CA2080002A1 - Composition for macrophage activation - Google Patents

Abstract

The present invention provides novel lipophilic disaccharide-dipeptide compounds. The compounds of the invention are preferably encapsulated into multilamellar liposomes, which can be formed from phosphatidyl choline and phosphatidyl glycerol. The compounds are effective in activating human monocytes with subsequent destruction of tumor cells. These compounds have acceptable toxicity in anticipated human dosages.

Description

2~ ?"
COMPOS ITION FOR MACROPH~GE ACT IVAT ION ;

Field of the I~vention The present invention provides novel lipophilic disaccharide-tripeptide compounds having improved - immunological efficacy, and a liposome encapsulated composition comprising said compounds.

Bac~qround of the Invention Intact microbial agents are known to have immunological effects. These effects include both immuno- adjuvant efficacy and antitumor effects, i~ both eYperimsntally induced and hllman malignanrles The active components, consisting of the peptidoglycar. cell wall skeleton and trehalose dimycolate, have been isolated from mycobacteria. These active components, ~.
especially when attached to mineral oil or squalene, are ~ -known to be as active as the intact microbial agents.
See, for example, E. Ribi et al., Ann. NY Acad. Science, `; U.S.A., 277, 228-236 (1976).
The cell wall skeleton of Nocardia rubra (N-CWS) is also known to activate macrophages. Given intravenously, oil-attached N-CWS can cure some rats ~,! 25 with experimental pulmonary metasta9es. See, for example, S. Sone et al., Cancer ImmunolooY
ImmunotheraPyl 12, 203-209 (1982). Smaller, water soluble monomeri~c units of the cell wall peptidoslycans have been demonstrated to be adjuvant active. Adjuvants -!, 30 are compounds causing stimulation of the immune system . of a human or other mammal which result in an increased production of antibodies and in an enhancement of the protective reaction of the organism, e.g., against :
infection. Such monomeric units have also shown --1 35 antitumor activity when given intravenously, for example, in mice bearing the Lewis lung carcinoma or the MCA mammary carcinoma. See, for example, G Sava et ~ --al., Cancer ImmunolooY Immunotherapy, 15, 84-86 (1983).

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WO91/16~7 PCT/US~1/016 2~ ?d 2 The active components of these organisms have been isolated, purified, and synthesized. These components are glycopeptides constituting a kroad class of organic compounds which include a sugar part and a peptide part.
Glycopeptides found in the cell are known to retain not only adjuvant activity, as evidenced by their ability to increase the antibody response, but also possess antitumor activity, as evidenced by their ability to activate macrophages to become cytotoxic and destroy lO tumor cells. For example, muramyl dipeptide (MDP) (e.g., N-acetylmuramyl-L-alanyl-D-isoglutamine) and a large number of ~DP derivatives are known to have antitumor macrophage ac.ivation properties.
~oth the in vitro and in vivo antitumor activity of 15 mono- and disaccharide peptides is increased by their incorporation into liposomes. Lipophilic derivatives of immunomodulators and/or antitumor agents are ~nown.
Such compounds are useful for efficiently incorporating ~ these agents into liposomes for targeting macrophages 3 20 and activating macrophages to the cytotoxic state.
Both the intact microbial agents and many MDP
analogues have shown an undesirable level of toxicity.
Intact microbial agents, used alone or in an oil-water emulsion, such as Freund's ad~uvant, can cause an 25 increased sensitivity to histamine, granulo-ma formation, and hyperplasia of the liver and spleer.. In particular, j the administration of N-acetylmuramyl-L-alanyl-D-isoglutaminyl-k--alanyl-phosphatidylethanolamine, when given in repeated doses, has caused the undesirable 3~ toxic reaction of generalized vasculitis (See, D.G.
Brown el al. in ImmunotoxicolooY, A. Belin et al., Ed;
..~
Martin Hijhof, Pub.; 1987, pp. 219-233).
Therefore, there is a need for novel glycopeptide ~ compounds which have improved adjuvant and/or antitumor -~ 35 activity, which are readily incorporated into liposomes, and which have acceptable toxicity in dosages exceedlng anticipated effective human dosages.

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WO91/16~7 PCT/US91/01~

. . .
-'t ' Summarv of the Invention . The pres?nt invention provides novel lipophilic ~
,, disaccharide-t_ipeptide compounds. The compound of the :
invention is preferably enc2psulated into muLtilamellar liposomes, whi_h can be formed from, for exam?la, - phocphatidyl choiine and phosphatidyl glycerol. The compounds zre e-~ective in activating human monocytes.
These compounds hava acceptable levels of toxicity in dosages exceeding anticipated human dosages.
lOThe compound of the invention has the formula ( T j:

HC ~ C~

i~ 15o I c 3~
, r~ C:" 1 2 :~ O O- ' '.' ~ 1l n . :
r--NH--CH--C.Y2--CY2-c-X--NH-c~--C.Y2--o--c--R3 J 20 C-NH2 C~2-0-C-R
O O
. ;i .~ . ;~

:: or the formula (II): , .
. .

$~ ~OH ( 3 0 0=5 R
0 101 :, Y--NH - c- y - -:l 2 - c :-lz - c - x - o - r l z - r ~! - c ;J 2 - o - c - ~ 3 ~ : 35 O O--r __ 4 .. ~ : - .
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.,; . ...

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WO91/16~7 PCT/US91/01~
2^..~
wherein Rl is a (Cl-C9)alkyl group, R is a (C -C5 ) alkyl group, R3 and R4 are individually (Cc-C30)alkyl groups having about 0-4 double bonds.
X is a spacer group that does not substantially adversely affect the activity or toxicity of the compound. X is particularly a single bond, or any peptidyl residue comprising one or more amino aci~s.
is preferably any amino acid residue of the general formula: -NH-CH-R.
More preferably X is any naturally occurring aminG acid residue or an enantio~orph of any naturally occurring , amino acid residue. Particularly useful compounds of the present invention include an X group selected from the group consisting of neutral naturally occurring amino acid residues, and more particularly neutral aliphatic naturally occurring amino acids, and the enan~iomorphs of these amino acid residues. L, D, or DL
mixtures of valine and alanine, are particularly l 25 preferred~
i5 Y is any amino acid residue of the general for~ula:
;. O
-NH-CH-C-Preferably Y is any naturally occurring amino acid residue or an enantiomorph of any naturally occurring amino acid residue. Particularly useful compounds of the present invention include a Y group selected from ; the group consisting of neutral naturally occurring ` amino acid residues, and more particularly neutral aliphatic naturally occurring amino acids, and the enantiomorphs of these amino acid residues. L, D, or DL
mixtures of threonine, alanine, valine, and serine are .~ .
- particularly preferred. L-threonine and L-alanine are most preferred.
.

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WO91/16~i7 PCTtUS91/01~
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The pharmaceutically acceptable salts of the ::
compounds, and a liposome comprising a compound of the above formulas, are also within the scope of the -~ invention. In addition to the L-enantiomorphs and the -~
D-enantiomorphs, DL-mixtures of amino acids can also be used in the present compositions.
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Detailed DescriPtion of the Invention ' ' Chemical Structure of ComPounds I and II
The compounds of the present invention (Compounds I
and II) are novel lipophilic disaccharide-tripeptides.
Compounds I and II include a giucosamine (Gic) derivative having an acyl group with about 2 to-10 ~i 15 carbons attached to the nitrogen. Preferably, the acyl ;~ group has 2 carbons (acetyl) forming N-acetylglucosamine ;
(GlcNAc).
i'~ The N-acylglucosamine moiety is attached to an N-acylmuramyl moiety. The acyl functionality attached to the nitrogen of the muramyl group has about 2 to 6 carbons, preferably 2 carbons, forming an N-acetylmuramyl group (NurNAc). The alternating disaccharide GlcNAc-NurNAc is a naturally occurring disaccharide, found in bacterial cell walls as part of a polymeric glycopeptide. See, U.S. Patent No. 4,395,399.
The disaccharide moiety of Compounds I and II, N-acylglucosamine-N-acylmuramate, is bonded to the N-~ terminus of a peptidyl moiety, Y, through the lactyl --;~ ether linkage at the number 3 position on the muramyl group. The amino acid residue, Y, is bonded to D-isoglutamine (isoGln), which is bonded through the C-terminus to the group, X.
Neutral naturally occurring amino acid residues for use in the X and Y positions are OI particular interest.
35 Neutral naturally occurring amino acids include neutral --aliphatic, neutral thioaliphatic, neutral aromatic, and neutral heterocyclic amino acids. Neutral aliphatic . ~ .
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WO91/16~7 PCT/~S9t/01~
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naturally occurring amino acids include: glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), and isoleucine (Ile). Neutral thioaliphatic amino acids include: cysteine (Cys), cystine (CyS-Cys), and methionine (Met). Neutral aromatic amino acids include phenylalanine (Phe) and tyrosine (Tyr). Neutral heterocyclic amino acids includ2: proline (Pro), hydroxyproline (Hyp), and ' tryptophan (Trp). ,Other naturally occurring amino acids, include: the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu); and the basic amino acids, histidine (His), lysine (Lys) and arginine (Arg).
PreIerred pep~idyi moieties include L-aianyl-D-isoglutaminyl-L-alanine (L-Ala-D-isoGln-k-Ala); D-alanyl-D-isoglutaminyl-k-aIanine (D-Ala-D-isoGln-L-Ala);
' and D-alanyl-D-isoglutaminyl-D-alanine (D-Ala-D-isoGln-,., D-Ala). The disaccharide-tripeptide portion of the '~ novel compound may be referred to as N-acylglucosaminyl-N-acyl~.uramyl-tripeptide.
Compounds I and II differ at the lipophilic end of the compounds. The lipophilic end of the compounds of the invention comprise a derivative of glycerol substituted with two acyl groups, the acyl groups individually having between 7 and 31 carbons, preferably 25 between 13 and 24 carbons (i.e., R3 and R4 are individually (C~ - C23)alkyl groups), and about 0 to 4 i ~ double bonds, preferably about 0 to 1 double bond.
.~ Preferably both acyl g-roups have 16 carbons [Cl6] (i.e., R3 and R~ are individually (C15)alkyl groups~ to form a dipalmitoyl-glycerol (DPG) derivative. In Compound I
. the glycerol derivative is attached to the C-terminus of y~ the terminal amino acid of X, through an amide linkage:

1 ~ ~ O
ll C-NH-~ attached tO the number two carbon of the glycerol .~ backbone. In Compound II the glycerGl derivative is :~'' ... .

WO91tl6~7 PCT/US91/01 2~ ?, .~ 7 attached to the C-terminus of the terminal amino acid of Y, through an ester linkage O '. : ' -C-O-attached to the number three (sn) carbon of the glycerol ;, backbone.
,; Novel compounds of the present invention can generally be described as N-acylglucosaminyl-N-acylmuramyl-tripeptide-diacyl-glycerol compounds.
Preferred compounds are GlcNAcMurNAc-L-Ala-D-isoGln-L-Ala-DPG; -' GlcNAcMurN~.c-k-Ala-D-iso~ln-D-Ala-DPG; GlcNAcMurNAc-D-15 Ala-D-isoGIn-D-Ala-DPG; GlcNAcMurNAc-L-Ala-D-isoGln-L- ~
Ala-NHDPG; GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-NHDPG; and -GlcNAcMurNAc-D-Ala-D-isoGln-D-Ala-NHDPG.
Compounds I and II may also be used as the pharmaceutically acceptable salts of the formulas above.
Such salts include the amine salts which are derived from the epsilon amino groups of lysine and the organic acids such as citrate, lactic, malic, methane sulfonic, p-toluene sulfonic, and the like; ac well as inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and the like. Salts such as (lawer) ~ alkyl sulfate and halides can also be used-. If X or Y
¦ have a free carboxyl side chain, such as with aspartic , or glutamic acid, such salts couId include those derived 9 '' from inorganic bases such as KOH, NaOH, and the like, from NH40H, or from organic amines. For isolation or purification of the compound, pharmaceutically ~; unacceptable salts may also be used. However, only the -~; pharmaceutically acceptable, nontoxic salts can be used therapeutically, and are therefore preferred.
Li~osomes The liposomes are generally produced from phospholipids or other lipid substances and are formed ~ ~ of mono- or multilamellar hydra~ed liquid crystals.
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W09l/16~7 PCT/US91/01~ -Z.

They are customarily used in dispersions in an aqueous carrier medium. The use of liposomes incorporating Compounds I and II results in an increase in the adjuvant and anti-tumor activity. Also, an increase in humoral and/or cellular mediation immune responses is often observed. Thus, Compounds I and II are preferabiy included in liposomes.
There are a number of conventional procedures to form liposomes. Any nontoxic, physiologically acceptable and metabolizable lipid, capable of forming liposomes, can be used. The most usual lipids are the phospholipids, and notably the phosphatidyl cholines (lecithins)~ both natural and synthetic. ~hospholipia_ may also be used, for examplè, the phosphatidyl serines, the phosphatidyl inositides, or the sphingomyelines.
Other lipids can also be used, which have been described, for example, by ~. R. Hargreaves and D. W.
Deamer (Conference on Liposomes and Their Vses in Biology and Nedicine, Sept. 14-16, 1977, New York Acad.
Sci.) and in iochem., 18, 3759 (1978).
Traditional techniques and apparatus can be employed to form the liposomes according to the invention. These techniques are described in, for example, Chapter IV of , the work entitled "Methods in Cell Biology", edited by ~- ~ 25 David M. Prescott, Volume XIV, 1976, Academic Press, New York, page 33, ~t seq. -i Another method of encapsulating the active Compounds I and II into a liposome involves casting a film of phospholipid (with or without a charged lipid) by 30 evaporation from a solution in an organic solvent, and c then dispersing the film in a suitable aqueous medium.
~ In the case of lipid-soluble, biologically active . j .
compounds, that is, those which associate with the lipid ~ layers rather than with the aqueous phase of the -~ 35 liposomes, the compound is usually cast as a film together with a phospholipid, using a common organic solvent. In the case of water-soluble, biologically ~ .

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WO9l/16~7 PCT/~IS91/0l~ -2s~
. 9 active compounds, the compound is typically encapsulated :
; in liposomes by dispersing a cast phospholipid film with an aqueous solution of the compound. The encapsulated compound is then separated from the free compound by cen~rifugation, chromatography or some other suitable procedure.
The lipophilic end of Compounds I and II enhance the incorporation of the compounds into liposomes. :
Compounds I and II are preferably incorporated into a liposome having a bilayer membrane consisting essentially of 1-palmitoyl-2-oleoyl-phosphatidyl choline (PC) and diolèoyl phosphatidyl glycerol (PG) in a weight ratio of about 5:1 to 1:1, preferably about 7:3. These compounds are commercially available from Avanti Polar .~ 15 Lipids, of Pelham, Alabama. ~ `
~ Preferred methods which can be used to encapsulale ;~ Compounds I and II into a liposome are described in U.S.
~ Patent No. 4,370,349 which is incorporated herein by .~ reference. The methods comprise either: (1) dissolving the necessary substances in a suitable solvent and then freeze-drying the solution, storing the resulting freeze-dried mixture, and, when desired, reconstituting .
~ it into an aqueous liposome preparation, or (2) .~ preparing an aqueous lLposome preparation by any known method and freeze-drying the preparation. When desired, the freeze-dried product can be made up into an aqueous liposome prepara~ion. The freeze-dried mixtures disperse easily when shaken with an aqueous medium, and ~ use of the freeze dried liposomes results in liposome .. 30 preparations having a narrower size distribution than a ~ corresponding preparation obtained by dispersing a cast film. This is advantageous to the reproducibility of the therapeutic effect of liposome preparations.
Generally, the compositions in the form of liposo~es can contain, in addition to Compounds I and II, any ;l constituents, stabilizers, preservztives, exc~pients, or other active substances capable of being used in the .-. .
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WO91/16~7 PCT/US91/01 Z.~ ~ ~?~ lO
injectable solutions or emulsions presented previously for administration of muramyl peptide compounds.

Deliverv Compounds I and II, preferably incorporated into `
liposomes, may be used for their adjuvant and!or antitumor activity, and may be administered orally or parenterally, preferably by injection.
The invention relates in particular to medicinal lO ad~uvant and antitumor compositions, containing Compounds I andjor II, in association with a pharmaceutically acceptable carrier vehicle.
Compositions of this type which are particuiarly preferred are constituted by the injectable solutions A 15 containing an effective immunomodulating amount of the product of the invention. Sterile solutions in an ; aqueous, preferably isotonic liquid, such as saline isotonic solutions or isotonic solutions of glucose, are advantageously used for this purpose. A simple solution 20 in distilled water can also be used. It is also possible to use injection media containing an oily phase, especially water-in-oil emulsions. Such emulsions are obtained in particular with metabolizable - vegetable oils, such as are described in the French Patent Applic~tion No. 75--04003. That French patent application corresponds to the U.S. co-pending patent ~ application Ser. No. 656,738 of Audibert et al., filed ~ -;~ on February 9, 1976, based on said French priority patent application Ser. No. 75-04003. The preferred 30 carrier vehicle is the freeze-dried liposomes described ~ above.
. The adjuvant and antitumor compositions of the `~ invention may also be administered in various forms, by using for this purpose vehicles suitable for the 35 selected method of administration. For example, unit dosage forms will be used in the form of cachets, -compressed tablets or hard or soft gelatine-capsules, . . ' . .

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WO91~16~7 PCT/US91/01~
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1 1 '" ~ ~ ' - for oral administration, and aerosols or gelc for the application to mucous membranes.
The compositions may also be in lyophilized form so as to permit the extemporaneous preparation of the adjuvant and antitumor compositions. A pharmaceutically advantageous form comprises unit doses of about 200 micrograms to lO milligrams of Compound I or II per meter~ of body surface area.

.. . .
Manufacture Compounds of the invention, for example, 4-0-[2-acetamido-2-deoxy-~-D-glucopyranosyl]-2-acetamido-2-deoxy-3-c)-LD-~-propanoyi-L-alanyi-~J-isogiulaminyi-L
alanyl-2,3-dipalmitoyl-sn-glycerol]-D-glucopyra~ose . 15 (GlcNAcMurNAc-k-Ala-D-isoGln-L-Ala-DPG (Compound IIA), j may be prepared from commercially available materials, in about nine major steps. The steps do not necessarily have to be performed in the order described as will become apparent from the description below.
The first step involves the preparation of a blocked amino acid-diacyl glycerol. This is the lipophilic portion of Compound II attached to the C-terminus of the spacer group X, e.g., an amino acid reqidue. For example, in one embodiment this residue would be ~ 25 blocked-L-alanyl-2,3-dipalmitoyl-sn-glycerol.
~ The blocked amino acids or peptides employed as starting materials in the synthesis are either ` commercially available in the blocked form or are obtained by known methods of peptide chemistry.
q 30 Blocking groups or protecting groups that can readily be .,~ .
split off are those known from peptide and sugar chemistry. For hydroxy groups the following are suitable examples: acyl radicals, for example lower alkanoyl radicals, such as acetyl; aroyl radicals, such as benzoyl; and especially radicals derived from 1~ carbonic acid derivatives, such as benzyloxycarbonyl or ;~ lower alkoxycarbonyl, or alkyl, especially tert-butyl, :.' ;.
. .,; .

WO91/16~7 PCT/US9t/016~
2~ 12 benzyl, optionally substituted by nitro, (lower) alkoxy or by halogen, triphenylmethyl or tetrahydropyranyl, each optionally substituted by halogen or by lower alkoxy such as methoxy, or optionally substituted alkylidene radicals that bond the oxygen atoms in ~he ~-and 6-position. Such alkylidene radicals are preferably a lower alkylidene radical, e.g., the methylidene, isopropylidene, or propylidene radicals, or alternatively, an optionally-substituted benzylidene radical.
For blocking C-terminal carboxy groups, suitable moieties include tert-butyl, benzyl, or benzhydryl. For `~
protection of free amino ~roups, tert-butyloxycarbonyl ; or benzyloxycarbonyl groups can be used.
These blocking groups can be cleaved in a manner known in the art, such as acid hydrolysis. Benzyl or t benzylidene radicals also can be removed by hydrogenolysis, for-example using hydrogen~in the presence of a noble metal catalyst, such as a palladium , 20 or platinum catalyst.
The second step in the preparation of a compound of the invention involves removal of the blocking group to form X-diacyl-glycerol, where X, for example, is an amino acid residue as described above, such as L-25 alanine. For example, a preferred component is L-alanine-2,3-dipalmitoyl-sn-g'ycerol (L-Ala-DPG).
The third step involves isolation of the disaccharide moiety GlcNAcMurNac. Several methods are ~! known in the art for obtaining this disaccharide moiety.
~ 30 One method involves the isolation from a suitable Y bacteria, for example Micrococcus lysodeikticus (dried cells are commercially available from Sigma Chemical ~ Co., St. Louis, MO). The disaccharide that is obtained - ~ is N-acetylglucosaminyl-N-acetylmuramate. The isolation 35 Gf this disaccharide from a biomass of Micrococcus 3 lysodeikticus is known and described in the literature.
~ It involves enzymatic hydrolysis of the cell walls ~ -.. .

WO91/16~7 PCT/US91/016~
2~

obtained from the biomass of ~icrococcus lysodeikticus by means of trypsin and lysozyme and a further purification in a column packed with Dowex~ 1 X 8 ~-(acetate form) 200-400 mesh (O. HoshinG, U. Zenavi, P.
Sinay, R. W. Jeanloz, J. Biol. Chem. 24~, No. 2, 381 ~- (1972); and N. Sharon, T. Osawa, ~. M. Flowers, R. W.
~; Jeanloz, J. ~iol. Chemis_rY, 241, 223 (1966). Also, see, U. S. Patent No. 4,427,659, which is incorporated herein by reference. A second method by which the disaccharide moiety GlcNAcMurNAc and disaccharide peptides may be obtained is ~y means of total chemical synthesis. See for example S. Kusomoto et al., Bull. Chem. Soc. Jpn., 55, 1414 (i986); ~. Kiso et 21.-, CarbohYdrale Res., iû~, 253 (1982); and A. Hasegawa et al., CarbohYdrate Res., 100, 234 (1982); S. Kusumoto et al., Tetahedron Letters, 45, 44û7 (1978); and R. Fhyuri et al., Aqric. Biol.
. Chem., 50 2561 (1986). A third method by which the disaccharide moiety, preferably linked to appropriate amino acid residues, may be produced is through the use '~! 20 of recombinant DNA technology.
Enzymatic methods have also been described, by which di~accharide peptides may be obtained. Such methods generally yield compounds containing the natural L-meso-2,5-diaminopimelic acid at the third amino acid posi~ion herein designated by "X". See, for example, S. Rawata et al., Aqric. Biol. Chem., 48, 1783 (1984); D. Keglevic , et al., Biochimica et BioPhYsica Acta, 585, 273 (1979).
; Through the use of an enzymatic hydrolysis using aamma- -D-glutamyl-meso-diaminopimelate endopeptidase I isolated from Bacillus sPhaericus 9602 or other appropriate bacterium, it is also possible to enzymatically isolate ~j~ the disaccharide dipeptide GlcNAc-~lurNAc-L-Ala-D-isoGln.
[See M. Guinand et al., Eur. J. Biochem., 143, 359 - (1984)]. Reference is also made tO U.S . Patent Nos.

4,395,339; 4,515,891; and 4,545,932; incorporated herein by reference.

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WOsl/16~7 PCT/US91/01 In the disaccharide isolated above, Rl and R2 areboth -CH3, forming acetyl groups on both the muramyl and ~ glucosamyl functionalities. The analogous compounds, - where Rl is a (C~-Cg)alkyl group and R2 is a (C!-C5)alkyl S group, can be prepared by methods known in the art. For example, the acetyl group can be hydrolyzed by a strong base, for example, as described in P. H. Gross and R. ~.
Jeanloz, J. Or~. Chem., 32, 2761 (1967). Then an acylating agent, corresponding to the Rl or R2 which is desired to be introduced, such as an acid anhydride or chloride, may be used to attach the desired Rl or R2 group to the muramyl or glucosaminyl functionality.
The next step involves the ~repdraLi~ f th~
dipeptide-Y-D-isoglutamine, which is blocked on both ends. For example, in Compound IIA where Y is L-alanine, BOC-L-alanyl-D-isoglutamine, commercially available from United States Biochemical Co. of Cleveland, Ohio (USLC), must be treated in a manner known in the art to terminate the C-terminus isoglutamine residue with a suitable blocking agent, such as a benzyl ester (-OBn). BOC refers to N-tert-! butoxycarbonyl, a blocking group. Thus, BOC-L-Ala-D-isoGln-OBn is preferably formed.
The next step involves the removal of the blocking ~5 group from the alanine by a known method to form, for example, L-Ala-D-isoGln-OBn. The next step involves coupling the N-acylglucosamine-N-acylmuramyl ; functionality with the alanine-isoglutamine moiety. The condensation reaction is conducted in an inert solvent medium, in the presence of a condensation agent, ~; preferably Woodward's Reagent X (N-ethyl-5-phenyl-l isoxazolium-3'-sulphonate), at a temperature of about Y 0C to 25C in one stage. See, U. S. Patent No.
~- 4,395,399-.~ 35 The next step involves removal of the blocking group by conventional means to form the unblocked ,~ disaccharide-dipeptide, for example, 4-0-[2-acetamido-2-`, .

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WO9l/16~7 PCT/US91/01 Z~

deoxy-~-D= glucopyranosyl]-2-acetamido-2-deoxy-3-0-[D-2-; propanoyl-L-alanyl-D-isoglutamine]-D-glucopyranose (GlcNAcMurNAc-L-Ala-D-isoGln).
The final step involves the coupling of the unblocke~ disaccharide-dipeptide, GlcNAcMurNAc-L-Ala-D-isoGln, with the amino acid-diacyl glycerol component by conventional techniques to _orm the Compound IIA.
A similar compound to Compound IIA, 4-0-[2-acetamido-2-deoxy-~-D-glucopyranosyl]-2-acetamido-2-10 deoxy-~-0-[D-2-propanoyl-L-alanyl- -isoglutaminyl-D-alanyl-2,3-dipalmitoyl-sn-glycerol]-D-glucop~ranose (GlcNAcMurNAc-k-Ala-D-isoGln-D-Ala-DPG) ~Compound IIB) is preparea by tne same procedure described for Compound - IIA, except that the final synthetic step involves the coupling of the disaccharide dipeptide, GlcNAcMurNAc-L--~ Ala-D-isoGln, to D-Ala-DPG rather than to k-Ala-DPG.
A compound according to Formula I, where the lipophilic group is attached via an amide linkage (designated NHDPG), for example, 4-0-[2-acetamido-2-deoxy-~-D-glucopyranosyl]-2-acetamido-2-deoxy-3-0-[D-2-propanoyl-L-alsnyl-D-isoglutaminyl-k-alanyl-2-(1,3-~ dipalmitoyloxy) propylamide~-D-glucopyranose, .i (GlcNAcMurNAc-L-Ala-D-isoGln-L-Ala-NHDPG) (Compound IA), is prepared by the same general app~oach used for the synthesis of compounds IIA and IIB, i.e., by the coupling of the disaccharide dipeptide, GlcNAcMurNAc-k-Ala-D-isoGln, to the lipophilic alanine amide, k-Ala-NHDPG. The disaccharide dipeptide is prepared as described in the synthesis of Compound IIA, while the ~;~j 30 required amide is prepared by the following three-step synthetic sequence: -~- (A) The blocked amino acid, BOC-k-Ala, is coupled with serinol (SerOH) by conventional means to give BOC-k-Ala-SerOH.
(B) BOC-k-Ala-SerOH is reacted with two moles of palmitic acid to yield the dipalmitate, BOC-L-i~ Ala-NHDPG.

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WO91/16~7 PCT/~S9l/~16~
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(C) The BOC-protecting group is removed by conventional means to form the unblocked lipophilic alanine amide, L-Ala-NHDPG.
The final synthetic step involves the coupling of ; 5 the GlcNAcMurNAc-k-Ala-D-isoGln with the lipophilic amide by conventional techniques to form the Compound IIA.
Compound I or II is preferably encapsulated into liposomes as described herein above. Preferably the compound of the invention is combined with phosphatidyl choline and phosphatidyl glycerol. Typically the phospholipids are dissolved in tert-butanol at a ; concentration OI about 100 mg per ml. Appropriate amounts of the PC and PG in tert-butanol are mixed to give a weight ratio of about 7:3. Compound I and/or II
is weighed out and added to a given volume of the lipids , to give a inal concentration of, for example, about 1 mg per 5 ml. ~he material is then passed through a millipore filter and the composition is dispensed into vials. The vials are frozen, typically at -20C and ~ ! . ' ~' lyophilized typically at about 20C for 18 hours. The -~ vials are then sealed under an inert gas, such as argon.
The present invention i9 further de~cribed by way of the following non-limiting examples:

PREPARATION OF GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-DPG
EXAMPLE 1 ~ -;` BOC-L-Ala-DPG
In a 25 ml-round bottomed flask (RBF) was placed --208.64 mg (1.103 n~ol) of BOC-L-alanine, 570.0 mg (1.002 mMol) of 1,2-dipalmitoyl-sn-glycerol (DPG, Sigma), 63.14 mg (0.517 mMol) of 4-dimethylaminopyridine (DMAP) (Aldrich Chemical Co., Milwaukee, WI), and 230.04 mg (1.200 mMol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiin~ide hydrochloride (EDCI).BOC is the abbreviation for N-tert-butoxycarbonyl, a blocking group. Methylene chloride (CH2C12) was added : ' . - ... . .

WO91~16~7 PCT/US91/01~

17 2~-$~
bringing the final volume to 14 ml. The mixture was stirred in an ice-water bath for one hour, then at room temperature (RT) overnight.
After stirring overnight, the solvent was removed oa a rotary evaporator under aspirator vacuum to yield a white solid, which was partitioned between 20 ml of ethyl acetate (EtOAc) and 10 ml of water. The water layer was extracted with another 20 ml of EtOAc. The organic fractions were combined and extracted with 2 x 20 ml of saturated aqueous sodium bicarbonate (NaHCO3) followed by 2 x 20 ml of water and then dried over sodium sulfate (Na2SO4). The solvent was removed on a rotary evaporator to yield 648 mg (h7~) of BOC-L-Ala-DP~
as a white solid. .
, 15 EXAMPLE 2 ~ -L-Ala-DPG
~ 630 mg (0.85 mMol) of BOC-L-Ala-DPG was dissolved in 3 15 ml of CH2Cl2 to which was added 5.0 ml of trifluoroacetic acid ~TFA). The solution was stirred at RT for two hours, then concentrated to dryness on a rotary evaporator to yield a tan oil that was dissolved ln 10 ml of hexane and concentrated to dryness on a 3 rotary evaporator. The process was repeated several times to remove the last traces of TFA. This material was then dried under high vacuum to yield 606.7 mg of L-- ~ Ala-DPG trifluoroacetate as an off-white solid.
: .

GlcNAcMurNAc Dried cells (15.0 grams) of Micrococcus l~sodeikticus (commercially available from Sigma Chemical Co., St. Louis, MO), was suspended in 200 ml of ;~ distilled water and disrupted by stirring at high speed 35 with 250 g of 0.1 mm glass beads for 90 minutes at 4cC.
The cell wall skeletons (CWS) were removed from the glass beads by decantation and then centrifuged at 1200 . , .
'' .

WO91/16~7 PCT/US9t/01 2~:'r,?~r~?~ . ..
1~ :
x g for 30 minutes. The supernatant was removed from the pellet ~intact cells), then centrifuged at 10,000 x g ~
for 50 minutes. The supernatant was removed and the ~ -resulting pellet (crude CWS) was washed three times by suspension in 100 ml of distilled H2O and centrifugation at 10,000 x g for 70 minutes. The resulting pellet was suspended in 150 ml of distilled water and then placed in a boiling water bath for 30 minutes.
After cooling to ambient temperature, the resulting slurry was centrifuged at 10,000 x g. The supernatant was removed and the pellet slurried in 60 ml of 0.5 M
ammonium acetate (NHLOAc) buffer (pH 7.60). The resulting slurry was treated with i0.G mg of porcine pancreas trypsin (Sigma, 14,600 BAC units/mg), and incubate~ at 37C for 20 hours. After several washes with distilled H2O, the CWS pellet was slurried in 60 ml of 0.05 M
NH40Ac buffer ~pH 6.30), treated with egg-white lysozyme -(Sigma, 56,000 units/mg, 10.0 mg), and incubated at 37C
~ for 19 hours.
`~ 20 The crude preparation was dialyzed to remove the enzymes and undigested cell walls. Final purification was achieved by ion exchange chromatography on Dowex~-1 j resin (acetate form) by elution with an acetic acid (HOAc) gradient. The column fractions were pooled based on W absorbance and thin layer chromatography (TLC) (silica gel, 50:39:8:3 CHCl3/MeOH/H2O/NH~OH, 5~ H2SO~/EtOH
I and heating). Positive identification of the -~ disaccharide product was obtained from colorimetric-analysis of muramic acid and total hexosamines, and fast atom bombardment mass spectrometry. Approximate yields were 120 mg GlcNAcMurNAc from 15 g of dried cells.
- :-~ EX~/IPLE 4 .~J BOC-L-Ala-D-isoGlneOBn .. ',`':.. .
~ 35 henzyl alcohol (77.0 mg, 0.71 ~ol), DMAP (33.0 mg, I 0.27 mMol), and BOC-L-alanyl-D-isoglutamine (VSBC, 159.0 mg, 0.50 mMolj were dissolved in 5 ml of CH2Cl2 and 2 ml .. . ... .

, .

WO9l/16~7 PCT/US91/0l~

1 9 2 f~3~
of N,N-dimethylformamide (DMF). This solution was cooled in an ice-water bath to 4C, treated with EDCI
(118.0 mg, 0.61 mMol) and stirred at 4'C for 30 minutes, -then at room temperature for 15 hours. After removing 5 the solvents in the rotary evaporator, the residue was partitioned between 20 ml of EtOAc and 10 ml of H2O. The layers were separated and the aqueous layer extracted with another 20 ml of EtOAc. The organic fractions were combined, then successively extracted with saturated NaHCO3 ~2 x 20 ml) and H2O (2 x 20 ml). After drying over Na~SO4, the solvent was removed on the rotary evaporator leaving a waxy solid, which was recrystallized from f Et~Ac-petroleum ether to yield 141 mg (69%) OL BOC-L-Ala-~-isoGln-OBn as white fluffy solid.

L-Ala-D-isoGln-OBn .. ~ . . .
BOC-L-Ala-D-isoGln-O~n (120 mg, 0.294 mMol) was treated with 10 ml of lN HCl/HOAc and the resulting solution stirred at RT for 2 hours. The solvent was then removed on the rotary evaporator to yield a colorless oil, which was taken up in 3 ml of MeOH, then precipitated by the dropwise addition of 20 ml of diethyl ether. After stirring for one hour at RT, the ~ 25 product wa~ collected on a filter,-washed with ether, f then dried under high vacuum to yield 88 mg of the ;~ hydrochloride salt of L-Ala-D-isoGln-OBn as a white solid.

30 EX~MPLE 6 CouPlina of GlcNAcMurNAc with L-Ala-D-isoGln-OBn A total of 200 mg of GlcNAcMurNAc (MW 496.47, 0.405 -~ mMol) was dissolved in 15 ml of DMF and then treated - with 0.95 ml of a solution containing 42.94 mg/ml of 35 triethylamine (TEA) in DMF (0.403 ~Mol). The solution was cooled with magnetic stirring in an ice bath and then treated with 139.63 mg t95~ pure, 0.524 mMol) i ff - ~ . - . ~ . . . . .
~r.f 6~7 PCT/US91/01 z~ 20 Woodward's Reagent K. The slurry was then stirred in an ice-water bath for one hour, then at room temperature -for 10 minutes. Then a solution containing 152.3 mg --(0.443 mMol) of the HCl salt of the L-Ala-D-isoGln-OBn in 8.0 ml of DMF to which W25 added 1.05 ml (0.443 ~Vol) of the TEA/D~F solution was added via a pressure ; -;
e~ualizing funnel over a period of 10 minutes. The solution was stirred at RT for 18 hours and then allowed ; to stand for an additional 96 hours. The reaction was followed during this time by TLC and allowed to go as far as possible to completion.
The D~F was removed in a rotary evaporator under high vacuum ~approximately 50 micronsj al 25 C IO yieid a reddish oil that was further dried under high vacuum; -; lS The oil was taken up in 5 ml of H20 and applie`d to a ;'? 1.7 x 7 cm column of Dowex~ 1 X 8 resin (200-4û0 mesh, acetate form). The column was washed with 50 ml of H2O
and the entire colorless eluate applied to a 1.7 x 7 cm ! column of Amberlite~ IR-120 P resin (16-20 mesh, Ht form). The column was washed with 50 ml of H~O and the eluate and washings were combined. This material was ~ taken to dryness in a rotary evaporator under aspirator r ~acuum at 25C to yield a colorless oil. This was dried under high vacuum (50-75 microns) overnight during which ~- 25 time it solidified to a glassy solid. This was taken up in 20 ml of ~2O and lyophilized to yield 181 mg of GlcNAcMurNAc-L-Ala-D-isoGln-OBn as a snow white fluffy -~
solid.
i . . .

GlcNAcMurNAc-L-Ala-D-isoGln . ~. ... .
1~ The protected material prepared (170 mg) in Example 6 was dissolved in a solution of H10 (30 ml) and acetic acid (1.0 ml). The solution was added to 100 mg of 5 Pd/C (by weight of the palladium, C is powdered charcoal, from Matheson, Coleman, and Bell of Norwood, Ohio) in a 500 ml Parr hydrogenation bottle and , ':

WO 91tl63"7 PCI /US91/01664 2r ~ ?

hydrogenated at 20 PSIG for 24 hours. The catalyst was removed and washed with water ~3 x 10 ml), and the filtrate and washings were combined and lyophilized to yield 150 mg (100~) of GlcNAcMurNAc-L-Ala-D-isoGln as a white solid. The product was further dried under high vacuum for 48 hours then tightly capped and stored at - 4C.
. ~
~: EXAMPLE 8 ..
Cou~llna of GlcNAcMurNAc-L-Ala-D-isoGln to L-Ala-DPG to .~ vield ~lcNAcMurNAc-~-Ala-D-isoGln-L-Ala-DPG ~IIA~
1-Hydroxybenzotriazole (HOBT) (31.35 mg, 0.232 mMol) and EDCI (44.26 m~, 0.231 mMol! were placed in a 5Q m]-RBF. To this was added a solution containing the . 15 disaccharide dipeptide as prepared in Example 7 (139.13 , mg, 0~20 mMol) in 7 ml DMF and 5 ml CH2Cl2. The resulting r.~ solution was stirred at RT for 30 minutes.
Y A triethylamine (TEA) solution was prepared by dissolving 202 mg (0.28 ml) of TEA in DMF and adjusting the final volume to 10 ml.
L-Ala-DPG (150.8 mg, 0.20 mMol) was dissolved in 1 : ml of CH2Cl2. DMF (1 ml) was added, followed by 1 ml of the TEA solution. The resulting solution was added to the activated di~accharide dipeptide solution, the - 25 vessel was securely capped and stirred for 72 hours.
: The reaction was followed by TLC and stopped at 72 hours. The reaction mixture was then split into two portions, one of 5 ml, the other of 10 ml. These samples were concentrated to dryness on a rotary evaporator at room temperature under high vacuum. They were then further dried in a desiccator for 24 hours during which time both samples dried to yellow-orange solids.
For purification, the smaller portion was partitioned between 25 ml H2O and 25 ml EtOAc. The layers were separated, and the organic layer was extracted with 2 x 10 ml of H~O and the washes added to the aqueous layer. The aqueous fraction was then washed with 25 ml of EtOAc, the layers were separated, and the .: .
.. i~ . .
.. , , :
~ .

, .. . . . . . . : . . - ': . ' ' . .

WO91/16~7 PCT/US91/01~
2 , J `' `' aqueous layer was concentrated to half volume on a - rotary evaporator, then extensively dialyzed against H~O through an Amicon YM-5 membrane at 30-35 psi.
TLC analysis of the inner dialysate showed a single spot. This material was then filtered through ~hatman~ -#2 paper then lyophilized to yield 35 mg of product as a cream colored solid.
.. ..
~ARGE SCA~E PREPARATION OF alcNAcMurNAc-L-Ala-D-isoGln-, L-Ala-DPG (IIA) BOC-~-Ala-DPG
1,2-Dipalmitoyl-sn-glycerol (Sigma, 2.845 g, 5.0 mMol), BOC-k-alanine (USBC, 966 m~, 5.1 mMol), and DMAP
(Aldrich, 357 mg, 2.93 mMol) were dissolved in 50 ml of $ CH2Cl2. EDCI (Sigma, 1.174 g, 6.12 mNol) was added, and the solution was stirred at RT for 17 hours. After removal of the solvent on a rotary evaporator, the residue was partitioned between 150 ml of EtOAc and 75 ml of H20, the layers separated and the organic layer extracted with saturated aqueous NaHCO3 ~3 x 50 ml), then ~ with H20 (3 x 75 ml). After drying over Na2SO~, the -~ sol~ent was removed on the rotary evaporator and the ¦ 25 residue further dried-under high vacuum to yield 3.59 g ! (97%) of product as a slightly off-white solid.
TLC (silica; CHCl3/MeOH/H20, 130:45:7; HCl spray, ~, then ninhydrin) of the product revealed a single spot of Rf 0.95.
:! 30 3~ L-Ala-DPG (IV~
The protected alanine ester (III) (2.50 g, 3.38 ~; mMol) was dissolved in 75 ml of CH2Cl2, then treated with 25 ml of TFA. After standing at room temperature for 35 two hours, the solvents were removed on the rotary ~:
evaporator to leave a tan oil that was repeatedly taken , up in 20 ml-portions of hexane then concentrated to dryness on the rotary evaporator. After extensive .i '.
` ' ` ' : ': ' ~ .: - .: .- . - . . . . i. : . :,, . . ,, :. , ,; . :.. : ,; . . , , -:

WO91/16~7 PCT/US~I/01~
. ~
23 Z~
- drying under high vacuum, 2.44 g (95.7%) of Compound IV
was obtained as its trifluoroacetate salt.
' ' BOC-L-Ala-D-isoGln-08n ~V) .
BOC-L-Ala-D-isoGln (USBC, 1.587 g, 5.0 mMol), benzyl alcohol (540.7 mg, 5.0 mMol), and DMAP (30s mg, 2.5 mMol) were dissolved in 40 ml of CH~Cl, and 10 ml of DMF, and the resulting solution was cooled in an ice-water bath to 4C with magnetic stirring. EDCI (1.150 g, 6.00 10 mMol) was added, and the reaction stirred in the ice bath for one hour, then at room temperature for 17 hours. After removal of the solvents on the rotary evaporator, the oily residue was par~itioned between 5G
ml of H2O and 150 ml of EtOAc, the layers separceed, and 15 the organic layer further extracted with saturatéd 1- aqueous NaHCO3 (3 x 50 ml) and H2O (3 x 50 ml). After i, drying over Na2SO4, the solvent was removed on the rotary evaporator to yield a colorless oil that was further dried under high vacuum, during which it solidified to a 3 20 waxy solid. Recrystallization from EtOAc-hexane yielded 1.318 g (65%) of Compound V as a snow-white solid.
3 A TLC (silica; EtOAc/pyridine/HOAc/H2O, 30:2:0.6:1;
~- HCl spray, then ninhydrin) of the product revealed a single spot of R~ 0.90.
1 L-Ala-D-~soGln-OBn (VI) :~ The protected dipeptide ester V (2.08 g, 5.10 mMol) was treated with 100 ml of lN HCl/HOAc, and the resulting solution was allowed to stand at room -30 temperature for two hours. After removal of the solvents on the rotary evaporator and further drying ~-~ under high vacuum, the product was crystallized from --8~ NeOH-ether to yield 1.68 g (9~.8%) of Compound VI as its ~ hydrochloride salt.
-~- 35 - Preferred Method of Preparation of GlcNAcMurNAc (VII
~i Cell Disruption ;~; - .:
':

? : - -WO91/16~7 PCT/US91/01~ ~
2~ 3~

Micrococcus lYsodeikticus (Sigma, 200 g), in the form of dried cells, was suspended in distilled water - for 24 hours, then disrupted with a Microfluidics Corporation laboratory Microfluidizer6 (Nodel M-llOY).
-; 5 This was driven by a Powerex~ GI-25 air compressor 2~ 2 normal operating air pressure of 82 PSIG, which resulted in a hydraulic pressure of l9,000 PSIG. Approximately ten passes through the interaction chamber at these operating conditions were required to effect nearly total (95-98~) disruption of the cells. The resulting slurry was then centrifuqed for 30 minutes at l000 x g, ` the supernatant (containing CWS) removed from the sma1l pellet (intact cellsj, th~n c~ntrifuged for t~o hours at ..
l0,000 x g. The resulting supernatant was carefu~y ; l5 removed by decantation and discarded. The white, somewhat greasy, pellet was suspended in 1,500 ml of distilled water, then carefully warmed with magnetic ;stirring to 90C and held at that temperature for 30 minutes. After cooling to RT, the slurry was 20 centrifuged at l0,000 x g for 2.5 hours, and the - -supernatant carefully removed and discarded. The resulting pellet ~containing CWS) was then washed once by suspension in a total of 2000 ml of water, followed by centrifugation at l0,000 x g for 2.5 hours.
Trypsin Treatment The pellet from above was suspended in 2000 ml of 0.05 M sodium phosphate buffer (pH 7.60) and the pH of the slurry corrected by 7.60 by the addition of l0 N
NaOH. Trypsin (porcine pancreas, type IX, 300 mg, 17,000 BAEE units/mg protein) and toluene (l0 drops) were added and the resulting slurry incubated at 37 C
for 20 hours. The pH was then corrected to 7.60 with l0 N NaOH, an additional l00 mg of trypsin was added, and .
~35 the mixture again incubated at 37C for 24 hours. The - slurry was then centrifuged at l0,000 x g for 2.5 hours, the supernatant carefully removed and discarded, and the .

.
: ~ .

, . . , . - . . ; . , , .. : .
. . . ......... . . . . . . .. . . . . . . . . .

WO91/16~7 PCT/US91/01 white pellet (containing deproteinized CwS) washed several times with water by suspension-centrifugation until a ninhydrin test of the supernatant was negative.

Lysozyme Diqestion The pellet (i.e., deproteinized CWS) from above was -suspended in 1500 ml of 0.1 M NH40Ac (pH 6.30), and the pH of the slurry was corrected to 6.30 with dilute HOAc.
Lysozyme (egg-white, 400 mg, 49,000 U/mg) and toluene (5 drops) were added, and the slurry was incubated at 37CC
for 24 hours, during which time the slurry cleared up considerably. Then, an additional 200 mg of lysozyme was added, and the mix~ure was incubated for anothe~ 24 hours at 37C. The reaction mixture was then 15 centrifuged at 10,000 x g for 2.5 hours. The clear, yellowish supernatant was removed from the small pellet, then dialyzed through an Amicon PM-10 membrane. The colorless outer dialysate was concentrated to dryness on q the rotary evaporator, and the xesulting residue repeatedly dissolved in water, then concentrated to dryness in order to remove the last traces of the NH6OAc buffer. The yellow syrupy residue was finally dissolved in 400 ml of water, then lyophilized to yield 20.8 g of a yellow, gummy solid.
` 25 , Purification of Disaccharide ~- The entir~ 20.8 g of the lysozyme digest from above : was dissolved in 100 ml of water, then applied to a 4.3 x 63 cm column of Dowex~ 1 (acetate form, 200-400 mesh) resin. The column was then eluted with a gradient comprised of 4000 ml of H2O (starting) and 4000 ml of 0.8 - N HOAc (final) at a flow rate of 3.5 ml/minute.
` Fractions of 21 ml were collected and analyzed by TLC as described in Example 3A. The desired fractions were 35 then pooled, concentrated to dryness on the rotary --evaporator, then redissolved in water and lyophilized to , .

: . : ~. ~ : . . . ,: .. . .

WO91/16~7 PCT/US91/01664 z ~ 26 yield 2.60 g of crude product as a slightly off-white solid.
For final purification, the 2.60 g of crude ~-disaccharide was dissolved in 25 ml of water, then --,5 applied to a 3 x 55 cm column of Dowex~ l (acetate form, ~
200-400 mesh) resin. The column was then eluted with a ~-gradient comprised of 2000 ml of H2O (starting) and 2000 --~
ml of 0.8 N HOAc (final) at a flow rate of 3.0 ml/minute. Fractions of ca. 2l ml were collected and were analyzed by TLC as above. The desired fractions were pooled, concentrated to dryness on the rotary evaporator, then redissolved in 50 ml of water and lyophilized to yield 2.243 g of pure product as a snow-white solid.
GlcNAcMurNAc-L-Ala-D-isoGln-OBn (VIII) Prior to use, the DMF was dried over 4A molecular sieves, then distilled from ninhydrin. The TEA was distilled from sodium hydroxide (NaOH) pellets.
Woodward's Reagent K was purified by dissolving 3.0 g of the commercial material (Aldrich) in 15 ml of lN HCl, ? filtering through Whatman #2 paper, then precipitating by the addition of 120 ml of acetone. After filtering and washing with l00 ml of acetone, the reagent was dried under high vacuum for several hours.
The disaccharide Compound vII (2.00 g, 4.028 mMol) , was dissolved in l00 ml of DMP, treated with TEA (0.62 d~ ml, 447.5 mg, 4.431 mMol), cooled in an ice-water bath to near 4C, then treated with Woodward's Reagent K
, f30 (95~, l.397 g, 5.24 mMol). The resulting slurry was stirred in the ice-water bath for one hour, then at room temperature for l0 minutes. Then, a solution containing ~ the dipeptide benzyl ester (VI) (1.523 g, 4.43 mMol) and j TEA (447.42 mg, 0.616 ml) in 50 ml of DMF was added via -a prèssure equalizing addition funnel over a period of ; 30 minutes. After the addition was completed, the reaction mixture was stirred at room temperature for a ~
,.i, ,:
~ ~.

WO91/16~7 _ ~P ~ ~S91/01 q total of 120 hours, during which the progress of reaction was monitored by TLC (silica;
CHCl3/MeOH/H2O/NH4OH, 50:25:4:2; 5~7 H7SO~/EtOH, heat).
The solvent was then removed on the rotary evaporator 5 and the oily residue further dried under high vacuum.
This was then taken up in 50 ml of H2O, then applied to a 2.5 x 17 cm column of DG~ex l x 8 resin (200-400 mesh, acetate form) and eluted with 500 ml of H2O. The entire eluate was concentrated to ca. 50 ml, then applied to a 2.5 x 17 cm column of Dowex 50 x 8 resin ~l00 mesh, H
form) and eluted with 500 ml of ~2O. The eluate was concentrated tO ca. 50 ml, then lyophilized to yield 2.25 g (71~) of Compound VIIl as a snow-white solid.
'.
: 15 GlcNAcMurNAc-L-Ala-D-isoGln (IX) The disaccharide dipeptide benzyl ester (VIII) (2.20 g, 2.80 m~ol) was dissolved in 150 ml of H2O and 3.0 ml ; of glacial acetic acid. To this was added 300 mg of 5%
Pd/C, and the resulting slurry was hydrogenated at room temperature and 40 PSIG for 40 hours. The catalyst was then removed by filtration through a Celite pad, washed with H2O (3 x l0 ml), and the filtrate and washings combined, concentrated to ca. 50 ml, then passed through a l ml column of Detoxi-Gel~ (Pierce) at a flow rate of 8 ml/hr. The column was washed with l0 ml of H2O, and the eluate and washings were combined, then lyophilized - to yield l.86 g (95.5~) of Compound IX as a white powder.

` 30 GlcNAcMurNAc-L-Ala-D-~soGln-L-Ala-DPG (IIA) The DMF and TEA used in this preparation were ` purified as described in the preparation of VIII. The ~i disaccharide dipeptide IX (1.531 g, 2.20 mMol) was r ' dissolved in 70 ml of DM.F, then diluted with 50 ml of CH2Cl2. To this was then added HOBT (Aldrich, 387.4 mg, 2.53 mMol) and EDCI (485 mg, 2.53 mMol), and the resulting solution was stirred 2t room temperature for .

... , . , .. ~ . .. - . . . -. . - .... . .

WO91/16~7 PCT/US9l/01~ -one hour. Then, a solution containing 1.659 g (2.2 mMol) of the ester (IV) and 225 mg (0.31 ml, 2.20 mMol) -of TEA in 20 ml of CH2Cl2 was added dropwise over a period of 5 min. The resulting solution was stirred at 5 room temperature for 24 hours, then treated ~ith an additional 100 mg of EDCI and stirred for another 48 ~ ~-hours. The solvents were removed on the rotary evaporator and the oily residue further dried under high vacuum for several hours, during which it solidified to 10 a yellow waxy material. This was then washed three times by suspension in 150 ml-portions of EtOAc and centrifugation at 200 x g. After drying under high vacuum, the pelle~ was suspended in 1000 mi of dis.illed H2O, then extensively dialyzed against distilled H,O in ;
15 an Amicon ultrafiltration cell through an Amicon YM-10 membrane. The inner dialysate was then diluted to 2000 ml with distilled H2O, filtered through a triple layer of paper (Labconco Corp. #fA-754448), concentrated to ca.
600 ml on the rotary evaporator, and lyophilized to 20 yield 1.60 g of Compound IIA as a white, electrostatic :
powder.
For final purification, 52.8 mg of the above product ~ was dissolved in 1.0 ml of CHCl3/MeOH/H2O, 2:3:1, then .~ applied to a 0.7 x 29 cm column of Sephadex LH-20-100 ;~~ 25 resin that had been swollen and packed in the same solvent. The column was eluted at a flow rate of 0.33 ml/min, and fractions of the eluate were collected and assayed by TLC (Merck silica; CHCl3/MeOH/H2O/NH~OH, 50:25:4:2; 5~ H2SO4/EtOH heat). The appropriate ~-. 30 fractions were combined, then applied directly to a 1 x -8 cm column of BioRad Cellex D resin (acetate form).
This column was then washed with 30 ml of solvent, and . the combined eluate and washings concentrated to near Y dryness on the rotary evaporator, treated with 75 ml of 35 H2O, and lyophilized to yield 35 mg of GlcNAcMurNAc-L-~ Ala-_-isoGln L-Ala-DPG (GMTP-DPG) =s = white powder.

., :
.

WO9l/16~7 PCT/US9l/016~
29 2`.. ~-$~
Analysis for product as the dihydrate:
.

::
- 5 C65Hll6N602! 2H10 , .
Calculated: C 57.67 H 8.93 N 6.21 Found: C 57.89 H 8.58 N 5.91 ~, FAB-MS, m/c 1340 (M + 23), 1318 (M + 1), 1300 ~M - 18 t 's 1 ) ' ' . . .

PreParation of L~osomes The compound (IIA) was encapsulated into li?osomes using the following procedure. One mg of IIA as i' prepared in Example 9 was combined with 175 mg of 1-palmitoyl-2-oleoyl phosphatidyl choline (PC) and 75 mg of 1,2-dioleoyl phosphatidyl glycerol ~PG), both ' commercially available from Avanti Polar Lipids, Pelham, Alabama. The PC and PG were previously dissolved in tert-butanol at a concentration of 100 mg lipid per ml, thus giving a 7:3 weight ratio of PC:PG in tert-butanol.
Tert-butanol wa5 then added to the 1 mg IIA; 175 mg PC;
` 75 mg PG to give a final volume of 5.0 ml. The IIA and ;~- lipid mixture was passed through a sterile millipore ,~ 0.22~ filter to remove any contaminants present. The - -filtrate was collected in a clean, sterile, round bottom -flask which was capped with aluminum foil after filling.
Five ml of the filtered mixture containing 1 mg of IIA
was dispensed into 10 ml vials. After the vials were filled, they were covered with sterile rubber serum stoppers. Each of the stoppers includes a slit in one side so that air can enter and leave the vial during l lyophilization and stoppering. Sterile aluminum foil was placed over the vials and the vials were transferred to the tray drying chamber of the lyophilizer. The i vials were then cooled to -20C until the tert-butanol lipid mixture was frozen (approximately 30 to 60 ~^' i :

.. .. .. . . . .. . . . . .. .. ... .. .. . . .. . .

- WO91/16~7 PCT/US91/01 z~ t~ ~J 30 minutes). The refrigeration was then turned off and the i tray heater set for 10C. The vials were then lyophilized for 18 hours. The lyophilizer containing the vials was purged with filtered sterile argon and 5 evacuated three times. The lyophilizer containing the vials was then purged again with argon and the vials stoppered under argon at atmospheric pressure.

: EXAMP~E ll lO Adiuvant Activitv of the Aqent ~n Sal~ne on Ant~bodv Produc~nq Cells ~n Combination with Particulate Antiaen -The efficacy of the compound of the invention in inducing antihody response was evaluated in an immuno-~ compromised model using aged Balblc mice and in normal S l; mice using a suboptimal dose of the immunogen.
Aged, Balb/c mice (18 months old), representing an .
immunodeficient animal, were immunized intraperitoneally with an optimal inoculum of l x 109 sheep red blood cells (SRBC) either alone or mixed with O-l mg of M~P or O.l i, 20 mg of Compound IIA. The ~pleen was removed on day 4 and assayed for plaque forming units.
The results (Table l) indicated a total of 66 x 103 pla~ue forming units (PFU) per spleen for controls, 198 x 103 PFU per spleen for mice rece~ving SRBC mixed with O.l mg MDP and 442 x 103 P~U for mice receiving SRBC
mixed with O.l mg. of a compound of the invention IIA.
Similarly, young Balb/c mice were immunized , intraperitoneally with a suboptimal dose of SRBC (1 x 107 ;~ cells) in saline or mixed with O.Ol of O.l mg of MDP or 30 IIA.
s The results (Table l) indicate that on a weight $ basis IIA was 3 to lO times more effective than MDP.
'Y ':
Table l.
COMPARISON OF THE ADJWANT ACTIVITY
Z . OF MDP AND IIA

.. . . . . .
.. AGE OF SRBC MDP IIA PFU :-.
~ .
~ :.

. .. . .

- 31 2f~
MICE INOCULUM (mcl/mouse)(mc~/mouse) x103 ;~ 18 months l x 109 --- --- 66 518 months 1 x 108 0 . 1 ___ 198 18 months 1 x 1 o8. ___ 0 . 1442 ,. :' 103 months 1 x 107 --- --- 75 3 months 1 x 107 0 . 0~ 00 3 months l x 10 7 0. ~ 144 3 months 1 x 107 --- 0 . 01184 3 months l x 107 --- 0.1 468 ~; Antitumor Activitv of TIA in Saline in the ~eth A
Sarcoma BALB/C female mice, age 7 weeks, were injected subcutaneousl~ with 1 x 106 Meth A tumor cells. Eight days later the animals were treated intravenously with ` either saline (control) or Compound IIA at a dose of 1, l~ 10, or 100 micrograms ~g). Each group consisted of 4 animals. Tumor measurements were taken every 2 days for 10 days and the mice followed for 60 days until cured or death due to tumor occurred.
The results indicated a 10 to 15% decrease in tumor size on day 6 after therapy with a single doss of 1 to ~ 35 10 yg of Compound IIA. A larger dose of 100 ~g resulted `.~a in a 50% decrease in tumor growth at day 6 after therapy - with one of four animals exhibiting complete regression of tumor.
: :,. : .

Activation of Human Peripheral 8lood Monocvtes to the Tumoricidal State bv IIA and LiPosome EncaPsulated IIA
-i Monocyte tumoricidal activity was determined by the method of Fogler and Fidler (W. E. Fogler and I. J.
Fidler, J. Immunol., 136; 2311-2317, 1986). Briefly, , .

- . . .,. . : .. .. . : - - .. --: . - .- .: . - . :

WOgl/16~7 PCT/VS91/01~
~f'~ f~ 3'7;~ ' human peripheral blood monocytes were isolated by gradient centrifugation on 46~ Percoll. Monocytes were then cultured in suspension for 18 hours in RP~I 1640 media containing 5% human sera with or without 1.0 ~gjml of Compound IIA at l x 106 monocytes/ml. After incubation monocytes were washed, and 1 x 105 or 5 x 10 monocytes allowed to attach to wells of a 96-well microplate for 1 hour, then the plate was wasned to ~-remove non-adherent cells; to this, 1 x 10~ labeled A-375 tumor cells were added. Monocytes were cultured with tumor cells for 72 hours. At the end of the 72 hour co-culture period, the plates were washed to remove non-adherent-non-viable tumor celis and Ihe remaining adherent viable I12s labeled tumor cells determined by lS lysing the cells with sodium dodecyl sulfate and counting radioactivity in a Gamma Counter.
Activation of human peripheral blood monocytes by liposomes consisting of PC and PG (7:3) containing Compound IIA was determined as described above.
The effector:target cell ratio was 10:1. The i'~ cultures contained a final concentration of 1.0 ~g/ml of ~ MDP, Compound IIA or Compound IIA in liposomes. The .~7; results of these tests (Table 2) indicate that Compound IIA is more effective than MDP when used as a saline ~ - 25 8uspension or when encapsulated in liposomes.

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~ 5 Table 2 -~ PERCENT CYTOTOXICITY-COMPOUND IIA2 ~ .
COMPO~ND IN
: EXPERIMENT MDP3~ 4_ LIPOSOMES
1 38~ 59% 73%
2 27~ 37~ 44 I Percent cytotoxicity = A-B x ln~ where A = CPM in wells with control monocytes; B = CPM in wells with treated i monocytes.
3 2 Compound IIA in liposomes at a concentration of 1 mg/ml had no detectable endotoxin as determined by LAL assay with a sensitivity of 0.06 endotoxin units per ml.
3 MDP purchased from Cal-Biochem.
4 Compound IIA at a concentration of 1 mg/ml had no detectable endotoxin as determined by LAL assay with a sensitivity of 0.06 endotoxin units per ml.
Liposomes composed of phosphatidyl cholins:
phosphatidyl glycerol 7:3 molar ratio.
~- 35 -, ;1 ' , i Inoreased effect of Com~ound IIA with LiPo~olvsaccharide -In-Vi~o BALB/C mice 7-8 weeks of age were injected subcutaneously with ~eth A sarcoma (1 x Io6 cells) and treated intravenously on day 3 with 10 yg of lipopolysaccharide (LPS) from Salmonella typhimurium ReG
30/21 alone or comoined with 1 or 10 ~g of MDP or ~,~ Compound IIA. Tumor growth was compared on day 6 'J~ ~ 45 following therapy. The animals were followed and ~ the percent cured determined at 60 days post-injection.
-~ The results noted in Table 3 indicate synergistic - activity of compound IIA and LPS.

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WO91/16~7 PCT~US91/016~

., ~'~, ''`"'~'' "
Table 3 ~-~
EFFECT OF CO~POUND IIA WITH LIPOPOLYSACCHARIDE : -J '" ' ' Percent Change in Complete ;
Average Tumor Area Regression GrouPl 6 Days Post Treatment at Day 60 Control l9l~ 0%
LPSIo 165% ~
15 LPS1~DP:,o 22~ 33%
LPS;0Compo-lnd IIA;,~ -56$ 5Q~i LPSi~iDp;o -56~ 75%
LPS~0Compound IIAIo -67~ l00%
,j .. _. _ . _. . .
' 20 The subscripts refer to the weight in micrograms. , -.~-,~ '. . ~' .

Acuto Toxicitv in M~ce and Gu~nea Pios Two mice weighing between 17 and 22 grams and two guinea pigs weighing les9 than 400 grams were given a :' single in~raperitoneal in~ection of 0.5 ml and 5.0 ml of !~ 30 a final clinical formulation consisti-ng of a total of l mg of Compound IIA, 175 mg of l-palmitoyl-2-oleoyl , phosphatidyl choline and 75 mg of dioleoyl phosphatidyl -~
glycerol per 5 ml. The animals were observed daily for weight and clinical signs of distress. The results , 35 showed an initial weight loss followed by a weight gain at 7 days in guinea pigs. Mice maintain their weight . and showed a gain at 7 days.

EXAMPLE 16 ;
Subacute Toxicitv in Mice A group of l0 mice were injected intravenously twice a week for four weeks with a dose of l,320 ~g of ~ compound IIA per Kg of body weisht. This is calculated .~
~ .
~I

WO91/16~7 PCT/US91/01 2~
to be equivalent to ten times an anticipated maximum human dose of 4 mg per meter squared. In the conversion from a meter squared to a kilogram basis an equivalency of 60 kilograms per l.73 meter squared body surface area was used instead of the usual equivalency of 70 Kg body mass for a l.73 meter squared body surface area to result in a somewhat higher dose for the toxicity studies. The results showed no weight loss over the four weeks of the test.
, 10 EXAMPLE l7 Subacute ~oxicitv in Rabbits Three rabbils were treated intravenously, daily for 14 days at a dose of 132 ~g of Compound IIA in liposomes per kilogram of body weight. Blood obtained by cardiac I puncture for clinical studies and complete autopsies for histological evidence of toxicity were performed on day 15. Blood was obtained by ear vein and by cardiac t puncture on three control rabbits.
The results of this study showed no pathological evidence of toxicity. Review of the blood chemistries from the treated rabbits in comparison to the controls ~ revealed a single rabbit with a significant increase in ;j the creatinine phosphokinase. This abnormal value is believed related to the trauma of the cardiac-puncture - as evidenced by the increase in the creatinine phosphoXinase in the control animals following cardiac puncture.

30 PREPARATION OF GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-DPG .
, ~IIB~ -.~. EX~MPLE 18 BOC-D-Ala-DPG .

~; BOC-D-Ala-DPG was prepared from BOC-D-alanine t 35 (Sigma) using the same procedure described for the L-~ isomer in Example l. Following workup and drying, the , '~ .

,'', ' ' , .. .
. ,. . . .; .. , . ~ ~,., ; .. , . .. ., -- .. .

.

WO91/16~7 PCT/US91/01 z~ ?d 36 product was obtained as a white amorphous solid in an 89% yield.

EXAMPLE l9 -Ala-DPG
A portion (657 mg) of BOC-D-Ala-DPG was deprotected with TFA following the procedure described in Example 2.
Following workup and drying under high vacuum, 650 mg of product was obtained as its trifluoroacetate salt.
.' 10 , .

GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-DPG (~IB) , GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-DPG was prepared i from GlcNAcMurNAc-L-Ala-D-isoGln (see Example 7) and ~-15 Ala-DPG following the same procedure described in -Example 8 for the k-isomer. The reaction was followed by TLC and stopped at 72 hours. The reaction mixture . was then concentrated to dryness on the rotary evaporator, then further dried under high vacuum to 20 yield the crude product as a yellow solid. This material was extracted three times by suspension in l5 ml-portions of EtOAc followed by centrifugation. The pellet was taken up in ca. 20 ml of H2/ then dialyzed . (Spectrapor 7, 2000 MWCO) three times against l000 ml of `~~ 25 H2O. The inner dialysate was then lyophilized to yield r 111 mg of product as a white electrostatic solid.
For further purification, a 99 mg-sample of the ` above product was dissolved in l.0 ml of CHCl3:MeOH:H2O ~
-~ (2:3:1), then applied to a l x 28 cm column of Sephadex -`~ 30 LH-20 resin that had been packed in the same solvent.
7 The column was eluted with solvent at a flow rate of 0.5 `~ ml/min and fractions of ca. 2.0 ml were collected and `~ analyzed by TLC (Merck silica; CHCl,:~7eOH:H2O:con ~H~OH, ~, 50:25:4:2; 5% H2SO4/EtOH, heat). The appropriate 35 fractions were combined, then applied directly to a l x 7 cm column of BioRad Cellex D resin (acetate form) that had been packed in the same solvent used above for the ., . . . . . . , . . . . . ~ . . . , . . . . - . ~ . . ` . . . ~ . , .

., ., . .; . ; . . ,.............. - ~ - . : , WO91/16~7 PCT/US91/01~

. , 37 2~ J ~
BH-20 chromatography. The column was washed with 50 ml of solvent, and the combined eluate and washings concentrated to near dryness on the rotary evaporator, treated with sterile H~O (50 ml), then lyophilized to yield pure product as a white electrostatic solid.
; Molecular Weight (HRMS): -(Molecular Ion + H) Calc: 1317.8272; Found: 1317.8274. ~ -, , :
PREPARATION OF GlcNAcMurNAc-L-Ala-D-isoGln-L-Ala-N~DPG (IA) . 10 ,, , EXAMPLE 21 : BOC-L-Ala-SerOH

BOC-k-Ala (567 mg, 2 mMoi) and HOBT-H2O (505 mg, 3.3 mMol) were dissolved in 5 ml CH2Cl2 and 5 ml DMF, then , 15 treated with 1,3-dicyclohexylcarbodiimide (DCC, 680 mg, ~; 3.3 mMol). The resulting solution was stirred at RT for . one hour, then treated dropwise with a solution that contained 421 mg (3.3 mMol) of serinol hydrochloride (Aldrich) and 333 mg (3.3 mMol) of N-methylmorpholine in 5 ml of DMF. The reaction mixture was stirred at RT for 20 hours, then treated with 0.5 ml of glacial acetic acid and stirred for another hour. The solid reaction ;~ by-products were removed by filtration and washed with 20 ml CH2Cl2. The combined filtrate and washings was ~ 25 concentrated to dryness on the rotary evaporator then .~ further dried under high vacuum. The oily residue was taken up in 50 ml of H2O, cooled at 4~C for several hours, then filtered. The filtrate was concentrated to .~ dryness on the rotary evaporator, then further dried ; 30 under high vacuum to yield the crude product as a yellow ~ ~ oil.
-~ For purification, the entire crude reaction product was dissolved in 25 ml of CHCl3, then applied to a 2.5 x 1 20 cm column of BioSil A (100-200 mesh) that had been -., ~
packed in CHCl3. The column was then successively eluted with (A) 200 ml of CHCl3, (B) 200 ml of 1% (v/v) MeOH in CHCl3, (C) 200 ml of 2% MeOH in CHC13, (D) 200 ml of 4~
. 1 , .
.

., . .. . . . .. . . . . , .. . .. , . . .. ~ , ~, WO91/16~7 PCT/VS91/01~ ~
2~ `3 ~ .

MeOH in CHCl3, (E) 200 ml of 6% MeOH in CHCl3, (F) 200 ml of 10% MeOH in CHCl3, and (6) 200 ml of l5t4 MeOH in CHCl3. Fractions of 50-l00 ml were collected and assayed by TLC (Eastman silica; ethyl acetate:pyridine:acetic acid:water, 30:2:0.6:1; ~Cl spray, then ninhydrin). The .
appropriate fractions (R~ 0.9) were combined, then :~
- concentrated to dryness to yield 739 mg (94%) of product as a white glassy solid.

EXAMP~E 22 30C-~-Ala-NHDPG
The entire reaction product from Example 2l (739 mg, 2.82 mMoij was dissolved in 50 ml of CH2Cl2, then treateà
with palmitic acid (1.59 g, 6.2 mMol), DMAP (344 ~g, 't, 15 2.82 mNol) and EDCI (l.l9 g, 6.2 mMol). The mixture was stirred at RT for 20 hours, and the reaction was : followed by TLC (Eastman silica; hexane:2-propanol, 9:l;
HCl spray, then ninhydrin). After removal of the solvent on the rotary evaporator, the resulting oily residue was partitioned between l00 ml of EtOAc and l00 ml of H~O, the layers separated, and the organic layer further washed with H2O (3 x 50 ml), then concentrated to ~ dryness in the rotary evaporator to yield a colorless ¦ oil. This was dissolved in ca. 25 ml of CHCl3:MeOH:H2O
, 25 (300:200:30), then applied to a 2.5 x 8 cm column of Whatman DE 52 resin (OH cycle) that had been packed in the same solvent. The column was washed with 500 ml of A solvent, the entire eluate collected, then concentrated -:~ to dryness on the rotary evaporator to yield an oil that ~' 30 solidified while drying under high vacuum. There was obtained 2.02 g (97%) of crude product as a white ~1 amorphous solid.
-~ For further purification, l.0 g of this product was dissolved in l0 ml of hexane, then applied to a l.5 x 28 ~' 35 cm column of hioSil A that had been packed in hexane.
The column was then eluted with (A) 50 ml of hexane, (B) l00 ml of 2~ (v/v) 1-PrOH in hexane, (C) l00 ml of 4% i-i. ' ~-~
. ~ .

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WO91/16~7 PcTtus9l/
39 Z~ F~ ?~
PrOH in hexane, and (D) 100 ml of 6% i-PrOH in hexane.
Fractions of ca. 20 ml were collected and assayed by TLC
- as above. The appropriate fractions were combined, -concentrated to dryness, then further dried under hish vacuum to yield 856 mg of pure product as a white amorphous solid.
, .

L-Ala-NHDPG
,, 850 mg (l.lS mMol) of the product from Example 22 was dissolved in 15 ml of CH~Cl2, then treated with l0 ml of TFA. After standing at RT for one hour, the solvents were removed on the rotary evaporator to ieave a colorless oil that was repeatedly taken up in l0 ml-portions of hexane then concentrated to dryness on the rotary evaporator. After extensive drying under high vacuum, 856 mg (100%) of solid product was obtained as :. its trifluoroacetate salt.
F, . ,, ':

GlcNAcMurNAc-L-Ala-D-isoGln-~-Ala-NHDPG (IA) The disaccharide dipeptide, GlcNAcMurNAc-~-Ala-D-isoGln, (see Example 7, 208.? mg, 0.3 mNol) and HOBT-H2O
(53.3 mg, 0.348 mNol) were dissolved in l0 ml of DNF, ; 25 then diluted with 7 ml of CH2Cl2. To this was added ~6.4 i mg (0.346 mMol) of EDCI, and the resulting solution was stirred at RT for 30 minutes.
A TEA solution was prepared by diluting 303 mg (0.42 ml) of TEA to l0 ml with CH2Cl2.
L-Ala-NHDPG (see Example 23, 226.2 mg, 0.3 mMol) was treated with l.5 ml of CH2Cl2, then with l.0 ml of the ~ -TEA solution. The resulting solution was added to the -; activated disaccharide dipeptide solution, and the reaction mixture was stirred at RT and the course of the reaction was monitored by TLC. The reaction was stopped after 20 hours, and the reaction mixture was filtered from the small amount of insoluble material, then ' ~, ., ~ .
, :

WO91/16~ PCTtUS91/01 2f ~ 'r3?~
concentrated to dryness on the rotary evaporator and further dried under high vacuum to yield a yellow semi-solid residue. This material was then purified by the .- procedure described in Example 20 (i.e., EtOAc 5 extraction and dialysis against sterile H2O) to yield 155 ., mg of a white electrostatic powder. For final purification, a 140 mg-sample was subjected to the same ; treatment as described in Example 20 (i.e., Sephadex LH-20 and Cellex D). Following the column treatments and , 10 lyophilization, the product was obtained as a white electrostatic powder. Molecular Weight (HRM~S):
(Molecular Ion + H) Calc: 1316.8432; Found: 1316.8389.
,', ' :'' PREPARATION OF GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-NHDPG ~IB
, 15 .v EXAMPLE 25 BOC-D-Ala-SerOH
BOC-D-Ala-SerOH was prepared on a 1.0 mMol-scale by ~, the DCC-HOBT mediated condensation of BOC-D-Ala and ,.~
A ^20 serinol following the procedure described in Example 21.
'; The crude reaction product was purified by chromatography on a 1.7 x 24 cm column of BioSil A by successive elution with ~A) 100 ml CHCl3, (B) 100 ml of 1% (v/v) MeOH/CHCl3, (C) 100 ml of 2% MeOH/CHCl3, (D) 100 `.~ 25 ml o 4% MeOH/CHCl3, (Ej 100 ml of 8% MeOH/CHCl3, and ~F) 100 ml of 12% MeOH/CHCl3. The appropriate column fractions were combined and concentrated to dryness on .', the rotary evaporator. The oily residue was then taken l up in 10 ml of CH2Cl2, filtered from the small amount of .j 30 insoluble material, and again concentrated to dryness.
The residue was further dried under high vacuum to yield ~:~ 168 mg (68%) of product as a colorless viscous oil.
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WO 91/163~7 PCr/US91/01664 41 :
E~AMPLE 2 6 BOC-D-Ala-NHDPG
BOC-D-Ala-NHDPG was prepared on a 0.64 ~Mol-scale by the EDCI/DMAP mediated condensation of BOC-D-Ala-SerOH
5 and palmitic acid following the procedure described in -Example 22. After the coupling reaction and the ensuing workup, the crude product was purified by ion exchange (Cellex D, OH-cycle) and silica qel (BioSil A) chromatography as described in Example 22. The column fractions containing pure product were combined, concentrated to dryness on the rotary evaporator, then further dried under high vacuum to yield 310 mg (65.5~) of product as a white amorphous solid.
.'; ' .
~ 15 EXAMPLE 27 $ D-Ala-NHDPG
310 mg (0.42 mMol) of the product from Example 26 was dissolved in 10 ml of CH2Cl2, then treated with 5 ml of TFA. After standing at RT for one hour, the reaction was worked up exactly as described in Example 23 for the L-isomer. After extensive drying under high vacuum, there was obtained 315 mg (99.7%) of the trifluoroacetate salt of the product as a white j amorphous solid.
~ EXAMPLE 28 ;~ GlcNAcMurNAc-L-Ala-D-isoGln-D-Ala-NHDPG (IB~
The EDCI/HOBT mediated condensation of GlcNAcMurNAc-L-Ala-D-isoGln (see Example 7) with D-Ala-NHDPG (see Example 27) was carried out on a 0.24 mMol-scale following the procedure described in Example 24.
Following the coupling reaction and the ensuing workup, -:~
-th~ product was purified by the procedure described in ' 3 Example 20 (i.e., EtOAc extraction and dialysis against distilled water) to yield, after lyophilization, 141 mg of a white powder.
! ~ . .

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. . ' ' WO91/16~7 PCT/US91/01~ ~

2~;,-~ 4 42 For final purification, a 125 mg-sample of the above product was subjected to the two column procedure (i.e., Sephadex LH-20 and Cellex D) described in Example 20.
After lyophilization, the pure product was obtained as a ;
5 white, electrostatic powder. Molecular Weight (HRMS): -(Molecular Ion + H) Calc: 1316.8432; Found: 1316.8375.
, ':
PREPARATION OF GlcNAcMurNAc-D-Ala-D-isoGln-D-Ala-NHDPG
( ~C) EXAMP~E 29 GlcNAcMurNAc-D-Ala-D-isoGln-OBn ., GlcNAcMurNAc-D-Ala-D-isoGln-OBn was prepared on a 0.75 m~ol-scale by the Woodwarà's Reagent K-media~ed coupling of GlcNAcMurNAc (VII) with D-Ala-D-isoGln-OBn (prepared as described in Example 4 and 5) using the ; procedure in Example 6. Following the 72 hour-coupling reaction and the ensuing workup and lyophilization, 332 ~ mg of product was obtained as a white powder.
::~
ESAMP~E 30 GlcNAcMurNAc-D-Ala-D-isoGln 1 The entire reaction product from Example 29 ~332 mg, 0.423 mMol) was deprotected by hydrogenation over a Pd/C
~i~ catalyst using the procedure described in Example 7.
Following workup and lyophilization, 313.1 mg of product was obtained as a glassy solid.

, EXAMP~E 31 GlcNAcMurNAc-D-Ala-D-isoGln-D-Ala-NHDPG (IC) .
The EDCI/HOBT mediated condensation of GlcNAcMurNAc-D-Ala-D-isoGln (see Example 30) with D-Ala-NHDPG (see ~-Example 27) was carried out on a 0.25 mMol-scale following the procedure detailed in Example 24.
j Following the coupling reaction and the ensuing workup, the product was purified by the procedure described in ; Example 20 (i.e., EtOAc extraction and dialysis against .'~ ..
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WO91/16~7 PCT/US91/01 z~

distilled water) to yield, after lyophilization, 102 mg of a white powder.
For final purification, a 90 mg-sample of the above product was subjected to the two column procedure (i.e., Sephadex LH-20 and Cellex D) described in Example 20.
After lyophilization, the pure product was obtained as a -white, electrostatic powder. Molecular Weight (HRMS): -(Molecular Ion + H) Calc: 1316.8432; Found: 1316.8444.
. , .
PREPARATION OF GlcNAcMurNAc-D-Ala-D-isoaln-D-Ala-DPG
(lIC~

; EXAMP~2 32 GlcNAcMurNAc-D-Ala-D-isoGlu-D-Ala-DPG (IICl The EDCI/HOBT mediated condensation of GlcNAc ~ MurNAc-D-Ala-D-isoGln (see Example 30) with D-Ala-DPG
$j ( see Examples 18 and 19) was caxried out as described in Examples 8 and 20. After the 72 hour-incubation, the reaction mixture was worked up as described in Example 20, i.e., by EtOAc extraction and extensive dialysis . ~ against distilled H2O. The crude lyophilized product was then further purified by the described two-column approach (i.e.' Sephadex LH-20 followed by BioRad Cellex D [acetate form]). After removal of the organic solvents, the column eluate was-lyophilized to yie].d Compound IIC as a white electrostatic powder.

-~ Activation of Monocvtes In-Vitro bv Drua Compounds as ;!
'`t 30 Measured bv the Production of Tumor Necrosis Factor Human peripheral blood monocytes were isolated by density gradient centrifugation as noted in Example 13.
; The cell number was adjusted to 5 x 105 cells per ml in .,~ RPM 1640 media containing 5% human AB negative serum.
One ml containing 5 x 105 cells were placed in individual wells of a 24-well Plastek tissue culture plate.
:~ Various drugs in a volume of 0.5 ml were added at time .~ zero. The cells were incubated a~ 37C in a 5% CO~
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atmosphere. Aliquots (0.1 ml) of the supernatant were removed at 24, 48, and 72 hours and frozen at -80'C.
These samples were subsequently assayed for tumor necrosis factor (TNF) activity using a commercially available ELISA Assay from T-Cell Sciences (Cambriage, MA). The results of a series of four similar experiments, set forth in Table 4, demonstrate significant levels of TNF production by all compounds as compared to a control.

Table 4. TNF Production by Monocytes Incubated with - Compounds Suspended in Saline.
Concentration Drug of Free Drug TNF Level Experiment ComPound (uq/ml) (Pqlml) . 1 1. Control -- 500 :.
2. Compound IIA 0.1 1760 1.0 1600 10.0 4550 2 5 2 1 . Control -- 170 2. Compound IIA 0.1 1450 $ 1.0 1580 10.0 '1630 ' 30 3. MDP 0.1 1450 1.0 1200 ~l 10.0 800 ~` - , ' .
~ 35 3 1. Control ~ -- 130 -~ 2. Compound IIA 0.1 1450 -` 1.0 1650 10.0 1800 ~ 3. Compound IIB 0.1 250 : ~ 1 . O 500 ;~ 10.0 1600 '.~ 45 4. Compound IA 0.1 280 1 . O 1 0 0 0 - 10.0 1680 '~
! 50 ~i -., .
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., ~ , . ., ' ~ : , WO91/16~7 PCT/US91/016 : -5. Compound IB O.l 180 . l.0 350 . lO.0 1980 56. Compound IC O.l 280 . l.0 300 . lO.0 250 ., - - . .

4 7. Control -- 50 8. Compound IIC O.l 40 . 15 l.0 llO
:` lO.0 450 `, . .
. 20 EXAMPLE 34 -: -~ Activation of Monoc~tes In-Vitro bv Dru~ ComPound -~ Incor~orated in Ll~osomes :, Human peripheral blood monocytes were isolated by ~ density gradient centrifugation as described in Example :i~ 25 13. ~he cells were plated in 24-well microliter plates :
: as described in Example 33. The compounds were prepared . in liposomes as described in Example lO, and assayed for ~:: TNF production, as described in Example 33. The results ... ..
~, of four separate experiments are set forth in Table 5, ¦ 30 and show significsnt levels of TNF compared tO controls.

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WO91/16~7 PCT/US9t/01 Table 5. TNF Production by Monocytes Incubated with Compounds Incorporated in Liposomes Concentration of Drug TNF Level Druq Com~oundt~a/ml1(Pq/ml~_ Control __ 70 Compound IIA 0.1 1270 1.0 850 10.0 1050 . ,~ , Compound IA O.i 430 1.0 800 -~
-~ 15 10.0 750 ;~

:~ ' ' :~" ,' EXAMP~E 35 Acti~atlon of Monocvtes In-V~tro as Moasured bv In-Vitro Tumor Coll CYtotoxlcitv Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on 46%
Percoll. 1 x 10~ of the mononuclear cells were plated , onto Falcon microliter plates and allowed to adhere for two hours. The non-adherent cells were removed and fresh media added. The cells were allowed to incubate -for 24 hours after which various concentrations of the .~ drug either alone or incorporated in liposomes was added. After 24 hours, the plates were washed and 1 x lO~ I125 labelled target cells were added. Following an - additional 72 hours of co-incubation, the plates were washed and the residual radioactivity~determined. The specific cytotoxicity was calculated from the formula:
% cytotoxicity = A-B x lO0 Where: A = initial radioactivity B = residual radioactivity : :j , .... .

W O 91/16347 P ~ /US91/01664 47 z ~ '?
The results of cytotoxicity assay of various compounds alone or incorporated in liposomes at various concentrations is given in Table 6. All of the tested compounds, either alone or incorporated in liposomes, . .
S demonstrated ability to activate monocytes to be cytotoxic. -:

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WO91/16~7 PCT/US91/01 Z~h~ 48 Table 6.
. In-Vitro Cytotoxicity of Various Compounds an~
: Formulations -, 5 :.
: Concentration Compound Formulation (ua/ml~ Cytotoxicitv (~) Control l.4 Compound IA PC/PG1O.l 7.0 l.0 7.0 lO.0 6.0 `~ 15 Compound IC PC/PG1O.l lO.0 : -l.0 4~0 lO.0 12.0 Compound IIA PC¦PS2 O.i l.û
~ 20 l.0 8.0 .
'' lO.0 12.0 ~ :
~ Compound IA PC/PS2O.l 8.0 :~
,.~ 1.0, 5.0 lO.0 l9.0 :: Compound IC O.l 5.0 . : l.0 16.0 lO.0 15.0 :i :~ 30 Compound IIA O.l 5.0 l.0 4.0 lO.0 3.0 35 Compound IA O.l l.0 .0 4.0 ~ lO 0 3 0 : MDP O.l ll.0 : 40 l.0 9-0 lO.0 7.0 ;--' lPC/PG: phosphatidyl choline/phosphatidyl glycerol ~i ~ 45 liposomes (7:3 molar ratio) PC/PS: phosphatidyl choline/phosphatidyl serine liposomes (7:3 molar ratio) .

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W091/16~7 ~ PCT/US91/01 49 Z ~'' PREPARATION OF GlcNAcMurNAc-L-Thr-D-isoGln-L-Ala-DPG ~:
(IID) EXAMPLE 36 BOC-D-isoGln-OBn Benzyl alcohol (476 mg, 4.4 mMol) BOC-D-isoGln (Sigma, 985.2 mg, 4.0 mMol) and DMA~ (2~4 mg, 2.~ m~ol were dissolved in 30 ml CH2Cl2 and 20 ml DMF, then treated with EDCI (920 mg, 4. 8 mMol), and the resulting , -solution stirred at RT for 24 hours. The reaction mixture was then concentrated to dryness on a rotary evaporator to yield an oily residue that was partitioned between 75 ml EtOAc and 25 ml H2O. The layers were ~eparated and the organic layer extracted with saturated NaHCO3 (3 x 25 ml) and ~2O (3 x 25 ml). After drying ~ over Na2SO~, the solvent was removed on the rotary : 15 evaporator to yield 1.206 g (90%) of a white solid, which was further purified by crystallization from ~ diethyl ether/hexane to yield pure product as a snow- -i white solid.
. .

~ D-isoGln-OBn -¦ A sample (1.0g, 2 . 9 8 mMol) of the product from Ex~mple 36 was dissolved in 50 ml of lN HC1/HOAc and the ~ resulting solution allowed to stand at RT for two hours. -1 2 5 The solvent was then removed on the rotary evaporator to --yield a white solid that was further dried under high vacuum, then crystallized from MeOH/ether to yield 720 mg (90~) of the pure product as its hydrochloride salt.
:; . .

BOC-L-Thr(OBn)-D-isoGln-OBn -BOC-L-Thr(OBn) (Sigma, 618.8 mg, 2.0 mMol), and N-hydroxysuccinimide (253.2 mg, 2.2 mMol) were dissolved in 5.0 ml DMF and 7.0 ml CH2Cl~. To this was added EDCI
i 35 (421.7 mg, 2.2 mMol), and the resulting solution stirred at RT for one hour. Then, a solution that contained 440.4 mg (1.62 mMol) of the product from Example 37 and -- - .

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WO91~ PCT/US91/016~

-164 mg (1.62 mMol) of N-methylmorpholine in 5.0 ml DMF
was added and the reaction mixture was stirred at ~T for 48 hours. The solvents were then removed on the rotary ~ -evaporator to yield an oily residue that was partitioned between 100 ml EtOAc and 50 ml H,O. The layers were ; separated, and the organic layer further extracted with saturated NaHCO3 (3 x 50 ml), then H2O (3 x 50 ml). The organic layer was concentrated to dryness on the rotary ~;l evaporator, then further dried under high vacuum to yield crude product as a yellow glassy solid.
For further purification, the entire product was dissolved in 10 ml CHC1~, then applied to a 2.5 x 18 cm column of BioSil A that had been packed in CHCl~. Tne column was then successively eluled with (A) 200 ml CHCl3, (B) 200 ml of 1~ MeOH/CHCl3, (C) 200 ml of ~%
MeOH/CHCl3, and (D) 200 ml of 3~ MeOH/CHCl3. Fractions of ca. 20 ml were collected, and aliquots (2ul) of each ,4 were assayed by ~LC as described in Example 21. The appropriate fractions were combined, concentrated to ' 20 dryness, then further dried under high vacuum to yield product as a white glassy solid.
For final purification, the entire product was crystallized from EtOAc/hexane to yield, after drying, 550 mg (65~ overall) of pure product as a white glassy , 25 solid.
- :~

1 L-Thr(OBn)-D-~soGln-OBn -~ The product from Example 38 (540 mg, 1.044 ' mMol) was dissolved in 50 ml of lN HCl/HOAc and the . resulting solution stirred at RT for 1.5 hours. After ~ removal of solvent on the rotary evaporator, the oily -j~ residue was further dried under high vacuum, then ~ crystallized from MeOH/ether to yield, after extensive ~ -;`~ 35 drying, 450mg of the hydrochloride salt of k-Thr(OBn)-D-~ isoGln-OBn as a white, amorphous solid.
` .

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W091/16~7 PCT/US91/01~

5l 2 .~

GlcNAcMurNAc-L-Thr(OBn~-D-isoGln-OBn GlcNAcMurNAc-L-Thr(OBn)-D-isoGln-OBn was - prepared on a 0.60 mMol-scale by the Woodward's ~eagent S K-mediated coupling of GlcNAcMurNAc (VII) with the product from Example 39 using the procedure described in Example 6. Following the 96 hour-coupling reaction and the ensuing workup and lyophilization, 388 mg of product was obtained as a white powder.

GlcNAcMurNAc-~-Thr-D-isoGln 263 mg OI the product from Exampie ~û was dissolved in 75 ml of 10% HOAc, then deprotected by hydrogenation over a Pd/C catalyst using the procedure described in Example 7. Following workup and lyophilization, 201 mg of product was obtained as a glassy tan solid.
.1 ' ' .

GlcNAcMurNAc-~-Thr-D-isoGln-L-Ala-DPG (IID) . .
. The EDCI/HOBT mediated condensation of GlcNAcMurNAc-L-Thr-D-isoGln (see Example 41) with L-Ala-DPG (see Example 2) was carried out on a 0.10 mMol-scale following the procedures described in Examples 8 and 20.
After the 48 hour-incubation, the reaction mixture was worked up as described in Example 20, i.e., by EtOAc extraction and extensive dialysis against distilled H2O.
The crude lyophilized product was then purified by the ~- 30 described two-column approach, i.e., Sephadex LH-20 followed by BioRad Cellex D (acetate form). After - removal of the organic solvents, the final column eluate was lyophilized to yield pure product as a white electrostatic powder. - -~-, 35 , , '' .

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W091~16~7 PCT/US9t/~16~
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PREPARATION OF GlcNAcMurNAc-L-Thr-D-isoGln-L-Ala-NHDPG
(ID~ EXAMPLE ~3 GlcNAcMurNAc-L-Thr-D-isoGln-L-Ala-NHDPG (ID) The EDCI/HOBT mediated condensation of GlcNAc~urNAc-L-Thr-D-isoGln (see Example 40) with L-Ala-NHDPG (see Example 23) was carried out on a 0.1 mMol-scale following the procedure detailed in Example 24.
After the 48 hour-coupling reaction, the reaction mixture was worked up, then purified as described in Example 24. The final column eluate was lyophilized to yield pure product as a white electrostatic powder.

, . . .
PREPARA~ION OF GlcNAcMurNAc-L-Ser-D-isoGln-L-Ala-DPG
(IIE) EXAMPLE 44 :.
15 BOC-L-Ser(OBn)-D-isoGln-OBn i This compound was prepared on a 1.0 mMol-scale ;. using the same procedure described in Example 38 for the synthesis of the threonine analog. After workup and ~ purification by both silica gel (BioSil A) .1~ 20 chromatography and crystallization from EtOAc/hexane, pure product was obtained in a 79% yield as a white, ~ amorphous solid.
:~, . .
:' EXAMP~E 45 ~i~ 25 ~-Ser(08n)-D-lsoGl~-OBn ;~
~ The product from Example 44 (300 mg, 0.6 mMoL) -~ was treated with 30 ml of lN HCl/HOAc, and the resulting solution was allowed to stand at RT for 2 hours. After removal of the solvent on the rotary evaporator, the é 30 oily residue was further dried under high vacuum, then crystallized form MeOH/ether (see ExampIe 5) to yield, after extensive drying, 234 mg of the hydrochloride salt of k-Ser(OBn)-D-isoGln-OBn as a white, amorphous solid.
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WO91/16~7 P~CT/US91tOl~f~

2' ~
EXA~PLE 46 GlcNAcMurNAc-L-Ser(OBn)-D-isoGln-O~n GlcNAcMurNAc-L-Ser(OBn)-D-isoGln-OBn was prepared on a 0.20 mMol-scale by the Woodward's Reagen_ K-mediated coupling of GlcNAcMurNAc (VII) with the product from Example 45 using the procedure described in Example 6. Following the 120 hour-coupling reaction and -the ensuing workup and lyophilization, ll0 mg of product ;
was obtained as a white powder.

GlcNAcMurNAc-L-Ser-D-isoGln - lG8 mg of the product from Exampie ~iÇ was dissolved in 50 ml of 10% HOAc, then deprotected by lS hydrogenation over a Pd/C catalyst using the procedure described in Example 7. Following workup and lyophilization, 95 mg of the crude product was obtained as a white glassy solid.
i, For further purification, the entire 95 mg of product was slurried in 2.0 ml of CHCl3, then treated with sufficient MeOH to effect solution. This was then applied to a 0.7 x 28 cm column of BioSil A that had 1 been packed in CHCl3. The column wa9 then ~ucce9sively eluted with (A) 50 ml of CHCl3, ~j 50 ml of 20%
NeOH/CHCl3, (C) l00 ml of 40% MeOH/CHCl3, (D) l00 ml of 60% MeOH/CHCl3, and (E) l00 ml of 80% MeOH/CHCl3.
Fractions of ca. 5.0 ml were collected and aliquots (2A) ~ of each were assayed by TLC (Merck silica;
-4' CHCl3:MeOH:H2O:con NH40H, 50:2S:4:2; 5% H2SO4/EtOH; heat).
The tubes containing material with Rf 0.0 were combined, `~. concentrated to dryness, then redissolved in l0 ml of H2O
and lyophilized to yield pure product as a white, electrostatic solid. ~
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WO 91tl6347 P~/US91/01664 2~ -- 5 4 ::

GlcNAcMurNAc-I.-Ser-D-isoGln-L-Ala-D?G (IIEL -, The EDCI/HOBT mediated condensation of GlcNAcMurNAc-L-Ser-D-isoGln (see Example 47) with L-Ala-5 DPG (see Example 2) was carried out on a 0.045 mMiol- ~ -scale followinq the procedures described in Examples 8 and 20. ~fter the 48 hour-reaction, the mixture was worked up as described in Example 20, i.e., by EtOAc extraction and extensive dialysis against distilled water. The crude lyophilized product was then purified by the described two column approach (i.e., Sephadex LH-20 followed by BioRad Cellex D (acetate form)). After removal of the organic solvents, the final coiumn eiuate was lyophilized to yield pure product as a white, - ~-^ 15 electrostatic powder.

PREPARATION OF GlcNAcMurNAc-~-Val-D-isoGln-L-Ala-DPG
~IIF) EXAMPLE g9 20 ~OC-~-Val-D-isoGln-OBn BOC-L-Val-D-isoGln-OBn was prepared on a 1.0 mMol-scale using the same procedure described in Example 38 for the synthesis of the threonine analog. After workup and purification by both silica gel (BioSil A) 25 chromatography and crystallization from the . EtOAc/hexane, pure product was obtained in a 60~ yield as a white, amorphous solid.
.

v 30 L-Val-D-~soGln-OBn The product from Example 49 (175 mg, 0.40 mMol) ~ was treated with 20 ml of lN HCl/HOAc, and the resulting 3 solution was allowed to stand at RT for 2 hours. After removal of the solvent on a rotary evaporator, the oily 35 residue was further dried under high vacuum, then crystallized from MeOH/ether (see Example 5) to yield, 'j . .

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after thorough drying, 134 mg of the hydrochloride salt of L-Val-D-isoGln-OBn as a white, amorphous solid.
EXAMPLE 5l GlcNAcMurNAc-L-val-D-isoGln-Osn GlcNAcMurNAc-L-Val-D-isoGln-OBn was prepared on a 0.20 mMol-scale by the Woodward's Reagent K-mediated coupling of GlcNAcMurNAc (VII) with the product from ;~ Example 50 using the procedure detailed in Example 6.
Following the 96 hour-coupling reaction and the ensuing workup and lyophilization, 95 m~ of product was obtained as a white powder.

;- GlcNAcMurNAc-L-Val-D-isoGln lS 95 mg of the product from Example 51 was dissolved in 50 ml of the 10% HOAc, then deprotected by hydrogenation over a Pd/C catalyst using the procedure described in Example 7. Following workup and , lyophilization, 87 mg of the crude product was obtained `;20 as a white glassy solid. This material was then purified by chromatography on a silica gel column exactly as described in Example 47. Fractions containing material with R- 0.05 were combined, concentrated to dryness, then redissolved in l0 ml of H,O
25 and lyophilized to yield pure product as a white powder.
.~` . ." ' -GlcNAcMurNAc-L-Val-D-isoGln-L-Ala-DPG (IIF) ~ The EDCI/HOBT mediated condensation of ;~30 GlcNAcMurNAc-L-Val-D-isoGln (see Example 52) wi~h L-Ala-DPG (see Example 2) was carried out on a 0.048 mMol-f scale following the procedures detailed in Examples 8 and 20. After the 48 hour-coupling reaction, the mixture was worked up exactly as described in Example 35 20, i.e., by EtOAc extraction followed by extensive dialysis against distilled water. The crude lyophilized product was then purified by the àescribed two column ;~, . -~
- .

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WO91/16~7 PCT/US9t/01 Z' approach and lyophilized to yield pure product as a white electrostatic powder.
:' PREPARATION OF GlcNAcMurNAc-L-Ala-D-isoGln-L-Val-DPG
5 (IIG) -BOC-L-Val-DPG
BOC-L-Val-DPG was prepared on a 1.O mMol-scale by the EDCI/D~AP condensation of BOC-L-Val (Sigma) ~ith l,2-dipalmitoyl-sn-glycerol using the procedure detzlIed in Example l for the preparation of BOC-L-Ala-DPG.
Following workup and extensive drying under high vacuum, there was obtained 703 mg (9l.5~) of product as an amorphous white solid.
: " , :

-i, L-Val-DPG
` The product from Example 54 (500 mg) was `~` 20 deprotected with TFA/CH2Cl2 following the procedure described in Example 2. After workup and extensive drying under high vacuum, there was obtained 485 mg of L-Val-DPG trifluoroacetate as an amorphous white solid.
:~
~ 25 EXAMPLE 56 ; GlcNAcMurNAc-L-Ala-D-isoGln-L-Val-DPG
The EDCI/HOBT mediated condensation of GlcNAcMurNAc-L-Ala-D-isoGln (see Example 7) with L-Val-~, DPG (see Example 55) was carried out on a 0.033 mMol-scale following the procedures detailed in Examples 8 ^ and 20. Following the 24 hour-coupling reaction, the ^ mixture was worked up as described in Example 20, i.e., ~-~. by EtOAc extraction followed by extensive dialysis ., against distilled water. The crude product was then purified by the described two column approach and lyophilized to yield pure product as a white electrostatic powder.

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EXAMPLE 57 :~.
Activation of Monocvtes In-Vitro by Druq Compounds and Formulations as Measured by the Production of Interleukin-l-beta and/or TNF

Human peripheral blood monocytes were isolated by density gradient centrifugation as noted in Example 13. The cell number was adjusted to 5 x lO~ cells per ml in RPM 1640 media containing 5% human AB negative serum.
One ml containing 5 x 105 cells were placed in individual wells of 24-well Plastek tissue culture plate. Various drugs in a volume of 0.5 ml were added at time zero.
The compounds were also formulated in liposomes as described in Example 10, and added in a volume of 0 .5 ml to individual wells of a 24-well Plastek tissue culture plate. The cells were incubated at 37C in a 5~ CO2 atmosphere. Aliquots (0.1 ml) of the supernatant were removed at 24 hours and frozen at -20C. These samples were subsequently assayed for TNF as described in Example 33 and Interleukin-1-beta (IL-1~) using a ii, 20 commercially available ELISA kit from Cistron ~Pine Brook, NJ).
The results of a series of two separate ! experiments set forth in Tables 7, 8, and 9 demonstrate significant levels of IL-1~ and/or TNF production by all compounds and formulations of the invention as compared to controls.
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Claims (33)

WHAT IS CLAIMED IS:

1. A compound of the formula:

(I) wherein R1 is (C1-C9)alkyl, R2 is (C1-C5)alkyl, R3 and R4 are individually (C6-C30)alkyl groups having about 0-4 double bonds, X is a single bond or any peptidyl residue comprising one or more amino acid residues; and Y is any amino acid residue; and the pharmaceutically acceptable salts thereof.

2. The compound of claim 1 wherein R1 is -CH3.

3. The compound of claim 2 wherein R2 is -CH3.

4. The compound of claim 1 wherein X is any amino acid residue.

5. The compound of claim 4 wherein X is any naturally occurring amino acid residue or an enantiomorph of any naturally occurring amino acid residue.

6. The compound of claim 5 wherein X is selected from the group consisting of L-valine, D-valine, L-alanine, and D-alanine.

7. The compound of claim 1 wherein Y is any naturally occurring amino acid residue or an enantiomorph of any naturally occurring amino acid residue.

8. The compound of claim 7 wherein Y is selected from the group consisting of threonine, alanine, valine, and serine.

9. The compound of claim 8 wherein Y is selected from the group consisting of L-alanine and L-threonine.

10. The compound of claim 1 wherein R3 and R4 are individually (C12-C23)alkyl groups having about 0-1 double bonds.

11. The compound or claim 10 wherein R3 is a (C15)alkyl group.

12. The compound of claim 11 wherein R4 is a (C15)alkyl group.

13. The compound of claim 10 wherein R1 and R2 are -CH3.

14. A compound of the formula:

(II) wherein R1 is (C1-C9)alkyl, R2 is (C1-C5)alkyl, R3 and R4 are individually (C5-C30)alkyl groups having about 0-4 double bonds, X is a single bond or any peptidyl residue comprising one or more amino acid residues, and Y is any amino acid residue; and the pharmaceutically acceptable salts thereof.

15. The compound of claim 14 wherein R1 is -CH3.

16. The compound of claim 15 wherein R2 is -CH3.

17. The compound of claim 14 wherein X is any amino acid residue.

18. The compound of claim 17 wherein X is any naturally occurring amino acid residue or an enantiomorph of any naturally occurring amino acid residue.

19. The compound of claim 18 wherein X is selected from the group consisting of L-valine, D-valine, L-alanine, and D-alanine.

20. The compound of claim 14 wherein Y is any naturally occurring amino acid residue or an enantiomorph of any naturally occurring amino acid residue.

21. The compound of claim 20 wherein Y is selected from the group consisting of threonine, alanine, valine, and serine.

22. The compound of claim 21 wherein Y is selected from the group consisting of L-alanine and L-threonine residue.

23. The compound of claim 14 wherein R3 and R4 are individually (C12-C23)alkyl groups having about 0-1 double bonds.

24. The compound of claim 23 wherein R3 is a (C15)alkyl group.

25. The compound of claim 24 wherein R4 is a (C15)alkyl group.

26. The compound of claim 23 wherein R1 and R2 are -CH3.

27. A composition C4 matter comprising an effective immunomodulating amount of the compound of claims 1 and/or 14 combined with a pharmaceutically acceptable liquid vehicle.

28. The composition of claim 27 further comprising a lipopolysaccharide.

29. A liposome comprising a compound of the formula:

(I) wherein R1 is (C1-C9)alkyl, R2 is (C1-C5)alkyl, R3 and R4 are individually (C6-C30)alkyl groups having about 0-4 double bonds, X is a single bond or any peptidyl residue comprising one or more amino acid residues, and Y is any amino acid residue; and the pharmaceutically acceptable salts thereof.

30. A liposome comprising a compound of the formula:

(II) wherein R1 is (C1-C9)alkyl, R2 is (C1-C5)alkyl, R3 and R4 are individually (C6-C30)alkyl groups having about 0-4 double bonds, X is a single bond or any peptidyl residue comprising one or more amino acid residues, and Y is any amino acid residue; and the pharmaceutically acceptable salts thereof.

31. A composition of matter wherein the liposome consists essentially of a liposome having a bilayer membrane consisting essentially of 1-palmitoyl-2-oleoyl-phosphatidyl choline and dioleoyl phosphatidyl glycerol in a weight ratio of about 5:1 to 1:1 and a compound of claims 1 and/or 14.

32. The composition of claim 31 wherein the weight ratio is about 7:3.

33. A method for stimulating the immune response of a mammal comprising administering an effective amount or the composition of claims 1, 14, 29, 30, or 31; itself or in combination with a pharmaceutically acceptable vehicle.