WO2001076626A2

WO2001076626A2 - Lipophilic aromatic aldehyde and ketone derivatives and the use thereof as immunostimulants and adjuvants

Info

Publication number: WO2001076626A2
Application number: PCT/US2001/011590
Authority: WO
Inventors: Dante J. Marciani; Jeffery B. Press
Original assignee: Galenica Pharmaceuticals, Inc.
Priority date: 2000-04-11
Filing date: 2001-04-10
Publication date: 2001-10-18
Also published as: AU2001251491A1; WO2001076626A3

Abstract

The invention in its braodest aspects relates to substituted aryl- and arylalkyl aldehyde derivatives that are useful in activating the immune system for use in prophylactic and therapeutic vaccines as well as general enhancement of the immune response. The compounds useful in this invention are broadly represented by Formula I: or a pharmaceutically acceptable salt thereof, wherein R1 through R5, and o are as defined in the specification.

Description

/

Lipophilic Aromatic Aldehyde and Ketone Derivatives and The Use Thereof as Immunostimulants and Adjuvants

Background of the Invention

Field of the Invention

The present invention is in the field of adjuvants and immunostimulating agents. More particularly, the invention relates to the use of lipophilic aromatic aldehyde and ketone derivatives as adjuvants and immunostimulating agents, and to novel classes of lipophilic aromatic aldehyde compounds.

Related Art

The immune system may exhibit both specific and nonspecific immunity

(Klein, J., et al, Immunology (2nd), Blackwell Science Inc., Boston (1997)). Generally, B and T lymphocytes, which display specific receptors on their cell surface for a given antigen, produce specific immunity. The immune system may respond to different antigens in two ways: (1) humoral-mediated immunity, which includes B cell stimulation and production of antibodies or immunoglobulins

[other cells are also involved in the generation of an antibody response, e.g. antigen-presenting cells (APCs; including macrophages), and helper T cells (Thl and Th2)]; and (2) cell-mediated immunity (CMI), which generally involves T cells including cytotoxic T lymphocytes (CTLs), although other cells are also involved in the generation of a CTL response (e.g., Thl and/or Th2 cells and

APCs).

Nonspecific immunity encompasses various cells and mechanisms such as phagocytosis (the engulfing of foreign particles or antigens) by macrophages or granulocytes, and natural killer (NK) cell activity, among others. Nonspecific immunity relies on mechanisms less evolutionarily advanced (e.g., phagocytosis, which is an important host defense mechanism) and does not display the acquired nature of specificity and memory, hallmarks of a specific immune response. Nonspecific immunity is more innate to vertebrate systems. In addition, cells involved in nonspecific immunity interact in important ways with B and T cells to produce an immune response. The key differences between specific and nonspecific immunity are based upon B and T cell specificity. These cells predominantly acquire their responsiveness after activation with a specific antigen and have mechanisms to display memory in the event of future exposure to that specific antigen. As a result, vaccination (involving specificity and memory) is an effective protocol to protect against harmful pathogens.

A critical component of inactivated vaccines, including subunit vaccines, is an adjuvant. Adjuvants are nonimmunogenic compounds, that when administered with an antigen (either mixed with, or given prior to the administration of the antigen) enhances or modifies the immune response to that particular antigen. Thus, the humoral and/or cell-mediated immune responses are more effective when an antigen is administered with an adjuvant. Furthermore, the adjuvant may alter the quality of the immune response by affecting the subclasses (isotypes) of immunoglobulins produced (IgGl, IgG2, IgG3, andIgG4 for human IgGs; IgGl, IgG2a, IgG2b, and IgG3 for mouse IgGs), as well as their affinities. A response regulated by Thl cells in mice will induce IgGl, IgG2a, IgG2b and to a lesser extent IgG3, and also will favor a CMI response to an antigen. If the IgG response to an antigen is regulated by Th2 type cells it will predominantly enhance the production of IgGl and IgA.

Adjuvants that have been used to enhance an immune response include aluminum compounds (all generally referred to as "alum"), oil-in-water emulsions (often containing other compounds), complete Freund's adjuvant (CFA, an oil-in- water emulsion containing dried, heat-killed Mycobacterium tuberculosis organisms), and pertussis adjuvant (a saline suspension of killed Bordatella pertussis organisms). These adjuvants generally are thought to have their mechanism of action by causing a depot of antigen and permitting a slow release of the antigen to the immune system, and by producing nonspecific inflammation thought to be responsible for their observed activity (Cox, J.C., et al, Vaccine 5:248-256 (1997)). Some saponins have been shown to have different types of immune stimulating activities, including adjuvant activity. These activities have been reviewed previously (Shibata, S., New Nat. Prod. Plant Pharmacol. Biol. Ther. Act., Proc. Int. Congr. 1st, 177-198 (1977); Price, K.R., et al. CRC Crit. Rev. Food Sci. Nutr. 26:27-135 (1987); Schopke, Th., & Hiller, K.„ Pharmazie

45:313-342 (1990); Lacaille-Dubois, M.A., et al, Phytomedicine 2:363-386 (1996)).

Several polysaccharides (carbohydrate polymers) of mannose (e.g. mannans), β(l,3) glucose (e.g. glucans), β(l,4) acetylated mannose (acemannans), β(l,4) N-acetyl-glucosamine (chitins), and heteropolysaccharides, such as rhamnogalacturonans (pectin), have been shown to stimulate the immune system. It has been shown that conjugation of a protein antigen to mannans under oxidizing conditions resulted in a cell-mediated immune response (Apostopoulos, V. et al, Vaccine 14:930 (1996)). However, protein antigens conjugated to mannans under non-oxidative conditions, i.e., without aldehyde formation, elicited only humoral immunity (Okawa, Y. et al, J. Immunol. Meth. 142:127 (1992) and Apostopoulos, V. et al, Proc. Natl. Acad. Sci. USA. 92:10128 (1995)).

WO 99/17783 discloses polysaccharide conjugates that comprise (i) a polysaccharide that binds to surface receptors present on Antigen Presenting Cells

(APCs), and (ii) one or more compounds having a stable carbonyl group (i.e., an aldehyde or a ketone group that is capable of reacting with amino groups to form an imine or Schiff base) wherein compounds (ii) are attached to the polysaccharide (i) through (iii) a direct covalent bond or covalently via the residue of a bifunctional linker. The conjugates are useful as adjuvant or immunostimulants.

PCT Published Application WO94/07479, published April 14, 1994 broadly discloses the use of low molecular weight compounds having an aldehyde, ketone or amino functional group to enhance the activity of a subject's immune system. The mechanism of action is described as Schiff base formation between a cell surface amino group with the carbonyl group of an administered compound.

Adjuvants have utility in activating the immune system to increase the efficacy of preventative and therapeutic vaccines. Immunoadjuvants have applications in: (1) the non-specific stimulation of host resistance against infection and cancer, (2) the potentiation of preventative vaccine immunogenicity, and (3) the potentiation of therapeutic vaccine immunogenicity. These adjuvants may selectively enhance cell-mediated immune responses (T cell responses, delayed hypersensitivity), humoral responses (B cell responses, antibody production), or both. Stimulation of humoral immunity is important for prevention of bacterial infection as well as in therapy of soft tissue and circulating cancers. Cellular immunity is of major importance for solid tumor cancer therapy and some viral diseases.

After an initial stimulation by a foreign agent or antigen (such as viruses, bacteria, or parasites), the immune system usually recognizes and reacts to the agent with an accelerated response upon re-exposure. This enhanced response forms the basis for the enormous success of vaccination for disease prevention. However, the initial immune response to a foreign antigen requires several days for full response, which is insufficient for protection against infections by highly virulent organisms. A way to achieve a faster protective immune response is by vaccination or immunization with a pathogen, which is usually attenuated or dead. However, in many cases immunization with killed microorganisms or with pure antigens elicits a poor short term immune response with weak or no cell-mediated immunity produced at all. In many cases this poor immune response can be modified by the addition of adjuvants to the antigen preparation.

Some adjuvants, such as insoluble inorganic salts, frequently elicit a response that is not very effective. Several carbohydrate containing plant and bacterial products have been found to improve the immune response. However, in some cases the elicited response is still too weak or their side-effects are too severe for consideration as useful products. New DNA vaccines,, although capable of stimulating a Thl immune response with formation of CTL, still need adjuvants to elicit an effective immune response. Thus, there is a need for new, improved adjuvants to activate the immune system for a variety of uses.

Summary of the Invention

The present invention is directed to enhancing the potentiation of an immune response in a vertebrate, comprising administering an effective amount of a compound of Formula / or a physiologically acceptable salt or ester thereof to enhance the immune response of a vertebrate to one or more antigens.

The present invention is directed to the method of treating diseases where there is a decrease in the host defense immunity, or to enhance activity to the immune system above normal levels by administering a compound of Formula / or a physiologically acceptable salt or ester thereof.

The present invention is directed to a method of treating or preventing cancer in mammals and acute and chronic viral infections by administering a compound of Formula / or a physiologically acceptable salt or ester thereof.

The present invention is directed to a method for treating conditions resulting from non-effective immune response, such as fungal infections, mycoplasma infections, tuberculosis, leprosy, and herpes simplex viral infections by administering a compound of Formula / or a physiologically acceptable salt or ester thereof.

A number of compounds useful in the present invention have not been heretofor reported. Thus, the present invention is also directed to novel lipophilic, aromatic compounds of Formula/; including compounds of Formulae HI-IX.

The present invention is also directed to a method of vaccination, comprising administering one or more antigens, and a compound of Formulae I-

IX.

The present invention is also directed to pharmaceutical and veterinary compositions comprismg one or more of the compounds of Formulae I-IX, and one or more pharmaceutically acceptable diluents, carriers or excipients. The compositions may further comprise one or more immunologically effective antigens or one or more polynucleotides encoding for one or more antigens. These compositions may be employed as immunopotentiators in animals and humans.

The present invention is also directed to vaccines comprising one or more antigens, and one or more compounds of Formulae I-IX.

The present invention is also directed to vaccines comprising one or more DNA sequences encoding for one or more specific antigens, and one or more compounds of Formulae I-IX.

Detailed Description of the Preferred Embodiments

The invention in its broadest aspects relates to the use of substituted aryl- and arylalkyl aldehyde and ketone derivatives for activating the immune system for use in prophylactic and therapeutic vaccines as well as general enhancement of the immune response.

The present invention relates to the use of amphipathic compounds or physiologically acceptable salts thereof for the manufacture of a medicament for the potentiation of an immune response. Without wishing to be bound by theory, compounds of the present invention form imines or Schiff bases with T-cell surface amine groups, and also may associate in a physical form such as a micelle or liposome.

Compounds useful in this aspect of the present invention are represented by Formula /:

or a physiologically acceptable salt or an ester thereof, wherein:

R¹ is -(L)_p- Z; wherein

L is a bifunctional linker selected from the group consisting of -O-; -NH-; -NCH₃-; -S-; -SO₂-; -X-NH-; -C(O)-O -X -; -X-C(O)-, preferably -O-C(O)-; -X-(CH₂) -C(O)-NH- preferably -O-(CH₂)_s-C(O)-NH- -O-(CH₂)_s-C (O)-N = ; -O CH₂CH₂N= ; -O-C ( O)-NH-

-O-CH₂CH₂-O-CH₂CH₂-O-; -OCH₂CH(OH)CH₂O-; -OCH₂CH(OH)- -X-(CH₂)_-, preferably -OCH₂-; -X-(CH₂) -Y- preferably -OCH₂CH₂O-; and an amino acid, wherein X and Y are independently selected from the group consisting of O, NH, NCH₃, S, and SO₂, and when an amino nitrogen is at the end of the linker, then two Z groups, which can be the same or different, are attached to the amino group, wherein s is 1 to 6;

Z is

(A) alkyl, alkoxy, or polyalkoxy, or

(B) aralkyl or aralkoxy, wherein the aryl group is_. substituted with at least one of alkyl, alkanoyl, alkoxy or polyalkoxy, wherein the alkyl, alkanoyl, alkoxy, or polyalkoxy is of sufficient chain length to allow the compound to form a micelle in aqueous solution with other like compounds, and wherein the carbon chain in the alkyl, alkanoyl, alkoxy or polyalkoxy is optionally interrupted with one or more oxygen, nitrogen or sulfur, and wherein

Z is optionally substituted with one or more of hydroxy, carboxy, alkoxy, sulfoxy, alkyl, amino, alkylamino, dialkylamino, alkylthio, alkylsulfonyl, pyridinium, imidazolinium, pyrimidinium, dialkylammonium, choline, halogen, cyano, alkanoyl, alkanoyloxy, -C(O)-O-alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy, sulfinic acid, sulfonic acid, 5-tetrazolyl, or alkylsulphonylcarbamoyl, all of which can be optionally substituted; R² is -(L)_p- Z or is selected from the group consisting of hydrogen, alkyl, alkoxy, alkoxyalkoxy, alkylaryl, arylalkyl, alkoxyaryl, alkoxyalkyl, hydroxy, alkylamino, dialkylamino, alkanoylamino, carboxy, -C(O)-O-alkyl, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium, and choline, all of which can be optionally substituted;

R³ and R⁴ are independently hydrogen, hydroxy, alkyl, halogen, alkoxy, carboxylic acid, sulfonic acid, cyano, 5-tetrazolyl, alkylsulfonylcarbamoyl or phosphonic acid;

R⁵ is hydrogen or methyl; o is 0 to 4, preferably 0 or 1; and p is 0 or 1; with the proviso that when o and p are both 0, R⁵ is H, R² is alkoxy or OH, R³ is alkoxy or H, R⁴ is H and Z is an alkoxy group substituted with carboxy, the alkyl chain in Z contains at least 7 continuous carbon atoms. When p is 0, Z is attached to the phenyl ring by a covalent bond.

The groups R¹, R², R³ and R⁴ can interchangeably be present at any position on the benzene ring to which they are attached. R¹ is preferably in apara position to the carbonyl containing group.

Preferably, the dialkylammonium group is dimethylammonium or diethylammonium.

Another group of useful compounds of this invention are those having the Formula //:

or a physiologically acceptable salt or ester thereof, wherein R¹ is -(L)_p - Z, wherein L is a bifunctional linker as defined above;

R² is -(L)_p- Z or selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, carboxy, alkylamino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline;

R³ and R⁴ are independently hydrogen, halogen, alkyl, alkoxy, hydroxy, carboxylic acid, sulfonic acid, cyano, 5-tetrazolyl, alkylsulfonylcarbamoyl or phosphonic acid;

R⁶ is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, cyano, carboxyl, 5-tetrazolyl, alkylsulfonylcarbamoyl, sulfonic acid and phosphonic acid;

R⁷ is selected from the group consisting of alkyl, alkanoyl, alkoxy, alkoxyalkyl, polyalkoxy, and alkoxyalkoxyalkyl, all of which can be optionally substituted; R⁸ and R⁹ are independently hydrogen, hydroxy, carboxy, -C(O)-O-alkyl, alkanoyl, alkylamino,alkanoyloxy, sulfoxy, alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy and alkoxyalkoxyalkyl, all of which can be optionally substituted; o is 0 to 4, preferably 0; p is O or 1; t is 1 to 6; and u, v and w are independently 0 to 6; with the provisos that 1) the R⁷ group or at least one of R⁸ or R⁹ together with the possible

CH₂ groups includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur; and

2) when o and p are both 0, R² is alkoxy or OH, R³ is alkoxy or H, R⁴ is H, R⁵ is H and Z is an alkoxy group substituted with carboxy, the alkyl chain in Z contains at least 7 continuous carbon atoms. Further another group of useful compounds of the invention are those having the Formula HI:

or a physiologically acceptable salt or ester thereof, wherein

R¹ is -L - Z, wherein L is a bifunctional linker as defined above;

R² is -(L)_p-Z or is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, carboxy, alkylarnino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline, preferably hydrogen, hydroxy, or carboxy;

R³ is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, hydroxy, carboxylic acid, sulfonic acid, cyano, 5-tetrazolyl, alkylsulfonylcarbamoyl or phosphonic acid; preferably hydrogen or hydroxy; R⁶ is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, cyano, carboxyl, 5-tetrazolyl, alkylsulfonylcarbamoyl and phosphonic acid;

R⁷ is selected from the group consisting of alkyl, alkanoyl, alkoxy, alkoxyalkyl, polyalkoxy, and alkoxyalkoxyalkyl optionally substituted with alkylarnino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline;

R⁸ and R⁹ are independently selected from the group consisting of hydrogen, hydroxy, carboxy, -C(O)-O-alkyl, alkanoyl, alkylamino,alkanoyloxy, sulfoxy, alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy and alkoxyalkoxyalkyl, all of which can be optionally substituted; o is 0 to 4, preferably 0; p is O or 1; t is 1 to 6; and u, v and w are independently 0 to 6; with the provisos that 1) the R⁷ group or at least one of R⁸ or R⁹ together with the possible

CH₂ groups includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur; 2) when R¹ is substituted or unsubstituted alkoxy or alkoxyalkoxy and R³ is OH, R² is different from R¹;

3) when R¹ is benzyloxy, R² is OH and R⁷ is alkoxy, one of R³ or R⁶ is other than H; and

4) R¹ is not alkenyloxy, when R² is OH and R³ is H.

Preferably, one of R² or R³ is hydroxy; more preferably R³ is a hydroxy group ortho to the aldehyde group (when o is 0) or to the aldehyde containing alkyl (when o is other than 0). Further, R² is preferably carboxy ortho or para to R³.

Preferred compounds of the present invention are as those, where two or more, preferably two or three, lipophilic chains are included in close proximity, since this promotes micelle formation an also allows for inclusion in liposomes. These side chains may be alkyl, alkenyl, aralkyl, alkoxy, or aralkoxy, among others. A benzaldehyde (preferably a 2-hydroxybenzaldehyde) moiety maintained on the "polar" side of the molecule, or any physical aggregation of molecules, is preferred.

There are several approaches to forming compounds of the present invention, for example:

( 1 ) two lipophilic chains independently attached to the benzaldehyde (Formula IV), (2) a branched lipophilic chain that is attached at a single location on the benzaldehyde (Formula V), or

(3) a propyl (or larger) side chain attached to the benzaldehyde with chains attached to positions 2- and 3- of the propyl moiety (Formula VI and Formula VII).

IV

V

VI

or a physiologically acceptable salt or ester thereof; wherein

R³ is as defined above for Formula ///, and is preferably hydrogen, hydroxy, alkoxy or carboxylic acid;

R¹⁰ and R¹¹ are alkyl, aralkyl, or alkanoyl;

X and Y are O, NH, NMe, S, or SO₂; except when the compound has Formula VII, then X is not S or SO₂; and

R¹² andR¹³ are alkyl, aralkyl, alkoxy, aralkoxy, alkylarnino, aralkylamino, alkanoyloxy or -C(O)-O-alkyl.

In a preferred embodiment, the molecules of Formulae IV, V, VI and VII possess the following structural features:

(1) two lipophilic chains (R¹⁰-R¹³ independently containing 6 to 22 carbons; (2) polar aldehydic linkage separate from lipophilic chain to promote micelle formation; and

(3) an ortho phenolic moiety to stabilize the aldehyde and its interactions with free amino groups.

Compounds of the invention can be synthesized by typical alkylation and acylation reactions. This "building block" approach allows maximum flexibility to prepare numerous compounds using the principals of combinatorial chemistry. Preferably in compounds of Formula IV, when X and Y are both O and R¹⁰ and R¹¹ are both alkyl chains, the alkyl chains are different.

In one aspect, preferred compounds of the present invention have the Formula VIII:

or a physiologically acceptable salt or ester thereof, wherein

R² is selected from the group consisting of hydrogen, hydroxy, or carboxy; R³ is hydrogen or hydroxy; and o, R⁶ and R⁷ are as defined for Formula III, with the provisos that 1) R⁷ includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur; and 2) when R² is OH, and R⁷ is an alkoxy at least one of R³ or R⁶ is other than H. Preferably, R² is OH or COOH in compounds of Formula VIII.

Preferably, a hydroxy group is ortho to the aldehyde group.

In one aspect, compounds useful in the present invention have Formula IX:

or a physiologically acceptable salt or ester thereof, wherein

R², R⁶, R⁷ and o are as defined above for Formula VIII and provided that the R⁷ group includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur.

One group of preferred compounds of the present invention include:

5-carboxy-4-decyloxy-2-hydroxybenzaldehyde;

5-carboxy-4-[4-decyloxybenzyloxy]-2-hydroxybenzaldehyde;

2-hydroxy-4-[l'-n-hexyloxy)ethoxy]-benzaldehyde; 2-hydroxy-4-[2'-( ,3 -di-n-hexyloxy)propyloxy]-benzaldehyde;

2-hydroxy-4- [2'-(l " ,3 "-ditetradecanoxy-2 "-ρropoxy)- l '- ethoxyjbenzaldehyde;

2-hydroxy-4-[2'-(l ",3"-ditetradecanoxy-2"-propoxy)- -ethoxy]- benzaldehyde-3-carboxylic acid; 2-hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-propoxy)-2'-hydroxy-l'- propoxyjbenzaldehyde;

2-hydroxy-4- [2 '-( 1 " ,3 ^M-ditetradecanoxy-2"-propoxy)- 1 -ethoxyethoxy]- benzaldehyde;

2-hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-propoxy)-2'-hydroxy- - propoxy]benzaldehyde-3-carboxylic acid; and

2-hydroxy-3-carboxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)- - ethoxyethoxy]-benzaldehyde-3-carboxylic acid.

Another group of preferred compounds of the present invention include:

4-[4'-decanoylbenzyloxy]-2-hydroxybenzaldehyde; 4-(l-formyl-2-hydroxyphenyl)-N-dodecylcarbamate;

4-dodecyloxyethyloxy-2-hydroxybenzaldehyde;

4-(2-hydroxy-dodecyloxy)-2-hydroxybenzaldehyde;

4-( 1 ',3 -didodecyloxy-propyl-2 -oxy)-2-hydroxybenzaldehyde;

4-[2-(2-octyl-dodecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde; 4-[2-(2-decyl-tetradecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde; 4-[2-(N,N-didecyl)ethyloxy]-2-hydroxybenzaldehyde; 4-(2-octyl-dodecyloxy)-2-hydroxybenzaldehyde; 4-(2-decyl-tetradecyloxy)-2-hydroxybenzaldehyde; and 4-(l-octyl-decyloxy)-2-hydroxybenzaldehyde.

Some compounds included in Formula / can form acid addition salts. In such salts the identity of the acid is of less importance although for use in medicine it must be physiologically acceptable to the recipient. Examples of physiologically acceptable acid addition salts include inorganic and organic acid addition salts, such as hydrochlori.de, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate, acetic acid, dichloroacetic acid and oxalate.

Selection of Lipophilic Groups

Selection of the substituents R¹, R², R³ and R⁴, especially R¹ and R², is critical to the performance of the compounds of this invention. The lipophilicity of R¹ will mainly control aggregation properties (ability to form micelles or lipid membranes) of the molecules. R² will contribute to the increased polarity and hydrophobicity of the aldehyde portion of the molecule. Selections of R² are made to optimize the polarity of the aldehyde-containing portion of the molecule. In a preferred embodiment of this invention, R¹ is alkoxy, polyalkoxy, alkyl or substituted aralkoxy, wherein the alkoxy, polyalkoxy and alkyl and the substituent on the aryl group of the aralkoxy group are of sufficient chain length that these molecules are capable of aggregating to form a micelle in aqueous media.

By sufficient chain length is meant that the group has a continuous chain of from 6 to 40 atoms, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur atoms. Preferably the chain length is from 6 to 28 atoms, more preferably from 8 to 20 atoms, most preferably from 10 to 18 carbon or oxygen atoms. Further, the polarity of the compounds of the invention may be adjusted by adding polar groups at R² or R⁷, such as carboxy, hydroxy, alkylsulfonyl, sulfinic acid, sulfonic acid, dialkylammonium, such as dimethylammonium and diethylammonium, pyridinium, imidazolinium, pyrimidinium, and choline to ensure a head to head alignment of the amphiphilic compound to form either micellar or lamellar structures. In this manner, a grouping of active carbonyl sites that are essential to produce enhanced activation of the T cells in the immune system will be established on the outer surface of a micelle or a lamellar vesicle.

Linkers

The compounds of Formula / may be directly linked to the lipophilic moiety (Z) or may be linked via a linking group. By the term "linking group" is intended one or more bifunctional molecules that can be used to covalently couple the compounds of Formula / to the lipophilic molecule. Bifunctional linkers are well known in the art for various applications (Hermanson, G.T., Bioconjugate Techniques, Academic Press 1996).

The linkers suitable in the present invention include -O-; -NH-; -NCH₃-; -S-; -SO₂-; -X-NH-; -C(O)-O-X-; -X-C(O)-, preferably -O-C(O)-; -X-(CH₂) -C(O)-NH-, preferably -O-(CH₂)_s-C(O)-NH- -0-(CH₂),-C (0)-N= ; -OCH₂CH₂N= ; -O-C (O)-NH- -O-CH₂CH₂-O-CH₂CH₂-O-; -OCH₂CH(OH)CH₂O-; -OCH₂CH(OH)-

-X-(CH₂)_S-, preferably -OCH₂-; -X-(CH₂) -Y-, preferably -OCH₂CH₂O-; and an amino acid, wherein X and Y are independently selected form the group consisting of O, NH, NCH₃, S, and SO₂, s is 1 to 6, and when an amino nitrogen is at the end of the linker, then two Z groups, which can be the same or different, are attached to the amino group.

When a carboxyl group of the lipophilic compound is employed as the conjugating group, one or more amino acids can be employed as the bifunctional linker molecule. Thus, an amino acid such as β-alanine or γ-aminobutyric acid, or an oligopeptide, such as di- or tri- alanine can be employed as a suitable linking molecule.

Further preferred bifunctional linkers include:

-NH-(CH₂)_y-NH- where y is from 2-5, -O-(CH₂)_y-NH-, where y is from 2-5,

-NH-CH₂-C(O)-,

-S-(CH₂)_y-C(O)-, where y is from 1-5,

-S-(CH₂)_y-NH- where y is from 2-5, and

-S-(CH₂)_y-O-, where y is from 1-5.

Definitions

The term "alkyl" as employed herein by itself or as part of another group refers to both straight and branched chain, saturated and unsaturated, radicals of up to 40 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl, tetracosyl, hexacosyl, octacosyl, ethenyl, propenyl, butenyl, 2-butenyl, pentenyl, hexenyl, 2-hexenyl, 2,3-hexedienyl, heptenyl, nonenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, eicosenyl, docosenyl, tetracosenyl, hexacosenyl, and octacosenyl. When alkyl refers to R¹ or R², the alkyl chain includes a continuous chain of 6 to 40 carbon atoms in length, preferably 6 to 28 carbon atoms in length, more preferably 8 to 20 carbon atoms in length, most preferably 10 to 18 carbon atoms in length. The alkyl can be branched symmetrically and asymmetrically. The alkyl groups at other R positions are preferably 1 to 6 carbon atoms in length. Useful alkoxy groups include oxygen substituted by one of the C_{λ iQ} alkyl groups mentioned above.

The term "aryl" as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 14 carbons in the ring portion, such as phenyl, biphenyl, naphthyl or tetrahydronaphthyl. Preferably, the aryl group contains 6-10 carbons in the ring portion.

Useful arylalkyl groups include any of the above-mentioned Cι_₃₀ alkyl groups substituted by any of the C₆.₁₄ aryl groups. Useful values include benzyl, phenethyl and naphthylmethyl.

Useful alkylaryl groups include any of the above-mentioned C₆.₁₄ aryl groups substituted by any of the C^ alkyl groups.

Useful aralkoxy groups include any of the above-mentioned C ₃₀ alkoxy groups substituted by any of the C₆.₁₄ aryl groups. Useful values include benzyloxy, phenethyloxy and naphthylmethyloxy.

Useful aryloxy groups include any of the above mentioned aryl groups attached to an oxy (-O-) group, for example, phenoxy.

A sulfonic acid is -SO₃H.

A sulfinic acid is -SO₂H. A carboxy or carboxylic acid is -COOH.

A phosphoric acid is -PO(OH)₂.

Useful alkanoyl groups include any of the above mentioned C ₃₀ alkyl groups substituted by a carbonyl (-C(O)-) group. Useful values include methanoyl, ethanoyl and propionyl. Useful alkoxyalkyl groups include any of the above-mentioned straight chain C ₃₀ alkyl groups substituted on the alkyl chain by any of the above mentioned C^ alkoxy groups.

Useful alkoxyalkoxy groups include any of the straight C ₃₀ alkoxy groups substituted on the alkoxy chain with any of the C^ alkoxy groups. The term "polyalkoxy" includes any of the straight C^ alkoxy groups substituted at the end of the alkoxy chain with one or more any of the Cι_₃₀ alkoxy groups, preferably 1-5 alkoxy groups.

Useful amino acid groups include -NH-R-C(O)-O- wherein R is straight chain or branched C ₆ alkyl. An alkylsulfonylcarbamoyl group is alkyl-SO₂-NH-C(O)- wherein the alkyl includes C^ alkyl groups.

Useful alkylarnino groups include any of the above-mentioned C^ alkyl groups attached to an amino nitrogen. Useful alkanoylamino groups include any of the above-mentioned C ₃₀ alkanoyl groups attached to an amino nitrogen.

Useful dialkylammonium groups include di(C₁_₄)alkylammonium groups such as dimethylammonium or diethylammonium.

Useful halogen groups include fluorine, chlorine, bromine and iodine. Optional substituents include any one of halogen, hydroxy(Cι_₆)alkyl, amino(C₁_₆)alkyl, C^ alkylarnino, di(C_j.6)alkylamino, hydroxy, nitro, C_h6 alkyl, C^g alkoxy, carboxy, amino, C ₆ alkanoyl, aminocarbonyl, pyridinium, imidazolinium, pyrimidinium, dialkylammonium, sulfinic acid, sulfonic acid, and choline.

Methods of Making

Substituted aromatic aldehyde derivatives useful in the present invention can be prepared by the procedures outlined in Schemes 1-22. Many modifications to these procedures will be apparent to one of ordinary skill in the art and, therefore, these syntheses should be considered as representative rather than all inclusive.

Scheme 1 illustrates the preparation of compounds of Formula/, wherein R¹ is alkoxy.

Scheme 1

A hydroxy- or dihydroxy-benzaldehyde (1) is reacted with an alkylating agent to form an alkoxy-hydroxybenzaldehyde (2). The resulting compound (2) is optionally reacted to add a further, highly-polar substitute, such as a carboxylic acid group (3). Useful benzaldehyde starting materials include, but are not limited to: 2-hydroxybenzaldehyde, 3 -hydroxybenz aldehyde, 4- hydroxybenzaldehyde,2,3-dihydroxybenzaldehyde,2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 2,6-dihydroxybenzaldehyde, 3,4- dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, vanillin (4-hydroxy-3- methoxybenzaldehyde), phloroglucinol carboxaldehyde. Illustrative alkylating agents (RX' ) that can be employed in step 1 include alkylbromides, such as octyl bromide, decyl bromide, dodecyl bromide, or tetradecyl bromide. Suitable intermediates can be prepared by reacting the benzaldehyde (1) with haloethers, such as 2-chloroethylethyl ether, 2-chloroethyl- methyl ether, 2-methoxyethoxymethyl chloride (MEM-Cl), 2-(2-ethoxy- ethoxy)ethyl chloride, di(ethyleneglycol)butyl ether chloride, di(ethyleneglycol)dodecyl ether chloride, and di(ethyleneglycol)hexyl ether chloride.

Scheme 2a illustrates the preparation of compounds of Formula VIII, where R⁷ is alkoxy or a polyalkyl ether.

Scheme 2a

A phenol (4) is reacted with an alkylating agent (RX to form an alkoxylated benzene derivative (5). Alkylating agents that can be employed include those agents described above for Scheme 1. Compound (5) is reacted with paraformaldehyde and hydrochloric acid to form a substituted benzyl chloride (6), which is thereafter reacted with a hydroxy moiety of dihydroxybenzaldehyde (1) to form product (7).

Hydroxy and dihydroxybenzaldehyde starting materials (1) are as described above for Scheme 1.

Scheme 2b describes the preparation of another preferred subset of compounds of Formula VIII, where R⁷ is alkyl.

Scheme 2b

An alkylated benzene (8) is reacted with paraformaldehyde or its equivalent and hydrochloric acid to form an alkylated benzyl chloride (9). The intermediate (9) is then reacted with a hydroxy- or dihydroxybenzaldehyde as described in Scheme 2a above to form the product (10).

Aromatic derivatives useful as starting materials include alkyl substituted benzenes, such as 1-phenylhexane, 1-phenyldecane, and 1-phenyldodecane.

Scheme 3 describes the preparation of compounds of Formula VIII, where R⁷ is an alkanoyl group. Scheme 3

Toluene (11) is reacted with an acylating agent (RCO₂H or RCOC1) to form an acylated toluene (12). Acylating agents include carboxylic acids or activated acid derivatives, such as acid chlorides. Examples include octanoic acid, decanoic acid, dodecanoic acid, ortetradecanoic acid. The intermediate (12) is brominated to form the corresponding acylbenzylbromide (13). Compound (13) is then reacted with a hydroxy or dihydroxybenzaldehyde to form product (14).

Scheme 4 illustrates the modification of the product (14) of Scheme 3 with a carboxylic acid group to adjust the polarity of the head group. Carboxylation may be accomplished by carbon dioxide/sodium hydroxide to direct ortho substitution or carbon dioxide/potassium hydroxide to direct para substitution (Kolbe Schmitt Reaction); or ortho lithiation using n-butyl lithium and subsequent reaction with carbon dioxide.

Scheme 4

Scheme 5 illustrates the preparation of compounds of Formula / wherein R¹ is alkyl, R² is carboxy, and R⁵ is H or methyl.

Scheme 5

Phenol derivatives as starting materials may include, but are not limited to, 4-octylphenol, 4-dodecylphenol and 4-dodecylresorcinol.

The phenol starting materials can be subjected to a number of reaction conditions and reagents, including for example: dimethylformamide/phosphorus oxychloride (Vilsmeier Reaction), HCN/hydrochloric acid (Gatterman Reaction),

CO/hydrochloric acid (Gatterman Koch Reaction), hexamethylene tetramine/hydrochloric acid (Reimer-Tieman Reaction), acetic acid/sulfuric acid, and acetyl chloride/aluminum chloride (Friedel-Crafts Reaction). Carboxylation may be accomplished by carbon dioxide/sodium hydroxide to direct ortho substitution or carbon dioxide/potassium hydroxide to direct para substitution (Kolbe Schmitt Reaction); or ortho lithiation using n-butyl lithium and subsequent reaction with carbon dioxide.

Schemes 6 and 7 illustrate the preparation of compounds of Formula VIII, wherein the second phenyl ring has added polarity (hydrophilicity) which may provide further directing influence to the formation of micelles.

The physical properties of these hydroxy substituted analogues of Scheme 6 may cause handling and purification problems arising from potential metal ion chelation arising from biphenolic coordination. Introduction of the carboxylic acid moiety in the carboxyalkoxy analogues of Scheme 7 may be accomplished under a variety of reaction conditions, some of which are identified herein.

Scheme 6

Scheme 7

Scheme 8 illustrates the preparation of compounds of Formula /// wherein R¹ is alkoxy para to the aldehyde group, R² is H or carboxy and R³ and R⁴ are independently H or OH. Sc heme 8

2,4-dihydroxybenzaldehyde

By a similar method compounds can be prepared where R¹ is alkoxyalkoxy, such as ethylene glycol analogues, for example,

using ethylene oxide and its derivatives followed by alkylation to give ethylene glycol analogues, and hydroxy ethylene glycol analogs, for example,

from 2,4,6-trihydroxybenzaldehyde.

Scheme 9 illustrates the preparation of compound of Formula / that lack an ort/io-hydroxy which participates in imine stabilization. In Scheme 9, R is alkyl, alkoxy, or glycol, R² is sulfonic acid which is further converted to an ester. A variety of R' groups can be used to alter the solubility of these compounds.

Scheme 9

Schemes 10, 11 and 12 can be employed to form compounds of Formula / wherein a lipophilic residue is attached to the benzaldehyde group via a thio-, sulfonyl, or sulfinyl linkage.

Scheme 10

Scheme 11

Scheme 12

Scheme 13 illustrates preparation of compounds of Formula/// wherein R⁹ is -C(O)-O-alkyl.

Scheme 13

Scheme 14 illustrates synthetic pathways to form compounds of the invention where the lipophilic group is attached to the benzaldehyde via a variety of different linking groups, including a carbonate, carbamate and a combination of ether and amide groups (aminocarbonylmethoxy).

Scheme 14

Scheme 15 illustrates preparation of compounds of the invention wherein the lipophilic group is attached to the benzaldehyde ring via a longer linking group (-O-(CH₂)₆-). Scheme 15

Methods of preparing compounds of Formulae IV-VII

Useful starting materials for incorporating the benzaldehyde moiety into compounds of the invention include, but are not limited to, 2,3,4- trihydroxybenzaldehyde, 2 ,3 -dihydroxybenzaldehyde, 2,4- dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 3 ,4- dihydroxybenzaldehyde, 5-chloro-2-hydroxybenzaldehyde, vanillin, ethylvanillin, 3-hydroxybenzaldehyde and 4-hydroxybenzaldehyde.

Useful starting materials for incorporating one or two lipophilic chains into the adjuvant molecules include: 1,2-epoxydodecane, 1,2-epoxyhexadecane, 1,2-epoxyhexane, l,2-epoxy-5-hexane, c -?-7,8-epoxy-2-methyloctadecane, 1,2- epoxyoctadecane, 1,2-epoxyoctane, l,2-epoxy-7-octene, 1,2-epoxypentane, 2,3- epoxypentane, 2,3-epoxypropylbenzene, 2,3-epoxy-l-propanol, 1,2- epoxytetradecane, 3 ,4-epoxytetrahydrothiophene- 1 , 1 -dioxide, 1 -hexanethiol, hexyl alcohol and hexylamine.

Compounds of Formula IV (where X and Y are both oxygen) can be synthesized by employing, for example, 2,3,4-trihydroxybenzaldehyde as a starting material. Controlled alkylation, acylation, or a combination thereof produces a compound of Formula IV wherein R¹⁰ and R¹¹ are the same or different. Suitable alkylating agents are among the group as listed above for Scheme 1. Schemes 16 and 17 illustrate synthetic pathways for preparing compounds of Formulae V-VI.

Useful starting materials to form linkers in compounds of Formulae For

VI , where R¹² is alkyl or aryl include epichlorohydrin, propylene sulfide, propyl ether, 1 ,2-epoxybutane, 1 ,2-epoxydecane, 1 ,2-epoxy-9-decene, and styrene oxide.

Useful starting materials to form linkers in compounds of Formulae For

VI, where R¹² is alkoxy, aralkoxy, or alkylarnino include glycidyl 4- methoxyphenyl ether, l ,2-epoxy-3-phenoxypropane, N-(2,3- epoxypropyl)phthalimide, (R)-(-)-glycidyl butyrate, and glycidol. A useful starting material to form linkers in compounds of Formula VII is 2,3-dichloropropionyl chloride.

In the preparation of compounds of Formulae V and VI, the order of addition will control whether the lipophilic chains are branched at the point of attachment to the aromatic moiety or not. Reaction of a variety of epoxides with anions will occur at the least hindered 3-position to form 2-hydroxyl intermediates. If the epoxide is reacted with an anion of alkyl alcohols, such as hexanol, heptanol, octanol, nonanol, decanol, dodecanol, intermediate (16-a) will form. Activation of the hydroxyl as a leaving group (tosylate, chloride, mesylate, etc.) and subsequent treatment with the anion of 2,4-dihydroxybenzaldehyde will produce compounds of Formula V where R¹³ is alkoxy. If the epoxide is reacted first with the 2,4-dihydroxybenzaldehyde, intermediate (17-a) will form.

Subsequent treatment with base and alkylation with an alkyl halide will produce compounds of Formula VI. If intermediate (17-a) is treated with an alkanoyl halide rather than an alkylating agent, compounds of Formula VI will form wherein R¹³ is alkanoyloxy rather than alkoxy.

Use of amine or mercaptan nucleophiles in this reaction will produce the analogous derivatives wherein X is ΝH, ΝMe or S in Formula V. Mild peroxide oxidation of compounds where X is S will provide the sulfone analogs (X is SO₂).

Compounds of Formula VII may be prepared by reaction of 2,3- dichloropropionoic acid derivatives with 2,4-dihydroxybenzaldehyde to form an ester at the 4-phenol hydroxyl group. Sequential displacement of the chlorides with alcohol, amine or mercaptan nucleophiles produces substitution wherein R¹² and R¹³ are the same or different.

Scheme 16

17

Based upon these considerations, the following compounds (18, 19, and 20) may be synthesized. Scheme 17

20

Scheme 18 illustrates the synthesis of compounds included in Formula VII, wherein m and n are independently 6 to 30. Scheme 18

Scheme 19 illustrates the synthesis of compounds included in compounds Formula V, wherein m and n are independently 6 to 30.

Scheme 19

Scheme 20 illustrates the synthesis of compounds included in compounds of Formulae V and VII.

Scheme 20

Methods for Preparing Compounds Having Two Lipophilic Chains Comprising at least 12 Carbon Atoms

Schemes 21 and 22 illustrate methods for forming compounds of the present invention that possess:

1. a lipophilic chain consisting of two at least 12-carbon alkyl chain;

2. polar aldehyde group distant from lipophilic chain to promote liposome formation;

3. ortho phenolic moiety to stabilize the aldehyde and activate it to membrane interactions;

4. optional carboxyl substitution on phenol for additional polarity.

These schemes utilize related chemistry to access multiple classes of compounds; use well-known chemical conversions to ensure high likelihood of successful conversion; and minimize number of chemical conversion steps. The standard laboratory reagents needed as starting materials include a variety of solvents including DMF as source of the aldehyde group in the Vilsmeier reaction, POCl₃, bases including sodium hydride and sodium hydroxide, and concentrated hydrochloric acid. The following experimental procedures provide one route of preparation of compounds of the present invention. Alternative procedures are also available including: European Patent Application EP 0,079,630 Al (1983); U.S. Patent 4,217,390 (1981); Voelkel etal, J. Chromatography 398:31 (1989); andChedini etal, Chem. Mater.3:752 (1991), all of which are fully incorporated by reference herein.

Scheme21 illustrates the preparation of four intermediates (21,22,23 and 24) that can be employed to form compounds of the invention. For example, myristyl alcohol (C₁₇H₂₉OH) can be employed as the alcohol to form (21) where n=17.

Scheme 21

where n is from 12 to about 30, preferably 12 to 18. The 3-position of the carboxy group in the phenyl group in compounds 26, 28 and 30 is only exemplary, and the carboxy group may be also in the 5- and 6-positions of the phenyl group.

Scheme 22

Methods of Using and Pharmaceutical and Veterinary Compositions

Compositions of the invention are useful as vaccines to induce active immunity towards antigens in subjects. Any animal that may experience the beneficial effects of the compositions of the present invention within the scope of subjects that may be treated. The subjects are preferably vertebrates, more preferably mammals, and more preferably humans.

The compounds of the present invention can be employed as a sole adjuvant, or alternatively, can be administered together with other adjuvants. Such other adjuvants useful with the present invention include oil adjuvants (for example, Freund's Complete and Incomplete), liposomes, mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), oligonucleotides containing unmethylated CpG dinucleotides, and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacteriumparvum, Bordetella pertussis, and members of the genus Brucella), bovine serum albumin, diphtheria toxoid, tetanus toxoid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A, choleragenoid, cholera toxin, pertussis toxin, viral proteins, and eukaryotic proteins such as interferons, interleukins, or tumor necrosis factor. Such proteins may be obtained from natural or recombinant sources according to methods known to those skilled in the art. When obtained from recombinant sources, the non-saponin adjuvant may comprise a protein fragment comprising at least the immunogenic portion of the molecule. Other known immunostimulatory macromolecules which can be used in the practice of the invention include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenyl- methane-3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (See Sela, M., Science 166:1365-1374 (1969)) or glycolipids, lipids or carbohydrates. The compounds of the present invention exhibit adjuvant effects when administered over a wide range of dosages and a wide range of ratios to one or more particular antigens being administered. The initial dose may be followed up with a booster dosage after a period of about four weeks to enhance the immunogenic response. Further booster dosages may also be administered.

The compounds of the present invention can be administered either individually or admixed with other substantially pure adjuvants to achieve an enhancement of immune response to an antigen.

The compounds of the present invention can be utilized to enhance the immune response to one or more antigens. Typical antigens suitable for the immune-response provoking compositions of the present mvention include antigens derived from any of the following: viruses, such as influenza, feline leukemia virus, feline immunodeficiency virus, HIN-1, HIV-2, rabies, measles, hepatitis B , or foot and mouth disease; bacteria, such as anthrax, diphtheria, Lyme disease, or tuberculosis; or protozoans, such as Babeosis bovis or Plasmodium.

The antigen can be proteins, peptides, polysaccharides, or mixtures thereof. The proteins and peptides may be purified from a natural source, synthesized by means of solid phase synthesis, or may be obtained means of recombinant genetics. The compounds of the present invention can be utilized to enhance the immune response to one or more antigens produced by the transient expression of the protein antigen(s) upon direct inoculation of the DΝA encoding for such antigen(s). Typical DΝA suitable for the immune-response provoking composition of the present invention include DΝA sequences coding for different viral proteins, such as hepatitis B, hepatitis C, HTV-1, HSV-1 and 2, different

HPVs, rabies, influenza, measles and others; bacterial, such as anthrax, diphteria, Lyme disease, and tuberculosis; protozoans, such as Babesia bovis and Plasmodium sp. or cancer cells, such as carcinoembryonic antigen, truncated EGF, prostate membrane surface antigen, and others. The DΝA may be purified from natural sources, synthesized by organic synthesis, or may be obtained by means of recombinant DNA methods.

The step of administering the DNA or RNA vaccine may be performed in vivo or ex vivo, the latter including the administration of the infected/transfected cells. In addition, where an antigen or tumor antigen is administered, the nucleic acid composition may also be designed to direct the expression of such antigens (either on the same or different vectors ormolecules). Immunogenic polypeptides can be used to elicit or enhance an immune response to an antigen coded by a DNA vaccine. DNA vaccines encode one or more immunostimulating antigens, such that the antigen is generated in situ. For instance, the DNA vaccine may encode a tumor antigen and, optionally, an immunogenic polypeptide. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an epitope of a prostate cell antigen on its cell surface. The DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al, PNAS 8-5:317-321 (1989); Flexner et al, Ann. N.Y. Acad. Sci. 569:86-103 (1989); Flexner et al, Vaccine 5:17-21 (1990); U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO

91/02805; Berkner, Biotechniques 6:616-627 (1988); Rosenfeld et al, Science 252:431-434 (1991); Kolls et al, PNAS 97:215-219 (1994); Kass-Eisler et al, PNAS 90:11498-11502 (1993); Guzman etal, Circulation 88:2838-2848 (1993); and Guzman etal, Cir. Res. 73:1202-1207 (1993). Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in published PCT application WO 90/11092, and Ulmer etal. , Science 259: 1745-1749 (1993), reviewed by Cohen, Science 259:1691-1692 (1993). The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

Compositions capable of delivering nucleic acid molecules encoding an immunogenic polypeptide or portion thereof include recombinant viral vectors, e.g., retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (see Berkner, Biotechniques 6:616-627 (1988); Li et al, Hum. Gene Tlier. 4:403-409, 1993; Vincent et al, Nat. Genet.

5:130-134 (1993); and Kolls et al, Proc. Natl. Acad. Sci. USA 91 :215-219 (1994)), pox virus (see U.S. Pat. No.4,769,330; U.S. Pat. No.5,017,487; and WO 89/01973)), naked DNA (see WO 90/11092), nucleic acid molecule complexed to a polycationic molecule (see WO 93/03709), and nucleic acid associated with liposomes (see Wang et al, Proc. Natl Acad. Sci. USA 84:7851 (1987)). In certain embodiments, the DNA may be linked to killed or inactivated adenovirus (see Curiel et al, Hum. Gene Ther. 3:147-154 (1992); Cotton et al, Proc. Natl. Acad. Sci. USA 89:6094 (1992)). Other suitable compositions include DNA-ligand (see Wu et al, J. Biol. Chem. 264: 16985-16987 (1989)) and lipid-DNA combinations (see Feigner et al, Proc. Natl. Acad. Sci. USA

84:7413-7417 (1989)). In addition, the efficiency of nakedDNA uptake into cells may be increased by coating the DNA onto biodegradable latex beads.

In an in vivo administration of a DNA or RNA vaccine, a polynucleotide operatively encoding for an immunogenic polypeptide in a pharmaceutically acceptable administrable carrier is administered in vivo into a tissue of a vertebrate, preferably mammal, suffering from cancer or pathogenic infection, wherem the polynucleotide is incorporated into the cells and a therapeutically effective amount of an immunogenic polypeptide is produced in vivo. The DNA or RNA formulation may further comprise a cationic vehicle such as cationic lipids, peptides, proteins or polymers, and are injected into muscle or other tissue subcutaneously, intradermally, intravenously, orally or directly into the spinal fluid. Of particular interest is injection into skeletal muscle. The tissue may also be skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, or connective tissue. An example of intramuscular injection may be found in Wolff et al, Science 247:1465-1468 (1990). Jet injection may also be used for intramuscular administration, as described by Furth et al. , Anal. Biochem. 205:365-368 (1992). The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun". Microparticle DNA vaccination has been described in the literature (see, for example, Tang et al, Nature 356:152-154). Gold microprojectiles are coated with the vaccine cassette, then bombarded into skin cells. Circular DNA molecules are preferred as they will persist longer than single-stranded polynucleotides, and they will be less likely to cause insertional mutation by integrating into the target genome.

The polynucleotide material delivered to the cells in vivo can take any number of forms. It may contain the entire sequence or only a fragment of an immunogenic polypeptide gene. It may also contain sequences coding for other polypeptide sequences. It may additionally contain elements involved in regulating gene expression (e.g., promoter, enhancer, 5'or 3'UTRs, transcription terminators, and the like). The polynucleotide may also comprise an immunostimulatory sequence that would enhance the immunogenicity of a given gene product, and/or it may comprise sequences that would enhance the delivery of the polynucleotide, such as by increasing cellular and/or nuclear uptake. Techniques for obtaining expression of exogenous DNA or RNA sequences in a host are known. See, for example, Korman et al, Proc. Nat. Acad. Sci. (USA) 84:2150-2154 (1987).

In addition to direct in vivo procedures, ex vivo procedures may be used in which cells are removed from an animal, modified, and placed into the same or another animal. Protocols for viral, physical and chemical methods of uptake are well known in the art.

DNA-based immunization refers to the induction of an immune response to an antigen expressed in vivo from a gene introduced into the animal. This method offers two major advantages over classical vaccination in which some form of the antigen itself is administered. First, the synthesis of antigen in a self-cell mimics in certain respects an infection and thus induces a complete immune response but carries absolutely no risk of infection. Second, foreign gene expression may continue for a sufficient length of time to induce strong and sustained immune responses without boost.

Several mammalian animal models of DNA-based immunization against specific viral, bacterial or parasitic diseases have been reported. These include influenza (Fynan et al, Proc. Nat'l Acad. Sci. USA 90:11478-11482 (1993); Montgomery et al. , DNA Cell Biol. 72:777-783 (1993); Robinson et al. , Vaccine 11:957-960 (1993); Ulmer etal, Science 259:1745-1749 (1993)), HIV (Wang et al. (1993)), hepatitis B (Davis et al, Hum. Molec. Genet. 2:1847-1851 (1993)), malaria(Sedagahet Z.,Rrøc. Nαt7Acα Sc/. f/SA9i:9866-9870 (1994)), bovine herpes (Cox etal., J. Virol 67:5664-5667 (1993)), herpes simplex (Rousse etal, J. Virol. 68:5685-5689 (1994); Manicken et al, J. Immunol. 155:259-265 (1995)), rabies (Xiang et al, Virology 199:132-140 (1994)), lymphocytic choriomeningitis (Yokoyama et al, J. Virol. (5964:2684-2688 (1995)) and tuberculosis (Lowrie et al, Vaccine 72:1537-1540 (1994)). In most of these studies a full-range of immune responses including antibodies, cytotoxic T lymphocytes (CTL), T-cell help and (where evaluation was possible) protection against challenge was obtained. In these studies naked DΝA was introduced by intramuscular or intradermal injection with a needle and syringe or by instillation in the nasal passages, or the naked DΝA was coated onto gold particles which were introduced by a particle accelerator into the skin.

Cancer cells often have distinctive antigens on their surfaces, such as truncated epidermal growth factor, folate binding protein, epithelial mucins, melanoferrin, carcinoembryonic antigen, prostate-specific membrane antigen, HER2-neu, which are candidates for use in therapeutic cancer vaccines. Because tumor antigens are normal or related to normal components of the body, the immune system often fails to mount an effective immune response against those antigens to destroy the tumor cells. To achieve such an immune response certain triterpenoid saponins and their derivatives, as well as the lipophilic aromatic aldehyde derivatives subject of the present invention can be utilized. Triterpenoid saponin adjuvants containing an aldehyde and a lipophilic side chain work by reacting with amino groups of certain receptor protein(s) present on T cells, and forming Schiff bases. As a result of this reaction, the T cell is stimulated to produce cytokines that bias the immune response toward a Thl type. The lipophilic side chain of these saponins, by interacting with the cell membrane, allows the delivery of exogenous proteins directly into the cytosol and their process by the endogenous pathway, leading to the production of cytolytic or cytotoxic T cells (CTLs). A similar result can be obtained by using liposomes containing an antigen and having a lipophilic aromatic aldehyde in their lipid composition as delivery systems. The targeting efficacy of liposomes can be enhanced by attaching to their external surface certain ligand, such as oligosaccharides, that may interact with specific cell surface receptors on antigen presenting cells. Liposomes, by fusing with the lipid bilayer of the cell membrane, are capable of delivering protein antigens directly into the cytosol for processing. However, although capable of delivering cytokines to stimulate the immune response, the currently used liposomes are not capable of providing the T cell with the costimulatory signal required for a Thl response. Inclusion of lipophilic aromatic aldehydes in the liposome lipid composition allows the liposomes to deliver their antigen load into the cell's cytosol as well as to provide the costimulatory signal required for a Thl response and CTL formation.The unique adjuvant effect of these compounds and formulations induces the production of antigen specific CTLs which seek and destroy these tumor cells carrying on their surface the tumor antigen(s) used for immunization. The compounds of the present invention can also be used with carbohydrate tumor antigens, such as gangliosides, the Thomsen-Friedenreich (T) antigen, and others. The compounds of the present invention can be administered with an antigen(s) or DNA sequence coding for an specific antigen(s), alone or in combination with therapeutic agents such as immunomodulators, such as

Ampligen (mismatched RNA) (DuPONT/HEM Research), colony stimulating factors including GM-CSF (Sandoz, Genetic Institute), interferon-α (Glaxo- Wellcome), interferon-γ, interleukin-1 (IL-1) (Hoffman-LaRoche, Lnmunex), interleukin-2 (IL-2) (Chiron Corporation), heat shock proteins (hsp), cytosine expressing plasmids, immunomodulatory oligonucleotides containing unmethylatedCpGdinucleotides (CpGLnmunoPharmaceuticals,Inc.), and RNA immunomodulator (Nippon Shingaku).

In another aspect of the invention, the present invention provides the use of a compound of Formula / or a physiologically acceptable salt or ester thereof for treating conditions resulting from non-effective immune response, such as fungal infections, mycoplasma infections, tuberculosis, leprosy, and herpes simplex viral infections.

In another aspect of the present invention provides for the use of the compound of Formula / or a physiologically acceptable salt or ester thereof for the manufacture of a medicament for the treatment and/or prophylaxis of cancer in mammals.

Examples of forms of cancers particularly suitable for treatment with compounds the present invention are: melanoma, breast cancer, colon cancer, cancer of the head and neck, gastric cancer, renal cancer, laryngeal cancer, rectal cancer, and non-Hodgkins lymphoma. Cancers that express turnout specific antigens or antigens rarely expressed or expressed at very low density on normal cells, are likely therapeutic targets. Cancers which contain turnout specific cytotoxic T-cells which are anergic or otherwise ineffective are likely targets for therapy. Surgically resetted tumors where there is a high risk of recurrence are also suitable for therapy with compounds of the present invention. Without wishing to be bound by theory, compounds of Formula / act by providing a co-stimulatory signal to cloned (partially) activated T-cells in vitro, thus maximally activating T-cells.

A further aspect of the present invention provides for the use, as a vaccine adjuvant, of a compound of Formula/ or a physiologically acceptable salt or ester thereof. A vaccine may therefore be prepared by formulating an antigenic component or a DNA encoding for specific antigen(s) with a compound of Formula /. Vaccines of the present invention can include one or more bacterial antigens from a particular bacteria. Bacteria for which vaccines can be formulated include: Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis,

Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholera, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejune, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphteria, Bordetella pertussis, Bordetella parapertussis, Bordetella brochiseptica, Hemophilus influenza, Escherichia coli,

Shigella spp., Erlichia spp., and Rickettsia spp.

Bacterial antigens can be native, recombinant, synthetic, or transiently expressed in vivo after inoculation with DNA sequences encoding for such antigens. Such bacterial antigens include, but are not limited to, selectins or lectins from bacteria that bind to carbohydrate determinants present on cell surfaces; and bacteria receptors for proteins, such as fibronectin, laminin, and collagens.

Vaccines of the present invention can include one or more antigens from a particular virus to form a vaccine. Viruses for which vaccines can be formulated include: Influenza virus, Mumps virus, Adenoviruses, Respiratory syncytial virus, Epstein-Barr virus, Rhinoviruses, Polioviruses, Coxsackieviruses, Echoviruses, Rubeola virus, Rubella virus, Varicell-zoster virus, Herpes viruses (human and animal), Herpes simplex virus, Parvoviruses (human and animal), Cytomegalo virus, Hepatitis viruses, Human papillomavirus, Alphaviruses, Flaviviruses, Bunyaviruses, Rabies virus, Arenaviruses, Filoviruses, FflV 1 , HTV

2, HTLV-1, HTLV-π, FeLV, Bovine LV, FelV, Canine distemper virus, Canine contagious hepatitis virus, Feline calicivirus, Feline rhinotracheitis virus, TGE virus (swine), and Foot and mouth disease.

Viral antigens can be native, recombinant, synthetic, or transiently expressed in vivo after inoculation with DNA sequences encoding for such antigens. Such viral antigens include, but are not limited to, viral proteins that are responsible for attachment to cell surface receptors to initiate the infection process, such as (i) envelope glycoprotein of retroviruses (FflV, HTLV, FeLV and others) and herpes viruses, and (ii) the neuramidase of influenza viruses. Additionally, peptides derived from such viral proteins can be employed either free, associated non-covalently, or conjugated covalently to a suitable carrier.

Vaccines of the present invention can include one or more tumor associated antigens. Tumor associated antigens can be native, recombinant, synthetic, or transiently expressed in vivo after inoculation with DNA sequences encoding for such antigens. Such tumor associated antigens include, but are not limited to, killed tumor cells and lysates thereof, MAGE-1 or MAGE-3 and peptide fragments thereof, Human chorionic gonadotropin (HCG) and peptide fragments thereof, Carcinoembryonic antigen (CEA) and peptide fragments thereof, Alpha fetoprotein (AFP) and peptide fragments thereof, Pacreatic oncofetal antigen and peptide fragments thereof, MUC-1 and peptide fragments thereof, CA 125, 15-3, 19-9, 549, 195 and peptide fragments thereof, Prostate- specific antigens (PSA) and peptide fragments thereof, Prostate-specific membrane antigen (PSMA) and peptide fragments thereof, Squamous cell carcinoma antigen (SCCA) and peptide fragments thereof, Ovarian cancer antigen (OCA) and peptide fragments thereof, Pancreas cancer associated antigen (PaA) and peptide fragments thereof, Herl/neu and peptide fragments thereof, gp-100 and peptide fragments thereof, mutant K-ras proteins and peptide fragments thereof, mutant p53 and peptide fragments thereof, truncated epidermal growth factor receptor (EGFR), and chimeric protein p210^BCR"ABL. Peptides that are derived from these tumor associated antigens can be employed, either free, or non-covalently associated, or conjugated covalently to a suitable carrier. Alternatively, gangliosides can be employed, either free, non- covalently associated or conjugated covalently to a suitable carrier, or oligosaccharide sequences that are specific or predominantly found in cancer cells can be employed either free, non-covalently associated or conjugated covalently to a suitable carrier.

DNA encoding for specific tumor associated antigens can be used to induce a transient expression of the immunogen(s) in vivo that stimulates an immune response against such an antigen(s). The DNA used for polynucleotide- mediated immunization can be isolated directly from the tumor cells, cloned and expressed as DNA plasmids, or prepared by organic synthesis. The DNA used can also encode for cytokines or carry unmethylated CpG dinucleotides to produce a synergistic adjuvant effect.

Adjuvant effect can be assessed by increase in antigen-specific antibody titers due to addition of potential adjuvant in the immunization formulation.

Increased titers result from increased antibody concentrations and/or increased antigen/antibody affinity. Adjuvant effects can be measured by increase in titer of neutralizing antibodies to foot-and-mouth disease vaccines in guinea pigs (Dalsgaard, K., Archiv. fur die gesamte Virusforschung 44:243-254 91974)), increase in titer of precipitating antibodies to BSA (as measured by radial immunodiffusion) in guinea pigs vaccinated with BSA/saponin mixtures (Dalsgaard, K., Acta Veterinaria Scandinavica 69:1-40 (1978)), as well as by the increase in titer of anti-keyhole limpet hemocyanin (KLH) antibody (measured by ELIS A) in mice immunized with KLH/saponin (Scott et al. Int. Archs. Allergy appl. Immun. 77:409-412 (1985)). A compound of the mvention or a physiologically acceptable salt thereof, may be used for the treatment of diseases where there is a defect in the immune system and/or an ineffective host defense mechanism, or to enhance activity of the immune system above normal levels. Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subject or in a test system to which the antigen is administered.

A compound of the invention or a physiologically acceptable salt thereof may be administered for the treatment or prophylaxis of immunodeficient mammals alone or in combination with other therapeutic agents, for example, with other antiviral agents, or with other anti-cancer agents.

By an "effective amount" is meant an amount of a compound of Formula / that will restore immune function to substantially normal levels, or increase immune function above normal levels in order to eliminate infection. By potentiation of an immune response is meant restoration of a depressed immune function, enhancement of a normal immune function, or both. Immune function is defined as the development and expression of humoral (antibody-mediated) immunity, cellular (T-cell-mediated) immunity, or macrophage and granulocyte mediated resistance. In this specification the term "immunodeficient patient" will be used to describe patients with a deficient or defective immune system. An immunodeficient patient can be characterised by means of a T-lymphocyte proliferation assay. Using this assay immunodeficient patients are characterised by a reduced ability of the T-cells to respond to stimulation by mitogens. An example of a mitogen commonly used in this assay is phytohaemagglutinin

(PHA).

Immunodeficiency and immunosuppression are thought to occur in many clinical situations where there are lesions in signalling to lymphocytes, particularly T-cells that orchestrate the immune response. T-cells require two signals in order to initiate an effective immune response: (i) occupation of the specific T-cell receptor for antigen, and (ii) stimulation through costimulatory receptors. In the absence of signal (ii), T-cells fail to respond and may also become chronically paralyzed, or anergic. Persistent viral and bacterial infections and neoplastic disease can produce T-cell hyporesponsiveness by interfering in various ways with the delivery of secondary signals and in this way evade the immune response. The compounds of Formula / appear to work by substituting or otherwise compensating for deficient costimulatory signals to T-cells.

There are a variety of circumstances in which the immune system may be defective or deficient. For example immune system deficiency is common in immature or premature infants (neonates). It may also result from suppression by certain drugs which may be deliberate e.g. as a side-effect of cancer chemotherapy. Disordered growth of one or more constituent parts of the immune system, e.g. as in certain forms of cancer, may also result in immunodeficiency. Immune deficiency can also be caused by viral infections, including human immunodeficiency virus (HIV).

A further aspect of the present invention provides for the use of a compound of Formula / or a physiologically acceptable salt or ester thereof for the treatment and/or prophylaxis of acute and chronic viral infections. Examples of acute viruses against which immunopotentiatory therapy with a compound of Formula/ or a physiologically acceptable salt or ester thereof may be used, preferably in conjunction with an antiviral agent, are: herpes viruses, influenza viruses, parainfluenza viruses, adenoviruses, coxsakie viruses, picorna viruses, rotaviruses, hepatitis A virus, mumps virus, rubella virus, measles virus, pox viruses, respiratory syncytial viruses, papilloma viruses, and entero viruses, arenavirus, rhinoviruses, poliovirus, Newcastle disease virus, rabies virus, and arbo viruses.

Examples of chronic viral infections against which immunopotentiatory therapy with a compound of Formula / or a physiologically acceptable salt or ester thereof may be used are persistent herpes virus infections, Epstein Barr virus infection, persistent rubella infections, papillovirus infections, hepatitis virus infections and human immunodeficiency virus infections.

Compounds of Formula / may be administered to a human recipient by a route selected from oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), rectal and inhalation. The size of an effective dose of a compound will depend upon a number of factors including the identity of the recipient, the type of immunopotentiation involved, the severity of the condition to be treated and the route of administration, and will ultimately be at the discretion of the attendant physician. For each of the aforementioned conditions, such an effective dose will generally be in the range of from about 0.05 to about 5 mL , preferably from about 0.1 to about 2.0 mL, containing between 1 to 5000 μg of antigen, preferably from 5 to 100 μg of antigen.

While it is possible for the compounds of Formula / to be administered as the raw chemical it is preferable to present them as a pharmaceutical composition preparation. The pharmaceutical compositions of the present invention comprise a compound of Formula /, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The compositions may further comprise one or more immunologically effective antigens or one or more polynucleotides encoding for one or more antigens.

The compounds of the present invention may be employed in such forms as capsules, liquid solutions, emulsions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions, emulsions or suspensions. Any inert carrier is preferably used, such as saline, or phosphate- buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the methods of the present invention. Formation of Emulsions

Activation of T-cells is necessary to produce cellular signaling within the T cell to produce immune response (Shearer, Nature 377:16-17 (1995); Rhodes et al, Nature 377:71-75 (1995)). Prior art emulsions of either "water in oil" or

"oil in water", although capable of depot formation, moderate targeting and stimulation of a Th2 immune response, are unable to provide the costimulatory signal needed for elicitation of a Thl response.

Because of their structures, the compounds of the present invention allow for a high activation of T-cells. Micelles containing these lipophilic aromatic aldehyde derivatives would have aldehyde groups on their external polar surfaces that are exposed to the aqueous environment. By gauging the micelle aggregate number and their relative concentration of lipophilic aromatic aldehyde derivatives, it would be possible to adjust the number of aldehyde groups proximate to the immune cell for cellular activation leading to a Thl response.

By selection of the R¹ and R² of these derivatives as well as the relative concentrations of these compounds and/or surfactant, such as Tween and non- ionic block polymers, or those of oil and water, both the micelle aggregate size and the relative density of their aldehyde groups can be controlled, thereby allowing for the optimization of T-cell activation leading to a Thl response. In addition, the compounds of the present invention can be used in conjunction with lipospheres and other similar vaccine carrier systems.

Formation of Liposomes and Lipid-DNA Complexes

The compounds of the present invention can be employed in association with liposomes, wherein the compound can be in one or both of the bilayers of the liposome, loosely-associated with lipid material in a liposome preparation

(where the conjugates are not within a bilayer, but otherwise associated with lipids), in some instances, entrapped within the bilayers of the liposomes. See, for example, U.S. Patent No. 4,235,877 to Fullerton.

Liposomes are single or multilamellar bilayer membrane vesicles typically composed of neutral or anionic lipids obtained from natural sources or prepared synthetically. The most commonly used lipids are cholesterol and phospholipids, which are extracted from natural sources, such as egg yolks, soy beans, and other sources. According to their lipid composition, liposomes can be conventional if they are composed of neutral or anionic lipids, and cationic if they have in their composition cationic lipids. Compounds that are hydrophilic, such as DNA, RNA, certain proteins, polysaccharide, and other organic molecules, are carried in the aqueous content of the liposome vesicles. Compounds that are either lipophilic or amphipathic, such as lipids, glycolipids, certain proteins, and other compounds, are carried as membrane-bound compounds. Because of the interactions between the negatively charged nucleic acids and the positively charged lipids , cationic liposomes appear to be a better delivery system for DNA,

RNA, and oligonucleotides in general. In addition, liposomes are readily taken up by macrophages which makes them potentially good targeting agents for delivery of antigens and DNA in vaccines. Moreover, because the liposomes' lipid bilayer membranes fuse with the cell membrane, they are capable of delivering their antigen or DNA payload directly into the cytosol for processing by the endogenous pathway in the case of protein antigens, or for transient expression of the protein antigen and its subsequent processing by the endogenous pathway in the case of DNA vaccines. Both processes may lead to the induction of a cytotoxic T cell (CTL) response. Lipid-DNA complexes do not always retain liposome structure. On suspension in an aqueous solvent comprising DNA, the lipid molecules can assemble themselves into primary lipid vesicles that are heterogenous in size. These primary vesicles may be reduced to selected mean diameter by means of a freeze-thaw procedure. Vesicles of uniform size can be formed prior to delivery according to methods for vesicle production known to those skilled in the art. For example, uniform size vesicles can be produced by sonication of a lipid solution as described by Feigner et al. (Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)) and U.S. Pat. No. 5,264,618.

Although liposomes have several characteristics desirable for use in vaccines, because they are not immunomodulating agents they do not elicit an enhanced immune response or select between the Thl and Th2 responses. This lack of the appropriate immunomodulation will fail to affect the selection of the IgG isotype produced, the type of other immunoglobulins which are produced, as well as the level of cell mediated immunity produced. These drawbacks can be compensated somewhat by incorporating hydrophilic or hydrophobic immunomodulators (such a cytokines and lipid A respectively) in the liposomes. Unfortunately, liposomes are better suited for amphipathic molecules, and incorporation of cytokines into liposomes is quite inefficient. In addition, because cytokines are expensive, unstable, toxic, as well as a potential source of autoimmunity, their use in commercial vaccines is rather questionable.

Hydrophobic immunomodulators, such as lipid A and lipophilic muramyl dipeptide derivatives also have serious drawbacks, such as high toxicity, which would preclude their practical use in vaccines. Consequently, there is a need for effective and safe immunomodulating compounds which are compatible with liposome formulations.

Ideally, immunomodulating agents which are suitable for incorporation in liposomes should have an amphipathic nature. Their hydrophobic moiety would be anchored in the liposome lipid bilayer, whereas the hydrophilic region carrying the groups responsible for immune stimulation would be exposed for interaction with the appropriate cellular receptors. The synthetic molecules subject of the present patent application fulfill these structural and functional requirements. Their alkyl, or similar hydrophobic chains, would facilitate their inclusion in the liposomes' lipid bilayer. To provide a better presentation of the immunomodulating aldehyde or ketone groups to the cell-surface-receptors, the hydrophilic moiety could be bound to two identical lipid chains instead of a single one. The moiety carrying the required aldehyde groups, such as hydrophilic aromatic cyclic derivative, would also have hydroxyl and/or carboxyl groups.

By incorporating costimulatory aldehyde groups on the liposomes' external surface, the compounds of the present invention would provide these delivery systems with an intrinsic immunomodulating capacity to stimulate cell mediated immunity, including the formation of CTLs. This costimulatory effect would act on either cells processing protein antigens delivered to their cytosol, or cells transiently expressing protein antigens encoded by DNA delivered by the liposome directly into the cell. Incorporation of the costimulatory aldehyde group on the liposome surface would obviate the need for high cost, short-lived cytokines or toxic immunomodulators. Moreover, in some cases it would be possible to obtain synergistic adjuvant effects by manipulating the composition and content of the liposomes.

The lipophilic compounds of the invention can be used together with cationic lipids described, for example, in U.S. Pat. Nos. 4,897,355; 4,946,787;

5,264,618; 5,279,833; 5,334,761; 5,429,127; 5,459,127; 5,589,466; 5,676,954; 5,693,622; 5,580,859; 5,703,055; and 5,578,475; and international publications WO 04/9469, WO 95/14381, WO 95/14651, WO 95/17373, WO 96/18372, WO 96/26179, WO 96/40962, WO 96/40963, WO 96/41873 and WO 97/00241, and documents cited therein.

Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide (DOGS), dipalmitoyl-phosphatidylethanolamine-5- carboxyspermiylamine(DPPES),3,5-(N,N-dilylsyl)-diaminobenzoyl-3-(DL-l,2- dioleyl-dimethylanunopropyl-β-hydroxyethylamine) (DLYS-DABA-DORI-ester), 3 , 5 -(N,N-dilysyl)diamino-benzoylglycyl-3 -(DL- l ,2-dioleoyl- dimethylaminopropyl-β-hydroxyethylamine) (DLYS-DAB A-GLY-DORI diester), (±)-N-(20-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanimium bromide (DMRIE), (±)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3- bis(dioleyloxy)-l-propanimium pentahydrochloride (DOSPA), and cationic cholesterol derivatives. The lipophilic compounds of the invention can also be used together with a mixture of neutral and cationic lipids. Examples of useful neutral lipids are cardiolipin, phosphatidylcholine, phosphatidylethanolamine, dioleylphosphatidylcholine, dioleylphosphatidyl-ethanolamine, 1 ,2-dioleyl-sn- glycerol-3-phosphatidylethanolamine (DOPE) and sphingomyelin, and mono-, di- or triacylglycerol.

Testing for Adjuvanticity

Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subject or in a test system to which the antigen is administered. Some antigens are weakly immunogenic when administered alone or are toxic to a subject at concentrations that evoke useful immune responses in a subject. An immune adjuvant can enhance the immune response of the subject to the antigen by making the antigen more strongly immunogenic. The adjuvant effect can also result in the ability to administer a lower dose of antigen to achieve a useful immune response in a subject.

The immunogen-inducing activity of compounds and compositions of the present invention can be determined by a number of known methods. The increase in titer of antibody against a particular antigen upon administration of a composition of the present invention can be used to measure immunogenic activity (Dalsgaard, K. Acta Veterinia Scandihavica 69:1-40 (1978)). One method requires injecting CD-I mice intradermally with a test composition that includes one or more exogenous antigens. Sera is harvested from mice two weeks later and tested by ELISA for anti-immunogen antibody. The adjuvant effects of the compounds and compositions of the present invention on DNA vaccines can be determined by measuring the antibody response against the antigen coded by the DNA used in the immunization and delivered in liposomes. The DNA can be synthetically produced or in a plasmid form. Cell mediated immunity can be assessed by measuring the lymphoproliferative response of lymphocytes derived from the immunized animals and stimulated in vitro with such an antigen. One method would require that mice are injected with DNA coding for a viral antigen, such as HSV-1 gD contained in liposomes, and their immune response measured by evaluation of the antibody response, and challenging the animals with the virus in question.

Kits

The invention also provides for a kit for the immunization of an individual comprising a carrier compartmentalized to receive in close confinement therein one or more container means wherein a first container contains a compound of the invention. The kit may also include at least one other container means which contains an adjuvant of the present invention or other adjuvant as described herein.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.

Example 1

5-Carhoxy-4-decyloxy-2-hydroxybenzaldehyde

4-Decyloxy-2-hydroxybenzaldehyde: 2,4-Dihydroxybenzaldehyde(MW 138)in water containing excess potassium carbonate was mixed with a solution of one equivalent of 1-bromodecane (MW 221) dissolved in ethanol. The reaction mixture was heated and the reaction was monitored by TLC. Upon completion of the reaction, the solvent was removed under reduced pressure and the product (MW 278) was purified by chromatography. Alkylation may also be accomplished using sodium hydroxide as the base. (Reaction of phenol with 1 ,3- dibromopropane, Organic Synthesis, Coll. Vol I, 435 (1964).)

5-Carboxy-4-decyloxy-2-hydroxybenzaldehyde: 4-Decyloxy-2- hydroxybenzaldehyde is dissolved in aqueous ethanol and treated with a five fold excess of potassium bicarbonate (MW 100) and a stream of carbon dioxide is passed through the mixture while is was stirred and heated for one hour using conditions similar to the preparation of β-resorcylic acid. (β-Resorcylic Acid, Organic Synthesis Coll. Vol.2, 557 (1943).) Hydrochloric acid is carefully added dropwise with stirring to the hot solution to adjust the pH to 3 and the mixture was allowed to cool to room temperature. Concentration, extraction with methylene chloride (3x) and evaporation of the organic extract layers provide the product (MW 322) as an oil that was purified by chromotography and crystallized upon standing.

Example 2 5-Carboxy-4-[4-decyloxybenzyloxy]-2-hydroxybenzaldehyde

p-Decyloxytoluene (method a): p-Cresol (MW 108) is dissolved in DMF and treated with an equimolar amount of sodium hydride (MW 24, 60% dispersion, prewashed with pentane to remove the mineral oil) and stirred for 15 minutes. A solution of an equivalent of 1-bromodecane (MW 221) dissolved in DMF is added dropwise to the resulting anion at room temperature. The reaction is monitored by quenching aliquots and analyzing by TLC. Upon complete reaction, water is added to quench the reaction and extraction with pentane or hexanes provides the product (MW 248), purifiable by distillation or chromatography.

p-Decyloxytoluene (method b): p-Cresol may be alkylated using sodium hydroxide in the manner of the alkylation of phenol (Reaction of phenol with 1,3- dibromopropane, Organic Synthesis, Coll. Vol.1, 435 (1964)). Briefly, equimolar mixtures of 1-bromodecane andp-cresol are dissolved in ethanol or water. The mixture is heated to boiling and treated dropwise with slightly less than one equivalent of sodium hydroxide dissolved in water. Less stringent conditions will work equally well. For example, alkylation of a chromosome derivative (Reaction of 5,7-dihydroxy-2,2-dimethyl-4- chromosome with 1-bromopentane, I. Heterocyclic Chem., 22:561 (1985)) with pentyl bromide was effected in acetone using potassium carbonate as the base. These mild conditions avoided the C-alkylation which competed with the desired O-alkylation when stronger bases such as sodium ethoxide were used. The specific conditions required equimolar amounts of the phenol derivative and pentyl bromide to be dissolved in dry acetone. The mixture was treated with excess anhydrous potassium carbonate and heated to reflux for three days (the progress of the reaction was monitored by TLC). The mixture was cooled to room temperature, filtered to remove inorganic salts and concentrated to provide the essentially pure product.

a-Bromo-p-decyloxytoluene (method a): Wolf Ziegler bromination (Reaction of o-nitrotoluene with NBS, Organic Synthesis, Coll Vol. V, 825 (1973)) provides the necessary bromide derivative for further substitution reaction. However, since /?-decyloxytoluene is a relatively activated aromatic system toward halogenation, careful control of the reaction conditions to avoid ring bromination is required. Such control includes the use of non-polar solvents to enhance radical reaction and to avoid cation formation.

A typical reaction consists of mixing -decyloxytoluene (MW 248) with slightly less than one equivalent of N-bromosuccinimide (MW 178) and a catalytic amount of benzoyl peroxide in carbon tetrachloride. The mixture is heated to reflux until reaction is complete as monitored by TLC analysis. Filtration to remove succinimide and concentration provides the crude product. The purification of the product (MW 327) may be accomplished by vacuum distillation or chromatography.

a-Bromo-p-decyloxytoluene (method b): Chloromethylation of 4- decyloxybenzene (Reaction naphthalene with paraformaldehyde, Organic Synthesis, Coll. Vol. Ill, 195 (1955)) is an excellent alternative to produce a desired an intermediate for subsequent reaction. In this case, alkylation of phenol (MW 94) with 1-bromodecane is described for -cresol provides the starting material. 4-Decyloxybenzene (MW 234) and paraformaldehyde are dissolved in glacial acetic acid: phosphoric acid: concentrated hydrochloric acid (volume ratio of 1.5:1:2.2) and the mixture and potassium carbonate and extracted with ether.

Distillation or chromatography provide the product α-chloro-p-decyloxytoluene (MW 282.5) for further reaction.

4-[4-Decyloxybenzyloxy]-2-hydroxybenzaldehyde: Formation of the title compound requires mild conditions since there are two potential phenolic reaction sites . Use of potassium carbonate in acetone (Reaction of 5 ,7-dihydroxy-

2,2-dimethyl-4-chromosome with 1-bromopentane, J. Heterocyclic Chem., 22:561 (1985) provides the product resulting from reaction at the phenol moiety not adjacent to the aldehyde group.

An equimolar or slight excess amount of 2,4-dihydroxybenzaldehyde (MW 138) is mixed with α-bromo- (or α-chloro-)p-decyloxytoluene (MW 327) in acetone containing excess potassium carbonate. The mixture is heated to reflux until the reaction is complete by monitoring with TLC analysis. Filtration to remove inorganic salts and concentration of the filtrate provides the product (MW 672) which may be purified by chromatography and crystallizes upon standing. 5-Carboxy-4-[4-decyloxybenzyloxy]-2-hydroxybenzaldehyde : 4- [4- Decyloxybenzyloxy]-2-hydroxybenzaldehyde (MW 672) is dissolved in aqueous ethanol and treated with a five fold excess of potassium bicarbonate (MW 100) and a stream of carbon dioxide is passed through the mixture while it was stirred and heated for one hour. Hydrochloric acid is carefully added dropwise with stirring to the hot solution to adjust the pH to 3 and the mixture is allowed to cool to room temperature. Concentration, extraction with methylene chloride (3x) and evaporation of the organic extract layers provide the product (MW 716) as an oil that crystallized upon standing.

Example 3

2-Hydroxy-4-[V-n-hexyloxy)ethoxy]-benzaldehyde

a. Hexyl alcohol (10.2 g, 0.1 mol) is dissolved in DMF (100 mL) and treated with sodium hydride (4.0 g of 60% dispersion in mineral oil, pre-washed with pentane to remove mineral oil prior to reaction, 0.1 mol) and stirred for 30 rnin. To this solution is added dropwise with stirring a solution of 1,2-epoxyoctane

(12.8 g, 0.1 mol) dissolved in DMF (100 mL) at a rate to maintain temperature of the reaction below reflux. The reaction is monitored by thin layer chromatography until reaction is complete and then quenched by addition to water (1 L). The aqueous layer is extracted with methylene chloride (3 x 250 mL) and the combined organic layers are dried over magnesium sulfate.

Concentration and chromatography on silica gel provides the alcohol product as an oil, expected yield 19.6 g (85%).

b. The alcohol from above (17.3 g. 0.075 mol) dissolved in methylene chloride (200 mL) is treated dropwise with thionyl chloride (12.0 g. 0.1 mol) dissolved in methylene chloride (10 mL) and the reaction is stirred until starting material is consumed by thin layer chromatographic analysis. The reaction is poured into aqueous sodium bicarbonate, the organic layer separated, dried over magnesium sulfate and concentrated to provide the chloride product, expected yield 17.7 g (95%).

c. 2,4-Dihydroxybenzaldehyde (9.93 g. 0.072 mol) and the above chloride

(17.7 g, 0.072 mol) are reacted in acetone (150 mL) containing anhydrous potassium carbonate (10.35 g. 0.075 mol) with stirring and heating to reflux.

Upon consumption of the starting materials, the reaction mixture is filtered and concentrated to give the crude product which is purified by silica gel chromatography. Expected yield is 18.9 g (75%).

Example 4 2-Hydroxy-4-[2'-(l^>,3^,-di-n-hexyloxy)propyloxy]-benzaldehyde

a. Hexyl alcohol (20.4 g, 0.2 mol) is dissolved in DMF (200 mL) and treated with sodium hydride (8.0 g of 60% dispersion in mineral oil, pre-washed with pentane to remove mineral oil prior to reaction, 0.2 mol) and stirred for 20 min. To this solution is added dropwise with stirring a solution of epichlorohydrin (9.25 g, 0.1 mol) dissolved in DMF (100 mL) at a rate to maintain temperature of the reaction below reflux. The reaction is monitored by thin layer chromatrography until reaction is complete and then quenched by addition to water (1 L). The aqueous layer is extracted with methylene chloride (3 x 250 mL) and the combined organic layers are dried over magnesium sulfate. Concentration and chromatography on silica gel provides the alcohol product as an oil, expected yield 22.1 g (85%).

b. The alcohol from above (20 g, 0.077 mol) dissolved in methylene chloride (200 mL) is treated dropwise with thionyl chloride (12.0 g, 0.1 mol) dissolved in methylene chloride (10 mL) and the reaction is stirred until starting material is consumed by thin layer chromatrographic analysis. The reaction is poured into ac. sodium bicarbonate, the organic layer separated, dried over magnesium sulfate and concentrated to provide the chloride product, expected yield 20.4 g (95%).

c. 2,4-Dihydroxybenzaldehyde (9.93 g. 0.072 mol) and the above chloride

(20.0 g, 0.072 mol) are reacted in acetone (150 mL) containing anhydrous potassium carbonate (10.35 g, 0.075 mol) with stirring and heating to reflux.

Upon consumption of the starting materials, the reaction mixture is filtered and concentrated to give the crude product which is purified by silica gel chromatography. Expected yield is 20.5 g (75%).

Example 5 2-Hydroxy-4-[2 '-(1 ",3 "-ditetradecanoxy-2 "-propoxy)-! '- ethoxyjbenzaldehyde

a. 1-Tetradecanol alcohol (42.8 g, 0.2 mol) is dissolved in DMF (300 mL) and treated with sodium hydride (8.0 g of 60% dispersion in mineral oil, pre- washed with pentane to remove mineral oil prior to reaction, 0.2 mol) and stirred for 30 min. To this solution is added dropwise with stirring a solution of epichlorohydrin (9.25 g, 0.1 mol) dissolved in DMF (50 mL) at a rate to maintain temperature of the reaction less than 75 °C (See Note). The reaction is monitored by thin layer chromatography until reaction is complete and then quenched by addition to water (1 L). The aqueous layer is extracted with methylene chloride (3 x 250 mL) and the combined organic layers are dried over magnesium sulfate.

Concentration and chromatography on silica gel provides the alcohol product Intermediate A as an oil, expected yield 41.2 g (85%).

Note: An excess of the enolate relative to epichlorohydrin is necessary to allow the second condensation of the enolate (forming Intermediate A) and to avoid potential self-condensation of intermediate. b. Intermediate A (40 g, 0.082 mol) is dissolved in DMF (250 mL) and treated with sodium hydride (3.2 g of 60% dispersion in mineral oil, pre-washed with pentane to remove mineral oil prior to reaction, 0.08 mol) and stirred for 30 min. To this solution is added dropwise with stirring and cooling, a solution of ethylene oxide (3.6 g, 0.082 mol) in DMF (50 mL) at a rate to prevent multiple condensations of the ethylene oxide with intermediates. Upon completion, the reaction is quenched by the addition of water (1 L), neutralized by careful addition of IN hydrochloric acid and extracted with methylene chloride (3 x 250 mL). Concentration and chromatography of the dried (magnesium sulfate) methylene chloride extracts gives Intermediate B, expected yield 36.9 g (85%).

Alternatively, two equivalents of ethylene oxide (7.2 g, 0.164 mol) dissolved in DMF (25 mL) may be added at a faster rate with longer reaction times to allow isolation of Intermediate D after work-up. Expected yield 33.9 g

(70%).

c. Intermediate B (35 g, 0.066 mol) dissolved in methylene chloride (200 mL) is treated dropwise with thionyl chloride (8.4 g, 0.07 mol) dissolved in methylene chloride (25 mL) and the reaction is stirred until starting material is consumed by thin layer chromatographic analysis. The reaction is poured into aqueous sodium bicarbonate, the organic layer separated, dried over magnesium sulfate and concentrated to provide the chloride, expected yield 34.3 g (95%). Note: Alternatively, the alcohol of Intermediate B may be activated for the subsequent displacement step d by formation of other leaving groups such as tosylate or by use of Mitsunobu conditions.

d. 2,4-Dihydroxybenzaldehyde (6.9 g, 0.05 mol) and the above chloride (27.4 g, 0.05 mol) are reacted in acetone (300 mL) containing anhydrous potassium carbonate (7.6 g, 0.055 mol) with stirring and heating to reflux. Upon consumption of the starting materials, the reaction mixture is filtered and concentrated to give the crude product which is purified by silica gel chromatography. Expected yield is 24.3 g (75%).

Example 6

2-Hydroxy-4-[2 '-(1 ",3 "-ditetradecanoxy-2 "-propoxy)-l '- ethoxy]-benzaldehyde-3-carboxylic acid

2-Hydroxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)-l '-ethoxy]- benzaldehyde-3 -carboxylic acid is prepared from 2-hydroxy-4-[2'-(l",3"- ditetradecanoxy-2"-propoxy)-l'-ethoxy]benzaldehyde by carboxylation procedures such as the Kolbe-Schmitt reaction (M. Nierenstein and D. A. Clibbens, Org. Synthesis Coll. Vol. II, 557 (1943)). Alternatively, palladium catalyzed carboxylation with carbon monoxide may provide may provide the desired carboxylic acid derivative (Sakakibara and Odaira, J. Org. Chem. 41, 2049 (1976)). The exact position of the carboxyl group is not critical to the activities of the compounds. Products with a carboxyl substitution in the 5- or 6- position are as useful as the 3 -carboxy substituted compound.

Example 7

2-Hydroxy-4-[3 '-(1 ",3 "-ditetradecanoxy-2 "-propoxy)-2 '-hydroxy- 1 '-propoxyjbenzaldehyde

a. Intermediate A from Example 5 (40 g, 0.082 mol) is dissolved in DMF (250 mL) and treated with sodium hydride (3.2 g of 60% dispersion in mineral oil, pre-washed with pentane to remove mineral oil prior to reaction, 0.08 mol) and stirred for 30 min. A solution of epibromohydrin (11.2 g, 0.082 mol) dissolved in DMF (50 mL) is added dropwise to provide after work-up, Intermediate C (39.8 g, 90%). Alternatively, a two step process using glycidol and subsequent reaction with N,N-dimethylformamide dimethyl acetyl will provide Intermediate C. Scheme 23

b. 2,4-Dihydroxybenzaldehyde (6.9 g, 0.05 mol) and Intermediate C (27.0 g, 0.05 mol) are reacted in acetone (300 mL) containing anhydrous potassium carbonate (7.6 g, 0.055 mol) with stirring and heating to reflux. Upon consumption of the starting materials, the reaction mixture is filtered and concentrated to give the crude product which is purified by silica gel chromatography. Expected yield is 25.4 g (75%).

Example 8 2-Hydroxy-4-[2 '-(1",3' '-ditetradecanoxy-2 ' '-propoxy)-

1 '-ethoxyethoxyj-benzaldehyde

a. Intermediate D from Example 5 (28.6 g, 0.05 mol) dissolved in methylene chloride (200 mL) is treated dropwise with thionyl chloride (7.0 g, 0.059 mol) dissolved in methylene chloride (25 mL) and the reaction is stirred. Upon consumption of the starting materials, the reaction mixture is poured into aqueous sodium bicarbonate, the organic layer separated, dried over magnesium sulfate and concentrated to provide the chloride.

Note: Alternatively, the alcohol of Intermediate D may be activated for the subsequent displacement step d by formation of other leaving groups such as tosylate or by use of Mitsunobu conditions. b. 2,4-Dihydroxybenzaldehyde (6.9 g, 0.05 mol) and the above chloride are reacted in acetone (300 mL) containing anhydrous potassium carbonate (7.6 g, 0.055 mol) with stirring and heating to reflux. Upon consumption of the starting materials, the reaction mixture is filtered and concentrated to give the crude product which is purified by silica gel chromatography. Expected yield is 26.0 g (75%).

Example 9 2-Hydroxy-4-[3 '-(1 ",3 "-ditetradecanoxy-2 "-propoxy)- 2 '-hydroxy-

1 '-propoxyJbenzaldehyde-3-carboxytic acid

2-Hydroxy-3-carboxy-4-[2'-(l", 3" -ditetradecanoxy-2" -propoxy)- 1 '-ethoxyethoxyj-benzaldehyde-3-carboxylic acid

2-Hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-propoxy)-2'-hydroxy-l'- propoxy]benzaldehyde-3-carboxylic acid and 2-hydroxy-4-[2'-(l",3"- ditetradecanoxy-2"-propoxy)-l'-ethoxyethoxy]-benzaldehyde-3-carboxylic acid are prepared from the corresponding non-carboxylated compounds of Examples 7 and 8, respectively, by carboxylation procedures, such as the Kolbe-Schmitt reaction (M. Nierenstein and D. A. Clibbens, Org. Synthesis Coll. Vol. II, 577 (1943)). Alternatively, palladium catalyzed carboxylation with carbon monoxide may provide the desired carboxylic acid derivative (Sakakibara and Odaira, I. Org. Chem., 41, 2049 (1976)). Products with a carboxyl substitution in the 5- or 6-position are as useful as the 3-carboxy substituted compounds.

Example 10 4-[4'-Decanoylbenzyloxy]-2-hydroxybenzaldehyde

a. 4-Decanoyl-l-methylbenzene: To a suspension of CuCN (320 mg, 3.7 mmol) in THF was added 1M solution of -tolylmagnesium bromide in ether (3.7 mL) at 0°C. The reaction mixture was slowly warmed up to room temperature and stirred for 2 hours. The reaction mixture was cooled to 0°C and to this solution was added lauroyl chloride (890 mg, 4.07 mmol) dropwise. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched by water and extracted with EtOAc (100 mL). The organic layer was washed with water (100 mL), brine (100 mL), and dried over Na^O^ After filtration, the organic solvent was evaporated and the residue was purified by silica gel chromatography to produce 4-decanoyl-l-methylbenzene

(yield 970.2 mg, 90%).Rf. 0.85 (5% EtOAc/hexane). Η NMR (CDC1₃): δ 0.88 (t, 3H, J=6.4Hz), 1.90-1.33 (m, 16H), 1.71-1.73 (m, 2H), 2.41 (s, 3H), 2.94 (t, 2H, J=7.6Hz), 7.21 (d, 2H, J=6.8Hz), 7.84 (d, 2H, J=6.8Hz).

b. l-(a-Bromomethyl)-4-decanoylbenzene: A mixture of 4-decanoyl-l- methylbenzene (970.2 mg, 3.3 mmol) and NBS (587.4 mg, 3.3 mmol) in CC1₄ (10 mL) was stirred for 4 hours while irradiated with a 500 W lamp. The mixture was concentrated in vacuum and the crude mixture was purified by silica gel chromatography to produce the product (yield 713 mg, 61%). Rf 0.83 (5% EtOAc/hexane). ΗNMR (CDC1₃): δ 0.89 (t, 3H, J=6.8Hz), 1.21-1.45 (m, 16H), 1.73-1.77 (m, 2H), 2.96 (t, 2H, J=7.5Hz), 4.52 (s, 2H), 7.48 (d, 2H, J=8.4Hz),

7.94 (d, 2H, J=8.4Hz).

c. 4-[4'-Decanoylbenzyloxy]-2-hydroxybenzaldehyde: To a solution of 2,4-dihydroxybenzaldehyde (2.02 mmol, 278.76 mg) in acetone (10 mL) was added K₂CO₃ (4.04 mmol, 557.62 mg) and l-(α-bromomethyl)-4- decanoylbenzene (2.02 mmol, 713 mg). The reaction mixture was stirred overnight. To the reaction mixture was added water (50 mL) and adjusted to pH 7. The mixture was extracted with EtOAc (3x50 mL) and the combined extracts were concentrated. The residue was purified by silica gel chromatography to yield 4-[4'-decanoylbenzyloxy]-2-hydroxybenzaldehyde (yield 746 mg, 90%). Molecular Formula: C₂₆H₃₄O₄, MS: 410; Rf 0.78 (30% EtOAc/hexane). ΗNMR

(CDC1₃): δ 0.88 (t, 3H, J=6.8Hz), 1.24-1.37 (m, 14H), 1.72-1.75 (m, 2H), 2.96 (t, 2H, J=7.6Hz), 5.18 (s, 2H), 6.55 (d, 1H, J=2.4Hz), 6.62 (dd, 1H, Jl=2.4, J2=8.8Hz), 7.46 (d, 2H, J=8.4Hz), 7.50 (d, 1H, J=8.44Hz), 7.99 (d, 1H, J=8.4Hz). Example 11

4-(l-Formyl-2-hydroxyphenyl)-N-dodecylcarbamate

2,4-Dihydroxybenzaldehyde (1 g, 0.072 mol) was dissolved in pyridine (100 mL) with stirring. Dodecyl isocyanate (1.5g, 0.072 mol, commercially available) was added and stirred at 100°C overnight. Upon completion of reaction as determined by TLC analysis, the reaction mixture was concentrated and the residue was purified by silica gel chromatography to provide the carbamate derivative (yield 500 mg, 20%). Rf 0.68 (25% EtOAc/hexane). ^!H NMR (CDC1₃): δ 0.88 (t, 3H, J=6.8Hz), 1.25-1.45 (m, 20H), 3.27 (q, 2H, J=6.8Hz, J=6.0Hz), 5.03 (bs, 1H), 6.62 (d, 1H, J=2.4Hz), 6.82 (dd, 1H, J=2.4Hz,

J=8.8Hz), 7.51(dd, J=2.4Hz, J=8.8Hz), 9.82 (s, 1H), 11.21 (s, 1H).

Example 12

4-Dodecyloxyethyloxy-2-hydroxybenzaldehyde

A mixture of ethylene glycol monododecylether (409 mg, 1.79 mmol), PPh₃ (489.7 mg, 1.87 mmol) and CBr₄ (620 mg, 1.87 mmol) in toluene was stirred for 4 hours at 60 °C. The mixture was concentrated and the residue was purified by silica gel chromatography to provide a bromide (529 mg, 96%). Rf :

0.5 (20% EtOAc/hexane); Η NMR (CDCl₃):δ 0.88 (t, 3H, J=6.8Hz), 1.22-1.56

(m, 18Hz), 1.56-1.62 (m, 2H), 3.45-3.50 (m, 4H), 3.74 (t, 3H, J=6.4Hz). 2,4-Dihydroxybenzaldehyde (236 mg, 1.71 mmol) and the bromide (529 mg, 1.71 mmol) prepared above were reacted in acetone (50 mL) containing anhydrous potassium carbonate (472 mg, 3.42 mmol) with stirring and heating to reflux. Upon consumption of the starting materials as determined by TLC analysis, the reaction mixture was filtered and concentrated to give the diether product which was purified by silica gel chromatography. Yield 50%. Rf:0.7

(30% EtOAc/hexane); ΗNMR (CDC1₃): δ 0.89 (t, 3H, J=6.8Hz), 1.22-1.30 (m,

16H), 1.60-1.63 (m, 4H), 3.53-3.55 (m, 2H), 3.78-3.82 (m, 2H), 4.16-4.19 (m, 2H), 6.45 (d, 1H, J=2.4Hz), 6.58 (dd, J=2.4Hz, J=8.8Hz), 7.42 (d, 1H, J=8.8Hz), 9.72 (s, 1H).

Example 13

4-(2-Hydroxy-dodecyloxy)-2-hydroxybenzaldehyde

a. To a solution of BnONa (10.66 g, 0.082 mol) in benzene (50 mL) was added 1,2-epoxydodecane (15.1 g, 0.082 mol). The reaction mixture was stirred for 24 hours, quenched with water (100 mL) and extracted with EtOAc (300 mL). The organic layer was concentrated and the residue was purified by silica gel chromatography to produce Intermediate A (yield 21.5 g, 90%). Rf: 0.3 (20% EtOAc/hexane) Η NMR (CDC1₃): δ 0.88 (t, 3H, J=6.8Hz), 1.26-143 (m, 18H),

2.32 (bs, 1H), 3.33 (dd, 1H, J=8Hz, J=9.2Hz), 3.51 (dd, 1H, J=2.8Hz, J=9.2Hz), 3.81 (bs, 1H), 4.56 (s, 2H), 7.298-7.36 (m, 5H).

b. To a solution of Intermediate A (8.4 g 0.035 mol) in CH₂C1₂ was added Et₃N (3.5 g 0.035 mol) following by MOM-C1 (2.8g, 0.035 mol). The reaction mixture was stirred overnight and concentrated. The residue was dissolved in hexane (50 mL) and passed a short bed of silica gel. Evaporation of the solvent gave Intermediate B (yield 11.7g,100%). Rf: 0.75 (20% EtOAc/hexane). *H NMR (CDCl₃):δ 0.88 (t, 3H, J=6.8Hz), 1.21-1.34 (m, 18H), 3.38 (s, 3H), 3.50 (d, 2H, J=7.2Hz), 3.75-3.79 (m, 1H), 4.58 (dd, 2H, J=2.8Hz, J=12Hz), 4.71 (dd, 2H, J=6Hz, J=10Hz), 7.30-7.36 (m, 5H).

c. A mixture of Intermediate B and palladium black in EtOAc was stirred under 1 atmosphere of hydrogen for 24 hours. The reaction mixture was filtered and the organic solvent was evaporated to give Intermediate C (yield 8.5 g, 99%). Rf: 0.3 (20% EtOAc/hexane). Η NMR (CDC1₃): δ 0.88 (t, 3H, 6.8Hz), 1.26-1.53 (m, 18H), 3.04 (dd, 1H, J=3.6Hz, J=8.8Hz), 3.43 (s, 3H), 3.48-3.62

(m, 3.53), 4.72 (dd, 2H, J=6.8Hz, J=20Hz). d. To a mixture of 2,4-dihydroxybenzaldehyde (571 mg, 4.1 mmol), Intermediate C ( 1 g, 4.1 mmol) and PPh₃ ( 1.08 g, 4.1 mmol) in toluene was added DEAD (713.4 mg, 4.1 mmol). The reaction mixture was stirred overnight and concentrated. The residue was purified by silica gel chromatography to give Intermediate D (yield 1.38g, 92%). Rf: 0.7 (30% EtOAc/hexane). Η NMR

(CDC1₃): δ 0.88 (t, 3H, J=6.8Hz), 1.21-1.52 (m, 16H), 1.63-1.65 (m, 2H), 3.40 (s, 3H), 3.88-3.97 (m, 1H), 4.04 (d, 2H, J=5.2Hz), 4.75 (dd, 2H, J=6.8Hz, J=24Hz), 6.43 (d, 1H, J=2Hz), 6.55 (dd, 1H, J=2Hz, J=8.4Hz), 7.43 (d, 1H, J=8.4Hz), 9.7 (s, 1H).

e. A solution of Intermediate D (1.08 g, 4 mmol) in MeOH (containing a trace of concentrated HC1) was heated to reflux for 2 hours. To the reaction mixture was added H₂O (100 mL) and diethyl ether (100 mL). The aqueous solution was extracted with ether (3x). The ether layers were combined, and evaporated to give 4-(2-hydroxy-dodecyloxy)-2-hydroxybenzaldehyde (yield 600 mg, 60%). Rf: 0.4 (30% EtOAc/hexane). Η NMR (CDC1₃): δ 0.89 (t, 3H,

J=6.8Hz), 1.26-1.29 (m, 18H), 3.88-3.90 (m, 1H), 4.02 (d, 2h, J=6.8Hz), 6.44 (d, 1H, J=2.4Hz), 6.58 (dd, 1H, J=2.4Hz, J-8.8Hz), 7.44 (d, 1H, J=9.2Hz), 9.73 (s, 1H).

Example 14 General Procedure for Preparing Compounds of Examples 15-21

To the mixture of alcohol (10 mmol), PPh₃(10 mmol) and 2,4- dihydroxybenzaldehyde (10 mmol) in toluene was added DEAD (10 mmol) with stirring at room temperature. Upon consumption of the starting materials as determined by TLC analysis, the reaction mixture was filtered and concentrated to give the desired product, which was purified by silica gel chromatography.

Yield: 70-90%. Example 15

4-(l 3 '-Didodecyloxy-propyl-2 '-oxy)-2-hydroxybenzaldehyde

a. A mixture of 1-decanol (10 g) and t-KOBu (401 mg, 3.58 mmol) was warmed to 70°C. To this solution was added dropwise epichlorohydrin (333.5 mg, 1.79 mmol). The reaction was monitored by TLC until reaction was complete and then quenched by addition of water (10 mL) to the reaction mixture. The aqueous layer was extracted with methylene chloride (3x250 mL) and the combined organic layers were dried over magnesium sulfate. Concentration and chromatography on silica gel provided 1.1 g of the diether alcohol product as a solid, yield 1.1 g. Rf: 0.5 (30% EtOAc/hexane). ΗNMR (CDC1₃): δ 0.89 (t, 6H,

J=6.8Hz), 1.2-1.4 (m, 20H), 2.48 (d, 1H), 3.42-3.51 (m, 8H), 3.92-3.97 (m, 1H).

b. Using the general procedure of Example 14, diether alcohol prepared above (610 mg, 1.6 mmol) and 2,4-dihydroxybenzaldehyde (220 mg, 1.6 mmol) provided 4-( ,3'-didodecyloxy-propyl-2'-oxy)-2-hydroxybenzaldehyde (yield 800 mg, 90%). Rf: 0.75 (30% EtOAc/hexane). Η NMR (CDC1₃): δ 0.88 (t, 6H,

J=6.8Hz), 1.23-1.42 (m, 4H), 3.44-3.47 (m, 4H), 3.64-3.68 (m, 4H), 4.59-4.64 (m, 1H), 6.51 (d, 1H, J=2.4Hz), 6.64 (dd, 1H, J=2.4Hz, J=8.8Hz), 7.44 (d, 1H, J=9.2Hz), 9.73 (s, 1H).

Example 16 4-[2-(2-Octyl-dodecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde

a. To a solution of 2-octyl-l-dodecanol (597.12 mg, 2 mmol) in CH₂C1₂ was added triethylamine (222 mg, 2.2 mmol) following by adding MsCl (252 mg,

2.2 mmol). The reaction mixture was stirred overnight and concentrated. The residue was purified by silica gel chromatography to provide the mesylate (yield 748 mg, 99%). Rf: 0.5 (20% EtOAc/hexane). Η NMR (CDC1₃): δ 0.89 (t, 3H, J=6.8Hz), 1.25-1.33 (m, 22H), 1.68-1.71 (m, 1H), 3.01 (s, 3H), 4.12 (d, 2H, J=5.6Hz).

b. To a solution of NaOCH₂CH₂OBn was added a solution of the mesylate prepared above (748 mg, 1.98 mmol) in benzene. The reaction mixture was stirred for 18 hours. The reaction mixture was quenched by adding 1 mL of

H₂O and extracted with EtOAc. Organic solution was dried over Na₂SO₄ and concentrated. The residue was purified by silica gel chromatography to provide benzyl protected the glycol ether derivative which was debenzylated using Pd-C hydrogenation to give glycol ether derivative (yield 470 mg). Rf: 0.4 (20% EtOAc/hexane). ΗNMR (CDC1₃): δ 0.88 (1, 3H, J=6.8Hz), 1.42-1.32 (m, 23H),

3.34 (d, 2H, J=5.6Hz), 3.51 (t, 2H, 4.8Hz), 3.70-3.73 (m, 2H).

c. Using the general procedure of Example 14, the mesylate prepared above (470 mg 1.24 mmol) and 2,4-dihydroxybenzaldehyde (171 mg, 1.24 mmol) gave 4- [2-(2-octyl-dodecyloxy)-l-ethyloxy] -2-hydroxybenzaldehyde (yield 800 mg, 90%). Rf: 0.6 (30% EtOAc/hexane). Η NMR (CDC1₃): δ 0.88 (t, 3H,

J=6.8Hz), 1.4-1.7 (m, 23H), 3.39 (d, 2H, 6.4Hz), 3.77 (2H, 6.8Hz), 4.16 (m, 2H), 6.44 (d, 1H, J=2.4Hz), 6.64 (1H, J=2.4Hz, J=8.8Hz), 7.44 (d, 1H, J=8.8Hz), 9.72 (s, 1H).

Example 17 4-[2-(2-Decyl-tetradecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde

4- [2-(2-Decyl-tetradecyloxy)-l-ethyloxy] -2-hydroxybenzaldehyde was prepared by the method described in Example 16.

a. mesylate: Rf: 0.5 (20% EtOAc/hexane) b.4-[2-(2-Decyl-tetradecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde : Rf :

0.4 (20% EtOAc/hexane). ΗNMR (CDC1₃): δ 0.88 (t, 6H, J=6.8Hz), 1.37-1.41 (m, 40H), 3.38 (d, 2H, J=6Hz), 3.77 (t, 2H, J=6Hz), 4.16 (t, 2H, J=6Hz), 6.44 (dd, 1H, J=2.4Hz), 6.63 (1H, J=8.8Hz, J=2.4Hz), 7.42 (d, 1H, J=8.8Hz). 9.72 (s, 1H).

Example 18

4-[2-(N,N-dodecyl)ethyloxy]-2-hydroxybenzaldehyde

a. A mixture of ethanolamine (100 mg, 1.62 mmol), K₂CO₃ (497 mg, 3.6 mmol) and 1-iododecane (966 mg, 3.6 mmol) in ethanol was heated to reflux for 24 hours. The mixture was concentrated and the residue was purified by silica gel chromatography to provide Intermediate (yield 497 mg, -90%). Rf : 0.5 (20%

EtOAc/hexane). Η NMR (CDC1₃): δ 0.89 (t, 6H, J=6.9Hz), 1.2-1.5 (m, 12H), 2.46 (t, 4H, J=7.6Hz), 2.59 (t, 2H, J=5.2Hz), 3.54 (t, 2H, J=5.2Hz).

b. Using the general procedure of Example 14, Intermediate (410 mg, 1.2 mmol) and 2,4-dihydroxybenzaldehyde (165 mg, 1.2 mmol) provided 4- [2-(N,N- dodecyl)ethyloxy] -2-hydroxybenzaldehyde (yield 500 mg, 90%). Rf : 0.78 (30%

EtOAc/hexane). ΗNMR (CDCl₃):δ 0.88 (t, 6H, J=6.8Hz), 1.25-1.34 (m, 28H), 1.43-1.47 (m, 4H), 2.50(t, 2H, J=7.6Hz), 2.86 (t, 2H, J=6.4Hz), 4.07 (t, 2H, J=6.4Hz), 6.42 (d, 1H, J=2.4Hz), 6.44 (d, 1H, J=2.4Hz, J=8.4Hz), 7.41 (d, 1H, J=8.4Hz), 9.71 (s, 1H).

Example 19

4-(2-Octyl-dodecyloxy)-2-hydroxybenzaldehyde

Using the general procedure of Example 14, 2-octyl-l-dodecanol (384 mg,

1.3 mmol) and 2,4-dihydroxybenzaldehyde (179 mg, 1.3 mmol) gave 4-(2-octyl- dodecyloxy)-2-hydroxybenzaldehyde (yield 500 mg, -90%). Rf: 0.75 (30% EtOAc/hexane). ΗNMR (CDC1₃): δ 0.89 (t, 6H, J=6.8Hz), 1.26-1.56 (m, 30H), 1.75-1.80 (m, 1H), 3.87 (d, 2H, J=5.6Hz), 6.42 (d, 1H, J=2.4Hz), 6.53 (dd, 1H, J=2.4Hz, J=8.8Hz), 7.41 (d,lH, J=8.8Hz), 9.70(s, 1H).

Example 20

4-(2-Decyl-tetradecyloxy)-2-hydroxybenzaldehyde

Using the general procedure of Example 14, 2-decyl-l-tetradecanol (372 mg, 1 mmol) and 2,4-dihydroxybenzaldehyde (138 mg, 1 mmol) provided 4-(2- decyl-tetradecyloxy)-2-hydroxybenzaldehyde (yield 500 mg, -90%). Rf: 0.75 (30% EtOAc)/hexane). ΗNMR (CDC1₃): δ 0.89 (t, 6H, J=6.8Hz), 1.25-1.56 (m, 40H), 1.76-1.80 (m, 1H), 3.87 (d, 2H), 6.41 (d, 1H, J=2.4Hz), 6.52 (d, 1H, J=2.4Hz, J=8.8Hz), 7.40 (d, 1H, J=8.8Hz), 9.72 (s, 1H).

Example 21

4-(l-Octyl-decyloxy)-2-hydroxybenzaldehyde

a. To a solution of decyl aldehyde (lOg, 64 mmol) in THF (200 mL) was added octylmagnesium bromide (32 mL, 2.0 M solution in THF, 64 mmol) at 0°C. The reaction mixture was stirred at room temperature overnight. The reaction was quenched by adding water (10 mL) and extracted with ether. The organic layer was washed with water and brine, dried with Na^O^ filtered, and evaporated. The residue was purified by silica gel chromatography to give Intermediate (yield 14 g, 85%); Rf.0.5 (20% EtOAc/hexane). Η NMR (CDC1₃): δ 0.89 (t, 3H, J=6.8Hz), 1.26-1.28 (m, 16H), 1.43-1.45 (m, 4H), 3.30-3.32 (m,

1H).

b. Using the general procedure of Example 14, Intermediate (372 mg, 1 mmol) and 2,4-dihydroxybenzaldehyde (138 mg, 1 mmol) provided 4-(l-octyl- decyloxy)-2-hydroxybenzaldehyde (yield 500 mg, -90%). Rf: 0.78 (30% EtOAc/hexane). Η NMR (CDC1₃): δ 0.88 (t, 6H, J=6.8Hz), 1.2-1.4 (m, 16H), 1.61-1.70 (m, 4H), 4.31 (t, 1H, J=6.6Hz), 6.44 (d, 1H, J=2.4Hz), 6.64 (dd, 1H, J=2.4Hz, J=8.8Hz), 7.44 (d, 1H, J=8.8Hz), 9.71 (s, 1H).

Example 22

Testing for Adjuvant Effect Using Ovalbumin (OVA) as Antigen

Assessment of adjuvant effect can be determined by increase in anti-OVA antibody titers following immunization with OV A/test compound compared with those titers from immunized animals in the absence of the test compound. The adjuvant activity of the test compounds is measured as follows: Swiss or CD-I mice (8-10 weeks old) are immunized subcutaneously or intramuscularly with 0.2 mL of the following formulation containing an adjuvant of the present invention prepared as follows: one volume of an emulsifier, e.g. glycerol monooleate (Myverol), dissolved in 4 volumes of n-hexadecane, and containing between 0 and 25 percent (v/v) of a lipophilic aromatic aldehyde derivative(s), are layered over 10 volumes of an isotonic phosphate buffered saline solution, pH 7.2 (PBS), containing the antigen OVA and emulsified using a high speed stirrer or similar instrument. The concentration of antigen should be adjusted to 50 μg OVA per mL of emulsion. Animals immunized with the emulsion minus the test compound provide the baseline value for antibody titers. Mice are immunized twice at two- week intervals with the different formulations. Control mice are injected with either PBS or PBS with OVA, plus 100 μg of aluminum hydroxide.

Sera is harvested two weeks post-immunization. Anti-OVA antibody is determined by ELIS A: Immulon II plates were coated overnight at 4°C with 100 μl fatty acid free OVA (10 μg/ml in PBS) in rows, A, C, E, and G. Plates are washed twice with PBS. Nonspecific binding is prevented by incubating for 1.5 hours at 37 °C with 100 μl diluent (2% casein acid hydrolysate (Oxoid, w/v) in

PBS) per well in all wells. Plates are washed four times with 0.05% Tween 20 surfactant in distilled water. Sera at dilutions of 10, 10², 10³ and 10⁴ is incubated in rows A + B, C + D, E + F and G + H, respectively (100 μl/well) for 1 hour at room temperature. Plates are washed as described above. Boehringer-Mannheim horse radish peroxidase conjugate goat anti-mouse antibody (1/5000 in 5% OVA in diluent) is incubated for 30 minutes at room temperature (100 μl per well, all wells). Plates are washed as described above. The extent of peroxidase reaction is determined by reaction with 2,2'-azido-bis(3-ethylbenzthiazoline)-6-sulfonate

(30 minute reaction at room temperature, absorbance measured at 410 nm) or with 3,3',5,5'-tetramethylbenzidine (10 min. reaction of nonspecific antibody binding to the total antibody binding is removed by subtraction of the absorbance of the antigen-negative well from the absorbance of the antigen-positive well for each sera dilution. The absorbance due to antigen-specific binding is plotted as a function of the logarithm of the sera dilution of 10 or less for immunization in the absence of adjuvant and are as high as 10³ in the presence of the test compound adjuvant.

A compound as prepared in Example 2 is tested for adjuvant effect and exhibits good adjuvant effect.

Example 23

Testing for Adjuvant Effect On T-Cell Immunity Using OVA as Antigen

In many viral vaccines, and likely in cancer vaccines, the adjuvant used with the protein antigens should elicit a strong specific cell-mediated immunity (CMI) or T-cell immune response with production of cytotoxic T lymphocytes

(CTL). Quillaja saponins are adjuvants capable of eliciting T-cell immunity (Newman et al, J. Immuno. 148:2357 (1992)). Most adjuvants, including muramyl dipeptides, glucans, immune modulators such as TL-2, and others, are only capable of stimulating a humoral immune response against exogenous proteins (Cod, J.C., and Coulter, A.R., Vaccine 75:248 (1997)), which would be of little value in the case of cancer and some viral vaccines. Because of their stimulation of humoral and T-cell immunity, as well as negligible toxicity, the compounds of the present invention are suitable for the preparation of viral or cancer vaccines. T-cell immunity induced by these adjuvants can be assayed in vitro by (i) blast transformation, which measures the proliferation response of sensitized T cells to antigens, or (ii) measurement of the enhancement of CTL priming to a protein antigen. The adjuvant effect on T-cell immunity is measured by a cell proliferation assay according to the following protocol. Six to eight week old female CD-I mice are immunized twice subcutaneously with the following formulation: 15 μg OVA (Sigma) and an adjuvant of the present invention or quillajasaponins (at doses ranging from 5-100 μg) in 200 μl PBS. The two immunizations are given at two week intervals. Control mice are injected with either PBS or PBS with

OVA, plus 100 μg of aluminum hydroxide. Two weeks after the second immunization, the spleens are removed and disrupted by extruding through a nylon mesh. The cells are washed and resuspended in RPMI 1640 medium with 10% heat-inactivated fetal calf serum. Four x 10⁵ spleen cells are dispensed in 100 μl volumes into microtiter plate wells, and 1 μg of OVA is added in 100 μl of cell culture media. After 72 hours in culture, the cells are pulsed with 1 μCi of tritiated thymidine (³H-thymidine, Amersham International) for 8 hours and harvested onto filters. The amount of label that is incorporated into cellular DNA is determined by liquid scintillation counting. Cell proliferation is expressed as cpm in the presence of antigen minus cpm in the absence of antigen. Spleen cells from mice immunized with OVA plus test compound or quillajasaponins and stimulated in vitro with OVA show a significantly higher level of stimulation. There is no significant proliferative response in cells from mice immunized with OVA plus aluminum hydroxide. Example 24

Testing for Adjuvant Effect of Costimulatory Liposomes Using Ovalbumin (OVA) as an Antigen

Assessment of the adjuvant effects of liposomes containing the compounds of the present invention can be done by comparing the immune responses after immunization with OVA-containing liposomes with and without the lipophilic aromatic aldehyde derivatives. Liposomes are prepared as follows: to egg lecithin (30 mg), cholesterol (4.4 mg), and phosphatidic acid (4.24 mg), is added from 0.1 mg to 25 mg of one or more of the two chain lipophilic aromatic aldehyde derivatives (Formulas IV- VII) and the mixture is dissolved in chloroform (3-5 mL) in a 50 mL round flask. After removal of the solvent at 37 °C under vacuum, the thin lipid layer is dispersed with 2 mL of a solution of OVA (20 mg/mL) in water and allowed to stand at room temperature for 2-6 hours to allow the liposomes to form. The suspension is then sonicated for 10-15 seconds and after 4-8 hours, the liposomes containing entrapped OVA are separated from the free OVA by gel filtration on a column of Sepharose 6B. The fractions containing the liposomes are collected, and the protein content per mL of the liposomes suspension is determined by one of the commonly used methods, such as amino acid analysis. Mice are inoculated subcutaneously with a dose of a liposome suspension equivalent to 20 μg of OVA, twice at a 14 day interval, and on the 21^st day after the first immunization the mice are bled and the spleens are removed. Humoral immunity is assessed using ELISA to determine the anti-OVA antibody titers. The production of OVA specific CTLs is determined as follows: spleen cells (2 x 10⁶/well) from control and immunized animals are added to 24 well plates containing 1 mL of complete medium plus 1 x 10⁵ E.G7-OVA cells that have been irradiated with 20,000 R. E.G7-OVA cells are derived from the EL4 cell line after being transfected with the OVA.gene. The cultures are incubated at 37 °C in humidified 5 % CO₂ for 6 days. Cells are harvested and washed to yield the effector (E) population. As target cells for the CTL assay, EL4 and E.G7-OVA cells that have been incubated for 1 hour with 300 μCi of ⁵¹Cr-labeled NaCrO₄ (ICN Biomedicals) and washed are used. Effector and target cells are added in various E:T ratios to round-bottom 96-well plates (each well containing 1 x 10⁴ target cells), centrifuged 30 seconds at 200 x g and incubated at 37 °C in humidified 5 % CO₂. After 6 hours the plates are harvested and the radioactivity in the supernatant is measured in a gamma counter. Control for the CTL assay consists of target cells that are lysed with 2 % Triton X-100 (maximum release) and target cells treated with medium only (spontaneous release). The percentage lysis for each E:T ration is calculated using the formula:

% lysis= [(experimental cpm - spontaneous cρm)/(maximum release cpm - spontaneous release cpm)] x 100

Efficacy of the costimulatory liposomes is assessed by comparing the degrees of lysis obtained with the aldehyde-containing liposomes relative to that obtained with the conventional liposomes.

Example 25 Testing for Adjuvant Effect of Costimulatory Liposomes Using DNA for Immunization

The lipophilic aromatic aldehyde and ketone derivatives of the present invention can also be used with cationic lipids and/or liposomes, such as those described in U.S. PatentNos.5,661,018 and 4,897,355, for effective intracellular delivery of polynucleotides such as DNA and RNA. Derivatives having structures similar to those shown in Formulae IV to VH are added in different proportions to cationic lipids dissolved in chloroform, and dried over a large surface to form a thin lipid layer. The lipid layer is then dispersed with an aqueous solution containing the polynucleotides to form liposomes with entrapped polynucleotides. The concentration of polynucleotide in the preparation can be determined using radioactively labeled material. These polynucleotide-liposome complexes are administered to mice either subcutaneously or intramuscularly, and the immune response is determined 2 weeks and 4 weeks post inoculation.

Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents and publications cited herein are fully incorporated by reference herein in their entirety.

Claims

What Is Claimed Is:

1. A method for enhancing the potentiation of an immune response in a vertebrate, comprising administering an effective amount of a compound of Formula /:

or a pharmaceutically acceptable salt or an ester thereof, wherein:

R¹ is -(L)_p-Z; wherein

L is a bifunctional linker selected from the group consisting of -O- -NH-; -NCH₃-; -S-; -SO₂--; -X-NH-; -C(O)-O -X -; -X-C(O)- -X-(CH₂)_s-C(O)-NH-;-O-(CH₂)_s-C(O)-N=; -OCH₂CH₂N=; -O-C(O)-NH-

-O-CH₂CH₂-O-CH₂CH₂-O-; -OCH₂CH(OH)CH₂O-; -OCH₂CH(OH)- -X-(CH₂)_S-; -X-(CH₂)_S-Y-; and an amino acid, wherein X and Y are independently selected form the group consisting of O, NH, NCH₃, S, and SO₂, and when an amino nitrogen is at the end of the linker, then two Z groups, which can be the same or different, are attached to the amino group, wherein s is from 1 to 6;

Z is

(A) alkyl, alkoxy, or polyalkoxy, or

(B) aralkyl or aralkoxy, wherein the aryl group is substituted with at least one of alkyl, alkanoyl, alkoxy or polyalkoxy, wherein the alkyl, alkanoyl, alkoxy, or polyalkoxy is of sufficient chain length to allow the compound to form a micelle in aqueous solution with other like compounds, and wherein the carbon chain in the alkyl, alkanoyl, alkoxy or polyalkoxy is optionally interrupted with one or more oxygen, nitrogen or sulfur, and wherein

Z is optionally substituted with one or more of hydroxy, carboxy, alkoxy, sulfoxy, alkyl, amino, alkylarnino, dialkylamino, alkylthio, alkylsulfonyl, pyridinium, imidazolinium, pyrimidinium, dialkylammonium, choline, halogen, cyano, alkanoyl, alkanoyloxy, -C(O)-O-alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy, sulfinic acid, sulfonic acid, 5-tetrazolyl, or alkylsulphonylcarbamoyl, all of which can be optionally substituted;

R² is -(L)_p- Z or is selected from the group consisting of hydrogen, alkyl, alkoxy, alkoxyalkoxy, alkylaryl, arylalkyl, alkoxyaryl, alkoxyalkyl, hydroxy, alkylarnino, dialkylamino, alkanoylamino, carboxy, -C(O)-O-alkyl, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium, and choline, all of which can be optionally substituted; R³ and R⁴ are independently hydrogen, hydroxy, alkyl, halogen, alkoxy, carboxylic acid, sulfonic acid, cyano, 5-tetrazolyl, alkylsulfonylcarbamoyl or phosphonic acid;

R⁵ is hydrogen or methyl; o is 0 to 4; and p is O or l; with the proviso that when o and p are both 0, R⁵ is H, R² is alkoxy or OH, R³ is alkoxy or H, R⁴ is H and Z is an alkoxy group substituted with carboxy, the alkyl chain in Z contains at least 7 continuous carbon atoms, in an amount sufficient to enhance the immune response of a vertebrate to one or more antigens.

2. The method according to claim 1, wherein the compound administered has the Formula //:

or a physiologically acceptable salt or ester thereof, wherein

R¹ is -(L)_p-Z, wherein

L is a bifunctional linker as defined in claim 1;

R² is -(L)_p- Z or selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, carboxy, alkylarnino, dialkylamino, alkylsulfonyl,sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline;

R⁷ is selected from the group consisting of alkyl, alkanoyl, alkoxy, alkoxyalkyl, polyalkoxy, and alkoxyalkoxyalkyl, all of which can be optionally substituted; R⁸ and R⁹ are independently hydrogen, hydroxy, carboxy, -C(O)-O-alkyl, alkanoyl, alkylamino,alkanoyloxy, sulfoxy, alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy and alkoxyalkoxyalkyl, all of which can be optionally substituted; o is 0 to 4; p is O or 1; t is 1 to 6; and u, v and w are independently 0 to 6; with the provisos that 1) the R⁷ group or at least one of R⁸ or R⁹ together with the possible

2) when o and p are both 0, R² is alkoxy or OH, R³ is alkoxy or H, R⁴ is H, R⁵ is H and Z is an alkoxy group substituted with carboxy, the alkyl chain in Z contains at least 7 continuous carbon atoms.

3. The method according to claim 2, wherein the compound administered has the Formula ///:

or a physiologically acceptable salt or ester thereof, wherein

R¹ is -L - Z, wherein L is a bifunctional linker as defined in claim 1;

R² is -(L)_p-Z or is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, carboxy, alkylarnino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline;

R³ is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, hydroxy, carboxylic acid, sulfonic acid, cyano, 5-tetrazolyl, alkylsulfonylcarbamoyl or phosphonic acid; R⁶ is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, cyano, carboxyl, 5-tetrazolyl, alkylsulfonylcarbamoyl, sulfonic acid and phosphonic acid;

R⁸ and R⁹ are independently selected from the group consisting of hydrogen, hydroxy, carboxy, -C(O)-O-alkyl, alkanoyl, alkylamino,alkanoyloxy, sulfoxy, alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy and alkoxyalkoxyalkyl, all of which can be optionally substituted; o is 0 to 4; p is O or 1; t is 1 to 6; and u, v and w are independently 0 to 6; with the provisos that 1) the R⁷ group or at least one of R⁸ or R⁹ together with the possible

4) R¹ is not alkenyloxy, when R² is OH and R³ is H.

4. The method according to claim 3, wherein L is selected from the group consisting of -O-, -O-C(O)-, -OCH₂- -O-(CH₂)_s-C(O)-N=; -OCH₂CH₂N= -O-(CH₂)_s-C(O)-NH-, -OCH₂CH₂O- and

-O-CH₂CH₂-O-CH₂CH₂-O- wherein s is from 1 to 6.

5. The method according to claim 3, wherem one of R² or R³ is hydroxy.

6. The method according to claim 3, wherein R² is selected from the group consisting of hydrogen, hydroxy and carboxy.

7. The method according to claim 6, wherein R² is carboxy ortho or para to R³.

8. The method according to claim 3, wherein R³ is selected from the group consisting of hydrogen and hydroxy.

9. The method according to claim 8, wherein R³ is a hydroxy group ortho to the aldehyde group when o is 0 or ortho to an alkyl containing the aldehyde group when o is other than 0.

0. The method according to claim 3 , wherein the compound administered is selected from the group consisting of compounds of Formula IV, V, VI and VII:

IV

VI

VII

or a physiologically acceptable salt or ester thereof, wherein R³ is as defined in claim 3; R¹⁰ and R^π are alkyl, aralkyl, or alkanoyl; X and Y are O, NH, NMe, S, or SO₂; except when the compound has

Formula VII, then X is not S or SO₂; and

11. The method according to claim 10, wherein R³ is hydrogen, hydroxy, alkoxy or carboxylic acid.

12. The method according to claim 3, wherein the compound administered is a compound of Formula VIII:

or a physiologically acceptable salt or ester thereof, wherein R² is selected form the group consisting of hydrogen, hydroxy, or carboxy; R³ is hydrogen or hydroxy; and o, R⁶ and R⁷ are as defined in claim 3, with the provisos that 1) R⁷ includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur; and 2) when R² is OH, and R⁷ is an alkoxy, then at least one of R³ or R⁶ is other than H.

13. The method according to claim 12, wherein R² is OH or COOH.

14. The method according to claim 1, wherein the compound administered has the Formula IX:

or a physiologically acceptable salt or ester thereof, wherein R⁶ is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, cyano, carboxyl, 5-tetrazolyl, alkylsulfonylcarbamoyl, sulfonic acid and phosphonic acid;

R⁷ is selected from the group consisting of alkyl, alkanoyl, alkoxy, alkoxyalkyl, polyalkoxy, and alkoxyalkoxyalkyl optionally substituted with alkylamino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline; and o is 0 to 4; with the proviso that the R⁷ group includes at least atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more • oxygen, nitrogen or sulfur.

15. The method according to claim 1 , wherein the compound administered is 5-carboxy-4-decyloxy-2-hydroxybenzaldehyde ;

5-carboxy-4-[4-decyloxybenzyloxy]-2-hydroxybenzaldehyde;

2-hydroxy-4- [ 1 -n-hexyloxy)ethoxy] -benzaldehyde ;

2-hydroxy-4-[2 -(1 ',3 -di-n-hexyloxy)propyloxy]-benzaIdehyde;

2-hydroxy-4- [2 '-( l " ,3 " -ditetradecanoxy-2"-proρoxy)- l '- ethoxy]benzaldehyde;

2-hydroxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)-r-ethoxy]- benzaldehyde-3-carboxylic acid;

2-hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-proρoxy)-2'-hydroxy-r- propoxyjbenzaldehyde ; 2-hydroxy-4-[2'-(l ",3 "-ditetradecanoxy-2"-propoxy)-l -ethoxyethoxy]- benzaldehyde;

2-hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-propoxy)-2'-hydroxy-l'- propoxy]benzaldehyde-3-carboxylic acid;

2-hydroxy-3-carboxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)-l'- ethoxyethoxy]-benzaldehyde-3-carboxylic acid;

4-[4 -decanoylbenzyloxy]-2-hydroxybenzaldehyde;

4-(l-formyl-2-hydroxyphenyl)-N-dodecylcarbamate;

4-dodecyloxyethyloxy-2-hydroxybenzaldehyde;

4-(2-hydroxy-dodecyloxy)-2-hydroxybenzaldehyde; 4-( ,3'-didodecyloxy-propyl-2'-oxy)-2-hydroxybenzaldehyde;

4-[2-(2-octyl-dodecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde;

4-[2-(2-decyl-tetradecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde;

4-[2-(N,N-didecyl)ethyloxy]-2-hydroxybenzaldehyde;

4-(2-octyl-dodecyloxy)-2-hydroxybenzaldehyde; 4-(2-decyl-tetradecyloxy)-2-hydroxybenzaldehyde; or 4-(l-octyl-decyloxy)-2-hydroxybenzaldehyde, or a physiologically acceptable salt or ester thereof.

16. The method according to claim 1, wherein the vertebrate is a mammal.

17. The method according to any one of claims 1-16, wherein diseases where there is a decreased host defense immunity are treated.

18. The method according to claim 17, wherein the activity of the immune system is enhanced above normal levels.

19. The method according to any one of claims 1-16, wherein conditions resulting from a non-effective immune response are treated.

20. The method according to claim 19, wherein fungal infections, mycoplasma infections, tuberculosis, leprosy, and herpes simplex viral infections are treated.

21. The method according to any one of claims 1-16, wherein cancer in mammals is treated or prevented.

22. The method according to any one of claim 1-16, wherein acute and chronic viral infections are treated or prevented.

23. A compound of the Formula ///:

or a physiologically acceptable salt or ester thereof, wherein

R¹ is -L-Z, wherein L is a bifunctional linker selected from the group consisting of -O

-NH-; -NCH₃-; -S-; -SO₂-; -X-NH-; -C(O)-O-X -; -X-C(O) -X-(CH₂)_s-C(O)-NH-; -O-(CH₂)_s-C(O)-N=; -OCH₂CH₂N=; -O-C(O)-NH-; -O-CH₂CH₂-O-CH₂CH₂-O-; -OCH₂CH(OH)CH₂O-; -OCH₂CH(OH)- -X-(CH₂)_S-; -X-(CH₂)_S-Y-; and an amino acid, wherein X and Y are independently selected form the group consisting of O, NH, NCH₃, S, and SO₂, and when an amino nitrogen is at the end of the linker, then two Z groups, which can be the same or different, are attached to the amino group, wherein s is from 1 to 6;

R² is -(L)_p-Z or is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, carboxy, alkylamino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium, and choline;

R³ is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, hydroxy, carboxylic acid, sulfonic acid, cyano, 5-tetrazolyl, alkylsulfonylcarbamoyl or phosphonic acid;

R⁷ is selected from the group consisting of alkyl, alkanoyl, alkoxy, alkoxyalkyl, polyalkoxy, and alkoxyalkoxyalkyl optionally substituted with alkylamino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline;

R⁸ and R⁹ are independently selected from the group consisting of hydrogen, hydroxy, carboxy, -C(O)-O-alkyl, alkanoyl, alkylamino,alkanoyloxy, sulfoxy, alkyl, alkoxyalkyl, alkoxyalkoxy, aralkyl, aralkylamino, aralkoxy and alkoxyalkoxyalkyl, all of which can be optionally susbtituted o is 0 to 4; p is O or 1; t is 1 to 6; and • u, v and w are independently 0 to 6; with the provisos that

1) the R⁷ group or at least one of R⁸ or R⁹ together with the possible CH₂ groups includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur;

2) when R¹ is substituted or unsubstituted alkoxy or alkoxyalkoxy and R³ is OH, R² is different from R¹;

3) when R¹ is benzyloxy, R² is OH and R⁷ is alkoxy, one of R³ or R⁶ is other than H; and 4) R¹ is not alkenyloxy, when R² is OH and R³ is H.

24. The compound according to claim 23, wherein L is selected from the group consisting of -O-, -O-C(O)-, -OCH₂-, -O-(CH₂)_s-C(O)-N=; -OCH₂CH₂N= -O-(CH₂)_s-C(O)-NH- -OCH₂CH₂O-, and -O-CH₂CH₂-O-CH₂CH₂-O- wherein s is from 1 to 6.

25. The compound according to claim 23 , wherein one of R² or R³ is hydroxy.

26. The compound according to claim 23, wherein R² is selected from the group consisting of hydrogen, hydroxy and carboxy.

27. The compound according to claim 26, wherein R² is carboxy ortho or para to R³.

28. The compound according to claim 23, wherein R³ is selected from the group consisting of hydrogen and hydroxy.

29. The compound according to claim 28, wherein R³ is a hydroxy group ortho to the aldehyde group when o is 0 or ortho to an alkyl containing the aldehyde group when o is other than 0.

30. The compound according to claim 23, wherein the compound has the Formula IV, V, VI or VII:

IV

V

VI

VII

or a physiologically acceptable salt or ester thereof, wherein R³ is as defined in claim 23; R¹⁰ and R¹¹ are alkyl, aralkyl, or alkanoyl; X and Y are O, NH, NMe, S, or SO₂; provided that when the compound has Formula VII, then X is not S or SO₂; and

R¹² andR¹³ are alkyl, aralkyl, alkoxy, aralkoxy, alkylamino, aralkylamino, alkanoyloxy or -C(O)-O-alkyl, with the proviso that when the compound has Formula TV, X and Y are O and R¹⁰ and R¹¹ are alkyl, said alkyl groups are different.

31. The compound according to claim 29, wherein R³ is hydrogen, hydroxy, alkoxy or carboxylic acid.

32. The compound according to claim 23, wherein the compound has the Formula VIII:

or a physiologically acceptable salt or ester thereof, wherein

R² is selected form the group consisting of hydrogen, hydroxy, or carboxy;

R³ is hydrogen or hydroxy; and o, R⁶ and R⁷ are as defined in claim 23, with the provisos that

1) R⁷ includes at least six atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur; and

2) when R² is OH, and R⁷ is an alkoxy, then at least one of R³ or R⁶ is other than H.

33. The compound according to claim 32, wherein R² is OH or COOH.

34. A compound having the Formula IX:

or a physiologically acceptable salt or ester thereof, wherein

R⁶ is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, cyano, carboxyl, 5-tetrazolyl, alkylsulfonylcarbamoyl, sulfonic acid and phosphonic acid; R⁷ is selected from the group consisting of alkyl, alkanoyl, alkoxy, alkoxyalkyl, polyalkoxy, and alkoxyalkoxyalkyl optionally substituted with alkylamino, dialkylamino, alkylsulfonyl, sulfinic acid, sulfonic acid, pyridinium, imidazolinium, pyrimidinium, dialkylammonium and choline; and o is 0 to 4; with the proviso that the R⁷ group includes at least atoms in a continuous chain, wherein the chain is a carbon chain optionally interrupted with one or more oxygen, nitrogen or sulfur.

35. The compound according to claim 23, wherein the compound is 5-carboxy-4-decyloxy-2-hydroxybenzaldehyde;

5-carboxy-4-[4-decyloxybenzyloxy]-2-hydroxybenzaldehyde;

2-hydroxy-4-[l'-n-hexyloxy)ethoxy]-benzaldehyde;

2-hydroxy-4- [2 '-( 1 ',3 '-di-n-hexyloxy)propyloxy] -benzaldehyde;

2-hydroxy-4- [2'-( l " , 3 " -ditetradecanoxy-2 " -propoxy)- l '- ethoxyjbenzaldehyde;

2-hydroxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)-l '-ethoxy]- benzaldehyde-3-carboxylic acid;

2-hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-propoxy)-2'-hydroxy-l'- propoxy]benzaldehyde; 2-hydroxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)-l'-ethoxyethoxy]- benzaldehyde;

2-hydroxy-4-[3'-(l",3"-ditetradecanoxy-2"-ρropoxy)-2'-hydroxy-l - propoxy]benzaldehyde-3-carboxylic acid;

2-hydroxy-3-carboxy-4-[2'-(l",3"-ditetradecanoxy-2"-propoxy)-l - ethoxyethoxy]-benzaldehyde-3-carboxylic acid;

4-[4'-decanoylbenzyloxy]-2-hydroxybenzaldehyde;

4-(l-formyl-2-hydroxyphenyl)-N-dodecylcarbamate;

4-dodecyloxyethyloxy-2-hydroxybenzaldehyde;

4-(2-hydroxy-dodecyloxy)-2-hydroxybenzaldehyde; 4-(l',3'-didodecyloxy-propyl-2'-oxy)-2-hydroxybenzaldehyde; 4-[2-(2-octyl-dodecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde; 4-[2-(2-decyl-tetradecyloxy)-l-ethyloxy]-2-hydroxybenzaldehyde; 4-[2-(N,N-didecyl)ethyloxy]-2-hydroxybenzaldehyde; 4-(2-octyl-dodecyloxy)-2-hydroxybenzaldehyde ;

4-(2-decyl-tetradecyloxy)-2-hydroxybenzaldehyde; or 4-(l-octyl-decyloxy)-2-hydroxybenzaldehyde, or a physiologically acceptable salt or ester thereof.

36. A vaccine, comprising one or more antigens, and one ormore compounds as defined in any one of claims 23-35.

37. A method of vaccinating a human or an animal, comprising administering one or more antigens, and one or more compounds as defined in any one of claims 23-35.

38. A vaccine comprising one or more polynucleotide sequences encoding for one or more antigens, and one or more compounds as defined in any one of claims 23-35.

39. A method of vaccinating a human or an animal, comprising administering one ormore polynucleotide sequences encoding for one ormore antigens, and one or more compounds as defined in any one of claims 23-35.

40. A pharmaceutical composition comprising one or more of the compounds as defined in any one of claims 23-35, . and one or more pharmaceutically acceptable diluents, carriers or excipients.

41. The pharmaceutical composition according to claim 40, wherein the composition further comprises one or more immunologically effective antigens or one or more polynucleotides encoding for one or more antigens.

42. The pharmaceutical composition according to claim 41, wherein the compounds form liposomes.