δ_H = 7.26 ppm, δ_c = 77.0), d₄-methanol (δ_H = 3.30 ppm, δ_c = 49.0) and D₂0 (δ_H = 4.80 ppm, δ_c (of CH₃CN) = 1.7 ppm) or TMS (δ_H = 0.00 ppm) are used as internal reference. Coupling constants are measured in Hertz (Hz). HRMS are recorded using FAB method in a NBA matrix doped with Nal or Csl. Infra-red spectra are recorded on a Perkin-Eimer FTIR 1620 spectrometer. Enantiomeric excess is determined by HPLC using a Daicel Chemical Industries CHIRALPAK AD column. Optical rotations are measured with an Optical Activity AA-1000 polarimeter. Melting points are taken on a Thomas Hoover capillary melting point apparatus and are uncorrected. Column chromatography is performed on Merck Kieselgel 60 (230-400 mesh). Analytical thin layer chromatography is performed using pre-coated glass-backed plates (Merck Kieselgel F₂₅ ) and visualised by cerium molybdophosphate or ninhydrin. Diethyl ether, THF and toluene are distilled from sodium-benzophenone ketyl, DCM and acetonitrile from calcium hydride. Other solvents and reagents are purified by standard procedures if necessary.

General procedure for coupling of C6-amine to a carboxylic acid.

To a stirred solution of azide (666 mg, 0.8 mmol) and Ph₃P (336 mg, 1.28 mmol) in 20 ml of THF, is added H₂O (23 ml, 1.28 mmol). After stirring overnight, tic analysis indicates the complete consumption of the starting material. To the reaction mixture is added a carboxylic acid (0.96 mmol), HOBt (130 mg, 0.96 mmol) and NMM (106 μl, 0.96 mmol) . After cooling the mixture to 0°C, EDC (184 mg, 0.96 mmol) is added. The cold bath is removed after 10 min and the mixture is vigorously stirred to dissolve the remaining solid material. After stirring overnight, the reaction is quenched with 5% aqueous citric acid and Et₂O is added. The aqueous portion is extracted with Et O three times. The combined organic extracts are washed with saturated aqueous NaHCO₃ solution and brine, dried over MgSO₄ and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using 5% MeOH in CH₂CI₂ as the eluent to provide amide.

General procedure for the preparation of MIMETIC-LINKER-LIPID

Fmo

Lipid

MIMETIC-LINKER-LIPID having amide bond connections

Step A [MIMETIC may or may not be attached to the optional linker, if not, then bypass this step and directly couple to the MIMETIC to the LIPID as described in step B, infra]: To a stirred 0°C solution of the MIMETIC carboxylic acid or LINKER carboxylic acid (1.0 mmol), HOBt (1.2 mmol), the LINKER-amine or MIMETIC amine (2.0 mmol) and NMM (2.5 mmol) in CH₂CI₂ (0.5 M) is added EDC (1.3 mmol). After stirring overnight and subsequent warming to RT, tic analysis indicates complete consumption of the starting acid. Saturated NaHCO₃ solution and CI₂CI₂ are added to the reaction mixture. The aqueous portion is extracted twice with CH₂CI₂. The combined organic fractions are washed with 1 N HCI and brine, dried over Na₂SO and concentrated. The crude residue is purified by flash chromatography in silica gel using ethyl acetate/hexane as the eluent to form MIMETIC-LINKER. Step B: The procedure is generally repeated for the attachment of the LIPID portion to MIMETIC-LINKER as follows: To a stirred 0°C solution of the MIMETIC carboxylic acid or LIPID (A) carboxylic acid (1.0 mmol; MIMETIC may or may not be attached to an optional linker as described supra), HOBt (1.2 mmol) , the LIPID-amine or MIMETIC amine (2.0 mmol) and NMM (2.5 mmol) in CH CI₂ (0.5 M) is added EDC (1.3 mmol). After stirring overnight and subsequent warming to RT, tic analysis indicates complete consumption of the starting acid. Saturated NaHCO₃ solution and CH₂CI₂ are added to the reaction mixture. The aqueous portion is extracted twice with CH₂CI₂. The combined organic fractions are washed with 1 N HCI and brine, dried over Na₂SO₄ and concentrated. The crude residue is purified by flash chromatograohy in silica gel using ethyl acetate/hexane as the eluent to synthesize the MIMETIC-LINKER-LIPID. MIMETIC-LINKER-LIPID having ester bond connections

Follow the General Procedure for MIMETIC-LINKER-LIPID having amide bond connections and replace the use of amines with free alcohol derivatives of the above compounds. Amides and esters may be intermixed, to form ester-amide derivatives, etc.

Example A1 : Synthesis of Lipid-N-hydroxy-succinimide succinate

(a) Synthesis of butanedioic acid bis(N-succinimidyl ester): A mixture of succinimide diacid (5 mmol), NHS (11 mmol), and EDC (11 mmol) are dissolved in dry 1 ,4-dioxane (25 ml), and the reaction mixture is stirred at RT overnight. The solvent is evaporated and the resulting residue is dissolved in EtOAc. The solution is washed with saturated NaHCO₃₍aq), brine, dried over MgSO₄, filtrated, and concentrated. The crude is purified by flash chromatography to give the desired product.

(b) A solution of butanedioic acid bis(N-succinimidyl ester) (0.27 mmol) in dry DMF (1 ml) is added to a solution of phosphotidylethanolamine, (0.13 mmol) in 1 ml dry DMF and dry NEt₃ (40 μl). The reaction mixture is stirred overnight at 0°C. After concentration under reduced pressure, the activated lipid derivative is purified by SGCC. Example A2: Synthesis of CBzHNCH₂(CH₂)_nCh₂NH₂

A solution of 4 mmol diamine in 30 ml ChCI₃ is stirred at RT under argon atmosphere. A solution of 2 mmol CBzCI in 30 ml of CHCI₃ is added dropwise. The solution is stirred for an additional 2 h and then the solvent is evaporated. The residue is diluted with 30 ml EtOAc and washed with NaHCO₃ and brine. The organic layer is dried over MgSO₄, filtrated and concentrated. The residue is purified by SGCC to give mono Cbz protected product.

Example A3: Synthesis of Compound 57

57

Compound 55 (1 g, 2.7 mmole; Avanti Polar Lipids, Inc.), N-hydroxy succinimide 56 (0.5 g, 4.3 mmole) and EDC (0.6 g, 3.1 mmole) are stirred in 65 ml of CH₂CI₂ at RT while shielded from light. After 6 h, the solution is washed with water, 1 % HCI, saturated aqueous NaHCO₃ and saturated aqueous NaCI. The organic phase is then dried with MgSO₄, filtered, and concentrated under reduced pressure to yield the desired succinimidyl ester 57 as a slightly yellow solid.

Example A4: Synthesis of compounds 63i, 64i, 66i, 67i, 70i and 71 i

To a solution of allyl mannopyranoside (6.6g, 30mmol; formed by refluxing neat allyl alcohol with mannose in the presence of 0.01 equivalents CSA) in dry pyridine (75 ml) at 0°C is added tosyl chloride (10.3 g, 54 mmol) dissolved in CH₂CI₂, and the reaction is stirred at 0°C for 16 h and at RT for 4 h. The reaction mixture is extracted with CH₂CI₂ and washed with 1 N HCI, saturated NaHCO₃, and brine, and purified by flash chromatography using EtOAc as eluent to yield allyl 6-tosyl-6-deoxy-D-mannopyranoside.

To a solution of allyl 6-tosyl-6-deoxy-D-mannopyranoside (1.6g, 4.3 mmol) in DMF (50 ml) is added 10 equivalents (4.1 g) of NaN₃, and this mixture is refluxed overnight, the solvent removed in vacuo, and the residue chromatographed in SiO using EtOAc as eluent to yield allyl 6-azido-6-deoxy-D-mannopyranoside.

A solution of above compound (482 mg, 2.2. mmol) and a catalytic amount of NaHCO₃ (10 mg) in CH₂CI₂/MeOH (4/1 , 40 ml) is cooled to -78°C. O₃ is bubbled through the solution until a blue color is observed (10 min), then N₂ is bubbled through the solution until it becomes colorless. Dimethyl sulfite (577 mg, 6.6 mmol) is added and the mixture is warmed to RT and stirred overnight. After filtration, the solvent is evaporated under reduced pressure. The residue is dissolved in water and washed with ether. The water layer is lyphilized to yield compound 62. The above compound (330 mg, 1.5 mmol) and DHAP (0.5 mmol) are dissolved in Tris buffer (50 mM, pH 7.4) 10 ml. The pH is adjusted to 7.0 by adding 1 N NaOH and then 200 U of RAMA (from Sigma) is added. The reaction mixture is shaked under N₂ at RT. The progress of the reaction is followed by DHAP consumption (UV assay) until > 90% of the DHAP has been consumed. The pH is adjusted to 7.5 and BaCI₂ • H₂O (1.0 M, 2 mmol) is added slowly. The cloudy mixture is kept at 4°C for 1 hr and the precipitates are removed by centrifuga- tion. To the supernatant 2 volumes of acetone are aded and the mixture is stored at 4°C for 2 h. The precipitates are collected by centrifugation and the supernatant is discarded. The pellet is treated with Dowex-50 H⁺ to pH 2 (the solid is dissolved) and the resin is filtered off. The pH of filtrate is adjusted to 7.0 - 7.5 by adding 0.2 N NaOH. Lyophilization yields compound as a white powder.

The above compound is dissolved in a mixture of THF/water (1/1 ) solution and hydrogen- ated at 1 atm in the presence of catalytic amount of Pd/C. After stirring at RT for 6 h, the reaction mixture is filtered off using celite pad, The solvent is removed under vacuum to give free amine compound 63i.

The following compounds are synthesized according to the above method:

Example A5: Synthesis of Compounds 68i, 69i and 72 i

To a solution of methyl 2,3,4, 6-tetra-O-benzyl-α-glycoside (0.69 mmol) in CH₃CN (1.4 ml) is added allyltrimethylsilane (1.45 mmol) at 0°C under argon. To the mixture is added TMSOTf (0.35 mmol) and the reaction is kept stirring under the same temperature overnight, then warmed up to 25°C. To the mixture is added acetic anhydride (1 ml) drop by drop. After 5 min the reaction is diluted with CH₂CI₂ (10 ml) and the resulting solution is quenched by saturated NaHCO₃. The aqueous layer is extracted with CH₂CI₂ (2 x 10 ml) and the combined organic layers are washed with brine, dried with MgSO₄, filtered, evaporated and purified by column chromatography to provide acetate.

To a solution of acetate (1.00 g, 2.11 mmol) is added 0.3 equivalents of sodium methoxide in 1.0 molar methanol and is stirred at 25°C for 6 h; the solution is concentrated and to the crude is added 95% NaH (0.1 Og, 2.74 mmol) at 0°C under argon. After 30 min, benzyl bromide (0.85 ml, 2.74 mmol) is added to the mixture followed by tetra-n-butylammonium iodide (37.4 mg, 0.11 mmol) and the resulting solution is warmed up to 25°C overnight. The reaction is quenched by H₂O (10 ml), then extracted with EtOAc (3 x 10ml) and the combined organic layers are washed with brine, dried with MgSO , filtered, evaporated and purified by column chromatography (hexane to EtOAc/hexane = 1/20) to yield ether. To a solution of acetate (1.00 g, 2.11 mmol) is added 0.3 equivalents of sodiummethoxide in 1.0 molar methanol and is stirred at 25°C for 6 h; the solution is concentrated and to the crude is added 95% NaH (0.10 g, 2.74 mmol) at 0°C under argon. After 30 min, to the mixture is added 1-bromohexadecane (0.84 ml, 2.74 mmol; alternatively, the following alkyl haiide or acyl halide may be used in lieu of 1-bromohexadecane wherein all of the following are commercially available: 1-chioro propane, 1-chloro butane, 1 -chloro pentane, 1 -chloro hexane, 1 -chloro heptane, 1-chloro octane, 1-chloro nonane, 1 -chloro decane, 1 -bromo tetradecane, 1-bromo pentadecane, 1 -bromo hexadecane, 1-bromo octadecane, 1 -bromo eicosane, 1-bromo docosane, 2-bromomethyl-naphthalene, 1-chloromethyl-naphthalene, 2- bromethyl-benzene, 1 -bromo-3-phenyl-propane, heptanoyi chloride, octanoyl chloride, nonaoyl chloride, decanoyi chloride, undecanoyl chloride, lauroyi chloride, myristoyl chloride, palmitoyl chloride, heptadecanoyl chloride and stearoyl chloride) followed by tetra- n-butylammonium iodide (37.4 mg, 0.11 mmol) and the resulting solution is warmed up to 25°C overnight. The reaction is quenched by H₂O (10 ml), then extracted with EtOAc (3 x 10 ml) and the combined organic layers are washed with brine, dried with MgSO₄, filtered, evaporated and purified by column chromatography (hexane to EtOAc/hexane = 1/20) to yield ether.

To a solution of the terminal olefin (500 mg, 0.887 mmol) in benzene (50 ml) is added PdCI₂ (catalytic) and the solution is heated to reflux for 24 h. The reaction mixture is filtered through Ceiite, evaporated, and the crude oil is purified by SGCC (EtOAc: Hexane 1 :9 to 1 :1) giving the internal olefin.

To a solution of olefin (230 mg, 0.407 mmol) in CH₂CI₂ (20 ml) at -78°C is bubbled O₃ until a blue color persists. To remove residual O₃, pure O₂ is bubbled through until the solution turns clear. DMS (1.0 ml) is added and the reaction mixture is warmed to 23°C and stirred for 24 h. The reaction mixture is concentrated under reduced pressure. The crude oil is used directly without further purification.

The aldehyde prepared above is dissolved in acetone (5 ml) and cooled to 0°C. Jones reagent is added drop-wise until an orange color persists. 'PrOH (1 ml) is added to quench any excess Jones reagent and the reaction mixture is then partitioned betweenEtOAc (50 ml) and 1 N HCI (50 ml). The aqueous layer is extracted with EtOAc (50 ml) and the combined organic phases are dried (MgSO₄), concentrated under reduced pressure, and purified by silica gel flash chromatography (EtOAc:Hexane:HOAc 3:1 :0.01 ) giving the carboxylic acid.

To a solution of methyl β-D-galactopyranoside (10 g, 51.5 mmol) in CH₃CN (250 ml) is added benzaldehyde dimethyl acetal (15.6 ml, 103 mmol) followed by CSA (1.19 g, 5.15 mmol). After 30 min Et₃N (1 ml) is added and the solvent is removed under reduced pressure and the crude solid is recrystallized from hot MeOH affording the benzylidene acetal. To a solution of the above galactose benzylidene acetal (4.7 g, 16.7 mmol) in toluene (55 ml) is added Bu₂SnO (4.56 g, 18.3 mmol) and the solution is dehydrated using a Dean-Stark trap (130°C, 2 h) . The reaction mixture is cooled to 70°C and TBAI (4.3 g, 1 1.7 mmol) is added followed by aliylbromide (2.18 ml, 25 mmol). The solution is stirred at 130°C for 24 h before being cooled to 23°C and partitioned between EtOAc (200 ml) and saturated NaHCO₃ (200 ml) . The aqueous layer is extracted with EtOAc (2 x vol) and the combined organic layers are dried (MgSO₄), concentrated under reduced pressure, and chromato- graphed (1 :1 to 100% EtOAc/Hexane) giving the product intermediate. To a solution of (COCI) (209 μl, 2.40 mmol) in CH₂CI₂ (4 ml) at -78°C is added DMSO (341 μl, 78.1 mmol). The reaction mixture is warmed to 0°C for 5 min and the re-cooled to -78°C. 3-Allyl-4,6-benzylidene-2-hydroxy β-D-methylgalactopyranoside (704 mg, 2.19 mmol) is dissolved in CH₂CI₂ (4 ml) and added slowly drop-wise. The reaction mixture is stirred for 30 min and DIPEA is added (1.48 ml, 10.9 mmol). The reaction mixture is warmed to 23°C, diluted with CH₂CI₂ (100 ml), washed with saturated NaHCO₃ (50 ml) and dried (MgSO₄). The crude product is used directly in the next step without further purification. To a solution of the above ketone in MeOH (20 ml) is added NH OAc until the solution is saturated. Sodium cyanoborohydride (1 16 mg, 2.19 mmol) is added and the reaction mixture is stirred for 48 h. The reaction mixture is partitioned between EtOAc (100 ml) and saturated NaHCO, (50 ml) and the aqueous layer is further extracted with EtOAc (2 x 50 ml). The combined organic layers are dried (MgSO₄), concentrated under reduced pressure and chromatographed (5% MeOH/ CH₂CI₂) giving the amine.

To a solution of the galactose amine (65 mg, 0.203 mmol), mannose carboxylic acid (150 mg, 0.264 mmol), NMM (45 μl, 0.407), and HOBt (41.1 mg, 0.305 mmol) in CH₂CI₂ (3 ml) at 0°C is added EDC (60.1 mg, 0.305 mmol) . The reaction mixture is warmed to 23°C and stirred for 24 h. The reaction mixture is diluted with EtOAc (50 ml) and washed successively with a 5% citric acid solution (20 ml) and saturated NaHCO₃ (20 ml) . The solvent is removed under reduced pressure, dried (MgSO₄), and the crude oil is purified by SGCC (EtOAc:Hexane 1 :3 to 3:1 ) giving the coupled product.

To a solution of olefin (100 mg, 0.114 mmol) in CH₂CI₂ (10 ml) at -78°C is bubbled O₃ until a blue color persists. To remove residual O₃ pure O is bubbled through until the solution turns clear. DMS (1.0 ml) is added and the reaction mixture is warmed to 23°C and stirred for 4 h. The reaction mixture is concentrated under reduced pressure. The crude oil is used directly without further purification.

The aldehyde prepared above is dissolved in acetone (5 ml) and cooled to O°C. Jones reagent is added drop-wise until a orange color persists. 'PrOH (1 ml) is added to quench any excess Jones reagent and the reaction mixture is then partitioned between EtOAc (50 ml) and 1 N HCI (50 ml). The aqueous layer is extracted with EtOAc (50 ml) and the com^¬ bined organic phases are dried (MgSO ), concentrated under reduced pressure, and purified by silica gel flash chromatography (EtOAc:HOAc 95:5) giving the carboxylic acid (two steps).

To a solution of the above protected mimic (33 mg, 0.037 mmol) in 80% HOAc/H₂0 (10 ml) is added a catalytic amount of Pd/C (Degussa type, 10% by wt). The solution is flushed with hydrogen for 30 min then stirred for 24 h under a H₂ atmosphere. The reaction mixture is filtered through Celite and evaporated down under reduced pressure. The crude oil is further evaporated with H₂O (2x15 ml) and finally lyophilized giving mimics a white hygroscopic solid of compound 69i.

To a solution of tetrabenzyl-C-allyl-mannose olefin (679 mg,1.20 mmol) in CH₂CI₂:MeOH (8 ml:4 ml) at -78°C is bubbled O₃ in O₂ until a blue color persists. To remove residual O₃ pure O₂ is bubbled through until the solution turns clear. DMS (1.7ml, 24.0 mmol) is added and the reaction mixture is warmed to 23°C and stirred for 24 h. The reaction mixture is evaporated and partitioned between a saturated NaHCO₃ solution (50 ml) and EtOAc (5 ml). The aqueous phase is extracted with EtOAc (2 x 30ml) and the combined organic phases are dried and concentrated under reduced pressure. The crude oil is used directly without further purification.

The aldehyde prepared above is dissolved in acetone (5 ml) and cooled to 0°C. Jones reagent is added drop-wise until an orange color persists. 'PrOH (1 ml) is added to quench any excess Jones reagent and the reaction mixture is then partitioned between EtOAc (50 ml) and 1 N HCI (50 ml). The aqueous layer is extracted with EtOAc (50 ml) and the combined organic phases are dried (MgSO₄), concentrated under reduced pressure, and purified by silica gel flash chromatography (EtOAc:Hexane: HOAc 3:1 :0.01 ) giving the carboxylic acid.

To a solution of carboxylic acid (50 mg, 0.086 mmol), H-Glu-(OBn)₂ ^• pTsOH (0.095 mmol), HOBt (12.8 mg, 0.095 mmol), and NMM (10.3 μl, 0.095 mmol) in CH₂CI₂ (500 μl) at 0°C is added EDC (18 mg, 0.095 mmol). The reaction is allowed to stir for 24 h before being diluted with CH₂CI₂ (50 ml) and washed successively with a 5% citric acid solution (25 ml), saturated NaHCO₃ solution (25 ml) , and brine (25 ml) . The organic phase is dried (MgSO₄), concentrated under reduced pressure, and purified by SGCC (EtOAc:Hexane 1 :1 ) giving the coupled product. To a solution of the benzyl protected glycine aminoglycoside (33 mg, 0.045 mmol) in 80% HOAc/H₂O is added a catalytic amount of Pd/C (Degussa type, 10% by wt). The solution is flushed with hydrogen for 30 min then stirred for 24 h under a H₂ atmosphere. The reaction mixture is filtered and evaporated down under reduced pressure. The crude oil is further evaporated with H₂O (2 x 5 ml) and finally lyophilized giving compound 72i as a white hygroscopic solid.

Step (a) To a solution of tribenzyl-C-allyl-C-6-substituted olefin (679 mg, 1.20 mmol) in CH₂CI₂:MeOH (8 ml:4 ml) at -78°C is bubbled O₃ in O₂ until a blue color persists. To remove residual O₃ pure O₂ is bubbled through until the solution turns clear. DMS (1.7 ml, 24.0 mmol) is added and the reaction mixture is warmed to 23°C and stirred for 24 h. The reaction mixture is evaporated and partitioned between a saturated NaHCO₃ solution (50ml) and EtOAc (5 ml). The aqueous phase is extracted with EtOAc (2x30 ml) and the combined organic phases are dried and concentrated under reduced pressure. The crude oil is used directly without further purification. The aldehyde prepared above is dissolved in acetone (5 ml) and cooled to 0°C. Jones reagent is added drop-wise until an orange color persists. 'PrOH (1 ml) is added to quench any excess Jones reagent and the reaction mixture is then partitioned between EtOAc (50 ml) and 1 N HCI (50ml) . The aqueous layer is extracted with EtOAc (50 ml) and the combined organic phases are dried (MgSO ), concentrated under reduced pressure, and purified by silica gel flash chromatography (EtOAc:Hexane: HOAc 3:1 :0.01 ) giving the carboxylic acid intermediate.

Step (b) To a solution of carboxylic acid intermediate (50 mg, 0.086 mmol), H-Glu-(OBn)₂ • pTsOH (0.095 mmol), HOBt (12.8 mg, 0.095 mmol), and NMM (10.3 μl, 0.095 mmol) in CH₂CI₂ (500 μl) at 0°C is added EDC (18 mg, 0.095 mmol). The reaction is allowed to stir for 24 h before being diluted with CH₂CI₂ (50 ml) and washed successively with a 5% citric acid solution (25 ml), saturated NaHCO₃ solution (25 ml), and brine (25ml). The organic phase is dried (MgSO₄) , concentrated under reduced pressure, and purified by SGCC (EtOAc:Hexane 1 :1 ) giving the coupled product.

To a mixture of benzyl ether (250 mg, 0.24 mmol), in HOAcTHF/H₂O (12 ml, 4/1/1 ) is added a catalytic amount of Pd/C (Degussa type, 10% by wt). The solution is flushed with hydrogen for 30 min then stirred for 24 h under a H₂ atmosphere. The reaction mixture is filtered and co-evaporated with toluene under reduced pressure. The crude product is recrystallized from chloroform and hexane to yield mimetic compound 68i as a white solid. Example A6: Synthesis of Compound 65i

To a solution of methyl 2,3,4,6-tetra-O-benzyl-α-glycoside (0.69 mmol) in CH₃CN (1.4 ml) is added allyltrimethylsilane (1.45 mmol) at 0°C under argon. To the mixture is added TMSOTf (0.35 mmol) and the reaction is kept stirring under the same temperature overnight, then warmed up to 25°C. To the mixture is added acetic anhydride (1 ml) drop by drop. After 5 min, the reaction is diluted with CH₂CI₂ (10 ml) and the resulting solution is quenched by saturated NaHCO₃. The aqueous layer is extracted with CH₂CI₂ (2 x 10 ml) and the combined organic layers are washed with brine, dried with MgSO₄, filtered, evaporated and purified by column chromatography to provide acetate.

To a solution of acetate (1.00 g, 2.11 mmol) is added 0.3 equivalents of sodium methoxide in 1.0 molar methanol and is stirred at 25°C for 6 h; the solution is concentrated and then at 0°C is added tosyl chloride (10.3 g, 54 mmol) dissolved in CH₂CI₂, and the reaction is stirred at 0°C for 16 h and at RT for 4 h. The reaction mixture is extracted with CH₂CI₂ and washed with 1 N HCI, saturated NaHCO₃, and brine, and purified by flash chromatography using EtOAc as eluent to yield allyl 6-tosyl-6-deoxy-D-mannopyranoside. To a solution of allyl 6-tosyl-6-deoxy-D-mannopyranoside (1.6 g, 4.3 mmol) in DMF (50 ml) is added 10 equivalents (4.1 g) of NaN₃, and this mixture is refluxed overnight, the solvent removed in vacuo, and the residue chromatographed in SiO₂ using EtOAc as eluent to yield allyl 6-azido-6-deoxy-D-mannopyranoside.

A solution of above compound (482 mg, 2.2 mmol) and catalytic amount of NaHCO₃ (10mg) in CH₂CI₂/MeOH (4/1 , 40 ml) is cooled to -78°C. O₃ is bubbled through the solution until a blue color is observed (10 min) then N₂ is bubbled through the solution until it becomes colorless. Dimethyl sulfite (577 mg, 6.6 mmol) is added and the mixture is warmed to RT and stirred overnight. After filtration, the solvent is evaporated under reduced pressure. The residue is dissolved in water and washed with ether. The water layer is lyphilized to yield compound 62.

The above compound (330 mg, 1.5 mmol) and DHAP (0.5 mmol) are dissolved in Tris buffer (50 inM, pH 7.4) 10 ml. The pH is adjusted to 7.0 by adding 1 N NaOH and then 200 U of RAMA (from Sigma) is added. The reaction mixture is shaked under N₂ at RT. The progress of the reaction is followed by DHAP consumption (UV assay) until >90% of the DHAP has been consumed. The pH is adjusted to 7.5 and BaCI₂ • H₂O (1.0 M, 2 mmol) is added slowly. The cloudy mixture is kept at 4°C for 1 h and the precipitates are removed by centrifuga- tion. To the supernatant 2 volumes of acetone are added and the mixture is stored at 4°C for 2 h. The precipitates are collected by centrifugation and the supernatant is discarded. The pellet is treated with Dowex-50 H⁺ to pH 2 ( the solid is dissolved) and the resin is filtered off. The pH of filtrate is adjusted to 7.0-7.5 by adding 0.2 N NaOH. Lyophiiization yields compound as a white powder.

The above compound is dissolved in a mixture of THF/water (1/1 ) solution and hydrogen- ated at 1 atm in the presence of a catalytic amount of Pd/C. After stirred at RT for 6 h, the reaction mixture is filtered off using celite pad. The solvent is removed under vacuum to give free amine compound 65i.

Example A7: Synthesis of Compound 73 i

wherein Ar is hydrogen, phenyl (styrene), benzyl (allylbenzyl), 4-methoxyphenyl (vinylanisole), 2,3,4,5, 6-pentafluorophenyl (2,3,4,5,6-pentafluorostyrene), 2-naphthyl (vinylnaphthalene), 2-fluorenyl (made from 2-fluorene carboxaldehyde), 4-biphenylyl (vinyl biphenyl) or dimer.

Ethyl (2S,3S)-2-azido-3-hydroxy-4-(tri-0-benzylr -L-fucopyranosyl)-butanoate is dissolved in a mixture of THF/water (1/1 ) solution and hydrogenated at 1 atm in the presence of a catalytic amount of Pd/C. After stirred at RT for 6 h, the reaction mixture is filtered off using celite pad. The solvent is removed under vacuum to give amine compound which can be used in next step without further purification.

To a solution of 1.1 equivalent allyl trimethyl silane in THF (50 ml) is added 1.0 equivalents of above amine compound and this mixture is stirred at 0°C for 12 h, the solvent is then diluted with ether and then extracted 3X with water, the organic layer is condensed, and the residue chromatographed in SiO₂ using EtOAc as eluent to yield allyl - free amine intermediate.

To a solution of above allyl - free amine intermediate (50 mg,0.086 mmol), compound 49 (0.095 mmol), HOBt (12.8 mg, 0.095 mmol), and NMM (10.3 μl, 0.095 mmol) in CH₂CI₂ (500 μl) at 0°C is added EDC (18 mg, 0.095 mmol) . The reaction is allowed to stir for 24 h before being diluted with CH₂CI₂ (50 ml) and washed successively with a 5% citric acid solution (25 ml), saturated NaHCO₃ solution (25 ml), and brine (25 ml). The organic phase is dried (MgSO₄), concentrated under reduced pressure, and purified by SGCC (EtOAc:Hexane 1 :1 ) giving the coupled product.

Metathesis: To a solution of above intermediate (50 mg, 0.086 mmol), a vinyl-aryl compound selected from the group consisting of styrene, aliylbenzene, vinylanisoie, 2,3,4,5, 6-pentafluorostyrene, vinylnaphthalene, vinyl biphenyl, hydrogen and 2-fulorene (0.095 mmol), [CI₂PCy₃)₂RuCHPH] (12.8 mg, 0.095 mmol) in CH₂CI₂ (500ml) at reflux for 24 h before being diluted with CH₂CI₂ (50 ml) and washed successively with a 5% citric acid solution (25 ml), saturated NaHCO₃ solution (25 ml), and brine (25ml) . The organic phase is dried (MgSO₄), concentrated under reduced pressure, and purified by SGCC (EtOAc:Hexane 1 :1 ) giving the coupled product.

Above compound is dissolved in a mixture of THF/water (1/1 ) solution and hydrogenated at 1 atm in the presence of a catalytic amount of Pd/C. After stirred at RT for 6 h, the reaction mixture is filtered off using celite pad. The solvent is removed under vacuum to give amine compound which can be used in the next step without further purification. A mixture of fucosyl amine 52 (0.5 mmole; Toronto Research Chemicals Inc.), above com^¬ pound (0.5 mmole) and CH₂CI₂ (0.068 molar) is cooled to 0°C. The coupling reagent HOBT (0.6 mmole) is added followed by EDC (0.6 mmole), and the solution is stirred at 0°C for 30 min. After being stirred at RT for 10 h, the solvent is evaperated and the residue is diluted with EtOAc. The resulting organic layer is washed with H₂O (2x10 ml, scaled accordingly, based on a 200-300 mg scale for either substrate), 1 N HCI (2x5 ml), saturated aqueous NaHCO₃ (2x5 ml), and saturated aqueous NaCI. The organic phase is dried over MgSO filtered, and concentrated. The residue is purified by flash column chromatography eluting with CHCI₃ MeOH (19:1 ) to afford compound.

Above Compound (256 mg, 0.2 mmole) is dissolved in ethanol H₂O dioxane : AcOH (2:1 :2:1 , 2 ml), and then a catalytic amount of Pd(OH)₂ (Degussa type; Aldrich) on carbon is added. Hydrogen is supplied to the reaction system through a ballon. After 6 h the mixture is filtered through celite and concentrated in vacuo. The crude product is purified by biogel P2 (water). The collected fractions are combined and freeze dried to afford compound 73i.

Example A8: Synthesis of Compound 74i, 75 i and 77i

(77i) wherein P is -[CH₂CH₂O]₃CH₂CH₂NH-

To a solution of L-fucose (10.4 g, 63.4 mmol) and 4-DMAP (390 mg, 3.2 mmol) in dry pyridine (25 ml) is added acetic anhydride (26.3 ml, 279 mmol) at 0°C dropwise within 25 min. The ice bath is removed and stirring is continued overnight. The reaction is taken up in ether (ca. 400 ml), extracted with 1 N HCI (5 x 50 ml), saturated NaHCO₃ solution (2 x 50 ml), brine (50 ml) and dried over MgSO₄. After removal of the solvent in vacuo the residue is directly used in the next step without further purification.

To a solution of L-fucose tetraacetate (20.3 g, 61.1 mmol) and trimethylallylsilane (19.4 ml, 122.2 mmol) in dry acetonitrile (110 ml) at 0°C are added BF₃Et₂O (7.75 ml, 122.2 mmol) and TMSOTf (2.36ml, 12.2 mmol) simultaneously within 25 min and the reaction mixture is stirred for 10 h at 0°C. After quenching with ice water, the mixture is taken up in ether and extracted with saturated NaHCO₃ solution and brine, the aqueous layer is reextracted with ether (2 x 40ml) and the combined organic phases are dried over MgSO₄. The solvent is removed in vacuo and the residue is purified by SGCC (gradient elution 20%→30%→50% ethyl acetate in hexanes) to give the triacetate of above as a slightly yellow oil. This material is dissolved in dry MeOH (100 ml), treated with NaOMe solution (1 ml, 25%w/w in dry MeOH) and stirred at 23°C for 15 h. The solution is neutralized by addition of Dowex H-50x resin, filtered and evaporated to dryness under reduced pressure. The remaining white solid is recrystallized from boiling ethyl acetate to yield two fractions of equally pure compound. A solution of above compound (4.7 g, 25 mmol), n-Bu₄NI (460 mg, 1.3 mmol) and benzyl- bromide (14.9 ml, 125.3 mmol) in dry THF (50 ml) cooled to 0°C and NaH (4.0 g, 100 mmol, 60 % w/w in mineral oil) is added followed by stirring at 23°C for 17 h. The reaction is quenched with icewater, taken up in ether and the mixture is acidified with 1 N HCI. The aqueous layer is extracted with ether (2 x 4.0 ml) and the combined organic layer is neutralized with saturated NaHCO₃ solution, treated with brine and dried over MgSO₄. Removal of the solvent in vacuo leaves a yellow oil, which is purified by silica gel flash column chromatography (gradient elution 0%→30%→50% ethyl acetate in hexanes) to yield the 3-(tri-O- benzyl-α-L-fucopyranosyl)-1 -propene.

A solution of 3-(tri-O-benzyl-α-L-fucopyranosyl)-1 -propene (2.00 g, 4.36 mmol) in CH CI₂ (18 ml) and MeOH (3.6 ml) is cooled to -78°C. O₃ is bubbled through the solution until a blue color appears, and then N₂ is bubbled until it becomes colorless. NaHCO₃ (733 mg, 8.72 mmol) and dimethylsulfide (3.2 ml, 43.6 mmol) are successively added to the reaction, and the mixture is stirred for 22 h at 23°C. The reaction is taken up in Et₂O, washed with water and brine and dried over MgSO . After removal of the solvent in vacuo the residual oil is purified by SGCC (gradient elution 20%→30% ethyl acetate in hexane) to give the aldehyde (1.81 g, 90%) as a slightly yellow oil.

A solution of 2-(tri-O-benzyl-α-L-fucopyranosyl)-acetaldehyde (1.57 mmol), glycine methyl ester hydrochloride (1.57 mmol), Et₃N (3.14 mmol) and MgSO₄ (1. 1 mmol) in dry CH₂CI (3.3 ml) is stirred at 23°C for 13 h. The solid remains are filtered off, washed with dry CH₂CI₂ and the solvent is removed in vacuo to give imine. The imine is dissolved in dry MeOH (9 ml) and cooled to 0°C, whereas NaBH₄ (3.9 mmol) is added in one portion. After 20 min the reaction mixture is concentrated under reduced pressure and the remaining oil is coevapo- rated with CH₂CI₂ (25 ml) to remove the rest MeOH. The residue is dissolved in CH₂CI₂ (15 ml) and Boc₂O (3.6 mmol) is added at 0°C. The reaction is warmed up to 23°C and stirred for 50 min. After diluting with Et₂O, the organic layer is washed with 5%w/v citric acid solution, saturated NaHCO₃ solution and brine, followed by drying over MgSO₄. The solvent is removed in vacuo and the remaining oil is purified by SGCC (gradient elution 20%→30%→- 35%→40% EtOAc in hexane) to give the Boc carbamate (252 μmol) which is deprotected using standard TFA deprotection conditions to give the intermediate amine. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.65 mmol) is added to a stirred solution of above intermediate amine (0.6 mmol), N-Boc amino acid (0.65 mmol) [all amino acids obtained according to Cappi et al, Bioorg. Med. Chem 5:283 (1997) and Woltering et al. Tetrahedron Lett. 37:9033(1996)], 1 -hydroxy benzotriazole (HOBt) (0.65 mmol) and 4-methyl morpholine (1.3 mmol ) in dry DMF (2 ml) under argon at -20°C. The resulting mixture is stirred at -20°C for 1 h and then allowed to warm slowly to 23°C. After 14 h, the reaction solution is quenched with a 5% citric acid solution (20 ml) and extracted with EtOAc (6 x 25 ml). The combined organic extracts are washed with saturated NaHCO₃ solution (50 ml), a saturated NaCI solution (50 ml), dried (MgSO₄) and evaporated down under reduced pressure. The residual oil is purified by silica gel flash chromatography to give the product.

Next, a solution of above olefin (6.5 mmol) and catalytic amount of TFA (0.3 g) in CH₂CI₂ (4/1 , 120 ml). is stirred at RT for 3 h. After washed with sodium bicarbonate, the solvent is evaporated under reduced pressure and the residue is purified by SGCC to obtain the desired compound.

Next, a solution of above compound (6.5 mmol), BnO₂CH₂(CH₂)_mCOSu (0.3 g, m = 1 ,2) in CH₂CI₂ (4/1 , 120 ml) is stirred at RT for 3 h. After washed with sodium bicarbonate, the solvent is evaporated under reduced pressure and the residue is purified by SGCC to obtain the desired compound.

Next, to a solution of above compound (386 mg, 0.4 mmol) in 40 ml of methanol (1 :1 ) is added Pd(PPh₃)₄ (45 mg, 0.04 mmol) and morphorine (0.4 mmol). After stirred at RT for 11 h, the solvent is evaporated and the residue is taken up in 50 ml of EtOAc. The resulting solution is washed three times with 30 ml of 1 N HCI, dried over MgSO₄, and concentrated in vacuo. The crude product is purified by flash column chromatography eluting with CH₃CI MeOH (9: 1 ) to afford the title compound.

Example A9: Synthesis of Compound 76i, 78i, 79i, 80i and 81 i

A solution of N-Fmoc threonine (541 mg, 1 .59 mmol) and cesium carbonate (258 mg, 0.5 eq) in DMF (20 ml) is stirred for 30 min at RT, and then allyl bromide (212 mg, 1 .1 eq) is added. After the reaction mixture is stirred for overnight at RT, the mixture is evaporated in vacuo. The residue is dissolved with EtOAc and washed with brine. The organic phase is dried over MgSO₄, filtered, and concentrated. The residue is purified by flash column chromatography eluting with hexane : EtOAc (1 :1 ) to obtain N-Fmoc threonine allyl ester (499 mg, 82%).

A suspension of dibenzyl 2,3,4-O-tribenzyl-α-L-fucopyranosyl phosphite (138 mg, 0.204 mmol), N-Fmoc threonine allyl ester (77.5 mg, 1 eq) and 4A molecular sieves (47; 0 mg) is stirred for 1 h at RT. To the mixture is added a solution of TMSOTf (4.5 mg, 0.1 eq) in CH₂CI₂ (1 ml) at -15°C. After stirring for 2 h at the same temperature, saturated aqueous NaHCO₃ is added to quench the reaction and the mixture is diluted with CH₂CI₂. The organic layer is separated, dried over MgSO₄, filtered, and concentrated. The residue is purified by flash column chromatography eluting with hexane : EtOAc (4:1 ) to obtain acid compound. To a solution of above compound (104 mg, 0.13 mmol) in CH₂CI₂ (5 ml) is added Et₂NH (0.72 ml, 53 eq) and the mixture is stirred for 3 h at RT. The solvent and the reagent are removed in vacuo and the residue is purified by SGCC (CH₂CI₂/MeOH=100/3) to give amine. A mixture of L-fucosyl threonine amine 48 (292 mg, 0.5 mmol), acid compound (218 mg, 0.5 mmol) [alternatively other acid compounds including hydroxy-pyridine base and tyrosine based acid compounds can be used as described in Lin et al. J. Am. Chem. Soc. 1 18:6826 (1996)] and CH₂CI₂ (6 ml) is cooled to 0°C. The coupling reagent HOBT (89 mg, 0.6 mmol) is added followed by EDC (127 mg, 0.6 mmol), and the solution is stirred at 0°C for 30 min. After being stirred at RT for 10 h, the solvent is evaporated and the residue is diluted with EtOAc. The resulting organic layer is washed with H₂O (2x10 ml), 1 N HCI (2x5 ml), saturated aqueous NaHCO₃ (2x5 ml), and saturated aqueous NaCI. The organic phase is dried over MgSO₄, filtered, and concentrated. The residue is purified by flash column chromatography eluting with CHCI₃ : MeOH (19:1 ) to afford the desired product. To a solution of above compound (386 mg, 0.4 mmol) in 40 ml of THF/MF (1 :1 ) is added tetrakis (triphenylphosphine) palladium (0) (4 5 mg, 0. 04 mmol) and morpholine (0.34 ml, 4.0 mmol). After stirred at RT for 11 h, the solvent is evaporated and the residue is taken up in 50 ml of EtOAc. The resulting solution is washed three times with 30 ml of 1 N HCI, dried over MgSO₄, and concentrated in vacuo. The crude product is purified by flash column chromatography eluting with CH₃CI : MeOH (9:1 ) to afford the title compound. A mixture of above acid (292 mg, 0.5 mmol), amine compound 52 (218 mg, 0.5 mmol) and CH₂CI₂ (6 ml) is cooled to 0°C. The coupling reagent HOBT (89 mg, 0.6 mmol) is added followed by EDC (127 mg, 0.6 mmol), and the solution is stirred at 0°C for 30 min. After being stirred at RT for 10 h, the solvent is evaporated and the residue is diluted with EtOAc. The resulting organic layer is washed with H₂O (2x10 ml), 1 N HCI (2x5 ml), saturated aqueous NaHCO₃ (2x5 ml), and saturated aqueous NaCI. The organic phase is dried over MgSO₄, filtered, and concentrated. The residue is purified by flash column chromatography eluting with CHCI₃ : MeOH (19:1) to afford the desired product.

To a solution of above compound (386 mg, 0.4 mmol) in 40 ml of THF/DMF (1 :1 ) is added tetrakis (triphenylphosphine) palladium (0) (45 mg, 0.04 mmol) and morpholine (0.34 ml, 4.0 mmol) . After stirred at RT for 11 h, the solvent is evaporated and the residue is taken up in 50 ml of EtOAc. The resulting solution is washed three times with 30 ml of 1 N HCI, dried over MgSO₄, and concentrated in vacuo. The crude product is purified by flash column chromatography eluting with CH₃CI : MeOH (9:1 ) to afford the title compound.

Example A10: Synthesis of Compound 82i, 83i, 84i and 85i

wherein R^B2 is -[CHgCHaOkCHsCHsNH-.

A solution of methyl 2,3,4,6-tetra-O-benzyl- -D-mannopyranoside (11.2 g, 20.3 mmol) in 32 ml of dry MeCN is cooled to 0°C under N₂. Allyltrimethylsilane (6.5 ml, 40.5 mmol) and trimethylsilyl triflate (1.9 ml, 10.2 mmol) are added. The solution is stirred for 20 h at 0°C. Acetic anhydride (8 ml, 80 mmol) is added and the resulting mixture is stirred for 30 min at RT. The deep-orange solution is diluted with 130 ml CH CI₂ and quenched with 70 ml of saturated NaHCO solution. The aqueous layer is washed with 30 ml CH₂CI₂ twice. The organic layers are combined and then dried over MgSO₄. The solvent is removed in vacuo, and the residue is purified by SGCC. A solution of above compound (5.3 g, 10.3 mmol) and NaOMe (0.23 g, 4.3 mmol) in 50 ml MeOH is stirred RT for 1 h. The solvent is evaporated under reduced pressure, and the residue is purified by SGCC to afford the desired compound.

To a solution of the above compound (3.2 g, 6.4 mmol) in THF (14 ml) at 0°C are added DEAD (1.0 ml, 6.4 mmol) and PPh₃ (1.74 g, 6.4 mmol), successively, and the mixture is stirred for 20 min at 0°C. Diphenylphosphoryl azide (1.4 ml, 6.4 mmol) is then added dropwise and the resulting mixture is stirred at RT for overnight. The solvent is evaporated under reduced pressure, and the residue is purified by SGCC.

A mixture of above compound (1.7 g, 3.5 mmol) and PPh₃ (1.83 mg, 7.0 mmol) is refluxed in a mixture of benzene (20 ml) and water (0.13 ml) for 5 h. The reaction mixture is evaporated in vacuo and the residue is applied to SGCC to give the title compound. EDC (0.65 mmol) is added to a stirred solution of 6-amino-L-galactopyranoside (0.6 mmol), N-Boc amino acid (0.65 mmol) [all amino acids obtained according to Cappi et al, Bioorg. Med. Chem. 5:283 (1997)], 1 -hydroxy benzotriazole (HOBt) (0.65 mmol) and 4-methyl morpholine (1.3 mmol) in dry DMF (2 ml) under argon at -20°C. The resulting mixture is stirred at -20°C for 1 h and then allowed to warm slowly to 23°C. After 14 h, the reaction solution is quenched with a 5% citric acid solution (20 ml) and extracted with EtOAc (6 x 25 ml). The combined organic extracts are washed with saturated NaHCO₃ solution (50 ml), a saturated NaCI solution (50 ml), dried (MgSO₄) and evaporated down under reduced pressure. The residual oil is purified by silica gel flash chromatography to give the product. Next, a solution of above olefin (6.5 mmol) and catalytic amount of NaHCO₃ (0.3 g) in CH₂CI₂/MeOH (4/1 , 120 ml) is cooled to -78°C. O₃ is bubbled through the solution until a blue color is observed (10 min) then N₂ is bubbled through the solution until it becomes colorless. PPh₃ (13.0 mmol) is added and the mixture is warmed to RT and stirred for overnight. After filtrated, the solvent is evaporated under reduced pressure and the residue is purified by SGCC to obtain the desired compound.

The aldehyde prepared above is dissolved in acetone (5 ml) and cooled to 0°C. Jones reagent is added drop-wise until an orange color persists, which indicates the oxidation had gone to completion. 'PrOH (1 ml) is added to quench any excess Jones reagent and the reaction mixture is then partitioned between EtOAc (50 ml) and 1 N HCI (50 ml). The aqueous layer is extracted with EtOAc (50 ml) and the combined organic phases are dried (MgSO₄)l concentrated under reduced pressure, and purified by silica gel flash chromatography (EtOAc:Hexane: HOAc 3:1 :0.01 ) giving the carboxylic acid. Next, to a solution of above compound (386 mg, 0.4 mmol) in 40 ml of methanol (1 :1 ) is added Pd-C (45 mg, 0.04 mmol; Aldrich).

After stirred at RT for 11 h under hydrogen balloon, the solvent is evaporated and the residue is taken up in 50 ml of EtOAc. The resulting solution is washed three times with 30 ml of 1 N HCI, dried over MgSO₄, and concentrated in vacuo. The crude product is purified by flash column chromatography eluting with CH₃CI : MeOH (9:1 ) to afford the title compound.

Example B1 : Synthesis of Compounds 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 and 85

Table 1 : Compounds of formula

w ere n r is as defined in Example A7; R is as defined above; and

^■ B2

R°' is -[CH₂CH₂O]₃CH₂CH₂NH-

To a suspension of compound 63i, 64i, 65i, 66i, 67i, 68i, 69i, 70i, 71 i, 72i, 73i, 74i, 75i, 76i, 77i, 78i, 79i, 80i, 81 i, 82i, 83i, 84i or 85i (31 mg, 0.05 mmol) in DMF (2.5 ml) and Et₃N (0.26 ml) is added 57 (24 mg, 0.05 mmol) at 0°C. The mixture is stirred for 30 min at 0°C and 48 h at RT. The solvent is removed in vacuo and the residue is dissolved in AcOEt (20 ml) and washed with water. The organic layer is dried over MgSO₄ and evaporated in vacuo. The residue is purified by gel-filtration on a Sephadex LH-20 column eluting with CHCI₃:MeOH (1 :1 ) to obtain compounds 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84 and 85.

Example C1 : Preparation of liposome

2.56 mg (0.0025 mmole) of lipid compound 81 of Table 1 and 17.79 mg (0.0475 mmole) of 57 are dissolved in C CyMeOH (10:1 ) solution. The solvent is evaporated and the residue is shielded from light and dried in vacuo. 5 ml of HEPES buffer (20 mM, PH=7.4) is added to yield a heterogeneous solution. To obtain a clear homogeneous solution, the lipid/buffer mixture is then sonicated with a probe-tip sonicator for at least 1 h at the maximum power setting at which no frothing occurs and at which there is minimal disturbance of the solution surface. The temperature is maintained above the gel-liquid crystal phase transition point (T_m=-64°C) with the heat generated from sonication.

Example C2: Preparation of liposome

The liposome solution of Example C1 is transferred to a petri dish resting on a bed of wet ice, cooled to 0°C, and irradiated at 254 nm for 1 h with a hand-held UV lamp placed in 1 cm about the petri dish, yields a dark-blue paramagnetic polymerized liposome (PPL). The PPL are then filtered through a 0.2 μm and collected [Procedure similar to that found in J.Am.Chem. Soc. 117:7301-7306 (1995)].

Example C3: Preparation of liposome

2.56 mg (0.0025 mmole) of lipid compound 81 of Table 1 and 17.79 mg (0.0475 mmole) of 61 (Avanti Polar Lipids Inc.)

are dissolved in CHCIs/MeOH (10:1 ) solution. The solvent is evaporated and the residue is shielded from light and dried in vacuo. 5 ml of HEPES buffer (20 mM, pH=7.4) is added to yield a heterogeneous solution. To obtain a clear homogeneous solution, the lipid/buffer mixture is then sonicated with a probe-tip sonicator for at least 1 h at the maximum power setting at which no frothing occurs and at which there is minimal disturbance of the solution surface. The temperature is maintained above the gel-liquid crystal phase transition point (T_m=64°C) with the heat generated from sonication.

Example C4: Preparation of liposome

The liposome solution of Example C3 is transferred to a petri dish resting on a bed of wet ice, cooled to 0°C, and irradiated at 254 nm for 1 h with a hand-held UV lamp placed in 1 cm about the petri dish, yields a dark-blue paramagnetic polymerized liposome (PPL). The PPL are then filtered through a 0.2 μm and collected (T_m=37°C, yellow PPL). Example D: Protocol for Assaying Biological Activity

This assay is performed as disclosed in WO 98/08,854 the contents thereof relating to this assay being incorporated hereinwith.

In this assay the liposomic sLe^x mimetic of formula I, VI and VP have an RIC₅₀ value of from

0.01 to 1.0.

RIC₅₀ means IC₅₀(test compound)/IC₅₀(control compound A).

¹NMR data of compound 69i:

¹NMR (D₂O, 400 MHz) δ 8.02 (d, J= 7.6 Hz, 1 H), 4.58 (s, 2 H), 4.51 (s, 1 H), 4.48 (s, 1 H),

4.24-4.16 (m, 2 H), 4.07 (s, 1 H), 3.84-3.73 (m, 6 H), 3.62 (dd, J = 3.2, 4.0 Hz, 1 H), 3.55-

3.45 (m, 5 H) NMR data of compound 72i:

¹NMR (D₂O, 400 MHz) δ 4.37-4.42 (m, 1 H), 4.32 (ddd, = 1.8, 5.0, 5.0 Hz, 1 H), 3.87 (t, J = 2.9 Hz, 1 H), 3.79 (dd, J = 3.3, 9.0 Hz, 1 H), 3.71 -3.77 (m, 2 H), 3.67 (dd, J = 9.2, 9.2 Hz, 1 H), 3.53-3.60 (m, 1 H), 2.80 (dd, J = 10.8, 14.8 Hz, 1 H), 2.55 (dd, J = 5.1 , 14.9 Hz, 1 H),

2.46 (dd, J = 7.0, 7.0 Hz, 2 H), 2.11-2.20 (m, 1 H), 1.90-2.02 (m, 1 H); ¹³C NMR (D2O/DMSO, 100 MHz) δ 192, 187, 181.8, 84.2, 83.7, 79.9, 76.2, 70.0, 44.3, 35.1 ; HRMS calculated for C₁₃H₂₂θ₁₀N (M + H), 352.1244, found 352.1238

Claims

WHAT IS CLAIMED IS:

1 . A liposomic sLe^x mimetic of formula I

MIMETIC-LINKER-LIPID (I) wherein MIMETIC is a sLe^x mimetic; LIPID is a lipid moiety; and LINKER is a conjugating element which links MIMETIC to LINKER with the proviso that when MIMETIC is a group of formula II

LIPID is not a group of formula III

2. The liposomic sLe mimetic according to claim 1 wherein MIMETIC is a radical derived from a compound of formulae lla, lib or lie

wherein

X is a direct bond, -NR³- or -O-; Y is a direct bond, -O- or C C₃alkylene;

R¹ is hydrogen, methyl, hydroxyl, -O(CH₂)_nCH -Ar; -O(CH₂)_nCHCH-Ar; -O(CH₂)nCONHCH(CH₂OCO(CH2)mCH₃)2 or -O(CH2)nCONHC(CH₂)_mCH₃; R² is hydrogen; -CONH₂; -COOH; -CONH(CH₂)nCONH(CH₂)mCH₃; -CONH(CH₂)_mCH₃;

-COOEt or -CONH(CH₂CH₂O)_pCH₂NH2; R³ is hydrogen, -(CH₂)_nCOOH or -(CH₂)nCONH(CH2)mCH₃; R⁴ is HO₂C(CH₂)_nCONHCH(CH(CH₃)(OH))-; HO₂C(CH₂)_nCONHCH(CH(OH)(CH₂OH))-;

HO₂C(CH₂)_nCONHC(CH₂╬╕H)₂-; H╬╕₂CCH(NH₂)CH₂CONHCH(CH₂Ph(OH))-; -CH2PO₃ ^2*;

-OPO₃ ^2" or a radical of formula lla' or lib'

R⁵ is -(CH₂)_n[CH(OH)]₂COCH₂╬╕P╬╕3Na₂; -(CH₂)_n[CH(OH)32COCH2CH₂OPO₃Na2;

-[CH(OH)]₂CONHCH₂COOH; -CH₂CONHPh-COOH; -[CH(OH)]2CO(CH2)nOPO3Na2;

-CONHCH(COOH)(CH₂)_nCOOH; -NHCO(CH₂)_nCO₂Bn; -NHCO(CH₂)_nCOOH;

-NHCOC[NHCO(CH₂)_nCOOH]CH(OH)(CH₂)_mCH₂OH; -CONHPh-(COOH); hydrogen;

-CH₂CONH(CH₂)₁₃CH₃; -NHCOC(CH₂OH)₂NHCO(CH₂)_nCOOH; -(CH₂),C(O)-R_x or

-[CH(OH)]₂CONHCHR_yCOOH; R⁶ is -NH₂, -CONH(CH₂CH₂O)_pCH₂NH₂; -CONH₂; -COOH, hydrogen, hydroxyl, -O-C C₆alkyl;

-OBn; -N₃; -OSO₃ ^2"; -OCO[CH₂]₂CONHCH(CH₂CO₂H)COOH or -NHR'; R⁷ is -NH₂, -CONH(CH₂CH₂O)_pCH₂NH₂; -CONH₂ or -COOH; R⁸ is -O-methyl, -O(CH₂)_nCH₂Ar; -O(CH₂)_nCHCHAr; -O(CH₂)_nCONHCH(CH2OCO(CH₂)_mCH3)2 or -O(CH₂)_nCONHC(CH₂)_mCH₃; R_x is butyl amine, amyl amine, hexyl amine, heptyl amine, octyi amine, nonylamine, decyl- amine, undecylamine, dodecylamine, tridecylamine, 1 -tetradecylamine, pentadecyl- amine, hexadecyl amine, octadecyl amine, 1 -amino naphthalene, 2-naphthalene,

2-amino-2-napthol perhydrochloride, 4-pentyl aniline, 4-hexyl aniline, 4-heptyl aniline,

4-octyl aniline, 4-decyl aniline, 4-dodecyl aniline, 4-tetradecyl aniline or 4-hexadecyl- aniline; R_y is a side chain of an amino acid selected from Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly,

Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His; R' is CrC₆alkyl, acyl, decanoyi, phenylacetyl, -CO[CH₂.₂COOH, hexanoyi, heptanoyi, octan- oyl, nonanoyl, decanoyi, undecanoyl, laurate, tridecanoyl, myristate, pentadecanoyl, pal- mitate, heptadecanoyl, stearate, nonadecanoyl, eicosanoyl, hexaicosanoyl, docosanoyl, tricosanoyl, tetracosanoyl, hexacosanoyl, heptacosanoyl, octacosanoyl, triacontanoyl, 4-phenylbutyroyl, 5-phenyl valerate, 6-phenyl hexanoyi, oleic acid, 3-trans-7-trans-far- nesoyl, 8-trans-10-trans-dodecandien-1-carboxy, 2-naphthanoyl, 1 -hydroxy-2-naphthan- oyl, 1 ,4-dihydroxy-2-naphthanoyl, -C(O)(CH₂)_uCH₃; -C(O)(CH₂)wNHCO(CH₂)_qC(O)OH; -C(O)(CH₂)_uC₆H₅ ╬╕r -C(O)(CH₂)_rCON(CH₂CONH(CH₂)CH3)HCONH(CH2)sCH₃;

3. The liposomic sLe^x mimetic according to claim 1 wherein LIPID is a group of formula Ilia or 1Mb

wherein 0 < m_x < 20; 0 < n_x < 10; and

R is a radical of formula lllb^a, lllb^b, lllb^c or lllb^d

(lllb^b);

(lllb^c);

(lllb^d)

wherein each n_y individually is a number of from 0 to 10.

4. The liposomic sLe^x mimetic according to claim 1 wherein LINKER is a group of formula IVa, IVb, IVe, IVd, IVe, IVf, IVg, IVh or IVi,

(IVd), ΓÇöNH ΓÇö PEG-NH- (IVe), ΓÇö OΓÇö PEG-NH- (IVf),

-0-PB3-0- (IVg), -_NH^^╬¢^ C ) or __o ^- _o_ ('VD

wherein 0 < v < 100.

5. A process for the preparation of the liposomic sLe^x mimetics according to claim 1 wherein a linker precursor is coupled to the corresponding MIMETIC precursor, followed by coupling to the LIPID precursor.

6. A cross-linked liposomic sLe^x mimetic of formula VI

ILIPID (Bj

IMIMETIC-LINKER-LIPID (A)| (VI)

wherein MIMETIC and LINKER are as defined in claims 2 and 4 and LIPID (A) is a group of formula Ilia or derived therefrom; and LIPID (B) is a compound selected from compounds of formulae Vila and Vllb or derived therefrom

7. A method for preparing the cross-linked liposomic sLe^x mimetic according to claim 6 comprising sonicating a lipid conjugate of formula XII

MIMETIC-LINKER-LIPID (A) (XII) wherein MIMETIC and LINKER are as defined in claim 6; and LIPID (A) is a group of formula Ilia according to claim 3, with a compound of formulae Vila or Vllb according to claim 6; and optionally irradiating the resulting product with UV light.

8. A method for preparing a cross-linked liposomic sLe^x mimetic of formula VP

ILIPID (B.

^JN

[ MIMETIC-LINKER-LIPID (Ap (VP)

wherein LINKER, LIPID (A) and LIPID (B) are as defined in claim 6; and MIMETIC is a radical derived from a compound of formula lla according to claim 2 wherein X, Y, R² and R⁴ are as defined in claim 2; and R¹ is hydrogen, methyl, hydroxyl, -O(CH₂)_nCH₂-Ar; -O(CH₂)_nCHCH-Ar;

-O(CH₂)_nCONHCH(CH₂OCO(CH₂)_mCH3)2, -O(CH₂)nCONHC(CH2)mCH₃; or a radical derived from a compound of formulae Vila or Vllb according to claim 6 comprising sonicating a lipid conjugate of formula XIP

MIMETIC-LINKER-LIPID (A) (XIP) wherein MIMETIC, LINKER and LIPID (A) have the above meanings; with a compound of formula Vila or Vllb; and optionally irradiating the resulting product with UV light.

9. A compound of formula I, VI or VP according to claim 1 , 6 or 8 or a pharmaceutically acceptable salt thereof for use as a pharmaceutical.

10. A method for preventing or treating conditions or diseases as indicated above in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound of formula I, VI or VP according to claim 1 , 6 or 8 or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising a pharmaceutically effective amount of the compound of formula I, VI or VP according to claim 1 , 6 or 8 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier.

12. A compound of formula I, VI or VP according to claim 1 , 6 or 8 or a pharmaceutically acceptable salt thereof for use in the manufacturing of a medicament for use in the method according to claim 10.